Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine / 药物分析学报

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2-5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90％-110％, and the inter-day and intra-day precision were acceptable (RSD<10％). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.

Protective effect of staphylococcal nuclease on 2, 4, 6-trinitro-benzene sulfonic acid-induced colitis in mice / 中国药科大学学报

@#To explore the improving effect and mechanism of staphylococcal nuclease(SNase)-mediated degradation of neutrophil extracellular traps(NETs)on 2, 4, 6-trinitro-benzene sulfonic acid(TNBS)-induced colitis in mice. The model of colitis in female BALB/c mice was established by intrarectal injection of 2. 5% TNBS solution, and SNase loaded by Lactococcus lactis(L. lactis)were orally administrated for 6 days. To investigate the effect of SNase-mediated degradation of neutrophil extracellular traps on colitis in mice, the experiment was divided into control group, TNBS model group, NZ900 group and L. lactis pCYT: SNase group. The daily body weight, stool consistency and bleeding of mice were observed. The pathological condition of HE in colon group was detected. The activity of MPO and the mRNA expression level of inflammatory cytokines in each group were measured, and the concentration of inflammatory factors in serum was detected. The expression of NETs level marker citH3 in colon tissue was determined by immunohistochemistry. The results showed that SNase loaded by lactis acid bacteria could alleviate the weight loss, disease activity index score, colonic length and pathological damage induced by TNBS in mice, and reduce the levels of inflammatory cytokines in serum and colonic tissue, inhibit the activity of MPO and the expression of Ly6G and citH3 in colon tissue. The preliminary mechanism showed that SNase could down-regulate the expression of inflammatory cytokines and reduce the content of NETs markers to alleviate colitis in mice.

Application of clustered regularly interspaced short palindromic repeats- associated protein 9 gene editing technology for treatment of HBV infection / 中华肝脏病杂志

A lack of effective drugs and technical means to eradicate hepatitis B virus (HBV) is a bottleneck that limits the ability to fully cure HBV infection. Recently, genome-editing technology based on clustered regularly interspaced short palindromic repeats -associated protein 9 is an emerging technique for editing specific gene loci, which can specifically target HBV covalently closed circular DNA, effectively inhibits HBV DNA replication and regulates HBV functional protein expression, and is expected to become a powerful gene therapy tool for the complete eradication of HBV. Considering this, it has become the focus of attention for scholars at home and abroad that how to use clustered regularly interspaced short palindromic repeats -associated protein 9 to accomplish modification of HBV genomes for complete eradication of HBV. This paper summarizes the latest progress based on the latest research results at home and abroad in the application of clustered regularly interspaced short palindromic repeats -associated protein 9 gene editing technology in anti-HBV infection treatment, and expounds its potential and challenges as a radical cure for HBV infection.

In vitro study of joint intervention of E-cad and Bmi-Ⅰ mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma / 中南大学学报(医学版)

Objective:To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors ofnasopharyngeal carcinoma cells.Methods:Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed,and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously.The integration of target genes were detected by PCR,the expressions of E-cad and Bmi-1 were detected by Western blot.The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay.T-he cell cycle and apoptosis were detected by flow cytometry.The cell migration and invasion were detected by Transwell assay.Results:The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells,the protein expression level of E-cad was up-regulated,and the protein expression level of Bmi-1 was down-regulated.The intervention of E-cad and Bmi-1 didn't affect the proliferation,cell cycle and apoptosis of CNE-2 cells,but it significantly inhibited the migration and invasion ability of CNE-2 cells.Furthermore,the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P＜0.01).Conclusion:The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro,which may lay the preliminary experimental basis for gene therapy of human cancer.

Coping with drought: stress and adaptive mechanisms, and management through cultural and molecular alternatives in cotton as vital constituents for plant stress resilience and fitness

Increased levels of greenhouse gases in the atmosphere and associated climatic variability is primarily responsible for inducing heat waves, flooding and drought stress. Among these, water scarcity is a major limitation to crop productivity. Water stress can severely reduce crop yield and both the severity and duration of the stress are critical. Water availability is a key driver for sustainable cotton production and its limitations can adversely affect physiological and biochemical processes of plants, leading towards lint yield reduction. Adaptation of crop husbandry techniques suitable for cotton crop requires a sound understanding of environmental factors, influencing cotton lint yield and fiber quality. Various defense mechanisms e.g. maintenance of membrane stability, carbon fixation rate, hormone regulation, generation of antioxidants and induction of stress proteins have been found play a vital role in plant survival under moisture stress. Plant molecular breeding plays a functional role to ascertain superior genes for important traits and can offer breeder ready markers for developing ideotypes. This review highlights drought-induced damage to cotton plants at structural, physiological and molecular levels. It also discusses the opportunities for increasing drought tolerance in cotton either through modern gene editing technology like clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), zinc finger nuclease, molecular breeding as well as through crop management, such as use of appropriate fertilization, growth regulator application and soil amendments.

