Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid protein is a potential antiviral drug target,serving multiple critical functions during the viral life cycle.However,the structural information of SARS-CoV-2 nucleocapsid protein remains unclear.Herein,we have determined the 2.7 A crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein.Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain,the surface electrostatic potential characteristics between them are distinct.Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside theβ-sheet core.Complemented by in vitro binding studies,our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain,guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

In vitro antitumor activity and targeted sites of two novel platinum-based(II) complexes on SW620 colon cancer cell line

Objective:The aim of our study was to evaluate the in vitro antitumor activity of two novel platinum-based(II) complexes(2.3-pyridinedicarboxylic acid dehydrate platinum and 2.3-pyrazinedicarboxylic acid dehydrate platinum),which were concurrently provided with hydrophilic carboxyl group and lipophilic pyrazinyl or pyridyl group,on SW620 colorectal cancer cell line and the impact of the two compounds on the cell cycle and apoptosis of the cells when compared with the oxaliplatin,desiring the new ligand combined with hydrophilic and lipophilic properties would facilitate the transportation and transmembrane of the drugs,showing a better antitumor activity.Methods:After SW620 cells were treated with different doses of the three platinum-based agents for 24,48 and 72 h,the cell proliferation inhibition rate was determined using methyl thiazolyl tetrazolium(MTT) assay;the morphology of cells were evaluated under inverted microscope;the changes in cell cycle were determined using flow cytometry;the percent apoptosis was measured using Annexin V/PI double staining and the micromorphology of the cells after drug exposure was evaluated using scanning electron microscopy.Results:The evaluation on the proliferation inhibition rate revealed that the three platinum-based agents inhibited the SW620 cells in a time-and dose-dependent manner and showed different strengths as pyridine > pyrazine > Oxa.Under optical microscope,the morphological changes such as cell shrinkage,round cells and dead cells were frequently observed after drug exposure.Cell cycle determination showed that all of the three agents could function to block the cells converting from phase S to phase G2M.Apoptosis evaluation revealed that the three agents promoted the apoptosis of SW620 cells in a time-and dose-dependent manner and showed different strengths as pyridine > pyrazine > Oxa.Typical early and late apoptotic morphological changes could be detected during electron microscopy.Conclusion:The two novel platinum-based(II) complexes showed a stronger antitumor effect on SW620 cells than oxaliplatin,with the targeted site at a certain phase of cell cycle and apoptosis.

Targeted montoring to surgical site infection and investigation of standard application of antibiotics during perioperative period

Objective To investigate current surgical site infection and perioperative antibiotics in inpatients and explore the controlling aim and methods. Methods The infection rates of surgical sites of 287 operated cases from May to Dec 31,2007 were studied and compared with

Bone infection site targeting nanoparticle-antibiotics delivery vehicle to enhance treatment efficacy of orthopedic implant related infection

Orthopedic implants account for 99%of orthopedic surgeries,however,orthopedic implant-related infection is one of the most serious complications owing to the potential for limb-threatening sequelae and mortality.Current antibiotic treatments still lack the capacity to target bone infection sites,thereby resulting in unsatisfactory therapeutic effects.Here,the bone infection site targeting efficacy of D6 and UBI29-41 peptides was investigated,and bone-and-bacteria dual-targeted nanoparticles(NPs)with D6 and UBI29-41 peptides were first fabricated to target bone infection site and control the release of vancomycin in bone infection site.The results of this study demonstrated that the bone-and-bacteria dual-targeted mesoporous silica NPs exhibit excellent bone and bacteria targeting efficacy,excellent biocompatibility and effective antibacterial properties in vitro.Furthermore,in a rat model of orthopedic implant-related infection with methicillin-resistant Staphylococcus aureus,the growth of bacteria was evidently inhibited without cytotoxicity,thus realizing the early treatment of implant-related infection.Hence,the bone-and-bacteria dual-targeted molecule-modified NPs may target bacteria-infected bone sites and act as ideal candidates for the therapy of orthopedic implant-related infections.

MicroRNA-regulated viral vectors for gene therapy

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

Detection of a novel panel of 24 genes with high frequencies of mutation in gastric cancer based on next-generation sequencing

