Abstract:Human microbiome is comprised of symbiotic microorganisms in the human body, whose dynamic balance is closely related to human health, and is recognized as important “organs” that can regulate immunity, metabolism and other aspects in human body, and is associated with functions of many organs including lung, intestine, vagina and brain, becoming a potential target for the treatment of cancer, coronary heart disease, neurological diseases and other difficult diseases. In recent years, with the rapid development of microbiome sequencing and analysis technology, it has become an international focus and forefront to discover the relationship between human microorganisms and many diseases, as well as target for new treatment methods. Thus, we organized this special issue and publish reviews on study methodology, human disease and microbiome as well as therapeutic strategies, and provide important information to advance microbiome research in China.

Abstract:The research on the relationship between gut microbiota and human health continues to be a hot topic in the field of life science. Culture independent 16S rRNA gene high-throughput sequencing is the current main research method. However, with the reduction of sequencing cost and the maturity of data analysis methods, shotgun metagenome sequencing is gradually becoming an important method for the study of gut microbiome due to its advantages of obtaining more information. With the support from the human microbiome project, 30 805 metagenome samples were sequenced in the United States. By searching NCBI PubMed and SRA databases, it was found that 72 studies collecting about 10 000 Chinese intestinal samples were used for metagenome sequencing. To date, only 56 studies were published, including 16 related to metabolic diseases, 16 related to infectious and immune diseases, and 12 related to cardiovascular and cerebrovascular diseases. The samples were mainly collected in Beijing, Guangzhou, Shanghai and other cosmopolitan cities, where great differences exist in sequencing platforms and methods. The outcome of most studies are based on correlation analysis, which has little practical value in guiding the diagnosis and treatment of clinical diseases. Standardizing sampling methods, sequencing platform and data analysis process, and carrying out multi center parallel research will contribute to data integration and comparative analysis. Moreover, insights into the functional verification and molecular mechanism by using the combination of transcriptomics, proteomics and culturomics will enable the gut microbiota research to better serve the clinical diagnosis and treatment.

Abstract:High cholesterol is one of the important factors inducing cardiovascular and cerebrovascular diseases. Drug therapy is the main method for reducing cholesterol, but has the disadvantages such as high cost and side effects. Studies have shown that intestinal bacteria play important roles in cholesterol metabolism. However, there are few reports on the screening and functional evaluation of cholesterol-lowering intestinal bacteria. In this study, 36 bile-tolerant bacteria were screened from healthy people stool through culturomics using bovine bile acid or artificial mixed bile acids as substrates. Taking Lactobacillus rhamnosus GG (LGG) as a positive control, three bile acid concentration groups (0 g/L, 0.3 g/L, 3 g/L) were set up to evaluate the cholesterol-lowering ability of bile-tolerant bacteria in vitro. Ten bacteria (including Proteus mirabilis, Providencia stuartii, Proteus vulgaris et al) were identified as the dominant cholesterol-lowering bacteria. Six of the above bacteria, Proteus mirabilis, Providencia stuartii, Proteus vulgaris, Proteus penneri, Wohlfahrtiimonas chitiniclastica, Providencia rettger, were evaluated for their ability to reduce triglycerides in vitro and tolerance to artificial gastric juice. Comparing with strain LGG, the six bacteria showed better triglyceride-lowering ability in vitro. With the decrease of pH value of artificial gastric juice and the increase of treatment time, the survival rate of six bacteria decreased. The above screening experiments and functional evaluation provide a basis for further development of potential cholesterol-lowering bacterial products.

Abstract:Coronary artery disease (CAD) is a chronic disease but causes the highest mortality and morbidity among the cardiovascular diseases worldwide. Correlations between CAD and gut microbiota have been observed. This suggests that the gut microbiota could become a vital diagnostic marker of CAD, and restoring the gut habitat may become a promising strategy for CAD therapy. The elevated level of trimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite, was found to be associated with the increased risk of cardiovascular disease and the all-cause mortality. Preclinical studies have shown that it has pro-arteriosclerosis properties. It is likely that regulating the production of TMAO by gut microbiota may become a promising strategy for anti-atherosclerosis therapy. This review summarizes the clinical and preclinical researches on the intervention of CAD by regulating the gut microbiota and the microbiota-derived metabolite TMAO, with the aim to provide new target for the therapy of CAD.

