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Abstract:Oncolytic viruses (OVs) have been investigated for a century and have become one of the most advanced types of tumor immunotherapy. It is primarily a natural or genetically modified viruses, including DNA and RNA viruses. In recent years, with the rapid development of genetic engineering technology, the gene-modified oncolytic viruses have made great progress in the field of tumor treatment, several types of viruses (including HSV, adenovirus, poxvirus, measles virus, reovirus etc.) are currently in preclinical studies, clinical trials or have been approved in clinic, showing good safety and clinical efficacy. It is generally believed that oncolytic viruses target and kill tumor cells by selectively replicating themselves in tumor cells, and finally lysing and killing tumor cells. At the same time, they can stimulate the immune responses of the body, thus enhancing the antitumor immunity, the tumor cells can be targeted and killed with no obvious side effects. The combination of oncolytic virus and immune checkpoint by gene recombination and the breakthrough of tumor immunotherapy have made the application of oncolytic virus more extensive, however, there are still some bottleneck problems such as virus targeting, security and administration route. This review provides a comprehensive and detailed overview of the development of oncolytic viruses. We list some viruses have been used as candidates for lysis of cancer cells and the clinical trials in the field of oncolytic virotherapy. And we discuss about the immunological mechanism of oncolytic virus targeting to kill tumors, and the challenges and prospects in the future.

Abstract:The force, speed and depth of microneedle array penetration process are usually used to evaluate the degree and efficiency of its penetration into the skin.The skin is the basis of its performance evaluation. The physical properties of the skin are mainly determined by the combination of keratin filaments, collagen fibers, elastic fibers and subcutaneous tissues, and reflect its impact on microneedle penetration from dimensions such as thickness, elasticity, hardness and toughness. Mechanical, permeable, tissue and barrier skin models are used to explain and simulate this aspect of real skin functions. Similarly, various skin mechanics models including constitutive models established after skin mechanics analysis also analyze the mechanical characteristics of the skin from the physical dimension. Real skin is complicated, with large differences, difficult access and storage, and some ethical issues. Skin model can replace real skin assisted to a certain extent for the design, development and performance evaluation of microneedle delivery system. The material involved in the model may be different from the physical properties of the real skin, it cannot fully simulate the chemical composition and distribution of the real skin, the tissue structure, and the interaction between the skin tissue and other tissues. However, the skin model can be easily adjusted by changing those characteristic materials comparable to the skin and form a simulation system. As for microneedle evaluation, it is more necessary to consider the mechanical properties of the skin and the skin model. The skin model needs to have a surface barrier that is difficult to puncture like the stratum corneum, and the hardness, elasticity and toughness under the barrier are similar to the real skin to simulate the reaction force after the microneedle penetration. In these aspects, the simulation of a single performance is relatively easy to achieve, but it is not easy to achieve a comprehensive performance similar to the real skin. The application of advanced methods with higher resolution, accurate quantitative and real-time dynamic evaluation of the penetration force and penetration rate of microneedle puncture can help us systematically and accurately analyze microneedle penetration behavior, and the development and application of 3D skin tissue engineering products that are closer to the composition, structure and physical properties of real skin can provide an effective solution path to help establish a more economical and applicable skin model. The establishment of a standardized evaluation model will undoubtedly help advance related research and promote the better industrialization and commercial application of microneedle array technology. In addition, the inherent hardness difference caused by different materials constructed by microneedles, such as metal, monocrystalline silicon and polymer materials, may have a corresponding difference in the penetration rate and penetration depth of real skin or skin model, but this can be solved by providing differentiated judgment methods for different rigid microneedles and formulating different indicators when setting skin model. Therefore, there is no need to design a special skin model to evaluate the puncture behavior of different microneedle.

