Abstract:Glycoprotein 96 (gp96) is a highly conserved and ubiquitous glycoprotein that belongs to the heat shock protein 90 (HSP90) family. It comprises 4 domains: N-terminal domain (NTD), middle domain (MD), C-terminal domain (CTD) and charged linker region (CR). Each domain performs a specific function. NTD containing the nucleotide binding site interacts with and hydrolyzes ATP. MD is involved in client protein recognition, and is the site of constitutive dimerization. The ATP hydrolytic activity of NTD requires cooperative action of CR and MD. The schematic representation of gp96 topology is shown in the main text. An increased expression of gp96 has been reported in multiple cancers. Its upregulation in tumors is closely correlated with poor prognosis and decreased overall survival of patients, indicating that gp96 serves as a potential diagnostic and prognostic biomarker. As a chaperone protein gp96 directs the folding and/or assembly of secreted and membrane proteins. It has a limited client protein profile that is involved in key processes linked with the hallmarks of cancer. Previous studies have shown that cellular gp96 physically interacts with and directs the folding and assembly of several client proteins, including insulin-like growth factors (IGF), integrins, epidermal growth factor receptor-2 (HER2) and Wnt co-receptor LRP6, which are involved in the regulation of cell multiplication, normal tissue differentiation, cancer progression and metastasis. It has been found that gp96 was only expressed on cell surface of malignant tumor but not benign tissues. Cell membrane gp96 is closely associated with cancer cell proliferation, invasion, and metastasis. We further demonstrated that gp96 on cell membrane interacts with HER2, urokinase-type plasminogen activator-receptor (uPAR) or ER-α36. Targeting gp96 by siRNA or a monoclonal antibody for gp96 led to decreased cell growth and invasion, increased apoptosis in vitro, and suppression of tumor growth in vivo, validating cell membrane gp96 as a therapeutic target. At present, the selective small-molecule inhibitors (NECA and PU-WS13), a gp96-specific monoclonal antibody (W9mAb), and an inhibitory gp96-targeted polypeptide (p37) are under development and their potential applications in the tumor targeting therapy are highlighted in this review.

Abstract:Phospholipase A₂ is an important enzyme with many family members. As a special member of the phospholipase A₂ superfamily, secretory phospholipase PLA2G5 is expressed in both immune and non-immune cells. Studies have shown that PLA2G5 is tissue and cell specific, and its biological functions are even related to species and environment. Many researchers have studied PLA2G5 in humans as well as in transgenic and knockout mice, and have gained a deeper understanding of its biological and pathophysiological effects. The PLA2G5 gene is located on mouse chromosome 4 and human chromosome 1. It encodes a protein with a molecular mass of 14 ku, highly conserved Ca²⁺ binding loop and enzyme binding site, and contains only 6 disulfide bonds without specific sulfur bonds of group Ⅰand Ⅱ sPLA₂, N-terminal propetide, insertion and C-terminal extension. PLA2G5 has a variety of biological functions. It can not only bind to heparan sulfate proteoglycans through a cluster of negatively charged residues, but also bind to membrane phosphatidylcholine with high affinity, release arachidonic acid, initiate eicosanoid cascade reaction, and then conduct signal transduction. Meanwhile, it can help against pathogens, present antigens, serve as Th2/M2 to regulate the innate immune response, and play an anti-inflammatory or pro-inflammatory role in different immune diseases. In addition, PLA2G5 can effectively hydrolyze phospholipids in lipoproteins, which helps to control the quality of lipids and participate in lipid metabolism. PLA2G5 induces airway inflammatory diseases such as asthma and acute respiratory distress syndrome. It is closely related to cardiac homeostasis and can reduce the risk of aortic dissection, but it can also aggravate atherosclerosis and has dual effects on low density lipoprotein and perivascular fibrosis. It is a potential biomarker for detecting the progression of knee osteoarthritis and can trigger inflammatory response in rheumatoid arthritis. It can improve metabolic syndrome, fight against adipose tissue inflammation, insulin resistance, hyperlipidemia and obesity. It has antibacterial activity against Gram-positive bacteria, and Pla2g5^-/- mice are more sensitive to Candida albicans and Escherichia coli infection. Besides, mutations in PLA2G5 gene site 45 produce extra cysteine, causing conformational changes that lead to the development of benign familial retina macular disease. Mutation or overexpression of PLA2G5 can also affect the occurrence and development of human malignant tumors. Studies have shown that overexpression of PLA2G5 is a marker of poor prognosis for gliomas. A tumor mutation load model based on PLA2G5 gene can accurately predict the recurrence risk of ovarian cancer. Furthermore, several synthetic inhibitors, including silicon-directed recognition inhibitors of indole-amide-biphenyl derivatives, phospholipid analogs, and potential natural inhibitors such as olanolic acid syrenic oregano, have certain inhibitory effects on PLA2G5. These results provide theoretical bases for novel therapeutic approaches targeting PLA2G5.

