Traditional development of small protein scaffolds has relied on display technologies and mutation-based engineering, which limit sequence and functional diversity, thereby constraining their therapeutic and application potential. Protein design tools have significantly advanced the creation of novel protein sequences, structures, and functions. However, further improvements in design strategies are still needed to more efficiently optimize the functional performance of protein-based drugs and enhance their druggability. Here, we extended an evolution-based design protocol to create a novel minibinder, BindHer, against the human epidermal growth factor receptor 2 (HER2). It not only exhibits super stability and binding selectivity but also demonstrates remarkable properties in tissue specificity. Radiolabeling experiments with ^99mTc, ⁶⁸Ga, and ¹⁸F revealed that BindHer efficiently targets tumors in HER2-positive breast cancer mouse models, with minimal nonspecific liver absorption, outperforming scaffolds designed through traditional engineering. These findings highlight a new rational approach to automated protein design, offering significant potential for large-scale applications in therapeutic mini-protein development.

OBJECTIVES: To investigate the therapeutic mechanism of Huoxue Shufeng Granules (HXSFG) for alleviating central sensitization in a mouse model of chronic migraine (CM). METHODS: We analyzed the main chemical components of HXSFG through literature review and explored their pharmacological mechanisms by bioinformatics analyses. In a male C57BL/6J mouse model of CM established by intraperitoneal injections of nitroglycerin (10 mg/kg) every other day (5 injections), the effects of gavage with low, and high doses of HXSFG or intraperitoneal injections of topiramate for ameliorating central sensitization were evaluated using Von Frey test and a hot plate apparatus; the changes in expressions of inflammatory factors, the proteins in the TLR4/NF‑κB signaling pathway, and activation of c-Fos and CGRP were detected using RT-qPCR, Western blotting and immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that the main active components in HXSFG for alleviating CM included formononetin, paeoniflorin, quercetin, and tanshinone. Gene Ontology (GO) enrichment analysis identified 492 GO entries, comprising 366 biological processes, 46 cellular components, and 80 molecular functions. KEGG pathway enrichment analysis indicated that the Toll-like receptor and NF‑κB signaling pathways were crucial in mediating the therapeutic effects of HXSFG on CM. In the mouse models of CM, both topiramate and HXSFG treatments alleviated the symptoms of central sensitization, evidenced by improved mechanical and thermal pain thresholds in the mice. HXSFG significantly reduced the expression of c-Fos and CGRP, improved inflammatory markers, and downregulated the expressions of TLR4, p-NF‑κB, IL-1β, and TNF‑α proteins in the mouse models. CONCLUSIONS HXSFG effectively alleviates central sensitization in CM mice by modulating the inflammatory pathways and inhibiting the TLR4/ NF-κB signaling pathway, suggesting its potential as a therapeutic option for CM.
Animals ; Toll-Like Receptor 4/metabolism* ; NF-kappa B/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Male ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Migraine Disorders/metabolism* ; Disease Models, Animal ; Inflammation

OBJECTIVES: To investigate the mechanisms of Modified Chaihu Guizhi Decoction (MCGD) for ameliorating anxiety- and depression-like behaviors in a mouse model of chronic unpredictable mild stress (CUMS). METHODS: The main chemical constituents of MCGD were identified through literature review, and network pharmacology analysis was performed to predict the potential pharmacological mechanisms of MCGD. For in vivo validation, male C57BL/6J mice were randomized into control group, CUMS model group, fluoxetine (FLX) treatment group, and low- and high-dose MCGD treatment groups (n=15), and in all but the control group, CUMS models were established by daily exposure to two randomized stressors for 28 consecutive days. Starting from 3 days prior to modeling, MCGD and fluoxetine treatments were administered daily via gavage and intraperitoneal injection, respectively. Depression- and anxiety-like behaviors of the mice were assessed using sucrose preference test, forced swim test, open field test and elevated plus maze test. The changes in mRNA expressions of the clock genes and inflammatory markers and expressions of the JAK2/STAT3 signaling proteins were detected using RT-qPCR and Western blotting, and immunofluorescence staining was used to detect microglia activation in the mice. RESULTS: The key active compounds in MCGD identified by network pharmacology analysis included quercetin, acacetin, formononetin, nobiletin, and baicalein. GO analysis identified 607 enriched pathways, and KEGG pathway enrichment revealed significant involvement of the JAK2/STAT3 and NF-κB signaling pathways. In the mouse models of CUMS, treatment with both fluoxetine and MCGD significantly alleviated anxiety- and depression-like behaviors. MCGD treatment significantly reduced Iba1 expression, improved the inflammatory markers, reversed the decrease in clock gene circadian rhythm amplitude, and obviously downregulated the expressions of JAK2, p-STAT3, p-NF-κB, IL-1β, and IL-6 proteins. CONCLUSIONS MCGD effectively alleviates anxiety- and depression-like behaviors in CUMS mice by modulating the inflammatory pathways and inhibiting the JAK2/STAT3 signaling pathway.
Animals ; Janus Kinase 2/metabolism* ; STAT3 Transcription Factor/metabolism* ; Signal Transduction/drug effects* ; Depression/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Male ; Mice, Inbred C57BL ; Anxiety/drug therapy* ; Stress, Psychological ; Disease Models, Animal