BACKGROUND Gastric cancer is a leading cause of cancer-related mortality worldwide.Many somatic mutations have been identified based on next-generation sequencing;they likely play a vital role in cancer treatment selection.However,nextgeneration sequencing has not been widely used to diagnose and treat gastric cancer in the clinic.AIM To test the mutant gene frequency as a guide for molecular diagnosis and personalized therapy in gastric cancer by use of next-generation sequencing.METHODS We constructed a panel of 24 mutant genes to detect somatic nucleotide variations and copy number variations based on a next-generation sequencing technique.Our custom panel included high-mutation frequency cancer driver and tumour suppressor genes.Mutated genes were also analyzed using the cBioPortal database.The clinical annotation of important variant mutation sites was evaluated in the ClinVar database.We searched for candidate drugs for targeted therapy and immunotherapy from the OncoKB database.RESULTS In our study,the top 16 frequently mutated genes were TP53(58%),ERBB2(28%),BRCA2(23%),NF1(19%),PIK3CA(14%),ATR(14%),MSH2(12%),FBXW7(12%),BMPR1A(12%),ERBB3(11%),ATM(9%),FGFR2(8%),MET(8%),PTEN(6%),CHD4(6%),and KRAS(5%).TP53 is a commonly mutated gene in gastric cancer and has a similar frequency to that in the cBioPortal database.33 gastric cancer patients(51.6%)with microsatellite stability and eight patients(12.5%)with microsatellite instability-high were investigated.Enrichment analyses demonstrated that high-frequency mutated genes had transmembrane receptor protein kinase activity.We discovered that BRCA2,PIK3CA,and FGFR2 gene mutations represent promising biomarkers in gastric cancer.CONCLUSION We developed a powerful panel of 24 genes with high frequencies of mutation that could detect common somatic mutations.The observed mutations provide potential targets for the clinical treatment of gastric cancer.

Toxicity of Magnetic Albumin Microspheres Bearing Adriamycin

Magnetic albumin microspheres bearing adriamycin (ADM MAM) is a novel chemotherapeutic compound with site specific drug delivery characteristics. The acute and subacute toxic tests of the compound, local irritating test and anaphylactic test were performed on mice and guinea pigs. The results showed there was no macroscopically and microscopically direct cytotoxic injuries of the compound to the animal organs or to the cells. The LD 50 value of the compound was higher than that of the single used adriamycin, indicating that the compound was less toxic than the single adriamycin and quite safe in its therapeutic dosage. Furthermore, there was also no side effects or toxic reactions to be observed on clinical patients with advanced carcinoma or gastric cancer.

Frequencies and mechanisms of pesticide resistance in Tetranychus urticae field populations in China

The two-spotted spider mite Tetranychus urticate is an important agricultural pest worldwide.It is extremely polyphagous and has developed resistance to many pesticides.Here,we assessed the pesticide resistance of seven field populations of T.urticae in China,their target site mutations and the activities of their detoxification enzymes.The results showed that abamectin and the traditional pesticides pyridaben,profenofos and bifenthrin had higher resistance or lower toxicity than more recently developed pesticides including chlorfenapyr,spinetoram,cyflumetofen,cyenopyrafen,bifenazate and B-azolemiteacrylic.The frequency of point mutations related to abamectin resistance,G314D in the glutamate-gated chloride channel 1(GluCl1)and G326E in GluC13,ranged 47%-70%and 0%-97%,respectively.The frequency of point mutations in A1215D and F1538I of the voltage-gated sodium channel gene(VGSC),which may increase resistance to pyrethroids,ranged 88%-100%and 10%-100%,respectively.For target sites related to organophosphate resistance,mutation frequencies ranged 25%-92%for G119S and 0%-23%for A201S in the acetycholinesterase gene(Ace).Mutation G126S in the bifenazate resistance-related cytochrome b gene(Cytb)was observed in three of the seven T.urticae populations.Higher activities of detoxification enzymes(P450,GST,CarEs and UGTs)were observed in two T.urticae populations,with significant difference in the XY-SX population.These results provide useful information on the status of pesticide resistance of T.urticae in China and suggest that T.urticae field populations may have multiple resistance mechanisms.

Limitations of RNAi of e6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The a6 nicotinic acetyl- choline receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking a6. RNAi was performed against the Da6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4-UAS system to examine if RNAi would yield results similar to those of a6 null mutants. These Da6-deficient flies were subject to spinosad contact bioassays to evaluate the role of the Da6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Da6 was reduced 60%-75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasct6. RNAi of Tcasa6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the a6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.

An insert in the Hpy region of hscp in Hefiothis virescens （Lepidoptera： Noctuidae） reveals a possible CORE-SINE of insects

A new putative transposon was identified in the tobacco budworm, Helio- this virescens. This transposon was characterized as a full length CORE-SINE （65 bp of ＂CORE＂ core specific nucleotide short interspersed elements） that resembled sequences from three other lepidopterans and humans. In particular, the A-box and B-box regions of this sequence most closely conformed to the signature of CORE-SINEs from widely divergent species. This CORE-SINE was present as a polymorphism in a hypervariable region of the gene hscp, which is the target of pyrethroid insecticides and other xenobiotics in the nerve axon. We described this new putative transposon as Noct-1 due to its presence in a noctuid moth. This is the first description of a full-length CORE-SINE with the A-box, B-box, target site duplication, and candidate core domain from an insect.