Abstract:Microtubule affinity-regulating kinase 4 (MARK4) is a Ser/Thr protein kinase, best known for its role in phosphorylating microtubule associated proteins, causing their detachment from microtubules thereby increasing microtubule dynamics and facilitating cell shape alterations, cell division, cell cycle control, regulating cell cycle, etc. The MARK4 gene encodes two alternatively spliced isoforms, L and S that differ in their C-terminal region. These isoforms are differentially regulated in human tissues including central nervous system. The isoform MARK4S is highly expressed in the normal brain and is presumably involved in neuronal differentiation and the isoform MARK4L is upregulated in hepatocarcinoma cells and gliomas. MARK4 exhibits are multi domain structure comprised of an N-terminal header, a catalytic kinase domain, a linker, UBA domain, spacer, and a kinase-associated domain at the C-terminal end. Over-expression of MARK4 is associated with the onset of neurodegenerative diseases such as Alzheimer’s disease, metabolism disorders and cancer. Therefore, MARK4 is being considered as a most suitable drug target for cancer, Alzheimer’s disease and other neurodegenerative diseases and its structural features are employed in the development of new therapeutic molecules. In this paper, the structure and biological functions of MARK4 and the related diseases mediated by MARK4 were reviewed, and the research progress of MARK4 inhibitors was summarized.

Abstract:Phosphorene, a single layer of black phosphorus, has a direct band gap, strong structural and functional anisotropy, high charge carrier mobility and has made great progress in the fields of biomedicine, drug delivery, biosensors, and disease diagnosis and treatment. Compared with other nanomaterials, phosphorene has better biocompatibility and biodegradability, and has a good application prospect in the field of biomedicine. Although there have been a large number of reports on the biological effects of phosphorene, the details of the interaction process between phosphorene and biological macromolecules, such as nucleic acids, lipids, and proteins, still lack systematic research. The dynamic process of the interaction between phosphorene and biomolecules is currently unobservable experimentally. Molecular simulation has unique advantages in capturing precise dynamic structures that cannot be obtained in experiments, and is widely used in the fields of nanomaterials and biology. This article summarizes the latest research progress in the interaction of phosphorene nanomaterials with biological macromolecules such as proteins, lipid membranes and DNA, based on computer simulation and experimental methods in recent years. We reviewed the current research on the biological toxicity of phosphorene and analyzed the problems that need to be solved in the future. It was found that phosphorene nanomaterials with higher concentrations and larger sizes are more cytotoxic, and different types of cells have different toxic reactions to phosphorene nanomaterials. Phosphorene nanosheets have both destructive and inhibitory effects on proteins, depending on with which amino acid residues phosphorene is combined. Research on the interaction between phosphorene nanosheets and DNA macromolecules is still lacking. Current research results show that phosphorene nanosheets do not significantly damage DNA macromolecules. Generally speaking, the biocompatibility of phosphorene is better compared with other nanomaterials, but it also has certain biological toxicity. Phosphorene could produce biological toxicity in the following ways: (1) extracting phospholipid molecules from the cell membrane which destroys the integrity of the lipid membrane and weaken the cell activity; (2) producing reactive oxygen species which makes cells lose their vitality; (3) destroying the structure of biomolecules which makes them lose their biological functions; (4) snatching the ligand and occupying the active site which blocks the signal pathway of the signal protein and results in the loss of its function. Everything has two sides. Phosphorene’s toxicity is harmful to organisms, but it can be used for sterilization and it is also beneficial to kill cancer cells. Phosphorene nanomaterials are unstable, but they are beneficial to their degradation in the organism. Phosphorene nanomaterials with weak biotoxicity and easy degradation will become ideal candidates for nanomedicine. How to effectively reduce the negative effects of phosphorene nanomaterials and maximize their biomedical functions will be an important issue to be faced in the future. This review will promote the basic research on the biological effects of phosphorene, and will also push forward the application of phosphorene nanomaterials in the field of biomedicine.

