Comparative characterization of microRNA-71 of Echinococcus granulosus exosomes.

Cystic echinococcosis (CE) is a global zoonotic disease caused by Echinococcus granulosus, posing a great threat to human and animal health. MiRNAs are small regulatory noncoding RNA involved in the pathogenesis of parasitic diseases, possibly via exosomes. Egr-miR-71 has been identified as one of the miRNAs in the blood of CE patients, but its secretory characteristics and functions remains unclear. Herein, we studied the secretory and biological activity of exosomal egr-miR-71 and its immunoregulatory functions in sheep peripheral blood mononuclear cells (PBMCs). Our results showed that egr-miR-71 was enriched in the exosome secreted by protoscoleces with biological activity. These egr-miR-71-containing exosomes were easily internalized and then induced the dysregulation of cytokines (IL-10 and TNF-α), nitric oxide (NO) and key components (CD14 and IRF5) in the LPS/TLR4 pathway in the coincubated sheep PBMCs. Similarly, egr-miR-71 overexpression also altered the immune functions but exhibited obvious differences in regulation of the cytokines and key components, preferably inhibiting proinflammatory cytokines (IL-1α, IL-1ß and TNF-α). These results demonstrate that exosomal egr-miR-71 is bioactive and capacity of immunomodulation of PBMCs, potentially being involved in immune responses during E. granulosus infection.

Title: Caractérisation comparative du microARN-71 des exosomes d'Echinococcus granulosus. Abstract: L'échinococcose kystique (EK) est une maladie zoonotique mondiale causée par Echinococcus granulosus, représentant une grande menace pour la santé humaine et animale. Les miARN sont des petits ARN régulateurs non codants impliqués dans la pathogenèse des maladies parasitaires, éventuellement via les exosomes. Egr-miR-71 a été identifié comme l'un des miARN présents dans le sang des patients atteints d'EK, mais ses caractéristiques et fonctions sécrétoires restent floues. Ici, nous avons étudié l'activité sécrétoire et biologique du egr-miR-71 exosomal et ses fonctions immunorégulatrices dans les cellules mononucléées du sang périphérique (CMSP) de mouton. Nos résultats ont montré qu'egr-miR-71 était enrichi dans l'exosome sécrété par les protoscolex ayant une activité biologique. Ces exosomes contenant egr-miR-71 ont été facilement internalisés et ont ensuite induit la dérégulation des cytokines (IL-10 et TNF-α), de l'oxyde nitrique (NO) et des composants clés (CD14 et IRF5) de la voie LPS/TLR4 dans les CMSP de mouton co-incubées. De même, la surexpression d'egr-miR-71 a également modifié les fonctions immunitaires mais a montré des différences évidentes dans la régulation des cytokines et des composants clés, inhibant de préférence les cytokines pro-inflammatoires (IL-1α, IL-1ß et TNF-α). Ces résultats démontrent que l'egr-miR-71 exosomal est bioactif et possède une capacité d'immunomodulation des CMSP, potentiellement impliquée dans les réponses immunitaires lors d'une infection à E. granulosus.

Understanding the Gut-Brain Axis and Its Therapeutic Implications for Neurodegenerative Disorders.

The gut-brain axis (GBA) is a complex bidirectional communication network connecting the gut and brain. It involves neural, immune, and endocrine communication pathways between the gastrointestinal (GI) tract and the central nervous system (CNS). Perturbations of the GBA have been reported in many neurodegenerative disorders (NDDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), among others, suggesting a possible role in disease pathogenesis. The gut microbiota is a pivotal component of the GBA, and alterations in its composition, known as gut dysbiosis, have been associated with GBA dysfunction and neurodegeneration. The gut microbiota might influence the homeostasis of the CNS by modulating the immune system and, more directly, regulating the production of molecules and metabolites that influence the nervous and endocrine systems, making it a potential therapeutic target. Preclinical trials manipulating microbial composition through dietary intervention, probiotic and prebiotic supplementation, and fecal microbial transplantation (FMT) have provided promising outcomes. However, its clear mechanism is not well understood, and the results are not always consistent. Here, we provide an overview of the major components and communication pathways of the GBA, as well as therapeutic approaches targeting the GBA to ameliorate NDDs.

