Abstract: Transient receptor potential vanilloid subfamily member 1 (TRPV1), also known as capsaicin receptor (VR1), is a kind of ligand gated non-selective cation channel which can be activated by capsaicin, heat (>43℃) and H+ (pH<6.0). TRPV1 is highly permeable to Ca2+. Previous studies found that TRPV1 mainly distributes in nervous system and mediates pruritus and pain response. Recent studies have shown that TRPV1 also widely distributes in non-nervous cells such as mast cells, bladder epithelial cells, monocytes, skin keratinized epithelial cells, islet cells and so on. TRPV1 has a wide range of functions and can mediate beneficial or harmful biological effects on the body. In the nervous system, TRPV1 related signal pathway mainly mediates itching and pain response. Relevant studies in pancreatic cells have shown that the upregulation of TRPV1 can alleviate the process of diabetes, but studies in pulmonary epithelial cells, pulmonary vascular endothelial cells, bronchial smooth muscle cells, etc. have shown that the upregulation of TRPV1 can accelerate the development of respiratory diseases. In addition, TRPV1 has dual effects of promotion or inhibition on the disease progression in cardiovascular system, digestive system and skin system. In cancer research, it was also found that the upregulation of TRPV1 played an important antineoplastic effect, which could inhibit the proliferation, invasion and migration of tumor cells in human tongue squamous cell carcinoma, prostate cancer, breast cancer and so on, arrest the cell cycle and induce cell apoptosis. At present, many studies have been carried out on the mechanism of TRPV1, among which the mechanism of TRPV1 mediating itching and pain is relatively in depth. TRPV1 has become a promising therapeutic target due to its extensive functions. New drugs targeted to TRPV1 have been developed to ameliorate diabetes, cardiovascular diseases, and some kinds of cancers. This paper introduces the latest progress in the distribution, structural characteristics and functions of TRPV1, and focuses on the research progress of pruritus and pain related signaling pathways mediated by TRPV1. We also introduced the Chinese herbal medicine with TRPV1 as the target, looking forward to providing theoretical guidance for taking TRPV1 as a potential therapeutic target by combination of traditional Chinese medicine and modern medicine.
Abstract: Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules with specific molecular recognition capability, obtained by a process called systematic evolution of ligands by exponential enrichment (SELEX). They have the advantages of high thermal stability, ease of chemical synthesis and modification, and low immunogenicity compared to antibodies, and have attracted widespread interest in many fields such as bioanalysis, biomedicine, and biotechnology. High-quality aptamers are the basis of applications, however, the number of them that meet requirements of practical applications is very limited. How to obtain aptamers with high affinity, high specificity, and high in vivo stability is the technical bottleneck in the field of aptamers. Firstly, this review briefly introduces the basic theory of SELEX and its critical experimental steps including design of nucleic acid library, monitoring selection process, preparation of secondary library, sequencing and screening of candidate aptamers. The six main research directions of SELEX during the past thirty years are then concluded. They are respectively (1) how to improve the specificity of aptamers, (2) how to improve the stability of aptamers against nuclease degradation, (3) rapid SELEX, (4) how to isolate aptamers for complex targets, (5) how to isolate small molecule-binding aptamers, and (6) how to isolate high affinity aptamers. The development of rapid SELEX technologies has attracted tremendous attention and almost all physical separation methods have been applied to improve the SELEX efficiency. Very recently, several methods involving the highly efficient chemical reactions have been reported, providing novel strategies for the rapid isolation of aptamers. The key research progresses of SELEX technologies suitable for the isolation of small molecule-binding aptamers are subsequently reviewed and the challenges of each method are critically commented. There are three types of SELEX methods including the target-immobilized SELEX, library-immobilized SELEX (Capture-SELEX), and homogeneous SELEX (GO-SELEX). Even though the target-immobilized SELEX suffers from many issues such as steric hindrance, it is still a popularly used method due to its simplicity. In recent years, Capture-SELEX has been widely applied. The experimental conditions of Capture-SELEX (concentration of positive-SELEX target, choice of negative-SELEX targets and their concentrations) and the affinity (KD,dissociation constant) and the specificity of the isolated aptamers for the 36 targets are listed in a table. Based on the information from the table, the effect of the experimental conditions on the affinity and the specificity is discussed. The statistical data indicates that the lower concentration of the positive-SELEX targets favors the isolation of the higher affinity aptamers, while it is not a necessary condition. Negative-SELEX is currently the dominant strategy to improve the specificity of aptamers. However, the specificity of many aptamers cannot meet the requirement for practical applications. The choice of negative-SELEX targets and their concentrations in each case are quite different. In 20 out of the 36 targets, no negative-SELEX was performed for the aptamer isolation. How to obtain the aptamers with high specificity is the most difficult challenge for small molecule targets. It is in urgent need to establish novel strategies beyond negative-SELEX to improve the specificity of aptamers. The experimental conditions of GO-SELEX and the KD and the specificity of the isolated aptamers for the 13 small molecule targets are also list for comparison. The comparison data shows the less numbers of the enrichment cycles required for GO-SELEX than Capture-SELEX, while the obtained aptamers all commonly have KD in the nanomolar range. The lower enrichment efficiency of Capture-SELEX should be due to the self-dissociation of the immobilized library. The affinity evaluation is the important part of the characterization of aptamer structure and performance. More than ten affinity assays are frequently used for aptamer characterization, which are roughly divided into three categories: separation-based, immobilization-based, and homogeneous methods. All techniques could generate false-positive and false-negative results. Taking gold nanoparticle-based colorimetric assay and isothermal thermal titration as examples, we review the technical progresses and comment on the fundamental reasons resulting in the inconsistent results when the different affinity assays are conducted. The final part of this review provides an outlook on the future trends of aptamer isolation technologies, affinity characterization techniques, and the technical standardization.
