Abstract:Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to international public health. The SARS-COV-2 gene continues to mutate in COVID-19 outbreaks. Mutation mainly manifests in 3 forms: point mutation, gene recombination and epigenetic modification. Viral mutations are driven by multiple factors, with mutation rates modulated at 3 levels, the nature of virus, host-virus interactions and natural selection. Therefore, it is particularly important to strengthen the monitoring of the global novel coronavirus genome and the protection of immunosuppressed populations. In the early stage of virus evolution, the mutant strains exhibit greater transmissibility and less virulence than the wild-type strain, although 5 variants of concern (VOCs) showed different stability, transmission capacity, adaptability and pathogenicity. So physical interventions need to be further strengthened. As herd immunity is established, novel mutant strains tend to mutate against vaccines and antibodies. In that case, VOCs, especially the prevailing Omicron variant, bring challenges to the prevention and control of COVID-19 worldwide. The existing and potential prevention, diagnosis and treatment approaches for COVID-19 were summarized. In the vaccination part, the protective efficacy of COVID-19 vaccine against VOCs and the factors influencing the efficacy of COVID-19 vaccine were analyzed. In the detection part, the detection methods based on nucleic acid, antigen and antibody were summarized in order to satisfy the requirements for point-of-care testing and timely recognition of novel variants. And in the treatment part, the potential therapeutic drugs and targets of SARS-CoV-2 were summarized. Drug targets are generally divided into extracellular targets and intracellular targets. In general, this review proposes possible countermeasures by analyzing the impact of mutations on global epidemic prevention and control, hoping to provide theoretical basis for possible large-scale epidemic prevention and control in the future.

Abstract:Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus is highly contagious and spreads rapidly, posing a serious threat to the health and safety of people around the world. Proteomics technology has the characteristics of high throughput and high sensitivity, and plays an important role in biomarker discovery, molecular mechanism research, and therapeutic target research. Proteomics technology has been widely used in the research of COVID-19. Herein, this study provides a comprehensive review of the research progress of COVID-19 based on proteomics techniques. In section 1, the genome structure of SARS-CoV-2 and the process of SARS-CoV-2 infecting host cells were summarized. In section 2, the currently commonly used mass spectrometry (MS)-based proteomics techniques including liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS were reviewed. In section 3, the application progress in the research of precise diagnosis, molecular mechanism and drug therapy targets of COVID-19 based proteomics technology was highlighted. Proteomics have been employed in biomedical research to uncover biomarkers associated with COVID-19, it also provides a comprehensive snapshot of virus-induced changes to the host following infection, invasion, persistence, and pathogenesis and can prime the identification of novel therapeutic targets for preventing or lessening disease severity. In section 4, the future development direction of proteomics was prospected. It’s hoped that this review can help to promote the development of proteomics technology in the precise diagnosis and treatment of diseases.

Abstract:Objective To investigate the effect of SARS-CoV-2 membrane protein on the processing of the 3" untranslated region (UTR) of the mRNA precursor (pre-mRNA) in host cells.Methods Based on the cell model of human lung epithelial cells A549, over-expression of the SARS-CoV-2 membrane protein was performed. The RNA-Seq high-throughput sequencing technique and bioinformatics methods was employed to analyze the systematic characterization of alternative polyadenylation (APA) events in host cells. Genes with significant APA events were uploaded to the Metascape database for functional enrichment analysis. In addition, alternative 3"UTR length of genes with APA events was verified by RT-qPCR. Then the target protein expression level was detected by Western blot.Results A total of 813 genes that were significant dynamic APA events in host cells that over-expressed SARS-CoV-2 membrane protein. These genes were enriched in cell biologicial processes such as the mitotic cell cycle and regulation of cellular response to stress. We further screened AKT1, which encodes a critical regulator involved in the above biological process, showing a 3"UTR lengthening in IGV software. RT-qPCR verified the trend of 3"UTR length changes of AKT1. Western blot showed the increased level of phosphorylated AKT1 protein in over-expressed group of M protein.Conclusion SARS-CoV-2 membrane protein potentially affects the 3" processing of host pre-mRNAs. AKT1, which is involved in a variety of viral biological processes, with 3"UTR lengthening, and its protein function was activated intracellularly.

