Tb^(3+)-nucleic acid probe-based label-free and rapid detection of mercury pollution in food

Mercury is a threatening pollutant in food,herein,we developed a Tb^(3+)-nucleic acid probe-based label-free assay for mix-and-read,rapid detection of mercury pollution.The assay utilized the feature of light-up fluorescence of terbium ions(Tb^(3+))via binding with single-strand DNA.Mercury ion,Hg^(2+)induced thymine(T)-rich DNA strand to form a double-strand structure(T-Hg^(2+)-T),thus leading to fluorescence reduction.Based on the principle,Hg^(2+)can be quantified based on the fluorescence of Tb^(3+),the limit of detection was 0.0689μmol/L and the linear range was 0.1-6.0μmol/L.Due to the specificity of T-Hg^(2+)-T artificial base pair,the assay could distinguish Hg^(2+)from other metal ions.The recovery rate was ranged in 98.71%-101.34%for detecting mercury pollution in three food samples.The assay is low-cost,separation-free and mix-to-read,thus was a competitive tool for detection of mercury pollution to ensure food safety.

Ultrathin metal-organic framework nanosheets (Cu-TCPP)-based isothermal nucleic acid amplification for food allergen detection

The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.

A brief review of novel nucleic acid test biosensors and their application prospects for salmonids viral diseases detection

Viral diseases represent one of the major threats for salmonids aquaculture.Early detection and identification of viral pathogens is the main prerequisite prior to undertaking effective prevention and control measures.Rapid,sensitive,efficient and portable detection method is highly essential for fish viral diseases detection.Biosensor strategies are highly prevalent and fulfill the expanding demands of on-site detection with fast response,cost-effectiveness,high sensitivity,and selectivity.With the development of material science,the nucleic acid biosensors fabricated by semiconductor have shown great potential in rapid and early detection or screening for diseases at salmonids fisheries.This paper reviews the current detection development of salmonids viral diseases.The present limitations and challenges of salmonids virus diseases surveillance and early detection are presented.Novel nucleic acid semiconductor biosensors are briefly reviewed.The perspective and potential application of biosensors in the on-site detection of salmonids diseases are discussed.

Non-exposed endoscopic wall-inversion surgery with one-step nucleic acid amplification for early gastrointestinal tumors:Personal experience and literature review

BACKGROUND Laparoscopic and endoscopic cooperative surgery is a safe,organ-sparing surgery that achieves full-thickness resection with adequate margins.Recent studies have demonstrated the safety and efficacy of these procedures.However,these techniques are limited by the exposure of the tumor and mucosa to the peritoneal cavity,which could lead to viable cancer cell seeding and the spillage of gastric juice or enteric liquids into the peritoneal cavity.Non-exposed endoscopic wallinversion surgery(NEWS)is highly accurate in determining the resection margins to prevent intraperitoneal contamination because the tumor is inverted into the visceral lumen instead of the peritoneal cavity.Accurate intraoperative assessment of the nodal status could allow stratification of the extent of resection.One-step nucleic acid amplification(OSNA)can provide a rapid method of evaluating nodal tissue,whilst nearinfrared laparoscopy together with indocyanine green can identify relevant nodal tissue intraoperatively.AIM To determine the safety and feasibility of NEWS in early gastric and colon cancers and of adding rapid intraoperative lymph node(LN)assessment with OSNA.METHODS The patient-based experiential portion of our investigations was conducted at the General and Oncological Surgery Unit of the St.Giuseppe Moscati Hospital(Avellino,Italy).Patients with early-stage gastric or colon cancer(diagnosed via endoscopy,endoscopic ultrasound,and computed tomography)were included.All lesions were treated by NEWS procedure with intraoperative OSNA assay between January 2022 and October 2022.LNs were examined intraoperatively with OSNA and postoperatively with conventional histology.We analyzed patient demographics,lesion features,histopathological diagnoses,R0 resection(negative margins)status,adverse events,and follow-up results.Data were collected prospectively and analyzed retrospectively.RESULTS A total of 10 patients(5 males and 5 females)with an average age of 70.4±4.5 years(range:62-78 years)were enrolled in this study.Five patients were diagnosed with gastric cancer.The remaining 5 patients were diagnosed with early-stage colon cancer.The mean tumor diameter was 23.8±11.6 mm(range:15-36 mm).The NEWS procedure was successful in all cases.The mean procedure time was 111.5±10.7 min(range:80-145 min).The OSNA assay revealed no LN metastases in any patients.Histologically complete resection(R0)was achieved in 9 patients(90.0%).There was no recurrence during the follow-up period.CONCLUSION NEWS combined with sentinel LN biopsy and OSNA assay is an effective and safe technique for the removal of selected early gastric and colon cancers in which it is not possible to adopt conventional endoscopic resection techniques.This procedure allows clinicians to acquire additional information on the LN status intraoperatively.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

