ROS-Induced Gingival Fibroblast Senescence: Implications in Exacerbating Inflammatory Responses in Periodontal Disease.

Periodontal disease is the pathological outcome of the overwhelming inflammation in periodontal tissue. Cellular senescence has been associated with chronic inflammation in several diseases. However, the role of cellular senescence in the pathogenesis of periodontal disease remained unclear. This study aimed to investigate the role and the mechanism of cellular senescence in periodontal disease. Using single-cell RNA sequencing, we first found the upregulated level of cellular senescence in fibroblasts and endothelial cells from inflamed gingival tissue. Subsequently, human gingival fibroblasts isolated from healthy and inflamed gingival tissues were labeled as H-GFs and I-GFs, respectively. Compared to H-GFs, I-GFs exhibited a distinct cellular senescence phenotype, including an increased proportion of senescence-associated ß-galactosidase (SA-ß-gal) positive cells, enlarged cell morphology, and significant upregulation of p16INK4A expression. We further observed increased cellular reactive oxygen species (ROS) activity, mitochondrial ROS, and DNA damage of I-GFs. These phenotypes could be reversed by ROS scavenger NAC, which suggested the cause of cellular senescence in I-GFs. The migration and proliferation assay showed the decreased activity of I-GFs while the gene expression of senescence-associated secretory phenotype (SASP) factors such as IL-1ß, IL-6, TGF-ß, and IL-8 was all significantly increased. Finally, we found that supernatants of I-GF culture induced more neutrophil extracellular trap (NET) formation and drove macrophage polarization toward the CD86-positive M1 pro-inflammatory phenotype. Altogether, our findings implicate that, in the inflamed gingiva, human gingival fibroblasts acquire a senescent phenotype due to oxidative stress-induced DNA and mitochondrial damage, which in turn activate neutrophils and macrophages through the secretion of SASP factors.

The Critical Role of Pyroptosis in Peri-Implantitis.

Background: Peri-implantitis (PI) is a prevalent complication of implant treatment. Pyroptosis, a distinctive inflammatory programmed cell death, is crucial to the pathophysiology of PI. Despite its importance, the pyroptosis-related genes (PRGs) influencing PI's progression remain largely unexplored. Methods: This study conducted histological staining and transcriptome analyze from three datasets. The intersection of differentially expressed genes (DEGs) and PRGs was identified as pyroptosis-related differentially expressed genes (PRDEGs). Functional enrichment analyses were conducted to shed light on potential underlying mechanisms. Weighted Gene Co-expression Network Analysis (WGCNA) and a pyroptotic macrophage model were utilized to identify and validate hub PRDEGs. Immune cell infiltration in PI and its relationship with hub PRDEGs were also examined. Furthermore, consensus clustering was performed to identify new PI subtypes. Protein-protein interaction (PPI) network, competing endogenous RNA (ceRNA) network, mRNA-mRNA binding protein regulatory (RBP) network, and mRNA-drugs regulatory network of hub PRDEGs were also analyzed. Results: Eight hub PRDEGs were identified: PGF, DPEP1, IL36B, IFIH1, TCEA3, RIPK3, NET7, and TLR3, which are instrumental in the PI's progression. Two PI subtypes were distinguished, with Cluster 1 exhibiting higher immune cell activation. The exploration of regulatory networks provided novel mechanisms and therapeutic targets in PI. Conclusion: Our research highlights the critical role of pyroptosis and identifies eight hub PRDEGs in PI's progression, offering insights into novel immunotherapy targets and laying the foundation for advanced diagnostic and treatment strategies. This contributes to our understanding of PI and underscores the potential for personalized clinical management.

Comparison of the osteogenic potential of fibroblasts from different sources.

OBJECTIVE: With the growing interest in the role of fibroblasts in osteogenesis, this study presents a comparative evaluation of the osteogenic potential of fibroblasts derived from three distinct sources: human gingival fibroblasts (HGFs), mouse embryonic fibroblasts (NIH3T3 cells), and mouse subcutaneous fibroblasts (L929 cells). MC3T3-E1 pre-osteoblast cells were employed as a positive control for osteogenic behavior. DESIGN: Our assessment involved multiple approaches, including vimentin staining for cell origin verification, as well as ALP and ARS staining in conjunction with RT-PCR for osteogenic characterization. RESULTS: Our findings revealed the superior osteogenic differentiation capacity of HGFs compared to MC3T3-E1 and NIH3T3 cells. Analysis of ALP staining confirmed that early osteogenic differentiation was most prominent in MC3T3-E1 cells at 7 days, followed by NIH3T3 and HGFs. However, ARS staining at 21 days demonstrated that HGFs produced the highest number of calcified nodules, indicating their robust potential for late-stage mineralization. This late-stage osteogenic potential of HGFs was further validated through RT-PCR analysis. In contrast, L929 cells displayed no significant osteogenic differentiation potential. CONCLUSIONS: In light of these findings, HGFs emerge as the preferred choice for seed cells in bone tissue engineering applications. This study provides valuable insights into the potential utility of HGFs in the fields of bone tissue engineering and regenerative medicine.