High mobility group box 1 (HMGB1), when released extracellularly, plays a pivotal role in the development of spinal cord synapses and exacerbates autoimmune diseases within the central nervous system. In experimental autoimmune encephalomyelitis (EAE), a condition that models multiple sclerosis, the levels of extracellular HMGB1 and interleukin-33 (IL-33) have been found to be inversely correlated. However, the mechanism by which IL-33 deficiency enhances HMGB1 release during EAE remains elusive. Our study elucidates a potential signaling pathway whereby the absence of IL-33 leads to increased binding of P300/CBP-associated factor with HMGB1 in the nuclei of astrocytes, upregulating HMGB1 acetylation and promoting its release from astrocyte nuclei in the spinal cord of EAE mice. Conversely, the addition of IL-33 counteracts the TNF-α-induced increase in HMGB1 and acetylated HMGB1 levels in primary astrocytes. These findings underscore the potential of IL-33-associated signaling pathways as a therapeutic target for EAE treatment.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; Astrocytes/metabolism* ; Interleukin-33/metabolism* ; HMGB1 Protein/metabolism* ; Acetylation ; Mice, Knockout ; Mice, Inbred C57BL ; p300-CBP Transcription Factors/metabolism* ; Mice ; Spinal Cord/metabolism* ; Cells, Cultured ; Female ; Signal Transduction

Objective:To explore the efficacy of long intramedullary nails versus short intramedullary nails in the treatment of AO/OTA 31-A3 intertrochanteric fractures.Methods:A retrospective analysis was conducted on 60 patients with AO/OTA 31-A3 intertrochanteric femur fractures treated between March 2019 and August 2022. The patients were randomly divided into two groups (the long nail group and the short nail group). Thirty-four patients were treated with long intramedullary nails, including 16 males and 18 females, aged 68.41±17.84 years old (range 31-96 years). Twenty-six patients were treated with short intramedullary nails, including 13 males and 13 females, aged 72.23±13.97 years old (range 31-90 years). The causes of injury, fracture classification (AO/OTA classification), intraoperative blood loss, operation time, fracture healing time, imaging indexes (fracture reduction quality, postoperative neck trunk angle, and medial support), Harris score of the hip joint at the last follow-up, one-year mortality rates and complications were compared between the two groups.Results:The follow-up time was 24.26±6.67 months in the long nail group and 24.31±5.60 months in the short nail group, and the general information of the two groups were comparable. Between the long nail and short nail group, the intraoperative blood loss was 281.47±235.28 ml vs. 121.92±84.14 ml and the operation time was 110.44±24.63 min vs. 81.15±28.54 min with significant differences ( P<0.05). While the length of hospital stay was 12.35±4.81 d vs. 10.89±4.30 d, the good rate of fracture reduction was 55.9% vs. 61.53%, the fracture healing time was 120.44±16.43 d vs. 128.07±18.33 d, the presence rate of medial support was 67.6% vs. 79.4%, and the excellent rate of Harris score was 65.4% vs. 65.4% with no significant difference between the two groups ( P>0.05). One-year mortality rates was 5.3% vs. 7.1% and complications was 11.7% vs. 15.4% with no significant difference between the two groups ( P>0.05). Conclusion:Both long intramedullary nails and short intramedullary nails are effective in the treatment of AO/OTA 31-A3 intertrochanteric femur fractures. However, surgical time and intraoperative blood loss was less in the short nail group.