Abstract:The human intestinal flora is a highly diverse ecosystem composed of trillions of bacteria. The imbalance of the flora is related to a variety of diseases. The intestinal flora interacts with the nervous system bidirectionally in many ways through the gut-brain axis. It causes neuroimmune inflammatory response, dysfunction of gut mucosa and blood-brain barrier, direct stimulation of the vagus nerve, spinal nerve of the enteric nervous system, and the neuroendocrine hypothalamus-pituitary-adrenal axis, causing neurological disorders. The metabolites of the intestinal microbial community also play a role. This article summarizes the characteristics of the altered intestinal flora and intervention measures in autism spectrum disorder, multiple sclerosis, Parkinson’s disease, epilepsy, Guillain-Barré syndrome, Alzheimer’s disease, neuromyelitis optica, hepatic encephalopathy, amyotrophic lateral sclerosis, schizophrenia, depression, chronic fatigue syndrome, Huntington’s disease and stroke. The current research on intestinal flora is still in its infancy, and very few studies were carried out on causality and the underlying mechanisms, which prevents the development of precise flora-based clinical intervention measures. It is expected the research on intestinal flora would lead to novel approaches for treatment of some neurological disorders.

Abstract:Intestinal microbes have an adjuvant therapeutic effect on neurological disorders (such as Parkinson’s, depression, and Alzheimer’s disease). It affects brain function and host behavior through the neural pathways, the immune pathways and the microbial metabolites, the so-called gut-brain axis. This article summarizes the recent advances in the role of the microbe-gut-brain axis in neurological disorders, in order to provide new ideas for the treatment of neurodegenerative diseases.

Abstract:Lung microbiota and gut microbiota are closely related to lung cancer. Studies have shown that the dysbiosis, i.e., the significantly altered composition and structure of gut and lung microbiota, usually occurs in patients with lung cancer. With the introduction of “Gut-Lung Axis”, an increasing attention has been paid to the close relationship between the lung and gut microbiota in human body. A deeper insight into this relationship would facilitate understanding the mechanisms behind the carcinogenesis and development of lung cancer. This article summarizes the composition of lung and gut microbiota in patients with lung cancer and the possible interaction mechanisms, highlighting the importance of the immune system in the Gut-Lung Axis. The effects of lung and gut microbiota on the clinical treatment of lung cancer were summarized, based on which the authors propose that the lung and gut microbiota can be used as novel targets for early diagnosis and treatment of lung cancer.

Abstract:The application of high-throughput sequencing technologies has greatly enhanced our understanding to the human microbiome. The causal relations between human microbiome and diseases have become a critical issue to elucidate disease development and develop precision medicine. Recently, the study about vaginal microbiome (the microbial flora that inhabits the female vagina) has received wide interests. It has been shown that dysbiosis of vaginal microbiome was closely related to the development of genital tract diseases. This article summarizes the interaction between vaginal microbiome and disease and the treatment for the dysbiosis of vaginal microbiome. The culturomics of virginal microbiome, engineered probiotics and synthetic microbiome were also proposed.

Abstract:Parkinson’s disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. Patients with PD often suffer from gastrointestinal symptoms in the early stage of the disease. Several studies have confirmed that gut microbiota is involved in the progress of PD. As one of the most effective ways to reconstruct the gut microbiota, fecal microbiota transplantation (FMT) has shown potential therapeutic effects on PD. This review summarizes the basic and clinical studies of FMT in the treatment of PD.

