Stresses induced by the deficiency or excess of trace mineral elements, such as manganese (Mn), represent a common limiting factor for the production of crops like Brassica napus. To identify key genes involved in Mn allocation in B. napus and elucidate the underlying mechanisms, a member of the metal tolerance protein (MTP) family obtained in the previous screening of cDNA library of B. napus under Mn stress was selected as the research subject. Based on the sequence information and phylogenetic analysis, it was named as BnMTP10. It belongs to the Mn-cation diffusion facilitator (CDF) subfamily. Expression of BnMTP10 in yeast significantly improved the tolerance of transformants to excessive Mn and iron (Fe) and reduced the accumulation of Mn and Fe. However, the yeast transformants exhibited no significant changes in tolerance to excess cadmium, boron, aluminum, zinc, or copper. The qRT-PCR results demonstrated that the flowers of B. napus had the highest expression of BnMTP10, followed by roots and leaves. Subcellular localization studies revealed that BnMTP10 was localized in the endoplasmic reticulum (ER). Compared with wild-type plants, transgenic Arabidopsis overexpressing BnMTP10 exhibited enhanced tolerance to excessive Mn stress but showed no significant difference under Fe stress. Correspondingly, under excessive Mn stress, the Mn content in the roots of transgenic Arabidopsis increased significantly. However, under excessive Fe stress, the Fe content in transgenic Arabidopsis did not alter significantly. According to the results, we hypothesize that BnMTP10 may alleviate excessive Mn stress in plants by mediating Mn transport to the ER. This study facilitated our understanding of efficient mineral nutrients, and provided theoretical foundations and gene resources for breeding B. napus.
Brassica napus/genetics* ; Manganese/metabolism* ; Plants, Genetically Modified/genetics* ; Plant Proteins/physiology* ; Arabidopsis/metabolism* ; Gene Expression Regulation, Plant ; Phylogeny ; Cation Transport Proteins/metabolism* ; Stress, Physiological

Objective To evaluate whether Vibrio vulnificus secreted exotoxin-hemolysin (VVH) can activate platelet, an important blood immune cell, and to explore the possible molecular mechanism of platelet activation by VVH. Methods Transcriptomics and immunohistochemistry were used to analyze whether Vibrio vulnificus infection caused platelet activation in mice. Then, flow cytometry was used to identify whether VVH was the main stimulator of platelet activation. Naturally expressed VVH toxin was purified and prepared. The effects of extracellular and intracellular Ca²⁺ signal inhibitors on VVH activated platelets were evaluated by flow cytometry and Western blotting. The immune activation effect of VVH in the early stage of Vibrio vulnificus infection was analyzed in vivo. Results VVH was the main stimulator of platelet activation in Vibrio vulnificus culture supernatant. Natural VVH can induce the increase of P-selectin (CD62P) on platelet surface, the formation of platelet-neutrophil complex (PNC), and the release of platelet microvesicles. The activation mechanism may be related to the VVH pore-dependent Ca²⁺-calmodulin (CaM) -myosin light chain kinase (MLCK) signaling pathway, which led to the release of platelet alpha particles and cascade activation of platelets. In a mouse model of ALD infected by Vibrio vulnificus gavage, VVH was strongly associated with platelet activation. Conclusion This study shows that VVH is an important platelet activating molecule in the early stage of Vibrio vulnificus infection, and its induction of platelet activation may be related to the pathogenic process.
Animals ; Platelet Activation/drug effects* ; Hemolysin Proteins/pharmacology* ; Vibrio vulnificus/metabolism* ; Mice ; Blood Platelets/drug effects* ; Vibrio Infections/immunology* ; P-Selectin/metabolism* ; Bacterial Proteins ; Female

The application of artificial intelligence(AI)in the medical field, particularly for predicting, diagnosing and treating retinal detachment(RD), has made remarkable achievements. This paper reviews the advancements in AI applications for RD across multiple dimensions, including predicting RD incidence, assessing surgical success rates, forecasting postoperative visual outcomes, and evaluating recurrence rates. In diagnostic support, AI technology has demonstrated significant value, especially in ophthalmic imaging, with applications in the intelligent analysis of ultra-wide-angle fundus photography, optical coherence tomography(OCT), ophthalmologic ultrasound images, and AI chatbots models. Furthermore, AI has proven uniquely beneficial in surgical decision-making, robotic-assisted surgical systems, and the assessment of surgical complications. This paper provides a comprehensive overview of the current state of AI applications in RD, underscoring its potential to address numerous challenges in clinical practice. It also explores existing limitations and offers insights into future directions for development in this field.

