Genetic transformation is a fundamental tool in molecular biology research of medicinal plants. Tailoring transgenic technologies to each distinct medicinal plant would necessitate a substantial investment of time and effort. Here, we present a simple hairy root transformation method that does not require sterile conditions, utilizing Agrobacterium rhizogenes strain K599 and the visible RUBY reporter system. Transgenic hairy roots were obtained for six tested medicinal plant species, roots or rhizomes of which have recognized medicinal value, spanning four botanical families and six genera (Platycodon grandiflorus, Atractylodes macrocephala, Scutellaria baicalensis, Codonopsis pilosula, Astragalus membranaceus, and Glycyrrhiza uralensis). Furthermore, two previously identified Glycyrrhiza uralensis UGTs that convert liquiritigenin into liquiritin in heterologous systems were studied in planta using the method. Our results indicate that overexpression of GuUGT1 but not GuUGT10 and Cas9-mediated knockout of GuUGT1 profoundly influenced the accumulation of liquiritin and isoliquiritin in licorice roots. Therefore, the method described here represents a simple, rapid and widely applicable hairy root transformation method that enables fast gene functional study in medicinal plants.

BACKGROUND:Diabetic ulcers are a common complication of diabetes mellitus,which is manifested as foot ulcers complicated with infection,long treatment cycle,high disability rate and mortality rate,and brings a heavy burden to patients and social care. OBJECTIVE:To review the mechanism of action and the latest treatment progress of traditional Chinese medicine(TCM)in the treatment of diabetic ulcers,and to provide a basis for further theoretical research and clinical application. METHODS:CNKI,WanFang Database and PubMed database were searched for relevant literature using the keywords of"diabetic ulcer,medicinal herb,inflammation,interleukin-1β,interleukin-6,tumor necrosis factor,hypersensitive C-reactive protein,γ-interferon,interleukin-4,interleukin-10"in Chinese and English,respectively.The relevant literature in recent years was searched,and finally 75 articles were included for review. RESULTS AND CONCLUSION:The high glucose environment of the body will increase the level of pro-inflammatory cytokines,so that diabetic ulcer wounds are in a state of chronic inflammatory response for a long time,and difficult to heal or even not heal.TCM has summed up a lot of experience in the long-term struggle with diabetic ulcer.At present,TCM divides diabetic ulcers into four syndrome types:dampness and heat poison syndrome,blood and blood stasis obstruction pattern,heat poison injury Yin pattern,and Qi and blood deficiency syndrome,as well as representative prescriptions for treatment.According to their clinical characteristics,diabetic ulcers can be also divided into three stages:primary,middle and late stages.Different treatment methods are proposed:"clear method,""warm and clear combined use"and"maintenance method."Under the guidance of dialectical typing and staging of TCM,TCM monomers,extracts and compounds inhibit the inflammatory response and promote the healing of diabetic ulcers by down-regulating the expression of pro-inflammatory factors and/or up-regulating the expression of anti-inflammatory factors.Compared with modern medicine,TCM has significant advantages in the treatment of diabetic ulcers.There are many TCM monomers,extracts and compounds for the treatment of diabetic ulcers,such as angelica,curcumin,improved Chonghe ointment,Sanhuang blood exhaustion prescription and sore-ulcer I.formula,etc.It has been found that TCM for the treatment of diabetic ulcers is mainly heat-clearing and detoxifying,invigorating blood circulation and removing blood stasis,and amassing sores and muscle-building drugs,and the frequency of use,treatment scope and therapeutic effect of TCM compounds are obviously better than those of TCM monomers and extracts.Among them,the most commonly used are the Sanhuang blood exhaustion prescription and the sore-ulcer I as well as prescription for the treatment of damp heat toxicity syndrome and Zizhu ointment for the treatment of non-ischemic diabetic ulcers.However,there are also some shortcomings in the treatment of diabetic ulcers with TCM.First,there are few clinical syndrome studies on diabetic ulcers.Secondly,there are a wide variety of TCM monomers,extracts and compounds for the treatment of diabetic ulcers,and the relevant research is insufficiently in-depth.Finally,the research on the mechanism underlying TCM treatment of diabetic ulcers is still in the preliminary exploration stage,and the mechanism of action still needs to be further explored.In the future,it is necessary to strengthen the research on the pharmacology of TCM and the clinical syndrome of diabetic ulcers,analyze the potential targets and related signaling pathways of TCM in the treatment of diabetic ulcers,give full play to the therapeutic advantages of TCM with multiple targets,multiple pathways,multiple levels and multiple systems,and develop TCM with significant efficacy,active ingredients and clear targets.