Abstract:Bacterial vaginosis (BV) is a disease caused by vaginal microbiota dysbiosis. The conventional antibiotic treatment can aggravate microbial dysbiosis, alter the acid environment of the vagina and lead to drug resistance, thus shows low cure rate and high recurrence rate. This poses significant physiological and psychological burden to the BV patients. Vaginal microbiota transplantation (VMT) is a novel live biotherapeutic approach. It directly engrafts the whole vaginal microbiota from healthy women to the vaginal tract of patients to rapidly reconstruct the vaginal microbiota environment and restore the vaginal health. This article summarizes the development, present challenges, and future directions of using VMT, with the aim to explore new strategies for treatment of BV and promote the clinical use of VMT.

Abstract:Psoriasis is considered as an inflammatory disease driven by T cells, and its pathogenesis is closely related to the imbalance of intestinal bacteria flora. It has been reported that Bacteroides fragilis could play an anti-inflammatory role by regulating the expression of cytokines in T cells. To date, there is no report using B. fragilis to treat psoriasis. In this study, we explored the therapeutic effect of B. fragilis BF839 on psoriasis. We selected 27 psoriasis patients who were treated in the Second Affiliated Hospital of Guangzhou Medical University from April to October 2019. The patients were given B. fragilis BF839 orally for 12 weeks while maintaining the original treatment. The psoriasis area and severity index (PASI) score was evaluated before and after the treatment. The rate of drug withdrawal and reduction after 12 weeks of treatment were calculated. Our results showed that the rate of 12-week trial completion was 96.3% (26/27). We used PASIN to define the proportion of people whose PASI score decreased more than or equal to N% after treatment. At 12 weeks, PASI30, PASI50, and PASI75 were 65.4%, 42.3%, and 19.2%, respectively. The PASI score was 9.1±5.9 and 5.8±4.9 before and after 12 weeks of treatment respectively, and the difference was statistically significant (P<0.01). The effective rate of the visual analog scale (VAS) score was 42.3% at 12 weeks, and the VAS score was 2.9±2.2 and 2.3±2.1 before and after 12 weeks of treatment, respectively, which had no statistically significant difference (P>0.05). The adverse reaction rate of patients was 3.8% (1/26) within 12 weeks of treatment, including 1 case of constipation, and the rate of drug withdrawal and reduction was 60.0%. The above results suggest that B. fragilis BF839 may be functional on the treatment of psoriasis by reducing the PASI score and the drug usage rate with few side effect, which deserves further study.

Abstract:As one of the three major nutrients, dietary lipids provide energy and nutrition for human. The quantity and quality of dietary lipids affect the composition of gut microbiota, which consequently may affect the host metabolic health. Development of disease animal models is an important approach to study the relationship between gut microbiota and human metabolic health. In this review, we discussed the types of dietary lipids, and summarized how dietary lipids affect the composition of gut microbiota and regulate the metabolic health of animal models. The clarification of potential underlying mechanisms will shed lights on future research in other live systems including human.

Abstract:With the cooperation of bacteria and the human body, the nutrients in food are deeply digested, utilized, and shared. In addition, symbiosis is formed between microorganisms and hosts. Such a delicate combination makes the microorganisms form the inherent flora in the human body. They obtain the biological basis for survival, and provide the necessary regulation and support for the host in terms of immunity and nutrition, through their functional metabolism and population signals. At present, most of the researches focus on the isolation and evaluation of the functional components of plants, such as plant polysaccharides, polyphenols, flavonoids, and other active functional components. However, in traditional Chinese medicine, plants are often used with whole food components. To date, studies have found that the dynamics of flora affecting human health are not fixed, nor dependent on the change of a single strain. The ecological competition and metabolic regulation between microorganisms are usually coevolved with the host. The regulatory effect of natural plants for both medicine and food mainly depends on their whole food components. This provides evidence to support the role of whole food components played in promoting the efficacy of traditional Chinese medicine from the perspective of microenvironment. Therefore, the development and utilization of medicinal and edible natural plant activities should be fully understood and evaluated with flora regulation.

