Microwave hyperthermia enhances radiosensitization by decreasing DNA repair efficiency and inducing oxidative stress in PC3 prostatic adenocarcinoma cells.

PURPOSE: Radiotherapy (RT) is the primary treatment for prostate cancer (PCa); however, the emergence of castration-resistant prostate cancer (CRPC) often leads to treatment failure and cancer-related deaths. In this study, we aimed to explore the use of microwave hyperthermia (MW-HT) to sensitize PCa to RT and investigate the underlying molecular mechanisms. METHODS: We developed a dedicated MW-HT heating setup, created an in vitro and in vivo MW-HT + RT treatment model for CRPC. We evaluated PC3 cell proliferation using CCK-8, colony experiments, DAPI staining, comet assay and ROS detection method. We also monitored nude mouse models of PCa during treatment, measured tumor weight, and calculated the tumor inhibition rate. Western blotting was used to detect DNA damage repair protein expression in PC3 cells and transplanted tumors. RESULTS: Compared to control, PC3 cell survival and clone formation rates decreased in RT + MW-HT group, demonstrating significant increase in apoptosis, ROS levels, and DNA damage. Lower tumor volumes and weights were observed in treatment groups. Ki-67 expression level was reduced in all treatment groups, with significant decrease in RT + MW-HT groups. The most significant apoptosis induction was confirmed in RT + MW-HT group by TUNEL staining. Protein expression levels of DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways significantly decreased in RT + MW-HT groups. CONCLUSION: MW-HT + RT treatment significantly inhibited DNA damage repair by downregulating DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways, leading to increased ROS levels, aggravate DNA damage, apoptosis, and necrosis in PC3 cells, a well-established model of CRPC.

Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

DNA-modified Prussian blue nanozymes for enhanced electrochemical biosensing.

Prussian blue nanoparticles exhibit the potential to be employed in bioanalytical applications due to their robust stability, peroxidase-like catalytic functionality, straightforward synthesis, and biocompatibility. An efficient approach is presented for the synthesis of nucleic acid-modified Prussian blue nanoparticles (DNA-PBNPs), utilizing nanoparticle porosity to adsorb nucleic acids (polyT). This strategic adsorption leads to the exposure of nucleic acid sequences on the particle surface while retaining catalytic activity. DNA-PBNPs further couple with functional nucleic acid sequences and aptamers through complementary base pairing to act as transducers in biosensors and amplify signal acquisition. Subsequently, we integrated a copper ion-dependent DNAzyme (Cu2+-DNAzyme) and a vascular endothelial growth factor aptamer (VEGF aptamer) onto screen-printed electrodes to serve as recognition elements for analytes. Significantly, our approach leverages DNA-PBNPs as a superior alternative to traditional enzyme-linked antibodies in electrochemical biosensors, thereby enhancing both the efficiency and adaptability of these devices. Our study conclusively demonstrates the application of DNA-PBNPs in two different biosensing paradigms: the sensitive detection of copper ions and vascular endothelial growth factor (VEGF). These results indicate the promising potential of DNA-modified Prussian blue nanoparticles in advancing bioanalytical sensing technologies.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Ultrasensitive 3D Stacked Silicon Nanosheet Field-Effect Transistor Biosensor with Overcoming Debye Shielding Effect for Detection of DNA.

Silicon nanowire field effect (SiNW-FET) biosensors have been successfully used in the detection of nucleic acids, proteins and other molecules owing to their advantages of ultra-high sensitivity, high specificity, and label-free and immediate response. However, the presence of the Debye shielding effect in semiconductor devices severely reduces their detection sensitivity. In this paper, a three-dimensional stacked silicon nanosheet FET (3D-SiNS-FET) biosensor was studied for the high-sensitivity detection of nucleic acids. Based on the mainstream Gate-All-Around (GAA) fenestration process, a three-dimensional stacked structure with an 8 nm cavity spacing was designed and prepared, allowing modification of probe molecules within the stacked cavities. Furthermore, the advantage of the three-dimensional space can realize the upper and lower complementary detection, which can overcome the Debye shielding effect and realize high-sensitivity Point of Care Testing (POCT) at high ionic strength. The experimental results show that the minimum detection limit for 12-base DNA (4 nM) at 1 × PBS is less than 10 zM, and at a high concentration of 1 µM DNA, the sensitivity of the 3D-SiNS-FET is approximately 10 times higher than that of the planar devices. This indicates that our device provides distinct advantages for detection, showing promise for future biosensor applications in clinical settings.

