Abstract:

Abstract:As a malleable and novel tool for antigen recognition and modulation,nanobodies have the advantages of small size,easiness of expression,screening and modification,as well as high affinity and stability.Nanobodies are capable of recognizing more cryptic antigenic epitopes that are difficult to be recognized by traditional antibodies,making them increasingly used in the diagnosis and treatment of various diseases and assays.Nanobodies are also playing an irreplaceable role in the basic research.This review summarized the recent development of nanobodies and their derivatives in the detection of small molecules,pathogenic microorganisms and diagnosis of diseases,as well as in the fields of targeted therapies,cellular and molecular imaging.Broad prospects of nanobodies in the field of protein conformation studies were also reviewed.

Abstract:Ectoine is an amino acid derivative and an important natural product in halophilic microorganisms.It plays an important role in protecting cells and stabilizing biological macromolecules,and can be widely used in biomedical fields such as drug preparation adjuvants,organ transplantation and preservation,skin wound repair and cosmetics.Due to the medical value and commercial market demand of ectoine,this article summarized the recent advances in the microbial production of ectoine,including the mutation and breeding of hyper-producing strains,construction of genetically and metabolically engineered strains,optimization of fermentation processes,and extraction and purification processes.The application of multi-omics technologies and computational biology to develop an ectoine producing cell factory was prospected,with the aim to provide a reference for ectoine overproduction.

Abstract:With the widespread application of genomics and transcriptomics in the genetics and cell biology of different species,synonymous codon usage bias has been gradually accepted and used to study the deep connection between biological evolution and biological phenotypes.It is an important part of the life activities that mRNA is expressed into proteins with normal biological activities.The synonymous codon usage patterns,which were named as ‘the second genetic codon’,can express genetic information carried by themselves at the levels of transcriptional regulations,translational regulations and metabolic activities through molecular mechanisms such as fine-tune translation selection.Some studies have shown that the length of mRNA half-life has significant impacts on mRNA activity and the process of transcription and translation.This review summarized the roles of synonymous codon usage patterns in transcription,translational regulation and post-translational modification,with the aim to better understand how organisms skillfully utilize the genetic effects caused by codon usage patterns to accurately synthesize different types of proteins,so as to ensure the growth or differentiation of the specific gene expression procedures to carry out smoothly and maintain the normal life cycle.

Abstract:Hepatitis B virus (HBV) infection is one of the most serious public health problems.HBV infection could lead to hepatitis B,and even further develop into hepatic cirrhosis and hepatocellular carcinoma.Interferon lambda (IFN-λ) is a member of the interferon (IFN) family and an important cytokine for antiviral defense.There are four members in IFN-λ family,including IFN-λ1,IFN-λ2,IFN-λ3,and IFN-λ4.The genetic polymorphisms in the IFN-λ genes are associated with HBV replication and treatment response of HBV patients.In this review,we summarized the roles of genetic polymorphisms of the IFN-λ genes played in HBV infection,disease progression and treatment,with the aim to better understand their function.This review could serve as a reference for the HBV prevention and treatment of HBV patients,as well as for future clinical usage.

Abstract:In recent decades,the treatment of autoimmune diseases has moved from the use of hormones and conventional immunosuppressive drugs to biological agents.B cell proliferation and maturation play crucial roles in the development of autoimmune diseases.The tumor necrosis factor superfamily ligand B cell activating factor (BAFF) and its receptor mediate B cell survival through regulating signaling pathways.Therefore,BAFF and its receptors are important therapeutic targets for the treatment of autoimmune diseases.This review describes the mechanism of BAFF and its receptor in the human body system and introduces the latest views on how over-activation of BAFF pathway promotes the development of autoimmune diseases including systemic lupus erythematosus,Sjogren’s syndrome,and rheumatoid arthritis.In connection to the treatment of the above three diseases,this review discusses the clinical trials and application status of three BAFF-targeting antibody drugs,including Belimumab,Tabalumab and Atacicept.Finally,this review proposes new strategies that targeting the BAFF pathway to provide a new treatment for autoimmune diseases.

