Abstract:Protein/peptide Detection and Disease Diagnostics and Therapeutics

Abstract:With the aging population increasing worldwide, neurodegenerative diseases are becoming a major public health crisis. TAR DNA-binding protein 43 (TDP-43) is one of the major components in the inclusion bodies containing aggregated proteins in affected patients with several types of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD). A large number of mutations in TDP-43 have been identified in familial cases of ALS. TDP-43 is an essential RNA/DNA binding protein critical for RNA-related metabolism, it shuttles between nucleus and cytoplasm, and undergoes phase transition to induce cytoplasmic and nucleoplasmic inclusion formation. Here, we summarized the recent advances in our understanding of protein aggregation and phase transition of TDP-43 in vitro and in vivo. Understanding the aberrant transition of TDP-43 will help identify potential therapeutic targets for neurodegenerative diseases.

Abstract:The increasing number of drug-resistant bacteria has brought great difficulties to clinical treatment, revealing the mechanism of drug resistance has become one of the basic ways to curb drug-resistant bacteria. Bacterial signaling system is the main channels of information exchange between bacteria and plays an important role in the regulation of bacterial drug resistance. This paper reviews the relationship between bacterial two-component system, quorum sensing signaling system, the second messengers, indole and other bacterial signal systems (molecules) and bacterial drug resistance, and summarizes the mechanisms and pathways of regulating drug resistance. It mainly includes regulating biofilm formation, regulating the activity of drug efflux system, activating antibiotic inactivation enzyme, improving drug-resistant gene expression level, promoting drug-resistant gene transfer, modifying cell wall structure, etc., which involves all links of bacterial drug resistance. Each signaling system can not only regulate drug resistance independently, but also cooperate with each other to form a regulatory network to regulate bacterial drug resistance at multiple levels. The above phenomenon indicates that bacterial signal transduction system plays a pivotal role in regulating bacterial drug resistance. Blocking or jamming the signaling system and cutting off the communication signals between bacteria could be a new strategy to curb the growing resistance of bacteria.

Abstract:Disinfectants can effectively inhibit or kill microorganisms on the surface of objects and transmission media, which are widely used in food, hygiene, health, pandemic prevention and other fields. During the COVID-19 pandemic, the global use of disinfectants increased sharply, which played an important role in effectively preventing and controlling the spread of the virus and preventing the spread of the pandemic. However, improper use of disinfectants will reduce its effectiveness and even induce microbial resistance, which may increase the risk of infectious disease transmission. The disinfectant resistance gene of microorganism will also aggravate its pollution and transmission risk through vertical reproduction or horizontal transfer between the same or different species, which seriously threatens public health safety. At present, the wide emergence of antibiotic resistance gene (ARG) has attracted global attention to public health, but the understanding of disinfectant resistance is very limited. This paper reviews the research on microbial resistance to disinfectants in recent years, focusing on the mechanism of microbial resistance by forming biofilm, reducing cell membrane permeability, over expressing efflux pump, producing specific enzymes to eliminate or attenuate disinfectants, and changing action targets. The formation of strong biofilm can effectively prevent disinfectants from approaching microorganisms, reduce microbial sensitivity and improve resistance; the reduction of cell membrane permeability depends on the changes of membrane protein, phospholipid and lipopolysaccharide, which can reduce the entry of disinfectants into microbial cells; the overexpression of efflux pump system is conducive to microorganisms to discharge harmful substances in cells; the action of specific enzymes can degrade the effective components of disinfectants or improve microbial immunity; the change of target can reduce the combination of disinfectant and action site, so as to reduce the disinfection effect. In addition, aiming at the acquisition and transmission of microbial disinfectant resistance, the chromosome and plasmid mediated resistance genes as well as the relationship between microbial disinfectant resistance and antibiotic resistance in the environment were discussed. Disinfectant resistance genes can be transferred and transmitted by transformation, transduction or conjugation through mobile genetic elements such as plasmids and phages, which puts forward new requirements for scientific disinfection.

