Effects of sleep deprivation on anxiety-depressive-like behavior and neuroinflammation.

BACKGROUND: Depression is defined by a persistent low mood and disruptions in sleep patterns, with the WHO forecasting that major depression will rank as the third most prevalent contributor to the global burden of disease by the year 2030. Sleep deprivation serves as a stressor that triggers inflammation within the central nervous system, a process known as neuroinflammation. This inflammatory response plays a crucial role in the development of depression by upregulating the expression of inflammatory mediators that contribute to symptoms such as anxiety, hopelessness, and loss of pleasure. METHODS: In this study, sleep deprivation was utilized as a method to induce anxiety and depressive-like behaviors in mice. The behavioral changes in the mice were then evaluated using the EZM, EPM, TST, FST, and SPT. H&E staining and Nissl staining was used to detect morphological changes in the medial prefrontal cortical (mPFC) regions. Elisa to assess serum CORT levels. Detection of mRNA levels and protein expression of clock genes, high mobility genome box-1 (Hmgb1), silent message regulator 6 (Sirt6), and pro-inflammatory factors by RT-qPCR, Western blotting, and immunofluorescence techniques. RESULTS: Sleep deprivation resulted in decreased exploration of unfamiliar territory, increased time spent in a state of despair, and lower sucrose water intake in mice. Additionally, sleep deprivation led to increased secretion of serum CORT and upregulation of clock genes, IL6, IL1ß, TNFα, Cox-2, iNOS, Sirt6, and Hmgb1. Sleep. CONCLUSIONS: Sleep deprivation induces anxiety-depressive-like behaviors and neuroinflammation in the brain. Transcription of clock genes and activation of the Sirt6/Hmgb1 pathway may contribute to inflammatory responses in the mPFC.

The failure behavior of coal-rock combined body under compression-shear loading.

As shallow coal reserves continue to deplete rapidly, deep mining has become an unavoidable course of action. In the process of deep coal mining, affected by blasting, mining, and excavation, the coal-rock interface often encounters the action of compression-shear composite load. The interface crack directly affects the stability of the coal-rock structure. Uniaxial compression experiments have been conducted on rock and coal-like material with pre-existing interfacial oblique cracks to study the crack propagation criterion of the rock and coal-like material interface. An image acquisition system is used to record the surface speckle field of the whole process of specimen failure. The strain fields and stress intensity factor of the sample at different times are obtained by digital image correlation. At the same time, the stress field near the crack tip is calculated. The results show that with the increase of loading, two strains of localization bands are formed on the surface of the specimen. One of the strain-localized bands starts at the lower tip of the prefabricated crack and propagates along the vertical interface. The other starts at the upper tip of the prefabricated damage and propagates along the interface direction. It can be seen that the lower tip of the prefabricated crack enters into the crack fracture process zone earlier than the upper tip. The strain localization band narrows gradually with the load increase, and then macro cracks appear. The initiation of two tips is suitable for different fracture criteria. The lower tip is dominated by the maximum circumferential tensile stress, and the upper tip is dominated by shear stress. The specimens eventually fracture along the interface. The evolution of the strain field during the failure process of rock and coal-like material can reflect the generation and propagation of the crack.

Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix.

Introduction: Apicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2). Methods: The cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein. Results: The EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel ß-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (P<0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of E. necatrix. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein. Discussion: EnApiAP2 protein encoded by ENH_00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study.

Comparative proteomic analysis of wall-forming bodies and oocyst wall reveals the molecular basis underlying oocyst wall formation in Eimeria necatrix.