Abstract:Group Ⅱ introns are self-splicing ribozymes,which insert directly into target sites in DNA with high frequency through “retrohoming”.They specifically and efficiently recognize and splice DNA target sites,endowing themselves with great potential in genetic engineering.This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification,and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the “retrohoming” characteristics and the dependence on high Mg²⁺ concentration.Finally,we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns,hoping to provide a reference for the application of group Ⅱ introns in biotechnology.

Abstract:Cartilage has poor self-recovery because of its characteristics of no blood vessels and high extracellular matrix.In clinical treatment,physical therapy or drug therapy is usually used for mild cartilage defects,and surgical treatment is needed for severe ones.In recent years,cartilage tissue engineering technology provides a new way for the treatment of cartilage defects.Compared with the traditional surgical treatment,cartilage tissue engineering technology has the advantages of small wound and good recovery.The application of microcarrier technology in the design of tissue engineering scaffolds further expands the function of scaffolds and promotes cartilage regeneration.This review summarized the main preparation methods and development of microcarrier technology in recent years.Subsequently,the properties and specific application scenarios of microcarriers with different materials and functions were introduced according to the materials and functions of microcarriers used in cartilage repair.Based on our research on osteochondral integrated layered scaffolds,we proposed an idea of optimizing the performance of layered scaffolds through microcarriers,which is expected to prepare bionic scaffolds that are more suitable for the structural characteristics of natural cartilage.

Abstract:Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics.The PCR includes multiple reaction stages (denaturation,annealing,and extension),and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30–40 cycles within at least 1–2 hours.Due to the complicated devices and the long amplification time,it is difficult to adopt conventional PCR in point-of-care testing (POCT).Comparing to conventional PCR,isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device.When isothermal amplification is combined with microfluidics,a more competent platform for POCT can be established.For example,various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses.This review summarized the recent development and applications of the microfluidics-based isothermal amplification.First,different typical isothermal amplification methods and related detection methods have been introduced.Subsequently,different types of microfluidic systems with isothermal amplification were discussed based on their characteristics,for example,functionality,system structure,flow control,and operation principles.Furthermore,detection of pathogens (e.g.SARS-CoV-2 viruses) based on isothermal amplification was introduced.Finally,the combination of isothermal amplification with other new technologies,e.g.CRISPR,has been introduced as well.

Abstract:Chromatography is a basic process in the current proteomics workflow,and the retention time alignment of the chromatogram is one of the important steps to effectively improve the identification and quantification accuracy.After years of development,a series of algorithms for retention time alignment have been developed.This review summarizes the advances of chromatographic retention time alignment algorithms and tools for proteomics analysis from the perspective of proteomics users,and discusses the development and future application directions.

Abstract:Human body can obtain energy from either carbohydrate or fat digestion.Although glucose metabolism derived from carbohydrate-based diets has long been utilized for energy supply,it has been recently discovered that shifting from glucose to fatty acid metabolism may become a novel way for improving human health especially when carbohydrate is deprived.In recent years,intermittent fasting and ketogenic diets have received a lot of attention in respect to favoring fatty acid metabolism.In all cases,fatty acid metabolism produces D-β-hydroxybutyrate (D3HB),which is a natural ketone body,as well as,a monomer of microbial poly-D-β-hydroxybutyrate (PHB).D3HB can be utilized by different cells of the body as an alternative energy fuel or an intracellular signaling molecule with multiple downstream signaling pathways.Usually,the serum level of D3HB is increased during ketogenic diets,however,requires a very long period of adaptation (over 3-months) and exhibits unwanted adverse effects.Hence,exogenous ketone supplements using D3HB have become a more effective approach to induce and maintain nutritional ketosis for subsequent functional effects.This review describes how D3HB is produced and metabolized within the body,the functional roles played by D3HB,and a detailed summary of the different applications of exogenous ketones that have been explored to date in both nutritional and therapeutical context.

