Double-stranded DNA Research Articles

FAN1 Deletion Variant in Basenji Dogs with Fanconi Syndrome.

Background: Fanconi syndrome is a disorder of renal proximal tubule transport characterized by metabolic acidosis, amino aciduria, glucosuria, and phosphaturia. There are acquired and hereditary forms of this disorder. A late-onset form of Fanconi syndrome in Basenjis was first described in 1976 and is now recognized as an inherited disease in these dogs. In part because of the late onset of disease signs, the disorder has not been eradicated from the breed by selective mating. A study was therefore undertaken to identify the molecular genetic basis of the disease so that dogs could be screened prior to breeding in order to avoid generating affected offspring. Methods: Linkage analysis within a large family of Basenjis that included both affected and unaffected individuals was performed to localize the causative variant within the genome. Significant linkage was identified between chromosome 3 (CFA3) makers and the disease phenotype. Fine mapping restricted the region to a 2.7 Mb section of CFA3. A whole genome sequence of a Basenji affected with Fanconi syndrome was generated, and the sequence data were examined for the presence of potentially deleterious homozygous variants within the mapped region. Results: A homozygous 317 bp deletion was identified in the last exon of FAN1 of the proband. 78 Basenjis of known disease status were genotyped for the deletion variant. Among these dogs, there was almost complete concordance between genotype and phenotype. The only exception was one dog that was homozygous for the deletion variant but did not exhibit signs of Fanconi syndrome. Conclusions: These data indicate that the disorder is very likely the result of FAN1 deficiency. The mechanism by which this deficiency causes the disease signs remains to be elucidated. FAN1 has endonuclease and exonuclease activity that catalyzes incisions in regions of double-stranded DNA containing interstrand crosslinks. FAN1 inactivation may cause Fanconi syndrome in Basenjis by sensitization of kidney proximal tubule cells to toxin-mediated DNA crosslinking, resulting in the accumulation of genomic and mitochondrial DNA damage in the kidney. Differential exposure to environmental toxins that promote DNA crosslink formation may explain the wide age-at-onset variability for the disorder in Basenjis.

Enzyme-free and highly sensitive detection of human epidermal growth factor receptor-2 based on MNAzyme signal amplification in breast cancer.

As a common cancer biomarker, human epidermal growth factor receptor-2 (HER2) is highly expressed in breast cancer. Consequently, developing a simple and accurate HER2 sensing platform is of great significance for early diagnosis and treatment of breast cancer. Herein, we developed a rapid enzyme-free fluorescent assay biosensor based on MNAzyme signal amplification for breast cancer biomarker, HER2. The MNAzyme consists of multiple parts, including complementary DNA (cDNA) and two parts of DNAzyme (partzyme A/B). Initially, cDNA is blocked by combining with the HER2 aptamer to form a double-stranded DNA. When HER2 is present, cDNA is released as a result of the binding between HER2 and its aptamer. Due to the complementary sequences among cDNA and partzyme A/B, the MNAzyme is successfully assembled to cleave the substrate, recovering the fluorescence output. The MNAzyme biosensor exhibited a low detection limit of 0.02 ng mL-1 and excellent selectivity. Furthermore, the proposed biosensor can also change the recognition element by changing the aptamer sequence to detect various biomarkers, holding great potential for cancer diagnosis and other related biomedical applications.

Abstract I003: Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the DHX9 helicase. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver. Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I003.