Abstract:The interferon (IFN) signaling pathway is an important cellular defense mechanism against microorganism invasion. By sensing pathogen-associated molecular patterns (PAMPs) and transmitting signaling through the downstream cascades, IFN is robustly induced in expression and secreted to activate numerous genes’ expression in self and neighboring cells. Products of these induced genes then participate in restricting infection and modulating the immune system to further respond. This process needs to be properly regulated, for its aberrant activation under non-infectious conditions results in inflammation and onset of autoimmune diseases in the host. The correct recognition of “self” and “non-self” is the first step to control. Given the fact that nucleic acids of microorganisms are important immunogenic sources to the IFN signaling, the endogenous DNA/RNA metabolisms then must be faithfully conducted and strictly regulated. A series of enzymes, using them as substrates, work at different pathways to maintain this homeostasis. Intensive investigations on mechanisms of autoimmune diseases highlighted the protective role of these enzymes. Take Aicardi-Goutières syndrome (AGS) as an example, a monogenic type I interferonopathy, 9 mutated genes have been identified separately in patients so far, including DNA metabolism involved genes TREX1, RNASEH2A, RNASEH2B, RNASEH2C, and SAMHD1, RNA-related genes ADAR1 and IFIH1, and two recently identified genes, LSM11 and RNU7-1 whose correct activity is required for histone expression. Aberrant DNA metabolism or damaged histone expression activates IFN signaling through the cGAS-STING axis, while RNA errors sensitize the MDA5-MAVS axis. Thus, despite these 9 mutations all leading to the aberrant activation of IFN signaling, they can rely on different mechanisms, implicating that even having the same symptoms clinically the optimized treatment can be different. We thus argue the importance and necessity of diagnosing at the genetic level to the treatment of complicated symptoms and hope this review benefits the understanding of the pathogenesis of autoimmune diseases.

Abstract:The prevention and treatment of kidney disease has been a focus of medical research.Eukaryotic translation initiation factor 2α (eIF2α) is a key factor in the metabolic stress response of mammalian cells, inducing inhibition of overall protein translation, restoring protein synthesis and controlling cell survival under different cellular metabolic stresses, and its kinase plays an important role in maintaining normal physiological functions of the body as well as in the development of tumor, immune and metabolism-related diseases. It is suggested that eIF2α kinase may be involved in the pathological process of several renal diseases. Therefore, this paper summarizes the research progress on eIF2α kinase family and its possible role in renal diseases, in order to provide a new theoretical basis and reference for the prevention and treatment of renal diseases.

Abstract:Chromatin accessibility refers to the level of physical compaction of chromatin, which is determined by the chromatin binding factors that hinder DNA contact, nucleosome occupancy and topological structure. The chromatin accessibility pattern will be changed dynamically with external stimuli and developmental cues. Analyzing the TF (transcription factor) binding sites in the regulatory regions within the accessible chromatin can provide insight into the lineage factors and gene regulatory networks of specific cell types. Combined with high-throughput sequencing technology, several biochemical methods have been developed to describe the accessibility of chromatin, including bulk and single-cell level analysis. Depending on the techniques, the using enzymatic cleavage (DNase/MNase), transposition (Tn5) or physical methods (FAIRE) to isolate the accessible chromatin and subsequently using the high-throughput sequencing provide a genome-wide panorama of chromatin organization. This review introduced the common techniques (DNase-seq, MNase-seq, FAIRE-seq, and ATAC-seq) for determining chromatin accessibility and nucleosome positioning. The advantages and disadvantages of these 4 chromatin accessibility analysis techniques were summarized and compared. Their principles and main experimental procedures were introduced in detail; the development and application of related technologies were briefly discussed. The ATAC-seq based single-cell chromatin accessibility analysis and the view of potential useful were specially introduced. Although the chromatin accessibility profile is very valuable for studying gene regulation, it only provides a partial view of this complex process. We envision that technological improvements including single-molecule, multi-omics and spatial methods will bring further insight into the secrets of genome regulation.

Abstract:Nrf3, which belongs to the CNC-bZIP (cap"n"collarbasic leucine zipper) family, is a key transmembrane-bound transcription factor. Compared with the most studied family members Nrf1 and Nrf2, there are still too many unknowns about the biological roles of Nrf3. In recent years, combined with the application of multi omics technology, the biological functions of Nrf3 has been gradually revealed. Nrf3 plays an important role in tissues development and function specialization, intracellular redox homeostasis, protein homeostasis, lipid metabolism homeostasis, energy metabolism, innate immune regulation and so on. With the application of knockout mouse model and clinical research, Nrf3 is found to be involved in physiological and pathological processes such as glucose metabolism, cholesterol metabolism, protein modification, endoplasmic reticulum stress, chronic inflammation and neurodegeneration, especially mediating the reprogramming of glucose and lipid metabolism in cancer development. In order to better understand the function of Nrf3, a briefly review concentrating on its molecular structure and biological functions is demonstrated in this work.

