Genome-wide association study of stem structural characteristics that extracted by a high-throughput phenotypic analysis "LabelmeP rice" in rice.

Stem is important for assimilating transport and plant strength; however, less is known about the genetic basis of its structural characteristics. In this study, a high-throughput method, "LabelmeP rice" was developed to generate 14 traits related to stem regions and vascular bundles, which allows the establishment of a stem cross-section phenotype dataset containing anatomical information of 1738 images from hand-cut transections of stems collected from 387 rice germplasm accessions grown over two successive seasons. Then, the phenotypic diversity of the rice accessions was evaluated. Genome-wide association studies identified 94, 83, and 66 significant single nucleotide polymorphisms (SNPs) for the assayed traits in 2 years and their best linear unbiased estimates, respectively. These SNPs can be integrated into 29 quantitative trait loci (QTL), and 11 of them were common in 2 years, while correlated traits shared 19. In addition, 173 candidate genes were identified, and six located at significant SNPs were repeatedly detected and annotated with a potential function in stem development. By using three introgression lines (chromosome segment substitution lines), four of the 29 QTLs were validated. LOC_Os01g70200, located on the QTL uq1.4, is detected for the area of small vascular bundles (SVB) and the rate of large vascular bundles number to SVB number. Besides, the CRISPR/Cas9 editing approach has elucidated the function of the candidate gene LOC_Os06g46340 in stem development. In conclusion, the results present a time- and cost-effective method that provides convenience for extracting rice stem anatomical traits and the candidate genes/QTL, which would help improve rice.

Critical Role of miR-130b-5p in Cardiomyocyte Proliferation and Cardiac Repair in Mice After Myocardial Infarction.

Poor proliferative capacity of adult cardiomyocytes is the primary cause of heart failure after myocardial infarction (MI), thus exploring the molecules and mechanisms that promote the proliferation of adult cardiomyocytes is crucially useful for cardiac repair after MI. Here, we found that miR-130b-5p was highly expressed in mouse embryonic and neonatal hearts and able to promote cardiomyocyte proliferation both in vitro and in vivo. Mechanistic studies revealed that miR-130b-5p mainly promoted the cardiomyocyte proliferation through the MAPK-ERK signaling pathway, and the dual-specific phosphatase 6 (Dusp6), a negative regulator of the MAPK-ERK signaling, was the direct target of miR-130b-5p. Moreover, we found that overexpression of miR-130b-5p could promote the proliferation of cardiomyocytes and improve cardiac function in mice after MI. These studies thus revealed the critical role of miR-130b-5p and its targeted MAPK-ERK signaling in the cardiomyocyte proliferation of adult hearts and proved that miR-130b-5p could be a potential target for cardiac repair after MI.

A LcDOF5.6-LcRbohD regulatory module controls the reactive oxygen species-mediated fruitlet abscission in litchi.

Reactive oxygen species (ROS) have been emerging as a key regulator in plant organ abscission. However, the mechanism underlying the regulation of ROS homeostasis in the abscission zone (AZ) is not completely established. Here, we report that a DOF (DNA binding with one finger) transcription factor LcDOF5.6 can suppress the litchi fruitlet abscission through repressing the ROS accumulation in fruitlet AZ (FAZ). The expression of LcRbohD, a homolog of the Arabidopsis RBOHs that are critical for ROS production, was significantly increased during the litchi fruitlet abscission, in parallel with an increased accumulation of ROS in FAZ. In contrast, silencing of LcRbohD reduced the ROS accumulation in FAZ and decreased the fruitlet abscission in litchi. Using in vitro and in vivo assays, we revealed that LcDOF5.6 was shown to inhibit the expression of LcRbohD via direct binding to its promoter. Consistently, silencing of LcDOF5.6 increased the expression of LcRbohD, concurrently with higher ROS accumulation in FAZ and increased fruitlet abscission. Furthermore, the expression of key genes (LcIDL1, LcHSL2, LcACO2, LcACS1, and LcEIL3) in INFLORESCENCE DEFICIENT IN ABSCISSION signaling and ethylene pathways were altered in LcRbohD-silenced and LcDOF5.6-silenced FAZ cells. Taken together, our results demonstrate an important role of the LcDOF5.6-LcRbohD module during litchi fruitlet abscission. Our findings provide new insights into the molecular regulatory network of organ abscission.

