Search Results (71)

CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls. Full article

Chloroplast biogenesis is a crucial biological process in plants. Endoribonuclease E (RNase E) functions in the RNA metabolism of chloroplast and plays a vital role for chloroplast development in Arabidopsis. However, despite sharing 44.7% of its amino acid sequence identity with Arabidopsis RNase E, the biological function of rice OsRNE (Oryza sativa RNase E) remains unknown. Here, we identified a white leaf and lethal 1 (wll1) mutant that displayed white leaves and died at the seedling stage. The causal gene OsRNE was isolated by MutMap+ method. CRISPR/Cas9-mediated knockout of OsRNE resulted in white leaves and seedling lethality, confirming OsRNE as the causal gene for the wll1 phenotype. The albino phenotype of osrne mutant was associated with decreased chlorophyll content and abnormal thylakoid morphology in the chloroplast. The absence of OsRNE led to a significant reduction in the Rubisco large subunit (RbcL), and the 23S and 16S chloroplast rRNAs were nearly undetectable in the osrne mutant. OsRNE transcripts were highly expressed in green tissues, and the protein was localized to chloroplasts, indicating its essential role in photosynthetic organs. Furthermore, transcriptome analysis showed that most of the genes associated with photosynthesis and carbohydrate metabolism pathways in the osrne mutant were significantly down-regulated compared with those in WT. Chlorophyll- and other pigment-related genes were also differentially expressed in the osrne mutant. Our findings demonstrated that OsRNE plays an important role in chloroplast development and chlorophyll biosynthesis in rice. Full article

Background: External guide sequences (EGSs) are small RNA molecules capable of hybridizing to a target mRNA and rendering the target RNA susceptible to degradation by ribonuclease P (RNase P), a tRNA processing enzyme. Methods: In this study, natural tRNA-originated and engineered variant EGSs were constructed to target the mRNA encoding human CC-chemokine receptor 5 (CCR5), an HIV co-receptor. Results: The EGS variant was about 100-fold more efficient in inducing RNase P-mediated cleavage of the CCR5 mRNA sequence in vitro than a natural tRNA-derived EGS. Furthermore, the expressed variant and natural tRNA-originated EGSs decreased CCR5 expression by 98% and 73–77% and reduced infection by the CCR5-tropic HIV_Ba-L strain in cells by more than 900- and 50-fold, respectively. By contrast, cells expressing these EGSs exhibited no change in the expression of CXCR4, another HIV co-receptor, and showed no reduction in infection by the CXCR4-tropic HIV_IIIB strain, which uses CXCR4 instead of CCR5 as the co-receptor. Thus, the EGSs specifically targeted CCR5 but not CXCR4. Conclusions: Our results demonstrate that EGSs are effective and specific in diminishing HIV infection and represent a novel class of gene-targeting agents for anti-HIV therapy. Full article

The immune system and neuroinflammation are now well established in the aetiology of neurodegeneration. Previous studies of transcriptomic and gene association studies have highlighted the potential of the 2′–5′ oligoadenylate synthetase 1 (OAS1) to play a role in Alzheimer’s disease. OAS1 is a viral response gene, interferon-induced, dsRNA activated enzyme, which binds RNase L to degrade dsRNA, and has been associated with COVID-19 response. This study explores whether a viral defence gene could play a vital role in neurodegeneration pathology. The genotyping of five SNPs across the OAS1 locus was conducted in the Brains for Dementia Research (BDR) Cohort for association with AD. RNA-sequencing data were explored for differences in OAS1 gene expression between phenotypes and genotypes. Finally, levels of dsRNA were measured in control cell lines, prior to and after exposure to amyloid oligomers and in cells harbouring a dementia-relevant mutation. No association of any of the OAS1 SNPs investigated were associated with the AD phenotype in the BDR cohort. However, gene expression data supported the previous observation that the minor allele haplotype was associated with higher levels of the OAS1 gene expression and the presence of an alternative transcript. Further to this, the presence of endogenous dsRNA was found to increase with exposure to amyloid oligomers and in the cell line with a dementia-relevant mutation. The data presented here suggest further exploration of the OAS1 gene in relation to dementia is warranted. Investigations of whether carriers of the protective OAS1 haplotype lower dsRNA presence and in turn lower inflammation and cell death are required to support the role of the gene as a moderator of neurodegeneration. Full article

