Update on functional analysis of long non-coding RNAs in common crops.

With the rapid advances in next-generation sequencing technology, numerous non-protein-coding transcripts have been identified, including long noncoding RNAs (lncRNAs), which are functional RNAs comprising more than 200 nucleotides. Although lncRNA-mediated regulatory processes have been extensively investigated in animals, there has been considerably less research on plant lncRNAs. Nevertheless, multiple studies on major crops showed lncRNAs are involved in crucial processes, including growth and development, reproduction, and stress responses. This review summarizes the progress in the research on lncRNA roles in several major crops, presents key strategies for exploring lncRNAs in crops, and discusses current challenges and future prospects. The insights provided in this review will enhance our comprehension of lncRNA functions in crops, with potential implications for improving crop genetics and breeding.

Enhanced laccase production by mutagenized Myrothecium verrucaria using corn stover as a carbon source and its potential in the degradation of 2-chlorophen.

Chlorophenols are widely used in industry and are known environmental pollutants. The degradation of chlorophenols is important for environmental remediation. In this study, we evaluated the biodegradation of 2-chlorophenol using crude laccase produced by Myrothecium verrucaria. Atmospheric and room temperature plasma technology was used to increase laccase production. The culture conditions of the M-6 mutant were optimized. Our results showed that corn stover could replace glucose as a carbon source and promote laccase production. The maximum laccase activity of 30.08 U/mL was achieved after optimization, which was a 19.04-fold increase. The biodegradation rate of 2-chlorophenol using crude laccase was 97.13%, a positive correlation was determined between laccase activity and degradation rate. The toxicity of 2-CP was substantially reduced after degradation by laccase solution. Our findings show the feasibility of the use of corn stover in laccase production by M. verrucaria mutant and the subsequent biodegradation of 2-chlorophenol using crude laccase.

Chrysin enhances antitumour immunity response through the IL-12-STAT4 signal pathway in the B16F10 melanoma mouse model.

Chrysin (CHR) is a flavonoid with extensive pharmacological activity. The molecular formula of CHR is C15 H10 O4 . CHR is reported to have antioxidative, antitumour and antiviral functions. To evaluate its potential function as a vaccine adjuvant, we prepared a melanoma vaccine using a soluble protein extract of B16F10 melanoma cells as antigen and CHR as an adjuvant. The melanoma model was developed after two immunizations, and it was discovered that combining B16F10 soluble protein antigen-mixed CHR vaccine could inhibit tumour growth in the mouse model, and the overall survival rate was higher than that of the B16F10 antigen vaccine alone. In vivo and in vitro experiments were conducted to determine whether CHR functioned as an adjuvant by activating antigen-presenting cells (APCs). We discovered that CHR activated APCs both in vivo and in vitro and may enhance Th1 cell function by activating the IL12-STAT4 signal pathway, thereby enhancing the antitumour response of cytotoxic T lymphocytes (CTLs) in vivo. Next, to verify the critical role of CD8+ T cells in suppressing melanoma development, we transplanted CD8+ T cells from immunized mice to B16F10 tumour-bearing mice and discovered that the survival rate of tumour-bearing mice was significantly prolonged. In summary, our experimental results indicate that CHR can be used as a potential adjuvant to enhance antigen immunogenicity, inhibit B16F10 tumour growth in mice and improve tumour immune response.

Cross-species opsonic activity of zebrafish fish-egg lectin on mouse macrophages.

Zebrafish Fish-egg lectin (zFEL) has been identified and proved to be a maternal factor with antibacterial and opsonic ability in fishes. In this study, we found that zFEL was capable of enhancing the phagocytosis of the bacteria by macrophages of mouse (RAW264.7 and mouse peritoneal macrophages), suggesting a cross-species function of zFEL in higher animals. Further studies showed that zFEL can active the antigen presentation ability by up-regulating the expression of CD80, CD86 and MHC II. Meanwhile, zFEL also promoted the polarization of macrophages to M1-type, which was confirmed by the increase of cytokines TNF-α and IL-6. The expression of p38 gene was up-regulated in macrophages preincubated with zFEL. Taken together, zFEL appears opsonic function in mammal macrophages and has potential application in immunomodulation.

Human Cytomegalovirus Immediate Early Protein 2 Protein Causes Cognitive Disorder by Damaging Synaptic Plasticity in Human Cytomegalovirus-UL122-Tg Mice.

