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 analyze the characteristics of drug resistance genes in a Klebsiella pneumoniae strain coproducing carbapenemases KPC-2 and NDM-5. Methods:Klebsiella pneumoniae KPN-hnqyy was separated from the stool specimen of a patient in the Hematology Department of Affiliated Cancer Hospital of Zhengzhou University. The strain was identified with a BD Phenix-M50 automated microbiology system and the minimum inhibitory concentration against the strain was measured as well. The genotypes of the carbapenemases were tested by enzyme immunochromatographic assay and PCR method. The transferability of related plasmids was analyzed by conjugation test. Whole-genome sequencing of the strain was conducted using PacBio and Illumina platforms. The MLST type, resistance gene and plasmid type of the strain were retrieved in BacWGSTdb. The genome and open reading frame sequence of the strain were compared using Easyfig_2.2.3. Visual cycle graphs were generated using BRIG v0.95. Results:Klebsiella pneumoniae KPN-hnqyy was resistant to carbapenem antibiotics. It belonged to ST11 and carried two carbapenemase genes of blaKPC-2 and blaNDM-5. The conjugant only harbored the blaKPC-2 gene. Whole-genome sequencing revealed that the strain contained one chromosome and three plasmids. Its chromosome genome shared more than 99.9% similarity with that of Klebsiella pneumonia KP69 and KP19-2029. Moreover, a similar IncR and IncFⅠ resistance gene fusion region was contained in different types of plasmids carried by them: the blaKPC-2 gene was located in a structure—which evolved from the Tn3-△Tn4401-Tn1721/Tn1722 sequence—inside this fusion region with its ends inserted into the transposase IS26 gene; the blaNDM-5 gene was located on a transposon containing the special plasmids of the insertion fragment in phages, with its ends inserted into the transposase IS26 gene too. Conclusions:The IncR and IncFⅡ resistance gene fusion region of blaKPC-2 carried by Klebsiella pneumoniae ST11 might be widely coexistent with the chromosomal genome. The blaNDM-5 gene carried by special plasmids might be accidentally obtained through gene recombination mediated by transposable element IS26. The wide transmission of Klebsiella pneumoniae ST11 carrying the blaKPC-2 gene in China and its ability to obtain other carbapenemase genes through transposable element IS26 were well worth attention.

Objective:To explore possible genetic causes associated with early pregnancy loss using chromosomal microarray analysis (CMA) with single nucleotide polymorphism (SNP) probes.Methods:A retrospective review was performed by the CMA of samples from 961 patients who spontaneously aborted in our hospital before the 20th week of pregnancy.Results:(1)The total chromosome abnormality rate in miscarriage samples was 54.44% (515/946), including single chromosome abnormality (39.53%), two chromosome abnormality (2.22%), multi-chromosome abnormality (0.42%), triploidy or hypertriploidy (4.86%), copy number variants (CNVs) in 41 cases (4.33%), regions of homozygosity (ROH, 0.74%), mosaic (2.22%) and chimera (0.11%) . (2) CNV analysis of 41 cases showed that 85.36% were pathogenic and likely pathogenic, 12.20% were classified as clinical significance unknown and 2.44% were interpreted as likely benign; (3) Among the cases of ROH, 2 cases shown whole-genome homozygosity and 1 case had completely homozygous at chromosome 21. The homozygous regions in 2 cases were located at the end of the short arm of chromosome 16, suggesting the mechanism of ROH in such cases could be the result of isodisomy.Conclusion:Chromosome abnormality is an important genetic factor causing pregnancy loss. The application of CMA with SNP probes can indeed improve the detection rate of chromosome abnormalities and evaluate the risk of reproductive fertility in patients with pregnancy loss.

With the extensive application of highly sensitive genetic techniques in the field of prenatal diagnosis, prenatal chromosomal mosaicisms including true fetal mosaicisms and confined placental mosaicisms are frequently identified in clinical settings, and the diagnostic criteria and principle of genetic counseling and clinical management for such cases may vary significantly among healthcare centers across the country. This not only has brought challenges to laboratory technician, genetic counselor and fetal medicine doctor, but can also cause confusion and anxiety of the pregnant woman and their family members. In this regard, we have formulated a consensus over the prenatal diagnosis and genetic counseling for chromosomal mosaicisms with the aim to promote more accurate and rational evaluation for fetal chromosomal mosaicisms in prenatal clinics.
Consensus ; Female ; Genetic Counseling ; Humans ; Mosaicism ; Placenta ; Pregnancy ; Prenatal Diagnosis/methods*