Abstract:Objective Voltage-gated sodium channels (VGSCs) are expressed in glioma U251 cells and affect the proliferation, invasion and apoptosis of U251 cells. It is reasonable to hypothesize that the cysteine-rich buccal gland protein (CRBGP), a VGSCs blocker isolated from the buccal glands of Lampetra japonica, may suppress the activity of U251 cells. Methods Firstly, the proliferation of U251 cells in the presence of CRBGP was detected by MTT assay. And the morphology, cytoskeleton and nucleus of U251 cells after treated with CRBGP were observed by Wright-Giemsa, FITC-phalloidin and Hoechst 33258 staining assays, respectively. Subsequently, extracellular matrix proteins such as collagen IV, fibronectin and laminin were used to detect the effect of CRBGP on U251 cells’ adhesion. In addition, the migration and invasion of U251 cells treated with CRBGP were detected by transwell assays. And the internal mechanisms of CRBGP on U251 cells’ apoptosis and mobility were explored by Western blot. Finally, the anti-inflammatory effect of CRBGP on U251 cells was detected by enzyme linked immunosorbent assay. Results CRBGP inhibited the proliferation of U251 cells by inducing apoptosis in a mitochondrial-dependent pathway and preventing the release of proinflammatory factor interleukin-6. Also, the VGSCs blocking and anti-inflammatory activities of CRBGP contributed to its inhibitory effects on the adhesion, migration and invasion of U251 cells. Finally, CRBGP affected the proliferation and mobility of U251 cells through the suppression of the FAK-AKT pathway. Conclusion Together, our data indicated that CRBGP could exhibit its anti-tumor activity probably by its VGSCs inhibitory property, providing a basis for the functional information of VGSCs to the gliomas.

Abstract:Objective Bradykinin and bradykinin B2 receptors (B2R) play important roles in the enteric nervous system. Bradykinin is usually involved in inflammation and neuroprotection, dependent on the bradykinin-induced formation of prostaglandins (PGs). Cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) catalyze the conversion of arachidonic acid to PGs. This study aimed to investigate the effect and the signaling mechanism of bradykinin stimulation on the release of prostaglandin E2 (pGE2) and the expression of COX2 in the enteric nervous system of guinea pigs. Methods Immunofluorescence was used to detecting the colocalization of COX2 with neural markers Anti-Hu and chAT in the primary cultured ileal submucosal plexus of guinea pigs. PCR and Western blot were used to detecting the effect of bradykinin evoking COX2 expression. Bradykinin B1 receptor (B1R) antagonist Leu-8 and B2R antagonist HOE-140 were preincubated before bradykinin stimulation. COX2 antagonist NS398 and COX1 antagonist FR12207 were used to observing the effect of bradykinin-induced pEG2 release. Results The results showed that COX2 was co-localized with neural markers Anti-Hu and chAT on ileal submucosal plexuses. Bradykinin induced COX2 expression was blocked by the B2R antagonist. The release of pGE2 by bradykinin stimulation in ileal submucosal plexuses was significantly decreased when incubating with the COX2 antagonist. Conclusion COX2 expression evoked by B2R signaling as an excitatory neurotransmitter in bradykinin stimulated pGE2 secretion, which provides a reasonable explanation for the role of bradykinin in intestinal inflammatory diseases.