Abstract:Undesirable pathological activities in sensory, motor or autonomic nerve are related to multiple neurological disorders, such as pain and spasticity. Kilohertz frequency alternating current (KHFAC) stimulation is an effective method for blocking the conduction of undesirable pathological activities in peripheral nerves, which has potentials for alleviating neurological disease symptoms in clinics. The nerve conduction block caused by KHFAC is influenced by kilohertz signal waveform and parameters, blocking electrode design and position as well as nerve fiber type and diameter, which is rapid, controllable, reversible, locally acting, and has less side effect. However, the target nerve is first activated to generate a burst of high-frequency firing by KHFAC before entering a state of complete conduction block. Such onset firing is likely to result in muscle contraction or painful sensation, which limits the clinical applications of KHFAC nerve block. Meanwhile, the conduction ability of target nerves usually requires a period of time to recover after the cessation of KHFAC, which is the carry-over effect produced by this technology. Since KHFAC stimulation has important potential applications in nerve conduction block, it is necessary to systematically review the developments of preclinical studies of this technology. In this paper, we first introduce the methods used in electrophysiological experiments and computational modeling simulations of KHFAC stimulation. Then, we present an exhaustive review on the main findings of KHFAC nerve block. For onset response, we describe its temporal characteristics and also review the existing methods proposed to reduce or eliminate such undesirable firing. For carry-over effect, we summarize the duration of poststimulation block in different target nerves and also review the underlying ionic mechanisms. For the effects of stimulus waveform and parameters, we focus on the minimal block frequency, block threshold, and KHFAC waveform. For the effects of blocking electrode and position, we focus on the electrode type, surface area, contact separation distance as well as electrode-fiber distance. For potential clinical applications, we summarize earlier explorations including the KHFAC block of vagus, sensory, motor, pudendal, and autonomic nerves in human trails. For the mechanisms of KHFAC nerve block, we introduce two biophysical explanations, which are K⁺ channel activation and Na⁺ channel inactivation caused by kilohertz signals. Finally, we raise several key issues on KHFAC stimulation of peripheral nerves that need to be addressed in the future. We highly suggest further determination of the effective stimulus parameters, nerve responses, and underlying mechanisms involved in different species for successful translation of KHFAC block, especially in human beings.

Abstract:It takes a long time for a drug to go from research and development to clinical application, and the investment cost during the period can reach one billion yuan. The combination of medicine and artificial and the development of big data of biochemistry contribute to sharply increasing drug activity data, and traditional experimental methods for drug activity prediction and discovery are hard to meet the needs of drug research and development. Algorithms are used to assist drug development and solve various problems during the process to significantly accelerate drug development. Traditional machine learning methods, especially random forests, support vector machines, and artificial neural networks, can improve drug activity prediction accuracy. Due to the multi-layer neural networks of deep learning, the model can process high-dimensional input variables and there is no need to limit the amount of input data characteristics manually. Deep learning can build a more complex function, and its application in drug research and development can further improve the efficiency of each step of drug research. Widely used deep learning models in drug activity are mainly DNN (deep neural networks), RNN(recurrent neural networks), and AE (auto encoder). GAN (generative adversarial networks) is often used in combination with other models for data enhancement due to its ability to generate data. Researches and applications of deep learning in drug molecule activity prediction in recent years showed that the accuracy and efficiency of deep learning models were higher than traditional experimental methods and traditional machine learning methods. Therefore, deep learning is expected to become the most critical auxiliary calculation model in drug research and development in the next decade.

Abstract:Objective Chemotaxis and electrotaxis are the primary mechanisms underlying directed cell migration, and they play important roles in the physiology and pathology of organisms. However, there are differences between the two. This paper presents a comparative study of the roles of Dictyostelium discoideum genes gbpC and gbpD in cell electrotaxis and chemotaxis in order to gain further insight into the differences between these two mechanisms of migration.Methods The gbpC/gefT- mutant strain and gbpD/gefU- mutant strain were placed in a 12 V/cm direct current (DC) electric field to investigate the direction and velocity of cell movement and the changes in cell electrotaxis; Lifeact-GFP (F-actin) was electroporated into cells and the distribution of F-actin during cell movement was observed under a fluorescence microscope; Western blot was used to quantify the phosphorylation of myosin regulatory light chain (RLC) in cells stimulated by DC electric fields.Results The gefT- mutant cells lost polarization but retained electrotaxis at a level similar to the wild-type cells; the gefU- mutant cells showed hyperpolarization but significantly reduced electrotaxis; in a DC field, F-actin was predominantly distributed in the pseudopods in both mutant and wild-type cells; there were differences in myosin RLC phosphorylation between the cell lines in electric fields. Phosphorylation was time-dependent in wild-type cells, whereas phosphorylation first decreased rapidly and then increased in gefT- mutant cells. Time-dependent dephosphorylation occurred in gefU- mutant cells.Conclusion Our findings indicate that gbpC and gbpD play differntal roles between the chemotaxis and electrotaxis of Dictyostelium discoideum and provide further evidence that electrical and chemical signals drive the directed migration of cells through different mechanisms.

