Abstract: Liposomes are hollow spheres composed of lipids bilayer membranes, which can encapsulate and deliver hydrophilic and hydrophobic substances. Liposomes are promising nano-drug delivery systems due to low immunogenicity, good stability, low toxicity and cost. Currently, a variety of liposome drugs for tumor treatment have been listed. Liposomes can accumulate in tumor tissues via enhanced permeability and retention effect (EPR) and are internalized into tumor cells by endocytosis or pinocytosis. Subsequently, liposomes are intracellularly cleaved to release drugs, thereby killing tumor cells. Liposomes that rely on the EPR effect are called passive targeting liposomes, which lack the ability to specifically recognize target tissues. However, active targeting liposomes can achieve targeting delivery via the specific binding between the targeting modifiers on the surface of liposomes and receptors on the surface of tumor cells. These receptors such as peptides, carbohydrates, ligands, antibodies and nucleic acid aptamers on the surface of tumor cells overexpress due to rapid growth of tumor cells and needs of nutrients and related growth factors. Thus, liposomes can be reasonably designed according to these specific receptors. Recent years, some studies have reported biomimetic liposomes by coating the cell membrane on the surface of liposomes, however, the research on biomimetic liposomes is still in its infancy, and there are still many problems to be solved. Additionally, since the length is limited, biomimetic liposomes are not reviewed in this paper. Taken together, liposomes as potential drug carriers, not only protect drugs, but also reduce side effects, importantly, they can precisely target tumor tissues through introducing targeting modifiers. In this work, we review the improvement of targeting function of liposome by five targeting modifiers including peptides, carbohydrates, ligands, antibodies and nucleic acid aptamers, and summarize the existing advantages and challenges of various targeted modifications. Finally, this review is expected to provide scientific reference for the LPs drug delivery system study and theoretical basis for the drug development.
Abstract: Nucleic acid metabolism processes such as the synthesis and degradation of DNA and RNA are the basic metabolic units to maintain the growth and development, metabolism, genetic variation and aging, and widely participate in the whole process of the body’s life activity. The enzyme activity related to nucleic acid metabolism is crucial for maintaining the stability of the intracellular environment, and the change of the activity may cause the occurrence and development of many diseases. The enzymes related to nucleic acid metabolism have become important targets for the study of various diseases and are indispensable tools in the field of biotechnology and bioengineering, such as polymerase chain reaction, site-directed mutagenesis, molecular cloning and DNA sequencing. Therefore, nucleic acid metabolism is the basis of all nucleic acid studies and related life science researches. In this paper, we introduce the common methods of enzymatic analysis of nucleic acid metabolism, and focus on the simple and fast real-time fluorescence method, classify and compare them according to their principles, development history and applications, and also prospect the future study and development of tools for enzymatic analysis of nucleic acid.
Abstract: Mass spectrometry-based proteomics aims to identify peptides and proteins to give direct proofs of gene expressions, analyze structures and functions of proteins, study the relationship between proteins and diseases, and provide targeted treatment options. All these studies are based on the credibility of identified peptides and proteins. However, it is impossible to manually check all identified peptides because a large number of identifications can be collected from one mass spectrometry experiment. Thus, target-decoy approach (TDA) is proposed and always used to control the quality of identified peptides and proteins, and has been expanded to subclasses of peptides (including ordinary subclasses of peptides, variant peptides, and modified peptides) and cross-linking peptides. However, TDA still has two limitations: (1) the estimation of false discovery rate (FDR) is inaccurate and (2) validation of single identification cannot be supported. Thus, the identification results that passed the TDA-based FDR control need to be further validated and other validation methods which are used after TDA-FDR filtration (referred to as Beyond-TDA methods) have been developed to enhance peptide validation. This paper reviews TDA and its extensions as well as Beyond-TDA methods and discusses the advantages and disadvantages of each method. In the first part of this paper, we introduce the goal of proteomics, the process of mass spectrometry acquisition and analysis, the validation problem, and the early statistical methods to evaluate the identification credibility. Then, in the second part of this paper, we describe in detail the ordinary TDA-FDR method, including the assumption that random matches are equally likely to appear in target and decoy databases, the construction methods to generate the decoy database, and the computational formula of TDA-FDR. We also introduce the extensions of TDA-FDR on ordinary subclasses of peptides, variant peptides, modified peptides, proteogenomics peptides, cross-linking peptides, and glycopeptides. However, TDA cannot model the homologous incorrect peptides, thus TDA-FDR underestimates the actual false rate. So, after TDA-FDR filtration, it is necessary to use more strict validation methods, i.e., Beyond-TDA methods, which are reviewed in detail in the third part of this paper, to control validation credibility. In this part, four kinds of methods are introduced, including validation methods based on search space (trap database validation and open search validation), spectra similarity (synthetic peptide validation and theoretical spectra prediction), chemical information (retention time prediction and stable isotopic labeling validation) and machine learning technology (Percolator, pValid, and DeepRescore). Lastly, we summarize the content of this paper and discuss the future improvement directions of validation methods.