Abstract:Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. The accurate diagnosis of AD, especially the early diagnosis, is urgently needed in clinic. In recent years, the technology of non-invasive in vitro diagnosis based on biomarkers has developed rapidly. In particular, taking advantage of the high surface activity, unique optoelectronic properties, good biocompatibility, easy surface modification, miniaturization and integration of nanomaterials and nanotechnology, a new nanotechnology for in vitro detection and diagnosis of AD has been developed. This greatly improves the sensitivity and accuracy of AD detection, and has the characteristics of simplicity and rapidness, so it plays an important role in early diagnosis, prognosis and curative effect evaluation of AD. This paper reviews the research progress of detection nanotechnology for AD protein biomarker. Here, we introduce the important role of nanomaterials in the enrichment of biomarkers, describe the photoelectric signal transduction technology based on nanomaterials and the methods to enhance the detection signal and improve the detection sensitivity. In addition, we also briefly introduced the application prospect of AD nano-detection technology in clinical diagnosis, prognosis and curative effect evaluation, summarized the advantages and challenges of AD in vitro diagnosis technology. It may provide reference information for the research of precision diagnosis and treatment of AD.

Abstract:Parkinson disease (PD) is an insidious and progressive neurodegenerative disease. It is the second most common neurodegenerative disease affecting 1%-3% of the population over the age of 65. While the etiology of PD is complex, α-synuclein (α-syn) misfolding and aggregation is identified as the hallmark of PD. Due to lack of the reliable biomarkers, early diagnosis of this disease is difficult. In this review, we discuss recent research progresses in the development of PD biomarkers based on the detection of α-synuclein from different samples, including body fluids (cerebrospinal fluid, blood, saliva) and peripheral tissues (skin, olfactory mucosa, salivary gland, gut mucosa). Furthermore, we summarize the recent advances on the therapeutic approaches targeting α-synuclein in treating PD.

Abstract:The amyloid cascade hypothesis is the core of etiology of Alzheimer’s disease (AD). However, in the past few years, immunotherapy targeting amyloid plaques has not been successful. In recent years, different forms of amyloid β peptides (Aβ) have attracted people’s attention. Here, we summarized the research progress of different Aβ mutants in the brains of early-onset AD patients and the truncated and modified Aβ isoforms that have been identified in the brains of sporadic AD patients, particularly focusing on the aggregation characteristics of these Aβ species and their impact on the process of AD. Single amino acid Aβ mutations associated with FAD (familiar AD) lead to alterations in the structure of Aβ, which are associated with differential aggregation kinetics, morphologies, and conformations. These differences may underlie the pathological polymorphism of FAD. Meanwhile, enzyme digestion and post-transnational modification of Aβ may contribute to sporadic AD. Targeting these peptides or related enzymes can be used as a new therapeutic mechanism or provide a new diagnostic method. The currently reported detection and analysis methods of Aβ species was also summarized, including traditional mass spectrometry, immunoassay methods, and electrochemistry methods. This review may contribute to the in-depth study of the role of different forms of Aβ and related molecules in the pathological process of AD, and provide insight for the development of new methods for the diagnosis and treatment of AD.