Dissecting the geometric and hydrophobic constraints of stapled peptides.

Stapled peptides are a promising class of molecules with potential as highly specific probes of protein-protein interactions and as therapeutics. Hydrocarbon stapling affects the peptide properties through the interplay of two factors: enhancing the overall hydrophobicity and constraining the conformational flexibility. By constructing a series of virtual peptides, we study the role of each factor in modulating the structural properties of a hydrocarbon-stapled peptide PM2, which has been shown to enter cells, engage its target Mouse Double Minute 2 (MDM2), and activate p53. Hamiltonian replica exchange molecular dynamics (HREMD) simulations suggest that hydrocarbon stapling favors helical populations of PM2 through a combination of the geometric constraints and the enhanced hydrophobicity of the peptide. To further understand the conformational landscape of the stapled peptides along the binding pathway, we performed HREMD simulations by restraining the peptide at different distances from MDM2. When the peptide approaches MDM2, the binding pocket undergoes dehydration which appears to be greater in the presence of the stapled peptide compared with the linear peptide. In the binding pocket, the helicity of the stapled peptide is increased due to the favorable interactions between the peptide residues as well as the staple and the microenvironment of the binding pocket, contributing to enhanced affinity. The dissection of the multifaceted mechanism of hydrocarbon stapling into individual factors not only deepens fundamental understanding of peptide stapling, but also provides guidelines for the design of new stapled peptides.

Molecular Mechanism of P53 Peptide Permeation through Lipid Membranes from Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.

A Novel Hydrated Iron Vanadate Cathode Material for Advanced Aqueous Nickel-Ion Batteries.

Aqueous nickel-ion batteries (ANIBs) as an emerging energy storage device attracted much attention owing to their multielectron redox reaction and dendrite-free Ni anode, yet their development is hindered by the divalent properties of Ni2+ and the lack of suitable cathode materials. Herein, a hydrated iron vanadate (Fe2V3O10.5∙1.5H2O, FOH) with a preferred orientation along the (200) plane is innovatively proposed and used as cathode material for ANIBs. The FOH cathode exhibits a remarkable capacity of 129.3 mAh g-1 at 50 mA g-1 and a super-high capacity retention of 95% at 500 mA g-1 after 700 cycles. The desirable Ni2+ storage capacity of FOH can be attributed to the preferentially oriented and tunnel structures, which offer abundant reaction active planes and a broad Ni2+ diffusion path, the abundant vacancies and high specific surface area further increase ion storage sites and accelerate ion diffusion in the FOH lattice. Furthermore, the Ni2+ storage mechanism and structural evolution in the FOH cathode are explored through ex situ XRD, ex situ Raman, ex situ XPS and other ex situ characteristics. This work opens a new way for designing novel cathode materials to promote the development of ANIBs.

MicroRNA482/2118 is lineage-specifically involved in gibberellin signalling via the regulation of GID1 expression by targeting noncoding PHAS genes and subsequently instigated phasiRNAs.

