RNA Technology Research Articles

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

Immune Gene Repertoire of Soft Scale Insects (Hemiptera: Coccidae).

Insects possess an effective immune system, which has been extensively characterized in several model species, revealing a plethora of conserved genes involved in recognition, signaling, and responses to pathogens and parasites. However, some taxonomic groups, characterized by peculiar trophic niches, such as plant-sap feeders, which are often important pests of crops and forestry ecosystems, have been largely overlooked regarding their immune gene repertoire. Here we annotated the immune genes of soft scale insects (Hemiptera: Coccidae) for which omics data are publicly available. By using immune genes of aphids and Drosophila to query the genome of Ericerus pela, as well as the transcriptomes of Ceroplastes cirripediformis and Coccus sp., we highlight the lack of peptidoglycan recognition proteins, galectins, thaumatins, and antimicrobial peptides in Coccidae. This work contributes to expanding our knowledge about the evolutionary trajectories of immune genes and offers a list of promising candidates for developing new control strategies based on the suppression of pests' immunity through RNAi technologies.

Silencing of Resistance Mechanism in Vancomycin-Resistance Enterococci Using Antisense RNA of vanA Gene

The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.

Probing the Effects of Retinoblastoma Binding Protein 6 (RBBP6) Knockdown on the Sensitivity of Cisplatin in Cervical Cancer Cells.

Cervical cancer is a major cause of death in women despite the advancement of current treatment modalities. The conventional therapeutic agent, cisplatin (CCDP), is the standard treatment for CC; however, resistance often develops due to the cancer's heterogeneity. Therefore, a detailed elucidation of the specific molecular mechanisms driving CC is crucial for the development of targeted therapeutic strategies. Retinoblastoma binding protein 6 (RBBP6) is a potential biomarker associated with cell proliferation and is upregulated in cervical cancer sites, exhibiting apoptosis and dysregulated p53 expression. Furthermore, RBBP6 has been demonstrated to sensitize cancer cells to radiation and certain chemotherapeutic agents by regulating the Bcl-2 gene, thus suggesting a crosstalk among RBBP6/p53/BCL-2 oncogenic signatures. The present study, therefore, investigated the relationship between cisplatin and RBBP6 expression in CC cells. Herein, we first explored bioinformatics simulations and identified that the RBBP6/p53/BCL-2 signaling pathway is overexpressed and correlated with CC. For further analysis, we explored the Genomics of Drug Sensitivity in Cancer (GDSC) and found that most of the CC cell lines are sensitive to CCDP. To validate these findings, RBBP6 was silenced in HeLa and Vero cells using RNAi technology, followed by measurement of wild-type p53 and Bcl-2 at the mRNA level using qPCR. Cells co-treated with cisplatin and siRBBP6 were subsequently analyzed for apoptosis induction and real-time growth monitoring using flow cytometry and the xCELLigence system, respectively. Cancer cells in the co-treatment group showed a reduction in apoptosis compared to the cisplatin-treated group. Moreover, the real-time growth monitoring revealed a reduced growth rate in RBBP6 knockdown cells treated with cisplatin. Although wild-type p53 remained unchanged in the co-treatment group of cancer cells, Bcl-2 was completely repressed, suggesting that RBBP6 is necessary for sensitizing cervical cancer cells to cisplatin treatment by downregulating Bcl-2. The Vero cell population, which served as a non-cancerous control cell line in this study, remained viable following treatment with both siRBBP6 and cisplatin. Findings from this study suggest that RBBP6 expression promotes cisplatin sensitivity in HeLa cells through Bcl-2 downregulation. Knockdown of RBBP6 limits apoptosis induction and delays cell growth inhibition in response to cisplatin. The knowledge obtained here has the potential to help improve cisplatin efficacy through personalized administration based on the expression profile of RBBP6 among individual patients.

Nematicidal Activity of 20-Deoxyingenol-3-angelate from Euphorbia peplus Latex Through Protein Kinase C Isotype TPA-1.