Abstract:Galanin (GAL) is a 29-amino acid neuropeptide that is co-expressed with neurotransmitters and neuropeptides, such as serotonin (5-HT), norepinephrine (NE), dopamine, and neuropeptide Y. GAL regulates various activities through three different G protein-coupled receptors (GalR1-3) and plays an important role in mental diseases, including depression. GAL and its receptors have been regarded as a possible therapeutic target for depression for a long time; however, there are still no widely used GAL-type antidepressants. Presently, various studies have revealed the link between the GAL system and depression. Activation of GalR1 and GalR3 induced depression-like behavior, and activation of GalR2 inhibited depression. GalR1 activation may induce depression-like behavior by inhibiting serotoninergic neurons in the dorsal raphe nucleus, noradrenergic neurons in the locus coeruleus, and glutamatergic neurons in the ventral periaqueductal gray and prefrontal cortex. GalR3 activation may induce depression-like behavior by inhibiting serotoninergic neurons in the dorsal raphe nucleus. GalR2 activation may reduce depression by activating serotoninergic neurons in the dorsal raphe nucleus and increasing the level of 5-HT. GAL N-terminal fragment GAL (1-15) mediates a stronger effect of regulating depression than GAL by causing an imbalance of receptor signal through the GalR1-GalR2 heteromer. Conversely, GAL (1-15) can enhance the antidepressant effect of 5-HT1A receptor agonists or selective serotonin reuptake inhibitors by its GalR1-GalR2 heteromer interacting with 5-HT1A receptors or forming GalR1-GalR2- 5-HT1AR heterotrimer. Besides the 5-HT system, GAL and its receptors can also interact with NE, neuropeptide Y, brain-derived neurotrophic factor, and dopamine to regulate depression. Although GAL and its receptors show great potential as a target for the treatment of depression, their regulatory effects on depression are different in different brain regions, and the mechanisms remain unknown. The development of GAL-type antidepressants also needs to be optimized. The following questions need to be addressed: first, whether the specific agonist SG2A for GALR2, which has a good antidepressant effect, will affect normal cognitive and emotional functions need to be explored. Second, as GAL and its receptors may have distinct sex-related effects, future research should distinguish the sex-related differences of GAL and its receptors on depression and further develop antidepressants accordingly. Third, and most important, in addition to designing agonists or antagonists targeting GAL receptors, the following will be of great significance for developing new antidepressants, enhancing the effects of traditional antidepressants, and improving their side effects: targeting enzymes that catalyze either the production of GAL (1-15) or GAL (1-16) or N-linked glycosylation and intracellular phosphorylation of GAL receptors, or factors affecting the formation of GalR1-GalR2 heteromer or GalR1-GalR2-5-HT1AR heterotrimer. This article reviews the regulatory effects of GAL and its receptors on depression, explores its possible mechanisms for regulating depression, and discusses the possibility of clinical application of antidepressant drugs developed for GAL and its receptors.

Abstract:Ischemic stroke is a kind of stroke in which the blood supply to the brain is disrupted, resulting in serious neurological deficits. Among stroke patients, about 87% of the cases are ischemic stroke. Neuroinflammation is one of the main pathological conditions of stroke injury. CKLF1 is a non-classical CC-type chemokine discovered in 2001, which showed strong chemotactic activity on monocytes, neutrophils, and lymphocytes. CKLF1 is most abundant in the fetal brain, but absent in the healthy adult. Growing evidence shows that CKLF1 is reactivated and expressed in adult stroke animal models and participates in multiple processing of neuroinflammatory responses. CKLF1 could activate the polarization of microglia to produce inflammatory mediators and trigger inflammation in the injured brain. And then they drove peripheral immune cells such as neutrophils to recruit into the injured brain. These stimulated the greater immune responses and destroyed the fragile blood-brain barrier (BBB). We believe that CKLF1 plays an important role in the course of ischemic stroke. However, the development of its biological activity and drug discovery lacks systematic literature reports. Thus, we collected the published data and made this review, to briefly describe the role of CKLF1 in ischemic stroke, and explained its mechanism of aggravating ischemic stroke. Moreover, some potential anti-stroke drugs have been discovered, indicating that CKLF1 is a potential target for the treatment of ischemic stroke.