Abstract:The accumulation of protein sequence and structure data allows researchers to obtain large amount of descriptive information, simultaneously it poses an urgent need for researchers to extract information from existing data efficiently and apply it to downstream tasks. Protein design enables the development of novel proteins that are no longer restricted by experimental conditions, which is of great significance for drug target prediction, drug discovery, and material design. As an efficient method for data feature extraction, deep learning can be used to model protein data, and further add a priori information to design novel proteins. Therefore, protein design based on deep learning has become a promising approach despite of many challenges. This review summarizes the deep learning-based modeling and design methods of protein sequence and structure data, highlighting the strategies, principle, scope of application and case studies, with the aim to provide a valuable reference for relevant researchers.

Abstract:In the application of CRISPR genome editing, direct cellular delivery of non-replicable Cas9/sgRNA may reduce unwanted gene targeting and integrational mutagenesis, thus offering greater specificity and safety. Cas9/sgRNA delivery system holds great potential for treating genetic diseases. This review summarizes the advances of Cas9/sgRNA delivery systems and its therapeutic applications, providing new understandings and inspirations for vector design and future clinical applications.

Abstract:Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats -associated protein (CRISPR/Cas) has been developed as a precise, efficient, affordable and sensitive nucleic acid detection tool due to its efficient targeted binding ability and programmability. At present, biosensors based on CRISPR-Cas system have shown excellent performance in the detection of nucleic acid of pathogens, which has attracted widespread attention, and is expected to replace the conventional detection methods. This review summarizes the latest research progress of biosensors based on CRISPR/Cas system for detecting nucleic acid of pathogens.

Abstract:Microfluidic chip technology integrates the sample preparation, reaction, separation and detection on a chip. It consists a network of microchannels, which controls the whole system through fluid. With the advantages of portability, high throughput, and the ability to simulate the microenvironment in vivo, it has a broad application prospect in the research of disease diagnosis, pathogenesis and drug screening. Pulmonary inflammatory disease is a common disease usually caused by bacterial, viral and fungal infections. Early pneumonia is often difficult to diagnose due to lack of obvious respiratory symptoms or the symptoms are mostly atypical, but the disease progresses rapidly. Recently, microfluidic chip technology has been increasingly used to the study of pulmonary inflammatory diseases. In particular, it has been used to develop a “lung-on-a-chip” model, which can reproduce the key structure, function and mechanical properties of human alveolar capillary interface (i.e., the basic functional unit of a living lung), and well simulate the alveoli in vitro. Compared with the cell and animal models, this multifunctional micro experimental platform has great advantages. This article summarizes the advances of using microfluidic chips for the research and diagnosis of pulmonary inflammatory diseases, with the aim to provide new ideas for researchers in this area.

Abstract:Targeted protein degradation (TPD) technology facilitates specific and efficient degradation of disease-related proteins through hijacking the two major protein degradation systems in mammalian cells: ubiquitin-proteasome system and lysosome pathway. Compared with traditional small molecule-inhibitors, TPD-based drugs exhibit the characteristics of a broader target spectrum. Compared with techniques interfere with protein expression on the gene and mRNA level, TPD-based drugs are target-specific, efficaciously rapid, and not constrained by post-translational modification of proteins. In the past 20 years, various TPD-based technologies have been developed. Most excitingly, two TPD-based therapeutic drugs have been approved by FDA for phase Ⅰ clinical trials in 2019. Despite of the early stage characteristics and various obstructions of the TPD technology, it could serve as a powerful tool for the development of novel drugs. This review summarizes the advances of different degradation systems based on TPD technologies and their applications in disease therapy. Moreover, the advantages and challenges of various technologies were discussed systematically, with the aim to provide theoretical guidance for further application of TPD technologies in scientific research and drug development.

