Methods: This retrospective study analyzed 32 consecutive IDS patients who underwent UEDD surgery. Clinical features, laboratory data (erythrocyte sedimentation rate and C-reactive protein), and treatment outcomes were analyzed. Results: Definite microorganisms were identified in 27 patients (84.3%), with 24 (88.9%) meeting cure criteria. The cure rate was significantly higher in the detected pathogen group compared to the undetected pathogen group (88.9% vs. 80%; χ²=19.36, p<0.0001). Metagenomic next generation sequencing (mNGS) provided faster diagnosis (41.72±6.81 hours) compared to tissue culture (95.74±35.47 hours, p<0.05). The predominant causative pathogen was Mycobacterium tuberculosis, followed by Staphylococcus aureus. Significant improvements were observed in Visual Analog Scale pain scores, from a mean of 7.9 preoperatively to 1.06 at 1 year postoperatively. The Oswestry Disability Index revealed a similar trend, showing significant improvement (p<0.05). Conclusions UEDD is a viable alternative to traditional open surgery for managing IDS in high-risk patients. UEDD offers a dual therapeutic-diagnostic advantage during the initial admission phase, enabling simultaneous debridement, neurological decompression, and targeted biopsy in a single intervention. Compared with traditional tissue culture, mNGS enables rapid microbiological diagnosis and extensive pathogen coverage.

The protein kinase A/protein kinase G/protein kinase C-family (AGC kinase family) of eukaryotes is involved in regulating numerous biological processes. The 3-phosphoinositide- dependent protein kinase 1 (PDK1), is a conserved serine/threonine kinase in eukaryotes. To understand the roles of PDK1 homologous genes in cell death and immunity in tetraploid Nicotiana tabacum, the previuosly generated transgenic CRISPR/Cas9 lines, in which 5-7 alleles of the 4 homologous PDK1 genes (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out, were used in this study. Our results showed that the hypersensitive response (HR) triggered by transient overexpression of active Pto (Pto^Y207D) or soybean GmMEKK1 was significantly delayed, whereas the resistance to Pseudomonas syrangae pv. tomato DC3000 (Pst DC3000) and tobacco mosaic virus (TMV) was significantly elevated in these partial knockout lines. The elevated resistance to Pst DC3000 and TMV was correlated with the elevated activation of NtMPK6, NtMPK3, and NtMPK4. Taken together, our results indicated that NtPDK1s play a positive role in cell death but a positive role in disease resistance, likely through negative regulation of the MAPK signaling cascade.
Nicotiana/virology* ; Disease Resistance/genetics* ; Plant Diseases/immunology* ; Plants, Genetically Modified/genetics* ; Gene Knockout Techniques ; Plant Proteins/genetics* ; CRISPR-Cas Systems ; Protein Serine-Threonine Kinases/genetics* ; 3-Phosphoinositide-Dependent Protein Kinases/genetics* ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Tobacco Mosaic Virus/pathogenicity*

Alopecia areata (AA) is an autoimmune-mediated, hair follicle-targeting, inflammatory, non-scarring hair loss. The treatment of severe AA has long been a focus and challenge. Currently, novel therapeutic agents, such as Janus kinase (JAK) inhibitors, have been increasingly employed in the treatment of severe AA. This review provides an overview of new small-molecule targeted drugs and biologics for AA.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Methods: This retrospective study analyzed 32 consecutive IDS patients who underwent UEDD surgery. Clinical features, laboratory data (erythrocyte sedimentation rate and C-reactive protein), and treatment outcomes were analyzed. Results: Definite microorganisms were identified in 27 patients (84.3%), with 24 (88.9%) meeting cure criteria. The cure rate was significantly higher in the detected pathogen group compared to the undetected pathogen group (88.9% vs. 80%; χ²=19.36, p<0.0001). Metagenomic next generation sequencing (mNGS) provided faster diagnosis (41.72±6.81 hours) compared to tissue culture (95.74±35.47 hours, p<0.05). The predominant causative pathogen was Mycobacterium tuberculosis, followed by Staphylococcus aureus. Significant improvements were observed in Visual Analog Scale pain scores, from a mean of 7.9 preoperatively to 1.06 at 1 year postoperatively. The Oswestry Disability Index revealed a similar trend, showing significant improvement (p<0.05). Conclusions UEDD is a viable alternative to traditional open surgery for managing IDS in high-risk patients. UEDD offers a dual therapeutic-diagnostic advantage during the initial admission phase, enabling simultaneous debridement, neurological decompression, and targeted biopsy in a single intervention. Compared with traditional tissue culture, mNGS enables rapid microbiological diagnosis and extensive pathogen coverage.