BACKGROUND: The durable oocyst wall formed from the contents of wall-forming bodies (WFBs) protects Eimeria parasites from harsh conditions and enhances parasite transmission. Comprehending the contents of WFBs and proteins involved in oocyst wall formation is pivotal to understanding the mechanism of the oocyst wall formation and the search for novel targets to disrupt parasite transmission. METHODS: Total proteins extracted from WFBs and the oocyst wall of Eimeria necatrix were subjected to comparative proteomic analysis using tandem mass tag in conjunction with liquid chromatography tandem-mass spectrometry techniques. After functional clustering analysis of the identified proteins, three proteins, including E. necatrix disulfide isomerase (EnPDI), thioredoxin (EnTrx) and phosphoglycerate kinase (EnPGK), were selected for further study to confirm their potential roles in oocyst wall formation. RESULTS: A total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively. Among these proteins, 1102 were identified as differentially expressed proteins, of which 506 were upregulated and 596 downregulated in the oocyst wall compared to the WFBs. A total of 108 proteins, including compositional proteins of the oocyst wall, proteases, oxidoreductases, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport, were proposed to be involved in oocyst wall formation. The approximate molecular sizes of native EnPDI, EnTrx and EnPGK proteins were 55, 50 and 45 kDa, respectively. EnPDI was present in both type 1 and type 2 WFBs, EnTrx was present only in type 2 WFB2 and EnPGK was present only in type 1 WFBs, whereas all of them were localized to the outer layer of the oocyst wall, indicating that all of them participate in the formation of the oocyst wall. CONCLUSIONS: To the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix. The data obtained from this study form a basis for deciphering the molecular mechanisms underlying oocyst wall formation of Eimeria parasites. They also provide valuable resources for future studies on the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection.

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

Exploring the therapeutic potential of natural compounds for Alzheimer's disease: Mechanisms of action and pharmacological properties.

Alzheimer's Disease (AD) is a global public health priority characterized by high mortality rates in adults and an increasing prevalence in aging populations worldwide. Despite significant advancements in comprehending the pathogenesis of AD since its initial report in 1907, there remains a lack of effective curative or preventive measures for the disease. In recent years, natural compounds sourced from diverse origins have garnered considerable attention as potential therapeutic agents for AD, owing to their anti-inflammatory, antioxidant, and neuroprotective properties. This review aims to consolidate the therapeutic effects of natural compounds on AD, specifically targeting the reduction of ß-amyloid (Aß) overproduction, anti-apoptosis, autophagy, neuroinflammation, oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Notably, the identified compounds exhibiting these effects predominantly originate from plants. This review provides valuable insights into the potential of natural compounds as a reservoir of novel therapeutic agents for AD, thereby stimulating further research and contributing to the development of efficacious treatments for this devastating disease.

Characterization of the novel glucose-methanol-choline (GMC) oxidoreductase EnOXIO1 in Eimeria necatrix.

Eimeria species are intracellular obligate parasites, among the most common pathogens affecting the intensive poultry industry. Oxidoreductases are members of a class of proteins with redox activity and are widely found in apicomplexan protozoans. However, there have been few reports related to Eimeria species. In this study, total RNA was extracted from the gametocytes of E. necatrix Yangzhou strain to amplify the EnOXIO1 gene using reverse-transcription polymerase chain reaction. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into Escherichia coli BL21(DE3), and the recombinant protein rEnOXIO1 was expressed by induction with isopropyl ß-D-1-thiogalactopyranoside. The full length EnOXIO1 gene was 2535 bp encoding 844 amino acids, and the EnOXIO1 protein had a molecular weight of about 100 kDa and was mainly expressed in inclusion bodies. Western blot analysis indicated that the rEnOXIO1 protein had good antigenicity and cross-reactivity and was specifically recognized by a 6 ×HIS labeled monoclonal antibody, mouse anti-recombinant protein polyclonal antibody, and recovery serum from chickens infected with E. necatrix, E. acervulina, and E. tenella sporulated oocysts. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein was mainly located on the wall-forming bodies in gametocytes and played an important role in the formation of the oocyst wall. Quantitative PCR analysis revealed that transcript levels of EnOXIO1 were highest in the gametocyte stage. Protein expression levels of EnOXIO1 were higher in the gametocyte stage than in other developmental stages according to western blot analysis. Vaccination of chickens against E. necatrix was achieved with recombinant protein rEnOXIO1, which triggered humoral immunity and antibody production, increased average body weight gain, reduced oocyst output and alleviated lesions after E. necatrix infection. The highest ACI value (172.36) was observed in chickens that received 200 µg rEnOXIO1 compared with other immunization groups.

Hesperetin attenuates cognitive dysfunction via SIRT6/NLRP3 pathway in scopolamine-induced mice.