Abstract:Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and is associated with increased cardiovascular morbidity and mortality. Recently, rather than general fat distribution, epicardial adipose tissue (EAT) gains a growing concern. EAT is the local adipose deposition between myocardium and pericardium. Accumulated evidence revealed several distinguishing characteristics of EAT. It lies contiguously with the myocardium and could infiltrate into myocardium, actively secrets cytokines and adipokines mediating inflammation or remodeling, and contains abundant ganglionated plexi. It is suggested that EAT is associated with the initiation, perpetuation and recurrence of AF, but the precise role of EAT in AF pathogenesis is not completely elucidated. Mechanisms involve adipocyte infiltration, profibrotic and pro-inflammatory paracrine effects, oxidative stress, neural mechanisms and genetic factors. This article reviews the characteristics of EAT, the relationship between imaging parameters and AF, the latest progress of molecular mechanism and treatment strategy in the occurrence and development of AF.

Abstract:Most of obese subjects show leptin resistance, characterized by abnormal increase of serum leptin but diminished effects of leptin on inhibiting appetite, enhancing energy expenditure and decreasing blood glucose. Reducing leptin resistance is considered as an effective strategy to treat obesity and obesity related diseases. Exercise decreases obesity, improves glycolipid metabolism and increases insulin sensitivity, which are closely related to exercise-induced decrease of serum leptin and alleviation of leptin resistance. This review mainly summarized the mechanisms of exercise-induced improvements of leptin resistance in obesity, including lowering hyperleptinaemia and decreasing central and peripheral leptin resistance. Alleviation of hyperleptinaemia in obesity by exercise attributes to decreased fat mass and improved inflammation. For the decrease of central leptin resistance in obesity, exercise modulates neurons activities (stimulating SF-1, POMC neurons and inhibiting AgRP neurons) and other molecules related to energy metabolic homeostasis and food intake (elevating hypothalamic IL-6), and regulates expressions of proteins involved in leptin signaling of hypothalamus (stimulating STAT3, and inhibiting PTP1B and SOCS3); while exercise-induced decrease of peripheral leptin resistance comes from improvements of body composition (decreased fat mass, increased brown adipose tissue and reduced inflammation) and regulation of protein expressions involved in leptin signaling in muscle and liver (inhibiting SOCS3 and PTP1B). This review could provide a new insight into the mechanism of exercise-mediated prevention and treatment of obesity.