Objective:To develop and validate a Self-care Ability Scale for Arteriovenous Fistula (AVF) in Maintenance Hemodialysis (MHD) patients.Methods:Guided by Orem's self-care theory, the initial item pool of the scale was developed through a literature review, semi-structured interviews, and group discussions. The initial scale was finalized after two rounds of expert consultation using the Delphi method. A convenience sampling method was used to recruit 418 MHD patients using AVF in January 2024 for item analysis, exploratory factor analysis and reliability testing. Another 293 MHD patients using AVF were recruited in March 2024 for confirmatory factor analysis.Results:The self-care ability scale for AVF in MHD patients included four dimensions: knowledge and skills of AVF self-care, willingness and attitude toward AVF self-care, recognition and prevention of AVF complications, and patient self-adjustment and adaptation, comprising 38 items. The content validity index at the scale level was 0.98. Exploratory factor analysis extracted four common factors with a cumulative variance contribution rate of 84.706%. Confirmatory factor analysis indicated good model fit, strong convergent validity, and ideal discriminant validity. The total Cronbach's α coefficient of the scale was 0.987; the split-half reliability coefficient was 0.902, and the test-retest reliability coefficient was 0.979.Conclusions:The Self-care Ability Scale for AVF in MHD patients demonstrates excellent reliability and validity, making it a suitable tool for assessing patients' ability to self-care for their AVF.

A 20-year old female patient diagnosed with right distal tibial giant cell tumor underwent a surgery of resection of distal tibial giant cell tumor, residual cavity liquid nitrogen and electrocauterization inactivation and local arthroplasty on March 17, 2023. Preoperatively a life-size distal tibia model was 3D printed using polylactic acid (PLA) material based on the CT data of patient's distal tibia. Tumor resection was simulated on the model, preserving the surrounding normal bone and articular cartilage unaffected by the tumor. The residual cavity was filled with bone cement and the distal tibial articular surface was shaped using the talar articular surface as a template. The 3D CT data of bone cement was collected and reconstructed. The irregular bone and cartilage defect data were trimmed to form a regular arc shape, which was used as the data for fabricating local arthroplasty prosthesis. The local arthroplasty prosthesis composed of a titanium base and a VE polyethylene liner was 3D printed. During the operation, the test models of titanium alloy base and VE polyethylene liner were used to test the matching degree with the bone and cartilage defect. Minor adjustments were made by removing a portion of the lateral wall of the residual cavity and modifying the base height to achieve proper alignment of the distal tibial articular surface with the talar surface. After confirming a satisfactory fit, the local arthroplasty prosthesis was implanted. Intraoperative fluoroscopic confirmed accurate placement of the prosthesis, good anatomical match with the defect, and restoration of the joint line. The postoperative follow-up was conducted at 2, 4, 12, 20, 48, 72 and 92 weeks. Wound healing was closely monitored, along with radiologic assessment for prosthesis bone ingrowth and local tumor recurrence. Functional evaluations were performed using the AOFAS and Kofoed scoring systems. Postoperatively, the patient experienced plantar numbness and sensory disturbance, which gradually resolved after three weeks. Assisted ambulation began at two weeks postoperatively, and the patient resumed a normal gait by 12 weeks. The Mayo ankle arthroplasty evaluation criteria at postoperative at 48 weeks were excellent. The AOFAS score and Kofoed score were 97 points and 94 points respectively, indicating excellent functional outcomes. Postoperative X-ray indicated that no bone ingrowth was observed at 2 weeks and 4 weeks after the operation, minor ingrowth at 12 weeks postoperatively, significant bone ingrowth at 20 weeks, and complete osseointegration by 48 to 64 weeks. Postoperative CT imaging at 92 weeks confirmed full prosthesis osseointegration, while MRI at 72 weeks showed no evidence of tumor recurrence.

