Abstract:

Abstract:Since the palivizumab for respiratory syncytial virus was approved in 1998, therapeutic antibodies against infectious diseases have been widely used in clinical treatment. Since the outbreak of COVID-19, plenty of neutralizing antibodies were developed and transferred into clinical trials, holding enormous promise for the treatment of COVID-19 under the context of emergency use authorization. This review summarizes the clinical progress of these drugs, in order to provide a reference for the research and development of neutralizing antibody drugs for the future.

Abstract:Natural killer (NK) cells are important innate effector cells serving as the first line of defense against certain infections and tumors. NK cells also play a role in regulating immune responses. NK cells develop from hematopoietic stem cells in the bone marrow. The development, maturation and function of NK cells depend on the regulation of microenvironment, intracellular transcription factors and post-transcriptional regulations. MicroRNA (miRNA) is a group of small non-coding RNAs that regulate the expression of target genes at the post-transcriptional level. It also plays important roles in various life processes of NK cells, including regulating the activation and effector function of NK cells. We showed that the miRNA profiles in NK cells changed with aging, which in turn affected the development and function of NK cells. Based on the regulatory effect of miRNA on the development and function of NK cells, miRNA may serve as a potential target to protect or restore the function of NK cells in patients, thereby treating related diseases. Here, we briefly summarized recent advances on the roles of miRNA in the developmental and functional regulation of NK cells.

Abstract:CRISPR-Cas systems are well known gene editing tools, among which CRISPR-Cas9 system targeting DNA is the most well developed. Compared with CRISPR-Cas9 system, CRISPR-C2c2/Cas13a system derived from TYPE VI of CRISPR family that can target RNA has attracted increasingly intense investigations in recent years. The CRISPR-Cas13a system is featured by specific recognition and binding of single stranded RNA sequences, thus playing a role in non-specific cleavage of RNA. This feature could be potentially applied to detect free nucleic acid in tumors or peripheral blood as a diagnostic approach. Since Cas13a specifically targets RNA, it can directly edit mRNA transcripts of genomic DNA to achieve the downregulation of target proteins without involving DNA editing. Therefore, Cas13a system could be used in tumor treatment. This review summarized the advances of using CRISPR-Cas13a for RNA targeting in tumor diagnosis and treatment, and prospected future applications.

Abstract:CRISPR/Cas9 is a cutting-edge gene-editing technology that has emerged as a promising tool for gene therapy. Nevertheless, the safe and efficient delivery of CRISPR/Cas9 is still an urgent issue in clinical application. Nanoparticles, such as lipid-based nanoparticles, polymer nanoparticles, gold nanoparticles, and biofilm nanoparticles, are expected to bring new opportunities for CRISPR/Cas9-based gene therapy because of their biocompatibility, safety and designability. This review briefly introduced the characteristics of nanoparticles and the development of CRISPR/Cas9 in gene therapy. Moreover, the application of nanoparticles in the delivery of different forms of CRISPR/Cas9 were elaborated. Finally, the challenges and safety of nanoparticle-based gene therapy were discussed.

Abstract:The intestinal microbiota is a complex micro-ecological system symbiotic with human body, which has attracted increasing attention in recent years. The intestinal microbiota plays important roles not only in maintaining normal physiological functions of the human body but also in the occurrence, development, diagnosis and treatment of tumors. This review summarized the relationship between the intestinal microbiota and tumor, highlighting the mechanisms by which intestinal microbiota modulates tumor occurrence, development and immunotherapy, particularly the immune checkpoint therapy. This review also summarized the currently available methods for enhancing the efficacy of tumor therapy through regulation of intestinal microbiota. Challenges in the field as well as future perspectives were also discussed.

Abstract:Parkinson's disease (PD) is the most common neurodegenerative disease. Along with the population aging of China, the increase of PD patients in China will result in serious economic and medical burdens. The typical pathological characteristics of PD are the death of dopaminergic neurons in the substantia nigra compacta and the pathological inclusion bodies formed by abnormally aggregated amyloid alpha-synuclein (α-Syn) in dopaminergic neurons, which is also named as Lewy body. Studies have found that the Lewy body exists not only in the central nervous system, but also in the peripheral nervous system. The abundant enteric nervous system in the gut is called the "second brain". The discovery of the gut-brain axis also proves that α-Syn can be transmitted bilaterally between the brain and the gut. The gut microbiota was shown to be involved in the formation and transmission of pathological α-Syn. Therefore, this article summarized the bilateral transmission relationship of α-Syn in the brain and the gut and illustrated the influence of gut microbiota on the abnormal aggregation of α-Syn. Combined with the current progresses on PD patients and animal models especially the non-human primate experiments, this article aimed to provide a reference for the screening and diagnosis of PD.

