Silence is not always golden: A closer look at potential environmental and ecotoxicological impacts of large-scale dsRNA application.

RNA interference (RNAi) technology has emerged as a pivotal strategy in sustainable pest management, offering a targeted approach that significantly mitigates the environmental and health risks associated with traditional insecticides. Originally implemented through genetically modified organisms (GMOs) to produce specific RNAi constructs, the technology has evolved in response to public and regulatory concerns over GMOs. This evolution has spurred the development of non-transgenic RNAi applications such as spray-induced gene silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence pest genes directly without altering the plant's genetic makeup. Despite its advantages in specificity and reduced ecological footprint, SIGS faces significant obstacles, particularly the instability of dsRNA in field conditions, which limits its practical efficacy. To overcome these limitations, innovative delivery mechanisms have been developed. These include nanotechnology-based systems, minicells, and nanovesicles, which are designed to protect dsRNA from degradation and enhance its delivery to target organisms. While these advancements have improved the stability and application efficiency of dsRNA, comprehensive assessments of their environmental safety and the potential for increased exposure risks to non-target organisms remain incomplete. This comprehensive review aims to elucidate the environmental fate of dsRNA and evaluate the potential risks associated with its widespread application on non-target organisms, encompassing soil microorganisms, beneficial insects, host plants, and mammals. The objective is to establish a more refined framework for RNAi risk assessment within environmental and ecotoxicological contexts, thereby fostering the development of safer, non-transgenic RNAi-based pest control strategies.

TAGAP activates Th17 cell differentiation by promoting RhoA and NLRP3 to accelerate rheumatoid arthritis development.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that can give rise to joint swelling and inflammation, potentially affecting the entire body, closely linked to the state of T cells. The T-cell activation Rho GTPase activating protein (TAGAP) is associated with many autoimmune diseases including RA and is directly linked to the differentiation of Th17 cells. The present study intends to investigate the influence of TAGAP on the RA progression and its mechanism to empower new treatments for RA. A collagen-induced-arthritis (CIA) rat model was constructed, as well as the extraction of CD4+ T cells. RT-qPCR, H&E staining and safranin O/fast green staining revealed that TAGAP interference reduced TAGAP production in the ankle joint of CIA rats, and joint inflammation and swelling were alleviated, which reveals that TAGAP interference reduces synovial inflammation and cartilage erosion in the rat ankle joint. Expression of inflammatory factors (TNF-α, IL-1ß, and IL-17) revealed that TAGAP interference suppressed the inflammatory response. Expression of pro-inflammatory cytokines, matrix-degrading enzymes, and anti-inflammatory cytokines at the mRNA level was detected by RT-qPCR and revealed that TAGAP interference contributed to the remission of RA. Mechanistically, TAGAP interference caused a significant decrease in the levels of RhoA and NLRP3. Assessment of Th17/Treg levels by flow cytometry revealed that TAGAP promotes Th17 cells differentiation and inhibits Treg cells differentiation in vitro and in vivo. In conclusion, TAGAP interference may decrease the differentiation of Th17 cells by suppressing the expression of RhoA and NLRP3 to slow down the RA progression.

Study on the relationship of Hsp70 with the temperature sensitivity of pedunsaponin A poisoning Pomacea canaliculata.

Previous studies have found that temperature influences molluscicidal the activity of pedunsaponin A (PA), which may be related to the expression of Hsp70, a cold-tolerance gene in Pomacea canaliculata. We determined the temperature effect of PA and the relationship between Hsp70 and temperature sensitivity of P. canaliculata poisoned by PA. Toxicity tests resulted in LC50 values of 17.7239 mg⋅L-1 at 10 °C, which decreased to 2.5774 mg⋅L-1 at 30 °C, implying a positive correlation between toxicity of PA and temperature. After Hsp70 being interfered, the mortality rate of P. canaliculata treated with PA for 72 h was 70%, which was significantly higher than that of snails treated with PA for 72 h without interfering (56.7%). Meanwhile, immune enzyme activities such as SOD, ACP and AKP were significantly increased in the interfered group and expression level of PcAdv in the gill was also significantly increased. These results suggest that deletion of Hsp70 promotes the activation of some immune enzymes of P. canaliculata and elevates the content of target proteins to cope with the dual stresses of low temperatures and molluscicides. These findings indicate that the Hsp70 plays an important role in influencing the temperature sensitivity of P. canaliculata when treated with PA.

Polycatechol-Derived Mesoporous Polydopamine Nanoparticles for Combined ROS Scavenging and Gene Interference Therapy in Inflammatory Bowel Disease.

