Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Establishing Ohmic Contact of a Radial Compressed CNT Bundle with High Work Function Metal.

Establishing low-resistance ohmic contact is critical for developing electronic devices based on traditional silicon and new low-dimensional materials. Due to unprecedented electronic and mechanical properties, the one-dimensional carbon nanotubes (CNTs) have been used as source/drain, gate, or tunnel to fabricate transistors. However, the mechanism causing low-resistance ohmic contact is not clear yet. Here, the hybrid atomic force microscopy-scanning electron microscopy (AFM-SEM) instrument was developed to establish lower-resistance ohmic contact between a radial compressed deformed multiwalled CNT bundle and high work function metal (platinum and gold). The radial compression structure under strong van der Waals attraction was in situ characterized through the SEM image to obtain the diameter and width and through AFM to get height and to perform nanoindentation, indicating that Pt has the smaller radial compression deformation. Molecular dynamics simulations exhibit that compared to Pt, a wider ribbon-like graphene layer formed when the radial compressed CNTs contacted with Au. The bond forming and electron orbital overlapping between C atoms of deformed CNTs and the high work function metal atom is beneficial for good electrical contact.

Research on the visual location method for strawberry picking points under complex conditions based on composite models.

BACKGROUND: Strawberry, being an important economic crop, requires a large amount of human labor for harvesting operations. Efficient and non-destructive harvesting by strawberry harvesting robots requires the precise location of the picking points. Current algorithms for locating picking points encounter significant issues with location errors and minimal effective information in complex situations. RESULTS: To improve the accuracy of the location of picking points, this study proposes a visual location method based on composite models. This method employs object detection and instance segmentation models to detect fruits and segment peduncles sequentially, thereby enabling the identification of picking points and inclination on the peduncle. Different object detection algorithms and instance segmentation models were validated to explore the optimal model combination, and the Convolutional Block Attention Module (CBAM) was integrated into YOLOv8s-seg to construct YOLOv8s-seg-CBAM. Test results show that the composite model built with YOLOv8s and YOLOv8s-seg-CBAM achieved a peduncle detection accuracy of 86.2%, with an inference time of 30.6 ms per image. CONCLUSION: The picking point visual location method based on YOLOv8s and YOLOv8s-seg-CBAM composite models can better balance accuracy and efficiency and can provide more accurate guidance for automated harvesting. © 2024 Society of Chemical Industry.

Isolation and pathogenicity evaluation of Escherichia coli O157:H7 from common carp, Cyprinus carpio.

Escherichia coli O157:H7 is the primary serotype of enterohaemorrhagic E. coli (EHEC), which can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. It is considered as a major health concern due to it being a zoonotic disease that is transmitted through food. In this study, a pathogenic bacterium was isolated from infected carp, which identified as E. coli O157:H7 named X21 through genetic sequencing, phylogenetic analysis, physiological and biochemical tests. In the experiment, crucian carp was used as a model to study the pathogenicity of the isolate, the pathological histological observations and cytokines expression of fish tissues were determined after bacterial challenge. The results showed that severe pathological damage observed in the liver, spleen, headkidney of fish infected with isolate X21. Besides, we found that accumulation of IgT+ B cells in the lamina propria of intestine, and up-regulation of SUCH-r, IL-1ß, IL-10, IL-11, MyD88, and TNF-α gene in various tissues. After challenged, the survivability of crucian carp infected with isolate X21 stands at a mere 14.27%. To our knowledge, this is the first report that E. coli O157:H7 infected the freshwater fish C. carpio, which indicates that this bacterium is a potential threat to public health and freshwater fish aquaculture.

Detection of changes in biological characteristics of Aeromonas veronii TH0426 after deletion of lsrB gene by homologous recombination.

