Objective To systematically evaluate robot-assisted gait training(RAGT)on motor function,ambulation and activities of daily living of patients after stroke and spinal cord injury(SCI),and to investigate the clinical value of differ-ent robotic technologies and control strategies.Methods In accordance with PRISMA guidelines,relevant randomized controlled trials(RCTs)published between 2020 and 2024 were identified from databases including Scopus,Web of Science,PubMed,Cochrane Library and CNKI.The PEDro scale was used to assess methodological quality,and a comprehensive analysis was performed on the therapeutic effects of RAGT on walking ability,balance,lower limb muscle strength and functional inde-pendence.Results Eight RCTs involving 702 participants were included,originating from countries such as China,Italy,India,Tur-key and Poland.The population consisted of adult patients with various subtypes of stroke or SCI.These studies were published in journals across geriatric neuroscience,biosciences,medicine and sports science.Interventions involved three categories of lower limb exoskeleton including treadmill-based systems(end-effector and exoskel-eton models),overground exoskeletons and specialized joint/platform-based robots.The training frequency was 20 to 45 minutes a time,once to twice a day,one to seven days a week,for a total of two to ten weeks.RAGT might significant improve gait parameters and lower limb muscle strength,though its impact on functional inde-pendence was heterogeneous.Adaptive control strategies(e.g.,assist-as-needed)proved superior to fixed-parame-ter modes.Treadmill-based systems(e.g.,Lokomat)were well-suited for early-stage rehabilitation,while over-ground exoskeletons(e.g.,EKSO-GT)better facilitated adaptation to real-world environments.Conclusion RAGT is an effective modality for improving gait and lower limb function of patients with stroke and SCI.The therapeutic outcome is contingent upon personalized setup of the exoskeleton and the implementation of adaptive control strategies.Different adaptive control modes have been developed for the three main types of lower limb exoskeleton.Rehabilitation training should consider the specific lower limb tasks with the robot's cor-responding adaptive movement and control modes.

Objective To investigate the incidence of functional bowel disease(FBD)and its relationship with depression,anxiety and sleep in navy submarine forces.Methods A questionnaire survey on the incidence of FBD was conducted on 364 naval soldiers who were enrolled according to Rome Ⅳ classification and diagnostic criteria.The risk factors of FBD were analyzed.The incidence of specific diseases of FBD was compared among soldiers with different jobs.The depression,anxiety,and sleep quality were investigated in FBD patients.Results In the 364 participants,132(36.3%)were diagnosed with FBD according to Roman Ⅳcriteria.Military rank and marital status might be risk factors for FBD.There was no significant difference in the overall incidence of FBD among the participants with various jobs(P>0.05).The incidence of functional constipation in the submariners was significantly higher than that in the land soldiers,while the incidence of functional diarrhea in the land soldiers was higher than that in the submariners(all P<0.05).The incidences of depression,anxiety and sleep disorder in the participants with FBD were higher than those in the participants without FBD.Conclusion There is a high incidence of FBD in submariners,which may be related to their psychological states.