Abstract:Long noncoding RNAs (lncRNAs) are a class of RNA molecules that are greater than 200 nt in length and do not have protein-coding capabilities or encode micropeptides only. LncRNAs are involved in the regulation of cell proliferation, differentiation, apoptosis and other biological processes, and are closely associated with the occurrence, recurrence and metastasis of a variety of malignant hematologic diseases. This article summarizes the function, regulatory mechanism and potential clinical application of lncRNAs in leukemia. In general, lncRNAs regulate the occurrence and development of leukemia and the multi-drug resistance in chemotherapy through epigenetic modification, ribosomal RNA transcription, competitive binding with miRNA, modulating glucose metabolic pathway, and activating tumor-related signaling pathway. Studies on lncRNAs provide new references for understanding the pathogenesis of leukemia, uncovering new prognostic markers and potential therapeutic targets, and addressing the problems of drug resistance and post-treatment recurrence in patients in clinical treatment of leukemia.

Abstract:The thymus is a pivotal immune organ of the human body, and it is the place where T cells differentiate and mature. The damage of thymus would easily induce autoimmune diseases and even malignant tumors. For years, researchers have been exploring the process of T cell development and revealing the mechanism of thymic injury and regeneration generally through the monolayer culture system of T cells in vitro. However, the classic monolayer culture system could neither reproduce the unique three-dimensional epithelial reticular structure of the thymus, nor provide the cytokines and growth factors required for the directed differentiation of hematopoietic stem cells into T cells. Thymic organoid technology utilizes cells with stem cell potential to simulate the anatomical structure of the thymus and the signaling pathway mediated by thymic epithelial cells in vitro through three-dimensional culture, which is particularly close to the microenvironment of the thymus in vivo. Thymic organoids show great potential in the study of T cell differentiation and development, thymus-related diseases, reconstruction of immune function, and cell therapy. This paper summarizes the methods for culturing thymic organoids, followed by comparing the advantages and disadvantages of the scaffolds used for culturing. The applications of thymic organoids in the disease model, tumor-targeting therapy, regenerative medicine, and organ transplantation were also discussed, with possible future research directions prospected.

Abstract:Novel model systems have provided powerful tools for the research of human biology. Despite of being widely used, the conventional research models could not precisely describe the human physiological phenomenon. Organoids are three-dimensional multicellular aggregates derived from stem cells or organ progenitors that could differentiate and self-organize to recapitulate some specific functionalities and architectures of their in vivo counterpart organs. Organoids can be used to simulate organogenesis because of their human origin. In addition, the genomic stability of organoids could be well maintained during long-term amplification in vitro. Moreover, organoids can be cryopreserved as a live biobank for high-throughput screening. Combinatorial use of organoids with other emerging technologies (e.g. gene editing, organ-on-a-chip and single-cell RNA sequencing) could overcome the bottlenecks of conventional models and provide valuable information for disease modelling, pharmaceutical research, precision medicine and regenerative medicine at the organ level. This review summarizes the classifications, characteristics, current applications, combined use with other technologies and future prospects of organoids.

Abstract:Sterols, a class of cyclopentane poly-hydrophenanthrene derivatives, are the predominant membrane constituent of eukaryotes. These substances have a variety of biological activities and have been widely used in food and pharmaceutical industries. The presence of endogenous ergosterol biosynthetic pathway in Saccharomyces cerevisiae cells make it an ideal chassis for the de novo synthesis of sterol and its derivatives. Most recently, the rational modification of organelles provides a novel strategy for the directed transportation and storage of target products and the ultimate enhanced product synthesis. This review summarizes the phenotypic responses of S. cerevisiae cells upon different physiological stimulations and the underlying molecular mechanisms. Reinforcement of sterol production through directed storage, transportation, and excretion of sterols offers a novel strategy for breaking the limitation of de novo biosynthesis of sterols in yeast.

Abstract:Kunitz-type serine protease inhibitors are a class of ubiquitous protease inhibitors, which play important roles in various life activities. The structures of such inhibitors are generally stable, and are usually characterized by the presence of one or several Kunitz domains in tandem, which are able to bind to serine proteases in a manner similar to substrate binding, thereby inhibiting enzyme activity. In terms of function, Kunitz-type serine protease inhibitors are involved in processes such as blood coagulation and fibrinolysis, tumor immunity, inflammation regulation, and resistance to bacterial and fungal infections. This article summarizes the advances of Kunitz-type serine protease inhibitors and provides new ideas for the development of novel Kunitz-type serine protease inhibitors.