Abstract: Molecular medicine focus on understanding the diseases based on molecular level, and developing personalized medicine strategies for diagnostics and therapeutics. However, powerful molecular recognition tool is still limited for cancer diagnosis and therapy, which impeding cancer research. Aptamers are generated from systematic evolution of ligands by exponential enrichment (SELEX) also known as in vitro selection, ranging from synthetic single-stranded DNA, RNA or XNA (enhanced modified nucleotides), HNA (nucleotides of specific structures such as G quadruplex). The main advantages of aptamers including high specificity, high affinity, simple and rapid synthesis, easy chemical modification, wide target range, good tissue penetration and low immunogenicity. As a molecular recognition tool in molecular medicine, aptamer shows wide applications in developing personalized prediction, diagnosis and therapeutics for its high specificity and high affinity against target. This review discusses the applications of aptamers in disease diagnosis, including aptamer-based tumor marker discovery, liquid biopsy, and molecular imaging, Moreover, the applications of aptamer-based cancer therapy are reviewed, including aptamer-based inhibitors, aptameric drug conjugates, nanomedicines, and aptamer-mediated immunotherapy. Finally, it is promising aptamer will be extensively employed in the future including fundamental research, diagnosis and therapeutics. However, following issues are still need to be addressed. First, the application scenarios-dependent SELEX procedures lack studying which limits the clinical applications of aptamers. Second, the structure of aptamer-target complex has not been fully elucidated, which restricts the precise regulation of aptamers. Third, aptamer is easily degraded by enzymes in vivo and has a short half-life period, which hinders the applications of aptamer-drug conjugates in the development of targeted drugs. With the advancement of screening technology and the further enhancement of aptamer performance, it is expected that aptamers will find more extensive utilization in the field of molecular medicine in the future.
Abstract: The structure of protein is determined by the sequence, and the function of protein is determined by its structure. The advent of accurate protein structure prediction tools has created new opportunities and challenges in the fields of structural biology, structural bioinformatics, drug discovery and many other fields of life sciences. The accuracy of single-chain protein structure prediction has reached a level comparable to that of experimental methods. In this review, we provide an overview of the theoretical basis, development history, and recent advances in the field of protein structure prediction. Additionally, we discuss how the large number of predicted protein structures and artificial intelligence-based methods affect experimental structural biology. Open questions and future research directions in the field of protein structure prediction are analyzed.