Abstract:Inflammatory bowel disease (IBD) is a chronic, recurrent inflammatory disease of the gastrointestinal tract that includes Crohn's disease and ulcerative colitis. IBD patients are numerous and a complete cure is difficult to achieve. Due to impaired digestive function, food is not easily absorbed and malnutrition often occurs in patients. Nutritional therapy is often used to overcome nutritional deficiency and change the inflammatory state in the clinic. Amino acids are adjuvant therapeutic candidates that may help maintain intestinal integrity in patients with IBD. It maypromote the treatment of IBD through reducing the level of inflammation, oxidative stress, and intestinal cell death. Recent studies in animals have demonstrated the potential of using amino acids for treating IBD. The supply and metabolism of amino acids may be a promising adjuvant therapy. This review summarized the immunomodulatory effects of specific amino acids such as glutamine, arginine, and glycine, with the aim to provide new ideas for the treatment of IBD.

Abstract:Listeria monocytogenes (LM) is a food-borne pathogen that can cause listeriosis. Pregnant women are main target population of listeriosis due to pregnancy-associated immune deficiency and unique intracellular infection ability of LM to non-phagocytic cells. LM can cross the placental barrier and cause significant harm to the fetus, including premature birth, miscarriage and even stillbirth. The role of placenta-specific virulence factors is particularly important for researchers to understand how it crosses the placental barrier and infects the fetus during LM infection. This review started by describing the listeriosis in pregnant women, followed by summarizing the advances in understanding the LM vertical transplacental infection and the mechanism of LM colonization in the placenta. Finally, recent advances in identifying placenta-specific virulence factors involved in LM infections were presented, with the aim to facilitate the control of LM transplacental infection and the improvement of food safety.

Abstract:In recent years, antibiotic resistance has become increasingly serious, and the number of cancer patients keeps increasing. There is an urgent need to develop new drugs with antibacterial and antitumor effects. Halophilic microorganisms are a special group of microorganisms living in extreme environment. They have the characteristics of metabolic diversity, low nutritional requirements and adaptability to harsh conditions, thus can serve as promising candidates for new drug discovery. To date, researchers have isolated a variety of metabolites and enzymes with antibacterial and/or antitumor activities from halophilic microorganisms. This review summarized the functions and potential biomedical applications of halophilic microorganisms and their related products, such as antibacterial, anti-inflammatory, antitumor, antioxidant, biomedical materials and drug carriers. In particular, novel antibacterial and antitumor substances recently discovered in halophilic microorganisms, as well as the biomedical applications of ectoine, a unique metabolite found in halophilic microorganisms, were introduced. Finally, future development and utilization of halophilic microorganisms in biomedical and industrial fields were prospected.

Abstract:Decellularized extracellular matrix (dECM) is designed to remove cells that cause immune rejection and retain the original tissue structure and composition. Since its structure and composition are similar to the original tissues and organs, it has attracted extensive attention in tissue engineering and biomedicine applications, and has become a promising tissue engineering material. dECM can be easily obtained from tissues and organs by appropriate decellularization methods. Here, we summarized the commonly used decellularization methods and reviewed the sterilization, cross-linking and storage methods of decellularized scaffold. In addition, we summarized the latest applications and developments of dECMs obtained from different tissues/organs in tissue engineering and biomedicine. Finally, we discussed the present challenges of dECM biomaterials and prospected future perspectives. With the development of tissue engineering and regenerative medicine technology, dECM biomaterials are expected to become a gold scaffold in the field of biomedicine and will receive wide applications.

Abstract:Monolignin alcohols (type H, type G and type S) are the basic units of lignin and lignans in plants, and their composition differences directly determine the chemical diversity and biological activity of lignin and lignans. Caffeic acid O-methyltransferase (COMT) catalyzes the methylation of oxygen atoms on the hydroxyl groups of phenylpropanoids, playing a critical role in the composition of different types of monolignin alcohols, and thus acting as a key enzyme involved in the biosynthesis pathway of lignin and lignans. A previous review published in 2010 mainly introduced the gene characteristics of COMT and its regulatory role in lignin biosynthesis. This article summarized the latest research progress of COMT in the past decade, including the gene characteristics, expression characteristics, structural characteristics of COMT and its regulatory effects, and prospected future research and application of COMT.