The latex of Euphorbia peplus and its major component 20-deoxyingenol-3-angelate (DI3A) displayed significant nematicidal activity against Caenorhabditis elegans and Panagrellus redivivus. DI3A treatment inhibited the growth and development of nematodes and caused significantly negative effects on locomotion behavior, reproduction, and accumulation of reactive oxygen species. Transcriptome analysis indicated that differential expression genes in DI3A-treated C. elegans were mainly associated with the metabolism, growth, and development process, which were further confirmed by RT-qPCR experiments. The expression level of TPA-1 gene encoding a protein kinase C isotype was obviously upregulated by DI3A treatment, and knockdown of TPA-1 by RNAi technology in the nematode could relieve the growth-inhibitory effect of DI3A. Metabolic analysis indicated that DI3A was hardly metabolized by C. elegans, but a glycosylated indole derivative was specifically accumulated likely due to the activation of detoxification. Overall, our findings suggested that DI3A from E. peplus latex exerted a potent nematicidal effect through the gene TPA-1, which provides a potential target for the control of nematodes and also suggests the potential application value of E. peplus latex and DI3A as botanical nematicides.

Silencing of B4Galnt1 Gene Prevents GM2 Accumulation in Tay-Sachs Cells

Introduction. The Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder resulting from genetic mutations in the HEXA gene encoding the α-subunit of β-hexosaminidase A leading to the accumulation of GM2 ganglioside in the central nervous system. Multiple therapeutical strategies have been investigated such as gene therapy for Tay-Sachs patients; however, there is still no cure. In the present study, we suggest a new approach for the treatment of the Tay-Sachs disease with the concept of substrate reduction therapy by using AAV9-mediated RNAi technology targeting the B4Galnt1 gene at the upstream of the enzymatic defect in TSD pathology to decrease GM2 biosynthesis and accumulation in cell models of TSD. Material and Methods. We employed AAV9-mediated shRNA transduction for mice and human Tay-Sachs cells. After transduction, expression levels of ganglioside metabolism genes were analyzed by RT-PCR and GM2 and lysosome-associated membrane protein 1 (LAMP1) protein levels were evaluated by immunocytochemistry analysis. Results. Here, we have shown that AAV9-shRNA transduction effectively reduced B4Galnt1 expression in TSD cells demonstrating a reduction in GM2 accumulation and LAMP1. Discussion. Our data shows that AAV-mediated B4Galnt1-shRNA transduction can ameliorate disease pathologies by decreasing the lysosomal accumulation of GM2 through selectively reducing B4Gant1 activity in cell models of the Tay-Sachs disease. Therefore, we suggest promising novel experimental therapy for this devastating disease using a mouse model in the future.

Harnessing RNA Technology to Advance Therapeutic Vaccine Antigens against Chagas Disease.

Chagas disease (CD) (American trypanosomiasis caused by Trypanosoma cruzi) is a parasitic disease endemic in 21 countries in South America, with increasing global spread. When administered late in the infection, the current antiparasitic drugs do not prevent the onset of cardiac illness leading to chronic Chagasic cardiomyopathy. Therefore, new therapeutic vaccines or immunotherapies are under development using multiple platforms. In this study, we assessed the feasibility of developing an mRNA-based therapeutic CD vaccine targeting two known T. cruzi vaccine antigens (Tc24─a flagellar antigen and ASP-2─an amastigote antigen). We present the mRNA engineering steps, preparation, and stability of the lipid nanoparticles and evaluation of their uptake by dendritic cells, as well as their biodistribution in c57BL/J mice. Furthermore, we assessed the immunogenicity and efficacy of two mRNA-based candidates as monovalent and bivalent vaccine strategies using an in vivo chronic mouse model of CD. Our results show several therapeutic benefits, including reductions in parasite burdens and cardiac inflammation, with each mRNA antigen, especially with the mRNA encoding Tc24, and Tc24 in combination with ASP-2. Therefore, our findings demonstrate the potential of mRNA-based vaccines as a therapeutic option for CD and highlight the opportunities for developing multivalent vaccines using this approach.

Assembling the RNA therapeutics toolbox.

From the approval of COVID-19 mRNA vaccines to the 2023 Nobel Prize awarded for nucleoside base modifications, RNA therapeutics have entered the spotlight and aretransforming drug development. While the term "RNA therapeutics" has been used in various contexts, this review focuses on treatments that utilize RNA as a component or target RNA for therapeutic effects. We summarize the latest advances in RNA-targeting tools and RNA-based technologies, including but not limited to mRNA, antisense oligos, siRNAs, small molecules and RNA editors. We focus on the mechanisms of current FDA-approved therapeutics but also provide a discussion on the upcoming workforces. The clinical utility of RNA-based therapeutics is enabled not only by the advances in RNA technologies but in conjunction with the significant improvements in chemical modifications and delivery platforms, which are also briefly discussed in the review. We summarize the latest RNA therapeutics based on their mechanisms and therapeutic effects, which include expressing proteins for vaccination and protein replacement therapies, degrading deleterious RNA, modulating transcription and translation efficiency, targeting noncoding RNAs, binding and modulating protein activity and editing RNA sequences and modifications. This review emphasizes the concept of an RNA therapeutic toolbox, pinpointing the readers to all the tools available for their desired research and clinical goals. As the field advances, the catalog of RNA therapeutic tools continues to grow, further allowing researchers to combine appropriate RNA technologies with suitable chemical modifications and delivery platforms to develop therapeutics tailored to their specific clinical challenges.