Abstract: Expansion microscopy (ExM) is a new super-resolution imaging technique. With the aid of expandable hydrogel, biological samples are uniformly physically amplified and can be imaged in super resolution by using conventional optical imaging microscopes. In ExM, after immunofluorescence staining, gel embedding, protease digestion and water swelling, the relative distance of fluorescent labeled molecules inside the biological samples was increased, so the sample can bypass the optical diffraction limit in conventional fluorescence microscope to achieve the super-resolution imaging. ExM is widely suitable for many types of biological samples such as cell and tissue sections. Proteins, nucleic acids, lipids and other biological macromolecules can also be imaged by ExM. ExM can be combined with confocal microscopy, light-sheet microscopy and super-resolution microscopy to further improve imaging resolution. In recent years, a variety of derivative technologies have been developed from base ExM, which further promotes the practical application of this technology. Protein retention expansion microscopy (proExM) can avoid complicated sample preparation process and directly image endogenous fluorescent proteins. Magnified analysis of the proteome (MAP) was suitable for super-resolution imaging in large biological samples. Iterative expansion microscopy (iExM) can increase the final expansion factor of biological samples to 16-22 times by changing the gel embedding steps. Cryo-expansion microscopy (Cryo-ExM) can provide better image fidelity. Expansion fluorescent in situ hybridization (ExFISH) and Click-ExM can achieve super-resolution imaging in nonprotein biomolecules, such as RNA, lipids, and polysaccharides. Expansion pathology (ExPath) can be used for clinicopathologic specimens imaging. The combination of ExM and light-sheet microscope can improve the image resolution to super-resolution level in the deep imaging depth. The application of ExM in super-resolution microscopy can further increase the resolution of images to 10-30 nm. In this paper, we reviewed the basic principles of ExM and its derivative technology, the research progress of combining ExM with different imaging technologies, the application progress of ExM in observing different types of biological samples, and the prospective of spreading ExM technology in the future.
Abstract: The high specificity and sustainability of enzymes make them widely used as green catalysts, and their stability and catalytic activity are vital for their practical applicability. Recently, enzymes have been endowed with desired physical and catalytic properties via using protein structural modification. From the protein structural point of view, enzyme thermal stability has been improved by modulation of non-covalent/covalent interactions (hydrophobic interaction, hydrogen bonding, salt bridges, aromatic interaction and disulfide bonds), loop truncation, C-/N-terminal engineering, introduction of proline with highest conformational rigidity in the flexible region, and substitution of glycine with highest conformational entropy. Meanwhile, the catalytic function has been enhanced or altered by various methods, including reducing steric hindrance, widening the binding pocket, moderating substrate binding affinity and active site flexibility. While, the generation of new features or improvement of the existing features often comes at the expense of the other ones. Thus, strategies include screening suitable mutation sites, co-selection for stability and activity, and using highly stable proteins as the parental backbones are also discussed to overcome the stability-activity trade-off. This review summarized recent advances in structural modification to improve the stability or/and catalytic activity of enzymes and further provided a brief prospect in the future developments.
Abstract: Antibody drug conjugate (ADC) is typically composed of a monoclonal antibody conjugated with a cytotoxic small molecule drug via a linker. It is an emerging and promising class of targeted cancer therapeutics, combining both the highly cytotoxic activity of chemical drugs and highly targeting ability and specificity of monoclonal antibody. Fourteen ADCs have been approved for marketing so far worldwide, and more than 140 ADC drug candidates have been investigated in clinical studies. Various ADC technologies have been well developed to manufacture these ADC drugs in commercial scale as well as clinical scale. In this review, we describe the molecular structure, mechanisms of action and development history of ADCs. We then provide an overview of the current landscape and recent advances in each key element of ADCs, including antibody, linker, payload and conjugation, and their advantages and disadvantages. Future directions in ADC development may encompass smaller sized forms of antibodies such as antibody fragments and nanobodies to improve the penetration and accumulation of ADCs in the solid tumors. Novel linkers are also being tested to enhance the stability in circulation systems and reduce off-target toxicities. Emerging payloads of new functional mechanisms are also explored in the construction of ADCs to overcome the drug resistance resulted from currently used payloads of marketed ADCs. Various site-specific conjugation technologies have been adopted to reduce the heterogeneity of drug-load species and optimize the pharmacokinetic properties of ADCs. This review article aims to enhance systemic understanding and careful considerations in designing an ADC drug with improved efficacy and safety.
Abstract: Accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen causes ER stress, which triggers the unfolded protein response (UPR) to restore protein homeostasis. So far, three UPR sensors have been identified, including IRE1, PERK, and ATF6. All of them are ER transmembrane proteins, which become activated and initiate downstream UPR signals under ER stress. Though first discovered during the study of how cells respond to ER stress, it remains unclear how the ER stress signal is sensed by the three UPR sensors. Structural studies provide insight into how direct binding of ER-localized peptides to the lumenal domain of IRE1 and PERK facilitates their oligomerization and thus activation. In another model, dissociation of the ER chaperone BiP is the key to the UPR activation. In addition, further studies reveal that the UPR not only functions in maintaining protein homeostasis and UPR sersors is not solely activated in response to the accumulation of unfolded proteins in the ER. Lipid bilayer stress, cytosolic factors or intercellular signals may initiate the UPR. Despite the importance of the UPR in physiology and pathophysiology, how the UPR is activated under physiological or pathophysiological conditions are largely unknown. Developing novel strategy to monitor the unfolded and misfolded proteins in the ER lumen will advance our understanding of the relationship between ER stress and the UPR. This paper introduces the discovery and the canonical pathways of the UPR, with a focus on the current mechanistic understanding of the UPR activation, and discusses the relationship between the UPR and ER stress as well as related questions.
