Objective: To investigate whether high glucose can aggravate oxygen deprivation/reoxygenation (OD/R) injury in neurons through regulating mitophagy and the specific molecular mechanism of regulating mitophagy. Methods: HT22 cells are used to establish a model of cellular oxygen deprivation/reoxygenation to simulate cerebral ischemia/reperfusion injury in vivo. And cells are treated with high glucose medium (HG 50 mmol/L). 3-TYP is given to inhibit the expression of SIRT3. CCK8 is used to examine cell viability; flow cytometry is used to detect cell apoptosis, and TMRE fluorescence detection kit is used to detect cell MMP; Western blot is used to detect the expression of mitochondrial division/fusion related protein (DRP1, OPA1, TOM20), autophagy related protein (LC3Ⅱ, Beclin-1), mitophagy related protein (PINK1, Parkin) and SIRT3. Results: Compared with OD groups, the cell density is further reduced, the cell contour is more blurred, the synapses are shortened, the floating cells increase in HG+OD groups, and cells in HG+OD groups have lower survival rate and higher apoptosis rate (P<0.05). Compared with OD groups, cells in HG+OD groups have lower MMP (P<0.05). Compared with OD groups, the expression of P-DRP1 in HG+OD groups is increased, but OPA1 and TOM20 are decreased (P<0.05); the expression of Beclin-1 and LC3Ⅱ as well as PINK1 and Parkin in HG+OD groups are also increased (P<0.05). Furthermore, SIRT3 protein and gene are further increased in HG+OD groups (P<0.05). 3-TYP treatment can further aggravate the OD/R injury of neurons in HG groups. And 3-TYP also increases the expression of DRP1, LC3Ⅱ and PINK1. Conclusions: High glucose can aggravate oxygen deprivation/reoxygenation injury in neurons by exacerbation of cell apoptosis, reduction of MMP and activation of mitophagy. In addition, SIRT3 can inhibit PINK1-Parkin pathway-mediated mitophagy and alleviate high glucose aggravated-neuronal OD/R injury.
In order to find more effective, simpler, less toxic, and more stable small peptides, Musca domestica cecropin (Mdc), a housefly antimicrobial peptide cloned in our laboratory, was used to design five derived peptides and analyze the specific structure and anticancer activities of them. Based on the analysis results of Mdc, five peptides derived from the conserved sequence of Mdc were redesigned:M1-6,M1-7,M1-8,M9-21 and M27-39. Bioinformatics tools were used to predict the physical and chemical properties and secondary structure. Proliferation effects of the derived peptides on ten different cancer cell lines and the toxic effects of them on normal cells were determined by MTT assay. The safety of the five polypeptides was verified by hemolysis test and normal cytotoxicity test. Five kinds of derived peptides were designed with Mdc as template, and they all had inhibitory effects on different cancer cells. M1-7, M1-8 and M27-39 showed good activity to several kinds of cancer cells and low hemolytic activity to human erythrocytes. Interestingly, M1-7 had significant inhibitory effect on PC-12 cells, M1-8 had effective biological activity on HepG2 cells, and M27-39 had obvious inhibitory effect on HCT116 cells. The small molecular peptides with corresponding anticancer activity were successfully screened, among which M1-7, M1-8 and M27-39 have obvious specific anticancer activities and may become promising anticancer agents.
Background: Tomato (Solanum lycopersicum) is one of the most valuable fruits and vegetables widely cultivated. Late blight disease of tomato reduces the quality and yield of tomatoes. MicroRNAs (miRNAs) area class of endogenous non-coding RNAs, which are widely involved in post-transcriptional regulation of genes. Recent evidence suggests that miR399 family participates in the regulation of plant disease resistance. Objective: Investigate the effect of sly-miR399 in tomato to late blight. Methods: The overexpressing and silencing vectors of sly-miR399 is constructed and transiently expressed in tomato leaves by the Agrobacterium-mediated method. Quantitative real-time PCR (qRT-PCR) is used to detect the related genes expression level. Trypan blue staining is used to analyze the lesion condition of tomato leaves with transient overexpressing (TO) and silencing (TS) sly-miR399. Results: Transient overexpressing sly-miR399 reduced the expression of its target gene UBC24 by 1.5 times and increased the expression of pathogenesis related proteins (PRs) SlPR1, SlPR2, SlPR3 and SlPR5 by 3.6 times, 2.2 times 2.3 times and 6.4 times, respectively. It increased the expression of JA related genes SlJA1, SlLOX1 and SlLOX2 by 1.3 times, 2.5 times and 1.5 times, respectively and reduced the expression of JA transcription repressor SlJAZ1 by 50%. After infection, the relative diseased area on TO leaves was reduced significantly. Transient silencing sly-miR399 increased the expression of its target gene by 1.8 times and reduced the expressions of PRs genes by 65%, 82%, 52% and 80%, and it decreased the expression of SlJA1, SlLOX1 and SlLOX2 by 84%, 50% and 65%,respectively and increased the expression of SlJAZ1 by 1.8 times. After infection, the relative diseased area on TS leaves increased significantly. Conclusion: sly-miR399 plays a positive regulatory role in tomato resistance to late blight.
