Genome-Wide Identification and Evolutionary Analysis of Receptor-like Kinase Family Genes Provides Insights into Anthracnose Resistance of Dioscorea alata.

Dioscorea alata, commonly known as "greater yam", is a vital crop in tropical and subtropical regions of the world, yet it faces significant threats from anthracnose disease, mainly caused by Colletotrichum gloeosporioides. However, exploring disease resistance genes in this species has been challenging due to the difficulty of genetic mapping resulting from the loss of the flowering trait in many varieties. The receptor-like kinase (RLK) gene family represents essential immune receptors in plants. In this study, genomic analysis revealed 467 RLK genes in D. alata. The identified RLKs were distributed unevenly across chromosomes, likely due to tandem duplication events. However, a considerable number of ancient whole-genome or segmental duplications dating back over 100 million years contributed to the diversity of RLK genes. Phylogenetic analysis unveiled at least 356 ancient RLK lineages in the common ancestor of Dioscoreaceae, which differentially inherited and expanded to form the current RLK profiles of D. alata and its relatives. The analysis of cis-regulatory elements indicated the involvement of RLK genes in diverse stress responses. Transcriptome analysis identified RLKs that were up-regulated in response to C. gloeosporioides infection, suggesting their potential role in resisting anthracnose disease. These findings provide novel insights into the evolution of RLK genes in D. alata and their potential contribution to disease resistance.

Achiral Solvent Inversed Helical Pathway and Cosolvent Controlled Excited-State "Majority Rule" in Enantiomeric Dansulfonamide Assemblies.

Achiral solvents are commonly utilized to induce the self-assembly of chiral molecules. This study demonstrates that achiral solvents can trigger helicity inversion in the assemblies of dansyl amphiphiles and control the excited-state "majority rule" in assemblies composed of pure enantiomers, through variation of the cosolvent ratio. Specifically, enantiomers of dansyl amphiphiles self-assemble into helical structures with opposite handedness in methanol (MeOH) and acetonitrile (MeCN), together with inversed circular dichroism and circularly polarized luminescence (CPL) signals. When a mixture of MeOH and MeCN is employed, the achiral cosolvents collectively affect the CPL of the assemblies in a way similar to that of "mixed enantiomers". The dominant cosolvent governs the CPL signal. As the cosolvent composition shifts from pure MeCN to MeOH, the CPL signals undergo a significant inversion and amplification, with two maxima observed at ≈20% MeOH and 20% MeCN. This study deepens the comprehension of how achiral solvents modulate helical nanostructures and their excited-state chiroptical properties.

Gliotoxin elicits immunotoxicity in the early innate immune system of ducks.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine (ETP) family, which is considered a crucial virulence determinant among the secondary metabolites produced by Aspergillus fumigatus. The metabolites are commonly found in food and feed, contributing to the invasion and immune escape of Aspergillus fumigatus, thereby posing a significant threat to the health of livestock, poultry, and humans. Heterophil extracellular traps (HETs), a novel form of innate immune defense, have been documented in the chicken's innate immune systems for capturing and eliminating invading microbes. However, the effects and mechanisms of GT on the production of duck HETs in vitro remain unknown. In this study, we first confirmed the presence of HETs in duck innate immune systems and further investigated the molecular mechanism underlying GT-induced HETs release. Our results demonstrate that GT can trigger typical release of HETs in duck. The structures of GT-induced HETs structures were characterized by DNA decoration, citrullinated histones 3, and elastase. Furthermore, NADPH oxidase, glycolysis, ERK1/2 and p38 signaling pathway were found to regulate GT-induced HETs. In summary, our findings reveal that gliotoxin activates HETs release in the early innate immune system of duck while providing new insights into the immunotoxicity of GT towards ducks.

Construction and validation of key genes-related prognosis model in children with acute myeloid leukaemia.