Abstract: R-loops are formed during transcription when the nascent RNA generated by RNA polymerases hybridizes with its complementary DNA template, giving rise to a region of DNA∶RNA hybrid and a displaced single-stranded DNA. R-loops are stable structures that have important beneficial physiological functions, but also could pose a threat to genomic stability. Unscheduled R-loops induce cell cycle checkpoint activation, DNA damage, and chromosome rearrangement in mammalian cells. R-loops expose unstable single-stranded DNA, which is prone to transcription-related mutations and recombination. R-loops may also directly block DNA replication, leading to DNA double strand breaks. Abnormal accumulations of R-loops have been found in some syndromes, human neurological disorders, and cancers. On the other hand, R-loops also play positive roles in physiological processes, such as epigenetic modification, DNA repair, gene regulation and mitochondrial stability. R-loops forming on transcription-termination regions, promote RNA polymerase pausing before termination. R-loops are regulated delicately in cells. Collisions between replication and transcription cause accumulation of R-loops. Replication stress, DNA damage and RNA Pol II pausing also induce R-loop formation. To resolve R-loops when they form, cell evolve numerous dissolution mechanisms. Ribonuclease RNase H1 and RNase H2 bind to R-loops and then catalyze the cleavage of RNA. Helicases, such as SETX, DHX9, DDX21, unwind the RNA from the R-loops. Defects in RNA processing factors, chromatin modulators, DNA repair proteins, cause accumulation of R-loops, suggesting they are involved in R-loop regulations. To detect R-loops, several methods have been developed and are mainly based on the S9.6 antibody and the HBD domain of RNase H1, however, both of them possess some issues. Understanding the regulatory mechanisms of R-loop formation and clearance could help us better know how cells maintain genomic stability and prevent disease development. In this review article, we summarized functions and regulations of R-loops. We also discussed methodologies used to detect R-loops. Finally, we proposed some future perspectives of R-loop research.
Abstract: Ubiquitylation, also termed ubiquitination, is one of the most important post-translational modifications in eukaryotic cells. It is a process by which a small signaling protein, called ubiquitin composed of 76 amino acids, is conjugated to protein substrates via an E1-E2-E3 enzymatic cascade. Ubiquitin can be attached to lysine, serine, threonine and cysteine residues of its substrates. Ubiquitin itself contains seven lysine residues, therefore, ubiquitylation can form various polyubiquitin chains to produce complex ubiquitin codes. Ubiquitylation can alter the fates of ubiquitylated proteins including kinase activation, alteration of protein localization and proteolysis via the 26S proteasome and is involved in nearly every aspect of biological activities in eukaryotic cells. Recent studies indicated that more complicated post-translational modifications can also be found on ubiquitin including sumoylation, ubiquitylation, phosphorylation and acetylation. These modifications largely increase the complexity of ubiquitin signals. Ser65 of ubiquitin is the first characterized phosphorylation site whose biological functions have been extensively studied in human cells. It has been shown that Ser65 phosphorylation by PINK1 kinase is critical for the activation of Parkin ubiquitin ligase during mitophagy induction. The researches on Ser65 phosphorylation of ubiquitin boosted the studies on biological significance of the rest phosphorylation sites of ubiquitin. Now it is clear that phosphorylation of Ser57 residue is involved in endocytosis and stress responses, including oxidative stress in yeast, whereas phosphorylation of Thr12 and Thr66 residues plays important roles in DNA damage response. In the case of Ser57 residue, members of the AMPK-related kinases phosphorylate it, however, mechanisms by which Ser57 phosphorylation regulates endocytosis or oxidative stress response are still unclear. Also, no experimental evidences are available in mammalian system yet. One interesting fact is that many AMPK-related kinases contain a ubiquitin-associated domain (UBA), although some studies suggested these UBA domains do not possess any binding capabilities to polyubiquitin chains. However, some of these kinases could phosphorylate the Ser57 residues on the M1 polyubiquitin chain, implying these UBA domains do recognize certain polyubiquitin chains. In this review article, we summarized the post-translational modification sites of ubiquitin, especially phosphorylation sites and highlighted the biological functions of Ser65, Ser57, Thr12, Thr66 phospho-ubiquitin proteins. We also discussed alternations of biophysical properties brought by the phosphorylation of ubiquitin. Finally, we proposed a few future research directions related to the phosphorylated ubiquitin.