Neuroinflammation is a critical feature in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). Hesperetin can exert anti-inflammatory, antioxidant and other neuroprotective effects. In this study, the scopolamine (SCOP)-induced cognitive dysfunction in mice model was used to evaluate the neuroprotective effects of hesperetin. Behavioral tests (Morris water maze, open field, and novel object recognition tests) were conducted to evaluate the effect of hesperetin on cognitive dysfunction behaviors. Nissl staining and Immunofluorescence were used to evaluate hippocampal neuronal damage and microglial activation in mice. The levels of proinflammatory factors, oxidant stress, and the cholinergic neurotransmitter were detected by real-time quantitative fluorescence PCR (RT-qPCR) or biochemical reagent kits. Western blotting was used to detect the relative protein expression of the sirtuin 6 (SIRT6) / NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway. Results showed that hesperetin could ameliorate SCOP-induced cognitive impairment and neuronal damage, and regulate the levels of cholinergic neurotransmitters in the hippocampal of AD mice. Hesperetin could also enhance antioxidant defense by regulating the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Hesperetin exerted anti-neuroinflammation effects through inhibiting of microglia activation and down-regulating the mRNA transcript levels of inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Meanwhile, hesperetin could attenuate the expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), thioredoxin-interacting protein (TXNIP), and caspase-1 p20 and upregulate the expression of SIRT6 in SCOP-induced mice. Overall, our study suggested that hesperetin might ameliorate SCOP-induced cognitive dysfunction by improving cholinergic system dysfunction and suppressing oxidative stress and attenuating neuroinflammation via SIRT6/NLRP3 pathway in mice.

iTRAQ-based proteomic analysis reveals invasion-related proteins among three developmental stages of Eimeria necatrix.

Eimeria necatrix is an obligate intracellular parasite that has a complex life cycle and causes significant economic losses to the poultry industry. To better understand the cellular invasion mechanism of E. necatrix and develop new measures against its infection, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis to investigate protein abundance across different life cycle stages, including unsporulated oocysts (UO), sporozoites (SZ) and second-generation merozoites (MZ-2). Our analysis identified a total of 3606 proteins, among which 1725, 1724, 2143 and 2386 were annotated by the Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and InterPro (IPR) databases, respectively. We also identified 388, 300 and 592 differentially abundant proteins in SZ vs UO, SZ vs MZ-2 and MZ-2 vs UO, respectively. Further analysis revealed that 118 differentially abundant proteins were involved in cellular invasion and could be categorized into eight groups. These findings provide valuable insights into protein abundance across the different life cycle stages of E. necatrix and offer candidate proteins for future studies on cellular invasion and other biological processes. SIGNIFICANCE: Eimeria necatrix is an obligate intracellular parasite results in huge economic losses to the poultry industry. Understanding proteomic variations across the life cycle stages of E. necatrix may offer proteins related to cellular invasion of E. necatrix, and provide resources for the development of new treatment and prevention interventions against E. necatrix infection. The current data provide an overall summary of the protein abundance across the three life cycle stages of E. necatrix. We identified differentially abundant proteins potential related to cellular invasion. The candidate proteins we identified will form the basis of future studies for cellular invasion. This work also will help in the development of novel strategies for coccidiosis control.

Network Proximity-based computational pipeline identifies drug candidates for different pathological stages of Alzheimer's disease.

Despite the massive investment in Alzheimer's disease (AD), there are still no disease-modifying treatments (DMTs) for AD. One major reason is attributed to the limitation of clinical "one-size-fits-all" approach, since the same AD treatment solely based on clinical diagnosis was unlikely to achieve good clinical efficacy. In recent years, computational approaches based on multiomics data have provided an unprecedented opportunity for drug discovery since they can substantially lower the costs and boost the efficiency. In this study, we intended to identify potential drug candidates for different pathological stages of AD by computationally repurposing Food and Drug Administration (FDA) approved drugs. First, we assembled gene expression data from three different AD pathological stages, which include mild cognitive impairment (MCI) and early and late stages of AD (EAD, LAD). We next quantified the network distances between drug target networks and AD modules by utilizing a network proximity approach, and identified 193 candidates that possessed significant associations with AD. After searching for previous literature evidence, 63 out of 193 (32.6%) predicted drugs were demonstrated to exert therapeutic effects on AD. We further explored the novel mechanism of action (MOA) for these drug candidates by determining the specific brain cells they might function on based on AD patient single cell transcriptomic data. Additionally, we selected several promising candidates that could cross the blood brain barrier together with confirmed neuroprotective effects, and subsequently determined the antioxidative activity of these compounds. Experimental results showed that azathioprine decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) levels and improved the superoxide dismutase (SOD) activity in APP-SH-SY5Y cells. Finally, we deciphered the potential MOA of azathioprine against AD via network analysis and validated several apoptosis-related proteins (Caspase 3, Cleaved Caspase 3, Bax, Bcl2) through western blotting. In summary, this study presented an effective computational strategy utilizing omics data for AD drug repurposing, which provides a new perspective for drug discovery and development.