INTRODUCTION: To identify the differentially expressed genes of acute myeloid leukaemia (AML) and construct and verify a survival prognosis model combined with patient survival information. METHODS: The TARGET database was searched to identify differentially expressed peripheral blood genes in children with AML and healthy children. A gene set functional analysis and pathway analysis were performed using gene ontology and the KEGG pathway. A prognostic model for children with AML was constructed using univariate Cox, LASSO Cox regression and multivariate Cox regression analyses. Time-dependent receiver operating characteristic (ROC) curves were adopted to assess the predictive capacity of the prognostic models. RESULTS: In total, 1640 differentially expressed genes were screened (1119 upregulated and 521 downregulated genes). The differentially expressed genes were mainly involved in nutrient metabolism and cytochrome P450 metabolism. Six key genes related to the prognosis of AML, FAM157A, GPR78, IRX5, RP4-800G7.1, RP11-179H18.5 and RP11-61N20.3, were identified. Kaplan-Meier curves indicated that 3-year and 5-year overall survival was significantly higher in the low-risk group than in the high-risk group. The area under the ROC curve was 0.722. At different stages of AML, FAM157A and RP4-800G7.1 exhibited significant differences in expression. The expression levels of FAM157A were significantly decreased in AML, whereas the expression levels of GPR78, IRX5, RP4-800G7.1, RP11-179H18.5 and RP11-61N20.3 were significantly increased in AML. CONCLUSION: A prognosis-related gene model of AML was successfully constructed, and the expression levels of the model genes varied with AML stage.

Handedness-Inverted and Stimuli-Responsive Circularly Polarized Luminescent Nano/Micromaterials Through Pathway-Dependent Chiral Supramolecular Polymorphism.

The precise manipulation of supramolecular polymorphs has been widely applied to control the morphologies and functions of self-assemblies, but is rarely utilized for the fabrication of circularly polarized luminescence (CPL) materials with tailored properties. Here, this work reports that an amphiphilic naphthalene-histidine compound (NIHis) readily self-assembled into distinct chiral nanostructures through pathway-dependent supramolecular polymorphism, which shows opposite and multistimuli responsive CPL signals. Specifically, NIHis display assembly-induced CPL from the polymorphic keto tautomer, which become predominant during enol-keto tautomerization shifting controlled by a bulk solvent effect. Interestingly, chiral polymorphs of nanofiber and microbelt with inverted CPL signals can be prepared from the same NIHis monomer in exactly the same solvent compositions and concentrations by only changing the temperature. The tunable CPL performance of the solid microbelts is realized under multi external physical or chemical stimuli including grinding, acid fuming, and heating. In particular, an emission color and CPL on-off switch based on the microbelt polymorph by reversible heating-cooling protocol is developed. This work brings a new approach for developing smart CPL materials via supramolecular polymorphism engineering.

Direct programming of human pluripotent stem cells into endothelial progenitors with SOX17 and FGF2.

Transcription factors (TFs) are pivotal in guiding stem cell behavior, including their maintenance and differentiation. Using single-cell RNA sequencing, we investigated TFs expressed in endothelial progenitors (EPs) derived from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Conversely, SOX17 knockdown via CRISPR-Cas13d significantly compromised EP differentiation. Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31- cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into endothelial cells. These findings underscore an undiscovered role of SOX17 in programming hPSCs toward an EP lineage, illuminating pivotal mechanisms in EP differentiation.

Combination of Sodium Nitroprusside and Controlled Atmosphere Maintains Postharvest Quality of Chestnuts through Enhancement of Antioxidant Capacity.