Abstract:Induced pluripotent stem cells (iPSCs) are a type of cells similar to embryonic stem cells but produced by reprogramed somatic cells. Through in vitro differentiation of iPSCs, we can interrogate the evolution history as well as the various characteristics of macrophages. iPSCs derived macrophages are not only a good model for drug screening, but also an important approach for immunotherapy. This review summarizes the advances, challenges, and future directions in the field of iPSCs-derived macrophages.

Abstract:Metal-organic frameworks (MOFs) are formed by self-assembly of metal ions or clusters with organic ligands, and are widely used in the fields of catalysis, sensing, energy and biomedicine. Recently, biological composites based on MOFs have attracted increasing attention. MOFs can be used as a platform for encapsulating bioactive substances due to the advantages such as large pore capacity, large specific surface area and diverse structure composition. These features can protect bioactive substances from adverse conditions, e.g. high temperature, high pressure, and organic solvents, thus improving the anti-adversity of bioactive substances. This review summarizes the advances of using MOFs as protective coatings to improve the anti-adversity of different bioactive substances, and introduces the synthesis strategy of MOFs-based biological composites, with the aim to promote the practical application of MOFs-based biological composites.

Abstract:Decellularized extracellular matrix (dECM), which contains many proteins and growth factors, can provide three-dimensional scaffolds for cells and regulate cell regeneration. 3D bioprinting can print the combination of dECM and autologous cells layer by layer to construct the tissue structure of carrier cells. In this paper, the preparation methods of tissue and organ dECM bioink from different sources, including decellularization, crosslinking, and the application of dECM bioink in bioprinting are reviewed, with future applications prospected.

Abstract:N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Abstract:The biocompatibility of nanomaterials has attracted much attention. Graphene oxide (GO) is a nanomaterial widely used in biomedicine, but its toxicity can not be ignored. In this study, the effect of GO on the blood system (the hemolysis rate, the fragility of erythrocyte, and acetylcholinesterase activity) was systematically investigated. The results showed that the hemolysis rate of erythrocytes was lower than 8% when the GO concentration was below 100 μg/mL (P<0.01). GO at low concentration levels (<5 μg/mL) had no significant effect on the fragility of erythrocytes, but GO at high concentration (10 μg/mL) increased the fragility of erythrocytes (P=0.01). Moreover, GO increased the activity of acetylcholinesterase on erythrocytes. The concentration of 20 μg/mL graphene oxide with the size >5 μm (LGO) increased the activity of acetylcholinesterase by 42.67% (P<0.05). Then molecular dynamics simulation was used to study how GO interacted with acetylcholinesterase and increased its activity. The results showed that GO was attached to the cell membrane, thus may provide an electronegative environment that helps the hydrolysate to detach from the active sites more quickly so as to enhance the activity of acetylcholinesterase.

Abstract:Photorhabdus is a Gram-negative bacterium from the family Enterobacteriaceae that lives in a symbiotic association with nematode or insects. In addition to the role of being insect pathogens, one species called Photorhabdus asymbiotica (Pa) causes human infection around the world. Nevertheless, how does this transkingdom infection occur remains elusive. Here we focus on one pathogenic determinant called Photorhabdus virulence cassette (PVC) that is founded in the Pa genome and many other pathogens. The RNA-seq and qPCR data showed that the NF-kB and MAPK pathways were drastically activated in the PVC-treated mammalian macrophages. Western blotting assays using samples treated with various inhibitors of the affected pathways confirmed the results we have observed for MAPK pathway previously. p65 translocation assays validated the NF-kB activation in the macrophages after PVC treatment. Moreover, the bacterial phagocytosis by macrophage was also promoted by PVC at the early stage, and this phagocytosis was inhibited by cytoskeleton inhibitors. Thus, the results indicated that PVC is involved in the bacterial invasion by activating NF-kB and MAPK signaling pathway, providing a new perspective for analyzing the pathogenicity of Pa in human infections.

