Search Results (56)

MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO₄³⁻ modified single-stranded DNAs (ssDNAs); the resulting 3′ hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 10⁸ fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy. Full article

The immune response triggered when plant cell surface receptors recognize pathogen-associated molecular patterns (PAMPs) is known as PAMP-triggered immunity (PTI). Several studies have demonstrated that extracellular plant ferredoxin-like protein (PFLP) can enhance PTI signaling, thereby conferring resistance to bacterial diseases in various plants. The C-terminal casein kinase II (CK2) phosphorylation region of PFLP is essential for strengthening PTI. However, whether phosphorylation at this site directly enhances PTI signaling and consequently increases plant disease resistance remains unclear. To investigate this, site-directed mutagenesis was used to generate PFLPT90A, a non-phosphorylatable mutant, and PFLPT90D, a phospho-mimetic mutant, for functional analysis. Based on the experimental results, none of the recombinant proteins were able to enhance the hypersensitive response induced by the HrpN protein or increase resistance to the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum ECC17. These findings suggest that phosphorylation at the T90 residue might be essential for PFLP-mediated enhancement of plant immune responses, implying that this post-translational modification is likely required for its disease resistance function in planta. To further explore the relationship between PFLP phosphorylation and endogenous CK2, the Arabidopsis insertion mutant cka2 and the complemented line CKA2R were analyzed under treatment with flg22_Pst from Pseudomonas syringae pv. tomato. The effects of PFLP on the hypersensitive response, rapid oxidative burst, callose deposition, and susceptibility to soft rot confirmed that CK2 is required for these immune responses. Furthermore, expression analysis of PTI-related genes FRK1 and WRKY22/29 in the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that CK2 is necessary for PFLP to enhance flg22_Pst-induced immune signaling. Taken together, these findings suggest that PFLP enhances A. thaliana resistance to bacterial soft rot primarily by promoting the MAPK signaling pathway triggered by PAMP recognition, with CK2-mediated phosphorylation being essential for its function. Full article

rAj-HRP30 is a recombinant peptide derived from the wild-type rAj-HRP of Apostichopus japonicus through a gene-shortening mutation. It has a high histidine content (53.3% in its primary structure) and a molecular weight of 3.919 kDa, classifying it as a histidine-rich peptide. The literature reports indicate that human histidine-rich peptides exhibit antitumor activity. Previous research by our group demonstrated similar properties in rAj-HRP, the precursor of rAj-HRP30. Therefore, this study used Panc01 (human) and Panc02 (mouse) cells—highly malignant models with limited targeted therapies—to investigate the antitumor activity and mechanisms of rAj-HRP30 and evaluate its potential for pancreatic cancer treatment. This study designed a gene-shortening strategy for rAj-HRP and artificially synthesized the gene sequence of rAj-HRP30. The cDNA sequence of rAj-HRP30 was cloned into the pET23b vector, and the recombinant plasmid pET23b-HRP30 was transformed into E. coli BL21 for expression. Following IPTG induction, the recombinant peptide was purified using nickel ion affinity chromatography, yielding rAj-HRP30 with a purity exceeding 95%. rAj-HRP30 markedly inhibited the adhesion, migration, and invasion of Panc01 and Panc02 cells. It also disrupted cellular morphology and cytoskeletal structure while inducing apoptosis. These effects were dose-dependent. After confirming the in vitro anticancer activity of rAj-HRP30, this study employed Panc02 cells as a model to investigate its inhibitory mechanisms using Western blot analysis. The results revealed that rAj-HRP30 reduced FGFR1 expression in Panc02 cells and inhibited the downstream FYN and FAK signaling pathways, subsequently blocking the PI3K/AKT signaling and apoptosis pathways. In the apoptotic pathway, rAj-HRP30 was able to downregulate the expression of Bcl-2, Caspase-9, Caspase-3, Caspase-7, and PARP1 and upregulate the expression of Bax, cleaved Caspase-9, cleaved Caspase-3, cleaved Caspase-7, and cleaved-PARP1 to induce apoptosis in Panc02 cells. Furthermore, rAj-HRP30 also downregulated the expression of MMP2 and MMP9, thereby inhibiting the migration and invasion of Panc02 cells. Conclusion: rAj-HRP30 exhibits significant inhibitory effects on pancreatic ductal adenocarcinoma Panc01 and Panc02 cells in vitro. Its mechanism involves FGFR1-related signaling and apoptosis pathways. rAj-HRP30 shows promise as a therapeutic agent targeting FGFR for pancreatic cancer. Full article