The purpose of this study was to clarify the effect of CA (controlled atmosphere, 2-3% O2 + 3% CO2) and NO (nitric oxide, generated by 0.4 nM sodium nitroprusside), alone or combined (CA + NO), on the physio-chemical properties, enzyme activities and antioxidant capacities of chestnuts during storage at 0 °C for 180 d. Compared with control (CT), CA and CA+NO both improved the storage quality of the samples, but only CA resulted in more ethanol production. Moreover, these improvements were further enhanced and ethanol synthesis was inhibited by the addition of NO. A spectrometer was used to assess the production of phenolic content (TPC) and activities of phenylalanine ammonia-lyase (PAL), superoxide dismutas (SOD), peroxidase (POD), catalase (CAT) and polyphenol oxidase (PPO) as influenced by CA or CA+NO treatments. Higher TPC, PAL, SOD, POD, CAT, and lower PPO were observed in CA alone, and more so in the combination with NO group. The increased antioxidant production and enhanced antioxidant activities contributed to scavenging reactive oxygen species (ROS) and reducing malondialdehyde (MDA). This study unveiled the correlations and differences between the effects of CA and CA+NO on storage quality, providing valuable insights into postharvest preservation and suggesting that the combination (CA+NO) was more beneficial for quality maintenance in chestnuts.

A hybrid photocatalytic system enables direct glucose utilization for methanogenesis.

Integration of methanogenic archaea with photocatalysts presents a sustainable solution for solar-driven methanogenesis. However, maximizing CH4 conversion efficiency remains challenging due to the intrinsic energy conservation and strictly restricted substrates of methanogenic archaea. Here, we report a solar-driven biotic-abiotic hybrid (biohybrid) system by incorporating cadmium sulfide (CdS) nanoparticles with a rationally designed methanogenic archaeon Methanosarcina acetivorans C2A, in which the glucose synergist protein and glucose kinase, an energy-efficient route for glucose transport and phosphorylation from Zymomonas mobilis, were implemented to facilitate nonnative substrate glucose for methanogenesis. We demonstrate that the photo-excited electrons facilitate membrane-bound electron transport chain, thereby augmenting the Na+ and H+ ion gradients across membrane to enhance adenosine triphosphate (ATP) synthesis. Additionally, this biohybrid system promotes the metabolism of pyruvate to acetyl coenzyme A (AcCoA) and inhibits the flow of AcCoA to the tricarboxylic acid (TCA) cycle, resulting in a 1.26-fold augmentation in CH4 production from glucose-derived carbon. Our results provide a unique strategy for enhancing methanogenesis through rational biohybrid design and reprogramming, which gives a promising avenue for sustainably manufacturing value-added chemicals.

DNase I rescues goat sperm entrapped by neutrophil extracellular traps.

Artificial insemination has been a predominant technique employed in goat husbandry for breeding purposes. Subsequent to artificial insemination, sperm can elicit inflammation in the reproductive tract, resulting in substantial the accumulation of neutrophils. Recognized as foreign entities, sperm may become entrapped within neutrophil extracellular traps (NETs) released by neutrophils, thereby exploiting their properties of pathogen elimination. Deoxyribonuclease I (DNase I), which is known for disintegrating NETs and causing loss of function, has been utilized to ameliorate liver and brain damage resulting from NETs, as well as to enhance sperm quality. This study investigated the mechanism of sperm-induced NETs and further explored the impact of DNase I on NETs. Sperm quality was evaluated using optical microscopy, while the structure of NETs was observed through immunofluorescence staining. The formation mechanism of NETs was examined using inhibitors and PicoGreen. The findings revealed that sperm induced the formation of NETs, a process regulated by glycolysis, NADPH oxidase, ERK1/2, and p38 signaling pathways. The composition of NETs encompassed DNA, citrullinated histone H3 (citH3), and elastase (NE). DNase I protects sperm by degrading NETs, thereby concurrently preserving the integrity of plasma membrane and motility of sperm. In summary, the release of sperm-induced NETs leads to its damage, but this detrimental effect is counteracted by DNase I through degradation of NETs. These observations provide novel insights into reproductive immunity in goats.

Mechanism of transport and toxicity response of Chlorella sorokiniana to polystyrene nanoplastics.