Heterologous expression of Vitreoscilla hemoglobin gene vgb in a variety of research and industrial fermentation bacteria can solve the problem of dissolved oxygen rate in high-density fermentation. Saccharomyces cerevisiae is a classic eukaryotic model and has an important application value in the fermentation industry. However, the impact of vgb in Saccharomyces cerevisiae on cell growth is unclear. In this study, a heterologous expression plasmid YEplac195-ADH1pr-vgb containing Vistreoscilla hemoglobin gene vgb was constructed with Adh1 as promoter, and transformed into Saccharomyces cerevisiae BY4741. Growth sensitivity test showed that the heterologous expression of vgb inhibited the growth of the strain in both fermentation and non fermentation carbon sources. Then, we found the accumulation of reactive oxygen species (ROS), the change of membrane permeability and lipid peroxidation in S. cerevisiae cells overexpressing vgb by DCFH-DA and PI staining and lipid peroxidation products assay. The results showed that the overexpression of vgb changed the oxidation state in cells, promoted the accumulation of reactive oxygen (ROS), and oxidative stress caused the growth inhibition of the strain.
Drug discovery is a very important and costly process. Computer-assisted methods for predicting drug-protein affinity can greatly speed up the process of drug discovery. The key to the prediction of drug target affinity lies in the accurate and detailed characterization of drug and protein information. In this paper, a prediction model for drug target affinity based on deep learning and multi-layered information fusion is proposed, in an attempt to obtain better prediction performance by integrating multi-layered information of drugs and proteins. Firstly, the drug is expressed as molecular graph and ECFP, GCN module and fully connected(FC) layer are used for learning, respectively. Secondly, protein sequence and K-mer feature of protein are input into CNN module and FC layer, respectively to learn potential protein features. Finally, the features learned from the four channels are concatenated and the FC layer is used for prediction. In this study, the availability of the proposed method is verified on the two benchmark datasets of drug-targets affinity and compared with other existing models. The results show that the proposed model can obtain better prediction performance than the baseline model, which indicates that the proposed strategy for predicting drug target affinity based on multi-layered information fusion of drug and protein is effective.
Photodynamic therapy (PDT) has the advantages of minimally invasive, controllable, low toxicity, and repeatable treatment, and has become an indispensable treatment method in clinical medicine. However, due to the self-protection mechanism of tumor cells, the efficacy of PDT is greatly reduced. This study uses PDT therapy while implementing pharmacological autophagy suppression strategies to cut off protective autophagy under severe oxidative damage due to photodynamic therapy. The porphyrin metal organic framework PCN-224 was synthesized by the oil bath heating method, and the autophagy inhibitor hydroxychloroquine sulfate (HCQ) was loaded on the PCN-224, and the scanning electron microscope (SEM), particle size test (DLS), UV visible spectral testing and other methods showed that the material was successfully synthesized, which enhanced the water solubility of the porphyrin photosensitizer, and significantly enhanced the toxicity to 4T1 mouse breast cancer cells after light, and it further improved the tumor killing ability after being loaded with HCQ. HCQ can be released in tumor cells to deacidify lysosomes and inhibit autophagy, cut off the protective autophagy under severe oxidative damage due to photodynamic therapy, and achieve the effect of killing tumors.
