Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

This study aims to analyze the main drug resistance mechanism of Salmonella enteritidis induced by polymyxin in vitro.In this study,the resistance of Salmonella enteritidis CMCC(B)50335(ZK)to polymyxin was induced in vitro,and the growth characteristics,exercise ability,ul-trastructure and sensitivity to 16 antimicrobial agents before and after induction were determined by turbidimetry,semi-solid agar method,transmission electron microscope and disk diffusion method,and the whole genome and single nucleotide polymorphism(SNP)were detected by Illu-mina NovaSeq PE150 method.RT-qPCR was used to detect the differences in the expression levels of six drug-resistant related genes.The recombinant strain △phoPE1-128-1,which induced drug-resistant strain E1-128-1,was constructed by homologous recombination of Red Escherichia coli,and its sensitivity to polymyxin was detected.The results showed that three strains of induced drug-resistant bacteria E1-128-1,E1-128-3 and E2-128-3 were screened out,and the MIC increased by 128,64 and 64 times respectively after drug resistance stability test.Induced drug resistance had no significant effect on the growth ability of the tested bacteria and the sensitivity of 16 antibacte-rial drugs.The exercise ability of E1-128-1was significantly increased,and the cell wall and plasma membrane obviously thicken.There was no significant difference in the genome components of E1-128-1 before and after induction,but eight missense mutations of six drug-resistance related genes,including phoP/phoQ,cpxP,lptD,csrA and acrB,were detected,including four missense mutation sites of phoP,namely Leu185Trp,His189Ser,Thr190Tyr and Ile191His.The corresponding genes were sequenced by PCR,and the results were consistent with those of SNP.RT-qPCR results showed that the expression levels of mutant genes of the three induced strains increased signifi-cantly.Compared with E1-128-1,the MIC of △phoP E1-128-1 decreased to 1 mg/L.It is sug-gested that the mutation and increased expression of phoP gene are important factors for inducing polymyxin resistance of Salmonella CMCC(B)50335 in vitro.

Objective:To investigate the role of group 3 innate lymphoid cells (ILC3) in skin wound healing, and to explore the underlying mechanisms.Methods:Twenty-four 5-week-old male C57BL/6 mice were randomly and equally allocated into 3 groups: the skin wound + ILC3 inhibitor group (referred to as ILC3 inhibitor group), the skin wound group, and the control group, with 8 mice in each group. Four days before the establishment of the wound model, mice in the ILC3 inhibitor group were intraperitoneally injected with 1 &mu;g of ILC3 inhibitor every 2 days for a total of 2 doses, mice in the skin wound group were injected with an equal volume of physiological saline solution, and mice in the control group were fed normally. To establish a mouse skin wound model, a full-thickness circular incision with a diameter of 0.6 cm was made around the midpoint of the dorsal midline using a biopsy punch after the intraperitoneal injection of anesthetics, which was histologically confirmed to be a full-thickness injury. The size of the wounds was observed and recorded, photographs of the wounds were taken on days 0, 1, 3, 5, 7, and 9 after wounding, and corresponding wound healing rates were calculated. On day 9 after wounding, tissue samples were collected from the wound edges, and subjected to flow cytometry analysis to quantify ILC3 infiltrating around the skin wound, and hematoxylin and eosin (HE) staining was performed to assess the healing status of the skin wounds. Real-time quantitative polymerase chain reaction (qRT-PCR) was conducted to determine the mRNA expression of vitamin D receptor (VDR), Notch1, tumor necrosis factor-alpha (TNF-α), interleukin (IL) -17A, IL-17F, and IL-22 in the wound-edge tissues, and Western blot analysis to determine their protein expression. Statistical analysis was carried out by using one-way analysis of variance and t test. Results:On day 9 after wounding, the skin wound group showed an increased number of ILC3 in the wound-edge tissues (5.31% ± 1.47% vs. 3.10% ± 0.54%, P < 0.01), increased mRNA and protein expression of TNF-α, IL-22, IL-17A, and IL-17F (all P < 0.05), but decreased mRNA and protein expression of VDR (both P < 0.05) compared with the control group; the protein expression of Notch1 was significantly higher in the skin wound group than in the control group ( P < 0.05), but there was no significant difference in its mRNA expression between the two groups ( P > 0.05). On days 1, 3 and 5, the wound healing rates were significantly higher in the ILC3 inhibitor group (45.17% ± 9.90%, 61.58% ± 11.61%, 75.61% ± 9.12%, respectively) than in the skin wound group (25.87% ± 10.96%, 47.78% ± 13.81%, 64.55% ± 10.29%, respectively, all P < 0.05). On day 9, the ILC3 inhibitor group showed a decreased number of ILC3 around the wound (2.69% ± 0.95%, P < 0.01), decreased mRNA and protein expression of TNF-α, IL-22, IL-17A, and IL-17F in the wound-edge tissues (all P < 0.05), but increased mRNA and protein expression of Notch1 and VDR in the wound-edge tissues (all P < 0.05) compared with the skin wound group. On day 9 after wounding, histopathological examination with HE staining revealed continuous and intact epithelial structure, as well as dense and neatly arranged collagen fibers in the ILC3 inhibitor group, and the structures of hair follicles, blood vessels, and sebaceous glands were similar to those in the control group. Conclusions:Skin ILC3 infiltrated local wounds and were involved in the skin wound healing process through inflammatory factors such as TNF-α, IL-17A, IL-17F, and IL-22. Downregulating the number of ILC3 may promote skin wound healing by activating VDR and Notch1, as well as inhibiting the TNF-α signaling pathway and the expression of downstream inflammatory factors.