Abstract IA007: Modulating cfDNA biology to enhance liquid biopsies

Abstract Liquid biopsies could transform cancer care but require higher sensitivity to detect early-stage tumors and minimal residual disease (MRD) and to enable tumor molecular profiling and therapy response monitoring for most patients. In this talk, I will focus on the significant biological bottlenecks upstream of cell-free DNA (cfDNA) testing. I will posit that in many circumstances, insufficient circulating tumor DNA (ctDNA) drawn in a blood tube cannot be overcome by assay technology alone, and that in vivo modulation of cfDNA biology may also be required to enhance liquid biopsies. Inspired by widespread use of diagnostic drugs in other areas of medicine, we sought to create the first “priming agents” for liquid biopsies that could be administered before a blood draw to recover more ctDNA. I will describe two such approaches—a monoclonal antibody against double-stranded DNA and a liposome nanoparticle—that briefly attenuate cfDNA clearance by shielding it from nuclease digestion and cellular uptake. These enabled >10x more ctDNA to be collected when administered within 1-2 hours of a blood draw in mice. This improved the analytical limit of detection for ctDNA testing by >10-fold, provided more complete representation of the tumor genome in each blood sample, and increased the sensitivity for detection of small tumors from <10% to >75% in mice. Envisioning a future where liquid biopsies are optionally enhanced with priming, I will also explore a unique potential pairing with our tumor-informed, whole-genome, mutation enrichment sequencing MRD test called MAESTRO, and how it stands to enable routine ctDNA detection below 1 part per million. Citation Format: Viktor Adalsteinsson. Modulating cfDNA biology to enhance liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA007.

Effect of Pregenomic RNA on the Mechanical Stability of HBV Capsid by Coarse-Grained Molecular Simulations.

Hepatitis B virus (HBV) is a double-stranded DNA virus, but its life cycle involves an intermediate stage, during which pregenomic RNA (pgRNA) is encapsulated in the capsid and then reverse-transcribed into the minus DNA strand. These immature HBV virions are the key target for antiviral drug discovery. In this study, we investigate the flexibility and mechanical stability of the HBV capsid containing pgRNA by employing residue-resolved coarse-grained molecular dynamics simulations. The results showed that the presence of pgRNA tends to decrease the overall flexibility of the capsid. In addition, the symmetrically arranged subunits of the capsid show asymmetry in the dominant modes of the conformational fluctuations with or without the presence of pgRNA. Furthermore, the simulations revealed that the presence of pgRNA enhances the overall mechanical stability of the virion particle. Electrostatic interactions between the disordered CTD of capsid and pgRNA were found to play a crucial role in modulating viral mechanical stability. Decreasing the electrostatic interactions by CTD phosphorylation or high salt concentration significantly reduces the mechanical stability of the HBV capsid containing pgRNA. Finally, the 2-fold symmetric sites have been proposed to be the most vulnerable to rupture during the initial stages of capsid disassembly. These findings could enhance our understanding of the physical basis of viral invasion and provide valuable insights into the development of antiviral drugs.

Characterization and Potential Application of Phage vB_PmuM_CFP3 for Phage Therapy Against Avian Pasteurella multocida.

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3's typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4-10 and temperatures of 30-40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3's specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera.

Synthetic Lesions with a Fluorescein Carbamoyl Group As Analogs of Bulky Lesions Removable by Nucleotide Excision Repair: A Comparative Study on Properties.

Mammalian nucleotide excision repair (NER), known for its broad substrate specificity, is responsible for removal of bulky lesions from DNA. Over 30 proteins are involved in NER, which includes two distinct pathways: global genome NER and transcription-coupled repair. The complexity of these processes, the use of extended DNA substrates, and the presence of bulky DNA lesions induced by chemotherapy have driven researchers to seek more effective methods by which to assess NER activity, as well as to develop model DNAs that serve as efficient substrates for studying lesion removal. In this work, we conducted a comparative analysis of model DNAs containing bulky lesions. One of these lesions, N-[6-{5(6)-fluoresceinylcarbamoyl}hexanoyl]-3-amino-1,2-propanediol (nFluL), is known to be efficiently recognized and excised by NER. The second lesion, N-[6-{5(6)-fluoresceinylcarbamoyl}]-3-amino-1,2-propanediol (nFluS), has not previously been tested as a substrate for NER. To evaluate the efficiency of lesion excision, a 3'-terminal labeling method was employed to analyze the excision products. The results showed that nFluS is removed approximately twice as efficiently as nFluL. Comparative analyses of the effects of nFluL and nFluS on the geometry and thermal stability of DNA duplexes - combined with spectrophotometric and spectrofluorimetric titrations of these DNAs with complementary strands - were performed next. They revealed that the absence of an extended flexible linker in nFluS alters the interaction of the bulky fluorescein moiety with neighboring nitrogenous bases in double-stranded DNA. This absence is associated with the enhanced efficiency of excision of nFluS, making it a more effective synthetic analog for studying bulky-lesion removal in model DNA substrates.

ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism

The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering.

Stabilization of a single-stranded DNA of adeno-associated virus by inverted terminal repeats

Parvoviruses have evolved to possess a linear single-stranded DNA (ssDNA) genome ranging from 4 to 6.3 kb. Adeno-associated virus (AAV), a member of the Parvoviridae family, contains approximately 5 kb of linear ssDNA within its capsid. This ssDNA features two 145-base inverted terminal repeats (ITRs) positioned at each end. ITRs have a T-shaped hairpin secondary structure, which plays a crucial role in viral replication. To investigate the impact of ITRs on ssDNA stability, we conducted a DNA denaturation-reannealing assay in 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate buffer at pH7.9. Conventional double-stranded DNA (dsDNA) fragments retain a reannealing capability of over 50% for sizes under 8.8 kb, gradually losing this capability as sizes increase; however, dsDNA fragments in rAAV ranging from 0.7 to 6.3 kb did not exhibit a reannealing profile. This suggests that the presence of ITRs at both ends hinders annealing between the complementary strands. These results indicate that ITR structures preferentially induce an ssDNA conformation less than 6.3 kb in size, and that the stability of AAV ssDNA contributes to the viral life cycle, including processes such as infection, replication, and packaging. Considering the size of parvovirus genomes, it appears that their genomes require reversible flexibility in complementary DNA strands; simultaneously, this adaptability needs to be regulated by specific palindromic ITRs at both ends.

Measuring Meiotic Recombination Frequency in Schizosaccharomyces pombe Using an Engineered Genetic Interval.

The fission yeast Schizosaccharomyces pombe has been used to elucidate meiotic recombination mechanisms for decades. Alongside the budding yeast Saccharomyces cerevisiae, research employing fission yeast has been instrumental in advancing our knowledge of double-stranded DNA break (DSB) formation and repair during meiosis. Genetic recombination assays are the workhorses of gene conversion and crossover frequency analysis; these have been employed to investigate cis and trans determinants of meiotic recombination. Here, I describe meiotic recombination assays engineered by the introduction of nutritional markers up- and downstream of the ade6 and ade7 genes. These particular setups enable a comprehensive assessment of reproductive success in a single assay because spore viability and the frequency of gene conversion, crossovers, and crossovers associated with gene conversion events are simultaneously measured.

CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.

Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.

Genome Characterization of Two Novel Lactococcus lactis Phages vL_296 and vL_20A.

Fermented dairy products are produced using starter cultures. They ferment milk to create products with a certain texture, aroma, and taste. However, the lactic acid bacteria used in this production are prone to bacteriophage infection. We examined the genomes of two newly discovered bacteriophage species that were isolated from cheese whey during the cheesemaking process. We have determined the species and the lytic spectrum of these bacteriophages. Phages vL_20A and vL_296 were isolated using lactococcal indicator cultures. They have unique lytic spectra: of the 21 possible identified host bacteria, only four are shared amongst them. The vL_20A and vL_296 genomes comprise linear double-stranded DNA lengths with 21,909 and 22,667 nucleotide pairs, respectively. Lactococcus phage bIL67 (ANI 93.3 and 92.6, respectively) is the closest to the phages vL_20A and vL_296. The analysis of the CRISPR spacers in the genomes of starter cultures did not reveal any phage-specific vL_20A or vL_296 among them. This study highlights the biodiversity of L. lactis phages, their widespread presence in dairy products, and their virulence. However, the virulence of phages is balanced by the presence of a significant number of bacterial strains with different sensitivities to phages in the starter cultures due to the bacterial immune system.