Janus silk fibroin/polycaprolactone-based scaffold with directionally aligned fibers and porous structure for bone regeneration.

To promote bone repair, it is desirable to develop three-dimensional multifunctional fiber scaffolds. The densely stacked and tightly arranged conventional two-dimensional electrospun fibers hinder cell penetration into the scaffold. Most of the existing three-dimensional structural materials are isotropic and monofunctional. In this research, a Janus nanofibrous scaffold based on silk fibroin/polycaprolactone (SF/PCL) was fabricated. SF-encapsulated SeNPs demonstrated stability and resistance to aggregation. The outside layer (SF/PCL/Se) of the Janus nanofiber scaffold displayed a structured arrangement of fibers, facilitating cell growth guidance and impeding cell invasion. The inside layer (SF/PCL/HA) featured a porous structure fostering cell adhesion. The Janus fiber scaffold containing SeNPs notably suppressed S. aureus and E. coli activities, correlating with SeNPs concentration. In vitro, findings indicated considerable enhancement in alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts and upregulation of genes linked to osteogenic differentiation with exposure to the SF/PCL/HA/Se Janus nanofibrous scaffold. Moreover, in vivo, experiments demonstrated successful critical bone defect repair in mouse skulls using the SF/PCL/HA/Se Janus nanofiber scaffold. These findings highlight the potential of the SF/PCL-based Janus nanofibrous scaffold, integrating SeNPs and nHA, as a promising biomaterial in bone tissue engineering.

Biphasic calcium phosphate recruits Tregs to promote bone regeneration.

The use of bone substitute materials is crucial for the healing of large bone defects. Immune response induced by bone substitute materials is essential in bone regeneration. Prior research has mainly concentrated on innate immune cells, such as macrophages. Existing research suggests that T lymphocytes, as adaptive immune cells, play an indispensable role in bone regeneration. However, the mechanisms governing T cell recruitment and specific subsets that are essential for bone regeneration remain unclear. This study demonstrates that CD4+ T cells are indispensable for ectopic osteogenesis by biphasic calcium phosphate (BCP). Subsequently, the recruitment of CD4+ T cells is closely associated with the activation of calcium channels in macrophages by BCP to release chemokines Ccl3 and Ccl17. Finally, these recruited CD4+ T cells are predominantly Tregs, which play a significant role in ectopic osteogenesis by BCP. These findings not only shed light on the immune-regenerative process after bone substitute material implantation but also establish a theoretical basis for developing bone substitute materials for promoting bone tissue regeneration. STATEMENT OF SIGNIFICANCE: Bone substitute material implantation is essential in the healing of large bone defects. Existing research suggests that T lymphocytes are instrumental in bone regeneration. However, the specific mechanisms governing T cell recruitment and specific subsets that are essential for bone regeneration remain unclear. In this study, we demonstrate that activation of calcium channels in macrophages by biphasic calcium phosphate (BCP) causes them to release the chemokines Ccl3 and Ccl17 to recruit CD4+ T cells, predominantly Tregs, which play a crucial role in ectopic osteogenesis by BCP. Our findings provide a theoretical foundation for developing bone substitute material for bone tissue regeneration.

Hi-Tag: a simple and efficient method for identifying protein-mediated long-range chromatin interactions with low cell numbers.

Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation. However, these techniques require complex experimental procedures and millions of cells per experiment, which limits their widespread application in life science research. Here, we develop a novel method, Hi-Tag, that identifies high-resolution, long-range chromatin interactions through transposase tagmentation and chromatin proximity ligation (with a phosphorothioate-modified linker). Hi-Tag can be implemented using as few as 100,000 cells, involving simple experimental procedures that can be completed within 1.5 days. Meanwhile, Hi-Tag is capable of using its own data to identify the binding sites of specific proteins, based on which, it can acquire accurate interaction information. Our results suggest that Hi-Tag has great potential for advancing chromatin interaction studies, particularly in the context of limited cell availability.