Abstract:Antimicrobial resistance is one of the critical public health issues in the world.There is an urgent need to develop effective broad-spectrum antibiotics to treat the infection of multi-drug resistant Gram-negative bacilli.Cefiderocol,developed by the Shionogi Inc.in Japan,is a new type of iron carrier cephalosporin antibiotics,which overcomes the drug resistance of Gram-negative bacilli due to the down-regulation of outer membrane pore protein and the up-regulation of efflux pump,and has good stability to serine-and metallo-carbapenemases.This drug has a broad spectrum and strong antibacterial activity against carbapenem-resistant Enterobacteriaceae (CRE),Pseudomonas aeruginosa,Acinetobacter baumannii,and Stenotrophomonas maltophilia.Cefiderocol can be used to treat complex urinary tract infections (including pyelonephritis),hospital-acquired pneumonia,and ventilator-associated pneumonia.By summarizing the chemical structure,antibacterial mechanism,in vitro antibacterial activity,pharmacokinetics,pharmacodynamics,and clinical treatment of cefiderocol,this review shows the application potential of cefiderocol as a new iron carrier cephalosporin in the treatment of multi-drug resistant Gram-negative bacilli infections.

Abstract:Triterpenoid saponins are widely used in medicine,health cares,cosmetics,food additives and agriculture because of their unique chemical properties and rich pharmacological activities.UDP-dependent glycosyltransferases (UGTs) are the key enzymes involved in triterpenoid saponin biosynthesis,and play important roles in the diversity of triterpenoid saponin structures and pharmacological activities.This review summarized the UGTs involved in plant triterpenoid saponin biosynthesis based on the sources of UGTs and the types of receptors.Moreover,the application of UGTs in heterologous biosynthesis of triterpenoid saponins based on synthetic biology was also discussed.

Abstract:In order to explore the antitumor effect and mechanism of different extracts of cultivated Phellinus vaninii fruit body on H₂₂ tumor bearing mice,150 ICR mice were randomly divided into blank group,model group,CTX group,P.vaninii water extract group,ethanol extract group,petroleum ether extract group and crude polysaccharide group.H₂₂ liver cancer cells were used to establish a solid tumor model and the mice were sacrificed on the 10th day after administration.The spleen and thymus organ index and tumor inhibition rate were calculated,the serum levels of TNF-α,INF-γ,VEGF,and hematoxylin-eosin were detected,and the immunohistochemical staining method was used to observe the pathological changes of tumor tissues,while Western blotting was used to detect the expression of tumor-related proteins.The high-dose petroleum ether extract group showed the best tumor inhibition rate (73.21%),increased serum levels of TNF-α,IFN-γ,and VEGF,as well as significantly promoted tumor necrosis and ablation.The immunohistochemistry of the water extract group showed negative regulation,indicating an insignificant tumor suppression.Western blotting showed the apoptosis genes Caspase-3,Caspase-9 and pathway genes NF-κB and JAK were all highly expressed in each administration group compared with the model group,and their expression levels gradually decreased with increasing doses.In summary,the petroleum ether extract of P. vaninii fruit body showed a significant anti-tumor effect which is presumably mediated through the mitochondrial pathway.The metabolism of drug in the body induces activation of Caspase-3 and Caspase-9 apoptotic proteins by Bax,Bcl-2,and TNF,which further caused nuclear chromatin or DNA to condense or degrade,and subsequently destroy the normal proliferation of tumor cells,thereby inducing their apoptosis and inhibiting tumor growth.

Abstract:Hepatitis B virus core protein (HBc) has become a hot spot in drug carrier protein research due to its natural particle self-assembly ability and ease of modification.The truncation of the C-terminal polyarginine domain (CTD,aa 151–183) of HBc does not affect the self-assembly of the particles.However,it does affect the internal and external charges of the particles,which may subsequently affect drug encapsulation.Thus,the truncated C-terminal polyarginine domain (CTD) of HBc and the inserted RGD peptide were selected to construct and express three HBc variants (RH) encapsulated with ICG (RH/ICG) with different C-terminal lengths to compare the stability and drug activity of their nanoformulations.RH160/ICG was found to have a great advantages in encapsulation efficiency and biological imaging.Compared with other HBc variants,RH160/ICG significantly improved encapsulation efficiency,up to 32.77%±1.23%.Cytotoxicity and hemolysis assays further demonstrated the good biocompatibility of RH160/ICG.Cell uptake and in vivo imaging experiments in mice showed that RH160/ICG could efficiently deliver ICG in tumor cells and tumor sites with good imaging effect.This research provides a new direction for further expanding the diagnosis and treatment application of ICG and development of HBc-based nanoparticle drug carrier platform.