Zinc improves the developmental ability of bovine in vitro fertilization embryos through its antioxidative action.

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 µg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 µg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 µg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Cooperative dynamics of DNA-grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami.

Magnetic nanoparticles (MNPs) provide new opportunities for enzyme-free biosensing of nucleic acid biomarkers and magnetic actuation by patterning on DNA origami, yet how the DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we performed surface functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry with a tunable grafting density, which enables the encapsulation of single MNPs inside a functional polymeric layer. We used several complementary methods to show that particle translational and rotational dynamics exhibit a sigmoidal dependence on the ssDNA grafting density. At low densities, ssDNA strands adopt a coiled conformation that results in minor alterations to particle dynamics, while at high densities, they organize into polymer brushes that collectively influence particle dynamics. Intermediate ssDNA densities, where the dynamics are most sensitive to changes, show the highest magnetic biosensing sensitivity for the detection of target nucleic acids. Finally, we demonstrate that MNPs with high ssDNA grafting densities are required to efficiently couple to DNA origami. Our results establish ssDNA grafting density as a critical parameter for the functionalization of MNPs for magnetic biosensing and functionalization of DNA nanostructures.

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259.

OBJECTIVE: Cardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. APPROACH AND RESULTS: We interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-ß-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. CONCLUSION: Downregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species.

DNA-binding with one finger (Dof) proteins comprise a large family that play central roles in stress tolerance by regulating the expression of stress-responsive genes via the DOFCORE element or by interacting with other regulatory proteins. Although the Dof TF has been identified in a variety of species, its systemic analysis in potato (Solanum tuberosum L.) is lacking and its potential role in abiotic stress responses remains unclear. A total of 36 potential Dof genes in potato were examined at the genomic and transcriptomic levels in this work. Five phylogenetic groups can be formed from these 36 Dof proteins. An analysis of cis-acting elements revealed the potential roles of Dofs in potato development, including under numerous abiotic stress conditions. The cycling Dof factors (CDFs) might be the initial step in the abiotic stress response signaling cascade. In potato, five CDFs (StCDF1/StDof19, StCDF2/StDof4, StCDF3/StDof11, StCDF4/StDof24, and StCDF5/StDof15) were identified, which are homologs of Arabidopsis CDFs. The results revealed that these genes were engaged in a variety of abiotic reactions. Moreover, an expression analysis of StDof genes in two potato cultivars ('Long10' (drought tolerant) and 'DXY' (drought susceptible)) of contrasting tolerances under drought stress was carried out. Further, a regulatory network mediated by lncRNA and its target Dofs was established. The present study provides fundamental knowledge for further investigation of the roles of Dofs in the adaptation of potato to drought stress, aiming to provide insights into a viable strategy for crop improvement and stress-resistance breeding.

Effect of Different Carbon Sources on Antioxidant Properties of Exopolysaccharides Produced by Scleroderma areolatum (Agaricomycetes).

Five kinds of exopolysaccharides (EPS) were obtained by fermentation of Scleroderma areolatum Ehrenb. with sucrose, glucose, maltose, lactose, and fructose as carbon sources. Antioxidant abilities of the obtained EPSs were evaluated by inhibiting AAPH, HO·, and glutathione (GS·) induced oxidation of DNA and quenching 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS· and galvinoxyl radicals. The effects of carbon sources on the antioxidant properties of EPSs could be examined. The results showed that five EPSs can effectively inhibit radicals induced oxidation of DNA, and the thiobarbituric acid reactive substances (TBARS) percentages were 44.7%-80.8%, 52.3%-77.5%, and 44.7%-73.3% in inhibiting AAPH, HO·, and GS· induced oxidation of DNA, respectively. All five EPSs could scavenge ABTS· and galvinoxyh, and exhibit superior activity in scavenging free radicals. Antioxidant abilities of EPS with fructose as carbon source were highest among five EPS.