Objective To explore the inhibitory effect of saikosonin a(SSa)on pentylenetetrazol-induced acute epilepsy seizures in a mouse model of depression and explore the mechanism mediating this effect.Methods Male C57BL/6J mouse models of depression was established by oral administration of corticosterone via drinking water for 3 weeks,and acute epileptic seizures were induced by intraperitoneal injection of a single dose of pentylenetetrazole.The effect of intraperitoneal injection of SSa prior to the treatment on depressive symptoms and epileptic seizures were assessed using behavioral tests,epileptic seizure grading and hippocampal morphology observation.ELISA was used to detect blood corticosterone levels of the mice,and RT-qPCR was performed to detect the pro-and anti-inflammatory factors.Microglia activation in the mice was observed using immunofluorescence staining.Results The mouse model of corticosterone-induced depression showed body weight loss and obvious depressive behaviors with significantly increased serum corticosterone level(all P<0.05).Compared with those with pentylenetetrazole-induced epilepsy alone,the epileptic mice with comorbid depression showed significantly shorter latency of epileptic seizures,increased number,grade and duration of of seizures,reduced Nissl bodies in hippocampal CA1 and CA3 neurons,increased number of Iba1-positive cells,and significantly enhanced hippocampal expressions of IL-1β,IL-10,TNF-α and IFN-γ.Pretreatment of the epileptic mice with SSa significantly prolonged the latency of epileptic seizures,reduced the number,duration,and severity of seizures,increased the number of Nissl bodies,decreased the number of Iba1-positive cells,and reduced the expression levels of IL-1β,IL-10,TNF-α,and IFN-γ in the hippocampus(P<0.05).Conclusion Depressive state aggravates epileptic seizures,increases microglia activation,and elevates inflammation levels.SSA treatment can alleviate acute epileptic seizures in mouse models of depression possibly by suppressing microglia activation-mediated inflammation.

Objective To analyze the risk factors for prolonged sedation recovery time in epileptic children.Methods A cross-sectional trial was conducted on the epileptic children who underwent electroencephalography(EEG)with oral administration of chloral hydrate and nasal dexmedetomidine for sedation in our hospital from November 2019 to December 2022.Their general information and sedation and medication data were collected.Prolonged sedation recovery time was defined as beyond the 75th percentile of awaking time,and then the children were divided into recovery time ≤1 h group and＞1 h group.Univariate and multivariable logistic regression analyses were used to identify the risk factors for recovery time in these children.Results A total of 1 346 children were enrolled,including 805 males and 541 females,with a median age of 6.03(4.42,8.56)years.There were 986 children assigned into recovery time ≤1 h group and 360 ones into recovery time＞1 h group.Univariate analysis showed that significant differences were observed in the proportions of age＜1 year(0.8％vs 2.2％,P=0.046),sedation more than 3 times(35.8％vs 42.2％,P=0.031),and treated with multiple antiepileptic drugs(AEDs)(25.5％vs 36.9％,P＜0.001)between the 2 groups.Multivariable logistic regression analysis indicated that age＜1 year(OR=2.943,95％CI:1.063～8.142,P=0.034),sedation times ≥3(OR=1.287,95％CI:1.003～1.649,P=0.047)and multiple use of AEDs(OR=1.718,95％CI:1.325～2.223,P＜O.001)were risk factors for prolonged recovery time.Conclusion Age＜1 year,sedation times ≥3 and multiple use of AEDs are risk factors for prolonged sedation recovery time in epileptic children undergoing EEG after oral chloral hydrate and nasal dexmedetomidine.

Objective:To investigate the effects of compound Duzhong Jiangu Granules on joint function and gut microbiota in patients with Kashin-Beck disease.Methods:A single group pre- and post-experimental design was conducted, the patients with Kashin-Beck disease were selected as the subjects in Xunyi County, Xianyang City, Shaanxi Province; and treated with oral administration of compound Duzhong Jiangu Granules (12 g/bag, 1 bag/time, 3 times/day) for a period of 1 month. The improvement of joint function was evaluated using the joint dysfunction index scoring method before and after treatment. Morning stool samples of patients were collected and the changes in gut microbiota were analyzed before and after treatment using 16S rDNA sequencing technology.Results:A total of 87 patients with Kashin-Beck disease were included, including 44 males and 43 females; the age was (60.38 ± 7.12) years old, and the body mass index was (23.67 ± 3.59) kg/m 2. The comprehensive scores of joint dysfunction index for patients with Kashin-Beck disease before and after treatment were (7.27 ± 2.05) and (5.86 ± 2.01) points, respectively, and the difference was statistically significant ( t = 5.88, P < 0.001). The sequencing results of gut microbiota showed that there were statistically significant differences in the alpha diversity (chao1, observed species index) and beta diversity of gut microbiota in patients with Kashin-Beck disease before and after treatment ( Z = - 5.08, - 5.03, R = 0.09, P < 0.001). In the distribution of gut microbiota, Firmicutes was the dominant phylum, with relative abundances of 50.21% and 52.09% before and after treatment, respectively; the Bifidobacterium was the dominant bacterial genus, with relative abundances of 16.83% and 18.81% before and after treatment, respectively. At the genus level, a total of 17 gut microbiota genera were screened out, among which the relative abundances of Hafnia-Obesumbacterium, Gammaproteobacteria_unclassified, Acinetobacter, Pantoea, Leuconostoc, and Akkermanisia were significantly higher than before treatment ( Z = - 2.40, - 2.24, - 2.06, - 3.59, - 2.24, - 2.11, P < 0.05). The relative abundances of Dubosiella, Selenomonas, Anaeroplasma, Lachnospiraceae_ NK4A136_group, Rikenella, Prevotella, Megasphaera, Lactobacillus, Prevotella-9, Phascolarctobacterium, and Desulfovibrio were significantly lower than before treatment ( Z = - 9.38, - 2.61, - 2.18, - 8.43, - 2.45, - 2.46, - 2.49, - 7.29, - 2.29, - 2.55, - 2.08, P < 0.05). Conclusions:Compound Duzhong Jiangu Granules can effectively improve the joint function of patients with Kashin-Beck disease, and alter the diversity and richness of the gut microbiota community. It may reduce clinical symptoms in patients by regulating the structure of gut microbiota.