Background/Aims: The shortage of donor liver hinders the development of liver transplantation. This study aimed to clarify the poor outcomes of functionally marginal liver grafts (FMLs) and provide evidence for the improvement of ischemia-free liver transplantation (IFLT) after FML transplantation. Methods: Propensity score matching was used to control for confounding factors. The outcomes of the control group and FML group were compared to demonstrate the negative impact of FMLs on liver transplantation patients. We compared the clinical improvements of the different surgical types. To elucidate the underlying mechanism, we conducted bioinformatic analysis based on transcriptome and single-cell profiles. Results: FMLs had a significantly greater hazard ratio (HR: 1.969, P=0.018) than did other marginal livers. A worse 90-day survival (Mortality: 12.3% vs. 5.0%, P=0.007) was observed in patients who underwent FML transplantation. Patients who received FMLs had a significant improvement in overall survival after IFLT (Mortality: 10.4% vs 31.3%, P=0.006). Pyroptosis and inflammation were inhibited in patients who underwent IFLT. The infiltration of natural killer cells was lower in liver grafts from these patients. Bulk transcriptome profiles revealed a positive relationship between IL-32 and Caspase 1 (R=0.73, P=0.01) and between IL-32 and Gasdermin D (R=0.84, P=0.0012). Conclusions FML is a more important negative prognostic parameter than other marginal liver parameters. IFLT might ameliorate liver injury in FMLs by inhibiting the infiltration of NK cells, consequently leading to the abortion of IL-32, which drives pyroptosis in monocytes and macrophages.

BACKGROUND: Implantation of bone marrow mesenchymal stem cells (BMSCs) is a potential alternative for promoting bone defects healing or osseointegration in osteoporosis. However, the reactive oxygen species (ROS) accumulated and excessive inflammation in the osteoporotic microenvironment could weaken the self-replication and multi-directional differentiation of transplanted BMSCs. METHODS: In this study, to improve the hostile microenvironment in osteoporosis, Poloxamer 407 and hyaluronic acid (HA) was crosslinked to synthetize a thermos-responsive and injectable hydrogel to load MnO2 nanoparticles as a protective carrier (MnO2 @Pol/HA hydrogel) for delivering BMSCs. RESULTS: The resulting MnO2 @Pol/HA hydrogel processed excellent biocompatibility and durable retention time, and can eliminate accumulated ROS effectively, thereby protecting BMSCs from ROS-mediated inhibition of cell viability, including survival, proliferation, and osteogenic differentiation. In osteoporotic bone defects, implanting of this BMSCs incorporated MnO2 @Pol/HA hydrogel significantly eliminated ROS level in bone marrow and bone tissue, induced macrophages polarization from M1 to M2 phenotype, decreased the expression of pro-inflammatory cytokines (e.g., TNFa, IL-1b, and IL-6) and osteogenic related factors (e.g., TGF-b and PDGF). CONCLUSION This hydrogel-based BMSCs protected delivery strategy indicated better bone repair effect than BMSCs delivering or MnO2 @Pol/HA hydrogel implantation singly, which providing a potential alternative strategy for enhancing osteoporotic bone defects healing.