Aeromonas veronii is a widespread pathogenic microorganism that can infect humans, animals, and a variety of aquatilia, at the same time, can cause diseases, mainly sepsis and ulcer syndrome. In this research, we first deleted the gene of lsrB's nucleotide sequences by homologous recombination. The results showed that the median lethal dose (LD50) of the mutant strain (ΔlsrB) for zebrafish was 1.28-times higher than that of the TH0426 strain. The toxicity of TH0426 to epithelioma papulosum cyprini (EPC) cells was 1.15-times and 1.64-times higher than that of ΔlsrB, 1 and 2 h after infection. The production ability of the biofilm of ΔlsrB decreased by 1.38-times, and the adhesion ability of ΔlsrB to EPC cells greatly decreased by 1.96-times than the TH0426. The result of motility detection pointed out that the swimming ability of ΔlsrB was down by 1.67-times. The results indicated that almost all of them lost their flagella after deleting the lsrB gene. In general, the virulence of TH0426 was reduced after deleting the lsrB gene. The final results point out that the lsrB gene of TH0426 is related to motility, biofilm formation, adhesion, and virulence.

Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp.

Aeromonas veronii is an important aquatic zoonotic, which elicits a range of diseases, such as haemorrhagic septicemia. To develop an effective oral vaccine against Aeromonas veronii infection in carp, the Aeromonas veronii adhesion (Aha1) gene was used as a target molecule to attach to intestinal epithelial cells. Two anchored recombinant. Lactic acid bacteria strains (LC-pPG-Aha1 1038 bp and LC-pPG-Aha1-LTB 1383 bp) were constructed by fusing them with the E. coli intolerant enterotoxin B subunit (LTB) gene and using Lactobacillus casei as antigen delivery vector to evaluate immune effects of these in carp. Western blotting and immunofluorescence were used to confirm that protein expression was successful. Additionally, levels of specific IgM in serum and the activities of ACP, AKP, SOD, LYS, C3, C4, and lectin enzymes-were assessed. Cytokines IL-10, IL-1ß, TNF-α, IgZ1, and IgZ2 were measured in the liver, spleen, kidney, intestines, and gills tissue by qRT-PCR, which showed an increasing trend compared with the control group (P < 0.05). A colonization assay showed that the two L. casei recombinants colonized the middle and hind intestines of immunized fish. When immunized carp were experimentally challenged with Aeromonas veronii the relative percentage protection of LC-pPG-Aha1 was 53.57%, and LC-pPG-Aha1-LTB was 60.71%. In conclusion, these results demonstrate that Aha1 is a promising candidate antigen when it is displayed on lactic acid bacteria (Lc-pPG-Aha1 and Lc-pPG-Aha1-LTB) seems promising for a mucosal therapeutic approach. We plan to investigate the molecular mechanism of the L. casei recombinant in regulating the intestinal tissue of carp in future studies.

Epidemiology and antifungal susceptibilities of clinically isolated Aspergillus species in South China.

Aspergillosis is a rising concern worldwide; however, its prevalence is not well documented in China. This retrospective study determined Aspergillus's epidemiology and antifungal susceptibilities at Meizhou People's Hospital, South China. From 2017 to 2022, the demographic, clinical, and laboratory data about aspergillosis were collected from the hospital's records and analysed using descriptive statistics, chi-square test, and ANOVA. Of 474 aspergillosis cases, A. fumigatus (75.32%) was the most common, followed by A. niger (9.92%), A. flavus (8.86%), and A. terreus (5.91%). A 5.94-fold increase in aspergillosis occurred during the study duration, with the highest cases reported from the intensive care unit (52.74%) - chronic pulmonary aspergillosis (79.1%) and isolated from sputum (62.93%). Only 38 (8.02%) patients used immunosuppressant drugs, while gastroenteritis (5.7%), haematologic malignancy (4.22%), and cardiovascular disease (4.22%) were the most prevalent underlying illnesses. In A. fumigatus, the wild-type (WT) isolates against amphotericin B (99.1%) were higher than triazoles (97-98%), whereas, in non-fumigatus Aspergillus species, the triazole (95-100%) WT proportion was greater than amphotericin B (91-95%). Additionally, there were significantly fewer WT A. fumigatus isolates for itraconazole and posaconazole in outpatients than inpatients. These findings may aid in better understanding and management of aspergillosis in the region.

Traditional methods and biosensors for detecting disinfection by-products in water: A review.