AGIDB: a versatile database for genotype imputation and variant decoding across species.

The high cost of large-scale, high-coverage whole-genome sequencing has limited its application in genomics and genetics research. The common approach has been to impute whole-genome sequence variants obtained from a few individuals for a larger population of interest individually genotyped using SNP chip. An alternative involves low-coverage whole-genome sequencing (lcWGS) of all individuals in the larger population, followed by imputation to sequence resolution. To overcome limitations of processing lcWGS data and meeting specific genotype imputation requirements, we developed AGIDB (https://agidb.pro), a website comprising tools and database with an unprecedented sample size and comprehensive variant decoding for animals. AGIDB integrates whole-genome sequencing and chip data from 17 360 and 174 945 individuals, respectively, across 89 species to identify over one billion variants, totaling a massive 688.57 TB of processed data. AGIDB focuses on integrating multiple genotype imputation scenarios. It also provides user-friendly searching and data analysis modules that enable comprehensive annotation of genetic variants for specific populations. To meet a wide range of research requirements, AGIDB offers downloadable reference panels for each species in addition to its extensive dataset, variant decoding and utility tools. We hope that AGIDB will become a key foundational resource in genetics and breeding, providing robust support to researchers.

CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.

Correction: Zhang et al. Genome-Scale CRISPR Knockout Screening Identifies BACH1 as a Key Regulator of Aflatoxin B1-Induced Oxidative Damage. Antioxidants 2022, 11, 1787.

In the original publication [...].

A Zinc- and Calcium-Rich Lysosomal Nanoreactor Rescues Monocyte/Macrophage Dysfunction under Sepsis.

Sepsis is a dysregulation of the immune response to pathogens and has high morbidity and mortality worldwide. However, the unclear mapping and course of dysregulated immune cells currently hinders the development of advanced therapeutic strategies to treat sepsis. Here, evidence is provided using single-cell RNA sequencing from peripheral blood mononuclear cells in sepsis that pathogens attacking monocytes/macrophages disrupt their immune function. The results reveal an enormous decline in monocytes/macrophages in sepsis and chart the evolution of their impaired phagocytosis (Pha) capabilities. Inspired by these findings, nanoparticles, named "Alpha-MOFs," are developed that target dysfunctional monocytes/macrophages to actively (A) lift (L) Pha by the release of lysosome-sensitive ions from a mineralized metal-organic framework (MOF). Alpha-MOFs have good stability and biosafety in peripheral blood and efficiently targeted monocytes/macrophages. They also release calcium and zinc ions into monocyte/macrophage lysosomes to promote the Pha and degradation of bacteria. Taken together, these results suggest that Alpha-MOFs rescue monocytes/macrophages dysfunction and effectively improve their survival rate during sepsis.

Designing polymersomes with inhomogeneous membranes by co-assembly of block copolymers for controlled morphological reversibility.

Polymersomes with inhomogeneous membranes in composition and structure have generated widespread interest for the preparation of functionalized nanocarriers. We propose a simple but versatile strategy to manipulate inhomogeneous subdomains on polymersome membranes by the co-assembly of block copolymer blends with varied molecular architectures and chemistries. Both binary and ternary copolymer blends are considered to construct polymersomes, and the subdomains of the membranes are formed by controlling the difference in the flexibility and rigidity of different blocks. This difference contributes to the formation of disk-like domains (by rigid blocks) and soft domains (by flexible blocks) on the membrane. An interesting effect of this structure is that in response to external stimuli, the soft membrane domain becomes worm-like or porous to "open" the polymersome for matter exchange, while the rigid domain stays undecomposed and acts like an anchor binding all flexible copolymers. Once the external stimuli disappear, all flexible copolymers can be pulled back to restore the original polymersome morphology (i.e., "close" the polymersome). The specific morphological reversibility of hybrid polymersomes holds great potential for practical applications where changeable membrane permeability or shape under environmental stimuli is highly needed.

Genome-Scale CRISPR Knockout Screening Identifies BACH1 as a Key Regulator of Aflatoxin B1-Induced Oxidative Damage.