Preliminary Investigation of a Potential Optical Biosensor Using the Diamond™ Nucleic Acid Dye Applied to DNA and Friction Ridge Analysis from Fingerprint Traces.

Developments in science and technology lead to an increasing use of scientific evidence in litigation. Interdisciplinary research can improve current procedures and introduce new ones for the disclosure and examination of evidence. The dactyloscopic trace is used for personal identification by matching minutiae (the minimum required may vary by country) or for extracting DNA material from the trace under investigation. The research presented in this article aims to propose the merging of two currently used personal identification methods, DNA analysis and dactyloscopic trace analysis, which are currently treated as separate forensic traces found at a crime scene. Namely, the forensic trace to be analyzed is the dactyloscopic trace containing DNA, and both sources of information needed for identification are examined as one. Promega's Diamond™ Nucleic Acid Dye, presented as a safe alternative to ethidium bromide, works by binding to single- and double-stranded DNA and is used to visualize the separation of material in a gel and to detect DNA in forensic samples. Spectroscopic studies as absorption and emission spectra and fluorescence microscopy observations presented in our research confirm that Diamond™ Nucleic Acid Dye can also be used to visualize fingerprints on non-absorbent surfaces and that combining the two methods into one can significantly increase the evidential value and contribute to the design of an innovative fast-acting optical biosensor.

Tandem-Controlled Dynamic DNA Assembly Enables Temporally-Selective Orthogonal Regulation of cGAS-STING Stimulation.

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem-regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)-connected blocking sequence to inhibit its self-assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)-triggered enzymatic cleavage of the AP site enables the reconfiguration and self-assembly of DNA motif into long double-stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2'3'-cyclic-GMP-AMP for STING stimulation. Importantly, we demonstrate that APE1-regulated DNA assembly allows for cell-selective activation of cGAS-STING signaling. Furthermore, by re-engineering the DNA motif with a photocleavable group, enzyme-triggered DNA assembly allows the cGAS-STING stimulation to operate (switched "ON''), whereas light-mediated fragmentation of the double-stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS-STING pathway in an orthogonally controlled manner.

A novel approach to double-strand DNA break analysis through γ-H2AX confocal image quantification and bio-dosimetry

DNA damage occurs in all living cells. γ-H2AX imaging by fluorescent microscopy is widely used across disciplines in the analysis of double-strand break (DSB) DNA damage. Here we demonstrate a method for the quantitative analysis of such DBSs. Ionising radiation, well known to induce DSBs, is used in this demonstration, and additional DBSs are induced if high-Z nanoparticles are present during irradiation. As a deliberate test of the methodology, cells are exposed to a spatially fractionated ionising radiation field, characterised by regions of high and low absorbed radiation dose that are only ever qualitatively verified biologically via γ-H2AX imaging. Here we validate our bio-dosimetric quantification method using γ-H2AX assays in the assessment of DSB enhancement. Our method reliably quantifies DSB enhancement in cells when exposed to either a spatially contiguous or fractionated irradiation fields. Using the γ-H2AX assay, we deduce the biological dose response, and for the first time, demonstrate equivalence to the independently measured physical absorbed dose. Using our novel method, we are also able quantify the nanoparticle DSB enhancement at the cellular level, which is not possible using physical dose measurement techniques. Our method therefore provides a new paradigm in γ-H2AX image quantification of DSBs, as well as an independently validated bio-dosimetry technique.

Precise Methylation Detection of Tumor Suppressor Gene Promoters by Magnetic Enrichment and Nano Silver Adduct-Promoted Surface-Enhanced Raman Scattering.