The toxicity of nanoparticles to freshwater microalgae is of significant importance in maintaining the overall stability of aquatic ecosystems. However, the transport mechanism and toxicity response of microalgae towards nanoplastics (NPs) remain to be further investigated. In this study, we examined the toxicity and internalization mechanisms of polystyrene nanoplastics (PS-NPs) in the microalga Chlorella sorokiniana. The results revealed that the PS-NPs inhibited algal cells' growth and disrupted cell integrity upon contact, leading to cell shrinkage or rupture. Moreover, amino-modified PS-NPs (Nano-PS-NH2) exhibited greater toxicity to C. sorokiniana than carboxyl-modified PS-NPs (Nano-PS-COOH). Furthermore, significant inhibition of PS-NPs internalization was observed when four different endocytosis-related inhibitors were used, indicating that internalized PS-NPs can enter algal cells through endocytic pathways. More importantly, C. sorokiniana exposed to Nano-PS-NH2 responded to the reduction in carbon sources and energy resulting from the suppression of photosynthesis by regulating the metabolism of carbohydrates. These findings elucidate the effects of PS-NPs on C. sorokiniana, including their impact on cell morphology and metabolism, while shedding light on the internalization mechanisms of NPs by C. sorokiniana which deepen our understanding of the toxicity of nanoplastics on algae and provide important theoretical support for solving such aquatic ecological environment problems.

Pathway-Dependent Self-Assembly for Control over Helical Nanostructures and Topochemical Photopolymerization.

Pathway-dependent self-assembly, in which a single building block forms two or more types of self-assembled nanostructures, is an important topic due to its mimic to the complexity in biology and manipulation of diverse supramolecular materials. Here, we report a pathway-dependent self-assembly using chiral glutamide derivatives (L or D-PAG), which form chiral nanotwist and nanotube through a cooperative slow cooling and an isodesmic fast cooling process, respectively. Furthermore, pathway-dependent self-assembly can be harnessed to control over the supramolecular co-assembly of PAG with a luminophore ß-DCS or a photopolymerizable PCDA. Fast cooling leads to the co-assembled PAG/ß-DCS nanotube exhibiting green circularly polarized luminescence (CPL), while slow cooling to nanofiber with blue CPL. Additionally, fast cooling process promotes the photopolymerization of PCDA into a red chiral polymer, whereas slow cooling inhibits the polymerization. This work not only demonstrates the pathway-dependent control over structural characteristics but also highlights the diverse functions emerged from the different assemblies.

Engineering of a Self-Regulatory Bidirectional DNA Assembly Circuit for Amplified MicroRNA Imaging.

The engineering of catalytic hybridization DNA circuits represents versatile ways to orchestrate a complex flux of molecular information at the nanoscale, with potential applications in DNA-encoded biosensing, drug discovery, and therapeutics. However, the diffusive escape of intermediates and unintentional binding interactions remain an unsolved challenge. Herein, we developed a compact, yet efficient, self-regulatory assembly circuit (SAC) for achieving robust microRNA (miRNA) imaging in live cells through DNA-templated guaranteed catalytic hybridization. By integrating the toehold strand with a preblocked palindromic fragment in the stem domain, the proposed miniature SAC system allows the reactant-to-template-controlled proximal hybridization, thus facilitating the bidirectional-sustained assembly and the localization-intensified signal amplification without undesired crosstalk. With condensed components and low reactant complexity, the SAC amplifier realized high-contrast intracellular miRNA imaging. We anticipate that this simple and template-controlled design can enrich the clinical diagnosis and prognosis toolbox.

Toxoplasma gondii-induced neutrophil extracellular traps are relevant to glycolysis, TLR2, and TLR4 MAPK signaling pathway in goats.