MicroRNA482/2118 (miR482/2118) is a 22-nt miRNA superfamily, with conserved functions in disease resistance and plant development. It usually instigates the production of phased small interfering RNAs (phasiRNAs) from its targets to expand or reinforce its silencing effect. Using a new high-quality reference genome sequence and comprehensive small RNA profiling, we characterized a newly evolved regulatory pathway of miR482/2118 in litchi. In this pathway, miR482/2118 cleaved a novel noncoding trans-acting gene (LcTASL1) and triggered phasiRNAs to regulate the expression of gibberellin (GA) receptor gene GIBBERELLIN INSENSITIVE DWARF1 (GID1) in trans; another trans-acting gene LcTASL2, targeted by LcTASL1-derived phasiRNAs, produced phasiRNAs as well to target LcGID1 to reinforce the silencing effect of LcTASL1. We found this miR482/2118-TASL-GID1 pathway was likely involved in fruit development, especially the seed development in litchi. In vivo construction of the miR482a-TASL-GID1 pathway in Arabidopsis could lead to defects in flower and silique development, analogous to the phenotype of gid1 mutants. Finally, we found that a GA-responsive transcription factor, LcGAMYB33, could regulate LcMIR482/2118 as a feedback mechanism of the sRNA-silencing pathway. Our results deciphered a lineage-specifically evolved regulatory module of miR482/2118, demonstrating the high dynamics of miR482/2118 function in plants.

Elevated type I IFN signalling directly affects CD8+ T-cell distribution and autoantigen recognition of the skeletal muscles in active JDM patients.

The link between type I IFN and adaptive immunity, especially T-cell immunity, in JDM still remained largely unclear. This study aimed to understand the effect of elevated type I IFN signaling on CD8+ T cell-associated muscle damage in juvenile dermatomyositis (JDM). This study used flow cytometry (FC) and RT‒PCR were used to examine the circulating cell ratio and type I IFN response. And scRNA-seq was used to examine peripheral immunity in 6 active JDM patients, 3 stable JDM patients, 3 juvenile IMNM patients and 3 age-matched healthy children. In vivo validation experiments were conducted using a mouse model induced by STING agonists and an experimental autoimmune myositis model (EAM). In vitro experiments were conducted using isolated CD8+ T-cells from JDM patients and mice. We found that active JDM patients showed an extensive type I IFN response and a decreased CD8+ T-cell ratio in the periphery (P < 0.05), which was correlated with muscle involvement (P < 0.05). Both new active JDM patients and all active JDM patients showed decreased CD8+ TCM cell ratios compared with age and gender matched stable JDM patients (P < 0.05). Compared with new pediatirc systemic lupus erythematosus (SLE) patients, new active JDM patients displayed decreased CD8+ T-cell and CD8+ TCM cell ratios (P < 0.05). Active JDM patient skeletal muscle biopsies displayed an elevated type I IFN response, upregulated MHC-I expression and CD8+ T-cell infiltration, which was validated in EAM mice. sc-RNAseq demonstrated that type I IFN signalling is the kinetic factor of abnormal differentiation and enhances the cytotoxicity of peripheral CD8+ T cells in active JDM patients, which was confirmed by in vivo and in vitro validation experiments. In summary, the elevated type I IFN signalling affected the differentiation and function of CD8+ T cells in active JDM patients. Skeletal muscle-infiltrating CD8+ T cells might migrate from the periphery under the drive of type I IFN and increased MHC I signals. Therapies targeting autoantigen-specific CD8+ T cells may represent a potential new treatment direction.

Increased DNA methylation of the splicing regulator SR45 suppresses seed abortion in litchi.

The gene regulatory networks that govern seed development are complex, yet very little is known about the genes and processes that are controlled by DNA methylation. Here, we performed single-base resolution DNA methylome analysis and found that CHH methylation increased significantly throughout seed development in litchi. Based on the association analysis of differentially methylated regions and weighted gene co-expression network analysis (WGCNA), 46 genes were identified as essential DNA methylation-regulated candidate genes involved in litchi seed development, including LcSR45, a homolog of the serine/arginine-rich (SR) splicing regulator SR45. LcSR45 is predominately expressed in the funicle, embryo, and seed integument, and displayed increased CHH methylation in the promoter during seed development. Notably, silencing of LcSR45 in a seed-aborted litchi cultivar significantly improved normal seed development, whereas the ectopic expression of LcSR45 in Arabidopsis caused seed abortion. Furthermore, LcSR45-dependent alternative splicing events were found to regulate genes involved in seed development. Together, our findings demonstrate that LcSR45 is hypermethylated, and plays a detrimental role in litchi seed development, indicating a global increase in DNA methylation at this stage.