Noninvasive liquid biopsies can be used for early tumor diagnosis by identifying the methylation level of the tumor suppressor genes (TSGs)-a reliable index for cancer evaluation. However, identifying trace circulating genes from specimens remains challenging. This work introduces a novel method that combines magnetic isolation and surface-enhanced Raman scattering (SERS) to concentrate and detect the methylated TSG promotors. A superparamagnetic iron oxide nanoparticle modified with streptavidin is prepared as a universal magnetic bead. Biotin-terminated probe single-strand DNA (ssDNA) is immobilized on the magnetic beads through biotin-streptavidin bioconjugation. Artificial target ssDNA fragments with various methylation levels are applied as a promoter gene model. Concentrated double-strand DNA (dsDNA) is produced by a hybridizing probe and target ssDNA on magnetic nanobeads, as well as an additional magnetic isolation process. The well-prepared DNA adduct, which consists of 3nm cisplatin-modified Ag nanoclusters, can specifically bind with guanine-cytosine base pairs of dsDNA. Ag-nanoparticle-induced localized SERS amplified signals of 5-methylcytosine (5-mC) from the dsDNA in Raman spectra, enabling accurate methylation level measurement in mixtures of 0-1µm methylated DNA, with a detection limit of 0.05µm. This method shows promise for enabling the methylation level evaluation of various TSGs and promoters in early cancer liquid biopsies.

Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope.

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription and repair. Dynamic nanoscale observations of DNA structural features are necessary for understanding these interactions. Here we introduce graphene energy transfer with vertical nucleic acids (GETvNA), a method to investigate DNA-protein interactions that exploits the vertical orientation adopted by double-stranded DNA on graphene. This approach enables the dynamic study of DNA conformational changes via energy transfer from a probe dye to graphene, achieving spatial resolution down to the Ångström scale at subsecond temporal resolution. We measured DNA bending induced by adenine tracts, bulges, abasic sites and the binding of endonuclease IV. In addition, we observed the translocation of the O6-alkylguanine DNA alkyltransferase on DNA, reaching single base-pair resolution and detecting preferential binding to adenine tracts. This method promises widespread use for dynamical studies of nucleic acids and nucleic acid-protein interactions with resolution so far reserved for traditional structural biology techniques.

Dual-signal ratiometric electrochemical aptasensor for Zearalenone detection based on magnetic-assisted enrichment and hybridization chain reaction

In this work, a dual-signal ratiometric electrochemical aptasensor based on the hybrid chain reaction (HCR) and streptavidin-modified magnetic beads (SA-MBs) was developed to rapidly detect zearalenone (ZEN). The HCR, as a powerful signal amplification technique to imporve the signal of sonser. When the target is present, they specifically bind with ZEN-Apt and release ZEN-cDNA to trigger HCR. Simultaneously, more double-stranded DNA causes the signal of Thi to be blocked. As a result, the two signals tend to change in the opposite direction as the ZEN concentration changes. Additionally, the peak current ratio of IThi/IFc showed a positive correlation with the ZEN concentration. Under optimal conditions, the constructed biosensor showed an excellent linear detection range (1.0 × 10−10 mol/L to 1.0 × 10−6 mol/L), a low detection limit (4.4 × 10−11 mol/L) and high specificity for ZEN. In addition, the detection method retains the characteristics of low cost and rapid detection of electrochemical detection, while improving the detection limit and detection accuracy via SA-MBs and internal reference signal. This provides a new idea for the practical detection of ZEN.

Bimetallic peroxide nanoparticles induce PANoptosis by disrupting ion homeostasis for enhanced immunotherapy.