Studies on preparation and properties of Staphylococcus nuclease / 中国药科大学学报

In order to study the relationship between Staphylococcal nuclease(SNase) and diabetes mellitus,genetic engineering bacteria E.coli BL21/pET28a-His-SNase was constructed,the expression of soluble extracellular protein SNase was induced and the a preliminary research was made on it.An expression vector pET28a-His-SNase plasmid containing the His-SNase gene was constructed and transformed into E.coli BL21 (DE3) competent cells.The protein was induced by lactose and purified by ultrasound destruction and Ni-affinity chromatography,respectively.It was then analyzed by SDS-PAGE and Western blot.The enzymatic properties for SNase has been preliminary studied as well.Results indicated that the purity of the correctly expressed fusion protein HisSNase was over 85％.SNase showed good activity within a wide range of pH and good heat resistance.This experiment might be a foundation work for the further study on the relationship between SNase and with diabetes.

SND1 protein co-localization with TIA-1 on stress granules under stress stimuli / 天津医药

Objective To analyze the association of staphylococcal nuclease domain-containing protein 1(SND1) and T-cell intracellular antigen 1(TIA-1) on stress granules, and the regulation of SND1 on stress granules under stress stimuli. Methods The immunofluorescence assay and laser scanning confocal microscopy were used to observe the co-localization of SND1 protein and TIA-1 protein under stress stimuli, and the over-expression plasmids of pEGFP vector were transfected into HeLa cells and to verify which domain of SND1 co-localized with TIA-1 under stress stimuli. RNA interference-mediated knockdown of the expression of SND1 protein in HeLa cells was measured by Western Blotting assay. Then whether the knockdown of SND1 affected the recruitment of TIA-1 on stress granules was observed. Heat shocks under different times were used to identify whether there were dynamic changes in transportation of SND1 and TIA-1 on stress granules. Results SND1 co-localized with TIA-1 on stress granules under stress stimuli, and the associated domain of SND1 were SN domain. TIA-1 still can be recruited on stress granules but a large amount of stress granules were reduced even though the expression of SND1 protein was decreased. And the transportation of SND1 on stress granules was laged behind TIA-1 under different-times of heat shocks. Conclusion SND1 protein co-localizes with TIA-1 on stress granules, and which co-regulates the cellular stress response under stress stimuli.

Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis / 生物工程学报

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Review of CRISPR/Cas9 system in the study of diabetes mellitus / 中国糖尿病杂志

Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)(CRISPR/Cas9),a revolutionary gene editing technology,has been applied in the researches of many diseases such as anemia,HIV,cardiovascular diseases and so on. Compared to other gene editing techniques such as zinc-finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs),CRISPR/Cas9 is much easier and effective to use. In the study field of diabetes, several researchers have tried to set up new diabetic animal models,modify diabetic associated gens or explore new diabetic therapy using CRISPR/Cas9. We review literatures related to the use of CRISPR/Cas9 in diabetes studies.

Surveyor assay to diagnose persistent Müllerian duct syndrome in Miniature Schnauzers

Persistent Müllerian duct syndrome (PMDS) is a pseudohermaphroditism in males characterized by the presence of Müllerian duct derivatives. As PMDS dogs often lack clinical symptoms, a molecular diagnosis is essential to identify the syndrome in these animals. In this study, a new molecular method using DNA mismatch-specific Surveyor nuclease was developed. The Surveyor nuclease assay identified the AMHR2 mutation that produced PMDS in a Miniature Schnauzer as accurately as that obtained by using the conventional method based on restriction digestion. As an alternative to the current molecular diagnostic method, the new method may result in increased accuracy when detecting PMDS.

Enzyme activity and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesis

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment.

Secretory expression and characterization of heat sensitive nuclease in Pichia pastoris / 生物工程学报

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.

Comparison of effects of staphylococcal nuclease A fused with different exogenous DNA fragments / 生物工程学报

Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.

Genome Editing Using Engineered Nucleases / 대한정형외과연구학회지

Genome editing is a useful research tool essentially applicable to gene therapy in the field of biotechnology, pharmaceutics and medicine. Scientists have developed three types of programmable nucleases for genome editing, and these include: Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system particularly derived from bacterial adaptive immune system. Programmable nucleaseses occur double strand breaks (DSBs) on target strand, and a repair mechanism of DSBs introduces either non-homologous end joining (NHEJ) or homology directed repair (HDR), where the pathway is determined by presence of donor DNA template. In this sense, we can generate gene insertion, gene correction, point mutagenesis and chromosomal translocations via endogenous repair mechanism. However, these nucleases exhibit several discrepancies in the aspects of their compositions, targetable sites, efficiency and other characteristics. Here, we discuss on various characteristics of three programmable nucleases and potential outcomes of DSBs. Acknowledging the distinctions among these programmable nucleases will help scientists to select appropriate tools in genome engineering.

