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The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVΔB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVΔB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals.

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone of reducing risk for atherosclerotic cardiovascular disease. Despite the approval of nonstatin therapies for LDL-C lowering over the past 2 decades, these medications are underused, and most patients are still not at guideline-recommended LDL-C goals. Barriers include poor adherence, clinical inertia, concern for side effects, cost, and complex prior authorization processes. With atherosclerotic cardiovascular disease-related mortality increasing globally, there remains a need for additional therapeutic options for lowering LDL-C as part of an atherosclerotic cardiovascular disease prevention strategy. Following the identification of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a promising therapeutic target, inclisiran was developed using the natural process of RNA interference for robust, sustained prevention of hepatic PCSK9 synthesis. Twice-yearly maintenance subcutaneous inclisiran (following initial loading doses at Day 1 and Day 90) reduces circulating LDL-C levels by ≈50% versus placebo when added to maximally tolerated statins. Long-term safety and tolerability of inclisiran have been assessed, with studies underway to evaluate the effects of inclisiran on cardiovascular outcomes and to provide additional safety and effectiveness data. In 2021,

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems. [ABSTRACT FROM AUTHOR]

Background: Saline-alkaline water aquaculture has become a key way to mitigate the reduction of freshwater aquaculture space and meet the increasing global demand for aquatic products. To enhance the comprehensive utilization capability of saline-alkaline water, it is necessary to understand the regulatory mechanisms of aquatic animals coping with saline-alkaline water. In this study, our objective was to elucidate the function of proline metabolism in the alkaline adaptation of Nile tilapia (Oreochromis niloticus). Results: Expose Nile tilapia to alkaline water of different alkalinity for 2 weeks to observe changes in its growth performance and proline metabolism. Meanwhile, to further clarify the role of proline metabolism, RNA interference experiments were conducted to disrupt the normal operation of proline metabolic axis by knocking down pycr (pyrroline-5-carboxylate reductases), the final rate-limiting enzyme in proline synthesis. The results showed that both the synthesis and degradation of proline were enhanced under carbonate alkalinity stress, and the environmental alkalinity impaired the growth performance of tilapia, and the higher the alkalinity, the greater the impairment. Moreover, environmental alkalinity caused oxidative stress in tilapia, enhanced ion transport, ammonia metabolism, and altered the intensity and form of energy metabolism in tilapia. When the expression level of the pycr gene decreased, the proline metabolism could not operate normally, and the ion transport, antioxidant defense system, and energy metabolism were severely damaged, ultimately leading to liver damage and a decreased survival rate of tilapia under alkalinity stress. Conclusions: The results indicated that proline metabolism plays an important role in the alkaline adaptation of Nile tilapia and is a key regulatory process in various biochemical and physiological processes. [ABSTRACT FROM AUTHOR]

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation. [ABSTRACT FROM AUTHOR]

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. We show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the 'gold-standard' delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated. [Display omitted] [ABSTRACT FROM AUTHOR]

The Chinese cordyceps, a unique parasitic complex of Thitarodes/Hepialus ghost moths and Ophiocordyceps sinensis fungus in the Tibetan Plateau, is a highly valuable biological resource for medicine and health foods in Asian countries. Efficient system for artificial cultivation of Chinese cordyceps relies on understanding the gene functions involved in the induction of growing blastospores into hyphae in the larval hemolymph of insect host, during O. sinensis infection. Transcriptome analysis and ribonucleic acid interference (RNA interference) method were employed to identify the key differentially expressed genes and to demonstrate their functions in Thitarodes xiaojinensis. Key larval genes critical for O. sinensis blastospore development or filamentation were identified. Nine of the 20 top upregulated genes encoded cuticles proteins, indicating that these proteins highly activated when the larval hemolymph was full of blastospores. Small interfering RNA (siRNA) knockdown of five larval genes such as Flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 significantly stimulated the dimorphic transition from blastospores to prehyphae in O. sinensis in the larval hemolymph after 120 h after injection. The expressions of these genes determined by quantitative real-time PCR were suppressed in various levels from 38.64% to 91.54%, compared to the controls. These results demonstrated that injection of the siRNAs of key upregulated genes into the larval hemolymph containing high load of blastospores caused the gene silence in T. xiaojinensis larvae and induced the fungal transition from blastospores to prehyphae, providing novel knowledge on the regulation of O. sinensis fungal dimorphism by Thitarodes host and cues for further study of Thitarodes biology and commercial cultivation of Chinese cordyceps. [ABSTRACT FROM AUTHOR]

Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology. Functional analysis reveals that targeting testisspecific serine/threonine protein kinase (TSSKs) is a feasible avenue for controlling invasive pest Cydia pomonella through genetic sterile insect technique (gSIT) technology. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is one of the frequent tumors that seriously endanger the physical and mental well-being in women with strong heterogeneity, and its pathogenesis involves multiple risk factors. Depending on the type of BC, hormonal therapy, targeted therapy, and immunotherapy are the current systemic treatment options along with conventional chemotherapy. Despite significant progress in understanding BC pathogenesis and therapeutic options, there is still a need to identify new therapeutic targets and develop more effective treatments. According to recent sequencing and profiling studies, non-coding (nc) RNAs genes are deregulated in human cancers via deletion, amplification, abnormal epigenetic, or transcriptional regulation, and similarly, the expression of many ncRNAs is altered in breast cancer cell lines and tissues. The ability of single ncRNAs to regulate the expression of multiple downstream gene targets and related pathways provides a theoretical basis for studying them for cancer therapeutic drug development and targeted delivery. Therefore, it is far-reaching to explore the role of ncRNAs in tumor development and their potential as therapeutic targets. Here, our review outlines the potential of two major ncRNAs, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) as diagnostic and prognostic biomarkers as well as targets for new therapeutic strategies in breast cancer. [ABSTRACT FROM AUTHOR]

Endometriosis is characterized by the ectopic proliferation of endometrial cells, posing considerable diagnostic and therapeutic challenges. Our study investigates AGPAT4's involvement in endometriosis pathogenesis, aiming to unveil new therapeutic targets. Our investigation by analyzing eQTL data from GWAS for preliminary screening. Subsequently, within the GEO dataset, we utilized four machine learning algorithms to precisely identify risk-associated genes. Gene validity was confirmed through five Mendelian Randomization methods. AGPAT4 expression was measured by Single-Cell Analysis, ELISA and immunohistochemistry. We investigated AGPAT4's effect on endometrial stromal cells using RNA interference, assessing cell proliferation, invasion, and migration with CCK8, wound-healing, and transwell assays. Protein expression was analyzed by western blot, and AGPAT4 interactions were explored using AutoDock. Our investigation identified 11 genes associated with endometriosis risk, with AGPAT4 and COMT emerging as pivotal biomarkers through machine learning analysis. AGPAT4 exhibited significant upregulation in both ectopic tissues and serum samples from patients with endometriosis. Reduced expression of AGPAT4 was observed to detrimentally impact the proliferation, invasion, and migration capabilities of endometrial stromal cells, concomitant with diminished expression of key signaling molecules such as Wnt3a, β-Catenin, MMP-9, and SNAI2. Molecular docking analyses further underscored a substantive interaction between AGPAT4 and Wnt3a.Our study highlights AGPAT4's key role in endometriosis, influencing endometrial stromal cell behavior, and identifies AGPAT4 pathways as promising therapeutic targets for this condition. [ABSTRACT FROM AUTHOR]