Abstract:Tetrodotoxin (TTX), a highly specific voltage-gated sodium channel blocker, is a deadly toxin but a valuable tool for life science research such as neurobiology and physiology. It provides a long nerve blockade without cardiovascular toxicity and is unable to penetrate the blood-brain barrier, making it an attractive candidate for local anesthetics. At present, conventional amino amide and amino ester local anesthetics are the mainstay in clinical practices such as local anesthesia, postoperative analgesia and cancer pain treatment. However, conventional local anesthetics have a relatively short action time and may cause systemic side effects in cardiovascular and nervous systems. To overcome these limitations, it is necessary to develop local anesthetics with long-lasting effect and high safety. The efficacy of TTX has been confirmed by clinical trials for patients with neuropathic pain, cancer pain and drug withdrawal syndrome. However, TTX is still limited in clinical application due to its systemic toxicity and scarcity of sources. The overdose of TTX can lead to mild suppression of motor and sensory functions, blood pressure changes and even respiratory muscle paralysis. Fortunately, researches are increasingly dedicated to improving the safety and efficacy of TTX. TTX has the potential to be a new type of anesthetic to replace amino esters and amino amides, without the abusability of opioids. TTX might be converted into a “potion” when used in tandem with vasoconstrictors, local anesthetics, polymer-drug conjugate and chemical permeation enhancers or when encapsulated with microparticles, liposomes, nanoparticles and other microparticle drug delivery systems (MDDS). Better results can be obtained by combining MDDS-TTX and external trigger energy (such as near-infrared light and ultrasound). These combinations produce repeatable and adjustable on-demand anesthesia, enabling patients to control the timing, intensity and duration of nerve block in a safe and non-invasive manner. This review summarizes the research progress of TTX adjuvant drugs, TTX sustained- and controlled-delivery systems, TTX modification techniques, and aims to provide reference for the development of new local anesthetic drugs and exploration of new ideas.

Abstract:As a new molecular marker, free DNA (cell free DNA, cfDNA) plays an increasingly important role in disease prediction, tumor prevention and treatment. However, there are still many obstacles before achieving a wide range of clinical application, mainly due to the lack of cfDNA-related standards, resulting in the lack of comparability of current research reports and the lack of stable repeatability of test results, thus reducing the reliability of clinical application. This review focuses on the related research progress of cfDNA in recent years, including sample source, sample preservation, sample extraction, qualitative and quantitative detection and application of cfDNA. We analyze and compare the various methods used in each link of cfDNA research and their advantages and disadvantages, analyze and summarize the problems existing in each link, and put forward the urgency of the formulation of cfDNA related standards.

Abstract:In bacteria, the acyl transmembrane modification of cell-surface polymers is a common feature to strengthen their pathogenic potential. The dlt operon-mediated D-alanylation of lipoteichoic acids (LTAs) is an important post-modification to adjust charge balance in Gram-positive bacteria. Four proteins of DltA/B/C/D were identified to be essential for LTA D-alanine incorporation. Though the process of D-alanine transfer by cytoplasmic DltA/DltC has been largely probed, transmembrane catalysis by MBOAT protein DltB and the terminal player DltD is yet to be defined. Here, the crystal structure of stDltD was determined from S. thermophilus at 2.94 ? resolution. On the basis of the structure comparison, DltD was considered as the terminal acyltransferase of the dlt operon, and it belonged to the SGNH-like family. An stDltD active center, including four blocks and a catalytic triad, conservatively exists in various Gram-positive pathogens. In addition, structural analysis showed that the stDltD catalytic center formed a strong positively charged groove docking with a glycerolphosphate molecule. Combined with previous reports, an updated working model was proposed for cross-membrane D-alanylation mediated by the dlt operon. The structural evidence provides more implications to clarify the biological function of stDltD and the process of transmembrane acyl modification.