Interplays of liver fibrosis-associated microRNAs: Molecular mechanisms and implications in diagnosis and therapy.

microRNAs (miRNAs) are a class of non-coding functional small RNA composed of 21-23 nucleotides, having multiple associations with liver fibrosis. Fibrosis-associated miRNAs are roughly classified into pro-fibrosis or anti-fibrosis types. The former is capable of activating hepatic stellate cells (HSCs) by modulating pro-fibrotic signaling pathways, mainly including TGF-ß/SMAD, WNT/ß-catenin, and Hedgehog; the latter is responsible for maintenance of the quiescent phenotype of normal HSCs, phenotypic reversion of activated HSCs (aHSCs), inhibition of HSCs proliferation and suppression of the extracellular matrix-associated gene expression. Moreover, several miRNAs are involved in regulation of liver fibrosis via alternative mechanisms, such as interacting between hepatocytes and other liver cells via exosomes and increasing autophagy of aHSCs. Thus, understanding the role of these miRNAs may provide new avenues for the development of novel interventions against hepatic fibrosis.

mmu-miRNA-342-3p promotes hepatic stellate cell activation and hepatic fibrosis induced by Echinococcus multilocularis infection via targeting Zbtb7a.

Liver fibrosis is one of the histopathological characters during Echinococcus multilocularis infection. The activation of hepatic stellate cells (HSCs) is a key event in the development of liver fibrosis. However, the molecular mechanism of HSC activation in the E. multilocularis infection-induced liver fibrosis remains largely unclear. Here, we reported that mmu-miR-342-3p was most dominantly expressed in HSCs and was upregulated in the HSCs in response to E. multilocularis infection. We further showed that mmu-miR-342-3p was able to bind to the 3' UTR of the Zbtb7a gene and regulated its expression. Moreover, mmu-miR-342-3p expression was negatively correlated with its target gene Zbtb7a in HSCs during E. multilocularis infection. Knockdown of mmu-miR-342-3p promoted the expression of Gfap in the activated HSCs in vitro. In the E. multilocularis-infected mice, knockdown of mmu-miR-342-3p suppressed the expression of α-Sma, Col1α1, and TGF-ß but promoted the expression of Gfap. Therefore, mmu-miR-342-3p is a key regulator for activation of HSCs, and inhibiting mmu-miR-342-3p to suppressed Zbtb7a-mediated TGF-ß signaling in activated HSCs could be a novel strategy to treat liver fibrosis induced by E. multilocularis.

microRNAs in parasite-induced liver fibrosis: from mechanisms to diagnostics and therapeutics.

Chronic parasite infections in the liver pose a global threat to human and animal health, often occurring with liver fibrosis that leads to cirrhosis, liver failure, and even cancer. Hepatic fibrogenesis is a complex yet reversible process of tissue repair and is associated with various factors, including immune cells, microenvironment, gut microbiome, and interactions of the different liver cells. As a profibrogenic or antifibrogenic driver, microRNAs (miRNAs) are closely involved in parasite-induced hepatic fibrosis. This article updates the current understanding of the roles of miRNAs in hepatic fibrogenesis by parasite infections and discusses the strategies using miRNAs as candidates for diagnostics and therapeutics.

Probiotics Supplementation Attenuates Inflammation and Oxidative Stress Induced by Chronic Sleep Restriction.