Objective:To investigate the role of microRNA-25-5p (miR-25-5p) in melanogenesis, and to explore its underlying mechanisms.Methods:Target genes of miR-25-5p were predicted using the TargetScan database. The interaction between miR-25-5p and the 3' untranslated region (3' UTR) of the RAB11B gene (a member of RAS oncogene family) was validated through a dual-luciferase reporter assay. Post-inflammatory hyperpigmentation (PIH) models were established in female C57BL/6J mice (6 - 8 weeks old) and female brown guinea pigs (4 - 6 weeks old) through daily broadband ultraviolet B (UVB) irradiation on the dorsal skin of the mouse ear or shaved dorsal skin of guinea pigs, while untreated mice and untreated dorsal skin areas of guinea pigs served as control groups. During modeling, these experimental animals received intradermal injections of a miR-25-5p agomir or a miR control agomir. Changes in skin pigmentation were observed, and skin tissue samples were harvested for further analysis after modeling. Melanin content in skin tissues was evaluated using Masson-Fontana staining. Expression of RAB11B and tyrosinase (TYR) in skin tissues was determined using immunohistochemical staining and quantitative real-time PCR (qPCR). Primary human melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision. Both primary human melanocytes and human MNT1 melanoma cells were transfected with miR-25-5p mimics or miR control mimics. Relative expression levels of miR-25-5p and RAB11B mRNA were quantified by qPCR using the 2 -ΔΔCt calculation method. In MNT1 cells, miR-25-5p and RAB11B were co-overexpressed to assess their effect on the mRNA expression of RAB11B and TYR. Statistical analysis was conducted using t test or one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons. Results:The bioinformatic prediction and dual-luciferase reporter assay confirmed a binding site for miR-25-5p in the 3′ UTR of the RAB11B gene. In both animal models, the treatment with the miR-25-5p agomir significantly reduced local skin pigmentation compared to the control groups; Masson-Fontana staining showed a marked decrease in the density of melanin granules in the epidermis and dermis in the miR-25-5p agomir groups compared with the miR control agomir groups (mice: 0.050 ± 0.005 vs. 0.087 ± 0.008; guinea pigs: 0.067 ± 0.015 vs. 0.110 ± 0.013; both P < 0.05). Immunohistochemical staining revealed significantly lower expression of RAB11B in mouse skin tissues in the miR-25-5p agomir group than in those in the miR control agomir group (both P < 0.05). qPCR revealed significantly lower mRNA expression of RAB11B and TYR in skin tissues of guinea pigs in the miR-25-5p agomir group than in those in the miR control agomir group (both P < 0.05). Similarly, RAB11B mRNA expression significantly decreased in the miR-25-5p mimics group compared with the miR control mimics group in primary human melanocytes and MNT1 cells (both P < 0.05). In human MNT1 melanoma cells, miR-25-5p overexpression could suppress TYR mRNA expression, whereas co-overexpression of miR-25-5p and RAB11B could reverse this suppression. Conclusion:Overexpression of miR-25-5p could alleviate UVB-induced post-inflammatory hyperpigmentation and inhibit melanogenesis, likely by targeted suppression of RAB11B expression.

The aim of this study is to explore the immunogenicity of African swine fever virus p37 recombinant protein in mice.C57BL/6J mice were immunized subcutaneously in the abdomen using p37 recombinant protein as antigen.The second immunization was performed 21 d after the first immunization.Serum-specific antibody levels were detected by ELISA;serum cytokine levels were detected using a multifactor assay technique;mice splenic lymphocytes were isolated 7 d after sec-ondary immunization,and the number of splenic lymphocytes secreting IFN-γ after recombinant protein stimulation was detected by ELISpot;and the ratio of CD4+T cells to CD8+T cells was detected by flow cytometry.The results of indirect ELISA showed that p37 recombinant protein could stimulate mice to produce high levels of specific antibodies;ELISpot showed that p37 recom-binant protein could significantly stimulate splenic lymphocytes to produce IFN-γ(P＜0.001)and activate cellular immune responses;the results of flow cytometry showed that it could signifi-cantly stimulate the differentiation of T-lymphocytes to CD4+T-lymphocytes(P＜0.001).In ad-dition,serum levels of IL-2,IL-4,IFN-γ,and TNF-α immune-related cytokines were significantly higher after the second immunization.Immunization of mice with p37 recombinant protein induced strong humoral and cellular immune responses with good immunogenicity,providing reference for the subsequent epitope identification and functional study of p37 protein and the antigen screening of ASF mRNA vaccine.

Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.