Objective:To search for valuable laboratory indexes of early diagnosis of anastomotic leakage after anterior resection for rectal carcinoma.Methods:From Sep 2017 to Jan 2019, 128 patients with colorectal cancer underwent anterior rectal resection at the Department of Colorectal & Anal Surgery, the 940 Hospital of the Joint Logistics Support Force.Results:Anastomotic leakage occurred in 16 of 128 patients (12.5%). Definite diagnosis of anastomotic leakage was made on between 2nd and 9th day, postopera tively averagign (6.13±2.00) days. Tumor location was a risk factor for anastomotic leakage with the incidence significantly lower when the distance from the lower edge of the tumor to the anal margin >7 cm than when the distance ≤7 cm ( χ 2=6.022, P=0.014). The percentage of increase in peripheral blood leukocytes, neutrophils, serum interleukin-6, C-reactive protein and procalcitonin in patients 3-5 days after surgery significantly related to the occurrence of anastomotic leakage (all P<0.05). The area under the working characteristic curve of the subjects with the percentage of C-reactive protein, procalcitonin, interleukin-6, leukocytes and neutrophils from the 3rd to the 5th day after operation was greater than 0.5. Conclusion:C-reactive protein, procalcitonin, interleukin-6, leukocyte and neutrophil percentage are risk factors predicting anastomotic leakage after anterior resection of rectal cancer.

OBJECTIVE: To compare the effectiveness of supramalleolar osteotomy (SMOT) and ankle arthrodesis (AA) in the treatment of inverted ankle osteoarthritis (OA) in Takakura 3A stage with talus tilt. METHODS: The clinical data of 41 patients with inverted ankle OA in Takakura 3A stage with talus tilt admitted between January 2016 to January 2020 and met the selection criteria were retrospectively analyzed, and they were divided into SMOT group (21 cases) and AA group (20 cases) according to the surgical method. There was no significant difference in baseline data such as gender, age, affected side, cause of injury, and preoperative talar tilt angle (TT), American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score, visual analogue scale (VAS) score, short-form 36 health survey scale (SF-36) score, and sagittal range of motion (ROM) between the two groups ( P>0.05). The operation time, intraoperative blood loss, partial weight-bearing time, and complications were recorded in the two groups. AOFAS ankle-hindfoot score, VAS score, SF-36 score, and sagittal ROM were used to evaluate the effectiveness. Bone healing was observed and the time of bony healing was recorded. In the SMOT group, the tibial lateral surface angle (TLS), TT, and the tibial articular surface angle (TAS) were measured on ankle joint weight-bearing anteroposterior and lateral X-ray films and compared with those before operation. And Takakura staging assessment was also performed. RESULTS: The operation time and intraoperative blood loss in AA group were significantly less than those in SMOT group ( P<0.05). Patients in both groups were followed up 24-36 months, with an average of 28.9 months. Incision infection occurred in 2 patients in SMOT group and 1 patient in AA group, respectively, and no vascular or nerve injury occurred in both groups. The partial weight-bearing time of SMOT group was significantly less than that of AA group ( P<0.05), but there was no significant difference in bony healing time between the two groups ( P>0.05). At last follow-up, the difference of VAS score and SF-36 score before and after operation of AA group were less than those of SMOT group, and the difference of sagittal ROM before and after operation in SMOT group was less than that of AA group, with significant differences ( P<0.05). The difference of AOFAS ankle-hindfoot score before and after operation in AA group was slightly greater than that in SMOT group, but the difference was not significant ( P>0.05). The above scores in both groups significantly improved when compared with those before operation ( P<0.05). Sagittal ROM in AA group was significantly less than that before operation ( P<0.05), while there was no significant difference in SMOT group ( P>0.05). In the SMOT group, 17 patients (81.0%) showed improvement in imaging staging, 2 patients (9.5%) showed no improvement in staging, and 2 patients (9.5%) showed stage aggravation. TLS, TAS, and TT significantly improved when compared with those before operation ( P<0.05). At last follow-up, 2 patients in SMOT group received AA due to pain and stage aggravation, and 1 patient with bone nonunion underwent bone graft. Subtalar joint fusion was performed in 1 case of subtalar arthritis in AA group. CONCLUSION For inverted ankle OA in Takakura 3A stage with talus tilt, both SMOT and AA can significantly releave pain, improve foot function and quality of life, but AA has more definite effectiveness and better patient satisfaction.