Identification of QTLs associated with very-long chain fatty acid (VLCFA) content via linkage mapping and BSA-seq in peanut.

KEY MESSAGE: Three major QTLs qA01, qB04.1 and qB05 for VLCFA content and their corresponding allele-specific markers will benefit peanut low VLCFA breeding, and a candidate gene Arahy.IF1JV3 was predicted. Peanut is a globally significant oilseed crop worldwide, and contains a high content (20%) of saturated fatty acid (SFA) in its seeds. As high level SFA intake in human dietary may increase the cardiovascular disease risk, reducing the SFA content in peanut is crucial for improving its nutritional quality. Half of the SFAs in peanut are very long-chain fatty acids (VLCFA), so reducing the VLCFA content is a feasible strategy to decrease the total SFA content. Luoaowan with extremely low VLCFA (4.80%) was crossed with Jihua16 (8.00%) to construct an F2:4 population. Three major QTLs including qA01, qB04.1 and qB05 for VLCFA content were detected with 4.43 ~ 14.32% phenotypic variation explained through linkage mapping. Meanwhile, three genomic regions on chromosomes B03, B04 and B05 were identified via BSA-seq approach. Two co-localized intervals on chromosomes B04 (100.10 ~ 103.97 Mb) and B05 (6.39 ~ 10.90 Mb) were identified. With markers developed based on SNP/InDel variations in qA01 between the two parents, the remaining interval was refined to 103.58 ~ 111.14 Mb. A candidate gene Arahy.IF1JV3 encoding a ß-ketoacyl-CoA synthase was found in qA01, and its expression level in Luoaowan was significantly lower than that in Jihua16. Allele-specific markers targeting qA01, qB04.1 and qB05 were developed and validated in F4 population, and an elite line with high oleic, low VLCFA (5.05%) and low SFA (11.48%) contents was selected. This study initially revealed the genetic mechanism of VLCFA content, built a marker-assisted selection system for low VLCFA breeding, and provided an effective method to decrease the SFA content in peanut.

The RNA-binding proteins regulate innate antiviral immune signaling by modulating pattern recognition receptors.

Viral infections pose significant threats to human health, leading to a diverse spectrum of infectious diseases. The innate immune system serves as the primary barrier against viruses and bacteria in the early stages of infection. A rapid and forceful antiviral innate immune response is triggered by distinguishing between self-nucleic acids and viral nucleic acids. RNA-binding proteins (RBPs) are a diverse group of proteins which contain specific structural motifs or domains for binding RNA molecules. In the last decade, numerous of studies have outlined that RBPs influence viral replication via diverse mechanisms, directly recognizing viral nucleic acids and modulating the activity of pattern recognition receptors (PRRs). In this review, we summarize the functions of RBPs in regulation of host-virus interplay by controlling the activation of PRRs, such as RIG-I, MDA5, cGAS and TLR3. RBPs are instrumental in facilitating the identification of viral RNA or DNA, as well as viral structural proteins within the cellular cytoplasm and nucleus, functioning as co-receptor elements. On the other hand, RBPs are capable of orchestrating the activation of PRRs and facilitating the transmission of antiviral signals to downstream adaptor proteins by post-translational modifications or aggregation. Gaining a deeper comprehension of the interaction between the host and viruses is crucial for the development of novel therapeutics targeting viral infections.

E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα.