Abstract:The prediction of tumor drug sensitivity plays an important role in clinically guiding patients' medication. In this paper, a multi-omics data-based cancer drug sensitivity prediction model was constructed by Stacking ensemble learning method. The data including gene expression, mutation, copy number variation and drug sensitivity value (IC₅₀) of 198 drugs were downloaded from the GDSC database. Multiple feature selection methods were applied for dimensionality reduction. Six primary learners and one secondary learner were integrated into modeling by Stacking method. The model was validated with 5-fold cross-validation. In the prediction results, 36.4% of drug models' AUCs were greater than 0.9, 49.0% of drug models' AUCs were between 0.8-0.9, and the lowest drug model's AUC was 0.682. The multi-omics model for drug sensitivity prediction based on Stacking method is better than the known single-omics or multi-omics model in terms of accuracy and stability. The model based on multi-omics data is better than the single-omics data in predicting drug sensitivity. Function annotation and enrichment analysis of feature genes revealed the potential resistance mechanism of tumors to sorafenib, providing the model interpretability from a biological perspective, and demonstrated the model's potential applicability in clinical medication guidance.

Abstract:Plasma exosome microRNAs (miRNAs) are closely related with the occurrence, diagnosis, and treatment of cancers. However, the underlying molecular mechanisms remain unclear. We herein investigated the solution for tackling the unspecific amplification of plasma exosome microRNAs from cancer patients during the construction of its cDNA library. For the restriction enzyme digesting method, the primers were degraded by exonuclease T (EXOT) and phi29 DNA polymerase. For the magnetic bead separation method, the templates and primers were separated through the DNA binding beads. The separation effects of magnetic beads were detected by agarose gel electrophoresis and modified polyacrylamide gel electrophoresis. The levels of plasma exosome miRNAs from cancer patients and various primers were assayed by RT-qPCR. The results indicated that the unspecific amplification stemmed from USR5SR. EXOT and phi29 DNA polymerase could degrade USR5SR, but the templates were also degraded simultaneously. Regarding the magnetic bead separation method, the best effect was achieved via precipitation of primer fragments by 9% PEG and precipitation of templates by 15% PEG. In conclusion, the magnetic bead separation method efficiently circumvented the unspecific amplification during the construction of cDNA library, and therefore led to the successful construction of cDNA library from plasma exosome miRNA of cancer patients and 293T cells.

Abstract:A TaqMan probe real-time quantitative PCR (RT-qPCR) approach was developed for rapid identification of SARS-CoV-2 main variants. Specific primers and probes were designed based on the sequence of the SARS-CoV-2 wild and main variants alpha (N501Y, HV69-70del), beta (E484K, K417N), gamma (K417T, V1176F), delta (L452R, T478K) and omicron (H655Y, N679K, P681H) genes. The specificity, sensitivity and performance of the RT-qPCR assay were tested. The assay can identify SARS-CoV-2 wild type and main variants efficiently, and has no crossover with a panel of respiratory pathogens (n=21), showing high specificity toward SARS-CoV-2 RNA. The assay's sensitivity was determined to be 2×10²copies/mL. In summary, we developed a simple, rapid and cost-effective RT-qPCR assay that enables identification of SARS-CoV-2 main variants. It can be used to monitor the variation of SARS-CoV-2 strain for accurate identification, prevention and control of outbreaks.

Abstract:For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 μmol/L Mpro and 5 μmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1,4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.

Abstract:Nanobodies derived from camelid single-chain antibodies have the advantages of being small, simple, highly soluble and stable. Nanobodies can be administered by inhalation and therefore is potentially valuable for the prevention and control of respiratory viruses. Trichoderma reesei is a food-grade protein expression host with a cellulase production capacity of up to 80 g/L, which can be employed for low-cost production of therapeutic proteins. In this study, a codon-optimized SARS-CoV-2 neutralizing nanobody Nb20 was expressed in T. reesei under a strong constitutive promoter Pcdna1. Nb20 protein was fused downstream of the N-terminal fragment of cellobiohydrolase Ⅰ, and the fusion protein can be intracellularly cleaved by the KEX2 protease to release Nb20. In a shake-flask fermentation using glucose medium, 47.4 mg/L Nb20 was detected in the culture after 48 h of cultivation. The expressed Nb20 showed the ability to interact with the receptor-binding domain of SARS-CoV-2 spike protein, suggesting that it can be used for the neutralization of SARS-CoV-2. The results indicate that T. reesei has the potential for recombinant production of nanobodies.