Background: Insufficient sleep is a serious public health problem in modern society. It leads to increased risk of chronic diseases, and it has been frequently associated with cellular oxidative damage and widespread low-grade inflammation. Probiotics have been attracting increasing interest recently for their antioxidant and anti-inflammatory properties. Here, we tested the ability of probiotics to contrast oxidative stress and inflammation induced by sleep loss. Methods: We administered a multi-strain probiotic formulation (SLAB51) or water to normal sleeping mice and to mice exposed to 7 days of chronic sleep restriction (CSR). We quantified protein, lipid, and DNA oxidation as well as levels of gut-brain axis hormones and pro and anti-inflammatory cytokines in the brain and plasma. Furthermore, we carried out an evaluation of microglia morphology and density in the mouse cerebral cortex. Results: We found that CSR induced oxidative stress and inflammation and altered gut-brain axis hormones. SLAB51 oral administration boosted the antioxidant capacity of the brain, thus limiting the oxidative damage provoked by loss of sleep. Moreover, it positively regulated gut-brain axis hormones and reduced peripheral and brain inflammation induced by CSR. Conclusions: Probiotic supplementation can be a possible strategy to counteract oxidative stress and inflammation promoted by sleep loss.

Bufalin induces apoptosis and autophagy via the Ca2+/CaMKKß/AMPK/Beclin1 signaling pathway in osteosarcoma cells.

Bufalin, a major cardiotonic compound of the traditional Chinese medicine Chanshu has been used for cancer treatment for several years. However, the molecular mechanisms of Bufalin-induced autophagy in osteosarcoma (OS) is not fully understood. In the present study, it was shown that Bufalin induced crosstalk between apoptosis and autophagy, which resulted in OS cell death. Mechanistically, Bufalin induced autophagy by increased the ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I, and inducing apoptosis via the caspase-dependent pathway. Inhibition of autophagy promoted Bufalin-induced cell death. In contrast, suppression of apoptosis enhanced Bufalin-induced autophagy. In addition, it was found that Bufalin activated the Ca2+ /calmodulin-dependent protein kinase ß/AMPK/Beclin1 pathway, which resulted in induction of autophagy. These findings provide a mechanistic understanding of the means by which Bufalin mediates autophagy and apoptosis in OS cells.

Analysis of sheep peripheral blood mononuclear cells in response to Echinococcus granulosus microRNA-71 overexpression.

Cyst echinococcosis, caused by Echinococcus granulosus, remains a zoonotic disease posing a great threat to public health and meat production industry. Sheep infected with E. granulosus show relatively high abundance of egr-miR-71 in the sera, but its role is unknown. Using bioinformatics and cell migration and Transwell assays, we comparatively analyzed the proteomes and cell invasion of sheep PBMCs in response to egr-miR-71 overexpression. The results showed that the egr-miR-71 induced a total of 157 proteins being differentially expressed and mainly involved in immune responses. In sheep PBMCs, egr-miRNA-71 overexpression induced significant downregulation of macrophage migration inhibitory factor (MIF) and accordingly promoted cell migration and invasion compared with the control. The results will provide a clue for further investigation of a role of circulating egr-miR-71 in immune responses during E. granulosus infection.

Immortalized Alzheimer's Disease Astrocytes: Characterization of Their Proteolytic Systems.

Alzheimer's disease (AD) is a progressive neurodegeneration with dysfunctions in both the ubiquitin-proteasome system (UPS) and autophagy. Astroglia participation in AD is an attractive topic of research, but molecular patterns are partially defined and available in vitro models have technical limitations. Immortalized astrocytes from the hippocampus of 3xTg-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively) have been obtained as an attempt to overcome primary cell line limitations and this study aims at characterizing their proteolytic systems, focusing on UPS and autophagy. Both 26S and 20S proteasomal activities were downregulated in 3Tg-iAstro, in which a shift in catalytic subunits from constitutive 20S proteasome to immunoproteasome occurred, with consequences on immune functions. In fact, immunoproteasome is the specific complex in charge of clearing damaged proteins under inflammatory conditions. Parallelly, augmented expression and activity of the lysosomal cathepsin B, enhanced levels of lysosomal-associated membrane protein 1, beclin1, and LC3-II, together with an increased uptake of monodansylcadaverine in autophagic vacuoles, suggested autophagy activation in 3Tg-iAstro. The two proteolytic pathways were linked by p62 that accumulated in 3Tg-iAstro due to both increased synthesis and decreased degradation in the UPS defective astrocytes. Treatment with 4-phenylbutyric acid, a neuroprotective small chemical chaperone, partially restored proteasome and autophagy-mediated proteolysis in 3Tg-iAstro. Our data shed light on the impaired proteostasis in 3Tg-iAstro with proteasome inhibition and autophagic compensatory activation, providing additional validation of this AD in vitro model, and propose a new mechanism of action of 4-phenylbutyric acid in neurodegenerative disorders.