Nucleic acid therapeutics are promising alternatives to conventional anti-cancer therapy, such as chemotherapy and radiation therapy. While conventional therapies have limitations, such as high side effects, low specificity, and drug resistance, nucleic acid therapeutics work at the gene level to eliminate the cause of the disease. Nucleic acid therapeutics treat diseases in various forms and using different mechanisms, including plasmid DNA (pDNA), small interfering RNA (siRNA), anti-microRNA (anti-miR), microRNA mimics (miRNA mimic), messenger RNA (mRNA), aptamer, catalytic nucleic acid (CNA), and CRISPR cas9 guide RNA (gRNA). In addition, nucleic acids have many advantages as nanomaterials, such as high biocompatibility, design flexibility, low immunogenicity, small size, relatively low price, and easy functionalization. Nucleic acid therapeutics can have a high therapeutic effect by being used in combination with various nucleic acid nanostructures, inorganic nanoparticles, lipid nanoparticles (LNPs), etc. to overcome low physiological stability and cell internalization efficiency. The field of nucleic acid therapeutics has advanced remarkably in recent decades, and as more and more nucleic acid therapeutics have been approved, they have already demonstrated their potential to treat diseases, including cancer. This review paper introduces the current status and recent advances in nucleic acid therapy for anti-cancer treatment and discusses the tasks and prospects ahead. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum is a widespread soil-borne fungal pathogen that can infect various plants, causing wilt and root rot diseases. The root rot disease of Atractylodes macrocephala caused by F. oxysporum is among the most serious diseases associated with continuous cropping, significantly hindering its sustainable development. In this study, we aimed to investigate the effect of exogenous application of double-stranded RNA (dsRNA) on silencing the F. oxysporum Tup1 gene to reduce its virulence and to evaluate its potential application in controlling root rot disease in A. macrocephala. The Tup1 gene was amplified from the F. oxysporum genome, and different lengths of Tup1-dsRNA were designed and synthesized. The uptake of dsRNA by the fungus was verified using Tup1-dsRNA labeled with fluorescein, and in vitro dsRNA treatment experiments were conducted to assess its impact on the growth and virulence of F. oxysporum. Additionally, Tup1-dsRNA was applied to the roots of A. macrocephala to evaluate its effectiveness in controlling root rot disease. The experimental results showed that F. oxysporum could effectively uptake exogenously applied Tup1-dsRNA, significantly reducing Tup1 gene expression. All lengths of Tup1-dsRNA inhibited fungal growth and caused morphological changes in the fungal hyphae. Further plant experiments and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis indicated that Tup1-dsRNA treatment significantly reduced the incidence of root rot disease in A. macrocephala, which was supported by the reduction in peroxidase (POD) and catalase (CAT) enzyme activities, malondialdehyde (MDA) content, and proline (Pro) levels in treated root tissues. This study demonstrated that exogenous dsRNA could reduce the virulence of F. oxysporum by silencing the Tup1 gene and effectively mitigate the root rot disease it causes in A. macrocephala. The successful application of Tup1-dsRNA provided strong evidence for the potential of RNA interference (RNAi) technology in plant disease control. Future research could further optimize the design and application of dsRNA to enhance its practical value in agriculture. [ABSTRACT FROM AUTHOR]

Background: Acute hepatic porphyria is a group of multisystem disorders of which acute intermittent porphyria is the most common subtype. Givosiran, a subcutaneously administered RNA interference therapeutic targeting liver ALAS mRNA, is approved for treating these disorders. This Phase 1/2 open-label extension study (NCT02949830) evaluated the long-term safety and efficacy of givosiran in adults with acute intermittent porphyria, with follow-up of up to 48 months, which is the longest follow-up of givosiran treatment to date. Participants were adults aged 18–65 years who completed part C of the Phase 1 givosiran study (NCT2452372). Methods: Enrollees received givosiran for up to 48 months. Primary and secondary endpoints included the incidence of adverse events, changes in urinary delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) levels, annualized rate of porphyria attacks, and annualized hemin use. Quality of life was assessed using the EQ-5D-5L instrument as an exploratory endpoint. Results: Sixteen patients (median age: 39.5 years) participated. Common adverse events included abdominal pain, nasopharyngitis, and nausea (50% each), with injection-site erythema (38%) and injection-site pruritus (25%) noted as frequent treatment-related reactions. Givosiran therapy reduced annualized rates of porphyria attacks and hemin use by 97% and 96%, respectively. From months > 33 to 48, all patients were free from attacks requiring significant medical intervention and did not use hemin. There were substantial reductions in median urinary ALA and PBG of 95% and 98%, respectively. Additionally, a clinically meaningful improvement in quality of life was observed. Conclusions: In the longest follow-up of givosiran-treated patients reported to date, the therapy maintained an acceptable safety profile and demonstrated sustained improvements in clinical outcomes over 4 years in patients with acute intermittent porphyria. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) shows great potential in plant defense against pathogens through RNA-mediated sequence-specific gene silencing. Among RNAi-based plant protection strategies, spray-induced gene silencing (SIGS) is considered a more promising approach because it utilizes the transfer of exogenous RNA between plants and microbes to silence target pathogen genes. The application of nanovesicles significantly enhances RNA stability and delivery efficiency, thereby improving the effectiveness of SIGS and further enhancing plant resistance to diseases and pathogens. This review explores the role of RNAi in plant protection, focusing on the cross-kingdom transport of small RNAs (sRNAs) via extracellular vesicles. It also explores the potential of nanotechnology to further optimize RNA-based plant protection, offering innovative tools and methods in modern plant biotechnology. [ABSTRACT FROM AUTHOR]

With the development of new technologies in recent years, researchers have made significant progress in crop breeding. Modern breeding differs from traditional breeding because of great changes in technical means and breeding concepts. Whereas traditional breeding initially focused on high yields, modern breeding focuses on breeding orientations based on different crops' audiences or by-products. The process of modern breeding starts from the creation of material populations, which can be constructed by natural mutagenesis, chemical mutagenesis, physical mutagenesis transfer DNA (T-DNA), Tos17 (endogenous retrotransposon), etc. Then, gene function can be mined through QTL mapping, Bulked-segregant analysis (BSA), Genome-wide association studies (GWASs), RNA interference (RNAi), and gene editing. Then, at the transcriptional, post-transcriptional, and translational levels, the functions of genes are described in terms of post-translational aspects. This article mainly discusses the application of the above modern scientific and technological methods of breeding and the advantages and limitations of crop breeding and diversity. In particular, the development of gene editing technology has contributed to modern breeding research. [ABSTRACT FROM AUTHOR]