Cell survival depends on precise gene expression, which is controlled sequentially. The guard proteins surveil mRNAs from their synthesis in the nucleus to their translation in the cytoplasm. Although the proteins within this group share many similarities, they play distinct roles in controlling nuclear mRNA maturation and cytoplasmic translation by supporting the degradation of faulty transcripts. Notably, this group is continuously expanding, currently including the RNA-binding proteins Npl3, Gbp2, Hrb1, Hrp1, and Nab2 in Saccharomyces cerevisiae. Some of the human serine–arginine (SR) splicing factors (SRSFs) show remarkable similarities to the yeast guard proteins and may be considered as functional homologues. Here, we provide a comprehensive summary of their crucial mRNA surveillance functions and their implications for cellular health. Full article

Grapes and grape products are rich in secondary metabolites such as phenolic compounds and anthocyanins, which have antioxidant properties. These compounds possess health-promoting attributes, including cardioprotective, antimicrobial, and anticancer effects. In recent years, biotechnological methods have been employed to produce high quantities and purity of secondary metabolites under in vitro conditions, aiming to elucidate their complex functions and optimize production methods. However, the potential effects of harpin proteins on the accumulation of secondary compounds in callus cultures have not been investigated thus far. Harpin proteins, encoded by the hrp gene clusters in Gram-negative phytopathogens, are known to trigger defense responses in various plant species by promoting the accumulation of secondary compounds. These findings suggest that harpin proteins may have the potential to enhance secondary metabolite accumulation in callus cultures. This study therefore investigated the potential of applying different concentrations of harpin protein (0, 0.1, 1, 10, and 100 ppm) to increase secondary metabolite production in calluses derived from petioles of the “Horoz Karası” grape cultivar. Our findings revealed that 1 and 10 ppm harpin treatments resulted in the highest anthocyanin accumulations, with 17.21 and 16.57 CV/g, respectively, representing 1.95- and 1.87-fold increases compared to control treatments, respectively. Total phenolic content peaked at 0.39 mg GAE g⁻¹ FW with the 1 ppm harpin treatment, representing a 4.33-fold increase over the control. Total flavanol levels reached their highest levels at 0.027 mg CE g⁻¹ FW with 1 and 10 ppm harpin concentrations, resulting in a 2.25-fold increase compared to the control. The highest averages for total flavonol content were recorded at 0.024 and 0.021 mg RE g⁻¹ FW with 1 and 10 ppm harpin concentrations, respectively, representing 1.5- and 1.3-fold increases over the control. Principal component analysis (PCA) corroborated the results obtained from the heatmap analysis, indicating that harpin applications at 1 and 10 ppm were the most effective concentration range for maximizing secondary metabolite synthesis, while very low or high concentrations diminished these effects. These findings offered valuable insights for optimizing the production of high-value bioactive compounds, which can be utilized in various fields such as medicine, pharmaceuticals, food, and cosmetics. These results are expected to serve as a valuable reference for elucidating the mechanisms by which harpin proteins, rarely used in vitro, exert their effects on grapevine calluses, contributing to the literature in this domain. Full article