Expression characteristics of chemotherapeutic related genes in non-small cell lung cancer / 重庆医学

Objective To analyze the expression characteristics of chemotherapeutic related genes (ERCC1 ,BRCA1 ,APE1 , RRM1 ,TUBB3 and TS) in non-small cell lung cancer(NSCLC) and their associations with the pathological types to provide the ex-perimental evidences for clinical individual chemotherapy .Methods The immunohistochemical SP method was used to determine the expression of ERCC1 ,BRCA1 ,APE1 ,RRM1 ,TUBB3 and TS genes in 733 patients with NSCLC .Chi-square test was performed to analyze the gene expression characteristics among different pathological types .Spearman relationship was conducted to evaluate the associations among the six genes and different pathological types .Results The expression of BRCA1 ,TUBB3 and TS had sta-tistical differences among three pathological types (P<0 .01);and these three genes showed a relationship with the pathological type (r=0 .107 ,-0 .229 and 0 .168 ,respectively ,P<0 .01) .ERCC1 vs .BRCA1 ,ERCC1 vs .APE1 and APE1 vs .RRM1 showed the pos-itive correlation(r=0 .214 ,0 .316 and 0 .222 ,respectively ,P<0 .01) .Conclusion The chemotherapeutic related gene expressions of NSCLC are related with the clinical pathological types .And the expressions of multiple markers are associated with each other .The combined detection of the chemotherapeutic related markers and the pathological types would be helpful to the decision making of individual chemotherapy in clinic .

Target-directed catalytic metallodrugs

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vs metal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements.

Preparation of lentivirus silencing SND1 and its influence on breast cancer cell proliferation and invasion / 中国肿瘤临床

Objective: This work aimed to construct stable MCF-7 cell sublines from which staphylococcal nuclease domain con-taining 1 (SND1) expression was interfered to analyze the effect of SND1 silencing on the proliferation and metastasis of MCF-7 cells. Methods: The lentivirus that could mediate SND1 silencing was prepared. MCF-7 cells were infected with the lentiviruses to construct stable sub-cell lines. Quantitative real-time polymerase chain reaction and Western blot analysis were employed to determine SND1 ex-pression level. MTS, wound healing, and transwell assays were applied to analyze the effect of SND1 silencing on the proliferation, mi-gration, and invasion of MCF-7 cells. Results: A lentivirus expression vector that contains sequences encoding shRNAs targeting SND1 and an shRNA negative control were successfully established. The lentiviruses (LV-SH1, LV-SH2, LV-SH3, and 和 LV-NC) were then collected and packaged. Stabilized MCF-7 sublines were prepared through infection with lentiviruses. The most efficient MCF-7 stable cell subline, MCF-SH3, was selected for SND1 silencing. Compared with the control cell, the proliferation, migration, and inva-sion potential of MCF-SH3 were significantly decreased. Conclusion: SND1 could promote the proliferation, migration, and invasion of breast cancer cells. Thus, silencing SND1 expression will inhibit such proliferation, migration, and invasion. These results indicated that the unusual expression of SND1 is associated with breast cancer and may participate in cancer progression by affecting prolifera-tion, migration, and invasion.

Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs) that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa) of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB) at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.

Nuclease activity and cytotoxicity to host cells of toxic protein VapC produced by Leptospira species / 中华微生物学和免疫学杂志

Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein.Methods Using genomic DNA of pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template,several PCRs were performed to amplify entire vapB,vapC and vapBC genes.Subsequently,the prokaryotic expression systems of vapB,vapC and vapBC genes were constructed.Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells were then determined.The changes of transcription and expression of vapB and vapC genes of L.interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay.The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of leptospiral VapB and VapC proteins on activity of host cells.Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes.The constructed prokaryotic expression systems could express rVapB and rVapC,respectively.rVapC displayed RNase avtivity but did not lyse DNA.When L.interrogans strain Lai infected THP-1 cells,the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells.The mass mortality was observed in HEK293 human renal tubular epithelial ceils containing the vapC gene through transfection.Conclusion VapC protein of L.interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.

Detection of Deoxyribonucleic Acid Using Cationic Fluorescent Conjugated Polymer and Nanoparticles / 分析化学

A novel method for DNA detection was developed based on the excellent fluorescence properties of cationic conjugated polymer ( CCP)and the target DNA enrichment,separation function of nanoparticles. First,the quencher-labeled DNA capture probes were modified on the surface of Au nanoparticles,and complementary DNA strands were captured. Second,S1 nuclease was added,and the capture probes that had not captured the complementary DNA were removed from the nanoparticles.Finally,the complementary double-stranded DNA was cut by Dnase I,the quenchers were dissociated from nanoparticle and the fluorescence of CCP was quenched by means of combination of quenchers and CCP.The results showed that this method is specific.In the range of 5. 0 -40 nmol/L,the concentration of target DNA was proportional to the fluorescence quenching and the detection limit was 3.7 nmol/L(S/N = 3).