Electrochemical Biosensor for Detection of Staphylococcus aureus Based on Nucleic Acid Aptamers

Staphylococcus aureus is a gram-staining positive cocci bacillus baterium and also one of the foodborne pathogens, which is a serious potential hazard to human health and food safety. We constructed an electrochemical biosensor for the detection of S. aureus based on nucleic acid aptamers to achieve highly specific detection of S. aureus. The detection of S. aureus was realized by using Aptamer (Apt) to capture S. aureus, which resulted in a change in the spatial conformation of Apt and a decrease in the electrochemical signal. Under the optimized experimental conditions, the detected electrochemical signals were positively correlated with the concentration of S. aureus with a linear range of 1 × 101 - 1 × 105 CFU/mL, a detection limit of 4.76 CFU/mL, and an experimental recovery of 97.43% - 99.37%. Therefore, we successfully constructed an electrochemical biosensor for the specific detection of S. aureus, which has the advantages of high specificity, sensitive detection and convenient operation.

Role of nucleic acid sensing in the pathogenesis of type 1 diabetes

During infections,nucleic acids of pathogens are also engaged in recognition via several exogenous and cytosolic pattern recognition receptors,such as the toll-like receptors,retinoic acid inducible gene-I-like receptors,and nucleotide-binding and oligomerization domain-like receptors.The binding of the pathogen-derived nucleic acids to their corresponding sensors initiates certain downstream signaling cascades culminating in the release of type-I interferons(IFNs),especially IFN-αand other cytokines to induce proinflammatory responses towards invading pathogens leading to their clearance from the host.Although these sensors are hardwired to recognize pathogen associated molecular patterns,like viral and bacterial nucleic acids,under unusual physiological conditions,such as excessive cellular stress and increased apoptosis,endogenous self-nucleic acids like DNA,RNA,and mitochondrial DNA are also released.The presence of these self-nucleic acids in extranuclear compartments or extracellular spaces or their association with certain proteins sometimes leads to the failure of discriminating mechanisms of nucleic acid sensors leading to proinflammatory responses as seen in autoimmune disorders,like systemic lupus erythematosus,psoriasis and to some extent in type 1 diabetes(T1D).This review discusses the involvement of various nucleic acid sensors in autoimmunity and discusses how aberrant recognition of self-nucleic acids by their sensors activates the innate immune responses during the pathogenesis of T1D.

Application of cationic liposomes for delivery of nucleic acids

Nucleic acid-based bioactive substances have recently emerged as a new class of nextgeneration therapeutics, but their development has been limited by their relatively weakdelivery into target cells. Cationic liposomes have been studied as a means to enhance thestability of nucleic acid therapeutics in the bloodstream and improve their cellular delivery.As nucleic acid therapeutics, siRNA and plasmid DNA have been extensively tested fordelivery using cationic liposomes. This review discusses recent progress in the applicationof cationic liposomes for the delivery of nucleic acid therapeutics.

Tetrahedral Framework Nucleic Acid-Based Delivery of Resveratrol Alleviates Insulin Resistance:From Innate to Adaptive Immunity

Obesity-induced insulin resistance is the hallmark of metabolic syndrome,and chronic,low-grade tissue inflammation links obesity to insulin resistance through the activation of tissue-infiltrating immune cells.Current therapeutic approaches lack efficacy and immunomodulatory capacity.Thus,a new therapeutic approach is needed to prevent chronic inflammation and alleviate insulin resistance.Here,we synthesized a tetrahedral framework nucleic acid(tFNA)nanoparticle that carried resveratrol(RSV)to inhibit tissue inflammation and improve insulin sensitivity in obese mice.The prepared nanoparticles,namely tFNAs-RSV,possessed the characteristics of simple synthesis,stable properties,good water solubility,and superior biocompatibility.The tFNA-based delivery ameliorated the lability of RSV and enhanced its therapeutic efficacy.In high-fat diet(HFD)-fed mice,the administration of tFNAs-RSV ameliorated insulin resistance by alleviating inflammation status.tFNAs-RSV could reverse M1 phenotype macrophages in tissues to M2 phenotype macrophages.As for adaptive immunity,the prepared nanoparticles could repress the activation of Th1 and Th17 and promote Th2 and Treg,leading to the alleviation of insulin resistance.Furthermore,this study is the first to demonstrate that tFNAs,a nucleic acid material,possess immunomodulatory capacity.Collectively,our findings demonstrate that tFNAs-RSV alleviate insulin resistance and ameliorate inflammation in HFD mice,suggesting that nucleic acid materials or nucleic acid-based delivery systems may be a potential agent for the treatment of insulin resistance and obesity-related metabolic diseases.