DNA Bloom Filter enables anti-contamination and file version control for DNA-based data storage.

DNA storage is one of the most promising ways for future information storage due to its high data storage density, durable storage time and low maintenance cost. However, errors are inevitable during synthesizing, storing and sequencing. Currently, many error correction algorithms have been developed to ensure accurate information retrieval, but they will decrease storage density or increase computing complexity. Here, we apply the Bloom Filter, a space-efficient probabilistic data structure, to DNA storage to achieve the anti-error, or anti-contamination function. This method only needs the original correct DNA sequences (referred to as target sequences) to produce a corresponding data structure, which will filter out almost all the incorrect sequences (referred to as non-target sequences) during sequencing data analysis. Experimental results demonstrate the universal and efficient filtering capabilities of our method. Furthermore, we employ the Counting Bloom Filter to achieve the file version control function, which significantly reduces synthesis costs when modifying DNA-form files. To achieve cost-efficient file version control function, a modified system based on yin-yang codec is developed.

Construction of a glycosylation-mediated fluorescent biosensor for label-free measurement of site-specific 5-hydroxymethylcytosine in cancer cells with zero background signal.

BACKGROUND: 5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. RESULTS: We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by ß-glucosyltransferase (ß-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10-14 to 1 × 10-8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SIGNIFICANCE: This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.

Species delimitation of tea plants (Camellia sect. Thea) based on super-barcodes.

BACKGROUND: The era of high throughput sequencing offers new paths to identifying species boundaries that are complementary to traditional morphology-based delimitations. De novo species delimitation using traditional or DNA super-barcodes serve as efficient approaches to recognizing putative species (molecular operational taxonomic units, MOTUs). Tea plants (Camellia sect. Thea) form a group of morphologically similar species with significant economic value, providing the raw material for tea, which is the most popular nonalcoholic caffeine-containing beverage in the world. Taxonomic challenges have arisen from vague species boundaries in this group. RESULTS: Based on the most comprehensive sampling of C. sect. Thea by far (165 individuals of 39 morphospecies), we applied three de novo species delimitation methods (ASAP, PTP, and mPTP) using plastome data to provide an independent evaluation of morphology-based species boundaries in tea plants. Comparing MOTU partitions with morphospecies, we particularly tested the congruence of MOTUs resulting from different methods. We recognized 28 consensus MOTUs within C. sect. Thea, while tentatively suggesting that 11 morphospecies be discarded. Ten of the 28 consensus MOTUs were uncovered as morphospecies complexes in need of further study integrating other evidence. Our results also showed a strong imbalance among the analyzed MOTUs in terms of the number of molecular diagnostic characters. CONCLUSION: This study serves as a solid step forward for recognizing the underlying species boundaries of tea plants, providing a needed evidence-based framework for the utilization and conservation of this economically important plant group.

Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy.

Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.

Target-Responsive DNA Nanoclaw for the On-Site Identification of Chinese Medicines with Naked Eye.

The identification of Chinese medicinal herbs occupies a crucial part in the development of the food and drug market. Although molecular identification based on real-time PCR offers good versatility and uniform digital standards compared with traditional methods, such as morphology, the dependence on large-scale equipment hinders spot detection and marketable applications. In this study, we developed a DNA nanoclaw for colorimetric detection and visible on-site identification of Chinese medicines. When specific miRNA is present, the DNAzyme is activated and cleaves the substrate strand, triggering the catalytic hairpin assembly (CHA) reaction and forming branched DNA junctions on AuNP-I. This can then capture AuNP-II through hybridization and facilitate their aggregation, resulting in a noticeable color change that is observable to the naked eye. By harnessing the dual amplification of DNAzyme and CHA, this highly sensitive nanoprobe successfully achieved specific identification of Chinese medicines. This offers a new perspective for on-site testing in the herbal market.