Objective: Benzoic acid decarboxylase can catalyze the carboxylation reaction to fix CO2. In order to obtain a benzoate decarboxylase that can efficiently fix CO2, it is required to screen a large number of mutants based on the high-throughput molecular cloning and mutant screening systems. Thus, it is essential to develop an efficient screening-evaluation method for obtaining mutants with high-efficiency of carboxylation. Methods: 2,3-dihydroxybenzoic acid decarboxylase catalyzes catechol with CO2 to produce 2,3-dihydroxybenzoic acid. A spectrometry-image grayscale method was established to high-throughput screen and evaluate the activities of mutants. The concentration of 2,3-dihydroxybenzoic acid was quickly quantified by spectrophotometry at 308 nm. Further, the spectrophotometric results were corrected using the high performance liquid chromatography (HPLC) method. The 2,3-dihydroxybenzoic acid concentration by the spectroscopy method was linearly correlated with the accurate concentration determined by the HPLC method (R2 = 0.996). The 2,3-dihydroxybenzoic acid concentration of the actual sample was determined by the correlation of HPLC-spectrometry. The grayscale average of protein standards and mutants were obtained by Image J software. The protein expression level of mutants was calculated by the standard curve using the grayscale method. The enzyme activities of mutants were compared based on the 2,3-dihydroxybenzoic acid concentration. Results: The 2,3-dihydroxybenzoic acid concentration was quantified by the absorbance value using linear equation C1=0.500A1-0.010 (R 2=0.996, purified enzyme) and C2=1.458A2+0.431 9 (R 2 =0.991, crude enzyme). Two mutants were screened out from 13 mutants, which carboxylation activities were 3.5-fold and 1.7-fold of WT, respectively. Conclusion: The high-throughput screening of benzoate decarboxylase for fixing CO2 can be achieved by the spectroscopy-image grayscale method. This method is suitable for screening of substrate selectivity, i.e. phenols with other substituents and salicylic acid analogs, which is catalyzed by benzoate decarboxylase with similar functions.
microRNAs (miRNAs) are a class of endogenous, small non-coding RNAs (about 22 nt) that have critical roles in gene expression. Functional studies have identified miRNAs dysregulation as a cause in many human diseases, including cancer, viral infection, and autoimmune disorders. Restoring or suppressing miRNAs function and activity has become an attractive therapeutic method for the management of cancer and other diseases. Several miRNA-based therapeutics have reached clinical development, including a mimic of the tumor suppressor miR-34 for treating cancer and anti-miRs targeted miR-122 for treating HCV infection. In this review, we summarize the recent advances in our knowledge of miRNA therapeutics in cancer and other diseases and discuss ongoing challenges to ensure the safety and efficacy of miRNA therapeutics in vivo.
Human diploid cells (HDCs) have attracted much attention as an important culture cell matrix for vaccine preparation. Since human diploid cells are the same as the human genome without exogenous factor, susceptible to a variety of viruses, and without potential tumorigenicity, and the preparation of human diploid cell vaccine (HDCV) has good immunogenicity and safety, they are suitable for industrial production of vaccine. Currently, the inactivated, attenuated or subunit vaccines used in the population all rely on primary cells, continuous cells and human diploid cells, of which WI-38, MRC-5, 2BS and KMB-17 cell lines are the main human diploid cells used in vaccine preparation. However, human diploid cells are limited cells, and there are some defects in cell sources and culture techniques, which further affect their application. In this paper, the research progress of human diploid cells used in vaccine production and vaccine preparation technology were reviewed, and the existing problems and improvement strategies were analyzed.
Long non-coding RNA (lncRNA) is a regulatory non-coding RNA with a length of more than 200 nucleotides, which can be used at the transcription level, post-transcriptional level, and epigenetic level to affect gene expression. Lipogenesis is a complex and orderly process. A large number of studies have shown that lncRNA plays an important role in the process of adipogenesis. It can affect various biological processes such as lipid metabolism and adipogenic differentiation, thereby indirectly affecting meat quality. This is of great significance for improving the quality of livestock and poultry meat, avoiding the waste caused by excessive conversion of feed into fat in the breeding industry, and preventing and treating diseases related to fat metabolism. This article reviews the basic characteristics of lncRNA and the progress of its role in animal fat deposition, in order to provide a theoretical basis for cultivating high-quality livestock and poultry, preventing and treating diseases related to fat metabolism.