Toxoplasma gondii (T. gondii) exhibits a significantly high prevalence of infection in goats, leading to adverse consequences such as abortion and stillbirth in ewes, thereby posing a substantial challenge to the goat farming industry. Neutrophil extracellular traps (NETs) have been shown to capture T. gondii in goats; however, the precise mechanisms underlying NET release in goats remain poorly understood. Therefore, the aim of our research was to elucidate the involved mechanism. We assessed the cytotoxicity of T. gondii on neutrophils using CCK-8 assay, visualized the structure of T. gondii-induced goat NETs through immunofluorescence, quantified ROS release during T. gondii-induced NET formation using fluorescence microplate analysis, and employed inhibitors targeting TLR 2, TLR4, NADPH oxidase, ERK1/2, and P38 MAPK signaling pathways as well as glycolysis to dissect the mechanisms underlying T. gondii-induced NET release. Within 1 h, T. gondii did not exhibit significant cytotoxicity towards neutrophils in our findings. The formation of typical NET structures induced by T. gondii involved DNA, citrullinated histone 3 (citH3), and neutrophil elastase (NE). Additionally, T. gondii significantly stimulated the release of NETs in goats. The process was accompanied by the production of reactive oxygen species (ROS) mediated through NADPH oxidase, p38, and ERK1/2 signaling pathways. Inhibition of these pathways resulted in a decrease in NET release. Moreover, inhibition of TLR 2, TLR4, and glycolysis also led to a reduction in T. gondii-induced NET release. Overall, our study demonstrates that T. gondii can induce characteristic NET structures and elucidates the involvement of various mechanisms including TLR2/TLR4 signaling pathway activation, NADPH oxidase activity modulation via ROS production regulation through p38 MAPK and ERK1/2 signaling pathways, and glycolysis regulation during the innate immune response against T. gondii infection in goats.

Regulating Lithium Transfer Pathway to Avoid Capacity Fading of Nano Si Through Sub-Nano Scale Interfused SiOx /C Coating.

Si nanoparticles (NPs) are considered as a promising high-capacity anode material owing to their ability to prevent mechanical failure from drastic volume change during (de)lithiation. However, upon cycling, a quick capacity fading is still observed for Si NPs, and the underlying mechanism remains elusive. In this contribution, it is demonstrated that the quick capacity fading is mainly caused by the generation of dead (electrochemically inert) Si with blocked electron conductivity in a densely composited Si/SEI (solid electrolyte interface) hybrid. This is due to the combined influence of electrolyte-related side reactions and the accompanied agglomeration of Si NPs. A compact, sub-nano scale interfused SiOx /C composite coating onto the Si NPs is constructed, and a highly stabilized electrochemistry is achieved upon long cycling. The SiOx /C coating with electron/ion dual transport paths and robust mechanical flexibility enables a fast and stable lithium ion/electron dual diffusion pathway towards the encapsulated Si. With fast reaction kinetics, stable SEI, and an antiagglomeration feature, the obtained Si@SiOx /C composite demonstrates a stable high capacity. This work unravels new perspectives on the capacity fading of Si NPs and provides an effective encapsulating method to remedy the structure degradation and capacity fading of nano Si.

Exploring the Core Parameters of CNC-Based Chiral Nematic Structures for Enhancing the Dissymmetry Factor of Right-Handed Circularly Polarized Luminescence.

The chiral nematic nanostructure formed from cellulose nanocrystal (CNC) self-assembly has shown great potential as a matrix for generating circularly polarized luminescent (CPL) light with a high dissymmetry factor. Exploring the relationship between the device composition and structure and the light dissymmetry factor is crucial to a common strategy for a strongly dissymmetric CPL light. In this study, we have compared the single-layered and double-layered CNC-based CPL devices with different luminophores, such as rhodamine 6G (R6G), methylene blue (MB), crystal violet (CV), and silicon quantum dots (Si QDs). We demonstrated that forming a double-layered structure of CNCs nanocomposites is a simple but effective pathway for enhancing the CPL dissymmetry factor for CNC-based CPL materials containing different luminophores. The |glum| values of double-layered CNC devices (dye@CNC5||CNC5) versus that of single-layered devices (dye@CNC5) are 3.25 times for Si QDs, 3.7 times for R6G, 3.1 times for MB, and 2.78 times for CV series. The different enhancement degrees of these CNC layers with a similar thickness may be due to the different pitch numbers in the chiral nematic liquid crystal layers whose photonic band gap (PBG) has been modified to match the emission wavelengths of dyes. Furthermore, the assembled CNC nanostructure has great tolerance to the addition of nanoparticles. Gold nanorods coated with the SiO2 layer (Au NR@SiO2) were added for enhancing the dissymmetry factor of MB in CNC composites (named MAS devices). When the strong longitudinal plasmonic band of the Au NR@SiO2 matched the emission wavelength of MB and the PBG of assembled CNC structures simultaneously, the increase in the glum factor and quantum yield of MAS composites was obtained. The good compatibility of the assembled CNC nanostructures makes it a universal platform for developing strong CPL light sources with a high dissymmetry factor.