In this study, new monolithic poly(9-anthracenylmethyl methacrylate-co-trimethylolpropane trimethacrylate (TRIM) columns, referred as ANM monoliths were prepared, for the first time, and were used for the separation media for biomolecules and proteomics analysis by nano-liquid chromatography (nano-LC). Monolithic columns were prepared by in situ polymerization of 9-anthracenylmethyl methacrylate (ANM) and trimethylolpropane trimethacrylate (TRIM) in a fused silica capillary column of 100 µm ID. Polymerization solution was optimized in relation to monomer and porogenic solvent. Scanning electron microscopy (SEM) and chromatographic analyses were performed for the characterization studies of ANM monoliths. The ANM monolith produced more than 46.220 plates/m, and the chromatographic evaluation of the optimized ANM monolith was carried out using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), allowing both strong hydrophobic and π-π interactions. Run-to-run and column-to-column reproducibility values were found as <2.91% and 2.9–3.2%, respectively. The final monolith was used for biomolecule separation, including both three dipeptides, including Alanine-Tyrosine (Ala-Tyr), Glycine-Phenylalanine (Gly-Phe), and L-carnosine and five standard proteins, including ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C), and myoglobin (Mb) in order to evaluate its potential. Both peptides and proteins were baseline separated using the developed ANM monolith in nano-LC. The ANM monolith was then applied to the protein and peptide profiling of MCF-7 cell line, which allowed a high-resolution analysis of peptides, providing a high peak capacity. Full article

During virus infection, the activation of the antiviral endoribonuclease, ribonuclease L (RNase L), by a unique ligand 2′-5′-oilgoadenylate (2-5A) causes the cleavage of single-stranded viral and cellular RNA targets, restricting protein synthesis, activating stress response pathways, and promoting cell death to establish broad antiviral effects. The immunostimulatory dsRNA cleavage products of RNase L activity (RL RNAs) recruit diverse dsRNA sensors to activate signaling pathways to amplify interferon (IFN) production and activate inflammasome, but the sensors that promote cell death are not known. In this study, we found that DEAH-box polypeptide 15 (DHX15) and retinoic acid-inducible gene I (Rig-I) are essential for apoptosis induced by RL RNAs and require mitochondrial antiviral signaling (MAVS), c-Jun amino terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) for caspase-3-mediated intrinsic apoptosis. In RNase L-activated cells, DHX15 interacts with Rig-I and MAVS, and cells lacking MAVS expression were resistant to apoptosis. RL RNAs induced the transcription of genes for IFN and proinflammatory cytokines by interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB), while cells lacking both DHX15 and Rig-I showed a reduced induction of cytokines. However, apoptotic cell death is independent of both IRF-3 and NF-kB, suggesting that cytokine and cell death induction by RL RNAs are uncoupled. The RNA binding of both DHX15 and Rig-I is required for apoptosis induction, and the expression of both single proteins in cells lacking both DHX15 and Rig-I is insufficient to promote cell death by RL RNAs. Cell death induced by RL RNAs suppressed Coxsackievirus B3 (CVB3) replication, and inhibiting caspase-3 activity or cells lacking IRF-3 showed that the induction of apoptosis directly resulted in the CVB3 antiviral effect, and the effects were independent of the role of IRF-3. Full article

The beneficial effect of milk kefir on respiratory heath has been previously demonstrated; however, water kefir and kefiran in the context of respiratory viral infections have not been investigated. Water kefir and kefiran could be alternatives to milk kefir for their application in persons with lactose intolerance or milk allergy and could be incorporated into vegan diets. Using mice models, this work demonstrated that the oral administration of water kefir or kefiran can modulate the respiratory Toll-like receptor (TLR3)-mediated innate antiviral immunity and improve the resistance to respiratory syncytial virus (RSV) infection. The treatment of mice with water kefir or kefiran for 6 days improved the production of interferons (IFN-β and IFN-γ) and antiviral factors (Mx2, OAS1, RNAseL, and IFITM3) in the respiratory tract after the activation of the TLR3 signaling pathway, differentially modulated the balance of pro- and anti-inflammatory cytokines, reduced RSV replication, and diminished lung tissue damage. Maintaining a proper balance between anti-inflammatory and pro-inflammatory mediators is vital for ensuring an effective and safe antiviral immune response, and the results of this work show that water kefir and kefiran would help to maintain that balance promoting a controlled inflammatory response that defends against infection while minimizing tissue damage. Full article