Unlocking the Potential of RNA Nanoparticles: A Breakthrough Approach to Overcoming Challenges in Colon Cancer Treatment.

Globally, one of the leading causes of cancer-related deaths is colon cancer. As this form of cancer has a tremendous potential to metastasize, effective treatment is complicated and sometimes impossible. Despite the improvement of conventional chemotherapy and the advent of targeted therapies, overcoming multi-drug resistance (MDR) and side effects remain significant challenges. As a therapeutic intervention for targeted gene silencing in cancer, RNA technology shows promise and certain RNA-based formulations are currently undergoing clinical studies. Various studies have reported that RNA-based nanoparticles have demonstrated substantial promise for targeted medication delivery, gene therapy, and other biomedical applications. However, using RNA as a therapeutic tool presents severe limitations, mainly related to its low stability and poor cellular uptake. Nanotechnology offers a flexible and tailored alternative due to the difficulties in delivering naked RNA molecules safely in vivo, such as their short half-lives, low chemical stability, and susceptibility to nuclease degradation. In addition to shielding RNA molecules from immune system attacks and enzymatic breakdown, the nanoparticle-based delivery systems allow RNA accumulation at the tumor site. The potential of RNA and RNA-associated nanomedicines for the treatment of colon cancer, as well as the prospects for overcoming any difficulties related to mRNA, are reviewed in this study, along with the current progress of mRNA therapeutics and advancements in designing nanomaterials and delivery strategies.

A pattern recognition receptor interleukin-1 receptor is involved in reproductive immunity in Macrobrachium nipponense ovary

The family of TIR domain-containing receptors includes numerous proteins involved in innate immunity. In this study, a member of this family was characterized from the ovary of the oriental river prawn Macrobrachium nipponense and identified as interleukin-1 receptor (MnIL-1R). Meanwhile, to elucidate the conservation of IL-1R, its orthologous were identified in several crustacean species as well. In addition, the expression pattern of MnIL-1R in various adult tissues and post different pathogen-associated molecular patterns (PAMPs) challenge in ovary was analyzed with qRT-PCR technology. Finally, the roles of MnIL-1R in the ovary were analyzed by RNAi technology. The main results are as follows: (1) MnIL-1R comprises a 1785 bp ORF encoding 594 amino acids and is structurally composed of five domains: a signal peptide, two immunoglobulin (IG) domains, a transmembrane region, and a TIR-2 domain; (2) the TIR domain showed a high conservation among analyzed crustacean species; (3) MnIL-1R is widely detected in all tested tissues including ovary; (4) MnIL-1R showed a positive response to challenges with LPS, PGN, and polyI:C in the ovary; (5) its IG domain showed strong binding ability to LPS and PGN, confirming its role as a pattern recognition receptor; (6) the expression patterns of several members of the Toll signaling pathway (Myd88, TRAF-6, Dorsal, and Relish) was similar to that of MnIL-1R after challenges with LPS, PGN, and polyI:C in the ovary; (7) the silencing of MnIL-1R resulted in down-regulation of theses gene’ (Myd88, TRAF-6, Dorsal, and Relish) expression level in the ovary. These results suggest that MnIL-1R can activate the Toll signaling pathway in the ovary by directly recognizing LPS and PGN through its IG domain, thereby contributing to the immune response in the ovary of M. nipponense.

The mRNACalc webserver accounts for the N1-methylpseudouridine hypochromicity to enable precise nucleoside-modified mRNA quantification

Nucleoside-modified messenger RNA (mRNA) technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260nm wavelength (A260). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5%-15% error, in an mRNA-sequence-dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260nm wavelength, the RNA composition of the mRNA, and the A260 of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs.

A C-type lectin (CTL2) mediated both humoral and cellular immunity against bacterial infection in Tribolium castaneum

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.