Abstract: The canonical functions of aminoacyl-tRNA synthetases (aaRS) are charging amino acids to their cognate tRNAs to ensure the precise protein synthesis. Over the course of evolution, aaRS progressively incorporated domains and motifs that have no essential connections to tRNA charging but play roles in cell signaling. These include mediating protein-protein interaction, protein subcellular localization and sensing and transmitting metabolites signals. Deregulated noncanonical aaRS functions are associated with array of human diseases. These all suggest that aaRS play roles beyond their tRNA charging activity, however, the underlying biochemical mechanisms remain to be elucidated. Recent studies revealed that aaRS have aminoacyl transferase activities. An amino acid can be specifically recognized and activated into aminoacyl-AMP by its cognate aaRS, and the formed aminoacyl-AMP in aaRS can modify lysines in proteins that physically interact with this aaRS. This aminoacylation senses and transmits amino acids abundance and side chain information into cell signaling network, provides opportunities to understand why additional domains are acquired by aaRS during evolution and how mutations in an aaRS causes specific human diseases. This review summarizes the noncanonical functions of aaRS and discusses how aaRS mutations may be linked to diseases.
Abstract: Single molecule fluorescence in situ hybridization (smFISH) is a method for imaging single mRNA molecule in fixed cell or tissue using oligonucleotide probes coupled with fluorophores. It can realize real-time study of interested transcripts by RNA localization and quantification. smFISH is widely suitable for many types of biological samples such as cell and tissue sections. It was invented in 1982 which opened up the application of visualizing single molecules. However, due to its shortcomings such as poor binding specificity, Raj et al. optimized this technique in 2008, using 48 independent probes that were separately coupled with fluorophores to locate transcripts. In contrast, methods using multiple labeled probes can distinguish false positive or false negative results due to a single probe misbinding or unbinding event. However, with the continuous application of the technique, it was found that the scheme still has many technical defects, such as low probe specificity, weak fluorescence intensity, low hybridization efficiency, and high background fluorescence. Since then, a series of derivative technologies have been developed. For example, HCR-FISH is a multi-fluorescence in situ hybridization method based on orthogonal amplification and hybridization chain reaction, which significantly improves the problem of weak signal. SeqFISH amplifies the signal and reduces nonspecific binding by continuously hybridizing the mRNA in the cell, imaging it, and stripping the probe in order to barcode RNA. MERFISH utilizes combination labeling, continuous imaging and other technologies to increase detection throughput, and uses binary barcodes to offset single-molecule labeling and detection errors, with more advanced built-in error correction functions to effectively improve the accuracy of results. ClampFISH uses biological orthogonal click chemistry to effectively lock the probe around the target and prevent the probe from disengaging in amplification microscopy. RNAscope amplifies its own signal while simultaneously suppressing the background by using novel probe design strategy and hybridization-based signal amplification system. Split-FISH uses splitting probes for signal enhancement to accurately detect single RNA molecule in complex tissue environments. AmpFISH achieves imaging of short RNA molecules by preparing long single-strand DNA concatemers through controlled rolling circle amplification. CircFISH uses two unique sets of probes (PC probes and PL probes) to distinguish between linear and circular RNAs. π-FISH rainbow enables simultaneous detection of DNA, RNA, and proteins at the single-molecule level with π-FISH target probes. HT-smFISH is more suitable for large or high throughput form of systematic experiments. With the development of technology, the subsequent data analysis process is particularly important. Different analysis software, such as dotdotdot and FISH-quant v2, also improve the process of smFISH. The excellent ability of smFISH to visualize single molecule of RNA makes that it is widely used in basic biological disciplines such as tumor biology, developmental biology, neurobiology, botany, virology. In this paper, we reviewed the basic principle of smFISH technology, its development process and improvement, limitations of smFISH technology and how to avoid them, its derivative technologies include HCR-FISH, SeqFISH, MERFISH, ClampFISH, RNAscope, Split-FISH, AmpFISH, CircFISH, π-FISH rainbow and HT-smFISH. The application progress of smFISH in different biological disciplines, such as developmental biology, tumor biology, neurobiology. Finally, the development prospect of smFISH technology is prospected.