Backgrounds: Neuropathic pain (NP) is a kind of chronic pain that has attracted much attention in clinical practice, characterized by high morbidity, complex mechanisms, and difficulties in clinical treatment, with which the activation of High mobility group box 1 (HMGB1) is closely related. The aim of this study was to investigate the effects of lentivirus-mediated RNA interference gene therapy targeting HMGB1 on neuropathic pain in rats with chronic dorsal root ganglion compression (CCD) and its specific mechanisms, so as to explore new pharmacological targets. Methods: Adult male Wistar rats were surgically subjected to chronic compression of the dorsal root ganglia (CCD). Behavioral tests were performed by calculating the paw withdrawal mechanical threshold (PWMT) and the thermal paw withdrawal latency (TPWL). Co-immunoprecipitation (CO-IP) was used to clarify protein interactions. Gene silencing was induced by injecting lentivirus expressing HMGB1 short hairpin RNA (shRNA) into rats. An LPS-inflammation-stimulated rat astrocyte model was established to validate the animal experiment results further. Western blot analysis and real-time quantitative PCR were used to detect pathway protein expression. Results: After first establishing the rat CCD model, both PWMT and PTWL were significantly reduced in rats, indicating that the model construction was successful. After lentiviral silencing of HMGB1 expression, NP was significantly alleviated in CCD rats. CO-IP experiments showed a link between HMGB1 and AQP1; After silencing HMGB1 expression, the expression of AQP1 was significantly reduced, and HMGB1 was able to modulate the effect of AQP1 on NP. Further use of an inhibitor of the HMGB1 receptor showed that after inhibition of RAGE, AQP1 was significantly reduced; HMGB1 may regulate AQP1 through its receptor RAGE to affect NP. Silencing of HMGB1 resulted in a significant decrease in NF-κB, and HMGB1 affects the inflammatory pathways it mediates. After silencing AQP1, NF-κB also decreased significantly, indicating that AQP1 is an upstream regulator of NF-κB. Conclusion: Lentivirus-mediated RNA interference (RNAi) silencing targeting HMGB1 may play a key role in the development of neuropathic pain in rats by regulating AQP1 expression via RAGE and ultimately activating NF-κB. [ABSTRACT FROM AUTHOR]

The ability of skeletal muscles to contract is derived from the unique genes and proteins expressed within muscles, most notably myofilaments and elastic proteins. Here we investigated the role of the sallimus (sls) gene, which encodes a structural homologue of titin, in regulating development, structure, and function of Drosophila melanogaster. Knockdown of sls using RNA interference (RNAi) in all body-wall muscle fibers resulted in embryonic lethality. A screen for muscle-specific drivers revealed a Gal4 line that expresses in a single larval body wall muscle in each abdominal hemisegment. Disrupting sls expression in single muscle fibers did not impact egg or larval viability nor gross larval morphology but did significantly alter the morphology of individual muscle fibers. Ultrastructural analysis of individual muscles revealed significant changes in organization. Surprisingly, muscle-cell specific disruption of sls also severely impacted neuromuscular junction (NMJ) formation. The extent of motor-neuron (MN) innervation along disrupted muscles was significantly reduced along with the number of glutamatergic boutons, in MNIs and MN-Ib. Electrophysiological recordings revealed a 40% reduction in excitatory junctional potentials correlating with the extent of motor neuron loss. Analysis of active zone (AZ) composition revealed changes in presynaptic scaffolding protein (brp) abundance, but no changes in postsynaptic glutamate receptors. Ultrastructural changes in muscle and NMJ development at these single muscle fibers were sufficient to lead to observable changes in neuromuscular transduction and ultimately, locomotory behavior. Collectively, the data demonstrate that sls mediates critical aspects of muscle and NMJ development and function, illuminating greater roles for sls/titin. [ABSTRACT FROM AUTHOR]

Background: Small RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions. Results: C. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs. Conclusions: We provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi. [ABSTRACT FROM AUTHOR]

Background: BCR-ABL is a constitutively active tyrosine kinase that stimulates multiple downstream signaling pathways to promote the survival and proliferation of chronic myeloid leukemia (CML) cells. The clinical application of specific BCR-ABL tyrosine kinase inhibitors (TKIs) has led to significantly improved prognosis and overall survival in CML patients compared to previous treatment regimens. However, direct targeting of BCR-ABL does not eradicate CML cells expressing T315I-mutated BCR-ABL. Our previous study revealed that inhibiting CREB binding protein (CBP) is efficacious in activating β-catenin/p300 signaling, promoting cell differentiation and inducing p53/p21-dependent senescence regardless of BCR-ABL mutation status. We hypothesize that the specific inhibition of CBP may represent a novel strategy to promote β-catenin/p300-mediated differentiation and suppress cancer cell proliferation for treating CML patients. Methods: The anticancer efficacy of PBA2, a novel CBP inhibitor, in CML cells expressing wild-type or T315I-mutated BCR-ABL was investigated in vitro and in vivo. Cell differentiation was determined by the nitroblue tetrazolium (NBT) reduction assay. The extent of cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-Gal) activity. Cytotoxicity was measured by MTS assay. RNA interference was performed to evaluate the cell proliferation effects of CBP knockdown. The interaction of β-catenin and CBP/p300 was examined by co-immunoprecipitation assay. Results: PBA2 exhibited significantly higher anticancer effects than imatinib in CML cells harboring either wild-type or T315I-mutated BCR-ABL both in vitro and in vivo. Mechanistically, PBA2 reduced CBP expression and promoted β-catenin-p300 interaction to induce cell differentiation and senescence. Conclusion: Our data supported the rational treatment of CML by inhibiting the β-catenin/CBP pathway regardless of BCR-ABL mutation status. [ABSTRACT FROM AUTHOR]