Kai-Xin-San protects against mitochondrial dysfunction in Alzheimer's disease through SIRT3/NLRP3 pathway.

BACKGROUND: Kai-Xin-San (KXS) has been reported to have a good curative impact on dementia. The purpose of the study was to determine whether KXS might ameliorate cognitive deficits in APP/PS1 mice and to evaluate its neuroprotective mechanism. METHODS: APP/PS1 mice were employed as an AD animal model; Aß1-42 and KXS-containing serum were used in HT22 cells. Four different behavioral tests were used to determine the cognitive ability of mice. Nissl staining was utilized to detect hippocampal neuron changes. ROS, SOD, and MDA were used to detect oxidative stress levels. Transmission electron microscopy and Western blot were used to evaluate mitochondrial morphology, mitochondrial division, and fusion state. Western blotting and immunofluorescence identified PSD95, BDNF, NGF, SYN, SIRT3, and NLRP3 inflammasome levels. RESULTS: The results indicated that KXS protected APP/PS1 mice against cognitive impairments. KXS suppressed neuronal apoptosis and oxidative stress among APP/PS1 mice. KXS and KXS-containing serum improved mitochondrial dysfunction and synaptic and neurotrophic factors regarding APP/PS1 mice. In addition, KXS and KXS-containing serum enhanced mitochondrial SIRT3 expression and reduced NLRP3 inflammasome expression in APP/PS1 mice. CONCLUSION: KXS improves cognitive dysfunction among APP/PS1 mice via regulating SIRT3-mediated neuronal cell apoptosis. These results suggested that KXS was proposed as a neuroprotective agent for AD progression.

Network Pharmacology and Mechanism Studies of the Protective Effect of Ginseng against Alzheimer's Disease Based on Aß Pathogenesis.

Alzheimer's disease (AD) is a critical neurodegenerative disease that manifests as progressive intellectual decline and is pathologically characterized by a progressive loss of neurons in the brain. Despite extensive research on this topic, the pathogenesis of AD is not fully understood, while the beta-amyloid (Aß) hypothesis remains the dominant one and only a few symptomatic drugs are approved for the treatment of AD. Ginseng has been widely reported as an effective herbal medicine for the treatment of neurodegenerative diseases such as dementia. Therefore, we explore the protective effects of ginseng in AD by a network pharmacological approach based on the pathogenesis of Aß. Twenty-one major ginsenosides are screened based on ultraperformance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) data. Among them, MAPK8, MAPK9, BACE1, FLT1, CDK2, and CCR5 are the core targets. By molecular docking and validation with the in vitro cell model APPswe-SH-SY5Y, we find that ginsenosides Rg3 and Ro have good neuroprotective effects and can reduce the expression of Aß 1 - 42 in APPswe-SH-SY5Y. Finally, through RT-qPCR experiment, we find that ginsenoside Rg3 targeted MAPK8, FLT1, and CCR5, while ginsenoside Ro targeted MAPK8, MAPK9, FLT1, and CCR5 for its potential anti-AD efficacy.

Ferroptosis regulator SLC7A11 is a prognostic marker and correlated with PD-L1 and immune cell infiltration in liver hepatocellular carcinoma.