Abstract:As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration),GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents.Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes,MsmEFG1(MSMEG_1400) and MsmEFG2(MSMEG_6535),fusA1(Rv0684) and fusA2(Rv0120c),respectively.MsmEFG1(MSMEG_1400) and fusA1(Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis.To investigate the biological function and characteristics of EF-G in mycobacterium,two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique.EF-G2 knockdown had no effect on bacterial growth,while EF-G1 knockdown significantly retarded the growth of mycobacterium,weakened the film-forming ability,changed the colony morphology,and increased the length of mycobacterium.It was speculated that EF-G might be involved in the division of bacteria.Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin,isoniazid,erythromycin,fucidic acid,capreomycin and other antibacterial agents,suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Abstract:In recent years,two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy.The protein bS22 is located near the decoding center of the 30S subunit,and the protein bL37 is located near the peptidyl transferase center of the 50S subunit.Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics,it is speculated that they might affect the binding of related drugs to these targets.Therefore,we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc²155 through homologous recombination,and then determined the growth curves of these mutants and their sensitivity to related antibiotics.The results showed that compared with the wild-type strain,the growth rate of these two mutants did not change significantly.However,mutant ΔbS22 showed increased sensitivity to capreomycin,kanamycin,amikacin,streptomycin,gentamicin,paromomycin,and hygromycin B,while mutant ΔbL37 showed increased sensitivity to linezolid.These changes in antibiotics sensitivity were restored by gene complementation.This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.

Abstract:To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1,we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line.We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles.SgRNA oligos based on the selected TrxR1 knockout sites were obtained.Next,we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector.After transfection of the plasmid into HCT-116 cells,TrxR1 knockout HCT-116 cells were selected using puromycin resistance.The TrxR1 knockout efficiency was identified and verified by DNA sequencing,immunoblotting,TRFS-green fluorescent probe,and cellular TrxR1 enzyme activity detection.Finally,the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay.The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells,and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116(HCT116-TrxR1-KO) cells.The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation.Based on these results,we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques,which may facilitate investigating the role of TrxR1 in various diseases.

Abstract:ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation,invasion and drug resistance,and it serves as a therapeutic target.However,only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present.Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor.The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc.The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged,characterized and amplified using AdMax^TM adenovirus packaging system.The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells.Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated.The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc,leading to significant inhibition of EGFR/ERK signaling pathway.Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.

Abstract:Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings.However,antibody-mediated immunity,which includes both natural and induced antibody responses,is a major challenge for the success of pig-to-primate xenotransplantation.Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation.In this study,we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1,3-galactosyltransferase gene GGTA1,which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation.Meanwhile,human leukocyte antigen-G5 gene HLA-G5,which acts as an immunosuppressive factor,was co-transfected with TALEN into porcine fetal fibroblasts.The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer.As a result,we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein.Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent.Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets.Functionally,the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs.These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Abstract:Influenza B virus is one of the causes for seasonal influenza,which can account for serious illness or even death in some cases.We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells,and then determined the immunogenicity of HA-ecto in mice.The gene sequence encoding influenza B virus HA-ecto,foldon sequence,and HIS tag was optimized and inserted into pCAGGS vector.The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS.The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine.Cell supernatant after transfection was collected after 96 h,and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography.Subsequently,the mice were immunized with HA-ecto protein,and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays.The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system.Moreover,trimeric HA-ecto protein,in combination with the adjuvant,induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice.Thus,the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Abstract:Transglutaminase 2(TGM2) is a ubiquitous multifunctional protein,which is related to the adhesion of different cells and tumor formation.Previous studies found that TGM2 is involved in the interaction between host cells and viruses,but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported.To explore the effect of TGM2 during H1N1 subtype influenza virus infection,a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed.The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus.The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus,while knockout of TGM2 up-regulated the expression of the NP and NS1 genes,and the expression of the NP at protein level was consistent with that at mRNA level.Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression.On the contrary,the virus titer in TGM2 knockout cells reached the peak at 48 h,which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells.By analyzing the expression of genes downstream of influenza virus response signaling pathway,we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway.The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.