Abstract:Autoimmune disease refers to a kind of chronic systemic disease in which the body is mistakenly attacked by the immune system, causing damages to cells, tissues and organs. Studies show that the incidence of autoimmune diseases now accounts for 7%-9% of the global population, and is increasing year by year. Current methods for the treatment of autoimmune diseases (such as immunosuppressant and plasma exchange) lack sufficient safety and effectiveness. Therefore, it is necessary to develop new treatment methods. Immunoadsorption (IA) is a method of selective or nonselective removal of autopathogenic antibodies to achieve the therapeutic effect on autoimmune diseases. Immunosorbent therapy can effectively avoid various side effects such as fever, nausea and infection caused by current treatment, and quickly improve the degree of disease in a short time. At present, the vast majority of IA columns in clinical application are nonselective adsorption, which may deplete the whole immunoglobulin spectrum and lead to serious adverse reactions and autoantibody rebound. Selective immunoadsorption can specifically remove pathogenic antibodies without immunoglobulin replacement, and the adsorption efficiency is higher. Thus, selective immunoadsorption is a better treatment option, but long-term research shows that the development of its adsorbent is also more difficult. However, with the in-depth study of the pathological mechanism of autoimmune diseases and the rapid development of biomedical engineering, more and more potential targets of in vitro immunoadsorption have been identified, and the research of selective immunoadsorption has entered a stage of rapid development. This paper introduces the research and clinical application of immunoadsorption in four autoimmune diseases: dilated cardiomyopathy (DCM), idiopathic membranous nephropathy (IMN), systemic lupus erythematosus (SLE) and myasthenia gravis (MG), discusses the effectiveness and safety of this treatment method, and emphasizes the need to strengthen the study on the treatment of selective immunoadsorption to remove pathogenic antibodies. At the same time, it is pointed out that in order to realize the clinical application of immunoadsorption in more diseases, more in-depth preclinical trials need to be done in terms of reliability, regionality and sample size.

Abstract:Plasma is the most frequently clinically used body fluid sample, characterized by noninvasive sampling, convenient to use, and rich contents, and is an important source sample for the excavation of potential biomarkers related to the disease. Low molecular weight proteins and peptides (LMWPs) in plasma include cytokines, growth factors, peptide hormones, and protein degradation products, which are favored by researchers because of their small molecular weight, measurable sequence information, and wide sources. In recent years, the studies on LMWPs have made new progress with the rapid development of high-resolution, high-sensitivity mass spectrometers and computational science, and the integration of new technologies and new methods. This review introduces the classification of plasma LMWPs, including small intact proteins, protein degradation products, neuropeptides, immunopeptides, carrier protein-bound peptides, and sORF-encoded peptides, based on the current research hotspots in this field. In addition, it also summarizes the research progresses on enrichment strategies, functional studies, and disease-related biomarkers. Finally, the challenges and future research direction of plasma LMWPs are discussed.

Abstract:Misfolding of native soluble proteins and peptides into insoluble amyloid aggregates is associated with a number of neurodegenerative disorders, including the most prevalent Alzheimer’s disease and Parkinson’s disease, which generally lead to cognitive decline and motor deficits. Although multiple therapies have made it to clinical trials, to date there are still no clinically effective treatments to cure these diseases or halt their progression. Development of inhibitors that prevent amyloid aggregation and recognize the toxic species to promote their clearance is one of the most important therapeutic strategies. Among different types of inhibitors, peptide-based molecules are promising candidates due to their high specificity, low toxicity, high chemical diversity, proteolytic stability and blood-brain barrier permeability after modification. Here we review the progress in research of peptide-based inhibitors towards the amyloid aggregation of Alzheimer’s disease related Aβ and Tau and Parkinson’s disease related α-synuclein. The peptide inhibitors summarized here have been mainly generated through rational design based on the amyloidogenic sequences and fibril core structures or through random selection from peptide libraries. These peptide molecules, both naturally occurring and synthetic, can inhibit protein aggregation, disassemble amyloid fibrils, reduce cytotoxicity and some have been shown to reduce brain damage and relieve cognitive and motor impairments in animal models. These studies demonstrate the advantages of peptide-inhibitors as anti-amyloid drugs and will facilitate the discovery of new therapeutic agents.