Background: Liver hepatocellular carcinoma (LIHC) is a complicated disease with poor survival and lack of viable treatment options. The roles of ferroptosis and immunotherapy in LIHC are increasingly prominent, but the interplay of ferroptosis with the tumor microenvironment (TME) in LIHC is currently under-investigated. Methods: In this study, we analyzed normal liver tissues and tumor tissues from the TCGA and GTEx databases to obtain differentially expressed ferroptosis-related genes (FRGs). We then clustered LIHC based on the expression levels of selected FRGs and acquired distinct subtypes with significant heterogeneity regarding survival prognoses, PD-L1 expression, and immune cell infiltration. The correlation of those FRGs with TME in LIHC and pan-cancer analysis was also investigated. GO functional annotations and KEGG pathway analyses were performed to investigate the potential reactions of the obtained differentially expressed genes (DEGs). Further external validation was performed using microarrays on the GEO database and the key ferroptosis regulator SLC7A11 expression between LIHC and normal cells was detected by Western blotting. Results: A large proportion of genes were upregulated in the LIHC group. Among three clusters, cluster 3 had the worst prognosis combined with the highest PD-L1 expression and was positively correlated with various immune cells. Subsequently, survival analysis and Cox regression analysis screened out SLC7A11 as an independent prognostic factor in LIHC featured strong PD-L1 expression and unfavorable survival time. We filter out SLC7A11 as an independent prognostic signature in LIHC patients with strongly associated PD-L1 expression and unfavorable survival probability. In the pan-cancer analysis, high expression of SLC7A11 showed poor overall survival in seven cancers, while the correlation between immune checkpoints (ICs) and SLC7A11 varied by cancer type, indicating the potential therapeutic effects of SLC7A11 in cancers other than LIHC. Western blot was further employed to verify the expression of SLC7A11 in LIHC in vitro. Conclusion: Ferroptosis and TME synergistically play key roles in oncogenesis and progression of LIHC, and SLC7A11 can be used as a predictive biomarker for customized immunotherapy.

[Exploration on mechanism of Polygalae Radix and Acori Tatarinowii Rhizoma in treating Alzheimer's disease based on network pharmacology and experimental verification].

This study aimed to explore the action targets and mechanisms of Polygala tenuifolia and Acorus tatarinowii in treating Alzheimer's disease(AD) based on network pharmacology, molecular docking, and animal tests. The AD-related targets were collec-ted from GeneCard and the main active ingredients and targets of P. tenuifolia and A. tatarinowii from the TCMSP. Cytoscape was applied to construct the "Chinese herb-active ingredient-target-disease" network, followed by the construction of protein-protein interaction(PPI) network using STRING. GO biological function and KEGG pathway enrichment analysis was performed by DAVID and Metascape. The main active components of P. tenuifolia and A. tatarinowii and their potential core targets were docking using AutoDock Vina. The effects of P. tenuifolia and A. tatarinowii on the cognitive function were verified in mice with scopolamine(SCOP)-induced cognitive impairment. A total of seven active ingredients including kaempferol, onjixanthone Ⅰ, and marmesin and 56 potential targets of P. tenuifolia and A. tatarinowii were screened out, with the core targets covering AKT1, PTGS2, TNF, and NF-κB inflammation pathway mainly involved. The results of molecular docking also showed that the main active components of P. tenuifolia and A. tatarinowii stably bond to the core targets predicted by network pharmacology. The new object recognition experiment suggested that P. tenuifolia and A. tatarinowii improved the learning and memory abilities of mice after SCOP induction. As revealed by pathological section observation and relevant kit assay, P. tenuifolia and A. tatarinowii reduced the damage of central cholinergic neurons and enhanced the antioxidant ability of SCOP-induced mice. Western blot confirmed that P. tenuifolia and A. tatarinowii down-regulated the protein expression levels of TLR4, NF-κB, and related inflammatory factors(TNF-α, IL-1ß, and IL-6). All these have suggested that P. tenuifolia and A. tatarinowii inhibits AD via multiple components, multiple targets, and multiple pathways, which has provided an experimental basis for the clinical application of P. tenuifolia and A. tatarinowii for the treatment of AD.