Human cytomegalovirus (HCMV) infection is very common in the human population all around the world. Although the majority of HCMV infections are asymptomatic, they can cause neurologic deficits. Previous studies have shown that immediate early protein 2 (IE2, also known as UL122) of HCMV is related with the cognitive disorder mechanism. Due to species isolation, a HCMV-infected animal model could not be established which meant a study into the long-term effects of IE2 on neural development could not be carried out. By establishing HCMV-UL122-Tg mice (UL122 mice), we explored the cognitive behavior and complexity of neuron changes in this transgenic UL122 mice that could consistently express IE2 protein at different ages (confirmed in both 6- and 12-month-old UL122 mice). In the Morris water maze, cognitive impairment was more pronounced in 12-month-old UL122 mice than in 6-month-old ones. At the same time, a decrease of the density of dendritic spines and branches in the hippocampal neurons of 12-month-old mice was observed. Moreover, long-term potentiation was showed to be impaired in 12-month-old UL122 mice. The expressions of several synaptic plasticity-regulated molecules were reduced in 12-month-old UL122 mice, including scaffold proteins postsynaptic density protein 95 (PSD95) and microtubule-associated protein 2 (MAP2). Binding the expression of IE2 was increased in 12-month-old mice compared with 6-month-old mice, and results of statistical analysis suggested that the cognitive damage was not caused by natural animal aging, which might exclude the effect of natural aging on cognitive impairment. All these results suggested that IE2 acted as a pathogenic regulator in damaging synaptic plasticity by downregulating the expression of plasticity-related proteins (PRPs), and this damage increased with aging.

Apoptosis Disorder, a Key Pathogenesis of HCMV-Related Diseases.

Human cytomegalovirus (HCMV) belongs to the ß-herpesvirus family, which is transmitted in almost every part of the world and is carried by more than 90% of the general population. Increasing evidence indicates that HCMV infection triggers numerous diseases by disrupting the normal physiological activity of host cells, particularly apoptosis. Apoptosis disorder plays a key role in the initiation and development of multiple diseases. However, the relationship and molecular mechanism of HCMV-related diseases and apoptosis have not yet been systematically summarized. This review aims to summarize the role of apoptosis in HCMV-related diseases and provide an insight into the molecular mechanism of apoptosis induced by HCMV infection. We summarize the literature on HCMV-related diseases and suggest novel strategies for HCMV treatment by regulating apoptosis.

Disruption of a key ligand-H-bond network drives dissociative properties in vamorolone for Duchenne muscular dystrophy treatment.

Duchenne muscular dystrophy is a genetic disorder that shows chronic and progressive damage to skeletal and cardiac muscle leading to premature death. Antiinflammatory corticosteroids targeting the glucocorticoid receptor (GR) are the current standard of care but drive adverse side effects such as deleterious bone loss. Through subtle modification to a steroidal backbone, a recently developed drug, vamorolone, appears to preserve beneficial efficacy but with significantly reduced side effects. We use combined structural, biophysical, and biochemical approaches to show that loss of a receptor-ligand hydrogen bond drives these remarkable therapeutic effects. Moreover, vamorolone uniformly weakens coactivator associations but not corepressor associations, implicating partial agonism as the main driver of its dissociative properties. Additionally, we identify a critical and evolutionarily conserved intramolecular network connecting the ligand to the coregulator binding surface. Interruption of this allosteric network by vamorolone selectively reduces GR-driven transactivation while leaving transrepression intact. Our results establish a mechanistic understanding of how vamorolone reduces side effects, guiding the future design of partial agonists as selective GR modulators with an improved therapeutic index.

The Risk of Gastrointestinal Hemorrhage in Low-Dose Aspirin Users with Diabetes Mellitus: Systematic Review and Meta-Analysis.

BACKGROUND: Our aim was to assess the risk of gastrointestinal (GI) hemorrhage associated with diabetes among patients taking low-dose aspirin (≤325 mg/day). METHODS: A systematic search was conducted for publication in English and Chinese using term equivalents for "GI hemorrhage", "aspirin", and "diabetes mellitus" up till April 2020. Electronic databases include PUBMED, EMBASE, Cochrane Library databases, Chinese National Knowledge Infrastructure (CNKI), Wanfang Database, and VIP Database. Two independent authors searched databases and reviewed abstracts for comprehensive studies keeping adequate study quality. Data of weighted odds ratios were statistically evaluated and potential bias was checked. RESULTS: Among 446 publications, eight case-control researches, including 1601 patients, were deemed for this meta-analysis. Patients with diabetes were associated with a higher risk of GI hemorrhage than patients without diabetes: the summary ORs were 3.10 (95% CI, 2.35-4.09). The heterogeneity of the reports was not significant (Chi2 = 3.39, P = 0.85; I 2 = 0%). CONCLUSION: The meta-analysis showed that aspirin users with diabetes were more likely to have GI hemorrhage. Hence, when treating diabetics with aspirin, the increased risk of GI bleeding should be taken in consideration.