Aflatoxin B1 (AFB1) is amongst the mycotoxins commonly affecting human and animal health, raising global food safety and control concerns. The mechanisms underlying AFB1 toxicity are poorly understood. Moreover, antidotes against AFB1 are lacking. Genome-wide CRISPR/Cas9 knockout screening in porcine kidney cells identified the transcription factor BTB and CNC homolog 1 (BACH1) as a gene required for AFB1 toxicity. The inhibition of BACH1 expression in porcine kidney cells and human hepatoma cells resulted in increased resistance to AFB1. BACH1 depletion attenuates AFB1-induced oxidative damage via the upregulation of antioxidant genes. Subsequently, virtual structural screening identified the small molecule 1-Piperazineethanol, α-[(1,3-benzodioxol-5-yloxy)methyl] -4-(2-methoxyphenyl) (M2) as an inhibitor of BACH1. M2 and its analogues inhibited AFB1-induced porcine and human cell death in vitro, while M2 administration significantly improved AFB1-induced symptoms of weight loss and liver injury in vivo. These findings demonstrate that BACH1 plays a central role in AFB1-induced oxidative damage by regulating antioxidant gene expression. We also present a potent candidate small-molecule inhibitor in developing novel treatments for AFB1 toxicity.

Protein interactions network of goat innate immune signalling pathway proteins and Haemonchus contortus excretory-secretory proteins.

Haemonchus contortus (H. contortus) has caused a huge impact on the animal husbandry economy in the world's tropical and subtropical regions. Innate immunity is the first-line of host defence. The host recognizes pathogen-associated molecular patterns (PAMPs) through a variety of pattern recognition receptors (PRRs) and activates downstream signalling pathways to resist pathogens invasion. Therefore, elucidating the immune interaction between host and pathogen is key to understanding how the host resists the pathogen. We identified 1516 protein-protein interactions (PPIs) between goat innate immune signal pathway proteins and H. contortus excretory-secretory proteins (ESPs) by Recombination-based "Library vs. Library" yeast two-hybrid system (RLL-Y2H) and constructed the PPIs network. Among them, the NLR and IL-17 signalling pathways have the most protein interactions. And there were more interaction proteins between NOD1 and MUC5AC proteins in the pathways. Combined with the differentially expressed genes (DEGs) of susceptible and resistant goats identified in the preliminary work of our laboratory, we selected the intersection genes to construct the PPIs network, and TRAF2 appeared as a key protein of goat innate immune signalling pathway. We initially studied the PPIs between goat and H. contortus ESPs, which provides valuable information for better understanding the immune interaction between the goats and the H. contortus.

Characterization and perturbation of CTCF-mediated chromatin interactions for enhancing myogenic transdifferentiation.

Expression of key transcription factors can induce transdifferentiation in somatic cells; however, this conversion is usually incomplete due to undefined intrinsic barriers. Here, we employ MyoD-induced transdifferentiation of fibroblasts as a model to illustrate the chromatin structures that impede the cell-fate transition. Focusing on the three-dimensional (3D) chromatin interactions, we show that MyoD directly establishes chromatin loops to activate myogenic transcriptional program. Similarly, dynamic changes of CTCF-mediated chromatin interactions are favorable for fibroblast-to-myoblast conversion. However, a substantial portion of CTCF-mediated chromatin interactions remain stable, and the associated genes are steady in expression and enriched for fibroblast function that may restrict cell-identity transformation. Temporal CTCF depletion can interrupt the resistant chromatin loops to enhance myogenic transdifferentiation in mice, pig, and chicken fibroblasts. Therefore, during induced transdifferentiation, the transcription factor can directly reorganize the 3D chromatin interactions, and perturbation of CTCF-mediated genome topology may resolve the limitations of cell fate transitions.

Enhancer-promoter interaction maps provide insights into skeletal muscle-related traits in pig genome.

BACKGROUND: Gene expression programs are intimately linked to the interplay of active cis regulatory elements mediated by chromatin contacts and associated RNAs. Genome-wide association studies (GWAS) have identified many variants in these regulatory elements that can contribute to phenotypic diversity. However, the functional interpretation of these variants remains nontrivial due to the lack of chromatin contact information or limited contact resolution. Furthermore, the distribution and role of chromatin-associated RNAs in gene expression and chromatin conformation remain poorly understood. To address this, we first present a comprehensive interaction map of nuclear dynamics of 3D chromatin-chromatin interactions (H3K27ac BL-HiChIP) and RNA-chromatin interactions (GRID-seq) to reveal genomic variants that contribute to complex skeletal muscle traits. RESULTS: In a genome-wide scan, we provide systematic fine mapping and gene prioritization from GWAS leading signals that underlie phenotypic variability of growth rate, meat quality, and carcass performance. A set of candidate functional variants and 54 target genes previously not detected were identified, with 71% of these candidate functional variants choosing to skip over their nearest gene to regulate the target gene in a long-range manner. The effects of three functional variants regulating KLF6 (related to days to 100 kg), MXRA8 (related to lean meat percentage), and TAF11 (related to loin muscle depth) were observed in two pig populations. Moreover, we find that this multi-omics interaction map consists of functional communities that are enriched in specific biological functions, and GWAS target genes can serve as core genes for exploring peripheral trait-relevant genes. CONCLUSIONS: Our results provide a valuable resource of candidate functional variants for complex skeletal muscle-related traits and establish an integrated approach to complement existing 3D genomics by exploiting RNA-chromatin and chromatin-chromatin interactions for future association studies.