Abstract:Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein,and its inhibitory activity on tumor cells and angiogenesis were assessed.luffin-α-NGR fusion gene was obtained by PCR amplification.The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR.The plasmid was transformed into E.coli BL21,and the target protein was isolated and purified by GST affinity chromatography.The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained,and the optimal soluble expression of the target protein was achieved under the conditions of 16℃,0.5 mmol/L IPTG after 16 h induction.SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa.Subsequently,the recombinant protein was de-tagged by precision protease digestion.The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α.The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization.The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.

Abstract:The α2δ-1 protein coded by Cacna2d1 is dramatically up-regulated in dorsal root ganglion (DRG) neurons and spinal dorsal horn following sensory nerve injury in various animal models of neuropathic pain.Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity.The α2δ-1-NMDAR interaction promotes surface trafficking and synaptic targeting of NMDARs in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury,as well as in other pathological conditions such as in the paraventricular nucleus (PVN) with neurogenic hypertension and in the brain with ischemic stroke.The lentiviral transfection method was used to construct a human embryonic kidney HEK293T cell line that could stably express α2δ-1-NMDAR complex.A stably transfected cell line was observed by florescence microscope,and identified by RT-qPCR and Western blotting.The results showed that the HEK293T cell line was successfully transfected and the genes could be stably expressed.Subsequently,the transfected cell line was successfully developed into a target drug screening system using patch clamp techniques.It provides a promising cell model for further research on the interaction mechanism of α2δ-1-NMDAR complex and drug screening for chronic pain and related diseases with low side effects.

Abstract:It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro.However,the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown.In this study,we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2(PC2) located on the primary cilia on the surface of ROBs.First,immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs,and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting.Subsequently,we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP),as well as the expression of Runx-2,Bmp-2,Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI),a PC2 blocker.Moreover,we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference.The results showed that PC2 was localized on the primary cilia of ROBs,and PEMFs treatment increased the expression of PC2 protein.When PC2 was blocked by AMI,PEMFs could no longer increase PC2 protein expression and ALP activity,and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset.After inhibiting the expression of PC2 using RNA interference,PEMFs can no longer increase the expression of genes related to bone formation.The results showed that PC2,located on the surface of primary cilia of osteoblasts,plays an indispensable role in perceiving and transmitting the physical signals from PEMFs,and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2.This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

Abstract:Opsin3(OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain.In recent years,it has been reported that OPN3 is also highly expressed in adipose tissue,and the protein is associated with the production of skin melanin.We found that the N82 site is the glycosylation site of OPN3.SNAP-tag^TM has diverse functions and can be applied to a variety of different studies.By constructing a SNAP-tagged OPN3 recombinant protein,the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface^® 549 and SNAP-Cell^® OregonGreen^®,which provides a new method for studying the function of OPN3.It also shows that SNAP-tag does not affect the function of OPN3.Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