Abstract: Ferroptosis is a kind of cell death triggered by the accumulation of iron-dependent lipid peroxidation products. Like apoptosis, necroptosis, it belongs to the regulated cell death. Numerous studies have shown that ferroptosis is linked to various diseases, such as cancer, neurodegenerative disease and stroke. Activation or inhibition of ferroptosis may play an important role in the treatment of related diseases. Regulating ferroptosis to intervene the occurrence and development of diseases has become a hotspot and focus of current research. Although people have made important discoveries in the molecular regulation of various cell death pathways, the differences in morphological characteristics have important practical significance for pathology departments to identify cell death types and guide the formulation of clinical treatment plans. As a new regulated form of cell death, ferroptosis has many different manifestations from other forms of cell death, among which cell morphological changes are markedly characterized. With the in-depth study of different cell death modes, further analysis and comparison of the morphological characteristics of different cell death forms, and exploration of their similarities and differences are of great significance for identifying cell death forms, judging the pathological process of diseases, and finding appropriate treatment options. This article focuses on the comparison of the morphological features of ferroptosis with other forms of cell death, such as apoptosis, necroptosis, autophagy and pyroptosis. The article shows that ferroptosis has the morphological characteristics of increased mitochondrial membrane densities, reduced or vanished mitochondria crista, rupture of outer mitochondrial membrane. It is obviously different from the morphological features of apoptosis (plasma membrane blebbing, cellular and nuclear volume reduction, mitochondria, Golgi and other organelles in cytoplasm condense, nuclear fragmentation, chromatin condensation and formed apoptotic bodies), autophagy (formation of double membraned autolysosomes), necroptosis (cells become round, swelling of the cytoplasm and organelles, moderate chromatin condensation and rupture of plasma membrane), and pyroptosis (cell edema and membrane rupture, karyopyknosis). We also highlight the involvement of ferroptosis in the major progression of stroke, neurodegenerative diseases and cancer. This paper provides an important basis for the identification and diagnosis of different pathological features.
Abstract: Because of its advantages of high sensitivity, simple method and easy operation, colorimetric biosensing technology has been widely used in many fields such as the detection of pollutants in the biological environment, the detection of important markers in the organism and cancer screening. The colorimetric biosensor based on nanozymes mainly uses the catalytic ability of nanozymes to simulate peroxidase-like activities, oxidize the chromogenic agent to form a colored solution, so as to realize visual detection, and obtain the content of related substances through the detection of its absorbance. Compared with colorimetric biosensors without nanozymes, colorimetric biosensors based on nanozymes have the advantages of higher selectivity, faster detection and higher sensitivity. Nanozymes, on the other hand, are more and more widely studied for their natural enzyme activity while also having the advantages of low cost, good stability, and easy synthesis. At present, colorimetric biosensors based on nanozymes have become an important method to assist related medical detection, and are also widely used in portable and real-time related detection, providing important support and guarantee for medical detection. In order to improve the sensitivity and application range of colorimetric biosensors, researchers are also working on increasing the variety of substances to be detected and diversifying the types of nanozymes. This paper mainly introduces the detection principle of nanozyme-based colorimetric biosensors, several typical used nanozymes, and the application and research progress of nanozyme-based colorimetric biosensors in the field of biomedical detection.
Abstract: Lower-cost genotyping technology has promoted the generation of large genetic datasets with the evolving next-generation sequencing technology. The emergence of genome-wide association studies (GWAS) has facilitated researchers’ understanding of common complex diseases. GWAS refers to finding the sequence variations present in the human genome and screening out disease-related single nucleotide polymorphisms (SNPs). These SNPs are considered as the basis for assessing the stability of complex diseases. However, a single variation is not sufficient to assess an individual’s risk of disease. Polygenic risk score (PRS) is an emerging genetic data analysis method for quantitatively estimating an individual’s genetic risk for complex diseases by comprehensively considering multiple genetic variation sites. A single-value estimate of an individual’s genetic risk for a certain phenotype can be calculated as the cumulative impact of multiple genetic variants by building a PRS model. The finally expected risk score is weighted by the strength and direction of association of each SNP with the phenotype based on the number of alleles carried by each SNP. With the continuous development of various PRS calculation methods and the constant accumulation of genomic data, PRS has received widespread attention in the field of genetics. So far, quite a few studies at home and abroad have shown that PRS is valuable in risk prediction of different types of human traits or complex diseases, and its effectiveness has been further verified in clinical applications. At present, many studies have established PRS models based on GWAS summary statistics to quantify the genetic risk of susceptibility loci and clinical characteristics on diseases such as lung cancer, breast cancer, coronary heart disease, diabetes and Alzheimer’s disease. The disease-susceptible populations can be recognized through comparing the relative risk and absolute risk of the disease in different risk groups according to the population risk stratification results. Additionally, individual-level genotype data and omics data can also be used as data sources for PRS analysis research, especially the latter can dynamically reflect the short-term or long-term effects of environmental factors on human gene expression, and has potential application value in building early warning models to assess health risks. Since the calculation of PRS involves a large amount of genomic data analysis, there are big differences in the methods for data selection, model building and validation. Different PRS construction methods and software have different performances in disease risk prediction, and even the performance of same algorithm varies across diseases. It is worth noting that the PRS model often needs to be re-evaluated and verified for different groups of people, because PRS is affected by race and region. This review combines currently published PRS-related research and algorithms to describe the basic principles of PRS, compares their construction and verification methods, and discusses their applications and prospects. As a powerful genetic risk assessment tool, PRS has great potential in analyzing the genetic code of complex diseases and achieving precise diagnosis and personalized treatment.