Heat shock protein 60 in parasitic helminths: A role in immune responses and therapeutic applications.

Heat shock protein 60 (HSP60) is an unique member of the heat shock protein family, being involved in parasite infections. To cope with harsh environments where parasites live, HSP60s are indispensable and involved in a variety of biological processes. HSP60s have relative low similarity among parasites, but their ATPase /Mg2+ active sites are highly conserved. The interactions of HSP60s with signaling pathway regulators in immune cells suggest a crucial role in immune responses, rendering them a potential therapeutic target. This paper reviews the current understandings of HSP60s in parasitic helminths in aspects of molecular characteristics, immunoregulatory responses and HSP60-based therapeutics.

Comparative transcriptomic analysis of the larval and adult stages of Dibothriocephalus dendriticus (Cestoda: Diphyllobothriidea).

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, some of which are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Genomic and transcriptomic data can be used to develop future diagnostic tools and epidemiological studies. The present work focuses on a comparative analysis of the transcriptomes of adult and plerocercoid D. dendriticus and the identification of their differentially expressed genes (DEGs). Transcriptome assembly and analysis yielded and annotated 35,129 unigenes, noting that 16,568 (47%) unigenes were not annotated in known databases, which may indicate a unique set of expressed transcripts for D. dendriticus. A total of 8022 differentially expressed transcripts were identified, including 3225 upregulated and 4797 downregulated differentially expressed transcripts from the plerocercoid and adult animals. The analysis of DEGs has shown that among the most differentially expressed genes, there are important genes characteristic of each stage. Thus, several genes are characteristic of D. dendriticus plerocercoids, including fatty acid-binding protein and ferritin. Among the most highly expressed DEGs of the adult stage of D. dendriticus is the Kunitz-type serine protease inhibitor, in two putative isoforms. The analyses of GO and KEGG metabolic pathways revealed that a large number of the DEGs of D. dendriticus are associated with the biosynthesis of various substances such as arginine and folate, as well as with various metabolic pathways such as galactose metabolism, selenocompound metabolism, and phosphonate and phosphinate metabolism. This will contribute to further research aimed at identifying targets for new generation drugs and the development of specific vaccines.

Evaluation of Protective Immune Responses Induced in BALB/c Mice and Goats by the Neospora caninum Surface SRS Proteins and Interleukin-18.

Neosporosis is caused by Neospora caninum (N. caninum), which mainly infects cattle and goats and severely threatens the animal industry. In this study, the inhibitory effects of polyclonal antiserum anti-NcSRS17, NcSRS2 and NcSRS52 were explored. Cytokines in mice or goat serum were detected after immunization. After infection, the survival of mice was recorded. The pathological changes and parasite loads were observed and detected in tissues. The results showed that anti-NcSRS2, NcSRS17 and NcSRS52 antibodies all inhibit the invasion and proliferation of N. caninum. The IFN-γ level in the NcSRS17 group was higher than that in the NcSRS2 and NcSRS52 groups, and higher in the NcSRS2-mIL-18 group than in the NcSRS2 group. The survival rates of mice were 16% in the positive control group, 67% in the SRS52 group, 83% in the SRS2 and mIL-18 groups and 100% in the SRS17 and SRS2-mIL-18 groups. Goats immunized with NcSRS17-gIL-18 developed high levels of IL-4, IL-12 and IFN-γ compared with those immunized with NcSRS-17. Parasite loads in the brains of animals in the NcSRS17 and NcSRS17-gIL-18 groups were significantly reduced, and were significantly lower in the NcSRS17-gIL-18 group (p ≤ 0.01). This study indicates that SRS17 may be an antigen candidate for vaccine development against neosporosis, and IL-18 can enhance the immune protective efficiency of antigen candidates.