The utilization of biomass ash in sustainable agriculture and increasing its fertilizing efficiency by biological agents, potentially sequestering CO₂, have become important issues for the global economy. The aim of this paper was to investigate the effects of ash from sorghum (Sorghum bicolor L. Moench) and Jerusalem artichoke (Helianthus tuberosus L.) biomass, a biogas plant digestate, and a Spirodela polyrhiza extract, acting alone or synergistically, on soil fertility and the development, health and physiological properties of sorghum plants. The results show novel information concerning differences in the composition and impact of ash, depending on its origin, soil properties and sorghum plant development. Sorghum ash was more effective than that from Jerusalem artichoke. Ash used alone and preferably acting synergistically with the digestate and Spirodela polyrhiza extract greatly increased soil fertility and the growth, biomass yield and health of sorghum plants. These improvements were associated with an increased chlorophyll content in leaves, better gas exchange (photosynthesis, transpiration, stomatal conductance), greater enzyme activity (acid and alkaline phosphatase, RNase, and total dehydrogenase), and a higher biomass energy value. The developed treatments improved environmental conditions by replacing synthetic fertilizers, increasing the sequestration of CO₂, solving the ash storage problem, reducing the need for pesticides, and enabling a closed circulation of nutrients between plant and soil, maintaining high soil fertility. Full article

Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans. Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium. Full article

The first line of defense against invading pathogens usually relies on innate immune systems. In this context, the recognition of exogenous RNA structures is primordial to fight, notably, against RNA viruses. One of the most efficient immune response pathways is based on the sensing of RNA double helical motifs by the oligoadenylate synthase (OAS) proteins, which in turn triggers the activity of RNase L and, thus, cleaves cellular and viral RNA. In this contribution, by using long-range molecular dynamics simulations, complemented with enhanced sampling techniques, we elucidate the structural features leading to the activation of OAS by interaction with a model double-strand RNA oligomer mimicking a viral RNA. We characterize the allosteric regulation induced by the nucleic acid leading to the population of the active form of the protein. Furthermore, we also identify the free energy profile connected to the active vs. inactive conformational transitions in the presence and absence of RNA. Finally, the role of two RNA mutations, identified as able to downregulate OAS activation, in shaping the protein/nucleic acid interface and the conformational landscape of OAS is also analyzed. Full article

2′-5′-oligoadenylate synthetase (OAS) is one of the proteins that act as a defense mechanism against foreign RNA in cells. OAS has two functions: an antiviral effect against a wide range of virus species via the OAS/RNase L pathway with synthesized oligoadenylates and inhibition of viral replication specific to viruses of the genus Flavivirus, which is independent of enzymatic activity. Several birds have been reported to possess only one type of OAS family member, OASL, which has both enzymatic activity and inhibitory effects on flaviviral replication. However, the ostrich has two types of OASs, OAS1 and OASL, which show different functions—enzymatic and anti-flaviviral activities, respectively. In this study, emu OASs were cloned to investigate their sequence and function and elucidate the role of OASs in emus. The cloning results showed that emus had OAS1 and OASL, suggesting that emu OASs were more closely related to ostrich than to other birds. Functional investigations showed that emu OAS1 and OASL had enzymatic and anti-flaviviral activities, respectively, similar to those of the ostrich. Emus and ostriches are evolutionarily different from most birds and may be more closely related to mammalian OAS diversity. Full article

Introduction: The interferon pathway plays a critical role in triggering the immune response to SARS-CoV-2, and these gene variants may be involved in the severity of COVID-19. This study aimed to analyze the frequency of three gene variants of OAS and RNASEL with the occurrence of COVID-19 symptoms and disease outcome. Methods: This cross-sectional study included 104 patients with SARS-CoV-2 infection, of which 34 were asymptomatic COVID-19, and 70 were symptomatic cases. The variants rs486907 (RNASEL), rs10774671 (OAS1), rs1293767 (OAS2), and rs2285932 (OAS3) were screened and discriminated using a predesigned 5′-nuclease assay with TaqMan probes. Results: Patients with the allele C of the OAS2 gene rs1293767 (OR = 0.36, 95% CI: 0.15–0.83, p = 0.014) and allele T of the OAS3 gene rs2285932 (OR = 0.39, 95% CI: 0.2–0.023, p = 0.023) have lower susceptibility to developing symptomatic COVID-19. The genotype frequencies (G/G, G/C, and C/C) of rs1293767 for that comparison were 64.7%, 29.4%, and 5.9% in the asymptomatic group and 95.2%, 4.8%, and 0% in severe disease (p < 0.05). Conclusions: Our data indicate that individuals carrying the C allele of the OAS2 gene rs1293767 and the T allele of the OAS3 gene rs2285932 are less likely to develop symptomatic COVID-19, suggesting these genetic variations may confer a protective effect among the Mexican study population. Furthermore, the observed differences in genotype frequencies between asymptomatic individuals and those with severe disease emphasize the potential of these variants as markers for disease severity. These insights enhance our understanding of the genetic factors that may influence the course of COVID-19 and underscore the potential for genetic screening in identifying individuals at increased risk for severe disease outcomes. Full article