The application of artificial intelligence(AI)in the medical field, particularly for predicting, diagnosing and treating retinal detachment(RD), has made remarkable achievements. This paper reviews the advancements in AI applications for RD across multiple dimensions, including predicting RD incidence, assessing surgical success rates, forecasting postoperative visual outcomes, and evaluating recurrence rates. In diagnostic support, AI technology has demonstrated significant value, especially in ophthalmic imaging, with applications in the intelligent analysis of ultra-wide-angle fundus photography, optical coherence tomography(OCT), ophthalmologic ultrasound images, and AI chatbots models. Furthermore, AI has proven uniquely beneficial in surgical decision-making, robotic-assisted surgical systems, and the assessment of surgical complications. This paper provides a comprehensive overview of the current state of AI applications in RD, underscoring its potential to address numerous challenges in clinical practice. It also explores existing limitations and offers insights into future directions for development in this field.

Objective To explore the clinical significance of miR-423-5p in ischemic stroke and its effects on SH-SY5Y cell apoptosis along with the underlying mechanisms.Methods Plasma samples were collected from three stroke-prone patients who subsequently developed acute ischemic stroke(AIS)within 24 hours.High-throughput sequencing was used to identify miR-423-5p as a key target.Venous blood samples were collected from 46 AIS pa-tients within 24 hours of onset and from 46 matched control cases.RT-qPCR was performed to measure the plasma expression of miR-423-5p in both groups.The diagnostic value of miR-423-5p for AIS was evaluated using receiver operating characteristic(ROC)curve.After overexpression and knockdown of miR-423-5p in SH-SY5Y cells,flow cytometry was performed to detect apoptosis in each group to analyze the potential relationship between miR-423-5p and SH-SY5Y cell apoptosis.Western blot was used to measure changes in the expression level of the ap-optotic protein cleaved caspase-3 and the anti-apoptotic proteins Bcl-2 and Bcl-xL.Results The expression of miR-423-5p in 3 AIS patients showed significant difference before and after onset.Plasma miR-423-5p expression was significantly elevated in AIS patients versus controls(P＜0.001).ROC analysis indicated its strong diagnostic potential for AIS.Overexpression of miR-423-5p increased the apoptosis rate and cleaved caspase-3 ex-pression of SH-SY5Y cells after OGD/R,and decreased the expression of Bcl-2 and Bcl-xL,while knocking down miR-423-5p had the opposite effect.Conclusions The expression of miR-423-5p is upregulated in the plasma of patients with ischemic stroke compared to healthy controls.Furthermore,miR-423-5p promotes OGD/R-induced apoptosis in SH-SY5Y cells by increasing the level of cleaved caspase-3 and suppressing the expression of Bcl-2 and Bcl-xL.