Introduction: Programmed death-ligand 1 (PD-L1) plays essential roles in the negative regulation of anti-tumor immunity. However, the regulatory mechanisms of PD-L1 expression need further exploration. MORC family CWtype zinc finger 3 (MORC3) is a transcriptional factor that regulates innate immune responses, but the expression and roles of MORC3 in cancers remain largely unknown. The present study explored the expression of MORC3 in cancers at both transcriptional and translational levels. Methods: The target genes and pathways were analyzed using RNA interference (RNAi), RNA sequencing (RNA-seq), and quantitative real-time polymerase chain reaction (qRT-PCR) technology in head and neck cancer cells. The expression of MORC3 and its target genes were also analyzed in single cancer cells. Results: MORC3 was significantly downregulated in multiple cancers, including head and neck cancer, and low expression of MORC3 was associated with poor overall survival. MORC3 knockdown significantly increased the expression of many immune-related genes, including interferon (IFN)-associated genes [MX dynamin like GTPase 2 (MX2), interferon induced protein with tetratricopeptide repeats 1 (IFIT1), interferon induced protein with tetratricopeptide repeats 2 (IFIT2), interferon regulatory factor 7 (IRF7), interferon regulatory factor 9 (IRF9), interferon induced protein 44 like (IFI44L), interferon induced transmembrane protein 1 (IFITM1), interferon induced transmembrane protein 3 (IFITM3), interferon induced protein 44 (IFI44), and interferon induced with helicase C domain 1 (IFIH1)]. MORC3 knockdown significantly upregulated PD-L1 and signal transducer and activator of transcription 1 (STAT1) expression. Moreover, the LINC00880 immune-related long non-coding RNA (lnc-RNA) was upregulated by MORC3 knockdown. Silencing LINC00880 attenuated PD-L1 expression. MORC3 knockdown also increased the expression of cellular proliferation-related genes and promoted cancer cell proliferation. Conclusion: The present study demonstrated that MORC3 regulates IFNassociated pathways and is a novel repressor of PD-L1 expression and cancer cell proliferation. [ABSTRACT FROM AUTHOR]

Salvia leucantha is a perennial herb of the genus Salvia in the family Labiatae, which has a wide range of biological activities, mainly including inhibition of acetylcholinesterase, antibacterial, and anti-inflammatory activity. To explore the protective effects and mechanism of action of S. leucantha on Alzheimer's disease (AD), the anti-AD activity of SLE (extracts of S. leucantha) was determined by using a transgenic Caenorhabditis elegans (C. elegans) model (CL4176). Analyses included paralysis assay, phenotypic experiments, transcriptome sequencing, RNA interference (RNAi), heat shock assays, and gas chromatography-mass spectrometry (GC-MS). SLPE (S. leucantha petroleum ether extract) could significantly delay CL4176 paralysis and extend the longevity of C. elegans N2 without harmful effects. A total of 927 genes were significantly changed by SLPE treatment in C. elegans, mainly involving longevity regulatory pathways—nematodes, drug metabolism—cytochrome P450, and glutathione metabolic pathways. RNAi showed that SLPE exerted its anti-AD activity through up-regulation of the gene gst-5; the most abundant compound in SLPE analyzed by GC-MS was 2,4-Di-tert-butylphenol (2,4-DTBP), and the compound delayed nematode paralysis. The present study suggests that active components in S. leucantha may serve as new-type anti-AD candidates and provide some insights into their biological functions. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) is a highly pathogenic avian infectious disease agent and also a promising oncolytic virus with broad application prospects. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery has been increasingly recognized for its crucial role in the life cycles of enveloped viruses, influencing processes such as viral entry, replication, and budding. In this study, we employed an RNA interference screening approach to identify key ESCRT components that regulate NDV replication in tumor cells. qPCR, immunofluorescence, and Western blot assays demonstrated that knockdown of HRS, CHMP4A, CHMP4B, and CHMP4C significantly impaired NDV replication in HeLa cells, with HRS exhibiting the most pronounced inhibitory effect. Additionally, HRS knockout significantly inhibited viral budding and suppressed NDV-induced cell death in HeLa cells. Notably, NDV infection was shown to significantly upregulate HRS gene and protein expression in a time-dependent manner. In conclusion, this study systematically identifies critical ESCRT components involved in NDV replication within tumor cells, with a particular focus on the role of HRS in promoting NDV's replication by promoting viral budding, offering new insights for the development of NDV-based oncolytic therapies. [ABSTRACT FROM AUTHOR]

Sirtuin 1 (SIRT1) is a key upstream regulator of lipid metabolism; however, the molecular mechanisms by which SIRT1 regulates milk fat synthesis in dairy goats remain unclear. This study aimed to investigate the regulatory roles of SIRT1 in modulating lipid metabolism in goat mammary epithelial cells (GMECs) and its impact on the adipose triglyceride lipase (ATGL) promoter activity using RNA interference (RNAi) and gene overexpression techniques. The results showed that SIRT1 is significantly upregulated during lactation compared to the dry period. Additionally, SIRT1 knockdown notably increased the expressions of genes related to fatty acid synthesis (SREBP1, SCD1, FASN, ELOVL6), triacylglycerol (TAG) production (DGAT2, AGPAT6), and lipid droplet formation (PLIN2). Consistent with the transcriptional changes, SIRT1 knockdown significantly increased the intracellular contents of TAG and cholesterol and the lipid droplet abundance in the GMECs, while SIRT1 overexpression had the opposite effects. Furthermore, the co-overexpression of SIRT1 and Forkhead box protein O1 (FOXO1) led to a more pronounced increase in ATGL promoter activity, and the ability of SIRT1 to enhance ATGL promoter activity was nearly abolished when the FOXO1 binding sites (FKH1 and FKH2) were mutated, indicating that SIRT1 enhances the transcriptional activity of ATGL via the FKH element in the ATGL promoter. Collectively, our data reveal that SIRT1 enhances the transcriptional activity of ATGL through the FOXO1 binding sites located in the ATGL promoter, thereby regulating lipid metabolism. These findings provide novel insights into the role of SIRT1 in fatty acid metabolism in dairy goats. [ABSTRACT FROM AUTHOR]

Summary: Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5′untranslated region (5′UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG).Here, we report an efficient gene silencing method by introducing a tailor‐designed uATG‐containing element (ATGE) into the 5′UTR of genes in plants, occupying the original start site to act as a new pATG.Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding.Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement. See also the Commentary on this article by Li & Wei, 243: 2052–2054. [ABSTRACT FROM AUTHOR]

Environmental stresses, including drought stress, seriously threaten food security. Previous studies reported that wheat F-box protein, TaFBA1, responds to abiotic stresses in tobacco. Here, we generated transgenic wheat with enhanced (overexpression, OE) or suppressed (RNA interference, RNAi) expression of TaFBA1. The TaFBA1-OE seedlings showed enhanced drought tolerance, as measured by survival rate and fresh weight under severe drought stress, whereas the RNAi plants showed the opposite phenotype. Furthermore, the OE plants had stronger antioxidant capacity compared to WT and RNAi plants and maintained stomatal opening, which resulted in higher water loss under drought stress. However, stronger water absorption capacity in OE roots contributed to higher relative water contents in leaves under drought stress. Moreover, the postponed stomatal closure in OE lines helped to maintain photosynthesis machinery to produce more photoassimilate and ultimately larger seed size. Transcriptomic analyses conducted on WT and OE plants showed that genes involved in antioxidant, fatty acid and lipid metabolism and cellulose synthesis were significantly induced by drought stress in the leaves of OE lines. Together, our studies determined that the F-box protein TaFBA1 modulated drought tolerance and affected yield in wheat and the TaFBA1 gene could provide a desirable target for further breeding of wheat. [ABSTRACT FROM AUTHOR]