Abstract:Targeted protein degradation based on the “event driven” mode of action directly targets and catalyzes the degradation of the target protein. Compared with the traditional small molecule inhibitors based on the “occupation driven” mode of action, there is no need to be interact with the target protein for a long time and high intensity. Low concentration compounds can achieve the efficient degradation of the target protein with high activity, high selectivity, targeting undruggable proteins and many other advantages. In addition, targeted protein degradation also effectively overcomes the limitations of irreversibility, poor druggability and off-target effects of traditional drug target identification and interference strategies such as DNA knockout and RNA interference. In recent years, a series of new targeted protein degradation technologies have been developed, including deGradFP, PROTAC, molecular glue, dTAG, AID and Trim-Away based on the ubiquitin-proteasome system, specific ubiquitination the protein of interest by recruiting ubiquitin-protein ligases (E3) and subsequent degradation by the 26S proteasome. The AUTAC and ATTEC technologies based on the autophagy pathway and the LYTAC technology based on the endosome-lysome pathway ultimately direct the target proteins to the lysosome for degradation. These strategies have successfully degraded a varitey of human disease-related proteins in vivo and in vitro, showing excellent prospects for drug design and development, especially many PROTAC drugs such as ARV-110 and ARV-471 have entered the clinical trial stage, showing good therapeutic effects in breast cancer, prostate cancer and other cancer diseases. This paper briefly introduces the research status of different targeted protein degradation technologies, systematically summarizes the advantages and disadvantages of each technology, and prospects the challenges faced by this technology in the field of drug research and development.

Abstract:Site-specific introduction of noncanonical amino acids (ncAAs) by genetic codon expansion enables manipulation of protein structure and function at the atomic level. More than 200 ncAAs have been incorporated into proteins by this method in different organisms, including plants and animals, and its application has been expanded to the development of new generation biotherapeutics. With the ability to introduce biorthogonal reactive groups into proteins site-specifically, genetic code expansion could not only improve the safety and efficacy of protein and peptide drugs, but also provide a pioneering solution for the prevention and treatment of human diseases. Here, we reviewed recent advances of genetic codon expansion and focused on its applications in protein and peptide drugs. Using genetic codon expansion, homogeneous antibody-drug conjugates with improved pharmacokinetic property could be constructed. Bispecific antibodies with flexible PEG-linkers constructed by ncAAs-based biorthogonal reaction could overcome the spatial arrangement limitation existed in the traditional methods, which generated the bispecific antibodies mainly relying on genetic fusion. Development of genetic codon expansion-based protein switch could improve the efficiency of cell therapy. Site-specifically modified cytokines with PEG and immobilized them on solid surface by genetic codon expansion could also improve their pharmacological properties. Incorporation of immunogenic ncAAs in proteins could elicit immune responses and be used to develop therapeutic protein vaccines. We believe that genetic code expansion could be widely used in the next generation protein and peptide drugs.

Abstract:Objective Lumbrokinase isozymes (LKIs), which were isolated from earthworm called Dilong in traditional Chinese medicine (TCM), have been used as the active ingredient of the enteric-coated capsule to treat clotting disease approximately 30 years. Recently, the study of LKIs on other critical diseases received much attention. Methods To demonstrate the efficacy of LKIs on hepatitis B proteins, we incubated surface antigen (HBsAg), core antigen (HBcAg) and e antigen (HBeAg) with LKIs at different concentrations for different time intervals, and then estimated their cleavage sites. HepG2.2.15 cells were incubated with LKIs and their HBsAg, HbeAg were determined by ELISA and Western blotting. HBV-transgenic mice (Balb/c) were gavaged with LKIs for 30 days. HBsAg and HBeAg in serum were detected by ELISA and Western blotting, and HBcAg in hepatic tissues were immunohistochemically stained. Hematoxylin-eosin (HE) staining was used to exhibit liver endolysis of LKI-treated HBV-transgenic mice. Serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were semi-quantitatively detected with ELISA. After intraperitoneal injection of LKIs into Sprague Dawley rats, LKIs in serum and liver tissue were assayed. Longyan sheldrakes (LYS) were gavaged with LKIs for 30 days, and their serum HBV DNA were assayed by PCR. Results We observed that the capsule ingredient is a compound drug containing 6 LKIs. By incubating with the HBV proteins, LKIs were probably estimated to degrade HBsAg at K141/P142 and R160/F161, HBcAg at R142/E143, and HBeAg at R122/E123. LKIs significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells. Levels of HBsAg and HBeAg in serum and those of HBcAg in hepatic tissues decreased in HBV-transgenic mice gavaged with LKIs, suggesting a suppression of viral assembly. Levels of GOT and GPT and the number of endolysis in liver exhibited by HE staining were decreased in the LKI-treated HBV-transgenic mice, demonstrating LKIs’ protecting mice hepatic cells. The activity of LKIs could be detected in serum and hepatic tissues of Sprague Dawley rats after being intraperitoneally injected with LKIs. After gavaged with LKIs, the ducks showed a decrease in their serum HBV DNA levels. Conclusion The current work indicates that LKIs degrade HBs, HBc and HBe proteins and may interfere with the virion assembly and release, leading to decrease in the virus transmission between hepatocytes, and to hepatic protection.