Pathological characteristics and congenital immunological responses of pigeons-infected with Neospora caninum.

Pigeons are natural intermediate host of Neospora caninum (N. caninum). In comparison to ruminants, N. caninum causes milder clinical symptoms and less financial loss to pigeons. Natural infectious rates and high prevalence of N. caninum in pigeons, and death cases of N. caninum-infected pigeons under experimental conditions have been reported, but the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum remain not well described. In this study, pigeons were infected intraperitoneally with 107 N. caninum tachyzoites. N. caninum in tissues was detected by qPCR. Pathological changes of tissues were examined by hematoxylin-eosin staining. Blood smears were prepared for counting eosinophils changes in blood. Heterophil extracellular traps (HETs) in vivo and in vitro were quantified by Pico Green. N. caninum-induced HETs structures were observed by immunofluorescence staining. The model of pigeons-infected with N. caninum was successfully established. Lung and duodenum were the main target organs of pigeons-infected with N. caninum. N. caninum caused hemorrhage, edema and inflammatory cell infiltration in liver, pulmonary congestion and hemorrhage, organizational destruction in lung, and shorter villi or even disappear in duodenum. N. caninum also increased the number of eosinophils in blood of pigeons. Moreover, N. caninum-induced HETs release in the congenital immunological system of pigeons were first demonstrated, and the HETs structures were consisted of DNA as the skeleton and modified with citH3 and elastase. N. caninum-induced HETs release was related with NADPH oxidase, TLR 2 and 4, ERK1/2 and p38 MAPK signaling pathways, and glycolysis. In summary, it is the first report on the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum, which may provide theoretical basis for the prevention and control of Neosporosis in pigeons.

Parental phubbing and academic burnout in adolescents: the role of social anxiety and self-control.

Based on the limited resource model of self-control, we construct a chain mediation model to examine the relationship between parental phubbing and adolescents' academic burnout, and whether social anxiety and self-control play a mediating role in it. We used 4 questionnaires to investigate parental phubbing, social anxiety, self-control, and adolescents' academic burnout among 828 high school students in Wuhu and Huangshan City, Anhui Province, China. The findings indicated that: (1) parental phubbing, social anxiety, and self-control all significantly predict adolescents' academic burnout directly and (2) parental phubbing could indirectly influence adolescents' academic burnout through three pathways: the separate mediating effect of social anxiety and self-control, and the chain mediating effect on both. The results of this study help parents understand how their phubbing actions affect adolescents' academic burnout and the mechanism of action.

Solvent-dependent supramolecular assembly behavior of a coumarin-headed amphiphile.

To study the effect of solvent on supramolecular self-assembly behaviors, a chiral courmarin-substituted glutamine amphiphile, L/DG-Cm, was synthesized for investigation. It was found that L/DG-Cm self-assembled into short nanotubes in toluene, while it formed longer nanotubes together with an obvious helix nanobelt structure for L/DG-Cm in DMSO, demonstrating that the nanotubes were formed by nanobelt rolling. The CD and CPL spectra revealed the same chiral property of the L/DG-Cm assemblies formed in toluene and DMSO. Theoretical calculations revealed that LG-Cm was prone to forming similar dimer structures in both DMSO and toluene. However, the distinct hierarchical packing ways in toluene and DMSO led to different nanostructures and chiroptical properties. Based on the temperature-dependent UV-visible and CD spectrometric measurements, LG-Cm was observed to aggregate in different supramolecular self-assembly modes, which was the cooperative (nucleation-elongation) mechanism in toluene and the isodesmic model in DMSO. This work proves that the solvent not only affects the self-assembly morphologies and properties but also determines the self-assembly pathways.