PANoptosis has recently emerged as a potential approach to improve the immune microenvironment. However, current methods for inducing PANoptosis are limited. Herein, through biological screening, the rational use of the nutrient metal ions Cu2+ and Zn2+ had great potential to induce PANoptosis. Inspired by these findings, we successfully developed hydrazided hyaluronic acid-modified zinc copper oxide (HZCO) nanoparticles as a PANoptosis inducer to potentiate immunotherapy. Bioactive HZCO actively delivered Cu2+ and Zn2+ while disrupting the cellular intrinsic ion metabolism pathway, resulting in double-stranded DNA release and organelle damage in cancer cells. Simultaneously, this process triggered the formation of PANoptosome and the activation of PANoptosis. HZCO-induced PANoptosis inhibited tumor growth and activated potent antitumor immune response, thereby enhancing the effectiveness of anti-programmed cell death 1 therapy. Overall, our work provides an insight into the development of PANoptosis inducers and the design of synergistic immunotherapy strategies.

Single-cell RNA-sequencing of BK polyomavirus replication in primary human renal proximal tubular epithelial cells identifies specific transcriptome signatures and a novel mitochondrial stress pattern.

BK polyomavirus (BKPyV) contributes to premature renal failure in 10%-20% of kidney transplant recipients. Current treatment relies on reducing immunosuppression to regain BKPyV-specific immune control. Subsequently, declining allograft function may result from persisting viral cytopathology, BKPyV-specific immune reconstitution, or alloimmunity/rejection, all being poorly distinguishable by current histological or molecular approaches. To reduce the complexity encountered in BKPyV-replicating kidneys, we analyzed differentially expressed genes (DEGs) in primary human renal proximal tubular epithelial cells at 24 and 48 h post-infection (hpi) using single-cell RNA-sequencing (10x-Genomics-3´ kit). At 24 hpi, viral transcript reads predominantly mapped to the early viral gene region (EVGR) and shifted to >100-fold higher late viral gene region (LVGR) levels at 48 hpi, matching the sequential bi-directional viral protein expression from the circular double-stranded BKPyV-DNA genome. Besides expected coverage "hills" at viral 3´-poly-A sites, unexpected "spike" and "pulse" reads resulted from off-target TSO priming. "Spike" and "pulse" patterns were rare for the mostly unidirectional reads mapping to the circular mitochondrial genome. Bioinformatic curation removed "spikes" and "pulses" and reclassified 10% of DEGs in renal proximal tubular epithelial cells (RPTECs). Up-regulated gene ontologies included S and G2/M phase, double-stranded DNA repair, proximal tubulopathy, and renal tubular dysfunction, whereas allograft rejection, antigen presentation, innate immunity, translation, and autophagy were down-regulated. BKPyV-LVGR expression induced a novel mitochondrial cell stress pattern consisting of discordant up-regulation and down-regulation of mitochondria-encoded and nucleus-encoded mitochondrial genes, respectively. We explored which top-scoring gene sets of late-phase BKPyV-replicating RPTECs can identify BKPyV-associated nephropathy in kidney transplant biopsies. The results should facilitate distinguishing BKPyV-associated pathology from other entities in kidney transplant biopsies.IMPORTANCEBK polyomavirus (BKPyV) infects more than 90% of the general population and then persists in the reno-urinary tract. Subsequently, low-level urinary shedding is seen in 10% of healthy BKPyV-seropositive persons, indicating that BKPyV replication occurs despite the presence of virus-specific cellular and humoral immunity. Notably, transplantation of donor kidneys with low-level BKPyV replication is a risk factor for progression to high-level BKPyV viruria, new-onset BKPyV-DNAemia and biopsy-proven BKPyV nephropathy. Here, we identify a short list of robust up- and down-regulated nucleus-encoded differentially expressed genes potentially allowing to discriminate viral from allograft immune damage. By carefully curating viral and mitochondrial transcriptomes, we identify a novel virus-associated mitochondrial stress pattern of up-regulated mitochondria-encoded and down-regulated nucleus-encoded mitochondrial transcripts which heralds the BKPyV-agnoprotein-mediated immune escape by breakdown of the mitochondrial membrane potential and network and mitophagy. The results may prove useful when assessing the role of BKPyV replication in kidney transplant patients with suspected acute rejection and/or BKPyV nephropathy.