Abstract:Objective Many mutant strains of SARS-CoV-2 have stronger infectivity and immune escape ability. The situation of epidemic evaluation, prevention and control is serious. The aim of the present paper is to track and predict the infectious transmission of COVID-19 through a theoretical model.Methods Based on the grid epidemic model, this paper discussed the relationship between the duration of infection and the effect of group immunity, and on this basis, established the theoretical model of infection transmission of COVID-19. The infectivity parameter A and the immune effect parameter B are introduced to predict the daily variation curve of infection. The parameter can be used to quantitatively compare the comprehensive infectivity of each mutant, and we also test the conjecture that the infection parameters A and B are not related to regional factors.Results Through the theoretical model of infection transmission of COVID-19, the infectious time was accurately predicted. By analyzing the infectivity and electrical changes of mutant strains, the internal relationship between the infectivity of mutant strains and the electrical changes of mutant residues was pointed out. The parameter changes of mutants were analyzed, and the comprehensive infectivity of each mutant was quantitatively compared. We also verified the conjecture that parameters A and B are only related to the nature of the virus itself and the coexistence of the virus and the human body, but not related to the region where the disease occurs and evaluated and compared the epidemic prevention level of each outbreak region.Conclusion This paper established a theoretical model of infection transmission of COVID-19, which can predict the duration of the epidemic, the number of new infections per day, and evaluate the infectivity of the virus, immune escape ability, comprehensive infectivity, and regional epidemic prevention level. It can also give some suggestions on epidemic prevention countermeasures according to the possible parameter changes caused by virus variation.

Abstract:Objective To detect the active ingredients in the traditional Chinese medicine prescription and its molecular mechanisms against SARS-CoV-2 by prescription mining and molecular dynamics simulations.Methods Herein, prescription mining and virtual screening of drugs were performed to screen the potential inhibitors against SARS-CoV-2. Molecular docking and molecular dynamics (MDs) simulations were further performed to explore the molecular recognition and inhibition mechanism between the potential inhibitors and SARS-CoV-2.Results The natural compounds library was constructed by 143 prescriptions of traditional Chinese medicine, which contained 640 natural compounds. Ten compounds were screened out from the natural compounds library. Among the 10 compounds, 23-trans-p-coumaryhormentic acid, the main active constituent of the Loquat leaf, showed the best binding affinity targeting the recognizing interface of SARS-CoV-2 S protein/ACE2. Upon binding 23-trans-p-coumaryhormentic acid, the key interactions between SARS-CoV-2 S protein and ACE2 were almost interrupted.Conclusion Ten compounds targeting SARS-CoV-2 S protein/ACE2 interface were screened out from natural compound library. And we inferred that 23-trans-p-coumaryhormentic acid is a potential inhibitor against SARS-CoV-2, which would contribute to the development of the antiviral drug for SARS-CoV-2.

Abstract:Internet gaming disorder (IGD) is a neurodevelopmental disorder characterized by dysfunctional reward processing. Recent studies combined experimental techniques of cognitive neuroscience to explore the neural mechanisms of IGD, and the existing results seem to indicate that the neural basis underlying IGD resembles those of drug addiction. However, the conclusions of the current research are still controversial. The differences were not surprising for two main reasons. One is that there is evidence of specificity to reward processing of different types, which means gaming addicts assign higher incentive value to gaming-related cues than other rewards. The appeal of game is that instant and unexpected rewards give the player a thrill that is hard to experience in reality. Besides, the stage of reward processing is another important factor. The game player can learn the game skill according to the feedback of practice again and again and achieve the upper level, in this way, reward processing can be decomposed into two phases: reward anticipation and outcome evaluation. Reward anticipation refers to the emotional and motivational states triggered by reward-related cues, while outcome evaluation refers to the hedonic experience when rewards are obtained. Thus, this review has compared different types of rewards and discussed different stages of reward processing among IGD patients based on the existing researches. We found the hyperactive reward system of IGD patients in reward anticipation may be associated with attention bias, emotional experience, and craving. In contrast, IGD patients show blunt activity to primary reward, especially the outcome evaluation. In order to conduct a more comprehensive and in-depth study on reward processing in IGD, further studies are required to eliminate comorbid subjects, use gaming rewards with higher ecological validity, and explore how the impairment of outcome evaluation contributes to the development of addictive behaviors.