Objective:To investigate the role of microRNA-25-5p (miR-25-5p) in melanogenesis, and to explore its underlying mechanisms.Methods:Target genes of miR-25-5p were predicted using the TargetScan database. The interaction between miR-25-5p and the 3' untranslated region (3' UTR) of the RAB11B gene (a member of RAS oncogene family) was validated through a dual-luciferase reporter assay. Post-inflammatory hyperpigmentation (PIH) models were established in female C57BL/6J mice (6 - 8 weeks old) and female brown guinea pigs (4 - 6 weeks old) through daily broadband ultraviolet B (UVB) irradiation on the dorsal skin of the mouse ear or shaved dorsal skin of guinea pigs, while untreated mice and untreated dorsal skin areas of guinea pigs served as control groups. During modeling, these experimental animals received intradermal injections of a miR-25-5p agomir or a miR control agomir. Changes in skin pigmentation were observed, and skin tissue samples were harvested for further analysis after modeling. Melanin content in skin tissues was evaluated using Masson-Fontana staining. Expression of RAB11B and tyrosinase (TYR) in skin tissues was determined using immunohistochemical staining and quantitative real-time PCR (qPCR). Primary human melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision. Both primary human melanocytes and human MNT1 melanoma cells were transfected with miR-25-5p mimics or miR control mimics. Relative expression levels of miR-25-5p and RAB11B mRNA were quantified by qPCR using the 2 -ΔΔCt calculation method. In MNT1 cells, miR-25-5p and RAB11B were co-overexpressed to assess their effect on the mRNA expression of RAB11B and TYR. Statistical analysis was conducted using t test or one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons. Results:The bioinformatic prediction and dual-luciferase reporter assay confirmed a binding site for miR-25-5p in the 3′ UTR of the RAB11B gene. In both animal models, the treatment with the miR-25-5p agomir significantly reduced local skin pigmentation compared to the control groups; Masson-Fontana staining showed a marked decrease in the density of melanin granules in the epidermis and dermis in the miR-25-5p agomir groups compared with the miR control agomir groups (mice: 0.050 ± 0.005 vs. 0.087 ± 0.008; guinea pigs: 0.067 ± 0.015 vs. 0.110 ± 0.013; both P < 0.05). Immunohistochemical staining revealed significantly lower expression of RAB11B in mouse skin tissues in the miR-25-5p agomir group than in those in the miR control agomir group (both P < 0.05). qPCR revealed significantly lower mRNA expression of RAB11B and TYR in skin tissues of guinea pigs in the miR-25-5p agomir group than in those in the miR control agomir group (both P < 0.05). Similarly, RAB11B mRNA expression significantly decreased in the miR-25-5p mimics group compared with the miR control mimics group in primary human melanocytes and MNT1 cells (both P < 0.05). In human MNT1 melanoma cells, miR-25-5p overexpression could suppress TYR mRNA expression, whereas co-overexpression of miR-25-5p and RAB11B could reverse this suppression. Conclusion:Overexpression of miR-25-5p could alleviate UVB-induced post-inflammatory hyperpigmentation and inhibit melanogenesis, likely by targeted suppression of RAB11B expression.

The aim of this study is to explore the immunogenicity of African swine fever virus p37 recombinant protein in mice.C57BL/6J mice were immunized subcutaneously in the abdomen using p37 recombinant protein as antigen.The second immunization was performed 21 d after the first immunization.Serum-specific antibody levels were detected by ELISA;serum cytokine levels were detected using a multifactor assay technique;mice splenic lymphocytes were isolated 7 d after sec-ondary immunization,and the number of splenic lymphocytes secreting IFN-γ after recombinant protein stimulation was detected by ELISpot;and the ratio of CD4+T cells to CD8+T cells was detected by flow cytometry.The results of indirect ELISA showed that p37 recombinant protein could stimulate mice to produce high levels of specific antibodies;ELISpot showed that p37 recom-binant protein could significantly stimulate splenic lymphocytes to produce IFN-γ(P＜0.001)and activate cellular immune responses;the results of flow cytometry showed that it could signifi-cantly stimulate the differentiation of T-lymphocytes to CD4+T-lymphocytes(P＜0.001).In ad-dition,serum levels of IL-2,IL-4,IFN-γ,and TNF-α immune-related cytokines were significantly higher after the second immunization.Immunization of mice with p37 recombinant protein induced strong humoral and cellular immune responses with good immunogenicity,providing reference for the subsequent epitope identification and functional study of p37 protein and the antigen screening of ASF mRNA vaccine.

Objective To systematically evaluate robot-assisted gait training(RAGT)on motor function,ambulation and activities of daily living of patients after stroke and spinal cord injury(SCI),and to investigate the clinical value of differ-ent robotic technologies and control strategies.Methods In accordance with PRISMA guidelines,relevant randomized controlled trials(RCTs)published between 2020 and 2024 were identified from databases including Scopus,Web of Science,PubMed,Cochrane Library and CNKI.The PEDro scale was used to assess methodological quality,and a comprehensive analysis was performed on the therapeutic effects of RAGT on walking ability,balance,lower limb muscle strength and functional inde-pendence.Results Eight RCTs involving 702 participants were included,originating from countries such as China,Italy,India,Tur-key and Poland.The population consisted of adult patients with various subtypes of stroke or SCI.These studies were published in journals across geriatric neuroscience,biosciences,medicine and sports science.Interventions involved three categories of lower limb exoskeleton including treadmill-based systems(end-effector and exoskel-eton models),overground exoskeletons and specialized joint/platform-based robots.The training frequency was 20 to 45 minutes a time,once to twice a day,one to seven days a week,for a total of two to ten weeks.RAGT might significant improve gait parameters and lower limb muscle strength,though its impact on functional inde-pendence was heterogeneous.Adaptive control strategies(e.g.,assist-as-needed)proved superior to fixed-parame-ter modes.Treadmill-based systems(e.g.,Lokomat)were well-suited for early-stage rehabilitation,while over-ground exoskeletons(e.g.,EKSO-GT)better facilitated adaptation to real-world environments.Conclusion RAGT is an effective modality for improving gait and lower limb function of patients with stroke and SCI.The therapeutic outcome is contingent upon personalized setup of the exoskeleton and the implementation of adaptive control strategies.Different adaptive control modes have been developed for the three main types of lower limb exoskeleton.Rehabilitation training should consider the specific lower limb tasks with the robot's cor-responding adaptive movement and control modes.