Benefiting from the evolution of nanotechnology, the combination therapy by gene interference and reactive oxygen species (ROS) scavenging are expected, which holds great potential in inflammatory bowel disease (IBD) therapy. However, the functional integration of different therapeutic modules through interface modification of gene vectors for safe and efficient treatment is urgently needed. Herein, we present a catechol chemistry-mediated core-shell nanoplatform for ROS scavenging-mediated oxidative stress alleviation and siRNA-mediated gene interference in a dextran sulfate sodium (DSS)-induced colitis model. The nanoplatform is constructed by employing mesoporous polydopamine nanoparticles (MPDA NPs) with surface modification of amines as the porous core for TNF-α-siRNA loading (31 wt %) and exerts an antioxidant function, while PDA-induced biomineralization of the calcium phosphate (CaP) coating is used as the pH-sensitive protective shell to prevent siRNA from premature release. The CaP layer degraded under weakly acidic subcellular conditions (lysosomes); thus, the synergistic integration of catechol and cation moieties on the exposed surface of MPDA resulted in an efficient lysosomal escape. Subsequently, effective ROS scavenging caused by the electron-donating ability of MPDA and efficient knocking down (40.5%) of tumor necrosis factor-α (TNF-α) via sufficient cytosolic gene delivery resulted in a synergistic anti-inflammation therapeutic effect both in vitro and in vivo. This work establishes the first paradigm of synergistic therapy in IBD by ROS scavenging and gene interference.

Identification and functional verification of the target protein of pedunsaponin A in the gills of Pomacea canaliculata.

BACKGROUND: Based on previous research indicating that pedunsaponin A (PA) can destroy the gills of Pomacea canaliculata, we chose the gill as the main research object, and identified the target protein of PA in the gills of P. canaliculata through proteomics and RNA interference (RNAi). RESULTS: Proteomics showed that 180 proteins were downregulated after PA treatment in P. canaliculata. Among them, we chose advillin (PcAdv), receptor type tyrosine protein phosphatase (PcRT) and unconventional myosin heavy chain 6 (PcUM) as candidate target proteins through bioinformatics analysis. The small interfering RNA (siRNA) with the best interference effect was identified through further screening. Gene interference rates were 97%, 98% and 82% for PcAdv, PcRT and PcUM, respectively. The results showed that after RNAi treatment, the mortality of P. canaliculata treated with PcAdv (60.0%) was significantly lower than that for the control (93.3%); histological analysis showed that the structure of the gill was intact, cilia shedding was reduced, and the survival rate of hemocytes had increased. CONCLUSION: These findings indicate that, when the protein was absent or suppressed, the channel for entry of PA into the hemocytes of P. canaliculata was blocked, which reduced PA binding to hemocytes, and that there is a close relationship between shedding of gill cilia and PA entry into hemocytes. PcAdv is thus the key protein in PA destruction of gill cilia. Locating the proteins in gills that interact with drugs and investigating their mode of action is of great importance in the development of new molluscicides to control P. canaliculata populations.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

CaMKKß regulates proliferation, apoptosis, and glycolysis of hepatocellular carcinoma via PI3K/AKT pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignancy of liver cancer. Calcium ions/calmodulins stimulate protein kinase kinases ß (CaMKKß) is a multifunctional protein kinase that is overexpressed in many types of cancer. This study aims to investigate the effect of CaMKKß interference on HCC in HepG2 cells and transplanted tumor mice. METHODS: CaMKKß gene was knocked out in HepG2 cells as an experimental group, empty vector lentivirus as a negative control (NC) group, and untreated HepG2 cells as a control group. Cell proliferation, cell cycle, apoptosis, invasion, and glycolysis potential assays were conducted, respectively. In addition, the expression of PI3K, p-PI3K, AKT, and p-AKT was quantified by Western blot. Finally, the effect of CaMKKß in vivo was investigated using a xenograft model. RESULTS: CaMKKß knockdown significantly suppressed HepG2 cell proliferation, cell cycle, invasion, EMT, and glycolysis, promoted cell apoptosis, and reduced the expression of hexokinase 2 (HK2), pyruvate kinase M (PKM2), and lactate dehydrogenase A (LDHA), p-PI3K, and p-AKT. Post the addition of AKT highly expression plasmid, glucose uptake, lactic acid production, and cell proliferation decreased, accompanied by an increase in apoptosis, which were substantially reversed. Notably, xenograft model experiments in vivo also confirmed that CaMKKß knockdown inhibited HCC growth. CONCLUSIONS: CaMKKß knockdown inhibited cell proliferation, invasion, and glycolysis through the PI3K/AKT pathway, heightened apoptosis, thus promoting the development of HCC. This might be a potential target for the diagnosis and treatment of HCC.

Cloning and characterization of the target protein subunit lst8 of rapamycin in Apostichopus japonicus.