Abstract:Sporosarcina pasteurii (S. pasteurii) is a kind of Gram-positive bacteria from soil. Various applications have been developed based on the efficient urease activity that can induce the precipitation of calcium carbonate. However, the metabolic mechanism related to biomineralization of S. pasteurii has not been clearly elucidated. Especially, there are few studies on the gene structure of urease, regulation mechanism of expression and associated metabolism, which play key roles in biomineralization. Nowadays, the uncontrollability and instability of biomineralization reactions in the applications of S. pasteurii root in the lack of research on urease metabolisms. Therefore, it is urgent to further reveal the gene information, expression regulation and related metabolism of urease in S. pasteurii.In this paper, we compared the growth and gene expression of S. pasteurii BNCC 337394 under four different culture conditions through high-throughput transcriptome analyses. The four medium conditions were: (1) control group with yeast extract of 20 g/L; (2) ammonium group with yeast extract of 20 g/L and ammonium chloride of 10 g/L; (3) urea group with yeast extract of 20 g/L and urea of 5.62 g/L; (4) ammonium Tris group with yeast extract of 20 g/L, ammonium chloride of 10 g/L and Tris of 15.75 g/L. Transcriptome data were analyzed by bioinformatics methods of differential gene expression analysis, GO enrichment analysis, KEGG enrichment analysis and operon prediction.The results showed that there were significant differences in the growth of S. pasteurii among the four conditions. The bacteria could not grow and reproduce normally in the control group. There was a growth delay in the ammonium Tris group. The growth rate and trend of the ammonium group and the urea group with the same nitrogen content were similar. A total of 3 090 genes were generated and expressed in S. pasteurii in the experiment. There were 1 152 differentially expressed genes (DEGs) between the ammonium group and the control group, of which 411 were up-regulated and 741 were down-regulated. There were 1 362 DEGs between the urea group and the control group, of which 736 were up-regulated and 626 were down-regulated. There were 803 DEGs between the ammonium group and the urea group, of which 279 were up-regulated and 524 were down-regulated. There were 794 DEGs between the ammonium group and the ammonium Tris group, of which 281 were up-regulated and 513 were down-regulated. Furthermore, it was found that the expression of urease was significantly enhanced in the control group which was short of a nitrogen source comparing with the ammonium group and the urea group.GO and KEGG analyses revealed that the control group needed to enhance its basal metabolism and express more flagellum in order to survive. The pathways of electron transfer activity and oxidative phosphorylation were different in the ammonium group and the urea group. It indicated that the synthesis of ATP was associated with the hydrolysis of urea in S. pasteurii. Meanwhile, ammonium stimulated the expression of urease and ATP synthase in the ammonium group which was more significant than that of the urea group. The results of the ammonium Tris group indicated that a stable and moderate alkaline pH environment favored a high level expression of urease. Finally, the double operon gene structure of urease was predicted based on the two key characteristics of gene expression similarity and gene spacing.

Abstract:This work aims to characterize the accuracy of decoded motor unit activities during multiple contraction conditions based on electromyography (EMG) decomposition techniques, and to evaluate the performance of extracted neural features for the estimation of muscle activation. Twelve healthy undergraduates participated in the experiments to perform the isometric contraction of knee extension with four levels. The high-density EMG signals were decomposed into motor unit spike trains based on convolution kernel compensation. Two neural features were extracted for the cross-correlation analysis with force. On average, (7±4) motor units were identified from the medial vastus muscle (MVM), while (9±5) motor units were identified from the lateralis vastus muscle (LVM). The average pulse-to-noise ratio (PNR) was 30.1 dB, corresponding to the decomposition accuracy of over 90%. The average correlation coefficient between the two neural features of MVM and the force was (0.79±0.08) and (0.80±0.08), respectively, while the average correlation coefficient of LVM was (0.85±0.05) and (0.85±0.06), respectively. These results demonstrate the feasibility of the identification of motor unit activities under various contraction conditions, and the strong correlation between neural features and force indicates the application of decomposition techniques in rehabilitation, exercise training, and human-machine interfacing.

Abstract:Existing research methods for biological effects of extremely low frequency electromagnetic fields (ELF-EMFs) are comparison between groups, with inability to exclude individual differences in cell sensitivity or changes in conditions during experiment. This work proposed a method for real-time effects observation of ELF-EMFs in the same cell and same condition. The stability domain was used to identify the stability of hippocampal neurons before ELF-EMFs exposure. At the time of electromagnetic field intervention (t=60 s), ELF-EMFs were exposed to hippocampal neurons at 0, 0.09, 0.38, 0.76, 7.33 and 14.78 mT, respectively. Reactive oxygen species (ROS) and Ca²⁺ fluorescence response curves of hippocampal neurons were recorded in real-time, and the autocorrelation function between ELF-EMFs and fluorescence response was established. The results showed as follows: (1) The step properties of fluorescence response were clear at the burst times of ROS and Ca²⁺, which were important indicator to identify the real-time response; (2) The response time of ROS and Ca²⁺ to ELF-EMFs were delayed and inconsistent; (3) ROS and Ca²⁺ had dose-dependent response to ELF-EMFs; (4) The response of ROS to ELF-EMFs was complex; (5) The response of Ca²⁺ to ELF-EMFs was asymptotically stable. When correlation function exceeded 0.3, the real-time response of ELF-EMFs and ROS/Ca²⁺ was correlated. It was concluded that a real-time response method of intracellular ROS and Ca²⁺ to ELF-EMFs exposure was feasible for the evaluation of electromagnetic bioeffects.