Humans ; Ankle ; Talus/surgery* ; Retrospective Studies ; Blood Loss, Surgical ; Quality of Life ; Ankle Joint/surgery* ; Osteoarthritis/surgery* ; Osteotomy/methods* ; Arthrodesis ; Pain ; Treatment Outcome

Objective:To detect the incidence and analyze the clinical significance of regions of homozygosity (ROH) through the single nucleotide polymorphism array (SNP array).Methods:The SNP array detection results of 5 116 pregnant women in the Third Affiliated Hospital of Guangzhou Medical University from January 2016 to December 2020 were retrospectively analyzed. The pregnant women with ROH (5 Mb as the threshold) were followed up to analyze the relationship between ROH and abnormal fetal phenotype. Whole exon sequencing was performed in 4 cases of consanguineous marriage to detect potential recessive causative genes in the ROH region.Results:(1) A total of 39 cases of ROH were detected, with a positive rate of 0.76% (39/5 116). Among them, 25 cases (64%, 25/39) were detected only on single chromosome, and chromosome 11 had the highest detection rate, suggesting the risk of uniparental disomy; fourteen cases (36%,14/39) were detected on multiple chromosomes, most commonly on chromosomes 11, 1, 3, 4 and 8. (2) The number of cases and detection rate of ROH detected by different prenatal diagnosis indicators were as follows: 12 cases (1.78%, 12/676) in pregnant women with abnormal non-invasive prenatal testing result, 12 cases (0.37%, 12/3 284) in pregnant women with ultrasound abnormality, 4 cases (4/4) in pregnant women with consanguineous marriage, 3 cases (0.92%, 3/326) in pregnant women with previous adverse pregnancy, 2 cases (1.15%, 2/174) in pregnant women with high risk of serology in screening, 2 cases (4.00%, 2/50) in pregnant women with abnormal fetal chromosomal karyotype, 2 cases (0.79%, 2/253) in pregnant women with advanced maternal age, 1 case (0.56%, 1/178) in pregnant women with related parental genetic factors and 1 case (0.58%, 1/171) in pregnant women with the other factors. (3) The follow-up results of 39 cases of prenatal ROH showed that there were 16 cases of term birth, 15 cases of termination of pregnancy, 2 cases of preterm births, 1 case of fetal death and 5 cases lost to follow-up.Conclusions:Chromosomal ROH phenomenon is not rare. By analyzing the detection rate of ROH in prenatal diagnosis, combined with the results of fetal phenotype and postpartum follow-up, the clinical characteristics of ROH are discussed, so as to better understand the relationship between ROH and its phenotype.

Objective:To explore the value of nomogram based on arterial spin labeling (ASL) MRI perfusion parameters and clinicopathological features in predicting the response to chemoradiotherapy (CRT) in advanced nasopharyngeal carcinoma (ANPC, stage Ⅲ and Ⅳ).Methods:From June 2018 to January 2021, 70 patients with ANPC confirmed by pathology were prospectively enrolled in Affiliated Hospital of Jiangnan University. Nasopharyngeal MRI plain scan, ASL and contrast-enhanced scan were performed before CRT, and routine MRI re-examination was performed within 1 week after the end of CRT. The pre-CRT perfusion parameter tumor blood flow (TBF) from ASL and clinicopathological features were recorded, and the maximum diameter (MD) of the tumor on T 1WI images was measured. The patients were divided into CRT effective group (48 cases) and ineffective group (22 cases) according to the response evaluation criteria in solid tumors. The independent sample t test was used to compare the differences of TBF, age and MD between effective group and ineffective group. The χ 2 test was used to compare the differences of gender, clinical stage and pathological type between the 2 groups. Using binary logistic regression analysis, clinicopathological model and TBF combined clinicopathological model were constructed, and the nomogram of combined model was constructed. The diagnostic efficacy of the models was obtained by receiver operating characteristic (ROC) curve analysis, and the area under the ROC curves (AUC) of the 3 models were compared by DeLong method. The