Abstract:Objective The present study was to clarify the improving effect of artemisinin on diabetes-induced cognitive deficit and the underlying mechanisms in mice.Methods Type 2 diabetes mellitus (T2DM) mouse models were established by a single dose of STZ injection (100 mg/kg, i.p.). Those animals were then treated with vehicle or artemisinin (40 mg/kg, i.p.) once daily for 4 weeks. Cognitive performances of the mice were evaluated by novel object recognition, Y maze test and Morris water maze test. After behavioral tests, the expressions of PI3K, Akt, SYN and PSD-95 proteins in the hippocampus were measured by Western blot. Changes in the synaptic ultrastructure of the hippocampal CA1 region were observed by transmission electron microscope.Results Our results indicated that artemisinin significantly ameliorated cognitive deficit in T2DM mice. Furthermore, PI3K and phosphorylated Akt protein levels in the hippocampus of T2DM mice treated with artemisinin were elevated, accompanied with increases in the number of hippocampal neurons, as well as the protein contents of SYN and PSD-95. Meanwhile, synaptic plasticity was also rescued, indicated by an increase in synapse number and synaptic curvature, the thickness of postsynaptic density, and a decrease in the width of synaptic cleft in the hippocampal CA1 region.Conclusion Taken together, these results demonstrate that artemisinin can protect T2DM mice against cognitive decline, at least partially through activating PI3K/Akt pathway to improve synaptic plasticity in the hippocampus. These findings demonstrate that artemisinin may serve as a novel therapeutic agent for diabetic cognitive impairment.

Abstract:Objective More than 200 million people are visually impaired or blind worldwide. Artificial vision system has been widely studied for a long time. There are two main technical paths for the research of artificial vision system, the first is implantable artificial vision device, and the second is non-implantable and wearable device. This study demonstrates a non-implantable system prototype based on a wearable touch-vision device designed for the head that can help blind people and visually impaired people complete complex tasks in life such as walking.Methods The image information front of the subjects is collected, and transmitted wirelessly to the operator. After analyzed and processed, it is wirelessly transmitted to wearable devices such as headgears in the form of tactile coding to trigger the multi-point head tactile sensation, thus the subject’s information or precise action instructions is obtained. The system also assists the subjects with voice information, allowing them to learn more about road conditions and environmental information.Results 5 healthy volunteers (2 males and 3 females) and 1 young blind person (male) were tested with the prototype. The results show that, the prototype makes full use of the head’s natural distinguishing ability for directions including front, back, left and right, and quick response ability to the touch, and the prototype can send clear instructions such as going straight, adjusting to the left or right, and turning. The test showed that the response time of subjects to tactile commands was within 0.5 s, and the mean value of the standard deviation of the deviation of walking for about 7 m is reduced to (16± 10) cm compared with the case without wearing the device.Conclusion Experimental results show that the system can provide environmental image information in a concealed and accurate manner, and help visually impaired people complete daily activities such as walking, avoiding obstacles, going up steps, entering cafes and other public places, fetching objects on the table, which meets expectations of assisting blind people improve their quality of life. In the future, the prototype system will be further improved, especially miniaturization and smart chip. The tactile devices are made into neck rings, belts, bracelets, foot rings, etc., which will be widely used in a variety of work scenarios such as travel assistance for the blind, night field trips, and deep sea diving.