RNA-seq analysis of chlorogenic acid intervention in duck embryo fibroblasts infected with duck plague virus

IntroductionChlorogenic acid, the primary active component in Chinese medicines like honeysuckle, exhibits anti-inflammatory and antiviral effects. It has been demonstrated that chlorogenic acid effectively prevents and treats Duck enteritis virus (DEV) infection. This study aims to further elucidate the mechanism by which chlorogenic acid prevents DEV infection.MethodsDuck embryo fibroblast (DEF) cells were pre-treated with chlorogenic acid before being infected with DEV. Cell samples were collected at different time points for transcriptomic sequencing, while qPCR was used to detect the proliferation of DEV. Additionally, 30-day-old ducks were treated with chlorogenic acid, and their lymphoid organs were harvested for histopathological sections to observe pathological damage. The proliferation of DEV in the lymphoid organs was also detected using qPCR Based on the transcriptomic sequencing results, NF-κB1 gene was silenced by RNAi technology to analyze the effect of NF-κB1 gene on DEV proliferation.ResultsCompared to the viral infection group, DEF cells in the chlorogenic acid intervention group exhibited significantly reduced DEV load (P < 0.05). Transcriptomic sequencing results suggested that chlorogenic acid inhibited DEV proliferation in DEF cells by regulating NF-κB signaling pathway. The results of RNAi silencing suggested that in the three treatment groups, compared with the DEV experimental group, there was no significant difference in the effect of pre-transfection after transfection on DEV proliferation, while both the pre-transfection after transfection and the simultaneous transfection group showed significant inhibition on DEV proliferation Furthermore, compared to the virus infection group, ducks in the chlorogenic acid intervention group showed significantly decreased DEV load in their lymphoid organs (P < 0.05), along with alleviated pathological damage such as nuclear pyretosis and nuclear fragmentation.ConclusionsChlorogenic acid effectively inhibits DEV proliferation in DEF and duck lymphatic organs, mitigates viral-induced pathological damage, and provides a theoretical basis for screening targeted drugs against DEV.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Identifying the Key Role of Plutella xylostella General Odorant Binding Protein 2 in Perceiving a Larval Attractant, (E,E)-2,6-Farnesol.

There is currently a lack of effective olfaction-based techniques to control diamondback moth (DBM) larvae. Identifying behaviorally active odorants for DBM larvae and exploring their recognition mechanisms can provide insights into olfaction-based larval control strategies. Through the two-choice assay, (E,E)-2,6-farnesol (farnesol) was identified as a compound exhibiting significant attractant activity toward DBM larvae, achieving an attraction index of 0.48 ± 0.13. PxylGOBP1 and PxylGOBP2, highly expressed in the antennae of DBM larvae, both showed high affinity toward farnesol. RNAi technology was used to knock down PxylGOBP1 and PxylGOBP2, revealing that the attraction of DBM larvae to farnesol nearly vanished following the knockdown of PxylGOBP2, indicating its critical role in recognizing farnesol. Further investigation into the PxylGOBP2-farnesol interaction revealed the importance of residues like Thr9, Trp37, and Phe118 in PxylGOBP2's binding to farnesol. This research is significant for unveiling the olfactory mechanisms of DBM larvae and developing larval behavior regulation techniques.

Combination of diflubenzuron and RNAi technology to improve the control effect of Helicoverpa armigera

Combination of diflubenzuron and RNAi technology to improve the control effect of Helicoverpa armigera

Determination of linearized pDNA template in mRNA production process using HPLC.

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.

A short review on CRP synthesis inhibition in cardiovascular disease

C-reactive Protein (CRP) is synthesized in the liver. Synthesis is stimulated via the IL-1ß/IL6 pathway. CRP activates the complement system via C1q and macrophages via Fcγ receptors. Since elevated CRP plasma levels are associated with increased cardiovascular risk, CRP may play a causal role in cardiovascular disease. One approach to transfer these observations into standard medical care would be to generate hepatic CRP synthesis inhibitors and use them in controlled clinical trials. Despite huge pharmacological efforts, the search for CRP synthesis inhibitors proved to be difficult. First, the antisense oligonucleotide RNA technology, although a promising idea, has not yet led to results feasible for clinical practice. Secondly, high throughput screening assays in search for hepatic CRP inhibitors were limited by the fact that primary human hepatocytes do not adequately grow in vitro. Use of genetically engineered hepatoma cells led to the observation that cardiac glycosides are capable of inhibiting CRP synthesis. Because of patent law considerations, however, pharmaceutical companies had limited interest in further pursuing this possible path. Upstream inhibition of IL-1ß and IL-6 by antibodies has shown positive results in cardiovascular clinical trials, but because of side effects none of these antibodies has yet received FDA approval. In contrast, long-term colchicine treatment, though not being a CRP-specific approach, has recently been approved by the FDA. Taken together, there is no compelling evidence until today that hepatic CRP synthesis can specifically, effectively and safely be inhibited in vivo in human medicine. Currently, other avenues appear more promising. Here, we summarize contemporary approaches to inhibit CRP synthesis and potential goals for future clinical trials.