calibration curve for the nomogram was generated, and the concordance index (C index) was acquired. Results:The TBF of the effective group and the ineffective group were (113±9) and (97±14) ml·100 g -1·min -1, with a statistical difference ( t=5.17, P<0.001). The MD value of the effective group was smaller than that of the ineffective group, with a statistical difference ( t=-2.24, P=0.028). There were statistical differences in clinical stage and pathological type between the 2 groups (χ 2 values were 12.21 and 12.95, respectively, both P<0.001). Three independent predictors, including TBF (OR=7.749), clinical stage (OR=0.129) and pathological type (OR=5.228), were included in logistic regression analysis. The AUC, sensitivity and specificity of TBF model in predicting the response to CRT were 0.843, 87.5% and 72.7%, of clinicopathological model were 0.822, 80.2% and 59.1%, of the nomogram model were 0.893, 81.2% and 90.9%. There was no statistical difference of AUC between the nomogram model and TBF model ( Z=1.23, P=0.215). However, the AUC of the nomogram model was greater than that of the clinicopathological model ( Z=2.47, P=0.031). The calibration curve showed that there was a good concordance index (C index=0.892) between the predicted value of nomogram and the actual clinical observation value. Conclusion:TBF, clinical stage and pathological type are independent predictors of the response to CRT in ANPC patients, and the nomogram based on these three factors has a good ability in predicting the response to CRT.

Objective:To evaluate the clinical value of 18F-FDG PET/CT findings in patients with T1-2 lung adenocarcinoma spread through air spaces (STAS). Methods:From June 2018 to June 2020, a total of 80 patients (36 males, 44 females; age: 19-84 (59.9±11.8) years) with surgically and pathologically confirmed T1-2 lung adenocarcinomas in Jiangmen Central Hospital were enrolled retrospectively. All patients underwent 18F-FDG PET/CT examination preoperatively and were divided into STAS positive and negative groups according to the histopathological diagnosis. Independent-sample t test, Mann-Whitney U test, χ2 test and Fisher exact test were used to analyze differences of gender, age, tumor biomarker, SUV max, SUV mean, features showed on high resolution CT (HRCT; including diameter, lesion location, morphology, density, lobulated sharp, spiculated sign, vacuole sign, air bronchgram sign, pleural traction and para-emphysema), and pathologic findings (micropapillary pattern, lymphvascular inversion, pleural inversion and lymph node metastasis) between the two groups, and then multivariate logistic regression was performed. The ROC curve was employed to evaluate the predictive value of parameters for STAS of T1-2 lung adenocarcinomas. Results:Among the 80 patients with T1-2 lung adenocarcinomas, 12 (15.0%) were STAS positive and 68 (85.0%) were STAS negative. Significant differences were shown in SUV max, SUV mean, micropapillary pattern, lymphvascular inversion and lymph node metastasis between the two groups ( z values: -2.60, -2.17; χ2 values: 29.56, 9.28, 17.40, P<0.001 or P<0.05). SUV max (odds ratio ( OR): 1.348 (95% CI: 1.071-1.695), P=0.011), micropapillary pattern ( OR=47.444 (95% CI: 4.592-490.214), P=0.001) and lymph node metastasis ( OR=8.201 (95% CI: 1.129-59.576), P=0.038) were independent risk factors for STAS positive in multivariation logistic regression analysis. The optimum cut-off value for SUV max was 3.85 in the ROC analysis with the AUC of 0.737 (95% CI: 0.614-0.859), the sensitivity of 11/12, the specificity of 55.9%(38/68) and the accuracy of 61.2%(49/80). The AUC of the SUV max combined with micropapillary pattern and lymph node metastasis was 0.945 (95% CI: 0.892-0.999) with the sensitivity of 11/12, the specificity of 88.2%(60/68) and the accuracy of 88.7%(71/80). Conclusions:The PET/CT characteristics may be useful in differentiating STAS status among patients with T1-2 lung adenocarcinoma. SUV max >3.85, pathological papillary pattern and lymph node metastasis are independent risk factors to predict STAS.