Abstract:In order to prepare insulin-like growth factor 1 (IGF-1) more economically and efficiently, the structure prediction and molecular docking of three IGF-1 fusion proteins were performed by computer simulation. The most suitable expression form of IGF-1 fusion protein was screened out. A prokaryotic expression vector of IGF-1 fusion protein was constructed and transformed into Escherichia coli Origami B(DE3) strain to obtain the recombinant strain. After induction with IPTG, the target protein was purified from the soluble fractions of the bacteria cell lysate by affinity chromatography, desalination, thrombin digestion and affinity chromatography of the enzyme digested products. An activity evaluation system was established by 3T3 cell proliferation method and the activity of the obtained IGF-1 was measured. The results showed that the sequence of the IGF-1 fusion protein prokaryotic expression vector was correct and the fusion protein was soluble upon 0.05 mmol/L IPTG induction at 25℃ for 16 h. After preliminary purification, thrombin digestion and re-purification, IGF-1 target protein with purity over 90% was obtained. Using the established activity evaluation system, the specific activity of IGF-1 was 2.47×10⁵ U/mg, which was close to the standard product available at the market. The preparation technology of IGF-1 developed in this study may facilitate the development and industrial production of IGF-1 drugs.

Abstract:Interleukin-6 (IL-6) is a pleiotropic cytokine which participates in the pathogenesis of a variety of clinical disorders, including many kinds of cancers. Anti-IL-6 antibody was proved to be useful for the immunotherapy of various inflammatory diseases. Plants are low-cost platforms for producing specific proteins of therapeutic interest. Two dependent transplastomic tobacco lines expressing murine anti-IL-6 single chain variable fragment (scFv) were generated after bombardment and regeneration, homoplasmy was then verified by Southern blotting analysis. The anti-IL-6 scFv gene was successfully expressed at both transcriptional and translational levels in transplastomic tobacco plants. Functional anti-IL-6 scFv accumulated to 1% of total soluble proteins, namely 41 mg/kg fresh weight. There was no obvious phenotypic difference between the wild-type and the transplastomic tobacco plants, including the growth rate, the height of mature plants and the number of siliques. The high-level expression of anti-IL-6 scFv indicates the potential for cost-effective production of scFV using transplastomic plants.

Abstract:As a new transdermal drug delivery technology, bubble microneedle could achieve painless and precise drug delivery, which has attracted great attention from researchers. In order to improve the utilization rate of the drug carried by microneedle, we proposed a method for preparing a tip-loaded bubble-soluble microneedle. During the molding process of the microneedle, air bubbles were formed in the needle body, and the drug was concentrated on the needle tip. The preparation process of the bubble microneedle was optimized. The effects of foaming agent concentration, drying temperature, and solution viscosity on the forming of bubble microneedles were explored. Furthermore, the transdermal effect of the product was analyzed. The experimental results showed that the bubble microneedle forming process was stable, with the forming rate above 90% and the forming cycle shortened to about 4 h. The drug was mainly concentrated on the tip of the microneedle, with a length of 180 μm, and the length of the bubble was 250 μm. Moreover, the microneedle array can create microchannels on the mouse skin, and the needle bodies can be rapidly dissolved within 5 min. The bubble microneedle could rapidly release about 48% of the drug within 1 min and about 91% of the drug within 5 min. The bubble microstructure of the microneedle array hindered the diffusion of the drug to the substrate, which improves the utilization rate of the drug. This study provides a technical basis for the practical application of microneedle for transdermal drug delivery.

Abstract:Proteolytic enzymes and lipopeptides contain broad-spectrum antimicrobial activities, which have great potential for research and development. A microbial strain X49 obtained from protease screening plate showed antifungal activities against six fungi. Biochemical analysis, 16S rRNA sequencing, API identification system, and electron microscope analysis were carried out to identify the bacterium. Azocasein method was used to analyze the protease activity. Lipopeptides were extracted for antifungal analysis. The result indicated that strain X49 grew in the range of 10-50℃ and pH 4.0-9.0. Moreover, it survived in 10% NaCl, showing good halotolerance. Strain X49 was identified as Bacillus velezensis. Genomic analysis of B. velezensis X49 revealed eleven genes encoding serine protease. The ID 1_894 belonging to S8 subtilisin family was 99% similar to the serine protease with known antifungal ability. On the other hand, thirty genes encoding non-ribosomal peptide synthetase involved in the lipopeptide biosynthesis, including surfactin, iturin, fengycin, bacitracin, and gramicidin, were identified. Part of the extracellular proteolytic activity remained under high temperature. After co-fermentation of B. velezensis X49 with Zingiber officinale Rosc., the antifungal activity of the lipopeptide extract from the co-fermentation was greatly improved. In conclusion, B. velezensis X49 showed clear inhibitory effect on both plant and human pathogens. The active substances co-fermented with Chinese herbs and microbes can be utilized for further drug development.