Abstract:Objective This study aimed to search for immunosuppressants with high-efficiency and low-toxicity from Chinese herbal medicine using calcineurin (CN) as the target enzyme. Methods Natural compound which could inhibit the activity of the target enzyme, CN, was screened and one effective compound was identified and isolated. The immunosuppressive effect and toxic side effects of the compound were evaluated at the cellular and animal levels. The underlying immunosuppressive mechanism was explored using fluorescence spectroscopy, molecular docking, Western blot, dual-luciferase reporter gene, real-time PCR, etc. Results Quercitrin inhibited CN activity both in vitro and in vivo. In addtion, it reduced the proliferation of splenocyte and ameliorated DTH symptoms in mice. Toxicological study showed that quercitrin had no or minimal toxic side effects on experimental animals. Fluorescence spectroscopy and molecular docking analysis showed that quercitrin may bind to two sites on the CN and regulate immune responses through the CN-NFAT pathway. Conclusion We identified a new CN inhibitor, quercitrin, which could be a useful novel immunosuppressant with low toxicity.

Abstract:Proteins are the executors of various kinds of metabolism and regulation in cells and they are also the most important target molecules of pathogenic factors and drugs. The study of protein expression is necessary to understand life, disease processes and drug effects. At present, routine protein detection methods in clinical practice require the support of large equipment. However, with the development of medical technologies and especially under the special background of the coronavirus (COVID-19) pandemic, point-of-care testing (POCT, also known as on-site testing and bedside testing) has become the current development trend. POCT can improve the ways of interaction between patients and doctors and create a positive approach to medical treatment. In addition to diagnosing and treating diseases, POCT has advantages in both on-site and remote detection for personnel engaged in emergency work. Therefore, it is very important to develop accurate, sensitive, simple and fast protein POCT. To develop miniature devices for POCT, many new approaches have been attempted in recent years, including microfluidics, electrochemical biosensors, smart phones, artificial intelligence, and wearable devices. Microfluidics deals with small quantities of samples and is a universal platform for integrating a variety of technologies, such as immunochemistry, electrochemistry, and mass spectrometry. With microfluidic technology, the sample size, reaction time, and detection limit could be easily improved to satisfactory levels. The use of electrochemistry in detecting proteins has opened a new field in POCT. Since the core of electrochemistry lies in the nanocatalyst development, the rapidly growing research on nanomaterials also has facilitated and expanded the areas of POCT applications. Furthermore, the current frontier technologies employing 5G, artificial intelligence, and wearable devices have not only generated new possibilities but also greatly inspired scientists to create novel POCT devices. In conclusion, with the increasing demand of people and the continuous progress of science and technology, the development direction of future protein detection methods will be focused on portability, intellectualization, rapidity, integration of diagnosis and treatment by multidisciplinary approaches for better application in clinical practice and applicability for bedside and home testing.