Comparative Metabolomics Analysis Reveals Key Metabolic Mechanisms and Protein Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most common progressive neurodegenerative diseases, accompanied by global alterations in metabolic profiles. In the past 10 years, over hundreds of metabolomics studies have been conducted to unravel metabolic changes in AD, which provides insight into the identification of potential biomarkers for diagnosis, treatment, and prognostic assessment. However, since different species may lead to systemic abnormalities in metabolomic profiles, it is urgently needed to perform a comparative metabolomics analysis between AD animal models and human patients. In this study, we integrated 78 metabolic profiles from public literatures, including 11 metabolomics studies in different AD mouse models and 67 metabolomics studies from AD patients. Metabolites and enrichment analysis were further conducted to reveal key metabolic pathways and metabolites in AD. We totally identified 14 key metabolites and 16 pathways that are both differentially significant in AD mouse models and patients. Moreover, we built a metabolite-target network to predict potential protein markers in AD. Finally, we validated HER2 and NDF2 as key protein markers in APP/PS1 mice. Overall, this study provides a comprehensive strategy for AD metabolomics research, contributing to understanding the pathological mechanism of AD.

Cluster analysis of splenocyte microRNAs in the pig reveals key signal regulators of immunomodulation in the host during acute and chronic Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that can cause a geographically widespread zoonosis. Our previous splenocyte microRNA profile analyses of pig infected with T. gondii revealed that the coordination of a large number of miRNAs regulates the host immune response during infection. However, the functions of other miRNAs involved in the immune regulation during T. gondii infection are not yet known. METHODS: Clustering analysis was performed by K-means, self-organizing map (SOM), and hierarchical clustering to obtain miRNA groups with the similar expression patterns. Then, the target genes of the miRNA group in each subcluster were further analyzed for functional enrichment by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway to recognize the key signaling molecules and the regulatory signatures of the innate and adaptive immune responses of the host during T. gondii infection. RESULTS: A total of 252 miRNAs were successfully divided into 22 subclusters by K-means clustering (designated as K1-K22), 29 subclusters by SOM clustering (designated as SOM1-SOM29), and six subclusters by hierarchical clustering (designated as H1-H6) based on their dynamic expression levels in the different infection stages. A total of 634, 660, and 477 GO terms, 15, 26, and 14 KEGG pathways, and 16, 15, and 7 Reactome pathways were significantly enriched by K-means, SOM, and hierarchical clustering, respectively. Of note, up to 22 miRNAs mainly showing downregulated expression at 50 days post-infection (dpi) were grouped into one subcluster (namely subcluster H3-K17-SOM1) through the three algorithms. Functional analysis revealed that a large group of immunomodulatory signaling molecules were controlled by the different miRNA groups to regulate multiple immune processes, for instance, IL-1-mediated cellular response and Th1/Th2 cell differentiation partly depending on Notch signaling transduction for subclusters K1 and K2, innate immune response involved in neutrophil degranulation and TLR4 cascade signaling for subcluster K15, B cell activation for subclusters SOM17, SOM1, and SOM25, leukocyte migration, and chemokine activity for subcluster SOM9, cytokine-cytokine receptor interaction for subcluster H2, and interleukin production, chemotaxis of immune cells, chemokine signaling pathway, and C-type lectin receptor signaling pathway for subcluster H3-K17-SOM1. CONCLUSIONS: Cluster analysis of splenocyte microRNAs in the pig revealed key regulatory properties of subcluster miRNA molecules and important features in the immune regulation induced by acute and chronic T. gondii infection. These results contribute new insight into the identification of physiological immune responses and maintenance of tolerance in pig spleen tissues during T. gondii infection.

Hierarchical Artificial Compound Eyes with Wide Field-of-View and Antireflection Properties Prepared by Nanotip-Focused Electrohydrodynamic Jet Printing.