Abstract:Icariin (ICA) is a small molecule drug capable of promoting cartilage repair and ameliorating inflammation. Loading ICA into a biomaterial scaffold for cartilage tissue engineering will thus potentially enhance the biological functionality of the engineered scaffold. In this study, short fibers processed from electrospun poly(l-lactide-co-caprolactone) (PLCL) fibers which were prior coated with polydopamine (PDA), were mixed with citric acid doped chitosan solution (CC) for preparing short fibers reinforced chitosan hydrogel (PDA@PLCL/CC) by a freeze-thawing combined freeze-drying method. Thereafter, ICA was loaded into the PDA@PLCL/CC scaffold through physical adsorption to generate a newly engineered biomimetic cartilage scaffold (ICA-PDA@PLCL/CC). Finally, ICA-mediated chondrogenic and ameliorated inflammatory effects of the ICA-PDA@PLCL/CC scaffold were examined in vitro using rabbit chondrocytes. The results showed that the ICA-free PDA@PLCL/CC scaffold possessed appropriate pore size and porosity (>80%), high water absorbance capacity and improved mechanical performance, and also promoted chondrocyte proliferation and adhesion. The ICA-laden ICA-PDA@PLCL/CC scaffold was evidenced to maintain cytomorphology, upregulate the expression of chondrogenic gene (sox-9), glycosaminoglycan gene (gag), and type Ⅱ collagen gene (col Ⅱ) as well as the synthesis of the cartilage matrix. In the presence of a simulated inflammation, the ICA-PDA@PLCL/CC scaffold was found to reduce chondrocyte fibrosis, effectively downregulate the expression of proinflammatory factors interleukin-6 (il-6), interleukin-1 (il-1), and inducible nitric oxide synthase (inos) in chondrocytes. It can also reduce matrix metalloproteinase-3 (mmp-3) expression and promote the synthesis of the extracellular matrix glycosaminoglycan (GAG) and type II collagen (Col II). The newly developed ICA-PDA@PLCL/CC scaffold may find applications in the regeneration and repair of cartilage defects.

Abstract:Fatty liver disease is a common chronic liver disease which is mainly induced by abnormal lipid metabolism. To find out the effect of GP73 on lipid metabolism in the liver, we constructed a high GP73 expression liver model through a tail vein injection of AAV-GP73 into eight-week-old C57BL/6J mice. Liver lipid metabolomics analysis showed that lipids in the liver of mice, especially the triglycerides, were significantly increased. In addition, kyoto encyclopedia of genes and genomes enrichment analysis showed that GP73 altered lipid metabolites profile that may further disturb many signaling pathways related to cellular metabolism. The diseases linked to type II diabetes, non-alcoholic fatty liver disease (NAFLD) and choline metabolism in cancer cells were more likely to be dysregulated. Thus, GP73 may induce fatty liver by regulating lipid metabolism and promoting lipid accumulation in the liver.

Abstract:The purpose of this study was to develop a fluorescence chromatography method for the detection of cartilage oligomeric matrix protein (COMP) in the auxiliary diagnosis of rheumatoid arthritis (RA). The principle of double antibody sandwich method was used to prepare immunochromatographic test strips, and the performance evaluation and methodological comparison were carried out. Through the detection of clinical samples, a receiver operating characteristic (ROC) curve was obtained, and the sensitivity, specificity, positive and negative predictive values of the test strip were calculated. The linear range was 0.39-50.00 ng/mL. The coefficients of variation inter and intra batches were less than 15%. The test strip was stable at 37℃ for 20 days, and the variation range of fluorescence signal intensity was within 15%. There was no cross reaction with rheumatoid factor (RF) and anti-cyclic citrulline peptide (anti-CCP) antibody. Forty-eight clinical serum samples were detected in parallel with ELISA kit, and the correlation was good. The test strip prepared in this study was used to detect the sample, the cut-off value of COMP between RA patients and healthy people was 22.55 ng/mL (sensitivity 0.821, specificity 0.842, positive predictive value 0.741, negative predictive value 0.895). At the same time, the same sample was tested with ELISA kit, the sensitivity and specificity of the two methods reached more than 80%. A quantitative COMP fluorescence chromatography test strip was developed, which has the advantages of celerity, simplicity and sensitivity, and may provide rapid auxiliary diagnosis for RA patients.