Abstract:Oncolytic virus therapy belongs to immunotherapy. It can achieve the purpose of killing tumors by virus-specific infection and activating tumor immunity. Compared with traditional therapies, it has the advantages of safety, efficiency, and less side effects. A variety of viruses have been studied, and commonly used viruses include adenovirus, vaccinia virus, herpes virus, reovirus, parvovirus, Newcastle disease virus, Coxsackie virus, etc. for oncolytic therapy, and have been used in clinical and achieved good therapeutic effect. With the continuous development of oncolytic viruses, there are currently 4 oncolytic viruses approved for marketing: Rigvir?, Oncorine?, Imlygic? and Delytact?. Currently many viruses used in tumor treatment. Since the influenza virus were first discovered in the 1900s as a “beneficial” virus to alleviate leukemia, there have been studies that prove that influenza viruses have the ability to kill tumor cells. Researchers have made many attempts in the process of fighting influenza. The process of developing inactivated vaccines, live attenuated vaccines, and subunit recombinant influenza vaccines accumulated valuable experience for further building oncolytic viruses. Influenza viruses were one of the first viruses to be used in tumor treatment. With the development of molecular biology, it is now possible to artificially intervene in many of the traits of viruses. The mechanism of oncolytic influenza virus become more and more in-depth, and the current mode of action of oncolytic influenza virus can be summarized as the following 3 points: (1) the virus directly lyses tumor cells; (2) viruses activate immunity to kill tumor cells; (3) viruses expressing exogenous genes as vectors improves both of these abilities. In this review, the modified influenza virus has stronger selectivity for pancreatic enzymes secreted by tumor cells with interferon-deficient RAS mutations to improve tumor targeting. The single-chain antibody encoding CTLA-4 or HER-2 enhances the anticancer specificity of influenza virus and acts as the carrier of foreign genes IL-2, IL-15, GM-CSF and anti-PD-1 to activate immunity are reviewed.

Abstract:DNA replication stress (RS) is a term that broadly defines DNA replication disorders, and usually refers to events that cause replication forks to slow down or stall. The excessive accumulation of replication stress is the main driver of tumorigenesis and genome instability. Cell chromosomes constantly expose to exogenous or endogenous replication stress in replication, telomeres and common fragile sites are some chromosome regions that are highly sensitive to replication stress. These regions are usually difficult to completely replicate under high replication stress. Recent studies have found that mitotic DNA repair synthesis (MiDAS) is different from S phase replication, which can help difficult-to-replicate regions to be able to replicate after entering mitosis. Therefore, MiDAS is also called “rescue mechanism of replication”. Since the maintenance of telomeres depends on telomerase activity and alternative lengthening of telomeres (ALT), ALT cells have more fragility of telomeres, and MiDAS shows high levels of activity, so this reviews the mechanism of MiDAS and how difficult-to-replicate telomeres respond to replication stress to complete DNA synthesis during mitosis under different telomere maintenance mechanisms.