In recent years, pollution caused by disinfection by-products (DBPs) has become a global concern. Initially, there were fewer contaminants, and the mechanism of their generation was unclear; however, the number of contaminants has increased exponentially as a result of rapid industrialization and numerous economic activities (e.q., during the outbreak of COVID-19 a surge in the use of chlorinated disinfectants was observed). DBP toxicity results in various adverse health effects and organ failure in humans. In addition, it profoundly affects other forms of life, including animals, plants, and microorganisms. This review comprehensively discusses the pre-treatment methods of traditional and emerging DBPs and the technologies applied for their detection. Additionally, this paper provides a detailed discussion of the principles, applicability, and characteristics of traditional large-scale instrumentation methods (such as gas/liquid/ion chromatography coupled with mass spectrometry) for detecting DBPs based on their respective detection techniques. At the same time, the design, functionality, classification, and characteristics of rapid detection technologies (such as biosensors) are also detailed and analyzed.

Transcriptomic insights into the immune response of the intestine to Aeromonas veronii infection in northern snakehead (Channa argus).

Intestinal inflammation is a protective response that is implicated in bacterial enteritis triggered by gastrointestinal infection. The immune mechanisms elicited in teleost against the infection of Aeromonas veronii are largely unknown. In this study, we performed a de novo northern snakehead (Channa argus) transcriptome assembly using Illumina sequencing platform. On this basis we performed a comparative transcriptomic analysis of northern snakehead intestine from A. veronii-challenge and phosphate buffer solution (PBS)-challenge fish, and 2076 genes were up-regulated and 1598 genes were down-regulated in the intestines infected with A. veronii. The Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes (DEGs) were enriched to 27, 21 and 20 GO terms in biological process, cellular component, and molecular function, respectively. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 420 DEGs were involved in 194 pathways. Moreover, 33 DEGs were selected for quantitative real-time PCR analysis to validate the RNA-seq data. The results reflected the consistency of the expression levels between qRT-PCR and RNA-seq data. In addition, a time-course analysis of the mRNA expression of 33 immune-related genes further indicated that the intestinal inflammation to A. veronii infection simultaneously regulated gene expression alterations. The present study provides transcriptome data of the teleost intestine, allowing us to understand the mechanisms of intestinal inflammation triggered by bacterial pathogens. DATA AVAILABILITY STATEMENT: All data supporting the findings of this study are available within the article and Supplementary files. The RNA-seq raw sequence data are available in NCBI short read archive (SRA) database under accession number PRJNA615958.

IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals.

The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON-NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

Oral vaccination with recombinant Lactobacillus casei expressing Aha1 fused with CTB as an adjuvant against Aeromonas veronii in common carp (Cyprinus carpio).

Aeromonas veronii (A. veronii) is a pathogenic that can infect human, animal and aquatic organisms, in which poses a huge threat to the health of many aquatic organisms such as Cyprinus carpio. In this study, Lactobacillus casei (L. casei) strain CC16 was used as antigen deliver carrier and fused with cholera toxin B subunit (CTB) as an adjuvant to construct the recombinant L. casei pPG-Aha1/Lc CC16(surface-displayed) and pPG-Aha1-CTB/Lc CC16(surface-displayed) expressing Aha1 protein of A. veronii, respectively. And the immune responses in Cyprinus carpio by oral route was explored. Our results demonstrated that the recombinant strains could stimulate high serum specific antibody immunoglobulin M (IgM) and induce a stronger acid phosphatase (ACP), alkaline phosphatase (AKP), C3, C4, lysozyme (LZM), Lectin and superoxide dismutase (SOD) activity in Cyprinus carpio compared with control groups. Meanwhile, the expression of Interleukin-10 (IL-10), Interleukin-1ß (IL-1ß), Tumor Necrosis Factor-α (TNF-α), immunoglobulin Z1 (IgZ1) and immunoglobulin Z2 (IgZ2) in the tissues were significantly upregulated compared with Lc-pPG or PBS groups, indicating that humoral and cell immune response were triggered. Additionally, recombinant L. casei could survive and colonize in fish intestine. Significantly, recombinant L. casei provides immune protection against A. veronii infection, which Cyprinus carpio received pPG-Aha1-CTB/Lc CC16 (64.29%) and pPG-Aha1/Lc CC16 (53.57%) had higher survival rates compared with the controls. Thus, we demonstrated that recombinant pPG-Aha1/Lc CC16 and pPG-Aha1-CTB/Lc CC16 may be the promising strategy for the development of an oral vaccine against A. veronii.

