Flavonoid 3-O-glucosyltransferase (3GT) is a key enzyme in the glucosidation of anthocyanins. To investigate the 3GT gene in rhododendron, we cloned an open reading frame (ORF) of 3GT gene (named Rh3GT) from Rhododendron hybridum Hort (Red cultivar) and then characterized this gene and the deduced protein in terms of the biochemical characteristics, expression level, and enzymatic function. The results showed that Rh3GT had a full length of 993 bp and encoded 330 amino acid residues. The deduced protein was hydrophilic, stable, weak acid, belonging to the glycosyltransferase family (GT-B type), with glutamine (Q) at position 44 in the PSPG box. The phylogenetic analysis showed that Rh3GT was most closely related to Vc3GT from Vaccinium corymbosum and Vm3GT from Vaccinium myrtillus. Rh3GT was expressed in the stems, leaves, and flowers and almost not expressed in the roots, with the highest expression level in petals during full blooming stage. Introduction of pCAMBIAL1302-Rh3GT into petals significantly up-regulated the expression level of Rh3GT and increased the total anthocyanin accumulation. Rh3GT was successfully expressed in Escherichia coli BL21 in the form of inclusion bodies with a size of about 36 kDa. The results of HPLC showed that the recombinant Rh3GT after denaturation, purification, and dilution could catalyze the synthesis of cyanidin and UDP-glucose to synthesize cyanidin 3-O-glucoside, indicating that the expressed protein had 3GT activity. This study provides basic data for further studying the molecular regulation mechanism of anthocyanin biosynthesis and theoretical support for molecular breeding of rhododendron.
Rhododendron/classification* ; Glucosyltransferases/metabolism* ; Cloning, Molecular ; Escherichia coli/metabolism* ; Recombinant Proteins/biosynthesis* ; Anthocyanins/biosynthesis* ; Phylogeny ; Plant Proteins/metabolism* ; Amino Acid Sequence

Objective:To verify the efficacy of VELscope autofluorescence examination technology in the early warning of potential oral malignant diseases.Methods:80 patients with suspected dysplasia were included and underwent oral examination,visually en-hanced lesion scope(VELscope)autofluorescence examination and histopathology examination respectively,the fluorescence imaging results were compared with the histopathological results.Results:Pathological reports showed 64 cases with mild or no dysplasia,9 cases with moderate or severe dysplasia,1 case with moderate dysplasia without malignant transformation and 6 cases with cancer.The results of VELscope showed that there were 73 positive cases and negative 7 cases of fluorescence deletion.The sensitivity and specificity of VELscope were 93.75％and 9.37％respectively,and the sensitivity could reach 100％in the cancerous tissue.Con-clusion:As a non-invasive examination method,VELscope is highly sensitive but lowly specific.