Abstract:Ionizing radiation can lead to DNA double-strand breaks, resulting in rapid phosphorylation of histone H2AX to γ-H2AX at the location of double-strand breaks. The number of focal points formed at γ-H2AX aggregations in cells can be applied to the evaluation of DNA double-strand breaks, and is correlated with the radiation dose. Therefore, γ-H2AX can be used as a biomarker to evaluate the mutagenicity of ionizing radiation. It can also be used as a biological dosimeter of ionizing radiation to estimate individual exposure dose. Conventional detection methods of γ-H2AX aggregations include enzyme linked immunosorbent assay (ELISA), Western blot and indirect immunofluorescence assay (IFA), the fluorescent focus of which can be detected by fluorescence microscopy and flow cytometry. Through the investigation of literatures in the past ten years, most of the research work focus on the decrease of sample volume, the development of super-resolution microscopy to obtain sharper images of γ-H2AX aggregations, various image analysis softwares to achieve automation detection and new detection techniques. Research on automation/portable devices is critical to realize point-of-care testing for radiation accident emergency response and medical treatment. In terms of rapid radiation dose estimation and radiation emergency triage, the establishment of a rapid, accurate, portable and automated high-throughput γ-H2AX biological dosimetry is one of the most important research directions. As a biomarker of ionizing radiation in the future, γ-H2AX detection technology has important application value in radiation biology research, radiation molecular epidemiology, radiation accident emergency response and medical treatment. This paper discusses the research progress and application prospect of detection methods based on ionizing radiation biomarker γ-H2AX.

Abstract:Deep brain stimulation has become an effective treatment for many neurological and psychiatric conditions. However, invasive electrode implantation carries the risk of surgical complications, and the stimulation target is difficult to change after implantation. Non-invasive stimulation methods such as transcranial magnetic stimulation and transcranial electrical stimulation offer new avenues for modulating brain function. However, these non-invasive brain stimulation methods have not been shown to directly modulate deep brain activity without affecting cortical neurons. Therefore, these methods are mainly used to modulate neural activity in the superficial regions of the brain. Temporal interference (TI) stimulation is a new non-invasive deep brain stimulation method that modulates neural activity through the interaction of two high-frequency electric fields to generate a low-frequency envelope. This approach is expected to address the need for non-invasive deep brain stimulation. This paper first introduces the concept and safety of TI stimulation, and then describes the evoked field analysis methods in the existing research on TI stimulation and discusses the physiological model modeling method and simulation platform related to TI stimulation analysis. Research progress and application progress in animals and humans also were introduced. Finally, based on the current research progress, we proposed future research directions for TI stimulation, to provide new research ideas for non-invasive deep brain stimulation research.

Abstract:Messenger RNA (mRNA) therapy is a novel anticancer treatment strategy based on in vitro transcription (IVT) mRNA, with promising potential for the treatment of malignant tumors. The outbreak of the COVID-19 pandemic in the early 21st century has greatly promoted the application of mRNA technologies in SARS-CoV-2 vaccines. Meanwhile, the research and development of the mRNA cancer vaccine has become a priority. A number of key technologies, including mRNA production strategies, delivery systems, anti-tumor immune strategies, etc., have made dramatic improvements and modifications. These technologies accelerated the research progress and clinical applications of mRNA therapy, thereby greatly overcoming the bottleneck problem, such as the instability, inefficient deliveries, and weak immunogenicity of the mRNA vaccines in the past. This review provides a detailed overview of the production, delivery systems, immunological mechanisms, and anti-tumor immune response strategies for mRNA cancer vaccines. We list some mRNA cancer vaccines that have been used as candidates for cancer treatment and the clinical trials in the field of tumor immunotherapy. In addition, we discuss about the immunological mechanism of the mRNA vaccines to destroy tumors, as well as the challenges and prospects for the future.

Abstract:As a new type of organoid model, skin organoids can reconstruct and modify different types of cells and appendages with specific functions such as umbilical cord blood stem cells, induced pluripotent stem cells, keratinocytes and fibroblasts, as well as sebaceous glands, sweat gland and hair follicles under special habitats, which can not only highly simulate the physiological structure and function of skin tissues, but also better restoring more realistic skin ecology under different in vitro environments. It can also be used in the fields of skin wound regeneration, skin tumor, immune and metabolic diseases, treatment of inflammatory diseases and drug screening. Meanwhile, skin organoids can not only make up for the deficiencies of existing in vitro skin models in terms of structure and function, but also enable high-throughput screening of drugs or raw materials, reducing the time and economic costs in the later stages of drug screening. However, due to the limitations of current technology, the types and functions of organoids cannot be fully realized to realistically simulate the physiological conditions in the body such as lipid metabolism and blood circulation. Moreover, issues such as consistency and standardization of mass-produced organoids need to be addressed, such as source cells, structure and function, which require the establishment of appropriate standards based on practical applications. Based on this, this paper details the cell sources involved in skin organoids construction and their applications in recent years and several skin appendages like organs that have been constructed and provides an outlook on the future development and optimization of skin organoids.