Xanthomonas oryzae pv. oryzicola (Xoc) is a notorious plant pathogen. Like most bacterial pathogens, Xoc has evolved a complex regulatory network to modulate the expression of various genes related to pathogenicity. Here, we have identified TfmR, a transcriptional regulator belonging to the TetR family, as a key player in the virulence mechanisms of this phytopathogenic bacterium. We have demonstrated genetically that tfmR is involved in the hypersensitive response (HR), pathogenicity, motility and extracellular polysaccharide production of this phytopathogenic bacterium. Our investigations extended to exploring TfmR’s interaction with RpfG and HrpX, two prominent virulence regulators in Xanthomonas species. We found that TfmR directly binds to the promoter region of RpfG, thereby positively regulating its expression. Notably, constitutive expression of RpfG partly reinstates the pathogenicity compromised by TfmR-deletion mutants. Furthermore, our studies revealed that TfmR also exerts direct positive regulation on the expression of the T3SS regulator HrpX. Similar to RpfG, sustained expression of HrpX partially restores the pathogenicity of TfmR-deletion mutants. These findings underscore TfmR’s multifaceted role as a central regulator governing key virulence pathways in Xoc. Importantly, our research sheds light on the intricate molecular mechanisms underlying the regulation of pathogenicity in this plant pathogen. Full article

The early diagnosis of malaria is crucial to controlling morbidity and mortality. The World Health Organization (WHO) recommends diagnosing malaria either using light microscopy or a malaria rapid diagnostic test (RDT). Most RDTs use antibodies to detect two P. falciparum histidine-rich proteins named PfHRP2 and PfHRP3. However, false-negative results are known to occur due to the poor performance of RDTs depending on the species and the deletion of the Pfhrp2 and Pfhrp3 genes. This study evaluated new malaria RDTs for the detection of the human Plasmodium species. The Acro Malaria P.f./P.v./Pan Rapid Test Cassette allows the qualitative detection of parasite antigens, such as PfHRP2 specific to Plasmodium falciparum, PvLDH specific to Plasmodium vivax, and/or panLDH Plasmodium genus lactate dehydrogenase, in the blood of infected individuals. This RDT was assessed against 229 samples collected from imported malaria cases, mainly from Africa. The samples were previously diagnosed using light microscopy and RDT (SD Malaria Ag P.f./Pan, SD Bioline Alere Abbott), then confirmed using real time PCR. The two RDTs were evaluated using a comparison with real time PCR as the reference method, and their performances were compared with each other. Compared to SD RDT, the Acro RDT showed a better sensitivity to P. falciparum (96.8% vs. 89.8%), P. vivax (78.6% vs. 64.3%), P. ovale (73.7% vs. 5.3%), and P. malariae (20.0% vs. 0%). The respective specificities of the Acro RDT and SD RDT are 90.7% vs. 95.3% to P. falciparum, 100% to P. vivax, and 100% vs. 100% to Plasmodium genus. Therefore, Acro RDT showed better performance in the identification of P. ovale and low parasitaemia of P. falciparum. In addition, Acro RDT has the advantage of detecting PvLDH-specific antigens. The Acro Malaria RDT presents the benefits of detecting a P. falciparum antigen (PfHRP2) and a P. vivax antigen (PvLDH) with high sensitivity (96.8% and 73.7%, respectively) and specificity (90.7% and 100%, respectively). Acro Malaria P.f./P.v./Pan rapid diagnostic tests could be effectively used in endemic areas, especially when microscopic examination cannot be performed. Full article

The ongoing COVID-19 pandemic, driven by persistent SARS-CoV-2 transmission, threatens human health worldwide, underscoring the urgent need for an efficient, low-cost, rapid SARS-CoV-2 detection method. Herein, we developed a point-of-care SARS-CoV-2 detection method incorporating recombinase polymerase amplification (RPA) and DNA–protein crosslinking chemiluminescence (DPCL) (RPADPCL). RPADPCL involves the crosslinking of biotinylated double-stranded RPA DNA products with horseradish peroxidase (HRP)-labeled streptavidin (SA-HRP). Modified products are captured using SA-labeled magnetic beads, and then analyzed using a chemiluminescence detector and smartphone after the addition of a chemiluminescent substrate. Under optimal conditions, the RPADPCL limit of detection (LOD) was observed to be 6 copies (within the linear detection range of 1–300 copies) for a plasmid containing the SARS-CoV-2 N gene and 15 copies (within the linear range of 10–500 copies) for in vitro transcribed (IVT) SARS-CoV-2 RNA. The proposed method is convenient, specific, visually intuitive, easy to use, and does not require external excitation. The effective RPADPCL detection of SARS-CoV-2 in complex matrix systems was verified by testing simulated clinical samples containing 10% human saliva or a virus transfer medium (VTM) spiked with a plasmid containing a SARS-CoV-2 N gene sequence or SARS-CoV-2 IVT RNA. Consequently, this method has great potential for detecting targets in clinical samples. Full article