Objective:To explore and compare the effect and prognosis of small bone window surgery at different timings in the treatment of hypertensive intracerebral hemorrhage (HICH).Methods:Using a retrospective analysis, HICH patients admitted to department of neurosurgery of Guihang Guiyang Hospital were selected from May 2021 to May 2023. According to the different surgical timings, the patients were divided into ultra-early group (time from onset to surgery <6 h) and early group (onset to surgery time ≥6~<24 h). Propensity score was used to match 51 cases in each group. The perioperative indicators (surgical time, intraoperative blood loss, hospital stay, hematoma clearance rate), incidence rates of complications, neurological function (National Institute of Health Stroke Scale (NIHSS), Barthel index), endothelin and arginine vasopressin (AVP) before surgery and after 7 days of surgery, clinical efficacy (Glasgow Outcome Scale (GOS)) at 1 month after surgery, rebleeding rate and mortality rate within 1 year after surgery were compared between both groups. Chi-square test was used for between-group comparison of enumeration data, t test was adopted for between-group comparison of measurement data with normal distribution, and non-parametric rank sum test was adopted for comparison of ranked data between groups.Results:The intraoperative blood loss and hospital stay in ultra-early group with (186.54±20.15) mL and (14.45±2.04) d were significantly less or shorter than (220.10±24.61) mL and (16.79±2.52) d in early group, the difference was statistically significant ( t values were 7.54 and 5.15; both P<0.001). At 7 days after surgery, the NIHSS ((14.55±1.57) and (16.14±2.13) points), endothelin ((69.14±6.37) and (73.48±6.83) mg/L) and AVP ((12.81±2.02) and (14.35±2.23) μg/L) in ultra-early group and early group were significantly lower than before ((32.67±3.81) and (32.38±3.53) points, (89.67±7.25) and (88.24±6.38) mg/L, (18.47±2.41) and (18.04±2.37) μg/L) while the Barthel index ((72.35±10.14) and (67.45±9.78) points) was significantly higher than before ((49.45±7.41) and (47.87±7.37) points),and the difference was statistically significant ( t values were 31.40, 28.13, 15.19, 11.28, 12.85, 8.10, 13.02, and 11.42, respectively; all P<0.001) and the NIHSS, endothelin and AVP in ultra-early group were significantly lower than in early group, while the Barthel index was significantly higher than early group, the difference was statistically significant ( t values were 4.29, 3.32, 3.66, and 2.48 respectively; P values were <0.001, 0.001, <0.001, and 0.015, respectively ). GOS grading (grade I-grade V: 1, 3, 9, 22, 16 cases) in ultra-early group at 1 month after surgery was significantly better than that in early group (grade I-V: 3, 5, 10, 26, 7 cases) ( Z=1.97, P=0.049). Conclusion:Both ultra-early and early small bone window surgery have good results in the treatment of HICH, but ultra-early surgery is more beneficial to the recovery of patients' neurological function and has better prognosis, which can be used as a reference.

Objective:To evaluate the value of ultrasound radiomics combined with machine learning for early diagnosis of seronegative Hashimoto’s thyroiditis (SN-HT) .Methods:This retrospective study included 164 patients from Liaoning Provincial People’s Hospital , Lixin County People’s Hospital, Linghai Dalinghe Hospital, Fengcheng Phoenix Hospital, who underwent thyroidectomy for solitary nodules with normal thyroid function between Nov. 2016 and Jan. 2024. Postoperative pathology confirmed Hashimoto’s thyroiditis (HT) in some cases, who were further categorized into antibody-positive and antibody-negative groups based on serum antibody status. Patients without Hashimoto’s thyroiditis served as the control group. A total of 298 ultrasound images were analyzed. Radiomics features were extracted from hypoechoic non-nodular areas within 0.5 cm surrounding the tumor. Two senior pathologists and two senior ultrasound physicians independently assessed lymphocytic infiltration, eosinophilic changes of follicular epithelium, and the proportion of hypoechoic areas in pathology and ultrasound images, respectively. A machine learning model, CCH-NET, was developed using linear regression and t-distributed stochastic neighbor embedding (t-SNE) techniques. The dataset was divided into a training set (80%) and a validation set (20%) to compare the diagnostic accuracy of CCH-NET with that of senior ultrasound physicians. Results:In internal validation, CCH-NET achieved a diagnostic accuracy of 88.89% for both antibody-positive and antibody-negative groups, significantly higher than the 66.67% accuracy of senior ultrasound physicians ( P<0.01). In external validation, CCH-NET achieved 75.00% and 66.67% accuracy for the two groups, compared to 50.00% by senior ultrasound physicians. For the control group, both methods achieved 93.33% accuracy. The AUC of CCH-NET was 0.848, outperforming senior ultrasound physicians (0.681) ,demonstrating superior diagnostic performance. Conclusion:The radiomics-based CCH-NET model, using non-nodular hypoechoic areas as a specific indicator, can accurately identify early SN-HT in euthyroid patients. It significantly outperforms senior ultrasound physicians, improving diagnostic accuracy and reducing missed diagnoses.