Background: Hereditary transthyretin (ATTRv) amyloidosis, a multifaceted disorder affecting multiple systems, substantially diminishes patients' physical capabilities and overall quality of life. Patisiran and Vutrisiran, two Ribonucleic acid (RNA) interference therapies, target reducing both pathogenic and wildtype transthyretin (TTR) protein levels. This systematic review assesses the effectiveness and safety of these treatments in managing ATTRv. Methods: A comprehensive, thorough literature search across databases including Embase, PubMed, Web of Science, Cochrane Central, and Google Scholar yielded 858 studies. Following removing duplicate and irrelevant articles, 676 distinct studies underwent review. These studies, conducted on a global scale, encompassed a range of methodologies, including clinical trials and indirect treatment comparisons. Results: Ten studies, spanning a total population of 756 patients, were selected for in-depth analysis. Patisiran and Vutrisiran consistently demonstrated significant improvements in primary and secondary endpoints related to neuropathy, quality of life, and cardiac function. Both medications were welltolerated, with primarily mild to moderate adverse events. Indirect treatment comparison studies indicated Vutrisiran's superiority over Tafamidis in treating ATTRv amyloidosis. Conclusion: This systematic review recommends using Patisiran and Vutrisiran to treat ATTRv amyloidosis. The findings suggest that these RNA interference therapies improve neuropathy, quality of life, and cardiac symptoms. The results indicate sustained benefits over prolonged treatment, with satisfactory safety profiles. However, potential biases, conflicts of interest in the studies, and limited follow-up periods in some trials necessitate cautious interpretation. Future research should address these limitations and provide more robust evidence for the long-term efficacy and safety of Patisiran and Vutrisiran in ATTRv treatment. [ABSTRACT FROM AUTHOR]

Holometabolous insects undergo morphological remodeling from larvae to pupae and to adults with typical changes in the cuticle; however, the mechanism is unclear. Using the lepidopteran agricultural insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the transcription factor RUNT-like (encoded by Runt-like) regulates the development of the pupal cuticle via promoting a pupal cuticle protein gene (HaPcp) expression. The HaPcp was highly expressed in the epidermis and wing during metamorphosis and was found being involved in pupal cuticle development by RNA interference (RNAi) analysis in larvae. Runt-like was also strongly upregulated in the epidermis and wing during metamorphosis. Knockdown of Runt-like produced similar phenomena, a failure of abdomen yellow envelope and wing formation, to those following HaPcp knockdown. The insect molting hormone 20-hydroxyecdysonen (20E) upregulated HaPcp transcription via RUNT-like. 20E upregulated Runt-like transcription via nuclear receptor EcR and the transcription factor FOXO. Together, RUNT-like and HaPCP are involved in pupal cuticle development during metamorphosis under 20E regulation. Author summary: Holometabolous insects develop pupal cuticle during metamorphosis; however, the mechanism is unclear. Using the lepidopteran agricultural insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the transcription factor RUNT-like regulates a pupal cuticle protein gene, HaPcp, expression therefore regulates the development of the pupal cuticle under insect molting hormone 20-hydroxyecdysonen (20E) regulation. HaPcp is an insect specific gene without homologue in humans, and the insect Runt-like is different from vertebrate, too. This work deeply addresses the mechanism that 20E regulates development of insect pupal cuticle and presents new gene targets to explore the new growth regulators to control the pest. [ABSTRACT FROM AUTHOR]

Background: Acute cholecystitis, inflammation of the gallbladder, can lead to serious complications if not promptly diagnosed and managed. Conventional therapies have limitations, necessitating newer personalized approaches. This review examines recent advances transforming cholecystitis care. Diagnostically, molecular techniques like next-generation sequencing rapidly identify causative microbes from gallbladder specimens, enabling targeted antimicrobial therapy. Regarding treatment, phage therapy uses viruses to lyse pathogenic bacteria. RNA interference and CRISPR-Cas9 gene editing silence microbial virulence factors. Probiotics competitively exclude pathogens. Robotics and fluorescence imaging refine surgical techniques. Additional emerging modalities include biosensors detecting inflammatory mediators, regenerative gallbladder tissue engineering using stem cells, and artificial intelligence for real-time decision support. However, the optimal integration of novel technologies with current best practices remains unknown. Further research is needed to validate and optimize personalized diagnostics and therapeutics for cholecystitis. Short conclusions: Advances in next-generation sequencing, CRISPR gene editing, robotics, and other biotechnologies promise to transform the precision and personalized management of cholecystitis when thoughtfully implemented. However, controlled trials are still required to define optimal integration with conventional supportive care and antibiotics. [ABSTRACT FROM AUTHOR]

Background: Haem is essential but toxic for metazoan organisms. Auxotrophic nematodes can acquire sufficient haem from the environment or their hosts in the meanwhile eliminate or detoxify excessive haem through tightly controlled machinery. In previous work, we reported a role of the unique transporter protein HRG-1 in the haem acquisition and homeostasis of parasitic nematodes. However, little is known about the haem efflux and detoxification via ABC transporters, particularly the multiple drug resistance proteins (MRPs). Results: Here, we further elucidate that a member of the mrp family (mrp-3) is involved in haem efflux and detoxification in a blood-feeding model gastrointestinal parasite, Haemonchus contortus. This gene is haem-responsive and dominantly expressed in the intestine and inner membrane of the hypodermis of this parasite. RNA interference of mrp-3 resulted in a disturbance of genes (e.g. hrg-1, hrg-2 and gst-1) that are known to be involved in haem homeostasis and an increased formation of haemozoin in the treated larvae and lethality in vitro, particularly when exposed to exogenous haem. Notably, the nuclear hormone receptor NHR-14 appears to be associated the regulation of mrp-3 expression for haem homeostasis and detoxification. Gene knockdown of nhr-14 and/or mrp-3 increases the sensitivity of treated larvae to exogenous haem and consequently a high death rate (> 80%). Conclusions: These findings demonstrate that MRP-3 and the associated molecules are essential for haematophagous nematodes, suggesting novel intervention targets for these pathogens in humans and animals. [ABSTRACT FROM AUTHOR]

This document discusses the role of LOXL2 in promoting the occurrence and progression of glioma, a type of brain tumor. LOXL2 activates autophagy, a cellular process that promotes tumor growth and resistance to chemotherapy. The specific mechanisms of LOXL2's relationship with histological grade and autophagy's regulation of epithelial-mesenchymal transition (EMT) are still unclear and require further study. The findings suggest that targeting LOXL2 in combination with chemotherapy may be an effective treatment strategy for glioma and potentially other tumors. [Extracted from the article]

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3–KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3–KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling. The multidomain scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) can bind GTP-bound Ras and Rap small GTPases. Here the authors show that, by binding active KRAS, SHANK3 maintains oncogenic KRAS/MAPK/ERK signaling at an optimal level while its depletion in KRAS-mutant cancer cell lines results in ERK signalling overdose and impaired cell proliferation. [ABSTRACT FROM AUTHOR]

Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns—high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation. [ABSTRACT FROM AUTHOR]

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post‐ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC‐layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post‐ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)‐cAMP‐PKA‐CREB signaling cascade, is necessary for mGC‐layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC‐layer stiffening", shedding light on the mechanism that prevents mGC escape from the post‐ovulatory follicle. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood. Methods: Public genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1β, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML. Results: Elevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms. Conclusion: Our findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients. [ABSTRACT FROM AUTHOR]

ABSTRACT Soybean rust (SBR), caused by an obligate biotrophic pathogen Phakopsora pachyrhizi, is a devastating disease of soybean worldwide. However, the mechanisms underlying plant invasion by P. pachyrhizi are poorly understood, which hinders the development of effective control strategies for SBR. Here we performed detailed histological characterization on the infection cycle of P. pachyrhizi in soybean and conducted a high‐resolution transcriptional dissection of P. pachyrhizi during infection. This revealed P. pachyrhizi infection leads to significant changes in gene expression with 10 co‐expressed gene modules, representing dramatic transcriptional shifts in metabolism and signal transduction during different stages throughout the infection cycle. Numerous genes encoding secreted protein are biphasic expressed, and are capable of inhibiting programmed cell death triggered by microbial effectors. Notably, three co‐expressed P. pachyrhizi apoplastic effectors (PpAE1, PpAE2, and PpAE3) were found to suppress plant immune responses and were essential for P. pachyrhizi infection. Double‐stranded RNA coupled with nanomaterials significantly inhibited SBR infection by targeting PpAE1, PpAE2, and PpAE3, and provided long‐lasting protection to soybean against P. pachyrhizi. Together, this study revealed prominent changes in gene expression associated with SBR and identified P. pachyrhizi virulence effectors as promising targets of RNA interference‐based soybean protection strategy against SBR. [ABSTRACT FROM AUTHOR]

Cotton plays a crucial role in shaping Indian economy and rural livelihoods. The cotton crop is prone to numerous insect pests, necessitating insecticidal application, which increases production costs. The advent of the expression of Bacillus thuringiensis (Bt) insecticidal protein in cotton has significantly reduced the burden of pest without compromising environmental or human health. After the introduction of transgenic cotton, the cultivated area expanded to 22 million hectares, with a 64% increase in adoption by farmers worldwide. Currently, Bt cotton accounts for 93% of the cultivated cotton area in India. However, extensive use of Bt cotton has accelerated resistance development in pests like the pink bollworm. Furthermore, the overreliance on Bt cotton has reduced the use of broad-spectrum pesticides, favouring the emergence of secondary pests with significant challenges. This emphasizes the urgent necessity for developing novel pest management strategies. The high-dose and refuge strategy was initially effective for managing pest resistance in Bt cotton, but its implementation in India faced challenges due to misunderstandings about the use of non-Bt refuge crops. Although gene pyramiding was introduced as a solution, combining mono toxin also led to instances of cross-resistance. Therefore, there is a need for further exploration of biotechnological approaches to manage insect resistance in Bt cotton. Advanced biotechnological strategies, such as sterile insect release, RNA interference (RNAi)-mediated gene silencing, stacking Bt with RNAi, and genome editing using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas), offer promising tools for identifying and managing resistance genes in insects. Additionally, CRISPR-mediated gene drives and the development of novel biopesticides present potential avenues for effective pest management in cotton cultivation. These innovative approaches could significantly enhance the sustainability and efficacy of pest resistance management in Bt cotton. [ABSTRACT FROM AUTHOR]

Background and purpose: Hepatocellular carcinoma (HCC) is a major disease seriously threatening human health. Poly (ADP-ribose) polymerase-1 (PARP1) is an enzyme that catalyzes poly ADP-ribosylation. Given the role of PARP1 in DNA damage repair, it is generally considered as an oncogene. However, the expression of PARP1 and its mechanism in HCC are not yet clear. This study aimed to investigate the role of PARP1 in the occurrence and development of HCC and its potential mechanisms. Methods: First, we analyzed the expression pattern of PARP1 in The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) HCC database, and identified the expression trend of PARP1 in our HCC cohort using real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. Then, the enzyme activity of PARP1 was inhibited by PJ34, an inhibitor of PARP1 and the expression of PARP1 in HCC cell lines was downregulated with small RNA interference technology. Based on these models, the following experiments were conducted: First, the effect of PARP1 on cell viability was assessed by cell counting kit-8 (CCK-8) assay and flow cytometry; Second, the expression levels of stemness-related genes in HCC cells were identified using RTFQ-PCR; Third, the effect of inhibition of PARP1 on migration and invasion of HCC cells was detected by migration and invasion assay (transwell assay). Finally, bioinformatic analysis was performed to identify new target genes and the pathways regulated by PARP1 in HCC progression. Rescue experiments were performed to determine whether PARP1 target genes were involved in the malignant phenotypes of HCC cells. Results: The expression of PARP1 was significantly up-regulated in HCC tissues in both TCGA and CPTAC database. RTFQ-PCR and Western blot assays showed that PARP1 was obviously up-regulated in HCC tissues compared to paracancerous tissues. Survival analysis showed that PARP1 expression was significantly negatively correlated with the prognosis of patients. The results of CCK-8, flow cytometry, RTFQ-PCR and transwell assay indicated that inhibition of PARP1 attenuated proliferation and activity of HCC cells, as well as weakened their stemness, migration and invasion. Bioinformatics analysis suggested that PARP1-regulated genes were enriched in the nuclear factor-κB (NF-κB) and necroptosis pathways, with POU class homeobox 2 (POU2F2) potentially being a target gene of PARP1. Correlation analysis, along with RTFQ-PCR and Western blot detection, confirmed that the expression of POU2F2 was regulated by PARP1, while not affected by PJ34, indicating the effect of nonenzymatic function of PARP1 on POU2F2. CCK-8, flow cytometry and RTFQ-PCR results showed that the reintroduction of POU2F2 enhanced proliferative capacity, increased activity, and promoted stemness of HCC cell lines with PARP1 knockdown. Conclusion: By positively regulating the expression of POU2F2, PARP1 promotes malignant phenotypes of HCC cells, providing new insights for clinical treatment and drug development for HCC. [ABSTRACT FROM AUTHOR]

Background: Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. Methods: The mouse model of IRI‐AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation‐mass spectrometry was performed to identify potential ANKRD1‐interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. Results: First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno‐associated virus (rAAV9)‐mitigated ischaemia/reperfusion‐induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl‐coenzyme A synthetase long‐chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti‐ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post‐translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63‐linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. Conclusions: Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI‐AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. Key Points/Highlights: Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro.ANKRD1 knockdown mitigates kidney damage and preserves renal function.Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI.ANKRD1 promotes acyl‐coenzyme A synthetase long‐chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway.The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1‐mediated ubiquitination of ACSL3. [ABSTRACT FROM AUTHOR]