Abstract:The type I toxin-antitoxin (TA) system is widespread in the bacterial genome and can play a variety of biological functions in the growth and survival of bacteria, including antibacterial function, red blood cell toxicity, promoting the formation of persistent bacteria, inhibiting bacterial growth, or causing bacterial dormancy, etc. The vast majority of type I toxin proteins target the cell membrane. One known mechanism of action is the formation of pores in the cell membrane, resulting in a decrease in membrane potential or destruction of the cell membrane, thereby inhibits the ATP synthesis or leads to the bacterial death; another possible mechanism of action is that the toxin proteins act on the cell membrane, changing the shape of the cell and causing it to enter a dormant state. The complexity of the type I toxin protein-membrane mechanism and the diversity of its biological functions are much more complex beyond our expectations. Therefore, it is important to analyze the assembly mechanism of type I toxin protein in different membranes and their structural characteristics, which is also the key to revealing its structure-function relationship. This article summarizes the reported structural characteristics and biological functions of the type I TA system, explores the formation of different structures in the cell membrane that affect its function combined with the prediction of its transmembrane domains, and analyzes the key factors affecting its mechanism of action. It brings opportunities for the treatment of drug-resistant bacteria, and also brings ideas for the research of new antibacterial drugs.

Abstract:Resilience, which is defined as the ability of a system to adjust its activity for retaining the basic functionality when errors, failures and environmental changes occur, is an essential and fundamental property for biomolecular networks. The studies of biomolecular network resilience attempt to answer the following 3 questions. (1) What is the potential mechanism of the resilience of biomolecular networks? (2) How the state of biomolecular network migrates from one stable steady state to another under the effect of resilience? (3) How to predict the tipping points of state transitions to prevent the system from evolving into undesirable states (such as disease states)? In view of the importance of resilience for biomolecular networks and its clinical application value, we systematically review the research progress focusing on 3 questions above in the past 20 years. As one of the important steady-state characteristics of resilience systems, bi-stability (or multi-stability) can help us to uncover the underlying mechanism of the resilience. Biomolecular networks consist of numerous repeated network motifs, and the steady-state response of almost all network motifs which contain feedback loops (e.g. positive autoregulation motif, mutual inhibition motif) is bi-stability (or multi-stability). Based on our numeric simulation, the network motifs with feedback loops have different steady-state response characteristics although they all bi-stability (or multi-stability), which result in the different biological functions they can describe. Many studies also indicated that stochastic noise from internal or external could affect the number of stable-steady states and hysteresis of the network motifs with bi-stability (or multi-stability). Furthermore, the bi-stable network motifs have been used to model many biological processes, such as cell cycle and embryonic development, to reveal their mechanism. The network motifs are too simple to model complex biological processes, which generally involved in interactions between lots of biomolecules. Potential function, as a powerful tool in the field of dynamical system, is widely used to uncover the state transition properties of high-dimensional biomolecular networks. Many methods have been proposed to reconstruct the potential function of equilibrium systems and non-equilibrium systems based on network dynamics. Using these methods, a vast number of studies revealed the state transition mechanism of various biological processes, such as cell differentiation, cancer initiation, etc. The state of biomolecular network generally migrates from one stable steady state to another abruptly and drastically under the effect of resilience. However, detecting tipping points of state transitions in system level is impossible based on network dynamic, due to the complexity and nonlinearity of biomolecular networks. To fill this gap, many indicators have been proposed to predict the upcoming tipping points from biological data under network perspective, such as dynamic network biomarker (DNB). And these indicators were also used to detect the critical transitions in the development of various diseases (e.g., diabetes, influenza, cancer). So far, the study of biomolecular network resilience has been helped us to understand the mechanism of state transitions in many biological processes. However, there are still some important issues that have not been resolved. (1) Studying the resilience of large-scale biomolecular networks which consist of thousands of biomolecules; (2) reconstructing potential function of large-scale biomolecular networks based on biological data; (3) control biomolecular network resilience.