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: Objective To study the anti-breast cancer effects and molecular mechanisms of syringin, and to provide a theoretical basis for the clinical application of syringin.Methods The inhibitory effect of syringin on the proliferation of breast cancer cells was measured with MTT assay. Trypan blue, TdT-mediated dUTP nick-end labeling (TUNEL), and Annexin V-FITC/PI staining were used to detect apoptosis. Caspase-3 activation was detected via Western blot to determine whether apoptosis occurred. The expression of apoptosis-associated protein B-cell lymphoma-2 (Bcl-2) was detected and the effect of syringin on the mitochondrial apoptosis pathway was investigated via JC-1 staining. The PI3K agonist Recilisib was used for comparison. qRT-PCR and Western blot were used to assess the role of syringin in regulating the PI3K/Akt/mTOR pathway and inducing the apoptosis of cancer cells.Results Syringin had a time- and dose-dependent inhibitory effect on the proliferation of breast cancer cells and induced their apoptosis. A further study showed that after syringin treatment, Caspase-3 was activated, Bcl-2 expression decreased, the mitochondrial membrane potential was significantly reduced, and the mRNA and protein expressions of PI3K, Akt, and mTOR were not significantly changed, but the protein phosphorylation levels were significantly decreased. Recilisib partially limits the effect of syringin on the apoptosis of breast cancer cells.Conclusion Syringin has a good inhibitory effect on MDA-MB-231 and MCF-7 breast cancer cells. It can inhibit cell proliferation and induce mitochondrial apoptosis by inhibiting the activation of the PI3K/Akt/mTOR signaling pathway. Syringin is a potential anti-breast cancer drug.
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: 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: 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: The CRISPR/Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). The system forms an adaptive immune system in archaea and bacteria. The inherent defense mechanism enables these microorganisms to protect themselves against the invasion of foreign genetic material. The system functions of immune response including three main stages: adaptation, expression/maturation, and interference, each stage needs specific Cas proteins encoded by Cas gene located near the CRISPR sequences, along with other auxiliary proteins. In 2015, Zhang et al. reported Cas12a (Cpf1) as a member of the Class II type V CRISPR/Cas12a system, which possesses endonuclease activity. This finding holds great promise for its application in the field of biotechnology. In 2018, Doudna’s team first applied the CRISPR/Cas12a system for detecting HPV nucleic acid. The system comprises the following essential components in vitro detection: Cas12a, the crRNA sequence complementary to the target DNA, the PAM sequence, and the ssDNA reporter. Cas12a possesses a typical RuvC domain, displaying a canonical bilobed architecture that consists of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe contains the REC1 and REC2 domains, and the NUC lobe includes RuvC, PAM-interacting (PI), Wedge (WED), and bridge helix (BH) domains. The mature crRNA for Cas12a has a length of 42-44 nt, consists of repeat sequence (19/ 20 nt) and spacer sequence (23-25 nt). The crRNA spacer sequence has been found to require a length of 18 nt to achieve complete cleavage activity in vitro. Additionally, mutation in the bases of crRNA can indeed affect the activity of Cas12a. The PAM sequence plays a critical role in the recognition and degradation of DNA by the CRISPR/Cas system, enabling the system to distinguish between self and non-self genomic materials. Cas12a can effectively target the spacer sequence downstream of a T-rich PAM sequence at the 5" end. LbCas12a and AsCas12a both recognize the PAM sequences of 5"-TTTN-3", while FnCas12a recognizes the PAM sequences of 5"-TTN-3". All of these PAM sequences are located upstream on the non-template strand (NTS) at the 5" end. Cas12a (Cpf1), guided by the crRNA, binds to the target DNA by recognizing the PAM sequence. It exhibits the ability to induce arbitrary cleavage of ssDNA within the system while cleaving the target ssDNA or dsDNA. According to this feature, an array of nucleic acid detection methods has been developed for tumor detection and infection diagnostics, such as the DETECTR (RPA-CRISPR/Cas12a method) and HOLMES (PCR-CRISPR/Cas12a method) in 2018. Then, in 2019, Cas12aVDet (one-step detection method), where Cas12a protein was immobilized on the upper wall of the reaction tube. This not only prevented contamination from opening the tube but also reduced the detection reaction time. In 2021, the dWS-CRISPR (digital warm-start CRISPR) was developed as a one-pot detection method. It serves as an accurate approach for quantitatively detecting SARS-CoV-2 in clinical specimens. With the innovation of scientific technology, the high-sensitivity signal transduction technology has also been integrated with the CRISPR/Cas12a system, enabling direct detection of nucleic acids, and eliminating the need for nucleic acid amplification