CLCN3 in mediating the proliferation of human ovarian cancer cells.

Chloride channel-3 (CLCN3), a crucial component of the voltage-gated chloride channel family, is implicated in numerous physiological and pathophysiological processes. This study aimed to investigate the characteristics of CLCN3 in pancancer and its influence on the immune response through the use of a range of databases. Concurrently, we assessed the impact of CLCN3 on the proliferation of ovarian cancer (OC) cells and explored its potential mechanisms. We employed the Tumor Immune Estimation Resource (TIMER) 2.0 and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to examine the messenger RNA (mRNA) and the protein expression of CLCN3 across various cancers. The prognostic significance of CLCN3 was evaluated using the Gene Expression Profiling Interactive Analysis 2.0 (GEPIA 2.0) database. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) facilitated the analysis of CLCN3 promoter methylation levels. The association between CLCN3 expression and tumor-infiltrating immune cells was investigated using various algorithms. The cBioportal database facilitated the analysis of CLCN3 mutations and mutation sites across various cancers. The Tumor-Immune System Interactions Database (TISIDB) database was employed to explore the correlation between CLCN3 expression and immune or molecular subtypes across a variety of cancer types. We collected ovarian tissue samples, encompassing both normal ovarian and OC tissues. The human OC cell lines, SKOV3 cells and OVCAR433 cells, were cultured. CLCN3 expression was determined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR), while phosphatidylinositol 3-kinase/Akt kinase (PI3K/AKT) expression was detected using Western blot. We utilized small interfering RNA (siRNA) technology to suppress CLCN3 expression. The proliferative capacity of SKOV3 and OVCAR433 cells was assessed using the Cell Counting Kit 8 (CCK-8) assay. CLCN3 demonstrated an aberrant expression in a number of cancer types and was markedly reduced in OC tissues. Poor prognosis in cervical squamous cell cancer and myeloid leukemia was linked to excessive expression of CLCN3. The examination of immune cell infiltration, which included CD8+ T cells, B cells, T regulatory cells, and cancer-associated fibroblast cells, showed a strong association with aberrant CLCN3 expression. Following the use of siRNA technology, the ability of the ovarian carcinoma cell line SKOV3 and OVCAR433 to proliferate as well as the expression of PI3K/AKT both increased. CLCN3 is a possible biomarker for immune-related processes and the prognosis of cancer, and the PI3K/AKT signaling pathway may affect OC cells' ability to proliferate.

Odorant binding protein 18 increases the pathogen resistance of the imported willow leaf beetle, Plagiodera versicolora.

Insect odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins. In the past few years, OBPs had been found to work as carriers of ligands and play a crucial role in olfaction and various other physiological processes, like immunity. A subset of insect OBPs had been found to be expressed differently and play a function in immunity of fungal infection. However, there are few studies on the role of OBPs in immunity of bacterial infection. To identify the immune-related OBPs of Plagiodera versicolora after infected by Pseudomonas aeruginosa, we determined the mortality of P. versicolora to P. aeruginosa and selected the time point of 50% mortality of larvae to collect samples for RNA-seq. RNAi technology was used to investigate the function of immune-related OBPs after P. aeruginosa infection. RNA-seq data shows that PverOBP18 gene significantly up-regulated by 1.8-fold and further RT-qPCR affirmed its expression. Developmental expression profile showed that the expression of PverOBP18 was highest in the pupae, followed by the female adults, and lower in the 1st-3rd larvae and male adults with lowest in eggs. Tissue expression profiling showed that PverOBP18 was dominantly expressed in the epidermis. RNAi knockdown of PverOBP18 significantly reduced the expression of bacterial recognition receptor gene PGRP and antibacterial peptide gene Attacin and reduced the resistance of P. versicolora to P. aeruginosa infection. Our results indicated that PverOBP18 gene increased the pathogen resistance of P. versicolora by cooperating with the immune genes and provided valuable insights into using OBPs as targets to design novel strategies for management of P. versicolora.