Anaerobic flora, Selenomonas ruminis sp. nov., and the bacteriocinogenic Ligilactobacillus salivarius strain MP3 from crossbred-lactating goats.

This study aimed to examine the distribution of anaerobic bacteria in the rumen fluid of Thai crossbred goats and to screen potential probiotic strains capable of producing antimicrobial compounds and inhibiting bacteria that cause milk fat depression. Thirty-four strains of bacteria from the rumen fluid were divided into 13 groups within 12 genera based on 16S rRNA gene sequences. The RF1-5 and RF5-12 were identified as Streptococcus luteliensis and Bacillus licheniformis, respectively, and demonstrated non-ropy exopolysaccharide. Furthermore, mPRGC5T was closely related to Selenomonas caprae JCM 33725 T (97.8% similarity) based on 16S rRNA gene sequences. It exhibited low average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values with related type strains ranging from 84.9 to 86.0%, 21.3 to 21.8%, and 73.8 to 76.1%, respectively. The genotypic and phenotypic characteristics of mPRGC5T strongly support this strain as a new species of the genus Selenomonas for which the name Selenomonas ruminis mPRGC5T was proposed. The type strain is mPRGC5T (= JCM 33724 T = KCTC 25177 T). Ligilactobacillus salivarius MP3 showed antibacterial activity against Cutibacterium acnes subsp. acnes DSM 1897 T and Kocuria rhizophila MIII. The enterolysin A cluster gene was identified in its genome. The auto-aggregation of L. salivarius MP3 was 93.6 ± 0.2%. Additionally, co-aggregation of L. salivarius MP3 with C. acnes DSM 1897 T and K. rhizophila MIII had 92.2 ± 3.4% and 87.3 ± 4.5%, respectively. The adhesion capacity of strain MP3 was 76.11 ± 2.2%. Probiogenomic analysis revealed that L. salivarius MP3 was nonhazardous to animal supplementation and included acid- and bile-tolerant ability. However, strain MP3 contained three antibiotic resistance genes. Thus, the supplementation of L. salivarius MP3 could increase the milk fat content by suppressing C. acnes DSM 1897 T with antibiotic resistance gene horizontal transfer awareness.

Transferrin maintains the motility rate, ATP content, and DNA integrity of common carp spermatozoa during short-term storage.

Short-term sperm storage is a straightforward and cost-effective method of managing logistics in large scale fish hatchery operations but may result in decline in sperm quality. For effective artificial reproduction of fish, use of an appropriate additive to optimize sperm storage conditions is essential. In this study, it was investigated the effect of purified seminal plasma transferrin (Tf) at 10 µg/ml on relevant parameters in common carp Cyprinus carpio sperm during short-term storage. We compared sperm motility and curvilinear velocity, adenosine triphosphate (ATP) content and DNA fragmentation of fresh spermatozoa to that stored for 24, 48, 72, and 144 h with or without Tf. The percentage of motile cells and the curvilinear velocity of spermatozoa in stored samples for 72 h with transferrin supplementation were greater compared to samples with no added protein. The ATP content in samples without added transferrin was reduced (P < 0.05) after 72 h of storage, in contrast to the levels observed in transferrin-supplemented sperm. A time-dependent increase in DNA fragmentation was observed. Significantly lower DNA damage, expressed as percent tail DNA (10.99 ±â€¯1.28) and olive tail moment (0.54 ±â€¯0.12), was recorded in Tf-supplemented samples stored for 48 h compared to that with no Tf. Hence, it is concluded that the beneficial effects of transferrin on common carp sperm could serve as an additional tool for developing and enhancing short-term sperm preservation procedures commonly used in aquaculture.