Tumors with a pathogenic BRCA1/2 mutation are homologous recombination (HR)-deficient (HRD) and consequently sensitive to platinum-based chemotherapy and Poly-[ADP-Ribose]-Polymerase inhibitors (PARPi). We hypothesized that functional HR status better reflects real-time HR status than BRCA1/2 mutation status. Therefore, we determined the functional HR status of 53 breast cancer (BC) and 38 ovarian cancer (OC) cell lines by measuring the formation of RAD51 foci after irradiation. Discrepancies between functional HR and BRCA1/2 mutation status were investigated using exome sequencing, methylation and gene expression data from 50 HR-related genes. A pathogenic BRCA1/2 mutation was found in 10/53 (18.9%) of BC and 7/38 (18.4%) of OC cell lines. Among BRCA1/2-mutant cell lines, 14/17 (82.4%) were HR-proficient (HRP), while 1/74 (1.4%) wild-type cell lines was HRD. For most (80%) cell lines, we explained the discrepancy between functional HR and BRCA1/2 mutation status. Importantly, 12/14 (85.7%) BRCA1/2-mutant HRP cell lines were explained by mechanisms directly acting on BRCA1/2. Finally, functional HR status was strongly associated with COSMIC single base substitution signature 3, but not BRCA1/2 mutation status. Thus, the majority of BRCA1/2-mutant cell lines do not represent a suitable model for HRD. Moreover, exclusively determining BRCA1/2 mutation status may not suffice for platinum-based chemotherapy or PARPi patient selection. Full article

Bacterial wilt is a significant soil-borne disease that poses a threat to mulberry production yield and quality of agricultural production worldwide. However, the disease resistance mechanisms dependent on root exudates are not well understood. In this present study, we investigated the antibacterial mechanisms of the main active substances (erucamide, oleamide, and camphor bromide) present in mulberry root exudates (MRE) against Ralstonia pseudosolanacearum (Rp), the causal agent of bacterial wilt. Our findings revealed that these three active substances inhibited the growth activity of Rp by affecting the cell morphology and extracellular polysaccharide content, as well as triggering a burst of reactive oxygen species. The active substances induced oxidative stress, leading to a decrease in Rp growth. Additionally, the expression levels of key genes in the hrp gene cluster (hrpB, hrpX, and hrpF) and other virulence-related genes (such as ripAW, ripAE, Rs5-4819, Rs5-4374, ace, egl3, and pehB) were significantly reduced upon treatment with the active substances. Further pathogenicity experiments demonstrated that root exudates (at a concentration of 1.5 mg·mL⁻¹) delayed or slowed down the occurrence of bacterial wilt in mulberry. These findings provide valuable insight into the antimicrobial mechanisms of MRE against Rp and lay a theoretical foundation for the development and application of biocontrol agents to control mulberry bacterial wilt. Full article

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in the food industry and cosmetics (glucose oxidase (GOx) and cellobiose dehydrogenase (CDH)), bioremediations (horseradish peroxidase (HRP) and laccase (LAC)), and medicine for biosensors and miniature biofuel cells (GOx, CDH, LAC, and HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with the heterologous expression of oxidoreductase enzymes, since there is a requirement not only for multiple OXR gene integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition and fermentation conditions during expression due to the need for transition metals (copper and iron) and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find the optimal formula using the above-mentioned approaches; most recently, researcher started using protein engineering and directed evolution to increase in the yield of recombinant enzyme production. In this review article, we will cover all the current state-of-the-art technologies and most recent advances in the field that yielded a high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increases in fermentation yield using cutting-edge technologies such as directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry, and microfluidics. Full article