Abstract:Objective To investigated the N-linked glycans in bladder cancer FFPE tissue sections, and explored abnormal N-linked glycans modification in bladder cancer FFPE tumor tissues sections.Methods An experimental procedure for in situ extraction of N-linked glycans based on FFPE tissue sections was developed. FFPE tissue sections were digested by PNGase F and liberated N-linked glycans. Permethylation was performed to modify the free end of the N-linked glycans. The relative intensity of N-linked glycans was detected by MALDI-TOF/TOF-MS. Database matching was performed to determine possible glycoforms of N-linked glycans. The accuracy of significantly different N-linked glycans as a predictive bladder cancer biomarker were predicted by ROC analysis.Results The data of MALDI-TOF/TOF-MS detection of premethylation-modified N-linked glycans showed that, in tumor and peritumoral FFPE tissue sections of 16 patients with bladder cancer, the relative intensity of N-linked glycans N2H6, N2H7, N2H8, N2H9 (high mannose) and N5H6F1 (complex) were increased significantly in the tumor tissues of bladder cancer. At the same time, N2H5 (high mannose), N3H5(hybrid) and type N3H4, N4H4, N5H6F1S2 (complex) N-linked glycans were significantly decreased. ROC analysis showed that the biantennary N-linked glycan N3H4 (AUC=0.90) and N4H4 (AUC= 0.91) were reliable in distinguishing tumor and peritumoral tissue of bladder cancer patients separately or combined together, and these N-linked glycans may become a potential biomarker for bladder cancer.Conclusion Abnormal N-glycosylation of proteins in bladder cancer FFPE tumor tissue, N-linked glycans N3H4 and N4H4 may be potential biomarkers of bladder cancer.

Abstract:Objective Molecular imaging technology has the characteristic of “early detection”, since aberrations at molecular levels occur much earlier than the changes at the anatomical level. In this study, intracellular photoacoustic molecular imaging (PMI) method was used to map the folic-acid-conjugated gold nanorods (FA-AuNRs) targeted on cancer cells.Methods We synthesized the FA-AuNRs and studied their properties including morphology, absorption spectrum, and biocompatibility. The conjugated folic acid has endowed the FA-AuNRs with the ability to target folate-receptor-expressing cancer cells. Then, PMI experiment was performed to study the target specificity of FA-AuNRs on cancer cells.Results FA-AuNRs were rod-shaped with a near-infrared absorption peak at ~800 nm. We observed the strong photoacoustic signals in the cytoplasm of cancer cells, while weak photoacoustic signals in the normal cells, indicating the selective uptake of FA-AuNRs inside cancer cells via folate-receptor-mediated endocytosis. The study demonstrated the ability of PMI to precisely map the FA-AuNRs targeted on cancer cells.Conclusion With the aid of specific targeting, information about the surface molecules of cancer cells can be obtained by PMI. This method is expected to visualize, characterize and quantify biological processes at cellular and molecular levels.