Objective:To explore the mechanisms of action of Huanglian Jiedu decoction combined with Xijiao Dihuang decoction (HLJDT-XJDH) in regulating fibroblasts in the treatment of psoriasis. Methods:A mouse model of psoriasis was established by topical application of imiquimod 5% cream on the shaved back; HLJDT-XJDH at different doses of 7.7 and 30.6 g/kg was administered via gavage for intervention, and methotrexate (2 mg/kg) served as a positive control; after 7 days, the severity of skin lesions was assessed using the psoriasis area and severity index (PASI), while histopathological changes of skin tissues were evaluated using hematoxylin-eosin (HE) staining and Baker scoring. For in vitro experiments, fibroblasts were divided into a control group, a model group, a low-dose (5% drug-containing serum) intervention group, and a high-dose (20% drug-containing serum) intervention group; cells in the control group were cultured with 20% normal rat serum for 24 hours; in the model group, cells cultured with 20% normal rat serum were stimulated with 5 ng/ml tumor necrosis factor (TNF) -α and 50 ng/ml interleukin (IL) -17A for 24 hours to mimic fibroblasts during the occurrence of psoriasis; cells in the low- and high-dose intervention groups received the same stimulation as the model group, and were cultured for 24 hours with 5% and 20% HLJDT-XJDH-containing serum, respectively, but not with the 20% normal rat serum. After the above treatment, these cells were co-cultured with keratinocytes (HaCaT cells) using a Transwell system. In addition, on the basis of the control group, fibroblasts were divided into the model group, 20% drug-containing serum intervention group, and 20% drug-containing serum intervention + OE-SFRP2 group; TNF-α and IL-17A were used to stimulate the cells to simulate the psoriatic state; the treatment in the 20% drug-containing serum intervention group was carried out as previously described; in the 20% drug-containing serum intervention + OE-SFRP2 group, cells were transfected with the vector for 48 hours to establish an overexpression model, followed by culture with 20% drug-containing serum for 24 hours, without co-culture with HaCaT cells.. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability, flow cytometry to measure apoptosis rates, enzyme-linked immunosorbent assay (ELISA) to detect levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) as well as chemokine ligand (CXCL) 1 and CXCL12 in mouse serum or cell culture supernatant, qPCR to determine the mRNA expression of inflammatory cytokines, chemokines, cell cycle- and proliferation-related factors, as well as SFRP2 in mouse skin tissues or cells, and Western blot analysis to determine the protein expression of SFRP2, Wnt3a, and β-catenin in fibroblasts. One-way analysis of variance was employed for intergroup comparisons, and post-hoc analysis was conducted using Tukey's test. Results:In vivo mouse experiments showed that compared with the normal control group, the model group exhibited typical psoriatic characteristics in skin morphology, including significant inflammatory infiltration in skin tissues and marked epidermal thickening; compared with the normal control group, the serum levels of TNF-α (531.16 ± 28.27 pg/ml vs. 239.58 ± 10.39 pg/ml), IL-1β (111.40 ± 5.16 pg/ml vs. 80.35 ± 3.87 pg/ml), and IL-6 (109.17 ± 4.84 pg/ml vs. 71.73 ± 2.04 pg/ml) significantly increased in the model group, along with their mRNA expression levels in mouse skin tissues (all P < 0.001) ; compared with the model group, the treatment group showed alleviated psoriatic manifestations, and significant reductions in the levels of inflammatory factors TNF-α (low-dose, high-dose, and positive control groups: 420.80 ± 29.30 pg/ml, 322.33 ± 9.40 pg/ml, 322.97 ± 12.16 pg/ml, respectively), IL-1β (98.69 ± 4.49 pg/ml, 89.02 ± 1.56 pg/ml, 88.88 ± 2.08 pg/ml, respectively), and IL-6 (94.07 ± 3.76 pg/ml, 80.54 ± 3.30 pg/ml, 83.21 ± 3.18 pg/ml, respectively), as well as in their mRNA expression levels (all P < 0.001). In in vitro fibroblast experiments, compared with the control group, the model group exhibited a significant elevation in the supernatant levels of IL-1β (126.42 ± 3.56 pg/ml vs. 34.81 ± 0.44 pg/ml), IL-6 (459.44 ± 9.35 pg/ml vs. 115.51 ± 7.26 pg/ml), CXCL1 (2 434.88 ± 127.63 pg/ml vs. 762.85 ± 30.60 pg/ml) and CXCL12 (3 542.14 ± 35.86 pg/ml vs. 2 095.86 ± 45.12 pg/ml), the expression levels of their mRNAs (all P < 0.001), as well as the protein expression levels of SFRP2, Wnt3a, and β-catenin; after intervention with HLJDT-XJDH-containing serum, all the above indices significantly decreased (all P < 0.001). However, when 20% drug-containing serum intervention was administered simultaneously, the expression of inflammatory factors and chemokines in fibroblasts was significantly higher in the SFRP2 overexpression group than in the non-overexpression group (all P < 0.01). When fibroblasts were co-cultured with HaCaT cells, the model group showed significantly increased cell viability but a decreased apoptosis rate of HaCaT cells compared with the control group, while the low- and high-dose intervention groups showed significantly decreased cell viability but increased apoptosis rates of HaCaT cells compared with the model group (all P < 0.05) . Conclusion:HLJDT-XJDH may exert therapeutic effects in psoriasis by downregulating the SFRP2/Wnt/β-catenin signaling pathway, thereby inhibiting fibroblast activation and inflammatory process, which subsequently suppresses the proliferation of keratinocytes and the activation of inflammatory cells.