steps. Here, we elaborated the detection principles of CRISPR/Cas12a in in vitro detection. We discussed the different stages leading to the catalytic pathway of target DNA, and the practical applications of Cas12a in nucleic acid detection. These findings revealed a target interference mechanism that originates from the binding of Cas12a-guided RNA complex to complementary DNA sequences within PAM-dependent (dsDNA) regions. The crRNA-DNA binding activates Cas12a, enabling site-specific dsDNA cleavage and non-specific ssDNA trans-cleavage. The release of Cas12a ssDNase activity provides a novel approach to enhance the sensitivity and specificity of molecular diagnostic applications. Before these CRISPR/Cas12a-based nucleic acid detection methods can be introduced into clinical use, substantial work is still required to ensure the accuracy of diagnosis. Nevertheless, we believe that these innovative detection tools based on CRISPR/Cas will revolutionize future diagnostic technologies, particularly offering significant assistance in pathogen infection diagnosis for developing countries with relatively poor healthcare conditions and high prevalence of infectious diseases.
Abstract: Point-of-care testing (POCT) is an innovative diagnostic technology that provides cost-effective and rapid analysis, as well as accurate diagnostics. It enables patients to obtain clinically relevant results through self-testing. This technology has played a vital role in clinical diagnosis, disease monitoring, and early detection of infectious diseases. Nucleic acid aptamers, which are molecular probes capable of specifically recognizing multiple targets, have emerged as valuable components in biomedical sensors for molecular recognition. They offer advantages such as easy synthesis, good stability, and signal amplification. In recent years, research on aptamer-based POCT technology has garnered widespread attention in the world. The key issues in current research include obtaining more high-affinity aptamers to meet the detection needs of various targets, improving detection sensitivity through signal amplification, and integrating with different sensors to meet the requirements of POCT products. In this review, we first briefly introduce the selection process and the targets used for systematic evolution of ligands by exponential enrichment (SELEX). We discuss new SELEX strategies that have been developed to improve the selection efficiency and enhance the affinity of aptamers. Furthermore, we analyze 4 commonly used signal amplification strategies in aptamer-based POCT sensors. Among these methods, nucleic acid signal amplification and self-assembly signal amplification techniques are commonly used due to their low cost and wide applicability. The combination of these two techniques has also been developed to improve detection sensitivity and shorten reaction time. Coupling aptamers with enzyme-based reactions is the simplest method to improve signal amplification in POCT sensors. Various nanomaterials, such as metal nanoparticles, graphene, carbon nanotubes, and metal-organic frameworks, are widely used to improve the detection sensitivity. The combination of multi-functional nanomaterials for signal amplification has also been introduced in this part. Additionally, we introduce a strategy that involves the use of aptamers to initiate the activation of CRISPR-associated proteins, resulting in the cleavage of DNA or RNA molecular beacons and leading to signal amplification. Furthermore, we also introduce the most recent advances in the development of various aptamer-based electrochemical sensors and optical sensors in the field of POCT. Aptamer-based electrochemical sensors offer advantages such as fast response, easy operation, and portability. In this part, we highlight a series of blood glucose meter based aptasensors used to quantify a variety of biomarkers. For the importance of research on continuous detection device, we review recent progress in the development of aptamer-based continues electrochemical testing devices. In aptamer-based optical POCT techniques, the recent development of colorimetry, lateral flow assay (LFA), fluorescence, surface-enhanced Raman scattering (SERS), surface plasmon resonance (SPR), and evanescent wave fiber optic sensors are introduced, with a focus on comparing the performance characteristics of each sensor. Finally, this review presents a summary and future challenges in the research and commercialization of aptamer-based POCT sensors. To simplify the aptamers selection process, it is crucial to invest in studying the molecular recognition mechanisms of aptamers and developing artificial intelligence-based methods for aptamer selection. Additionally, integrating aptamers with advanced sensor technologies like microfluidic chips and wearable devices can greatly enhance detection sensitivity and stability. From a commercial perspective, current aptamer-based POCT products mostly comprise fluorescent or colorimetric assay kits and lateral flow test strips. However, to garner more attention in the competitive POCT market, aptamer-based POCT sensors have an edge in small molecules detection and multi-channel detection.