DICER1 syndrome, a rare autosomal dominant genetic disorder, stems from mutations in the DICER1 gene, disrupting RNA interference and leading to various tumors. These tumors, affecting organs like the lung, kidney, ovaries, and brain, pose diagnostic challenges due to diverse presentations. Understanding DICER1-associated tumors, including pleuropulmonary blastoma, ovarian Sertoli–Leydig cell tumors, and others, is vital for early detection and management. Surgical resection, chemotherapy, and targeted therapies are primary treatment modalities, with genetic counseling playing a crucial role. Multidisciplinary care is essential for optimal management, offering hope for improved outcomes in affected individuals. [ABSTRACT FROM AUTHOR]

The relationship of Amyotrophic Lateral Sclerosis, Parkinson's disease, and other age-related neurodegenerative diseases with mitochondrial dysfunction has led to our study of the mitochondrial fission gene Drp1 in Drosophila melanogaster and aspects of aging. Previously, the Drp1 protein has been demonstrated to interact with the Drosophila Bcl-2 mitochondrial proteins, and Drp1 mutations can lead to mitochondrial dysfunction and neuronal loss. In this study, the Dopa decarboxylase-Gal4 (Ddc-Gal4) transgene was exploited to direct the expression of Drp1 and Drp1-RNAi transgenes in select neurons. Here, the knockdown of Drp1 seems to compromise locomotor function throughout life but does not alter longevity. The co-expression of Buffy suppresses the poor climbing induced by the knockdown of the Drp1 function. The consequences of Drp1 overexpression, which specifically reduced median lifespan and diminished climbing abilities over time, can be suppressed through the directed co-overexpression of pro-survival Bcl-2 gene Buffy or by the co-knockdown of the pro-cell death Bcl-2 homologue Debcl. Alteration of the expression of Drp1 acts to phenocopy neurodegenerative disease phenotypes in Drosophila, while overexpression of Buffy can counteract or rescue these phenotypes to improve overall health. The diminished healthy aging due to either the overexpression of Drp1 or the RNA interference of Drp1 has produced novel Drosophila models for investigating mechanisms underlying neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Simple Summary: The control of insect pest species, mainly belonging to Orthoptera, Hemiptera, and Coleoptera orders, can be based on novel emerging species-specific pesticides. These consist of dsRNA molecules delivered by feeding to insect larvae or adults, which suppress vital gene functions by RNA-RNA sequence complementarity and RNA interference. However, fewer studies of dsRNA feeding have been performed in dipteran pest insects. Two studies in Orthoptera and Coleoptera species have shown that suppressing intestinal enzymes degrading exogenous dsRNA can improve insect mortality rates. Ceratitis capitata (Tephritidae), the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on beneficial pollinators. Previous attempts to induce mortality by adult dsRNA feeding in this and other Tephritidae species, such as Bactrocera tryoni and B. dorsalis, showed some effectiveness, but were often limited. We improved this method by simultaneously silencing two intestinal nucleases and a vital gene. We found a mix of three dsRNAs able to induce much higher mortality (79%) within one week, following only three days of adult feeding. Ceratitis capitata, known as the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A more sustainable and species-specific alternative strategy may be based on double-stranded RNA (dsRNA) delivery through feeding to disrupt essential functions in pest insects, which is poorly reported in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by specific nucleases in the intestinal lumen is among the major obstacles to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of the ATPase vital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness. In contrast, by simultaneously feeding dsRNA against the CcVha68-1, CcdsRNase1, and CcdsRNase2 genes, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest management. [ABSTRACT FROM AUTHOR]

Simple Summary: Hippodamia variegata is a key natural enemy of cotton-growing regions in northwestern China. In this study, transcriptome sequencing analysis was performed on H. variegata, after beetles were exposed to different temperatures (from 32 to 38 °C) for 24 h, using high-throughput sequencing technology. We found the largest number of differentially expressed genes (DEGs) in the 35 °C vs. 32 °C group (1151); the fewest DEGs were found in the 38 °C vs. 35 °C group (901), indicating that H. variegata expressed the largest number of newly mobilized genes under medium-high temperature (35 °C). Gene functional analysis showed that the DEGs were mainly involved in "Catalytic activity", "Oxidoreductase activity", "Metabolic pathways", and "Longevity regulating pathway-multiple species" gene groups. The results from qRT-PCR were consistent with the transcriptome data, confirming their reliability. Finally, the RNAi results showed that adult survival, longevity, and fecundity were lower in the group in which gene expression of the heat shock proteins (Hsp70-01 and Hsp68) was suppressed than in the control group (injection ds-GFP) at all the experimental temperatures (32, 35, and 38 °C). Our results indicate the important role of the heat shock proteins (Hsp70-01 and Hsp68) in resistance to high-temperature stress in H. variegata and provide a molecular basis for analyzing the thermotolerance mechanism of H. variegata. Previous studies have shown that the survival and reproduction of Hippodamia variegata are increasingly harmed by progressive increases in temperature (from 32 °C to 35 °C and 38 °C). In this study, transcriptome sequencing analysis was performed on H. variegata, after being exposed to different temperatures (from 32 to 38 °C) for 24 h, using high-throughput sequencing technology. We found the largest number of differentially expressed genes (DEGs) in the 35 °C vs. 32 °C group (1151) followed by the 38 °C vs. 32 °C group (1054) and then the 38 °C vs. 35 °C group (901), indicating that H. variegata expressed the largest number of newly mobilized genes under medium-high temperature (35 °C). Gene functional analysis showed that a large number of DEGs were involved in "Catalytic activity", "Oxidoreductase activity", "Metabolic pathways", and "Longevity regulating pathway-multiple species" gene groups. We randomly selected nine DEGs for validation using qRT-PCR. The results of qRT-PCR were consistent with the transcriptome data, confirming their reliability. Finally, the RNAi results showed that adult survival, longevity, and fecundity were lower in the group in which gene expression of the heat shock proteins (Hsp70-01 and Hsp68) was suppressed than in the control group (injection ds-GFP) at all the experimental temperatures (32, 35, and 38 °C). Our results indicate the important role of the heat shock proteins (Hsp70-01 and Hsp68) in resistance to high-temperature stress in H. variegata and provide a molecular basis for analyzing its thermotolerance mechanism. [ABSTRACT FROM AUTHOR]