Abstract: The UV cross-linking immunoprecipitation (CLIP) technique was first established in 2003. Sequences of target RNAs and binding sites of specific RNA-binding proteins (RBPs) were identified within the entire transcriptome by UV cross-linking, immunoprecipitation, reverse transcription, and subsequent high-throughput sequencing. Over the last 20 years, CLIP has been continuously modified and improved. Advanced operability and accuracy have extended its application category. Currently, the widely used CLIP technologies include high-throughput sequencing with crosslinking-immunoprecipitation (HITS-CLIP), photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP), individual nucleotide resolution CLIP (iCLIP), enhanced CLIP (eCLIP), infrared-CLIP (irCLIP), etc. HITS-CLIP combines high-throughput sequencing with UV cross-linking immunoprecipitation. The 254 nm UV cross-linking and RNAase digestion steps allow the technology to capture transient intracellular RBP-RNA interactions. However, there are limitations in the efficiency of UV cross-linking, with low resolution and high intrinsic background noise. For PAR-CLIP, photoactivatable ribonucleoside was incorporated into RNA molecules, and RBP cross-linked with RNA by 365 nm UV light to improve cross-linking efficiency and resolution. Cross-linking mediated single-base mutations provide more accurate binding site information and reduce interference from background sequences. Long-term alternative nucleotide incorporation, on the other hand, can be cytotoxic and may skew experimental results. iCLIP can identify RBP-RNA cross-linking sites at the single nucleotide level through cDNA circularization and subsequent re-linearization steps, but it has more experimental procedures, and partial cDNAs lost in the circularization step are inevitable. eCLIP discards the radioisotope labeling procedure and reduces RNA loss by ligating adaptors in two separate steps, greatly improving the library-building efficiency, and reducing bias associated with PCR amplification; however, the efficiency of immunoprecipitation cannot be visually assessed at the early stage of the experiment. The irCLIP technique replaces radioisotopes with infrared dyes and greatly reduces the initial number of cells required for the experiment; however, an infrared imaging scanner is essential for the irCLIP application. To address more particular scientific issues, derivative CLIP-related techniques such as PAPERCLIP, cTag-PAPERCLIP, hiCLIP, and tiCLIP have also been developed in recent years. In practice, the aforementioned CLIP approaches have their advantages and disadvantages. When deciding on a technical strategy, we should take into account our experimental objectives and conditions, such as whether we need to precisely define the RNA site for binding to RBP; whether we have the necessary experimental conditions for working with radioisotopes or performing infrared imaging; the amount of initial sample size, and so on. In addition, the CLIP technique has a relatively large number of procedures and can be divided into several successive experimental modules. We can try to combine modules from different mainstream CLIP technologies to meet our experimental requirements, which also gives us more opportunities to improve and refine them and to build more targeted derivative CLIP technologies according to our research objectives.