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: Peroxisome is a kind of organelle conserved in eukaryotes, which is involved in many biochemical metabolic processes, including β-oxidation of fatty acids, production and degradation of reactive oxygen species, etc. Peroxisome biogenesis has growth and division model and de novo biogenesis model, which involves the import of peroxisome matrix and membrane proteins. Under normal physiological conditions, the proliferation and degradation of peroxisomes are balanced. While the matrix protein and membrane proteins in the peroxisome are misfolded and excessively accumulated, or the peroxisome is under environmental stress, such as high reactive oxygen species (ROS) concentration was exhibited, the peroxisomes homeostasis will be unbalanced. In order to maintain homeostasis in the biogenesis process and environmental stress, the peroxisome through division and degradation for quality control. What’s more, peroxisome has evolved multiple degradation pathways, including pexophagy, the receptor accumulation and degradation in the absence of recycling (RADAR) depending on ubiquitin-proteasome system (UPS) and so on. These pathways of peroxisomal degradation are significant for maintaining the integrity of cell structure and function. As the metabolic hub of eukaryotic cells, peroxisomes exchange substances and transmits signals with other organelles through peroxisomal membrane contact sites (MCSs), such as mitochondria, endoplasmic reticulum, lysosome and so on. These peroxisomal MCSs play a vital role in metabolic functions and homeostasis regulation, including lipid metabolism, peroxisome division, autophagy and other biological processes. In recent years, the maintenance of peroxisome homeostasis and MCSs have become research hotspots at home and abroad. The quantity change and spatio-temporal distribution of peroxisome are regularly dynamic to maintain the organism’s normal life activities, while the homeostasis imbalance of peroxisome will result in serious physiological dysfunction of cells. In humans, more and more diseases have been confirmed to be related to the imbalance of peroxisome homeostasis or mutations of peroxisome membrane contact sites, including cancer, diabetes, Alzheimer’s disease and Parkinson’s disease. In plant pathogenic fungi, recent studies have proved that the key genes of peroxisome biogenesis play an important role in pathogenicity, such as of rice blast fungus. This paper reviews the recent advances in the mechanism of peroxisome homeostasis and MCSs.
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: Marine macroalgae (including brown algae, red algae, and green algae) exhibit several features of an excellent feedstock for biorefinery, such as high yield of biomass, no occupation of arable land, and no requirement of fresh water. In 2021, the production of brown algae in China was 1.9 million tons, which was much higher than other economic algae. It is worth noting that the carbohydrate content of brown algae is as high as 60%, and three sugars, including alginate, fucoidan and laminarin are unique to brown algae. Amongst them, alginate is a linear anionic polysaccharide which consists of 1,4-linked C-5-epimers β-D-mannuronic acid (M) and α-L-guluronic acid (G). The decomposition of alginate is catalyzed by alginate lyases via β-elimination of glycosidic bonds. They produce various oligosaccharides with unsaturated uronic acid at the non-reducing end, or 4,5-unsaturated uronic acid monomers mannuronate (ΔManUA) and guluronate (ΔGulUA). Fucoidans usually consist of a backbone of α-1,3-L-fucopyranose residues or alternating α-1,3-linked and α-1,4-linked L-fucopyranosyls, and side branches containing glucose, galactose, rhamnose, xylose, mannose or glucuronic acid. The fucopyranose residues may be substituted with sulfate. The highly modified structure of fucoidans can significantly affect the cleavage of glycosidic linkages. Therefore, hydrolases that act on a branched chain and sulfatases are required for the primary degradation. Subsequently, L-fucoses are produced by a series of sulfatases and fucosidases belonging to GH29, GH95, GH107, GH141, GH151, or GH168 families. Laminarin, the storage polysaccharide in algae, is composed of a linear backbone of 20-30 residues of β-1,3-linked-D-glucopyranose and a branched chain of β-1,6-linked-D-glucopyranose. The glycosidic bond in its backbone can be broken by endo-β-1,3-laminarinases (EC 3.2.1.6 and EC 3.2.1.39) and exo-β-1,3-glucanases (EC 3.2.1.58). The β-1,6-glucanase (EC 3.2.1.75) releases glucose by breaking the glycosidic bond in the branched chain of laminarin. Algae-derived polysaccharides and their oligosaccharides have shown health beneficial effects, such as immunomodulatory, antitumor, anti-inflammatory, and other activities, which possess great potential as alternative, renewable resources in cosmetics and functional foods. In this review, we mainly focus on the efficient degradation of brown algae, and summarize the mechanisms adopted by these enzymes for catalysis and conformation changes of substrate specific recognition. Furthermore, it will provide insights for the precise customization of oligosaccharides and the construction of industrial biorefinery platform, thereby promoting the efficient conversion of brown algae.