Autophagy plays an important role in the immune defense systems of vertebrates through the interaction between the lethal with SEC13 protein 8 (lst8) and the mechanistic target of rapamycin. In the present study, a novel invertebrate lst8 homologue is identified from Apostichopus japonicus (designated as Ajlst8) via polymerase chain reaction. The full-length complementary DNA of Ajlst8 comprises a 5'-untranslated region (UTR) of 78 base pair (bp), a 3'-UTR of 479 bp, and a putative open reading frame of 951 bp; hence, 316 amino acids are encoded. Structural analysis shows that the deduced amino acid of Ajlst8 shares six typical WD40 domains (28 aa-248 aa). Spatial expression analysis indicates that Ajlst8 is ubiquitously expressed in all the examined tissues, with a larger magnitude in coelomocytes. Vibrio splendidus infection in vivo and lipopolysaccharide exposure in vitro can significantly upregulate the messenger RNA expression of Ajlst8 by 2.39-fold and 1.93-fold compared with the control group, respectively. LPS exposure could also significantly induced the protein level of Ajlst8 to 2.38-fold and the autophagy level was markedly increased by 3.08-fold under same condition. The RNA interference of Ajlst8 in primary coelomocytes also reduces the relative expression of autophagy with a 0.71-fold decrease in the ratio of LC3-II/LC3-I compared with that in the control group. These results indicate that Ajlst8 is a novel immune regulator that may be involved in the antibacterial response process of sea cucumber by regulating autophagy.

In Situ SiRNA Assembly in Living Cells for Gene Therapy with MicroRNA Triggered Cascade Reactions Templated by Nucleic Acids.

The in situ generation of siRNAs in living cells can greatly enhance the specificity and efficiency of gene therapy. Inspired by the natural molecular machines that organize different compartments sequentially in a limited space to facilitate cellular process, this work constructs a DNA nanomachine (DNM) by alternately hybridizing two pairs of DNA/RNA hybrids to a DNA scaffold generated by rolling circle amplification for highly efficient in situ siRNA assembly in living cells. After target cell-specific delivery of DNM, intracellular specific microRNA can work as a trigger to operate the DNM by initiating DNA cascade displacement reaction between DNA/RNA hybrids along the scaffold for continuous generation of siRNAs. Using miR-21 as a model, efficient siRNAs generation is achieved via DNA templated cascade reaction, which demonstrated impressive suppressions to VEGF mRNA and protein expressions in cells and in vivo tumor growth and indicated promising application of the designed strategy in gene therapy.

Stimuli-Responsive Nanocarrier for Co-delivery of MiR-31 and Doxorubicin To Suppress High MtEF4 Cancer.

Gene interference-based therapeutics represent a fascinating challenge and show enormous potential for cancer treatment, in which microRNA is used to correct abnormal gene. On the basis of the above, we introduced microRNA-31 to bind to 3'-untranslated region of mtEF4, resulting in the downregulation of its messenger RNA and protein to trigger cancer cells apoptosis through mitochondria-related pathway. To achieve better therapeutic effect, a mesoporous silica nanoparticle-based controlled nanoplatform had been developed. This system was fabricated by conjugation of microRNA-31 onto doxorubicin-loaded mesoporous silica nanoparticles with a poly(ethyleneimine)/hyaluronic acid coating, and drug release was triggered by acidic environment of tumors. By feat of surface functionalization and tumor-specific conjugation to nanoparticles, our drug delivery system could promote intracellular accumulation of drugs via the active transport at tumor site. More importantly, microRNA-31 not only directly targeted to mtEF4 to promote cell's death, but had synergistic effects when used in combination with doxorubicin, and achieved excellent superadditive effects. As such, our research might provide new insights toward detecting high mtEF4 cancer and exploiting highly effective anticancer drugs.

[Effect of silencing the VDR gene on the migration and invasion of prostate cancer cells].

OBJECTIVE: To investigate the effect of small interfering RNA silencing the vitamin D receptor (VDR) on the biological behavior of prostate cancer PC-3 cells. METHODS: We constructed the VDR-shRNA lentiviral vector and determined the mRNA and protein expressions of VDR by RT-PCR and Western blot. Using scratch wound healing and Transwell chamber assays, we detected the changes in the migration and invasiveness of the PC-3 cells after silencing VDR. RESULTS: The VDR-shRNA plasmid significantly interfered the VDR expression and successfully screened the cell lines with stable VDR-shRNA interference. The rate of scratch wound healing was markedly lower in the VDR interference group than in the blank control and LV3 negative control groups (59% vs 73.6% and 77.8%, P <0.05), but with no statistically significant difference between the latter two (P >0.05), and so was the count of permeable cells (P <0.05), but with no significant difference between the latter two groups, either (P >0.05). The migration ability and invasiveness of the VDR-treated cells were remarkably decreased as compared with those of the control cells. CONCLUSIONS: Down-regulated expression of the VDR gene may reduce the migration and invasiveness of prostate cancer cells.