Dietary supplementation of probiotics fermented Chinese herbal medicine Sanguisorba officinalis cultures enhanced immune response and disease resistance of crucian carp (Carassius auratus) against Aeromonas hydrophila.

Aeromonas hydrophila, a Gram-negative bacterium, is one of the major pathogens causing bacterial sepsis in aquatic animals due to drug resistance and pathogenicity, which could cause high mortality and serious economic losses to the aquaculture. Sanguisorba officinalis (called DiYu in Chinese, DY) is well known as herbal medicine, which could inhibit the growth of pathogenic bacteria, hemostasis and regulate the immune response. Moreover, the active ingredients in DY could remarkably reduce drug resistance. In this study, we investigated the effects of probiotic fermentation cultures on A. hydrophila through in vitro and in vivo experiments. Three lactic acid bacteria, including Lactobacillus rhamnosus (LGG), Lactobacillus casei (LC) and Lactobacillus plantarum (LP), were selected to ferment the Chinese herbal medicine DY. The assays of antagonism showed that all three fermented cultures could influence the ability of A. hydrophila growth, among which L. rhamnosus fermented DY cultures appeared to be the strongest inhibitory effect. In addition, the biofilm determination revealed that L. rhamnosus fermented DY cultures could significantly inhibit the biofilm formation of A. hydrophila compared to the other groups. Furthermore, protease, lecithinase and urease activities were found in the three fermentation cultures. Three probiotics fermented DY cultures were orally administration with crucian carp to evaluate the growth performance, immunological parameters and pathogen resistance. The results showed that the three fermentation cultures could promote the growth performance of crucian carp, and the immunoglobulins, antioxidant-related enzymes and immune-related genes were significantly enhanced. Besides, the results showed that crucian carp received L. rhamnosus (60.87%), L. casei (56.09%) and L. plantarum (41.46%) fermented DY cultures had higher survival rates compared with the control group after infection with A. hydrophila. Meanwhile, the pathological tissue results revealed that the probiotic fermented cultures could largely improve the tissues damage caused by the pathogenic bacteria. In conclusion, this study proved that the fermentation cultures of three probiotics could effectively inhibit the growth of A. hydrophila, regulate the level of immune response and improve the survival rate against A. hydrophila in crucian carp. The present data suggest that probiotic fermented Sanguisorba officinalis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection.

Aeromonas veronii virulence and adhesion attenuation mediated by the gene aodp.

Aeromonas veronii (A. veronii) is an opportunistic pathogen of fish-human-livestock, which poses a threat to the development of aquaculture. Based on our previous studies on proteomics and genomics, we found out that the aodp gene may be related to the virulence of A. veronii TH0426. However, aodp gene encodes a hypothetical protein with an unknown function, and its role in A. veronii TH0426 is not clear. Here, we first constructed a mutant strain (△-aodp) to investigate the functional role of aodp in A. veronii TH0426. Compared with the wild strain A. veronii TH0426, the growth rate of strain △-aodp was slower and was resistant to neomycin and kanamycin, but sensitive to cephalexin. The swimming and swarming ability of △-aodp strain decreased, and the pathogenicity to mice decreased by 15.84-fold. Besides, the activity of caspase-3 in EPCs infected with △-aodp strain was 1.49-fold lower than that of the wild strain. We examined 20 factors closely related to A. veronii virulence, among them 17 genes were down-regulated as a result of aodp deficiency. This study laid a foundation for further studies on the pathogenesis of A. veronii.

Functional analysis of fis in Aeromonas veronii TH0426 reveals a key role in the regulation of virulence.