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: Diabetic cognitive dysfunction refers to the impairment of cognitive function in diabetic patients, which is a common complication of diabetes. This is especially true for elderly patients with type 2 diabetes mellitus. Studies have shown that adipokines, such as adiponectin (APN) and leptin (LEP) secreted from adipose tissue are implicated not only in the regulation of energy metabolism, but also in the development and progression of diabetic cognitive dysfunction. APN and LEP may serve as biomarkers for diabetes-related cognitive dysfunction. They can cross the blood-brain barrier, enter the brain, and regulate multiple physiological processes such as hippocampal neurogenesis, synaptic plasticity, neuroinflammation, oxidative stress, and neuronal apoptosis by binding to the receptors on neurons or glial cells (e.g., microglia and astrocytes), and activating or inhibiting downstream intracellular signaling pathways, including p38MAPK, AMPK, ERK, JAK2/STAT3, PI3K/AKT, and SIRT1/PGC-1α, etc., and subsequently regulate cognitive function. Importantly, APN and LEP may also act as key mediators in the improvement of diabetic cognitive dysfunction by physical exercise. This study aimed to open up ideas for further enriching the theoretical system of “fat-brain” crosstalk, and developing and refining the diagnosis and treatment strategies of diabetic cognitive dysfunction through analyzing the relationship between APN or LEP and diabetic cognitive dysfunction, sorting out the underlying biological mechanism of APN and LEP regulating cognitive function, and exploring the possible mechanism of exercise-mediated APN and LEP in improving diabetic cognitive dysfunction.
Abstract: Piezo1 is a newly discovered mechanosensitive ion channel in mammals, which plays important functions in different tissues and organs, including bone, urinary tract, eyeball, and artery. However, abnormal Piezo1 mechanical transmission can cause a variety of diseases and promote the course of disease. Fibrotic disease can occur in almost any tissue and organ, and its main feature is excessive cross-linking and accumulation of collagen and other extracellular matrix components, which eventually leads to increased stiffness of tissues and organs and affected physiological functions. At present, more and more studies have shown that Piezo1 plays an important regulatory role in the occurrence and development of fibrotic diseases, which is closely related to the change of matrix mechanical state. This paper describes the structure and activation mechanism of Piezo1, and systematically summarizes the research progress of Piezo1 in fibrotic diseases of the heart, kidney, pancreas, liver and other organs, in order to provide a new perspective and strategy for the treatment of fibrotic diseases.
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: 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: Natriuretic peptides (NPs) have been discovered for 30 years, and the clinical use of B-type natriuretic peptides (BNP) and N-terminal pro-B type natriuretic peptide (NT-proBNP) precursors have been a landmark in the management of cardiovascular disease, particularly in heart failure. The BNP has a powerful cardioprotective effect, but the BNP that rises dramatically after heart failure does not show corresponding activity, which is known as the “natriuretic peptide paradox”. In recent years, with the use of mass spectrometry and nuclear magnetic resonance techniques, “natriuretic peptide paradox” is being revealed through novel metabolic findings and testing technology. There are many different biologically active BNP isoforms in the peripheral circulation, and BNP metabolism after heart failure is different from that in the physiological state. Although the significant increase of BNP is detected after heart failure, it is essentially false positive due to bottlenecks in conventional the assay reagents of cross-react to various BNP isoforms, and therefore the bioactive levels of BNPs have been overestimated. So, we believe that it is necessary to strengthen the understanding of BNP in different pathophysiological conditions , and establish sensitive and specific detection methods by biochemical means to identify BNP1-32, BNP1-30, BNP3-32 and pro-B-type natriuretic peptide (proBNP). Accurate detection of BNPs will help us understand the deeper pathophysiological mechanisms of heart failure, and make precise clinical decision on the diagnosis and treatment.
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