Simple Summary: Pyridaben is an efficient, low-toxicity, broad-spectrum insecticide that impairs the predatory capabilities of predatory mites, but the specific mechanisms that affect the predatory functions remain underexplored. This study elucidated that pyridaben significantly diminished the predatory efficiency and searching behavior of the predatory mite Neoseiulus womersleyi. A gene linked to olfactive functions, NwNPC2a, was cloned from N. womersleyi. Post-treatment with pyridaben at LC30 and LC50 concentrations resulted in a substantial downregulation of NwNPC2a expression. Silencing NwNPC2a in N. womersleyi females led to significant reductions in predatory functions. The decrease in predatory performance at LC30 and LC50 concentrations was attributable to the suppression of NwNPC2a gene expression. Thus, the underlying molecular mechanism through which pyridaben compromises the predatory function of N. womersleyi likely involves the downregulation of NwNPC2a expression. Pyridaben is a widely utilized, broad-spectrum contact acaricide, which has notable sublethal effects that impair the predatory capabilities of predatory mites, but the specific mechanisms that affect the predatory functions remain underexplored. When predatory mites hunt for prey, they may rely on Niemann–Pick-type C2 (NPC2) proteins to collect herbivore-induced plant volatiles (HIPVs) and other odor molecules to locate and pursue their prey. This study elucidated that pyridaben significantly diminished the predatory efficiency and searching behavior of the predatory mite Neoseiulus womersleyi. Key metrics, including predatory capacity (a/Th) and predation rate (a) on various developmental stages of Tetranychus urticae, were markedly reduced in treated mites compared to controls. The searching efficiency (S) also declined proportionally with the increased sublethal dose of pyridaben. A gene linked to olfactive functions, NwNPC2a, was cloned from N. womersleyi. Post-treatment with pyridaben at LC30 and LC50 concentrations resulted in a substantial downregulation of NwNPC2a expression by 60.15% and 58.63%, respectively. Silencing NwNPC2a in N. womersleyi females led to significant reductions in the attack rate (a), handling time (Th), predation efficiency (a/Th), and maximum predation rate (1/Th). The searching efficiency (S) was also lower than that of the control group, displaying a slight decline with the increasing prey density. The findings revealed that pyridaben exerted inhibitory effects on both the predatory function and searching efficiency of N. womersleyi populations. The decrease in predatory performance at LC30 and LC50 concentrations was attributable to the suppression of NwNPC2a gene expression. RNA interference (RNAi) studies corroborated that the NwNPC2a gene plays a critical role in the predation process of N. womersleyi. Thus, the underlying molecular mechanism through which pyridaben compromises the predatory function of N. womersleyi likely involves the downregulation of NwNPC2a expression. [ABSTRACT FROM AUTHOR]

Simple Summary: Insects rely on innate immunity to defend against invading pathogens in nature. BmToll9-1 is a receptor in the immune pathway of silkworms. BmToll9-1 is abundantly present in the midgut tissue of silkworm larvae. Silencing of BmToll9-1 results in smaller larval and cocoon bodies. Concomitantly, the expression of most downstream genes in the immune pathways is reduced. The antibacterial activity of hemolymph is also inhibited. The findings reveal that BmToll9-1 plays a positive role in regulating the development and immune response in silkworm. Toll receptors are involved in the development and innate immunity of insects. BmToll9-1 is an important immune receptor in the Toll pathway. Previous studies have focused on its role as a receptor in immune response. In this study, we aimed to investigate the role of BmToll9-1 as a regulator in the immune response. The expression profiles demonstrated that BmToll9-1 was predominantly expressed in the midgut. RNA interference (RNAi) of BmToll9-1 was found to be effective in the midgut via the injection of dsRNA, which resulted in smaller and lighter larvae and cocoons. Most signaling genes in the Toll pathway and downstream effector genes were downregulated after the RNAi of BmToll9-1. The hemolymph from BmToll9-1-silenced larvae showed decreased antibacterial activity against Escherichia coli, either in growth curve or inhibition zone experiments. The above results indicate that BmToll9-1 might be positively involved in the immune pathway of silkworm. As a positive regulator, BmToll9-1 might function mainly in the gut to maintain microbial homeostasis to regulate the growth of silkworms. Silencing of BmToll9-1 downregulates the signaling genes in the Toll pathway and antimicrobial peptide (AMP) production, resulting in decreased antibacterial activity in the hemolymph. [ABSTRACT FROM AUTHOR]

Biological barriers remain a major obstacle for the development of innovative therapeutics. Depending on a disease's pathophysiology, the involved tissues, cell populations, and cellular components, drugs often have to overcome several biological barriers to reach their target cells and become effective in a specific cellular compartment. Human biological barriers are incredibly diverse and include multiple layers of protection and obstruction. Importantly, biological barriers are not only found at the organ/tissue level, but also include cellular structures such as the outer plasma membrane, the endolysosomal machinery, and the nuclear envelope. Nowadays, clinicians have access to a broad arsenal of therapeutics ranging from chemically synthesized small molecules, biologicals including recombinant proteins (such as monoclonal antibodies and hormones), nucleic-acid-based therapeutics, and antibody-drug conjugates (ADCs), to modern viral-vector-mediated gene therapy. In the past decade, the therapeutic landscape has been changing rapidly, giving rise to a multitude of innovative therapy approaches. In 2018, the FDA approval of patisiran paved the way for small interfering RNAs (siRNAs) to become a novel class of nucleic-acid-based therapeutics, which—upon effective drug delivery to their target cells—allow to elegantly regulate the post-transcriptional gene expression. The recent approvals of valoctocogene roxaparvovec and etranacogene dezaparvovec for the treatment of hemophilia A and B, respectively, mark the breakthrough of viral-vector-based gene therapy as a new tool to cure disease. A multitude of highly innovative medicines and drug delivery methods including mRNA-based cancer vaccines and exosome-targeted therapy is on the verge of entering the market and changing the treatment landscape for a broad range of conditions. In this review, we provide insights into three different disease entities, which are clinically, scientifically, and socioeconomically impactful and have given rise to many technological advancements: acquired immunodeficiency syndrome (AIDS) as a predominant infectious disease, pancreatic carcinoma as one of the most lethal solid cancers, and hemophilia A/B as a hereditary genetic disorder. Our primary objective is to highlight the overarching principles of biological barriers that can be identified across different disease areas. Our second goal is to showcase which therapeutic approaches designed to cross disease-specific biological barriers have been promising in effectively treating disease. In this context, we will exemplify how the right selection of the drug category and delivery vehicle, mode of administration, and therapeutic target(s) can help overcome various biological barriers to prevent, treat, and cure disease. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a process in which small RNA molecules (such as small interfering RNAs or siRNAs) bind to specific messenger RNAs (mRNAs), leading to its degradation and inhibition of protein synthesis. Our studies have shown that RNAi can effectively silence genes involved in the replication of the Chikungunya virus (CHIKV) in cells. However, these investigations were performed only in laboratory settings and have yet to be tested in human clinical trials. Researchers need to conduct more research to determine the safety and efficacy of RNAi-based therapies as a therapeutic agent to treat viral infections. In this review, the history of evolution of siRNA as an inhibitor of protein synthesis, along with its current developments, is discussed based on our experience. Moreover, this review examines the hurdles and future implications associated with siRNA based therapeutic approaches. [ABSTRACT FROM AUTHOR]