Abstract:Objective Enzyme linked immunosorbent assay (ELISA) has been widely used for detection of antibodies or antigens, and is regarded as the gold standard in clinical diagnosis, which can provide relatively reliable, sensitive and specific results. The essence of ELISA is the specific interaction between antigens and the corresponding antibodies. However, the inherent instability of natural antibodies is the Achilles heel of ELISA, which may lead to poor reproducibility or even false results. Previously, our group created a nova gold nanoparticle-based artificial antibody, denoted as goldbody. Goldbody can specifically interact with antigens like natural antibodies, but has much better stability than that of natural antibodies. The excellent stability of goldbody makes it a potential replacement of natural antibodies in ELISA assays. Methods Herein, we demonstrate with a series of experiments that goldbody is indeed a good replacement of natural antibodies in ELISA for antigen detection. Results After necessary optimization and conjugation of horseradish peroxidase (HRP), the 13 nm HRP-labeled anti-hen egg white lysozyme (HEWL) goldbody can be used in ELISA assay to detect HEWL in the range of 1-16 mg/L. Conclusion Goldbodies can indeed replace natural antibodies in ELISA for a more accurate and consistent detection of antigens.

Abstract:Objective Vibrio anguillarum (V. anguillarum) is an important conditional pathogenic bacterium, which can infect many aquacultural animals, and cause huge losses to aquaculture industry every year. Study on the pathogenic mechanism of V. anguillarum and rapid detection of the bacterium are necessary for the prevention of the disease. Aptamers show good application potential in many fields such as target analysis, detection of bacterium and research on pathogenic mechanism due to their high affinities and specificities. Therefore, selection of the aptamers against V. anguillarum and analysis of the related sites of the pathogen by its aptamers, can not only provide a new way for the identification of V. anguillarum, but also have important significance to explore the role of those sites in the disease control. Methods Aptamers against V. anguillarum were selected from the high frequency sequences by SELEX with sequencing in each selection round. Affinity of aptamer was measured by the ssDNA concentration method and the affinity and specificity of aptamers against the pathogen were also studied based on the measurement. The affinity constant (K_d) and maximum affinity (A_m) of aptamer were obtained by the nonlinear fitting according to the hyperbola function of the software Origin. Binding protein of aptamer H5 was isolated by magnetic separation and purified by polypropylene acyl amine gel electrophoresis (PAGE). The binding protein was identified by mass spectrometry. Its spatial structures and subcellular location were analyzed online by the websites of Prabi, Phyre2 and Psortb 3.0. Results An efficient selection method for aptamers was established based on each round of sequencing and high frequency sequences. A series of aptamers (H1, H5, H6, H12, H25, H26, H28, H33, H38 and H42) with good affinities and specificities towards the target bacterium V. anguillarum were selected by the efficient method. The K_d and A_m of 6 aptamers (H1, H5, H25, H26, H33, H38) were measured, and their K_d were (78.77±10.99), (180.65±23.01), (121.14±21.43), (276.42±51.23), (89.24±10.84), (167.12±23.73) nmol/L, respectively, and their A_m were (229.4±8.35), (891.04±50.14), (647.20±41.51), (720.85±75.35), (510.65±33.89), (576.06±38.73) nmol/L, respectively. The binding protein of aptamer H5 was identified as E1 component of pyruvate dehydrogenase in the cytoplasm of V. anguillarium. The main skeleton of the binding protein was composed of α-helix and β-fold, and the loops of random coil were mostly distributed outside of the protein. The interaction regions of the aptamer and its binding protein were also analyzed and speculated. Conclusion It was proved that the selection method based on each round of sequencing and high frequency sequences was quite efficient. Apparent affinity of aptamer was dependent on both K_d and A_m, and the A_m also played an important role in aptamer’s apparent affinity. Aptamer H5 entered into V. anguillarum probably by endocytosis, and then bound to E1 component of pyruvate dehydrogenase in the cytoplasm. The present study proved that aptamers could enter bacteria and bind to the corresponding targets, which provides a new idea for prevention of the disease caused by V. anguillarum and for development of novel aptamer medicines.