Objective:To explore and compare the effect and prognosis of small bone window surgery at different timings in the treatment of hypertensive intracerebral hemorrhage (HICH).Methods:Using a retrospective analysis, HICH patients admitted to department of neurosurgery of Guihang Guiyang Hospital were selected from May 2021 to May 2023. According to the different surgical timings, the patients were divided into ultra-early group (time from onset to surgery <6 h) and early group (onset to surgery time ≥6~<24 h). Propensity score was used to match 51 cases in each group. The perioperative indicators (surgical time, intraoperative blood loss, hospital stay, hematoma clearance rate), incidence rates of complications, neurological function (National Institute of Health Stroke Scale (NIHSS), Barthel index), endothelin and arginine vasopressin (AVP) before surgery and after 7 days of surgery, clinical efficacy (Glasgow Outcome Scale (GOS)) at 1 month after surgery, rebleeding rate and mortality rate within 1 year after surgery were compared between both groups. Chi-square test was used for between-group comparison of enumeration data, t test was adopted for between-group comparison of measurement data with normal distribution, and non-parametric rank sum test was adopted for comparison of ranked data between groups.Results:The intraoperative blood loss and hospital stay in ultra-early group with (186.54±20.15) mL and (14.45±2.04) d were significantly less or shorter than (220.10±24.61) mL and (16.79±2.52) d in early group, the difference was statistically significant ( t values were 7.54 and 5.15; both P<0.001). At 7 days after surgery, the NIHSS ((14.55±1.57) and (16.14±2.13) points), endothelin ((69.14±6.37) and (73.48±6.83) mg/L) and AVP ((12.81±2.02) and (14.35±2.23) μg/L) in ultra-early group and early group were significantly lower than before ((32.67±3.81) and (32.38±3.53) points, (89.67±7.25) and (88.24±6.38) mg/L, (18.47±2.41) and (18.04±2.37) μg/L) while the Barthel index ((72.35±10.14) and (67.45±9.78) points) was significantly higher than before ((49.45±7.41) and (47.87±7.37) points),and the difference was statistically significant ( t values were 31.40, 28.13, 15.19, 11.28, 12.85, 8.10, 13.02, and 11.42, respectively; all P<0.001) and the NIHSS, endothelin and AVP in ultra-early group were significantly lower than in early group, while the Barthel index was significantly higher than early group, the difference was statistically significant ( t values were 4.29, 3.32, 3.66, and 2.48 respectively; P values were <0.001, 0.001, <0.001, and 0.015, respectively ). GOS grading (grade I-grade V: 1, 3, 9, 22, 16 cases) in ultra-early group at 1 month after surgery was significantly better than that in early group (grade I-V: 3, 5, 10, 26, 7 cases) ( Z=1.97, P=0.049). Conclusion:Both ultra-early and early small bone window surgery have good results in the treatment of HICH, but ultra-early surgery is more beneficial to the recovery of patients' neurological function and has better prognosis, which can be used as a reference.

Objective:To evaluate the value of ultrasound radiomics combined with machine learning for early diagnosis of seronegative Hashimoto’s thyroiditis (SN-HT) .Methods:This retrospective study included 164 patients from Liaoning Provincial People’s Hospital , Lixin County People’s Hospital, Linghai Dalinghe Hospital, Fengcheng Phoenix Hospital, who underwent thyroidectomy for solitary nodules with normal thyroid function between Nov. 2016 and Jan. 2024. Postoperative pathology confirmed Hashimoto’s thyroiditis (HT) in some cases, who were further categorized into antibody-positive and antibody-negative groups based on serum antibody status. Patients without Hashimoto’s thyroiditis served as the control group. A total of 298 ultrasound images were analyzed. Radiomics features were extracted from hypoechoic non-nodular areas within 0.5 cm surrounding the tumor. Two senior pathologists and two senior ultrasound physicians independently assessed lymphocytic infiltration, eosinophilic changes of follicular epithelium, and the proportion of hypoechoic areas in pathology and ultrasound images, respectively. A machine learning model, CCH-NET, was developed using linear regression and t-distributed stochastic neighbor embedding (t-SNE) techniques. The dataset was divided into a training set (80%) and a validation set (20%) to compare the diagnostic accuracy of CCH-NET with that of senior ultrasound physicians. Results:In internal validation, CCH-NET achieved a diagnostic accuracy of 88.89% for both antibody-positive and antibody-negative groups, significantly higher than the 66.67% accuracy of senior ultrasound physicians ( P<0.01). In external validation, CCH-NET achieved 75.00% and 66.67% accuracy for the two groups, compared to 50.00% by senior ultrasound physicians. For the control group, both methods achieved 93.33% accuracy. The AUC of CCH-NET was 0.848, outperforming senior ultrasound physicians (0.681) ,demonstrating superior diagnostic performance. Conclusion:The radiomics-based CCH-NET model, using non-nodular hypoechoic areas as a specific indicator, can accurately identify early SN-HT in euthyroid patients. It significantly outperforms senior ultrasound physicians, improving diagnostic accuracy and reducing missed diagnoses.