Construction and identification of multiple genes Co silence of plasmid shRNA.

Objective To construct and identify the eukaryotic vector expressing shRNA (Plasmid-1), which expressed the VEGF, C-myc, Survivin and hTERT gene at the same time. To detect its interference effects on the nasopharyngeal carcinoma cell line (CNE-2Z) compared with single gene plasmid VEFG (Plasmid-2). Methods According to the sequence of VEGF, C-myc, Survivin and hTERT gene, we designed 2 oligonucleotide sequences and synthesized a complementary DNA chain, then inserted it into the eukaryotic vector expressing pGenesil 1. The cell proliferation activity was detected by MTT method. The interference efficacy on human nasopharyngeal carcinoma cell line (CNE-2Z) in the level of mRNA and protein were detected by RT-PCR and Western-bolt. The inhibitory effect of plasmid on tumor in nude mice was also observed in vivo. Results The restriction enzyme digestion and sequencing technologies confirmed the construction of recombinant eukaryotic vector expressing was correct. The plasmid was transfected into CNE-2Z cells, green fluorescence can be seen clearly in the single gene and multi gene transfected cells under fluorescent microscope. MTT showed that the proliferation of cell was inhibited, the invasive ability was decreased in vitro, and the inhibition effects of single gene plasmid on the growth and proliferation of cells were lower than multi gene. Real-time-PCR and Western-bolt confirmed that the expression of target gene was decreased in the level of mRNA and protein, and the interference effect of multi gene was better than the single gene. The nude mice experiment showed that the interference effect of shRNA plasmid on the growth of tumor cell was better than single gene plasmid Conclusion We constructed a shRNA plasmid encoded four different genes successfully. After transfected with nasopharyngeal carcinoma cells, it can interfere the expression of VEGF, C-myc, Survivin and hTERT gene at the same time. And the interference effect was better than silence VEGF alone. Out results may provide experimental basis for multi gene therapy in the treatment of nasopharyngeal carcinoma.

LIM homeobox transcription factor 1B expression affects renal interstitial fibrosis and apoptosis in unilateral ureteral obstructed rats.

LIM homeobox transcription factor 1B (LMX1B) is a transcription factor of the LIM homeodomain type and has been implicated in the development of diverse structures such as limbs, kidneys, eyes, and the brain. Furthermore, LMX1B has been implicated in nail-patella syndrome, which is predominantly characterized by malformation of limbs and nails, and in 30% of patients, nephropathy, including renal fibrosis, is observed. Since no reports were available that studied the link between LMX1B expression and renal interstitial fibrosis, we explored if LMX1B affects typical markers of fibrosis, e.g., extracellular matrix components, profibrotic factors, and apoptosis as the final detrimental consequence. We recently showed that LMX1B acts as a negative regulator of transforming growth factor-ßl, collagen type III, fibronectin, cleaved caspase-3, and the cell apoptosis rate in a renal tubular epithelial cell system under hypoxic conditions. Here, we confirmed these results in unilateral ureteral obstructed rats. Furthermore, LMX1B was distinctly expressed throughout the glomerulus and tubule lining, including epithelial cells. Knockdown of LMX1B aggravated the expression of fibrosis markers, oxidative stress, and apoptosis compared with the already increased levels due to unilateral ureteral obstruction, whereas overexpression attenuated these effects. In conclusion, reduced LMX1B levels clearly represent a risk factor for renal fibrosis, whereas overexpression affords some level of protection. In general, LMX1B may be considered to be a negative regulator of the fibrosis index, transforming growth factor-ßl, collagen type III, fibronectin, cleaved caspase-3, cell apoptosis, ROS, and malondialdehyde (r = -0.756, -0.698, -0.921, -0.923, -0.843, -0.794, -0.883, and -0.825, all P < 0.01).

Characterization of an aphid-specific, cysteine-rich protein enriched in salivary glands.

Aphids secrete saliva into the phloem during their infestation of plants. Previous studies have identified numerous saliva proteins, but little is known about the characteristics (physical and chemical) and functions of these proteins in aphid-plant interactions. This study characterized an unknown protein (ACYPI39568) that was predicted to be enriched in the salivary glands of pea aphid. This protein belongs to an aphid-specific, cysteine-rich protein family that contains 14 conserved cysteines. ACYPI39568 is a monomeric globular protein with a high beta strand extent. The binding stoichiometric ratios for Zn(2+) and ACYPI39568 were approximately 3:1 and 1:1 at two binding sites. ACYPI39568 was predominantly expressed in the first instar stage and in the salivary glands. Aphids required more ACYPI39568 when feeding on plants than when feeding on an artificial diet. However, the interference of ACYPI39568 expression did not affect the survival rate of aphids on plants.