Abstract:We compared ultracentrifugation, sucrose gradient centrifugation, improved ultracentrifugation, and polyethylene glycol (PEG) precipitation in the extraction of plasma exosomes from human umbilical cord blood, aiming at screening out a stable and efficient method. The morphology, structure, and size of exosomes were observed based on transmission electron microscopy and dynamic light scattering. Total protein content of exosomes was determined by bicinchoninic acid (BCA) assay, and the expression of exosome markers CD63 and HSP70 and exosome negative marker GM130 (Golgi marker) by Western blotting. Results showed that sucrose gradient centrifugation was more stable and yielded exosomes of uniform particle size compared with ultracentrifugation which had been considered as the "gold standard" for exosome extraction. However, sucrose gradient centrifugation had the limitations of complex operation and time-intensiveness. The improved ultracentrifugation featured ease of implementation and the extracted exosomes were of high purity. PEG precipitation extracted the most exosomes in a shorter timeframe, but the purity of the exosomes was low. In conclusion, all the four methods can separate exosomes from human umbilical cord blood plasma, but they are different in operation time, product purity, and product content. Therefore, the method for extracting plasma exosomes from human umbilical cord blood should be selected based on the experimental purpose and specific requirements.

Abstract:SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1,2,3,4,6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.

Abstract:DLP4 (defensin-like peptide 4) is a novel insect defensin, which has strong antibacterial activity against Gram-positive bacteria and is not susceptible to develop drug resistance. In this study, an elastin-like polypeptide (ELP) and an intein fusion system were used for production and purification of DLP4, which combined the characteristics of the phase transition of ELP and the C-cleavage of the intein. A recombinant expression plasmid pET-ELP-I-DLP4 was constructed and transformed into Escherichia coli. Subsequently, DLP4 was purified by simple centrifugation, alternation of pH and temperature. However, the C-cleavage of the intein occurred unexpectedly during the process of expression and purification. To solve this problem, the intein was split into N-intein (I0_N) and C-intein (I0_C), and fused with ELP or DLP4 to construct two recombinant expression plasmids pET-ELP-I0_N and pET-ELP-I0_C-DLP4, respectively. These two plasmids were transformed into E. coli separately. The mixture of the two cultures of E. coli strains restored the C-cleavage activity of the intein. This operation yielded DLP4 of about 1.49 mg/L. Antibacterial test confirmed that the purified DLP4 exhibited expected activity. Thus, this approach can be used as an effective way for DLP4 expression and purification in the prokaryotic system.

Abstract:Salmonella enteritidis (SE) has been recognized as an important zoonotic pathogen, and the prevention and control of salmonellosis has long been a conundrum. However, glycoconjugate vaccines seem to be a promising solution. Glycoproteins are conventionally synthesized by chemical cross-linking which features complex procedure and cost-intensiveness. Therefore, a stable biosynthesis method at lower cost is in urgent need. For the biosynthesis of SE O-antigen-based glycoproteins, we used CRISPR/Cas9 to develop the waaL-deleted SE strain ∆waaL. The synthesis of lipopolysaccharide (LPS) was detected based on silver staining. Circular polymerase extension cloning (CPEC) was employed to construct the plasmids expressing glycosyltransferase PglL, recombinant Pseudomonas aeruginosa exotoxin A (rEPA), and cholera toxin B subunit (CTB). Meanwhile, PilE^S45-K73glycosylation motif was added to the N-terminal and C-terminal of rEPA and CTB, respectively. The recombinant plasmids were transformed into SE ∆waaL. After induction, the synthesis of glycoprotein was verified by Western blotting and the synthesized glycoprotein was purified by Ni-NTA column. The results showed that waaL deletion blocked the LPS synthesis of SE, and that rEPA and CTB proteins were expressed in SE ∆waaL. In addition, obvious glycosylation occurred to rEPA and CTB when PglL was expressed, and the glycosylated part was SE O antigen polysaccharide. In summary, after waaL deletion in SE, PglL can transfer its own O antigen polysaccharides (OPS) to the carrier proteins rEPA and CTB, resulting in OPS-rEPA and OPS-CTB glycoproteins. The result lays a basis for the biosynthesis of SE glycoprotein.