Objective To construct a Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)long-fragment fusion protein plasmid;investigate the expression,extraction,and purification of CaMK Ⅱ;and identify its binding to the Cav1.2 channel.Methods The extracted pGEX-6p-1/CaMK Ⅱ long-fragment plasmid was transformed into Escherichia coli BL21 receptor cells and cultured in a shaking incubator for 12 h.Isopropyl β-D-thiogalactoside was added to promote GST fusion protein expression.Next,the GST-CaMK Ⅱ long frag-ment was isolated and purified with GS-4B using dithiothreitol(DTT)combined with ultrasonic crushing.After treatment with the PreScis-sion protease,the GST label was removed to obtain the CaMK Ⅱ long-fragment protein.The molecular weight and relative purity of the CaMKⅡ long-fragment protein were determined using 15％sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The concentration of the purified protein was determined using the Bradford method.The binding ability of the CaMK Ⅱ long-fragment pro-tein to the Cav1.2 channel protein was evaluated using the pull-down method combined with Western blotting.Results The sequencing results showed that the CaMK Ⅱ long fragment was successfully constructed.A CaMK Ⅱ long-fragment protein with high purity and con-centration was obtained using DTT combined with ultrasonic crushing.This protein can bind to the CT1 protein of cardiac Cav1.2 calcium channel.Conclusion In this study,we successfully constructed a CaMKⅡ long-fragment plasmid.The CaMKⅡ long-fragment protein was extracted and purified,and was determined to bind to Cav1.2 channel proteins and exhibit biological activity.Collectively,this study provides a basis for further study of the function of CaMK Ⅱ.

Objective:To survey the child health service capacity of community health service institutions in Shenzhen city.Methods:This was a cross-sectional study. An online survey was conducted among 559 community health service centers, stations and clinics in Shenzhen from January 23, 2024 to February 3, 2024. The questionnaire contents included the development of child health management, vaccination, diagnosis and treatment of common diseases in children, management of children′s chronic diseases, appropriate technology, availability of children′s special drug dosage forms, provision of special Chinese patent medicine and appropriate technology of traditional Chinese medicine for children, referral and remote consultation in 2019 and 2023. The reasons of unavailable service items were asked in the questionnaire and the questionnaire also contained an open-ended question about the suggestions for improving the capacity of child health services.Results:A total of 559 valid questionnaires were collected, accounting for 64.48% (559/867) of the community health service centers, stations and health clinics in Shenzhen in 2023. Compared to 2019, there was a significant increase in the rate of pediatric health services provided in 2023, including the diagnosis and treatment of common diseases in children under 6 years (96.06%, 537 institutions), nebulized inhalation therapy (96.60%, 540 institutions), influenza and other respiratory pathogen detection (90.70%, 507 institutions), rotavirus and other intestinal pathogen detection (34.53%, 193 institutions), allergen detection (81.75%, 457 institutions), blood oxygen saturation monitoring (84.44%, 472 institutions), pediatric-specific formulations of Western medicine (90.52%, 506 institutions), pediatric-specific formulations of traditional Chinese medicine (89.27%, 499 institutions), appropriate Chinese medicine techniques (88.19%, 493 institutions), relatively fixed referral hospitals (95.17%, 532 institutions), and remote consultation service (19.14%, 107 institutions); and the differences were statistically significant ( P<0.01). The reasons for unavailable service items were lack of space and personnel for the basic public health services, lack of space, personnel and a deficiency in knowledge and technical capabilities for diagnostic tests, and lack of equipment and personnel for remote consultation service. Suggestions made by 394 respondent institutions (70.48%) for further improvement included: conducting and strengthening various training (44.42%, 175/394), consultation and teaching by expert visiting (18.53%, 73/394), increasing the space and equipment (9.39%, 37/394), and further study at higher-level units (98.88%, 35/394). Conclusions:The child health services have been greatly improved in community health service institutions in Shenzhen, but there are still rooms for further improvement such as the ability of child health management, vaccination, chronic disease managements and provision of remote consultation.