Objective:To explore the mechanisms of action of Huanglian Jiedu decoction combined with Xijiao Dihuang decoction (HLJDT-XJDH) in regulating fibroblasts in the treatment of psoriasis. Methods:A mouse model of psoriasis was established by topical application of imiquimod 5% cream on the shaved back; HLJDT-XJDH at different doses of 7.7 and 30.6 g/kg was administered via gavage for intervention, and methotrexate (2 mg/kg) served as a positive control; after 7 days, the severity of skin lesions was assessed using the psoriasis area and severity index (PASI), while histopathological changes of skin tissues were evaluated using hematoxylin-eosin (HE) staining and Baker scoring. For in vitro experiments, fibroblasts were divided into a control group, a model group, a low-dose (5% drug-containing serum) intervention group, and a high-dose (20% drug-containing serum) intervention group; cells in the control group were cultured with 20% normal rat serum for 24 hours; in the model group, cells cultured with 20% normal rat serum were stimulated with 5 ng/ml tumor necrosis factor (TNF) -α and 50 ng/ml interleukin (IL) -17A for 24 hours to mimic fibroblasts during the occurrence of psoriasis; cells in the low- and high-dose intervention groups received the same stimulation as the model group, and were cultured for 24 hours with 5% and 20% HLJDT-XJDH-containing serum, respectively, but not with the 20% normal rat serum. After the above treatment, these cells were co-cultured with keratinocytes (HaCaT cells) using a Transwell system. In addition, on the basis of the control group, fibroblasts were divided into the model group, 20% drug-containing serum intervention group, and 20% drug-containing serum intervention + OE-SFRP2 group; TNF-α and IL-17A were used to stimulate the cells to simulate the psoriatic state; the treatment in the 20% drug-containing serum intervention group was carried out as previously described; in the 20% drug-containing serum intervention + OE-SFRP2 group, cells were transfected with the vector for 48 hours to establish an overexpression model, followed by culture with 20% drug-containing serum for 24 hours, without co-culture with HaCaT cells.. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability, flow cytometry to measure apoptosis rates, enzyme-linked immunosorbent assay (ELISA) to detect levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) as well as chemokine ligand (CXCL) 1 and CXCL12 in mouse serum or cell culture supernatant, qPCR to determine the mRNA expression of inflammatory cytokines, chemokines, cell cycle- and proliferation-related factors, as well as SFRP2 in mouse skin tissues or cells, and Western blot analysis to determine the protein expression of SFRP2, Wnt3a, and β-catenin in fibroblasts. One-way analysis of variance was employed for intergroup comparisons, and post-hoc analysis was conducted using Tukey's test. Results:In vivo mouse experiments showed that compared with the normal control group, the model group exhibited typical psoriatic characteristics in skin morphology, including significant inflammatory infiltration in skin tissues and marked epidermal thickening; compared with the normal control group, the serum levels of TNF-α (531.16 ± 28.27 pg/ml vs. 239.58 ± 10.39 pg/ml), IL-1β (111.40 ± 5.16 pg/ml vs. 80.35 ± 3.87 pg/ml), and IL-6 (109.17 ± 4.84 pg/ml vs. 71.73 ± 2.04 pg/ml) significantly increased in the model group, along with their mRNA expression levels in mouse skin tissues (all P < 0.001) ; compared with the model group, the treatment group showed alleviated psoriatic manifestations, and significant reductions in the levels of inflammatory factors TNF-α (low-dose, high-dose, and positive control groups: 420.80 ± 29.30 pg/ml, 322.33 ± 9.40 pg/ml, 322.97 ± 12.16 pg/ml, respectively), IL-1β (98.69 ± 4.49 pg/ml, 89.02 ± 1.56 pg/ml, 88.88 ± 2.08 pg/ml, respectively), and IL-6 (94.07 ± 3.76 pg/ml, 80.54 ± 3.30 pg/ml, 83.21 ± 3.18 pg/ml, respectively), as well as in their mRNA expression levels (all P < 0.001). In in vitro fibroblast experiments, compared with the control group, the model group