Abstract:Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91–120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312–80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.

Abstract:The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.

Abstract:Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Abstract:Human induced pluripotent stem cells (hiPSCs) have the potential to differentiate into multiple cell types. Motor neurons (MNs) differentiated from hiPSCs are important models of many motor neuron diseases. To simplify the identification of MNs, lentivirus vectors were used to transfer MNs-specific promoter HB9 and red fluorescent protein (RFP) gene into hiPSCs-derived human neural stem cells (hNSCs). Stable positive cells hNSCs-HB9-RFP-Puro were obtained after antibiotic selection. Subsequently, the positive cell line was infected with lentiviruses LV-Ngn2-Sox11-GFP and LV-Isl1-Lhx3-Hygro, which overexpressed the MNs differentiation transcription factor, and differentiated to MNs directly. Differentiated mature MNs showed neuron-like structure, expressed RFP and neuron-related markers β-tubulin and choline acetyltransferase (ChAT) under the control of the MNs-specific promoter HB9. The fluorescence reporter system provides a visual method for directed differentiation and identification of MNs, and may promote the applications of MNs in disease models and drug screening.

Abstract:The abundance of proteins in human urine is low and easily to be masked by high-abundance proteins during mass spectrometry analysis. Development of efficient and highly selective enrichment methods is therefore a prerequisite for achieving deep coverage of urine protein markers. Notably, different experimental methods would affect the urine protein enrichment efficacy and the coverage of urine proteome. In this study, ultrafiltration, nitrocellulose membrane enrichment and saturated ammonium sulfate precipitation were used to process 10 mL urine samples from five healthy volunteers and five bladder cancer patients. The urine proteins were enriched and separate by SDS-PAGE to compare the purification efficiency of different methods. Moreover, the peptide identification effects of different purification methods were analyzed by mass spectrometry to determine the best method for enriching urine protein histones. Saturated ammonium sulfate precipitation method outperformed the ultrafiltration and the nitrocellulose membrane enrichment methods in terms of the protein enrichment efficacy and quality. The interference of highly abundant albumin was reduced, whereas the amount of low-abundance protein was increased, and the sensitivity of mass spectrometry identification was increased. The saturated ammonium sulfate precipitation method may be applied for large-scale urine processing for screening clinical diagnostic markers through proteomics.

Abstract:In case/control gene expression data, differential expression (DE) represents changes in gene expression levels across various biological conditions, whereas differential co-expression (DC) represents an alteration of correlation coefficients between gene pairs. Both DC and DE genes have been studied extensively in human diseases. However, effective approaches for integrating DC–DE analyses are lacking. Here, we report a novel analytical framework named DC&DEmodule for integrating DC and DE analyses and combining information from multiple case/control expression datasets to identify disease-related gene co-expression modules. This includes activated modules (gaining co-expression and up-regulated in disease) and dysfunctional modules (losing co-expression and down-regulated in disease). By applying this framework to microarray data associated with liver, gastric and colon cancer, we identified two, five and two activated modules and five, five and one dysfunctional module(s), respectively. Compared with the other methods, pathway enrichment analysis demonstrated the superior sensitivity of our method in detecting both known cancer-related pathways and those not previously reported. Moreover, we identified 17, 69, and 11 module hub genes that were activated in three cancers, which included 53 known and three novel cancer prognostic markers. Random forest classifiers trained by the hub genes showed an average of 93% accuracy in differentiating tumor and adjacent normal samples in the TCGA and GEO database. Comparison of the three cancers provided new insights into common and tissue-specific cancer mechanisms. A series of evaluations demonstrated the framework is capable of integrating the rapidly accumulated expression data and facilitating the discovery of dysregulated processes.

Abstract:The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG? M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.