Fibroblasts Mediate Ectopic Bone Formation of Calcium Phosphate Ceramics.

Heterogeneity of fibroblasts directly affects the outcome of tissue regeneration; however, whether bioactive ceramics regulate bone regeneration through fibroblasts is unclear. Ectopic bone formation model with biphasic calcium phosphate (BCP) implantation was used to investigate the temporal and spatial distribution of fibroblasts around ceramics. The effect of BCP on L929 fibroblasts was evaluated by EdU assay, transwell assay, and qRT-PCR. Further, the effect of its conditioned medium on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was confirmed by ALP staining. SEM and XRD results showed that BCP contained abundant micro- and macro-pores and consisted of hydrogen-apatite (HA) and ß-tricalcium phosphate (ß-TCP) phases. Subsequently, BCP implanted into mice muscle successfully induced osteoblasts and bone formation. Fibroblasts labelled by vimentin gathered around BCP at 7 days and peaked at 14 days post implantation. In vitro, BCP inhibited proliferation of L929 fibroblast but promoted its migration. Moreover, expression of Col1a1, Bmp2, and Igf1 in L929 treated by BCP increased significantly while expression of Tgfb1 and Acta did not change. ALP staining further showed conditioned media from L929 fibroblasts treated by BCP could enhance osteogenic differentiation of BMSCs. In conclusion, fibroblasts mediate ectopic bone formation of calcium phosphate ceramics.

CD301b+ macrophages mediate angiogenesis of calcium phosphate bioceramics by CaN/NFATc1/VEGF axis.

Calcium phosphate (CaP) bioceramics are important for tissue regeneration and immune response, yet how CaP bioceramics influence these biological processes remains unclear. Recently, the role of immune cells in biomaterial-mediated regeneration, especially macrophages, has been well concerned. CD301b+ macrophages were a new subset of macrophages we have discovered, which were required for bioceramics-mediated bone regeneration. Nevertheless, the impact of CD301b+ macrophages on angiogenesis, which is a vital prerequisite to bone formation is yet indistinct. Herein, we found that CD301b+ macrophages were closely correlated to angiogenesis of CaP bioceramics. Additionally, depletion of CD301b+ macrophages led to the failure of angiogenesis. We showed that store-operated Ca2+ entry and calcineurin signals regulated the VEGF expression of CD301b+ macrophages via the NFATc1/VEGF axis. Inhibition of calcineurin effectively impaired angiogenesis via decreasing the infiltration of CD301b+ macrophages. These findings provided a potential immunomodulatory strategy to optimize the integration of angiogenesis and bone tissue engineering scaffold materials.

CD301b+ Macrophages as Potential Target to Improve Orthodontic Treatment under Mild Inflammation.

Due to improvements of quality of life and the demand for aesthetics, more and more people are choosing orthodontic treatments, resulting in a surge in adult orthodontic patients in recent years. However, a large amount of clinical evidence shows that many orthodontic patients have mild periodontitis in the periodontal tissues, which affects the efficacy of the orthodontic treatment or aggravates the periodontal condition. Therefore, it is important to identify the key factors that affect orthodontic treatments in this inflammatory environment. The aim of this study was to investigate the role of macrophages in orthodontic treatments under inflammatory environments. By analyzing the functional groups of macrophages in the orthodontic rat model of periodontitis, we found that macrophages with high expression levels of CD301b could improve the periodontal microenvironment and improve the efficiency of the orthodontic tooth movement. CD301b+ macrophages transplanted into the model can promote osteogenesis around orthodontic moving teeth, improve bone remodeling during orthodontic treatment, and accelerate orthodontic tooth movement. Considered together, these results suggest that CD301b+ macrophages may play an active role in orthodontic treatments in inflammatory environments and may serve as potential regulatory targets.

Rapid Visual CRISPR Assay: A Naked-Eye Colorimetric Detection Method for Nucleic Acids Based on CRISPR/Cas12a and a Convolutional Neural Network.

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.