Until 2020, Djiboutian health authorities relied on histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) to establish the diagnosis of Plasmodium falciparum. The rapid spread of P. falciparum histidine-rich protein-2 and -3 (pfhrp2/3) gene-deleted parasite strains in Djibouti has led the authorities to switch from HRP2-based RDTs to lactate dehydrogenase (LDH)-based RDTs targeting the plasmodial lactate dehydrogenase (pLDH) specific for P. falciparum and P. vivax (RapiGEN BIOCREDIT Malaria Ag Pf/Pv pLDH/pLDH) in 2021. This study was conducted with the primary objective of evaluating the diagnostic performance of this alternative RDT. Operational constraints related, in particular, to the implementation of this RDT during the COVID-19 pandemic were also considered. The performance of BIOCREDIT Malaria Ag Pf/Pv (pLDH/pLDH) RDT was also compared to our previously published data on the performance of two HRP2-based RDTs deployed in Djibouti in 2018–2020. The diagnosis of 350 febrile patients with suspected malaria in Djibouti city was established using two batches of RapiGEN BIOCREDIT Malaria Ag Pf/Pv (pLDH/pLDH) RDT over a two-year period (2022 and 2023) and confirmed by real-time quantitative polymerase chain reaction. The sensitivity and specificity for the detection of P. falciparum were 88.2% and 100%, respectively. For P. vivax, the sensitivity was 86.7% and the specificity was 100%. Re-training and closer supervision of the technicians between 2022 and 2023 have led to an increased sensitivity to detect P. falciparum (69.8% in 2022 versus 88.2% in 2023; p < 0.01). The receiver operating characteristic curve analysis highlighted a better performance in the diagnosis of P. falciparum with pLDH-based RDTs compared with previous HRP2-based RDTs. In Djibouti, where pfhrp2-deleted strains are rapidly gaining ground, LDH-based RDTs seem to be more suitable for diagnosing P. falciparum than HRP2-based RDTs. Awareness-raising and training for technical staff have also been beneficial. Full article

The root architecture of a range of host plants is altered in response to Ralstonia solanacearum infection. This work aimed to identify host genes involved in root development during R. solanacearum infection. A deficient mutant of the type III secretion system regulator hrpB was created in R. solanacearum GMI1000. The hrpB mutant was impaired in virulence but showed a similar suppressive effect as wild-type GMI1000 on tomato root development. Based on comparative transcriptome analysis, 209 genes were found that showed the same changed expression pattern in GMI1000 and hrpB mutant infected roots relative to uninoculated roots. Among them, the wall-associated receptor kinase WAKL20 was substantially downregulated in GMI1000 and hrpB mutant infected roots. Knockdown of WAKL20 led to a shorter primary root length and fewer lateral roots in tomato as well as in Nicotiana benthamiana. The WAKL20 is a pivotal target suppressed by R. solanacearum to shape the altered root development during infection. Full article

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence. Full article

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. We previously conducted a screening for reduced virulence using Tn5 transposon mutants and identified one of the transcriptional factors, HexR, as a potential Pcal virulence factor. However, the role of HexR in plant pathogenic Pseudomonas virulence has not been investigated well. Here, we demonstrated that the Pcal hexR mutant showed reduced disease symptoms and bacterial populations on cabbage, indicating that HexR contributes to Pcal virulence. We used RNA-seq analysis to characterize the genes regulated by HexR. We found that several type three secretion system (T3SS)-related genes had lower expression of the Pcal hexR mutant. Five genes were related to T3SS machinery, two genes were related to type three helper proteins, and three genes encoded type three effectors (T3Es). We also confirmed that T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, were also down-regulated in the Pcal hexR mutant both in culture and in vivo by using RT-qPCR. T3SS functions to suppress plant defense in host plants and induce hypersensitive response (HR) cell death in non-host plants. Therefore, we investigated the expression profiles of cabbage defense-related genes, including PR1 and PR5, and found that the expressions of these genes were greater in the Pcal hexR mutant. We also demonstrated that the hexR mutant did not induce HR cell death in non-host plants, indicating that HexR contributes in causing HR in nonhost plants. Together, these results indicate that the mutation in hexR leads to a reduction in the T3SS-related gene expression and thus an impairment in plant defense suppression, reducing Pcal virulence. Full article