First High-Resolution Crystal Structures of the Glucocorticoid Receptor Ligand-Binding Domain-Peroxisome Proliferator-Activated γ Coactivator 1-α Complex with Endogenous and Synthetic Glucocorticoids.

Both synthetic and endogenous glucocorticoids are important pharmaceutic drugs known to bind to the ligand-binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor (NR) superfamily. Ligand binding induces conformational changes within GR, resulting in subsequent DNA binding and differential coregulator recruitment, ultimately activating or repressing target gene expression. One of the most crucial coregulators is peroxisome proliferator-activated γ coactivator 1-α (PGC1α), which acts to regulate energy metabolism by directly interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α senses GR conformation to drive transcription are not completely known. Here, an ancestral variant of the GR (AncGR2) LBD was used as a tool to produce stable protein for biochemical and structural studies. PGC1α is found to interact more tightly and form a more stable complex with AncGR2 LBD than nuclear receptor coactivator 2. We report the first high-resolution X-ray crystal structures of AncGR2 LBD in complex with PGC1α and dexamethasone (DEX) or hydrocortisone (HCY). Structural analyses reveal how distinct steroid drugs bind to GR with different affinities by unique hydrogen bonds and hydrophobic interactions. Important charge clamps are formed between the activation function 2 and PGC1α to mediate their specific interactions. These interactions lead to a high level of protection from hydrogen-deuterium exchange at the coregulator interaction site and strong intramolecular allosteric communication to ligand binding site. This is the first structure detailing the GR-PGC1α interaction providing a foundation for future design of specific therapeutic agents targeting these critical metabolic regulators. SIGNIFICANCE STATEMENT: High-resolution structures of AncGR2 LBD bound to DEX and HCY in complex with PGC1α reveal the molecular mechanism of PGC1α binding to AncGR2 LBD as well as the distinct affinities between DEX and HCY binding. Identifying the structural mechanisms that drive drug affinity is of pharmacologic interest to the glucocorticoid receptor field as an avenue to guide future drug design targeting GR-PGC1α signaling, which plays a crucial role in controlling hepatic glucose output.

Gastrodin, a traditional Chinese medicine monomer compound, can be used as adjuvant to enhance the immunogenicity of melanoma vaccines.

Gastrodin (GAS) is a Chinese medicine with wide application for the treatment of nervous system disease. Previous studies reported that GAS exhibited non-specific immunomodulatory activities. To explore the effects of GAS as a vaccine adjuvant, the expression levels of CD80, CD86, MHCI and MHCII activated markers were detected after GAS treatment in vitro and in vivo, and the expression levels of IL-2 and TNF-α in splenocytes were detected after GAS treatment in vivo. Besides, the expression levels of IL-2 and IFN-γ in CD4+T cells and perforin, TNF-α and IFN-γ in CD8+T cells were detected. The effects of GAS on the survival rate and tumor size of tumor-challenged mice and the effect of cytotoxicity on CD8+T cells were also investigated. Our data showed that GAS ameliorated CD8+T cell mediated immune response and significantly improved protection of tumor-challenged animals. The results demonstrated that GAS is a potential adjuvant contributing to anticancer immunomodulation.

The hepatic lectin of zebrafish binds a wide range of bacteria and participates in immune defense.

C-type lectins (CTLs) have a diverse range of functions including cell-cell adhesion, immune response to pathogens and apoptosis. Asialoglycoprotein receptor (ASGPR), also known as hepatic lectin, a member of CTLs, was the first animal lectin identified, yet information regarding it remains rather limited in teleost. In this study, we identified a putative protein in zebrafish, named as the zebrafish hepatic lectin (Zhl). The zhl encoded a typical Ca2+-dependent carbohydrate-binding protein, and was mainly expressed in the liver in a tissue specific fashion. Challenge with LPS and LTA resulted in significant up-regulation of zhl expression, suggesting involvement in immune response. Actually, recombinant C-type lectin domain (rCTLD) of Zhl was found to be capable of agglutinating and binding to both Gram-negative and Gram-positive bacteria and enhancing the phagocytosis of the bacteria by macrophages. Moreover, rCTLD specifically bound to insoluble lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), which were inhibited by galactose. Interestingly, Zhl was located in the membrane, and its overexpression could inhibit the production of pre-inflammatory cytokines. Taken together, these results indicate that Zhl has immune activity capable of defending invading pathogens, enriching our understanding of the function of ASGPR/hepatic lectin.

Identification and bioactivity analysis of a newly identified defensin from the oyster Magallana gigas.