Abstract:Mesenchymal stem cells (MSCs) have broad application potentials in regenerative medicine and translational medicine.Obtaining large quantities of primary-cultured MSCs and select the most suitable cell origin for targeted diseases are critical to research.To select the most suitable seed cells of MSCs from different origins for clinical treatment and research,biological characteristics of MSCs from human umbilical cord and placenta were compared.These include cell morphology,surface marker expression,differentiation and karyotype.Transcriptome sequencing of four MSCs from fetus were performed and the results were analyzed from the perspective of proliferation and cytokine secretion.The results revealed that MSCs from umbilical cord (UC),amniotic membrane (AM),chorionic membrane (CM),chorionic villi (CV) and deciduae (DC) met the minimum standards of the International Society of Cell Therapy (ISCT) in 2006 and had the general characteristics of stem cells.Karyotype analysis showed that MSCs derived from UC,AM,CM and CV were all from fetus except that the DC-MSCs were from mother.Transcriptome sequencing analysis showed that hMSCs from umbilical cord and placenta had similar gene expression patterns,while different expression patterns were observed in specific genes involved in cell cycle,cell division,cell death,cell growth and development.These genes play important roles in transcriptional regulation,DNA repair,DNA replication and chromosome stability,which were momentous components of cellular or subcellular fraction movement,cell communication,cell tissue protrusions,cytokine secretion and hormone metabolism.Transcriptome sequencing analysis explained the differences in biological characteristics among MSCs from different sources,while verification experiments based on the transcriptome sequencing results showed that the proliferation and cytokine secretion capabilities of MSCs from different sources were significantly different.In all,UC-MSCs and CV-MSCs with stronger proliferation and higher levels of paracrine factors secretion may show their respective advantages in treating diseases.

Abstract:8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive and stable biomarker for evaluating DNA oxidative damage.A rapid and sensitive colloidal gold immunochromatographic strip was developed for 8-OHdG detection by a competitive method.The sample pad (glass cellulose film),bonding pad (glass cellulose film),nitrocellulose film and absorbent pad were pasted on the polyvinyl chloride (PVC) base plate to construct the test strip.Colloidal gold (AuNPs) was prepared by the reduction of chloroauric acid with sodium citrate.8-OHdG antibody (Ab) was coated on the outer layer of AuNPs to form Ab@AuNPs as a probe.Bovine serum albumin (BSA) and 8-OHdG were conjugated with carbodiimide hydrochloride to prepare an artificial antigen,which was used as the coating antigen of detection line.Goat anti mouse polyclonal antibody IgG was used as the coating antibody of control line.The experimental parameters were optimized including the type of nitrocellulose membrane,the formula of loading solution,and the spraying amount of gold labeled antibody.The results showed that the appropriate nitrocellulose membrane was CN 95.The optimal loading solution included BSA (1%),Tween-20(3%),sucrose (3%) and NaCl (0.9%).The optimal spraying amount of gold labeled antibody was 4 μL.8-OHdG can be detected by the strip under visible light,and the level of 8-OHdG in urine can be preliminarily determined by comparing the color intensity of T line and C line.The 8-OHdG concentration in urine was further calculated by the gray value of T line and the threshold of detection was 2.55 μg/L.This colloidal gold immunochromatographic strip is simple,rapid and specific for detecting 8-OHdG in human urine to preliminarily evaluate the human status.

Abstract:Recombinant HLA-Ⅰ molecules/antigenic peptide complexes (pHLA complexes) are applied in the research of human T cell-specific immune responses.The preparation of pHLA complex is based on genetic engineering and protein in vitro dilution and folding-refolding technology.In an in vitro refolding system,recombinant HLA-Ⅰ molecules correctly fold and bind with antigenic peptides to form complexes.In this study,ultrafiltration-high performance liquid chromatography (ultrafiltration-HPLC) was used for quantitative determination of the antigenic peptides in recombinant pHLA complexes,especially for those in a small amount of prepared products.By adding the recombinant HLA-Ⅰ molecules and antigenic peptides into the refolding buffer,the heavy chain (HC) and light chain (β2m) of recombinant HLA-Ⅰ molecules were refolded and bond with the VYF antigenic peptide containing anchor residues to form a pHLA complex.The unbound free antigenic peptide VYF was removed by ultrafiltration to retain the complex.Finally,the pHLA complex was treated by acid to destroy its interaction,thus releasing the antigenic peptide.The results showed that the prepared recombinant pHLA complex was recognized by HLA-Ⅰ molecule specific antibody W6/32,which indicated that the recombinant HLA-Ⅰ class molecule had correct folding and was identified as pHLA complex.The antigen peptide VYF contained in the pHLA complex was also detected by ultrafiltration-HPLC,so it is feasible to apply ultrafiltration-HPLC for determination of pHLA complex.Compared with Western blotting,the concentration of antigenic peptides detected by ultrafiltration-HPLC was 0–9 μg/mL.The binding conditions can be optimized according to the amount of antigenic peptides bound in the complex in order to improve the folding efficiency of HLA-Ⅰ molecules and promote the binding of HLA-Ⅰ molecules to antigenic peptides.The production rate of pHLA complexes in the refolding system can also be calculated according to the content of antigenic peptides bound by pHLA complexes.Therefore,ultrafiltration-HPLC in this study can be used for the quality control of the preparation process of pHLA complexes,and may facilitate the research of T cell-specific immunity,artificial antigen-presenting cells,and development of specific tetramer probe applications.