In prokaryotes and eukaryotes, the regulatory systems are very different and they usually change with the environment. Environmental factors play an important role in gene expression in prokaryotes because every cell in a population of prokaryotes is in direct contact with the environment, which leads to the adaptation by turning certain genes on or off. Therefore, environmental factors are often regulatory effectors, and transcription must be rigorously regulated to ensure that cells respond effectively and adequately to environmental changes. Thus, the regulation of gene expression is crucial to the overall fitness of the cell. The expression of prokaryotic genes is regulated by many factors, and transcription can be roughly divided into three main stages: initiation, extension and termination. For most bacteria, the key steps to regulate gene expression are promoter recognition in the process of transcription initiation and RNA polymerase to initiate transcription. The formation of the transcription initiation complex between RNA polymerase and promoter DNA is the most important regulatory process of bacterial gene expression. Promoters control the transcription of all genes, and the regulation of most promoters is complex. Promoter activity is determined by sequence elements that optimize promoter strength, and strong promoters usually have elements similar to the common sequence. Since the expression level of most transcription units is dependent on environmental inputs, many transcription factors are involved. Transcription factors involve RNA polymerases in different states, promote desired RNA polymerase function and regulate RNA polymerase activity. Each transcription unit is controlled by a regulatory region containing at least one promoter, usually located upstream of the first readable frame. These regulatory regions contain sequence elements that interact with RNA polymerases, as well as binding targets for different transcription factors involved in regulation. Transcription initiation requires the interaction of RNA polymerase with promoter DNA and the formation of an open complex(in order for transcription to occur, the closed promoter complex DNA is double-stranded and must be isomerized into an open promoter complex where the DNA melts into a transcription bubble and the single-stranded template DNA is delivered to the RNA polymerase active site).In bacterial cells, promoter recognition of RNA polymerase is a multi-step process involving multiple protein-DNA interactions and several structural and kinetic intermediates. Therefore, the transcription process of genes can be optimized to adapt to different environmental changes by adjusting the activity of RNA polymerase and changing the binding of RNA polymerase to promoter. Regulation is mediated by a combination of sigma factors, activators, repressors, and transcription factors, as well as by changes in DNA super-helix and local nucleoprotein structure. In this paper, various factors involved in the transcription regulation of bacterial cells have been summarized, and the molecular mechanisms of these factors regulating gene expression are expounded from the aspects of the effects of these factors on promoters, the effects of RNA polymerase and their interactions. This paper aims to summarize the role of various regulation mechanisms, and deepen the understanding of transcription initiation process, in the hope of providing ideas for the future regulation of transcription initiation process to achieve the efficient expression of target genes and the inhibition of the expression of adverse genes, and to provide a basis for the future modification of industrial strains.
Quorum sensing (QS) is an intercellular communication system found in many microorganisms. The autoinducers accumulate as the bacteria population grows. It can promote bacteria to acclimate current population density, regulate the formation of biofilm, and modulate the expression of specific genes. In recent years, researchers have been working on the principle of operation as well as the key elements of QS system and now we understand much more about them. These results would help us in designing a promising multi-system work using multiple methods of synthetic biology, and it can help researchers to take control of intercellular communication dynamically, which is particularly important. On the basis that quorum sensing is a cell-cell communication system, the research progress of the joint design of multiple quorum sensing systems in the automatic regulation of bio based chemicals production is reviewed. The application of quorum sensing system to realize two-way biological information exchange in the field of bioelectrochemical transformation is summarized. At the same time, this article summarizes the research progress of the combination of the dynamic regulation function of quorum sensing system and the diagnosis and treatment of various diseases in the medical field, and the expectation of development prospect.
The past decade has seen breakthrough progress in bispecific antibody development worldwide, with four products approved for marketing and multiple products entering clinical and preclinical studies. With a unique biological mechanism that distinguishes them from mAbs, dual antibodies hold promise as the next generation of biologic therapies against cancer, autoimmune, and infectious diseases, but the development of dual antibodies is more complex and has higher technical barriers. In this paper, we analyze the global progress in the overall development of dual antibody, the landscape of corporate R & D, and the progress of product R & D in the future, in order to provide references for the choice of direction of dual antibody R & D for related firms and the regional industrial decision.
Biotechnology is the core of the new round of scientific and technological revolution and industrial change since the 21st century, while the biotechnology base platform is an important scientific and technological innovation body to carry out biotechnology research, production and services, and is an important part of the development of biotechnology. This paper analyzes the overall scale and construction of biotechnology base platform in the world’s major developed countries and China, draws lessons from international experience, and puts forward policy suggestions to improve the construction of biotechnology base platform in China for reference.