IL-3/STAT5 signaling pathway is crucial for the development and activation of immune cells, contributing to the cellular response to infections and inflammatory stimuli. Dysregulation of the IL-3/STAT5 signaling have been associated with inflammatory and autoimmune diseases characterized by inflammatory cell infiltration and organ damage. IL-3 receptor α (IL-3Rα) specifically binds to IL-3 and initiates intracellular signaling, resulting in the phosphorylation of STAT5. However, the regulatory mechanisms of IL-3Rα remain unclear. Here, we identified the E3 ubiquitin ligase RNF128 as a negative regulator of IL-3/STAT5 signaling by targeting IL-3Rα for lysosomal degradation. RNF128 was shown to selectively bind to IL-3Rα, without interacting with the common beta chain IL-3Rß, which shares the subunit with GM-CSF. The deficiency of Rnf128 had no effect on GM-CSF-induced phosphorylation of Stat5, but it resulted in heightened Il-3-triggered activation of Stat5 and increased transcription of the Id1, Pim1, and Cd69 genes. Furthermore, we found that RNF128 promoted the K27-linked polyubiquitination of IL-3Rα in a ligase activity-dependent manner, ultimately facilitating its degradation through the lysosomal pathway. RNF128 inhibited the activation and chemotaxis of macrophages in response to LPS stimulation, thereby attenuating excessive inflammatory responses. Collectively, these results reveal that RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα. This study uncovers E3 ubiquitin ligase RNF128 as a novel regulator of the IL-3/STAT5 signaling pathway, providing potential molecular targets for the treatment of inflammatory diseases.

LncRNA SOX1-OT V1 acts as a decoy of HDAC10 to promote SOX1-dependent hESC neuronal differentiation.

Long noncoding RNAs (lncRNAs) are abundantly expressed in the nervous system, but their regulatory roles in neuronal differentiation are poorly understood. Using a human embryonic stem cell (hESC)-based 2D neural differentiation approach and a 3D cerebral organoid system, we show that SOX1-OT variant 1 (SOX1-OT V1), a SOX1 overlapping noncoding RNA, plays essential roles in both dorsal cortical neuron differentiation and ventral GABAergic neuron differentiation by facilitating SOX1 expression. SOX1-OT V1 physically interacts with HDAC10 through its 5' region, acts as a decoy to block HDAC10 binding to the SOX1 promoter, and thus maintains histone acetylation levels at the SOX1 promoter. SOX1 in turn activates ASCL1 expression and promotes neuronal differentiation. Taken together, we identify a SOX1-OT V1/HDAC10-SOX1-ASCL1 axis, which promotes neurogenesis, highlighting a role for lncRNAs in hESC neuronal differentiation.

Antenna effect enhanced ECL immunoassay using microfloral europium porphyrin coordination polymers based on Eu3+ and TCPP for the detection of chloramphenicol in foods.

The "antenna effect" is one of the most important energy transfer modes in lanthanide light-emitting polymers. In this study, novel luminescent nanostructured coordination polymers (Eu-PCP) were synthesized in one step using Eu3+ as the central metal ion and 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) as the organic ligand. The unique "antenna effect" observed between Eu3+ and TCPP leads to a substantial improvement in the electrochemiluminescence (ECL) emission efficiency. Eu-PCP exhibits good cathodic ECL characteristics. Additionally, Au@SnS2 nanosheets exhibit favorable electrical conductivity, biocompatibility, and a significant specific surface area. This makes them a suitable choice as substrate materials for the modification of electrode surfaces and capturing antigens. Being well known, the development of sensitive and rapid methods to detect chloramphenicol is essential for food safety. Based on this, we report a novel competitive electrochemiluminescence immunoassay to achieve ultra-sensitive and highly specific detection of chloramphenicol. The linear range was 0.0002-500 ng mL-1 and the detection limit was 0.09 pg mL-1. Apart from that, the experimental results proved that it provided a new analytical tool for the detection of antibiotic residues in food safety.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples.

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC50 and LOD of the ELISA for ZEN were 0.37 ng mL-1 and 0.04 ng mL-1, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB1, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, ß-zearalanol, zearalanone, α-zearalenol, ß-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R2 = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.

Increased risk of antibiotic resistance in surface water due to global warming.