Nucleic acid vaccines: A taboo broken and prospect for a hepatitis B virus cure

Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.

Construction and expression of nucleic acid vaccine pVAX1-h-alpha S_(1-140) coding human alpha-synuclein

BACKGROUND： The deposition of α -synuclein （ α -syn） aggregates is a neuropathological feature of Parkinson＇s disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson＇s disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson＇s disease. OBJECTIVE： To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING： The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University ＆ Chongqing Key Laboratory of Neurology. MATERIALS： The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS： The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES： Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS： The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION： Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.

Development of Nucleic Acid Sequence-Based Amplification Assay for Detection of Macrobrachium rosenbergii Nodavirus

A nucleic acid sequence-based amplification（NASBA）assay was established for the detection of Macrobrachium rosenbergii Nodavirus（MrNV）.The specific primers were designed according to the high conserved region of RNA2 sequence of MrNV.The 224 bp specific amplification product was obtained in positive sample determined with 3%agarose gel electrophoresis,while no product was generated from shrimp infected with other viruses including DNA viruses（IHHNV,WSSV）and RNA viruses（TSV,IMNV,YHV）.The detecting limit of the assay was 8pg nucleic acid,which is more sensitive than that of PCR method.

Electronic Structures and Spectra of the Bases and Base Pairs of Nucleic Acids

The present paper covers electronic structures and spectra of the bases and the base pairs of nucleic acids calculated by using the INDO/S method. For free bases we give the energy levels of ground states and transition energies of low-lying excited states and discuss the band characters. The results indicate that the calculated spectra are in good agreement with experimental values. On the other hand, our calculations for A-T and G-C pairs are very beneficial to understanding hydrogen bond properties of these pairs.

Spectroscopic Study of the Interactionbetween New Fluorescent Dyes with Nucleic Acids

A series of new dansylamide derivatives have been synthesized and the specific bindingaffinity of such fluorophores to nucleic acids has been investigated by using absorption, circulardichroism (CD), fluorescence and atomic force microscopy (AFM). The results indicate that thepositive charge of the ligand and the stacking between the dansy1 part of the ligand and theDNA base pair may play an important role when binding to polynucleotides.

Synthesis of Peptoid Nucleic Acid with Thymine as Nucleic Base

The synthesis of peptoid nucleic acid bearing thymine as nucleobase has been achieved. This modified oligonucleotide showed good hybridization with DNA.

Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

To investigate the effects of anti-sense peptide nucleic acids （PNAs） targeting Ki-67 gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations （1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L）. The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and ^3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide （ASODNs） targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs （16.9±0.7） was significantly inhibited as compared with that of the control groups （28.6±0.4） （P〈0.01）. The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs （42.1 ±2.2） was significantly reduced when compared with that of the control groups （83.6± 1.4） （P〈0.01）. Proliferation of 786-0 cells treated with anti-sense PNAs （20.7 ± 1.5） was significantly inhibited as compared with that of the control groups （58.6± 1.4） （P〈0.01）. The apoptosis rate of 786-0 cells treated with anti-sense PNAs （28.7 ± 2.3） was significantly increased higher compared with that of the control groups （13.8 ±1.0） （P〈0.01）. From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Construction of Prokaryotic Expressing Vector of Antisense Nucleic Acid of lasR and Its Effect on the Virulence of Pseudomonas Aeruginosus

To construct a pUCP18/lasR^antisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus, LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18. The recombinant pUCP18/lasR^antisense was verified by enzyme digestion, PCR and sequencing. The biological effects of pUCP18/lasR^antisense were examined by using RT-PCR, NAD method and the assay of pyocyanin. Our results showed that the expected full length lasR fragment （721 bp） was extended from Pseudomonas aeruginosus gene with PCR. And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank （No. NC_002516）. The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus. The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.

Acid-base and lipophilic properties of peptide nucleic acid derivatives

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid(PNA)derivatives,including eleven PNA monomers and two PNA decamers,is described.The acidity constants(pKa)of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution,and these values were found to be in agreement with those obtained by MoKa software.These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers.In addition,immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers,which showed the PNA derivatives had poor affinity towards membrane phospholipids,and confirmed their scarce cell penetrating ability.Overall,our study not only is of potential relevance to evaluate the pharmacokinetic properties of PNA,but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

Synthesis of Two New Chiral Blocks for the Construction of Peptide Nucleic Acid (PNA)

Protected (L) and (D)-lysine were used respectively as starting materials to synthesize two new types of chiral blocks for the construction of PNA. Nucleobase was linked to alpha-NH2 of lysine via -CH2C (O)- spacer in type I, and -C (O)- was used in type II. The corresponding oligomers were constructed in solution.