Artificial compound eyes (ACEs) endowed with durable superhydrophobicity, wide field-of-view (FOV), and antireflection properties are extremely appealing in advanced micro-optical systems. However, the simple and high-efficiency fabrication of ACEs with these functions is still a major challenge. Herein, inspired by moth eyes, ACEs with hierarchical macro/micro/nano structures were fabricated using the combination of nanotip-focused electrohydrodynamic jet (NFEJ) printing and air-assisted deformation processes. The NFEJ printing enables the direct and maskless fabrication of hierarchical micro/nanolens arrays (M/NLAs) without intermediate steps. The introduction of M/NLAs on the eye surface significantly improves the water hydrophobic performance with a water contact angle of 161.1° and contact angle hysteresis (CAH) of 4.2° and generally decreases the reflectance by 51% in the wavelength range of 350-1600 nm in comparison to the macroeye without any structures. The contact angle remains almost unchanged, and the CAH slightly increases from 4.2° to 8.7° after water jet impact for 20 min, indicating a durable superhydrophobicity. Moreover, the results confirm that the durable superhydrophobic ACEs with antireflection properties exhibit excellent imaging quality and a large FOV of up to 160° without distortion.

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing.

BACKGROUND: Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. METHODS: Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. RESULTS: SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. CONCLUSIONS: We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control.

Full-length transcriptome sequence analysis of Eimeria necatrix unsporulated oocysts and sporozoites identifies genes involved in cellular invasion.

Eimeria necatrix is one of the most pathogenic chicken coccidia and causes avian coccidiosis, an enteric disease of major economic importance worldwide. Eimeria parasites have complex developmental life cycles, with an exogenous phase in the environment and an endogenous phase in the chicken intestine. Oocysts excreted by chickens rapidly undergo meiosis and cell division to form eight haploid sporozoites (SZ). SZ liberated from sporocysts in the chicken intestine migrate to their preferred site of development to initiate cellular invasion. To date, almost nothing is known about the proteins that mediate parasite invasion in E. necatrix. In order to discover genes with functions involved in cellular invasion, the transcriptome profiles of E. necatrix unsporulated oocysts (UO) and SZ were analyzed using a combination of third-generation single-molecule real-time sequencing (TGS) and second-generation sequencing (SGS) followed by qRT-PCR validation. Correction of TGS long reads by SGS short reads resulted in 34,932 (UO) and 23,040 (SZ) consensus isoforms. After subsequent assembly, a total of 4949 and 4254 genes were identified from UO and SZ libraries, respectively. A total of 8376 genes were identified as differentially expressed genes (DEGs) between SZ and UO. Compared to UO, 4057 genes were upregulated and 4319 genes were downregulated in SZ. Approximately 1399 and 1758 genes were defined as stage-specific genes in SZ and UO, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 2978 upregulated SZ genes were clustered into 29 GO terms, and 857 upregulated SZ genes were associated with 26 KEGG pathways. We also predicted a further 50 upregulated SZ genes and 73 upregulated UO genes encoding microneme proteins, apical membrane antigens, rhoptry neck proteins, rhoptry proteins, dense granule proteins, heat shock proteins, calcium-dependent protein kinases, cyclin-dependent kinases, cGMP-dependent protein kinase, and glycosylphosphatidylinositol-anchored surface antigens. Our data reveal new features of the E. necatrix transcriptional landscape and provide resources for the development of novel vaccine candidates against E. necatrix infection.

Preparation of aligned nanofibers using parallel inductive-plates assisted electrospinning.

Electrospinning is a simple, cost-effective, and versatile technique for fabrication of nanofibers. However, nanofibers obtained from the conventional electrospinning are typically disordered, which seriously limits their application. In this work, we present a novel and facile technique to obtain aligned nanofibers with high efficiency by using parallel inductive-plates assisted electrospinning (PIES). In this new electrospinning setup, the electrostatic spinneret is contained in a pair of parallel inductive-plates, which can change the shape and direction of the electric field line during the electrospinning so as to control the flight trajectory and spatial alignment of the spinning nanofibers. This electrospinning setup can divide the electric field line into two parts which are respectively directed to the edge of the upper and lower inductive-plates. Then the nanofibers move along the electric field line, suspend and align between the parallel inductive-plates. Finally, the well aligned nanofibers could be easily transferred onto other substrates for further characterizations and applications. The aligned nanofibers with an average diameter of 469 ± 115 nm and a length as long as 140 mm were successfully achieved by using PIES technique. Moreover, nanofiber arrays with different cross angles and three-dimensional films formed by the aligned nanofibers were also facilely obtained. The novel PIES developed in this work has been proved to be a facile, cost-effective and promising approach to prepare aligned nanofibers for a wide range of applications.