Abstract:Objective To investigate the toxic effect and molecular mechanism of silicon dioxide nanoparticles (SiO₂NPs) on mouse Sertoli cells (TM4), TM4 cells were exposed to medium containing various concentrations of SiO₂NPs (0, 1, 10, 100 mg/L) for 24 h.Methods After treatment, the morphology and viability of TM4 cells were detected by optical microscope and cell counting kit (CCK-8). The level of intracellular reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA, and the content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were detected by kits according to the manufacturers’ protocol. The percentage of apoptotic cells in TM4 cells was detected by Annexin V-FITC/PI kit, and the expression levels of apoptotic molecules including Fas, FasL, Caspase-8, Caspase-3, Bax and Bcl-2 were detected by Western blot.Results The results showed that SiO₂NPs inhibited the cell survival rates, decreased cell concentrations and changed cell morphology of TM4 cells in a dose-dependent manner. In addition, the level of ROS in TM4 cells was significantly up-regulated after exposure, followed by an increase in the content of MDA and the activity of SOD. Further study found that SiO₂NPs increased cell apoptosis rate and activated the pro-apoptotic signaling pathway mediated by Fas/FasL. Interestingly, inhibition of oxidative stress by NAC in TM4 cells can alleviate the cell injure and apoptosis induced by SiO₂NPs.Conclusion In summary, SiO₂NPs induce TM4 cells apoptosis through oxidative stress and activation of Fas/FasL signaling pathway.

Abstract:Objective To prepare and evaluate the biocompatibility of placental acellular matrix and to explore its feasibility as tissue repair material.Methods The waste placental tissue from delivery was handled by virus inactivation, acellular treatment and freeze-drying to obtain the acellular spongy matrix material of placenta. The effect of acellular was observed by HE staining and the microstructure of the materials was observed by scanning electron microscope. At the same time, 39 healthy male SD rats with body mass ranging from 120 g to 150 g were selected, randomly divided into experiment 1 group, experiment 2 group and control group. The subcutaneous implantation of the constructed matrix material was performed in rats, experimental group 1 implanted matrix materials, experimental group 2 implanted matrix materials and umbilical cord mesenchymal stem cells, the control group was the sham operation group. Routine blood tests were performed on the 3rd, 5th and 7th day after surgery to analyze the number of lymphocytes, granulocytes and other inflammatory cells. At 1, 2, 4, 8 and 9 week after the operation, the tissue samples of the implanted material and surrounding tissues were taken for HE staining analysis.Results The constructed acellular matrix of placenta appears milky white spongy in macroscopic view, HE staining indicated no cell residue. Under electron microscope, the internal void of the material was obvious, and the crosslinking degree of the material was good. The total porosity was (77.54±2.53)%. After subcutaneous implantation, the incision healed well, and no obvious inflammatory cell increase was observed in blood routine examination. Seven days after the operation, blood vessel formation can be observed by HE staining in the sections of the implanted materials. Moreover, the addition of umbilical cord mesenchymal stem cells can accelerate the fusion of the materials with the body, promote the in-depth growth of cells and vascularization.Conclusion Placental acellular sponge matrix material has good biocompatibility and can be an ideal source of tissue engineering materials.

Abstract:Objective Cell mechanics plays an important role in regulating cellular physiological and pathological processes and investigating cell mechanics is meaningful for revealing the underlying mechanisms guiding life activities. The advent of atomic force microscopy (AFM) provides a novel powerful tool for single-cell studies and AFM-based indentation assay has become an important method for characterizing cell mechanics in the field of life sciences, yielding numerous new insights into single-cell behaviors. However, current studies of cell mechanics by AFM are commonly performed in static environment, while cancerous cells are in flow environment of vascular fluids during tumor metastasis, and thus the results obtained in static environment cannot completely reflect the real behaviors of cancerous cells in fluidic condition. Particularly, so far knowledge of the mechanical mechanisms guiding the interactions between fluidic microenvironment and cancerous cells in the process of tumor metastasis is still limited. Here, based on AFM, a method allowing measuring the mechanical properties of single cells in fluidic environments is developed.Methods A fluidic cell culture medium device was established on a petri dish with openings on both sides with the use of an injection pump and an extraction pump. The fluidic cell culture medium device was integrated with AFM to measure the mechanical properties of cells in fluidic environments. The fluidic cell culture medium device was also integrated with inverted optical microscope which had a heating plate to observe the growth states of cells in fluidic environments. MCF-7 cells (human breast cancer cell) and HGC-27 cells (human gastric undifferentiated carcinoma cell) were used for the experiments. The fluidic cell culture medium device was used to grow cells to examine the effects of medium flow rate and flow time on cell proliferation and cell mechanics. Under the guidance of optical microscopy, AFM probe was moved to cells grown in static culture medium or fluidic culture medium to perform indentation assay to record force curves, and then Hertz-Sneddon model was applied to analyze the force curves to obtain the Young’s modulus of cells. Calcein fluorescein was used to stain live cells and PI fluorescein was used to stain dead cells. The cytoskeleton changes of MCF-7 cells after growth in fluidic culture medium were observed by confocal fluorescence microscopy.Results The effects of fluidic culture medium on the growth of cells were firstly analyzed. Experimental results on cell growth show that cell culture medium fluidic environment could better promote cell growth and proliferation compared with cell culture medium static environment. Then the mechanical properties of cells grown in static culture medium and fluidic culture medium were measured respectively. Experimental results show that, when the growth condition of cancerous cells changed from static to fluidic, the Young’s modulus of cancerous cells decreased significantly and cytoskeletons reorganized, indicating the influence of fluidic environment on the mechanics of cancerous cells.Conclusion Combining AFM with fluidic control techniques allows detecting the mechanical properties of single cells grown in fluidic environments, providing a novel way to investigate the mechanical cues involved in the interactions between fluidic microenvironments and cancerous cells during tumor metastasis.