Abstract: Lactic acid (C3H6O3), also known as 2-hydroxypropionic acid, propanoic acid, is a type of hydroxy acid. It is an essential metabolite of human and microbial cells. In diagnosis and medical management, determination of lactate level in serum is greatly required, and it is also important to measure lactate in fermentative foods to access their quality. Therefore, how to detect lactic acid in different samples with high throughput has become the focus of different researches. The traditional lactic acid detection methods are complicated, time-consuming and laborious, or requires expensive detection equipments. However, the electrochemical enzymatic L-lactate biosensors combining the robustness of electrochemical techniques with the specificity of biological recognition processes showed great advantages over the conventional analytical techniques in size, cost, sensitivity, selectivity, response speed and sample pre-treatment, which show a broad application prospects. There are two main types of lactate biosensors based on L-lactate oxidase (L-LOD) and L-lactate dehydrogenase (L-LDH). Designing a successful enzyme-based L-lactate biosensor requires assembling the enzyme onto a solid carrier and selecting an appropriate transduction strategy between the enzyme and the electrode. Due to the restriction of enzyme molecular structures, reaction mechanism and electrode materials, the traditional lactate biosensors have some limitations in sensitivity, selectivity and stability. Therefore, an increased research was performed to improve the performance of lactate sensors according to the characteristic of the enzymes and the electron transfer type. In this paper, we provide an overview of the structural characteristics, origin and catalytic mechanism of L-LOD and L-LDH, and discuss three strategies, including electrode material modification, enzyme immobilization and enzyme engineering modification, to improve the performance of enzyme electrode based lactate biosensors. In addition, the lactate biosensors were compared and analyzed on the basis of different carriers including membrane, transparent gel matrix, hydrogel carrier, nano-particles, etc. Finally, we comprehensively described the merits and demerits of current commercial lactate sensors and preconceive how emerging new technologies may benefit to future lactate biosensor design.
Abstract: Transient receptor potential vanilloid 1 (TRPV1) channel, belonging to transient receptor potential (TRP) channel superfamily, is a ligand gated non-selective cation channel which can be activated by multiple physical and chemical stimuli. The abnormal irritation and expression of TRPV1 is involved in pathogenesis of various diseases, so that TRPV1 channel is one of the important targets for drug research and development. For a long time, TRPV1 channel has attracted much attention because of the excellent analgesic effect of TRPV1 modulators. Due to the recognition of the research work of receptors for temperature and touch by the 2021 Nobel Prize in physiology or medicine, TRPV1 channel has become the focus of attention once again. It has been more than 20 years of the research for TRPV1, but the gating mechanism and drug development are still the difficulties. TRPV1 agonists can only be used for topical administration, and the antagonists could be used for oral administration. However, the problem with antagonists is that they cause hyperthermia and damage to noxious heat detection, which is the result of TRPV1 antagonists simultaneously affecting capsaicin, H+ and heat gating. Studies have shown that there are common processes of the three gating mechanisms, but no way to affect a single gating mechanism. From the angles of physiological function, gating mechanism and drug discovery, this paper reviews the distribution and expression, functions and features as well as structural characteristics of TRPV1 channel. This paper focuses on three gating mechanisms and the progress of TRPV1 modulators in drug discovery. TRPV1 modulators are a exceptional analgesia drug, and have been studied in cardiovascular diseases, itch, cough, psychiatric disorders and diabetes. With the emerging of artificial intelligence (AI)-assisted drug design and the continuous exploration of gating mechanism, we should have confidence in the future of TRPV1 modulators.
Abstract: Objective As a powerful tool in correcting genomic point mutations, base editors (BEs) show a promising prospect for biotechnology development and therapeutic applications. While editing the target single-nucleotide variant (SNV), it’s primary to select competent BEs and design single guide RNA (sgRNA). Currently, although there are multiple sgRNA design tools, no tools are available for integrating the design of sgRNA with the assessment of the specificity of BEs.Methods 27 cytosine base editors (CBEs) and 12 adenine base editors (ABEs) were used to design base editing schemes. BE-Hive, a third-party tool, was provoked to predict the editing efficiency. The off-target profiles of editing schemes were evaluated by using a combination of multiple off-target prediction tools. Finally, all possible off-target editing products were calculated by considering both base editor types and off-target sites, and then ANNOVAR, a variant annotation tool, was called for functional analysis of off-target products.Results We propose a comprehensive tool, BE-dot, which enables the complete process from a given SNV to designing sgRNAs, predicting off-target profiles, and annotating off-target products’ functions. Besides providing precise correction schemes at DNA level, in order to expand the range of editable SNVs, BE-dot can perform synonymous corrections at protein level by degeneracy. When predicting off-target profiles of single base editing systems, BE-dot integrates multiple tools such as Cas-OFFinder, CALITAS, CFD, uCRISPR and BEdeepoff, which allows BE-dot to evaluate the specificity of single base editing systems more comprehensively and provide users with consultations about BEs and sgRNA selection. In addition, BE-dot can automatically analyze all possible editing products at off-target sites, and convert them into avinput format for functional annotation by ANNOVAR, avoiding the tedious manual annotation.Conclusion BE-dot designs editing schemes for applying base editing to correct or introduce SNVs, and comprehensively evaluates the editing scheme in terms of editing efficiency, off-target profile, and off-target functional impact.