Objective:To verify the efficacy of VELscope autofluorescence examination technology in the early warning of potential oral malignant diseases.Methods:80 patients with suspected dysplasia were included and underwent oral examination,visually en-hanced lesion scope(VELscope)autofluorescence examination and histopathology examination respectively,the fluorescence imaging results were compared with the histopathological results.Results:Pathological reports showed 64 cases with mild or no dysplasia,9 cases with moderate or severe dysplasia,1 case with moderate dysplasia without malignant transformation and 6 cases with cancer.The results of VELscope showed that there were 73 positive cases and negative 7 cases of fluorescence deletion.The sensitivity and specificity of VELscope were 93.75％and 9.37％respectively,and the sensitivity could reach 100％in the cancerous tissue.Con-clusion:As a non-invasive examination method,VELscope is highly sensitive but lowly specific.

ObjectiveTo observe the clinical efficacy of modified Zuojinwan granules in treating reflux esophagitis （RE） and functional dyspepsia （FD） with the same syndrome with disharmony between liver and stomach）. MethodA randomized double-blind placebo-controlled clinical trial was conducted to enroll 144 patients with disharmony between liver and stomach， including 72 patients with RE and 72 patients with FD. These patients were then randomly divided into observation and control groups， with 36 patients in each group. The observation group was given modified Zuojinwan granules orally， and the control group was given placebo granules orally. They both were treated with two packs each time， twice a day， for four weeks. The traditional Chinese medicine （TCM） syndrome scores， cerebrointestinal peptides ［calcitonin gene-associated titanium （CGRP）， vasoactive intestinal peptide （VIP）， 5-hydroxytryptamine （5-HT）， and substance P （SP）］， inflammatory factors ［tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）］， common gastrointestinal related hormones ［gastrin （GAS） and motilin （MTL）］， and other indicators in the two groups were compared before and after treatment， and the curative effect of TCM syndromes and the occurrence of adverse reactions were determined. At the same time， the changes in the above indicators and the curative effect of TCM syndromes in the two groups of patients with the same disease were analyzed. ResultAfter treatment， CGRP， VIP， 5-HT， SP， TNF-α， IL-6， GAS， MTL， and TCM syndrome scores in the observation group and control group were significantly improved （P<0.05）. After treatment， the improvement of CGRP， VIP， 5-HT， SP， TNF-α， IL-6， GAS， MTL， and TCM syndrome scores in the observation group was better than that in the control group （P<0.05）. After treatment， CGRP， VIP， 5-HT， SP， TNF-α， IL-6， GAS， MTL， and TCM syndrome scores in both groups of RE patients and FD patients were significantly improved （P<0.05）. After treatment， the improvement of CGRP， VIP， 5-HT， SP， TNF-α， IL-6， GAS， MTL， and TCM syndrome scores in RE patients and FD patients in the observation group were better than that in the control group （P<0.05）. In the observation group and the control group， the incidence of nausea， vomiting， fatigue， dry mouth， and other adverse reactions was lower， and there was no statistical significance. ConclusionModified Zuojinwan granules can effectively improve the TCM syndromes of disharmony between liver and stomach of RE and FD， brain and intestinal peptide， gastrointestinal hormone， and inflammatory factors and provide evidence for the clinical application of TCM theory of "treating different diseases with the same method".

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.
Humans ; HEK293 Cells ; CRISPR-Cas Systems ; DEAD Box Protein 58/metabolism* ; Signal Transduction ; Receptors, Immunologic/metabolism* ; Gene Knockout Techniques ; Transfection ; DEAD-box RNA Helicases/metabolism* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Interferon-Induced Helicase, IFIH1/metabolism* ; Transcription Factor RelA/metabolism* ; Interferon-beta/metabolism*

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe²⁺ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.
Arabidopsis/metabolism* ; Rhododendron/metabolism* ; Amino Acid Sequence ; Anthocyanins/metabolism* ; Phylogeny ; Flavonoids/metabolism* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*