Transcribed single nucleotide polymorphism: Ideal markers for detecting gene imprinting by 5' nuclease assay

Objective: To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the genotype of a transcribed single nucleotide polymorphism (SNP) rs705(C>T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lym-phoblast cell lines. Results: Allele discrimination showed a clear monoallelic expression pattern of SNRPN, which was confirmed by RT-PCR based restriction fragment length polymorphism (RFLPs). Pedigree analysis verified the paternal origin of expressed allele, which was in consistency with previous report. Conclusion: Transcribed SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach also may be used to discover differential allele expression of non-imprinted genes, finding out gene cis-acting functional polymorphism.

The Distribution and Substrate Specificity of Extracellular Nuclease Activity in Marine Fungi

The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium, Scopulariopsis, Wardomyces, Periconia, have implied its important function in the organic phosphorus and nitrogen circle in the Ocean. The fungal nucleases of 64 isolates tested were more or less specific for single-stranded DNA with a high preferential specificity towards poly-U substrate with forming of 5’-phosphate mononucleotides. A couple of the nucleases were capable of RNA digesting. The highest level of extracellular nucleolytic ability was observed in Penicillium spp. isolates. The tight correlation found between extracellular nuclease activity and the rate of thymidine uptake by actively growing and sporulating marine fungus Penicillium melinii suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

Synthesis, chemical nuclease activity, and in vitro cytotoxicity of benzimidazole-based Cu(Ⅱ)/Co(Ⅱ) complexes

In this study, novel mono/di-nuclear Cu（p-2-bmb）（OH）（Cl O4）（1） and Co2（p-2-bmb）2Cl4（2）（p-2-bmb = 1-（（2-（pyridin-2-yl）-benzoimidazol-1-yl）methyl）-1H-benzotriazole） complexes with the nitrogen heterocyclic benzimidazole-based ligand were synthesized and characterized. The two complexes showed antiproliferative effects in various carcinoma cell lines, especially complex 1 in the SMMC7721 tumor cell line. Complex 1 was also able to pass through the cell membrane and enter the nucleus and mitochondrion. An analysis of in vitro chemical nuclease activity revealed that complex 1 partially intercalated to calf thymus DNA and exhibited strong unwinding activity against p BR322 superhelical plasmid DNA. The comet assay and flow cytometry analysis confirmed that 1 caused extensive DNA damage and arrested SMMC7721 tumor cells at G2/M phase of the cell cycle, leading to loss of mitochondrial membrane potential and apoptosis. These results suggest that these benzimidazole-based metal complexes could be potential anti-cancer agents.

Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay(NPA-SH)

Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c...

Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay

Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.

Gene editing nuclease and its application in tilapia

Gene editing nucleases including zinc-finger nucleases(ZFNs), transcription activator like effector nucleases(TALENs) and clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated(Cas) system(CRISPR/Cas9) provide powerful tools that improve our ability to understand the physiological processes and their underlying mechanisms. To date, these approaches have already been widely used to generate knockout and knockin models in a large number of species. Fishes comprise nearly half of extant vertebrate species and provide excellent models for studying many aspects of biology. In this review, we present an overview of recent advances in the use of gene editing nucleases for studies of fish species. We focus particularly on the use of TALENs and CRISPR/Cas9 genome editing for studying sex determination in tilapia.

Preparation of human decellularized peripheral nerve allograft using amphoteric detergent and nuclease

Animal studies have shown that amphoteric detergent and nuclease(DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents(Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review.

Genome engineering and disease modeling via programmable nucleases for insulin gene therapy;promises of CRISPR/Cas9 technology

Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases,zinc finger nucleases,transcription activator-like effector nucleases and RNA-guided engineered nucleases(RGENs),which create double-strand breaks at specific target sites in the genome,and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism.A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype,without the need for the reengineering of the specific enzyme when targeting different sequences.CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function.RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes,as summarized and exemplified in this manuscript.

Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine

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.

Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.

Spectroscopic Characterization of Staphylococcal Nuclease Mutants with Tryptophan at Internal Sites

Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type （WT）. Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.

Integration of the nuclease protection assay with sandwich hybridization (NPA-SH) for sensitive detection of Heterocapsa triquetra

Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization（NPA-SH） probe that targets the large subunit of ribosomal RNA（LSU r RNA）. We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H.triquetra at a concentration of 1.5×10^4 cells/m L, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015（3.0×10^4 cells/m L）. We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H.triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.

Blocking Translation of Oncogenic mRNA

Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.

Structure and mechanism for DNA lesion recognition

A fundamental question in DNA repair is how a lesion is detected when embedded in millions to billions of normal base pairs. Extensive structural and functional studies reveal atomic details of DNA repair protein and nucleic acid interactions. This review summarizes seemingly diverse structural motifs used in lesion recognition and suggests a general mechanism to recognize DNA lesion by the poor base stacking. After initial recognition of this shared structural feature of lesions, different DNA repair pathways use unique verification mechanisms to ensure correct lesion identification and removal.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

Modulation of mitochondrial bioenergetics as a therapeutic strategy in Alzheimer＇s disease

Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.

Gene therapeutic approaches to inhibit hepatitis B virus replication

Acute and chronic hepatitis B virus(HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellularcarcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV.

Combination of CRISPR/Cas9 System and CAR-T Cell Therapy:A New Era for Refractory and Relapsed Hematological Malignancies

Chimeric antigen receptor T(CAR-T)cell therapy is the novel treatment strategy for hematological malignancies such as acute lymphoblastic leukemia(ALL),lymphoma and multiple myeloma.However,treatment-related toxicities such as cytokine release syndrome(CRS)and immune effector cell-associated neurotoxicity syndrome(ICANS)have become significant hurdles to CAR-T treatment.Multiple strategies were established to alter the CAR structure on the genomic level to improve efficacy and reduce toxicities.Recently,the innovative gene-editing technology-clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease9(Cas9)system,which particularly exhibits preponderance in knock-in and knockout at specific sites,is widely utilized to manufacture CAR-T products.The application of CRISPR/Cas9 to CAR-T cell therapy has shown promising clinical results with minimal toxicity.In this review,we summarized the past achievements of CRISPR/Cas9 in CAR-T therapy and focused on the potential CAR-T targets.

Genome engineering technologies for targeted genetic modification in plants

Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspaced short palindromic repeats and associated protein system-based editing （CRISPR/Cas） are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future.

Cleavage of Nucleotides by Lanthanide Metal Complexes

The present work revealed that the praseodymium()complex of 2carboxyethylgermanium sesquioxide(Ge132)promotes the hydrolysis of the phosphodiester linkages of 3,5cyclic adenosine monophosphate(cAMP),3,5cyclic deoxyadenosine monophosphate(dcAMP),5adenosine monophosphate(5AMP)and 5deoxyadenosine monophosphate(5dAMP)under mild conditions.Both cAMP and dcAMP were hydrolyzed sitespecifically,yielding predominantly 3monophosphates,the main products of the cleavage of 5AMP and 5dAMP included adenosine(Ado),deoxyadenosine(dAdo)and free phosphates respectively.A hydrolytic mechanism was proposed for cAMP,dcAMP,5AMP and 5dAMP.