Objective:To evaluate the role of the CC chemokine ligand 2/CC chemokine receptor 2 (CCL2/CCR2) signaling pathway in electroacupuncture (EA)-induced reduction of spinal cord injury (SCI) in rats.Methods:Sixty clean-grade healthy adult female Sprague-Dawley rats, weighing 210-250 g, were divided into 5 groups ( n=12 each) using a random number table method: sham operation group (group S), group SCI, SCI+ Anti-CCL2 group (group SCI+ A), SCI+ EA group (group SCI+ EA), and spinal cord injury+ EA+ rCCL2 group (group SCI+ EA+ R). The SCI model was established using the Allen method in anesthetized animals. Group S only underwent spinous process and laminectomy without damaging the spinal cord. In SCI+ A group, CCL2 neutralizing antibody 50 μg/kg was intrathecally injected at 0, 3 and 6 days after successful development of the SCI model. On the 7th day after the successful development of the SCI model, Jiaji, Dazhui and Mingmen acupoints were stimulated with a depth of 2 mm, voltage of 12-15 mV and frequency of 2 Hz for 30 min once a day for 7 consecutive days in SCI+ EA group. In SCI+ EA+ R group, recombinant rat CCL2 2.5 μg/kg was intrathecally injected at the site of injury at 0, 3 and 6 days after successful development of the SCI model, and the remaining treatments were similar to those in SCI+ EA group. At 1 day before developing the model, 0, 3, 7, 14, 21 and 28 days after developing the model, the mechanical paw withdraw threshold (MWT) and thermal paw withdrawal latency (TWL) were measured, and the motor function was assessed by BBB score. The rats were sacrificed after the final behavioral testing, and their spinal cord tissues were obtained for determination of the expression of CCL2 and CCR2 protein and mRNA (by Western blot or quantitative real-time polymerase chain reaction), the expression of GFAP (by immunofluorescence), contents of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay) and for examination of the pathological changes (using HE staining). Results:Compared with S group, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model, the expression of CCL2 and CCR2 protein and mRNA and GFAP was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI group ( P<0.05). Compared with SCI group, the MWT and BBB scores were significantly increased, and the TWL was prolonged at 7 days after developing the model in SCI+ A group, the MWT and BBB scores were significantly increased, and the TWL was prolonged at 14 days after developing the model in SCI+ EA group, and the expression of CCL2 and CCR2 protein and mRNA and GFAP was significantly down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased in SCI+ A and SCI+ EA groups ( P<0.05). Compared with SCI+ EA group, the MWT and BBB scores were significantly decreased at 14 days after developing the model, the TWL was shortened, the expression of CCL2 and CCR2 protein and mRNA and GFAP was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI+ EA+ R group ( P<0.05). Compared with SCI+ A and SCI+ EA groups, the histopathological injury were significantly attenuated in SCI group, and the histopathological injury was aggravated in SCI+ EA+ R group. Conclusions:The CCL2/CCR2 signaling pathway is involved in the process by which EA reduces SCI, and the mechanism is related to the inhibition of astrocyte activation, thereby reducing the inflammatory response.

Objective:To evaluate the effect of electroacupuncture (EA) on ionotropic purinergic receptor 4 (P2X4R)/nuclear factor-kappa B (NF-κB) signaling pathway during spinal cord injury (SCI) in rats.Methods:Thirty-six clean-grade healthy adult female Sprague-Dawley rats, weighing 210-250 g, were divided into 3 groups ( n=12 each) using a random number table method: sham surgery group (S group), SCI group, and SCI+ EA treatment group (SCI+ EA group). The SCI model was established by the Allen′s method in anesthetized animals. In group S, only the spinous processes and vertebral laminae were resected, but the spinal cord was not injured. On the 7th day after developing the model, EA of Jiaji, Dazhui, and Mingmen lasting 30 min was performed once a day for 7 consecutive days, with a depth of 2 mm, intensity of 12-15 mV, frequency of 2 Hz, in SCI+ EA group. The mechanical paw withdraw threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before developing the model and 3, 7, 14, 21 and 28 days after developing the model, and the motor function was assessed using the Basso-Beattie-Bresnahan (BBB) score. The recovery of motor function was assessed using footprint analysis at 28 days after developing the model. After the final behavioral testing, the rats were sacrificed, and spinal cord tissues were harvested to observe the pathological changes of the spinal cord tissues using hematoxylin-eosin staining, to detect the expression of P2X4R and phosphorylated NF-κB p65 (p-NF-κB p65) (by immunohistochemical analysis and Western blot) and to determine contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with the baseline measured at 1 day before developing the model, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model in SCI group and SCI+ EA group ( P<0.05). Compared with S group, the MWT and BBB scores were significantly decreased and the TWL was shortened at each time point after developing the model, the expression of P2X4R and p-NF-κB p65 in spinal cord tissues was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in SCI group ( P<0.05). Compared with SCI group, the MWT and BBB scores were significantly increased and the TWL was prolonged at 14, 21 and 28 days after developing the model, the expression of P2X4R and p-NF-κB p65 in spinal cord tissues was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased ( P<0.05), and the pathological damage of spinal cord tissues was alleviated and footprints were reduced in SCI+ EA group. Conclusions:The mechanism by which EA alleviates SCI may be related to the inhibition of the activation of the P2X4R/NF-κB signaling pathway and the reduction in the inflammatory response in rats.

Objective To evaluate the practical application of the Media Fill Test(MFT)in assessing aseptic compounding competency of personnel in Pharmacy Intravenous Admixture Services(PIVAS).Methods A multicenter controlled study was conducted across six tertiary hospitals(center ①-⑥)in China.Participants were divided into an inexperienced group(Group A,n=118)and an experienced group(Group B,n=118),each performing five MFT operations.Positive controls validated medium efficacy.Contamination rates and pass rates were analyzed using chi-square and Fisher's exact tests.Results Valid samples included 584 for Group A and 588 for Group B.The sample pass rate was 66.78%(390/584)in Group A and 91.67%(539/588)in Group B,while personnel pass rates were 46.15%(54/117)and 80.51%(95/118),respectively,with significant intergroup differences(both P<0.01).All centers except Center ⑥ showed significantly higher pass rates in Group B(all P<0.05).Conclusion MFT effectively differentiates technical proficiency levels and is suitable for training evaluation of novice PIVAS staff.