Targeting Proteolysis with Cyanogenic Glycoside Amygdalin Induces Apoptosis in Breast Cancer Cells.

BACKGROUND: Breast cancer is the most diagnosed cancer among women, and its incidence and mortality are rapidly growing worldwide. In this regard, plant-derived natural compounds have been shown to be effective as chemotherapeutic and preventative agents. Apricot kernels are a rich source of nutrients including proteins, lipids, fibers, and phenolic compounds and contain the aromatic cyanogenic glycoside amygdalin that has been shown to exert a cytotoxic effect on cancer cells by affecting the cell cycle, inducing apoptosis, and regulating the immune function. METHODS: Here, we describe a previously unexplored proapoptotic mechanism of action of amygdalin in breast cancer (MCF7) cells that involves the modulation of intracellular proteolysis. For comparative purposes, the same investigations were also conducted upon cell treatment with two apricot kernel aqueous extracts from Prunus armeniaca L. RESULTS: We observed that both the 20S and 26S proteasome activities were downregulated in the MCF7 cells upon 24 h treatments. Simultaneously, the autophagy cascade resulted in being impaired due to cathepsin B and L inhibition that also contributed to a reduction in cancer cell migration. The inhibition of these proteolytic systems finally promoted the activation of apoptotic events in the MCF7 cells. CONCLUSION: Collectively, our data unveil a novel mechanism of the anticancer activity of amygdalin, prompting further investigations for potential application in cancer preventative strategies.

The Effects of Phosphate Impurity on Recovered LiNi0.6Co0.2Mn0.2O2 Cathode Material via a Hydrometallurgy Method.

From portable electronics to electric vehicles, lithium-ion batteries have been deeply integrated into our daily life and industrial fields for a few decades. The booming field of battery manufacturing could lead to shortages in resources and massive accumulation of battery waste, hindering sustainable development. Therefore, hydrometallurgy-based approaches have been widely used in industrial recycling to recover cathode materials due to their high efficiency and throughput. Impurities have always been a great challenge for hydrometallurgical recycling, introducing challenges to maintain the consistency of product quality because of potential unintended effects caused by impurities. Herein, after comprehensive investigation, we first report the impacts of phosphate impurity on a recycled LiNi0.6Co0.2Mn0.2O2 ("NCM622") cathode via a hydrometallurgy method. We demonstrate that a passivation layer of Li3PO4 is formed at grain boundaries during sintering, which significantly raises the activation barrier and hinders lithium diffusion. In addition, the distinct degradation of cathode electrochemical properties is observed from poor particle morphology and high cation mixing as a result of phosphate impurity. Cathode powders with 1 at. % phosphate impurity retain a capacity of 146 mAh/g after 100 cycles at 0.33C, 6% less than that of a virgin cathode. Furthermore, cathodes with higher phosphate concentrations perform even worse in electrochemical tests. Therefore, phosphate impurities are detrimental to the hydrometallurgical recycling of NCM cathode materials and need to be excluded from the recycling process.

Proteomic profiling of serum extracellular vesicles identifies diagnostic markers for echinococcosis.

Echinococcosis is a parasitic disease caused by the metacestodes of Echinococcus spp. The disease has a long latent period and is largely underdiagnosed, partially because of the lack of effective early diagnostic approaches. Using liquid chromatography-mass spectrometry, we profiled the serum-derived extracellular vesicles (EVs) of E. multilocularis-infected mice and identified three parasite-origin proteins, thioredoxin peroxidase 1 (TPx-1), transitional endoplasmic reticulum ATPase (TER ATPase), and 14-3-3, being continuously released by the parasites into the sera during the infection via EVs. Using ELISA, both TPx-1 and TER ATPase were shown to have a good performance in diagnosis of experimental murine echinococcosis as early as 10 days post infection and of human echinococcosis compared with that of control. Moreover, TER ATPase and TPx-1 were further demonstrated to be suitable for evaluation of the prognosis of patients with treatment. The present study discovers the potential of TER ATPase and TPx-1 as promising diagnostic candidates for echinococcosis.