Abstract:Objective To investigate the effects of lactoferrin (LF) deficiency during lactation and LF supplementation from different sources on intestinal development of newborn mice.Methods Female LF knockout mice were used to produce LF deficiency milk for newborn mice intake during lactation, and 100 mg/kg bovine Lactoferrin (bLF), recombinant human Lactoferrin (rhLF) and bovine serum albumin (BSA) were artificially fed every day from the 3rd day to the 21st day. Samples were taken at the age of 21 days to determine the small intestine development index of mice in each group.Results In this experimental cycle, rhLF supplementation significantly increased the villus length/crypt depth of ileum (P<0.05), up-regulated the expression of Occludin and ZO-1 in ileum (P<0.05) and increased the ratio of maltase activity/lactase activity in duodenum (P<0.05), jejunum (P<0.05) and ileum (P<0.05) of mice. In addition, the supplementation of bLF only significantly increased the ratio of maltase activity/lactase activity in ileum (P<0.05), indicating that the supplementation of LF during lactation can promote the intestinal development of newborn mice.Conclusion For newborn mouse without LF intake during lactation, the supplement of LF during lactation can enhance the intestinal digestion and absorption of nutrients, promote the development and maturity of the intestinal tract, and enhance the intestinal barrier function. Moreover, rhLF plays a more effective role than bLF.

Abstract:Objective Quick TarSeq integrated system developed by our groups for on-field fast analysis is a fully automated and integrated system, which can be applied to the InDel ancestry inference, and complete the “sample-in result-out” rapid automatic InDel typing in about 2 h. In this paper, the performance of the InDel ancestry inference microfluidic chip amplification system was studied, hoping to provide a reference for practical application.Methods The system was validated by sensitivity, inhibitor tests, success rate, concordance tests, sizing precision and accuracy, peak height balance, case sample tests, and the accuracy of infering ancestral origins.Results The microfluidic chip amplification system was concordant with traditional methods, with 95.65% success rates and 98.85% concordance rates for 138 testing samples. The complete InDel profiles can be obtained from more than 5 ng of template DNA input. The best collection of buccal swab was the donor who performed 8 swipes inside of cheek. The best collection of dried blood spot samples was 6 pieces (Φ=2 mm). The average heterozygous peak height ratio across all locus was 0.86. The standard deviation of allelic ladder for 10 runs was within 0.3 bp. The size differences between allele and corresponding allele in allelic ladder was within 0.5 bp.Conclusion Microfluidic chip amplification system enable providing reliable InDel profiles from buccal swabs, dried blood spot, dried saliva spot samples and cigarette butt with accurately ancestral inference. It is possible to yield “sample-in result-out” results of InDel typing in 2 h. This system can meet the requirements of on-site testing and can be used for forensic casework.

Abstract:Objective As a new type of transcranial magnetic stimulation, the intermittent theta burst stimulation (iTBS) has been widely used to explore brain cognitive function and neuroregulation, but its electrophysiological regulation mechanism is still unclear. Therefore, exploring the underlying electrophysiological mechanism and the effect of iTBS on the brain’s cognitive functions is of great significance for the treatment of brain diseases and clinical application of magnetic stimulation.Methods In this study, the model rats were treated with iTBS, and the local field potential (LFPs) signals in the ventral hippocampus (vHPC) and medial prefrontal lobe (mPFC) were collected and recorded during the working memory (WM) tasks in rats. The effect of iTBS on neural network coordination and information interaction across brain regions was investigated by using Granger causality network.Results The results suggested that iTBS enhanced the WM ability and the behavioral performance of the rats, reducing the time to complete the WM task by (2.67±1.63) d (P<0.05). At the same time, iTBS enhanced the self-causal network connections in vHPC and mPFC during WM, and increased the network connection strength, connection density and global efficiency (P<0.05). In addition, iTBS enhanced the cross-brain network connection between vHPC and mPFC, and increased the node degrees and causal flow (P<0.05) from vHPC to mPFC.Conclusion iTBS has a significant positive effect on the behavior of WM and neural network in related brain areas of rats. iTBS can promote the cognitive ability of rats and improve the efficiency of information interaction and transmission of neural network. The neural regulation mechanism of iTBS may be to enhance the WM ability by enhancing the network connection and information interaction between vHPC and mPFC.