As the pace of global warming accelerates, so do the threats to human health, urgent priority among them being antibiotic-resistant infections. In the context of global warming, this review summarises the direct and indirect effects of rising surface water temperatures on the development of bacterial antibiotic resistance. First, the resistance of typical pathogens such as E. coli increased with average temperature. This is not only related to increased bacterial growth rate and horizontal gene transfer frequency at high temperatures but also heat shock responses and cumulative effects. Secondly, the acceleration of bacterial growth indirectly promotes antibiotic residues in surface water, which is conducive to the growth and spread of resistant bacteria. Furthermore, the cascading effects of global warming, including the release of nutrients into the water and the resulting increase of bacteria and algae, indirectly promote the improvement of resistance. Water treatment processes exposed to high temperatures also increase the risk of resistance in surface water. The fitness costs of antibiotic resistance under these dynamic conditions are also discussed, concluding the relationship between various factors and resistance persistence. It was expected to provide a comprehensive basis for mitigating antibiotic resistance in the face of global warming.

Development of multiplex allele-specific RT-qPCR assays for differentiation of SARS-CoV-2 Omicron subvariants.

Quick differentiation of current circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmissions. However, the widely applied gene sequencing method is time-consuming and costly especially when facing recombinant variants, because a large part or whole genome sequencing is required. Allele-specific reverse transcriptase real time RT-PCR (RT-qPCR) represents a quick and cost-effective method for SNP (single nucleotide polymorphism) genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 5 multiplex allele-specific RT-qPCR assays targeting 20 key mutations for quick differentiation of the Omicron subvariants (BA.1 to BA.5 and their descendants) and the recombinant variants (XBB.1 and XBB.1.5). Two parallel multiplex RT-qPCR reactions were designed to separately target the prototype allele and the mutated allele of each mutation in the allele-specific RT-qPCR assay. Optimal annealing temperatures, primer and probe dosage, and time for annealing/extension for each reaction were determined by multi-factor and multi-level orthogonal test. The variation of Cp (crossing point) values (ΔCp) between the two multiplex RT-qPCR reactions was applied to determine if a mutation occurs or not. SARS-CoV-2 subvariants and related recombinant variants were differentiated by their unique mutation patterns. The developed multiplex allele-specific RT-qPCR assays exhibited excellent analytical sensitivities (with limits of detection (LoDs) of 1.47-18.52 copies per reaction), wide linear detection ranges (109-100 copies per reaction), good amplification efficiencies (88.25 to 110.68%), excellent reproducibility (coefficient of variations (CVs) < 5% in both intra-assay and inter-assay tests), and good clinical performances (99.5-100% consistencies with Sanger sequencing). The developed multiplex allele-specific RT-qPCR assays in the present study provide an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants. KEY POINTS: • A panel of five multiplex allele-specific RT-qPCR assays for quick differentiation of 11 SARS-CoV-2 Omicron subvariants (BA.1, BA.2, BA.4, BA.5, and their descendants) and 2 recombinant variants (XBB.1 and XBB.1.5). • The developed assays exhibited good analytical sensitivities and reproducibility, wide linear detection ranges, and good clinical performances, providing an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants.

Endoplasmic reticulum stress caused by traumatic injury promotes cardiomyocyte apoptosis through acetylation modification of GRP78.

Cardiomyocyte apoptosis is an important cause of trauma-induced secondary cardiac injury (TISCI), in which the endoplasmic reticulum stress (ERS)-mediated apoptosis signaling pathway is known to be first activated, but the mechanism remains unclear. In this study, rat models of traumatic injury are established by using the Noble-Collip trauma device. The expression of glucose-regulating protein 78 (GRP78, a molecular chaperone of the cardiomyocyte ER), acetylation modification of GRP78 and apoptosis of cardiomyocytes are determined. The results show that ERS-induced GRP78 elevation does not induce cardiomyocyte apoptosis in the early stage of trauma. However, with prolonged ERS, the GRP78 acetylation level is elevated, and the apoptosis of cardiomyocytes also increases significantly. In addition, in the early stage of trauma, the expression of histone acetyl-transferase (HAT) P300 is increased and that of histone deacetylase 6 (HDAC6) is decreased in cardiomyocytes. Inhibition of HDAC function could induce the apoptosis of traumatic cardiomyocytes by increasing the acetylation level of GRP78. Our present study demonstrates for the first time that post-traumatic protracted ERS can promote cardiomyocyte apoptosis by increasing the acetylation level of GRP78, which may provide an experimental basis for seeking early molecular events of TISCI.