exhibited a significant elevation in the supernatant levels of IL-1β (126.42 ± 3.56 pg/ml vs. 34.81 ± 0.44 pg/ml), IL-6 (459.44 ± 9.35 pg/ml vs. 115.51 ± 7.26 pg/ml), CXCL1 (2 434.88 ± 127.63 pg/ml vs. 762.85 ± 30.60 pg/ml) and CXCL12 (3 542.14 ± 35.86 pg/ml vs. 2 095.86 ± 45.12 pg/ml), the expression levels of their mRNAs (all P < 0.001), as well as the protein expression levels of SFRP2, Wnt3a, and β-catenin; after intervention with HLJDT-XJDH-containing serum, all the above indices significantly decreased (all P < 0.001). However, when 20% drug-containing serum intervention was administered simultaneously, the expression of inflammatory factors and chemokines in fibroblasts was significantly higher in the SFRP2 overexpression group than in the non-overexpression group (all P < 0.01). When fibroblasts were co-cultured with HaCaT cells, the model group showed significantly increased cell viability but a decreased apoptosis rate of HaCaT cells compared with the control group, while the low- and high-dose intervention groups showed significantly decreased cell viability but increased apoptosis rates of HaCaT cells compared with the model group (all P < 0.05) . Conclusion:HLJDT-XJDH may exert therapeutic effects in psoriasis by downregulating the SFRP2/Wnt/β-catenin signaling pathway, thereby inhibiting fibroblast activation and inflammatory process, which subsequently suppresses the proliferation of keratinocytes and the activation of inflammatory cells.

Objective To investigate the effect of glycine N-methyl transferase (GNMT)on intrauterine adhesion (IUA)fibrosis and its related mechanism.Methods In vivo experiment:A total of 36 healthy female SD rats (SPF grade,6～8 weeks old and weighing from 180～220 g)were subjected in this study.IUA model of SD rats and IUA model of GNMT overexpressed rats were established.RT-qPCR and immunofluorescence assay were applied to detect GNMT expression level in normal uterus and model group.RT-qPCR and Western blotting were used to detect the mRNA and protein levels of fibrosis-related molecules and the activation of TGF-β1/Smad3 signaling pathway in each group.The number of endometrial glands in each group was observed by HE staining.Masson staining was used to analyze the severity of endometrial fibrosis in each group.In vitro experiment:transformed human endometrial stromal cells (THESCs)fibrotic phenotype model was constructed using TGF-β1,and THESCs stably transfected with GNMT overexpression lentvirus were treated with TGF-β1.RT-qPCR and Western blotting were used to detect the mRNA and protein expression of fibrosis-related molecules.The expression of TGF-β1/Smad3 signaling pathway was detected by Western blotting.TGF-β1/Smad3 signaling pathway was activated by TGF-β1/Smad signaling pathway activator (SRI-011381),and the expression of TGF-β1/Smad3 signaling pathway and key molecular proteins of fibrosis phenotype was measured with Western blotting.Results In vivo experiment,the mRNA and protein expression levels of GNMT were significantly decreased in the IUA rats than the control rats (P<0.05).Overexpression of GNMT decreased the mRNA and protein levels of fibrosis related molecules,Collagen Ⅰ,Collagen Ⅲ and FN in the IUA rats (P<0.05),and decreased the phosphorylation levels of TGF-β1 and its downstream Smad3 protein (P<0.05).HE and Masson staining showed that overexpression of GNMT could increase the number of endometrial glands and reduce the severity of fibrosis in the IUA rats (P<0.05).In vitro experiments:overexpression of GNMT decreased the mRNA and protein levels of Collagen Ⅰ,Collagen Ⅲ and FN associated with fibrotic phenotype of THESCs (P<0.05),and reduced the phosphorylation level of Smad3 protein,downstream of TGF-β1 (P<0.05).After activation of TGF-β1/Smad3 signaling pathway,the protein levels of TGF-β1/Smad3 signaling pathway and downstream fibrosis phenotype molecules,Collagen Ⅲ and FN,were significantly decreased in the LV-GNMT+SRI-011381 group.Conclusion Overexpression of GNMT can inhibit endometrial fibrosis by regulating TGF-β1/Smad3 signaling pathway,thus achieving therapeutic effect on IUA.