Toxoplasma gondii triggers heterophil extracellular traps via NADPH oxidase, ERK1/2 and P38 signalling pathways, glycolysis and autophagy in chickens.

Toxoplasma gondii is a zoonotic parasite with a global distribution. Heterophil extracellular traps (HETs) are a novel innate immune mechanism of chickens against pathogens, but whether T. gondii can induce HETs release in chickens has not been reported. The effects of T. gondii on heterophils viability were assessed by using Cell Counting Kit-8. T. gondii-induced HETs were observed and analysed by the immunofluorescence method. T. gondii-induced reactive oxygen species (ROS) was determined by the DCFH-DA method. The mechanisms underlying T. gondii-triggered HETs were investigated by inhibitors and fluorescence microplate reader. T. gondii did not significantly affect heterophils viability at a 1:1 ratio within 1 h. It was demonstrated for the first time that T. gondii could induce HETs release in chicken, and the structure of HETs was comprised of DNA, elastase and citrullinated histone 3 (citH3). T. gondii increased ROS production in a dose-dependent manner. Inhibitors of NADPH oxidase, extracellular signal-regulated kinase 1/2 (ERK1/2 ) and P38 signalling pathways, glycolysis and autophagy significantly decreased the release of T. gondii-induced HETs. Taken together, T. gondii can induce HETs release in chickens, and ROS, NADPH oxidase, ERK1/2 and P38 signalling pathways, glycolysis and autophagy participate in the process of HETs release, which provides new insights into the innate immune mechanism of chickens against T. gondii infection.

STAMP-Based Digital CRISPR-Cas13a for Amplification-Free Quantification of HIV-1 Plasma Viral Loads.

Quantification of HIV RNA in plasma is critical for identifying the disease progression and monitoring the effectiveness of antiretroviral therapy. While RT-qPCR has been the gold standard for HIV viral load quantification, digital assays could provide an alternative calibration-free absolute quantification method. Here, we reported a Self-digitization Through Automated Membrane-based Partitioning (STAMP) method to digitalize the CRISPR-Cas13 assay (dCRISPR) for amplification-free and absolute quantification of HIV-1 viral RNAs. The HIV-1 Cas13 assay was designed, validated, and optimized. We evaluated the analytical performances with synthetic RNAs. With a membrane that partitions ∼100 nL of reaction mixture (effectively containing 10 nL of input RNA sample), we showed that RNA samples spanning 4 orders of dynamic range between 1 fM (∼6 RNAs) to 10 pM (∼60k RNAs) could be quantified as fast as 30 min. We also examined the end-to-end performance from RNA extraction to STAMP-dCRISPR quantification using 140 µL of both spiked and clinical plasma samples. We demonstrated that the device has a detection limit of approximately 2000 copies/mL and can resolve a viral load change of 3571 copies/mL (equivalent to 3 RNAs in a single membrane) with 90% confidence. Finally, we evaluated the device using 140 µL of 20 patient plasma samples (10 positives and 10 negatives) and benchmarked the performance with RT-PCR. The STAMP-dCRISPR results agree very well with RT-PCR for all negative and high positive samples with Ct < 32. However, the STAMP-dCRISPR is limited in detecting low positive samples with Ct > 32 due to the subsampling errors. Our results demonstrated a digital Cas13 platform that could offer an accessible amplification-free quantification of viral RNAs. By further addressing the subsampling issue with approaches such as preconcentration, this platform could be further exploited for quantitatively determining viral load for an array of infectious diseases.