Abstract:In order to develop a simple and efficient site-directed mutagenesis solution,the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations,with the aim to simplify the overlap extension PCR.The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR.Meanwhile,an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments.The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal,single-reaction,creating a recombinant plasmid.After the recombinant plasmids were transformed into competent Escherichia coli DH5α,several clones were screened from each group.Through restriction analysis and DNA sequencing,it was found that the randomly selected clones were 100% target mutants.Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation,and there was no need to digest the original plasmid,this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis.Hence,genes with single or multiple mutations could be cloned easily and efficiently.In summary,the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol,making it a good solution for site-directed mutagenesis.

Abstract:In the “Tutorial for outline of the healthy China 2030 plan”,biomedicine was listed as a key planning and development area.Shanghai government also lists biomedicine as an emerging pillar industry.The rapid development of biomedicine industry put higher requirement for talents.Taking the idea of cross integration,mutually beneficial development,inheritance and innovation,the School of Biotechnology of East China University of Science and Technology organically integrates bioengineering and pharmaceutical majors to develop a new undergraduate engineering program of biomedicine,which specially reforms the talent training practice from the aspects of developing a “trinity teaching” standard system,a “three integration,three convergence” curriculum system,and a “three comprehensive education” innovative talent training system.We put forward the trinity of “value guidance,knowledge system,technology and non-technical core competence literacy” to foster emerging biomedicine engineering talents,and developed a comprehensive innovative talents training mode featured by “covering class-in and class-out,covering every student,and covering ideology and curriculum”.Moreover,we established effective connections between courses and training goals,between general education courses and professional courses,and between top-notch talent training systems and training programs.Based on the achievements of teaching reform of the emerging engineering program “intelligent bio-manufacturing”,the experience we obtained may provide ideas for development of the first-class bioengineering major in China.

Abstract:Innovation and entrepreneurship training through higher education sector is an important way to foster innovative talents and enhance their social adaptation abilities.We reformed and optimized the experimental teaching of human anatomy and animal physiology with the aim to promote the integration of students’ theory learning with practice,to promote students’ ability to apply anatomical and physiological knowledge to medicine,pharmacy,and life practice.Last but not least,students’ innovative consciousness of applying scientific research to serve the society could also be enhanced.These practices would enhance the practical ability of the students through integrating the innovation education and professional education.

Abstract:Natural medicinal chemistry is one of the important courses for students in pharmacy majors.Its experimental teaching focuses on fostering comprehensive experimental skills and innovative abilities of undergraduates.Liaoning University has explored ways to promote the experimental teaching of natural drug chemistry based on the graduate employment and practical teaching experience in the past decade.These explorations include three aspects,such as synchronizing experimental teaching with theoretical teaching,fostering students’ awareness of experimental safety,and improving experimental teaching methods in natural drug chemistry experiments.The practices showed that the reform has achieved a good effect.A teaching system that can achieve the three expected aspects has been established,which improved the teaching effect and quality of natural medicinal chemistry experimental courses for undergraduates.Furthermore,these explorations may facilitate fostering pharmacy specialists who can meet the opportunities of developing Chinese medicine and natural drug research and meet the requirements of employment.