Objective To isolate the Japanese encephalitis virus carried by Culex tritaeniorhynchus in Dongchuan District of Yunnan Province and analyze its molecular characteristics, so as to provide insights into the prevention and control of Japanese encephalitis in Yunnan Province. Methods Mosquito specimens were collected using mosquito-trapping lamps from pig farms in Batang Village and Xiaoxin Village, Dongchuan District, Kunming City, Yunnan Province in July 2016, and the mosquito species was identified according to the mosquito morphology. Then, 60 to 100 mosquitoes of each species served as a group and were ground. Baby hamster kidney-21 (BHK-21) cells and Aedes albopictus clone C6/36 cells were used for virus isolation, and positive isolates were identified using flavivirus primers. The positive isolates were amplified using reverse transcription polymerase chain reaction (RT-PCR) assay with 15 pairs of specific primers covering the full length of the genotype I Japanese encephalitis virus, and DNA sequence assembly was performed using the software SeqMan in the DNASTAR package. The obtained sequences were aligned with the complete sequences of 38 Japanese encephalitis virus downloaded from the GenBank with the software MegAlign, and the nucleotide and amino acid homology analyses of the obtained sequences were performed. The difference in amino acid sites was analyzed with the software GeneDoc, and phylogenetic trees were created based on the sequences of the coding region and E protein of the isolated Japanese encephalitis virus with the software Mega X. In addition, the secondary and tertiary structures of the E protein of the Japanese encephalitis virus were predicted using the online tool SOPMA and the software Swiss-Model. Results A total of 5 820 mosquitoes were collected and 3 843 Cx. tritaeniorhynchus (66.03%) were identified according to the mosquito morphology. A positive virus isolate, termed YNDC55-33, was isolated from Cx. tritaeniorhynchoides following batches of virus isolation from mosquito specimens, and cytopathic effect was observed following inoculation into BHK-21 and C6/36 cells. The YNDC55-33 virus isolate was successfully amplified with the flavivirus primes, and a long sequence containing 300 nucleotides was obtained. Following sequence alignment using the BLAST tool, the sequence of the YNDC55-33 virus isolate had high homology with that of the genotype I Japanese encephalitis virus. A long sequence with 10 845 nucleotides in length, which encoded 3 432 amino acids, was obtained by splicing the full sequence of the YNDC55-33 virus isolate. Phylogenetic analysis based on the whole-genome sequence and E gene sequence of the YNDC55-33 virus isolate showed that the new YNDC55-33 virus isolate was most closely related to the genotype I Guizhou isolate (GenBank accession number: HM366552), with nucleotide homology of 98.5% and amino acid homology of 99.4%, and the YNDC55-33 virus isolate shared 97.96% ± 0.33% nucleotide homology and 99.35% ± 0.08% amino acid homology with other genotype I Japanese encephalitis virus isolates, and < 90% nucleotide homology and < 98% amino acid homology with other genotypes of Japanese encephalitis virus. The YNDC55-33 virus isolate and the live attenuated virus vaccine candidate SA14-14-2 isolate differed at 16 amino acid sites on E gene, and 7 out of 8 key amino acid sites related to neurovirulence. The secondary and tertiary structures of the E protein of the YNDC55-33 virus isolate were predicted to be characterized by random coils. Conclusions A genotype I Japanese encephalitis virus was isolated from Cx. tritaeniorhynchus in Dongchuan District, Kunming City. This virus isolate and the live attenuated virus vaccine candidate SA14-14-2 isolate does not differ at antigenic epitopes-related key amino acid sites, and the major protein structure of the virus isolate is random coils. This study adds new data for the epidemiological distribution of Japanese encephalitis virus in Yunnan Province, which may provide insights into the prevention and control of Japanese encephalitis in the province.