Abstract:Objective Cryopreservation of testicular tissue for later transplantation is another effective way to maintain male fertility.Methods In this paper, the procedure of slow freezing of massive testicular tissue was optimized by shortening the loading time of cryoprotectant (CPA), increasing the freezing rate at the first stage, and directly plunging into liquid nitrogen at the second stage. The mouse testis was cryopreserved by modified two-step freezing. In addition, ice seeding procedure was applied at different temperatures in order to reduce CPA concentration required for cryopreservation of testicular tissue.Results The results showed that negative rate of apoptosis of germ cells in frozen tissues with modified two-step method was significantly higher than commonly used slow freezing method, and had no significant difference with control group. Among them, the negative rate of spermatogonial cells was 98.4%, that of spermatoblast cells was 99.2%, that of sperm cells were 88.4%, and that of sertoli cells was 98.1%. Compared with non-seeding group, seeding at -10℃ can improve the survival rate of testicular tissue that frozen with 5% DMSO. The negative rate of apoptosis were 92.1% (spermatozoa), 93.2% (spermatocytes) and 88.9% (Sertoli), which are not significantly different from that of 10% DMSO group. This indicates ice seeding can reduce the CPA concentration and toxic damage.Conclusion Modified two-step freezing and ice seeding improve the quality of mouse testicular tissue after freezing, and provide a reference for freezing of human testis in clinical.

Abstract:Objective Protein chromatography is a fundamental technique in protein sciences. Difficulties were always met in experimental teaching for this technique due to its complexity and tedious details. Therefore, we would like to find new strategies for experimental teaching of such technique.Methods We used the 3D simulation techniques to setup an on-line Protein Chromatography Laboratory. We designed the teaching contents, core equipments, the scoring systems, user interface etc. based on our every-day teaching experiences.Results We took the advantage of the 3D simulation techniques, and designed an on-line Protein Chromatography Laboratory exactly reproduced from the equipment-setups of Protein Facility, Zhejiang University School of Medicine. This virtual simulation teaching platform for protein chromatography contains 3 modules regarding the teaching, practicing and on-line exams, so that the efficiency and quality of teaching could be largely improved. In this VR platform for teaching, a new but commonly used type of protein chromatography equipment was chosen in order to complete such shortages in VR teaching for protein purification. Furthermore, fluorescence-detection size-exclusion chromatography (FSEC) and several case studies were also included in the “advance teaching” section.Conclusion Experimental teaching of protein chromatography using virtual simulation was more advantageous than the “old-fashion” ways of teaching. Students could be involved deeply in learning and practicing of such techniques anywhere beside the chromatography laboratory; and based on our teaching experiences, their learning efficiency and quality were indeed improved.