Aeromonas veronii is a comorbid pathogen that can infect humans, and animals including various aquatic organisms. In recent years, an increasing number of cases of A. veronii infection has been reported, indicating serious risks. This bacterium not only threatens public health and safety but also causes considerable economic loss in the aquaculture industry. Currently, some understanding of the pathogenic mechanism of A. veronii has been obtained. In this study, we first constructed the A. veronii TH0426 fis gene deletion strain Δfis and the complementation strain C-fis through homologous recombination technology. The results showed that the adhesion and invasion ability of the Δfis strain towards Epithelioma papulosum cyprini (EPC) cells and the cytotoxicity were 3.8-fold and 1.38-fold lower, respectively, than those of the wild-type strain. In the zebrafish infection model, the lethality of the deleted strain is 3-fold that of the wild strain. In addition, the bacterial load of the deletion strain Δfis in crucian carp was significantly lower than the wild-type strain, and the load decreased with time. In summary, deletion of the fis gene led to a decrease in the virulence of A. veronii. Our research results showed that the deletion of the fis gene significantly reduces the virulence and adhesion ability of A. veronii TH0426. Therefore, the fis gene plays a vital role in the pathogenesis of A. veronii TH0426. This preliminary study of the function of the fis gene in A. veronii will help researchers further understand the pathogenic mechanism of A. veronii.

NLRP3 inflammasome and IL-1ß pathway in type 2 diabetes and atherosclerosis: Friend or foe?

Type 2 diabetes and atherosclerosis have gradually garnered great attention as inflammatory diseases. Previously, the fact that Interleukin-1ß (IL-1ß) accelerates the development of type 2 diabetes and atherosclerosis has been proved in animal experiments and clinical trials. However, the continued studies found that the effect of IL-1ß on type 2 diabetes and atherosclerosis is much more complicated than the negative impact. Nucleotide-binding oligomerization domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome, whose activation and assembly significantly affect the release of IL-1ß, is a crucial effector activated by a variety of metabolites. The diversity of NLRP3 activation mode is one of the fundamental reasons for the intricate effects on the progression of type 2 diabetes and atherosclerosis, providing many new insights for us to intervene in metabolic diseases. This review focuses on how NLRP3 inflammasome affects the progression of type 2 diabetes and atherosclerosis and what opportunities and challenges it can bring us.

Terbinafine Hydrochloride Combined With Itraconazole for Fungal Skin Diseases: A Randomized Controlled Trial.

BACKGROUND: Terbinafine and itraconazole are the common antifungal drugs in clinic. In vitro experiments proved that terbinafine combined with itraconazole achieves better antifungal effects. However, clinical study addressing this issue was still scarce. STUDY QUESTION: Terbinafine combined with itraconazole achieves better therapeutic effects in fungal skin diseases. STUDY DESIGN: Approximately 178 patients with fungal skin diseases from Meizhou People's Hospital, China, between October 2016 and October 2017 were admitted to this study. Patients were randomly distributed to 3 groups by randomly selecting random numbers and were treated with terbinafine, itraconazole, monotherapy, or combined therapy. Both patients and study investigators were unaware of grouping situations during experiments. Fifteen patients were excluded due to poor compliance, and 11 patients were excluded due to incomplete data. Finally, 152 patients were analyzed for this study. MEASURES AND OUTCOMES: The therapeutic effects were evaluated by clinic symptom scores, mycology examination, the cure rate, and the cure time. Adverse events, relapse of disease, and patient's satisfaction level were recorded during follow-up. RESULTS: In the terbinafine + itraconazole group, at 14 days after treatment, the symptom scores were significantly decreased, compared with the terbinafine or itraconazole group (P1 < 0.05, P2 < 0.05). At 28 days after treatment, the fungal infection of 37 patients was eradicated, which were significantly more than 26 patients in the terbinafine group and 19 patients in the itraconazole group (P1 < 0.05, P2 < 0.05). The terbinafine + itraconazole group also exhibited 100% cure rate of patients with fungal skin diseases, shorter cure time, and increased number of cured patients during the same treatment period, which was better than terbinafine or itraconazole monotherapy (P1 < 0.05, P2 < 0.05). In addition, no adverse events and no relapse of fungal disease were reported in the terbinafine + itraconazole group during follow-up. Ninety-eight percent patients were satisfied with the therapeutic effects of combined treatment. CONCLUSIONS: Compared with terbinafine or itraconazole monotherapy, terbinafine + itraconazole combined treatment achieves better therapeutic effects in fungal skin diseases.

Comparative genomic analysis of different virulence strains reveals reasons for the increased virulence of Aeromonas veronii.