Abstract:Preservation of living lung cancer tissues will provide more complete sample information for in vitro experimental studies, such as lung cancer genetic screening and targeted drug screening. This article studied the vitrification method of living lung cancer tissue. Firstly, the needle immersion method was used to vitrify a single piece of lung cancer tissue, and the concentration and equilibrium time of the required cryoprotectant were optimized. Secondly, multiple lung cancer tissues were vitrified in cryotubes, and the volume of cryoprotectant solution and equilibrium time were optimized. Finally, the effects of the traditional slow freezing, rapid freezing without cryoprotectant, and vitrification methods were compared, and low-temperature microscope was used to analyze the damage mechanism of ice crystal. The results showed when 20% EG+20% DMSO+0.5 mol/L trehalose was used as the cryoprotectant, the equilibration solution and the vitrification solution are loaded for 3 min and 1 min, respectively, the needle immersion method and vitrification in a 0.25 ml cryotube have the highest tissue viability (79.96% and 80.44%) after recovery. Immunohistochemistry showed that the tissue structure of lung cancer after vitrification was less damaged, only a few positive expression of TUNEL in the cells, compared with slow freezing and fast freezing without cryoprotectants. Low-temperature microscopy showed that only a few small ice crystals appeared in and around the vitrified tissue, while obvious ice crystals appeared in slow freezing and rapid freezing.

Abstract:To evaluate the commutability between HER2 genomic DNA reference materials (RM) and clinical samples, ddPCR and qPCR were used to study the commutability of HER2 genomic DNA RM, which can provide traceable RM with commutability for clinical laboratory testing. 29 clinical samples were randomly measured among the 5 levels of RM by real time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The averaged copy number ratio of HER2 to RPPH1 was calculated. According to the guideline EP30 of American Society for clinical laboratory standardization (CLSI) and the guideline WS/t356-2011 for Guideline for Evaluation of Matrix Effects and Commutability of the People’s Republic of China, a regression curve was drawn with the result of ddPCR as abscissa and the result of qPCR as ordinate, and the commutability of RM was evaluated by Deming regression method. If the test results of the proposed RM fall within the 95% confidence interval of its predicted, it is considered that the analyzed RM is communtable; if it falls outside the range, it is considered that the analyzed RM is not communtable. In addition, the results were compared with the ratio of copy number concentration when CEP17 gene was used as reference gene. HER2/RPPH1 of the five RMs determined by ddPCR and qPCR were: 1.91, 1.86; 5.70, 4.45; 16.94, 12.21; 22.38, 17.19; 35.38, 28.84, respectively, which fall in the prediction range, indicating the five RMs are communtable. Moreover, this was confirmed by the ratio of HER2/CEP17 determined by the two methods. However, the regression coefficient of HER2/RPPH1 (R² = 0.97) was better than that of HER2/CEP17 (R² = 0.78) and even one group of clinical sample fell outside the 95% confident interval. We used FISH to detect the cell lines used to prepare HER2 genomic DNA RM. The results showed that in the same nucleus, part of chromosome 17 showed HER2 amplification in the short arm, and part of chromosome 17 showed no HER2 amplification. Additionally, CEP17 was amplified in some cells, which indicates it is not suitable to be used as reference gene as this will cause false negative results. This confirms the necessity of using RPPH1 gene as reference gene when diagnostic of HER2. In conclusion, the five RMs are communtable, which can be used for the method validation and quality control in analyzing of HER2 copy number variation in clinical laboratories.