The relatively conserved sequences of signal peptides and proregions that antimicrobial peptides (AMPs) contain have been successfully used to search for and identify novel AMPs from databases within the same lineages of fish and amphibians and across different animal classes. If such an approach is applicable to invertebrate species such as oyster has not yet been tested so far. In this study, we found a cDNA from the digestive gland of the oyster Magallana gigas, designated Mgdefdg, which contains two exons interspaced by one intron. Mgdefdg coded for a protein with features characteristic of defensins. The mature peptide had the cysteine-stabilized α-helix/ß-sheet motif (CSαß) and the consensus pattern C-X5-6-C-X3-C-X4-6-C-X3-4-C-X7-8-C-X-C-X2-C forming potential disulfide linkages C1-C5, C2-C6, C3-C7 and C4-C8 in the predicted tertiary structure. Functional assays revealed that recombinant mature MgDefdg (rmMgDefdg) was able to kill the Gram-negative bacterium Aeromonas hydrophila and the Gram-positive bacterium Staphylococcus aureus, and to induce bacterial membrane/cytoplasmic damage. ELISA showed that rmMgDefdg had high affinity to both A. hydrophila and S. aureus as well as the microbe-associated molecular pattern molecules LPS and LTA. Moreover, rmMgDefdg was capable of causing bacterial membrane permeabilization and depolarization, and intracellular ROS increase. Additionally, rmMgDefdg was not cytotoxic to human red blood cells and murine RAW264.7 cells. Taken together, our results indicate that MgDefdg is a previously uncharacterized defensin with membrane selectivity towards bacterial cells. It also shows that the use of conserved sequences of signal peptides of defensins can be an effective tool to identify potential defensins across different animal genera in invertebrates.

Identification and expression of carbonic anhydrase 2, myosin regulatory light chain 2 and selenium-binding protein 1 in zebrafish Danio rerio: Implication for age-related biomarkers.

Proteomic study has determined age-related changes in synthesis of carbonic anhydrase 2, myosin regulatory light chain 2 and selenium-binding protein 1 in muscle of post-menopausal women. However, little information is available regarding the expression and role of these proteins in early development and life span. In this study we showed that zebrafish ca2, myl2a, myl2b and selenbp1 were highly identical to their mammalian counterparts in primary and tertiary structures as well as genomic organization and syntenic map. They displayed distinct spatiotemporal expression patterns in embryos and larvae of zebrafish. Moreover, their transcription levels in the respective tissues were obviously remodeled in an age-dependent fashion, i.e. some mRNA levels were increased, while others remained unchanged or even decreased, suggesting that CA2, MYL2a, MYL2b and SELENBP1 can be used as aging biomarkers. Our study also lays a foundation for further illumination of the functions of these genes in early development and aging processes.

Augmentation of the antibacterial activities of Pt5-derived antimicrobial peptides (AMPs) by amino acid substitutions: Design of novel AMPs against MDR bacteria.

The ever-growing concerns on multi-drug resistant (MDR) bacteria lead to urgent demands for novel antibiotics including antimicrobial peptides (AMPs). Pt5, a peptide consisting of the C-terminal 55 residues of zebrafish phosvitin, has been shown to function as an antibacterial agent. Here we used Pt5 as a template to design new AMPs by shortening the sequence and substituting with tryptophan (W) and lysine (K) at selected positions. Among the resultant Pt5-derived peptides, Pt5-1c showed the strongest antimicrobial activity against both Gram-negative and Gram-positive bacteria, including MDR bacteia, with the minimum inhibitory concentrations (MICs) ranging from 1.2 µM to 4.8 µM. Electron microscopic examination showed that Pt5-1c was able to kill the bacteria directly. ELISA revealed that Pt5-1c possessed high affinity to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Importantly, Pt5-1c was able to disrupt the bacterial membrane by a combined action of membrane depolarization and permeabilization, with little cytotoxicity to mammalian cells. Taken together, these findings suggest that Pt5-1c has considerable potential for future development as novel peptide antibiotics against MDR bacteria.

Identification and functional characterization of fish-egg lectin in zebrafish.

Fish-egg lectins (FELs) are identified in several species of fishes, but their activity and mode of action remain largely unknown in early life stages. Here we showed that zebrafish FEL (zFEL) was a maternal factor, which was capable of interacting with Gram-negative and Gram-positive bacteria and enhancing the phagocytosis of the bacteria by macrophages. Interestingly, microinjection of purified native zFEL into the embryos (resulting in the increase of zFEL in the embryos) markedly promoted the resistance of the embryos to the pathogenic Aeromonas hydrophila. Taken together, zFEL appears a maternal immune-relevant molecule capable of defending the developing embryos/larvae from pathogenic attacks.