Objective:To explore the independent risk factors for bleeding in patients following percutaneous liver biopsy examination.Methods:The clinicopathological data of patients who underwent percutaneous liver biopsy examination at Nanjing Second Hospital from January 2012 to December 2021 were retrospectively collected. Univariate and multivariate logistic regression analysis were used to investigate the effect of age, gender, lesion type (diffuse liver parenchymal lesions, focal liver lesions), number of biopsies, tissue length, presence or absence of cirrhosis, presence or absence of portosystemic shunt, erythrocytes, white blood cells, hemoglobin, platelets, prothrombin time, fibrinogen, international normalized ratio, and liver biochemical indicators on bleeding following liver biopsy, as well as to screen independent risk factors.Results:A total of 3 331 patients were examined by percutaneous liver biopsy, and 3 060 cases were actually included by excluding 271 cases who took consultation from other hospitals. The overall postoperative hemorrhagic rate was 1.6% (49/3 060). Of which, forty-four cases (1.4%) had overt bleeding (hemodynamic changes or hemoglobin decreased by more than 20 g/L), five cases (0.2%) had minor bleeding, three cases had subcapsular hepatic hemaotma, and two cases had local bleeding from liver biopsy. Among the overt bleeding cases, two cases were in the off-label group (platelet<50×10 9/L or international normalized ratio>1.5), and the rest were in the non-off-label group. The results of univariate analysis showed that factors such as focal liver lesions, portosystemic shunt, prolonged prothrombin time, increased international normalized ratio, bilirubin, and alkaline phosphatase were associated with bleeding after liver biopsy in the non-off-label group. The multivariate collinearity diagnosis revealed statistically significant differences for the indicators. Multivariate logistic regression analysis finally included factors such as lesion type, portosystemic shunt, international normalized ratio, total bilirubin, and alkaline phosphatase. The results showed that patients with focal liver lesions were more prone to bleed after surgery than patients with diffuse liver parenchymal lesions ( OR=3.396, P=0.002, 95% CI: 1.596-7.228). Patients with portosystemic shunt were more prone to bleed than those without portosystemic shunt ( OR=3.301, P=0.018, 95% CI: 1.232-8.845). Patients were more likely to experience bleeding following liver biopsy when their total bilirubin levels were elevated ( OR=1.006, P<0.001, 95% CI:1.003-1.008). Conclusion:Focal liver lesions, portosystemic shunts, and elevated total bilirubin are independent risk factors for bleeding after percutaneous liver biopsy.

Purpose To investigate the clinical pathologic features of five SMARCA4-deficient non-small lung cancers(SMARCA4-dNSCLCs).Methods Five cases of SMARCA4-dNSCLC was underwent by HE,immunohistochemical staining,and molecular detection,analyzed their clinicopathological char-acteristics and reviewed relevant literatures.Results All 5 ca-ses were male,and mean age was 66 years.Five patients had a history of smoking,three patients were treated with cough and blood in sputum as the first symptom,one was treated with a history of pulmonary tuberculosis combined with limb mobility disorder,and one was diagnosed with pulmonary nodules by physical examination.Under microscopic observation,tumor cells were poorly differentiated,with solid nest sheet distribu-tion,some with glandular structure,tumor cells had abundant e-osinophilic or transparent cytoplasm,vacuolar nuclear chroma-tin,nucleoli was visible,and nuclear mitosis was common.In-flammatory cell infiltration and sheet of necrosis were seen in the stroma.Immunohistochemical staining showed 5/5 diffuse ex-pression of CK(AE1/AE3)and CK7,5/5 loss expression of BRG1,1/5 diffuse expression of p40 and CK5/6,and Ki67 proliferating index ranged from 20％to 90％.FISH tests showed that 4/4 SMARCA4 genes missed.Five patients were followed up for 2-15 months,3 patients died and 2 patients survived.Conclusions SMARCA4-dNSCLC can have extensive morphologi-cal features,high degree of malignancy,and complicated treat-ment.BRG1 deficiency is helpful for diagnosis.Deepening the understanding of SMARCA4-dNSCLC can help the clinical cor-rect choice of treatment strategies and accurately evaluate patient prognosis.