Efficient delivery of Echinococcus multilocularis miRNAs using chitosan nanoparticles.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease, a great threat to human health due to limited interventions. microRNAs are a type of small non-coding RNA that plays a key role in many diseases and is considered as a potential therapeutic target for control of parasitic diseases. However, naked miRNAs are difficult to enter into cells and are easily degraded in both external and internal environments. Chitosan (CS) has recently been used as a promising vehicle for delivery of nucleic acids. Therefore, we prepared miRNA-bearing CS nanoparticles and investigated the physicochemical properties as well as the delivery efficiency. We found that CS nanoparticles was relatively stable, offered miRNA strong protection from degradation and had low cytotoxicity with no significant effects on cell proliferation and apoptosis. CS nanoparticles were shown to be easily absorbed by cells and have remarkable liver tropism. Furthermore, CS nanoparticles were used to efficiently deliver E. multilocularis miR-4989 in vitro and in vivo and caused a significant reduction in the expression of UBE2N in the liver, a potential target of emu-miR-4989, at both mRNA and protein levels. Our data demonstrate that CS nanoparticles can act as a vehicle for efficient liver-targeted delivery of miRNAs and for development of miRNA-based therapeutics against E. multilocularis infection.

Integrative Analysis of RNA Expression and Regulatory Networks in Mice Liver Infected by Echinococcus multilocularis.

The larvae of Echinococcus multilocularis causes alveolar echinococcosis, which poses a great threat to the public health. However, the molecular mechanisms underlying the host and parasite interactions are still unclear. Exploring the transcriptomic maps of mRNA, miRNA and lncRNA expressed in the liver in response to E. multilocularis infection will help us to understand its pathogenesis. Using liver perfusion, different cell populations including the hepatic cells, hepatic stellate cells and Kupffer cells were isolated from mice interperitoneally inoculated with protoscoleces. Their transcriptional profiles including lncRNAs, miRNAs and mRNAs were done by RNA-seq. Among these cell populations, the most differentially-expressed (DE) mRNA, lncRNAs and miRNAs were annotated and may involve in the pathological processes, mainly including metabolic disorders, immune responses and liver fibrosis. Following the integrative analysis of 38 differentially-expressed DEmiRNAs and 8 DElncRNAs, the lncRNA-mRNA-miRNA networks were constructed, including F63-miR-223-3p-Fbxw7/ZFP36/map1b, F63-miR-27-5p-Tdrd6/Dip2c/Wdfy4 and IFNgAS1-IFN-γ. These results unveil the presence of several potential lncRNA-mRNA-miRNA axes during E. multilocularis infection, and further exploring of these axes may contribute to better understanding of the pathogenic mechanisms.

circRNA Expression Pattern and circRNA-miRNA-mRNA Network in HCs, HSCs, and KCs of Murine Liver After Echinococcus multilocularis Infection.

Caused by Echinococcus multilocularis (E. multilocularis), alveolar echinococcosis is reported every year around the world and severely threatens the safety of human beings and animals. However, the molecular interaction relationships between host and E. multilocularis still remains unclear. With multiple functions, circRNA plays a crucial role in regulating the development of a parasitic disease. With that in mind, the main purpose of this study was to reveal the circRNA expression profiles and circRNA-miRNA-mRNA network relationships in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) of murine liver after E. multilocularis infection. After sequencing, 6,290 circRNAs were identified from 12 hepatic cell samples. Based on the subsequent analysis, 426 and 372 circRNAs were significantly different in HC expression at 2 and 3 months after E. multilocularis infection, and similar results were also demonstrated in HSCs (426 and 372 circRNAs) and KCs (429 and 331 circRNAs), respectively. Eight candidate circRNAs were randomly selected to identify the accuracy of the sequencing results by using qRT-PCR. Additionally, three circRNAs-miRNA-mRNA networks in HCs, HSCs, and KCs were constructed. Taken together, our study provided a systematic presentation of circRNAs in murine liver cells after E. multilocularis infection, and these networks are essential for research in circRNAs associated with E. multilocularis infection.