Aeromonas veronii is an important zoonotic and aquatic agent. More and more cases have shown that it has caused huge economic losses in the aquaculture industry in addition to threatening human health. But the reasons for the increasing virulence of A. veronii are still unclear. In order to further understand the reasons for the increased virulence of A. veronii, we conducted a comparative analysis of the genomes of A. veronii with different virulence. The analysis revealed that there are multiple virulence factors, such as those related to fimbriae, flagella, toxins, iron ion uptake systems and type II, type III and type VI secretion systems in the virulent strain TH0426 genome. And comparative analysis showed that there were two complete type III secretion systems (API1 and API2), of which the API2 and iron ion transport system were unique to the TH0426 strain. In addition, TH0426 strain also has unique functional gene clusters, which may play important roles in terms of resisting infection, adapting to different environments and genetic evolution. These particular virulence factors and gene clusters may be the important reasons for the increased virulence. These insights will provide a reference for the study of the pathogenesis of A. veronii.

Bilobalide reversibly modulates blood-brain barrier permeability through promoting adenosine A1 receptor-mediated phosphorylation of actin-binding proteins.

Bilobalide, one of the key bioactive components of Ginkgo biloba leaves, exerts prominent neuroprotective properties in central nervous system (CNS) disease. However, the effect of bilobalide on blood-brain barrier (BBB) permeability remains unknown. In this study, we investigated the effect of bilobalide on BBB permeability and its potential mechanism involved. Both the in vitro and in vivo results showed that significant enhancement of BBB permeability was found following bilobalide treatment, evidenced by the reduced transendothelial electrical resistance (TEER), the increased fluorescein sodium (Na-F) penetration rate in vitro and the leakage of FITC-dextran in vivo. Transmission electron microscope (TEM) images demonstrated that bilobalide modulated BBB permeability by changing the ultrastructure of tight junctions (TJs). In addition, actin-binding proteins ezrin, radixin and moesin (ERM) and Myosin light chain (MLC) phosphorylation was observed following bilobalide treatment. Moreover, the effect of bilobalide on TEER reduction and ERM/MLC phosphorylation was counteracted by adenosine A1 receptor (A1R) siRNA. The current findings suggested that bilobalide might reversibly modulate BBB permeability by the alteration of TJs ultrastructure through A1R-mediated phosphorylation of actin-binding proteins.

Nucleoside Diphosphate Kinases (ndk) reveals a key role in adhesion and virulence of Aeromonas veronii.

Aeromous veronii is a severe pathogen that can infect aquatic organisms and mammals also causes irreparable damage to fish aquaculture. Analysis of the results of epidemiological investigations have revealed that its tolerance to drugs and the virulence of A. veronii have increased in recent years. Most of the researches on A. veronii focuse on the strain isolation, identification, and drug susceptibility. However, we do not know so much about the molecular mechanism of the pathogenesis on A. veronii. Here we identified and obtained the highly expressed TH0426 Nucleoside Diphosphate Kinases (NDK) of A. veronii. We first constructed a mutant strain (△-ndk) by generating an in-frame deletion of the ndk gene, to investigate the functional role in A. veronii TH0426. The ability in the adhesion and invasion of EPC cells and biofilm formation significantly reduced of the △-ndk strain. The motility test showed that the ndk gene affected on the swimming ability, while did not affect the swarming motility. Compared with the wild-type strain TH0426, the pathogenicity of △-ndk strain to zebrafish reduced severely. Besides, the ndk gene has affected the apoptosis rate of A. veronii TH0426. These results would help to demonstrate the function of ndk further and realize the pathogenesis on A. veronii.

Construction and immune efficacy of recombinant Lactobacillus casei strains expressing Malt from Aeromonas veronii.

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1ß, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Results also showed that recombinant L. casei could stimulate the mucosa through colonization of the intestine, resulting in increased transcription of IL-10, IL-1ß, TNF-α, and IFN-γ. Immunity and colonization assays also showed that after 34 days of fasting, recombinant L. casei were still present in the intestines of the immunized fish. Common carp that received Lc-MCS-Malt(53.3%) and Lc-pPG-Malt (46.7%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our findings suggested that recombinant L. casei can adequately protect fish and improve immunity, providing a theoretical basis for the future development of an oral Lactobacillus vaccine for use in aquaculture.