Chandipura virus (CHPV) is a vesiculovirus that is a member of Rhabdviridae and is an encephalitic pathogen responsible for numerous epidemics in Central and Western India. The virus affects the brain and central nervous system mainly in children under 15 age of years, leading to neurological dysfunctions. Vectors that include sand flies, mosquitoes, and ticks are the main culprits in the transmission of CHPV. Five structural proteins (N, P, M, G, and L) encode the viral genome. The nucleocapsid protein N (N protein) encapsulates the viral genomic RNA in an RNase-resistant state, which plays a crucial role in the viral life cycle. Currently, no effective vaccine or therapeutics are available to treat the viral infection, and therefore efficient interventions are urgently needed. The repurposing of drugs is one of the best possible ways to controlCHPV infections in India and other parts of the world. In this study, we used a structure-based virtual screening approach by using FDA-approved drugs against the nucleocapsid protein of CHPV. The docking process identified a few drug candidates, which showed potent binding affinity towards the N protein. We used the Schrödinger Desmond v3.0 module; to compute the relative binding energies of ligands, we used the premier mm-GBSA module. Based on a short molecular dynamics simulation and prime MM-GBSA analysis, we identified Adrabetadex, Hydroxypropyl betadex, Beta-1,2,3,4,6-penta-O-Galloyl-D-Glucopyranose, thio-maltohexaose, and Indium-III pentetreotide as potent drug candidates for CHPV. Our computational results provide suggestions for in vitro and in vivo testing of these drugs against CHPV. Full article

Self-incompatibility is a widespread genetic mechanism found in flowering plants. It plays a crucial role in preventing inbreeding and promoting outcrossing. The genes that control self-incompatibility in plants are typically determined by the S-locus female determinant factor and the S-locus male determinant factor. In the Solanaceae family, the male determinant factor is often the SLF gene. In this research, we cloned and analyzed 13 S₂-LbSLF genes from the L. barbarum genome, which are located on chromosome 2 and close to the physical location of the S-locus female determinant factor S-RNase, covering a region of approximately 90.4 Mb. The amino acid sequence identity of the 13 S₂-LbSLFs is 58.46%, and they all possess relatively conserved motifs and typical F-box domains, without introns. A co-linearity analysis revealed that there are no tandemly repeated genes in the S₂-LbSLF genes, and that there are two pairs of co-linear genes between S₂-LbSLF and the tomato, which also belongs to the Solanaceae family. A phylogenetic analysis indicates that the S₂-LbSLF members can be divided into six groups, and it was found that the 13 S₂-LbSLFs are clustered with the SLF genes of tobacco and Petunia inflata to varying degrees, potentially serving as pollen determinant factors regulating self-incompatibility in L. barbarum. The results for the gene expression patterns suggest that S₂-LbSLF is only expressed in pollen tissue. The results of the yeast two-hybrid assay showed that the C-terminal region of S₂-LbSLFs lacking the F-box domain can interact with S-RNase. This study provides theoretical data for further investigation into the functions of S₂-LbSLF members, particularly for the identification of pollen determinant factors regulating self-incompatibility in L. barbarum. Full article

Ionizing radiation (IR) induces severe hematopoietic injury by causing DNA and RNA damage as well as activating the immune responses, necessitating the development of effective therapeutic strategies. Ribonuclease L (RNase L) as an innate immune response pathway is triggered by exogenous and endogenous abnormal dsRNA under viral infection and dyshomeostasis, thereby activating the immune responses. Thus, we investigated the effect of RNase L on irradiation-induced bone marrow damage using RNase L knockout (RNase L^−/−) mice. Phenotypic analysis revealed that RNase L knockout mitigates irradiation-induced injury in the bone marrow. Further investigation into the mechanism of RNase L by RNA-seq, qRT-PCR, and CBA analysis demonstrated that RNase L deficiency counteracts the upregulation of genes related to immune responses induced by irradiation, including cytokines and interferon-stimulated genes. Moreover, RNase L deficiency inhibits the increased levels of immunoglobulins in serum induced by irradiation. These findings indicate that RNase L plays a role in the immune response induced by irradiation in the bone marrow. This study further enhances our understanding of the biological functions of RNase L in the immune response induced by irradiation and offers a novel approach for managing irradiation-induced bone marrow injury through the regulation of RNase L activation. Full article