Effects of dietary palmitic acid and oleic acid ratio on milk production, nutrient digestibility, blood metabolites, and milk fatty acid profile of lactating dairy cows.

Adequate energy supply is a crucial factor for maintaining the production performance in cows during the early lactation period. Adding fatty acids (FA) to diets can improve energy supply, and the effect could be related to the chain length and degree of saturation of those FA. This study was conducted to evaluate the effect of different ratios of palmitic acid (C16:0) to oleic acid (cis-9 C18:1) on the production performance, nutrient digestibility, blood metabolites, and milk FA profile in early lactation dairy cows. Seventy-two multiparous Holstein cows (63.5 ± 2.61 days in milk) blocked by parity (2.39 ± 0.20), body weight (668.3 ± 20.1 kg), body condition score (3.29 ± 0.06), and milk yield (47.9 ± 1.63 kg) were used in a completely randomized design. Cows were divided into 3 groups with 24 cows in each group. Cows in the 3 treatment groups were provided iso-energy and iso-nitrogen diets, but the C16:0 to cis-9 C18:1 ratios were different: (1) 90.9% C16:0 + 9.1% cis-9 C18:1 (90.9:9.1); (2) 79.5% C16:0 + 20.5% cis-9 C18:1 (79.5:20.5); and (3) 72.7% C16:0 + 27.3% cis-9 C18:1 (72.7:27.3). Fatty acids were added at 1.3% on a dry matter basis. Although the dry matter intake fat-corrected milk yield and energy-corrected milk yield were not affected, the milk yield, milk protein yield, and feed efficiency increased linearly with increasing cis-9 C18:1 ratio. The milk protein percentage and milk fat yield did not differ among treatments, whereas the milk fat percentage tended to decrease linearly with the increasing cis-9 C18:1 ratio. The lactose yield increased linearly and lactose percentage tended to increase linearly with increasing cis-9 C18:1 ratio, but the percentage of milk total solids and somatic cell count decreased linearly. Although body condition scores were not affected by treatments, body weight loss decreased linearly with increasing cis-9 C18:1 ratio. The effect of treatment on nutrient digestibility was limited, except for a linear increase in ether extract and neutral detergent fiber digestibility with increasing cis-9 C18:1 ratio. There was a linear increase in the concentration of plasma glucose, but the triglyceride and nonesterified FA concentrations decreased linearly with increasing cis-9 C18:1 ratio. As the cis-9 C18:1 ratio increased, the concentration of de novo FA decreased quadratically, but the mixed and preformed fatty acids increased linearly. In conclusion, increasing cis-9 C18:1 ratio could increase production performance and decrease body weight loss by increasing nutrient digestibility, and the ratio that had the most powerful beneficial effect on early lactation cows was 72.7:27.3 (C16:0 to cis-9 C18:1).

Development of a flow injection chemiluminescence immunoassay based on DES-mediated CuCo2O4 nanoenzyme for ultrasensitive detection of zearalenone in foods.

Nanoenzymes have been widely used to construct biosensors because of their cost-effectiveness, high stability, and easy modification. At the same time, the discovery of deep eutectic solvents (DES) was a great breakthrough in green chemistry, and their combination with different materials can improve the sensing performance of biosensors. In this work, we report an immunosensor using CuCo2O4 nanoenzyme combined with flow injection chemiluminescence immunoassay for the automated detection of zearalenone (ZEN). The immunosensor exhibited excellent sensing performance. Under the optimal conditions, the detection range of ZEN was 0.0001-100 ng mL-1, and the limit of detection (LOD) was 0.076 pg mL-1 (S/N = 3). In addition, the immunosensor showed excellent stability with a relative standard deviation (RSD) of 2.65% for  15 repetitive  injections. The method has been successfully applied to the analysis of real samples with satisfactory recovery results, and can hence provide a reference for the detection of small molecules in food and feed.