Abstract:Objective In order to prepare ubiquitin samples with different chain types, different chain lengths and phosphorylation modifications.Methods This study mainly uses the biological enzymatic method to describe the preparation routes of the above samples. There are two main methods, the first method is to add ubiquitin monomer one by one and then extend the ubiquitin chain; the second is to prepare mixed polyubiquitin chains through an enzymatic reaction, and then purify and separate these mixed chains.Results The experimental results show that both methods can achieve the purpose of preparing polyubiquitin chains. Further, phosphorylated ubiquitin samples were prepared by phosphorylation of ubiquitin. K11/K48 branched chain ubiquitin was prepared by K11 and K48 ubiquitinase.Conclusion In short, based on the above preparation route of the ubiquitin chain, it is possible to further perform post-translational modifications such as phosphorylation modification on different subunits of different link forms. The site-specific attachment of a prosthetic probe and isotope labeling on different subunits allowed us to perform NMR and FRET measurements. In summary, these methods presented here can provide reference and help for scientists involved in Ub research.

Abstract:Objective The total saponins of Baibiandou (Lablab Semen Album) are prepared from the traditional Chinese medicine Lablab Semen Album through extraction, separation and purification steps. Although there are studies on the targeting and treatment of cancer with Xiaobiandou (Lens culinaris) lectin in nude mice with human liver cancer transplants, there is a lack of research on how the total saponins of Lablab Semen Album affect the growth of the prostate cancer cell line PC-3. Therefore, it is necessary to explore the mechanism through which total saponins of Lablab Semen Album impact the growth of the prostate cancer cell line PC-3.Methods In this paper, the CCK8 method was adopted to test the effect of different concentrations of Lablab Semen Album total saponins on the growth of PC-3 cells. Transcriptomics was used to analyze the molecular mechanism of the inhibition of the growth of PC-3 cells by Lablab Semen Album total sponins, and the related differential gene expression was verified by real-time quantitative PCR and Western blot experiments. Western blot and CCK8 assays were used to detect the survival rate of total saponins in Lablab Semen Album-treated PC-3 cells overexpressing ALDH7A1.Results With increasing concentration of Lablab Semen Album total saponins, the survival rate of PC-3 cells was reduced significantly, and the IC₅₀ value of Lablab Semen Album total saponins was 1 086 mg/L. The transcriptomics sequencing results showed that compared to the control group, there were 2 360 differentially expressed genes in the cells treated with Lablab Semen Album total saponins, of which 1 982 genes were upregulated and 378 genes were downregulated. The GO results showed that the differentially expressed genes were significantly enriched in biological processes closely related to cancer onset and development. In addition, the KEGG analysis results also showed that the differentially expressed genes were enriched in the signaling pathway of tumor metabolism. When the differential genes were further verified, the results showed that compared to the control group, the protein expression levels of ALDH7A1, GCAT and PGAM4 in the prostate cancer cells treated with Lablab Semen Album total saponins were obviously reduced (P<0.05), whereas the protein expression levels of DMGDH and CBSL were significantly increased (P<0.001). According to the results of in vitro cell experiments, the downregulated expression of ALDH7A1 in prostate cancer cells by total saponins of Lablab Semen Album inhibited their growth.Conclusion Lablab Semen Album total saponins may inhibit the growth of prostate cancer cells in vitro through the expression of ALDH7A1.

Abstract:Objective Flipped classroom teaching mode can improve students’ academic performance and cognition, and develop students’ initiative and innovation ability, but there are obvious deficiencies in systematic knowledge mastery and education. However, the traditional teacher-centered teaching concept has its unique advantages in systematic knowledge learning and education. In recent years, there have been reports on the reform of the blended teaching mode combining flipped classroom and traditional teaching mode, but no relevant research has been carried out in the Biochemistry teaching of nursing and clinical medicine majors.Methods We introduced Biochemistry blended teaching mode based on the combination of flipped classroom with the traditional teaching mode into classes 2020 of nursing (31 students) and clinical medicine (245 students divided into 2 classes) in Cheeloo Medical School, Shandong University. Compared to traditional teaching mode for classes 2019, students’ academic performance and self-cognition were analyzed.Results Compared with traditional teaching mode, the students who received blended teaching mode achieved higher scores than those who received traditional teaching mode (P<0.01); the students’ cognition also improved significantly (P<0.01 or P<0.05). The effect of Biochemistry blended teaching mode in small class (nursing major) is better than that in large class (clinical medicine major). This study provides reference for related teaching and research in medical colleges and universities.Conclusion The blended teaching mode of Biochemistry is beneficial to the teaching reform of medical colleges.