Medication-related osteonecrosis of the jaw (MRONJ) is a refractory disease that can lead to severe destruction of the jaw. As there is no standard protocol for treating MRONJ, various treatments have been studied. Teriparatide has been used as an adjunct therapy for MRONJ. However, its effectiveness has not been sufficiently demonstrated for use as a standard treatment for MRONJ. This study aimed to demonstrate the efficacy of teriparatide in treating MRONJ by presenting two successfully treated cases. Each patient received teriparatide therapy with surgical intervention. The appropriateness of teriparatide use was evaluated based on the patient’s systemic condition, and the administration of teriparatide was supervised by a physician.Complete resolution of the lesion was observed clinically and radiographically in both patients. The first patient underwent implant placement at the lesion site. Due to its anabolic properties and ability to stimulate bone remodeling, teriparatide is an effective adjunctive pharmacological treatment for bone healing before and after surgery with associated beneficial effects on bone and mucosal healing.

Medication-related osteonecrosis of the jaw (MRONJ) is a refractory disease that can lead to severe destruction of the jaw. As there is no standard protocol for treating MRONJ, various treatments have been studied. Teriparatide has been used as an adjunct therapy for MRONJ. However, its effectiveness has not been sufficiently demonstrated for use as a standard treatment for MRONJ. This study aimed to demonstrate the efficacy of teriparatide in treating MRONJ by presenting two successfully treated cases. Each patient received teriparatide therapy with surgical intervention. The appropriateness of teriparatide use was evaluated based on the patient’s systemic condition, and the administration of teriparatide was supervised by a physician.Complete resolution of the lesion was observed clinically and radiographically in both patients. The first patient underwent implant placement at the lesion site. Due to its anabolic properties and ability to stimulate bone remodeling, teriparatide is an effective adjunctive pharmacological treatment for bone healing before and after surgery with associated beneficial effects on bone and mucosal healing.

Medication-related osteonecrosis of the jaw (MRONJ) is a refractory disease that can lead to severe destruction of the jaw. As there is no standard protocol for treating MRONJ, various treatments have been studied. Teriparatide has been used as an adjunct therapy for MRONJ. However, its effectiveness has not been sufficiently demonstrated for use as a standard treatment for MRONJ. This study aimed to demonstrate the efficacy of teriparatide in treating MRONJ by presenting two successfully treated cases. Each patient received teriparatide therapy with surgical intervention. The appropriateness of teriparatide use was evaluated based on the patient’s systemic condition, and the administration of teriparatide was supervised by a physician.Complete resolution of the lesion was observed clinically and radiographically in both patients. The first patient underwent implant placement at the lesion site. Due to its anabolic properties and ability to stimulate bone remodeling, teriparatide is an effective adjunctive pharmacological treatment for bone healing before and after surgery with associated beneficial effects on bone and mucosal healing.

Objective To explore the effectiveness of virtual autopsy-based postmortem computed tomography(PMCT)liver three dimensional slicer(3D slicer)artificial intelligence(AI)volume reconstruction to assist forensic practice.Methods Twenty cases of the deceased who underwent both virtual autopsy and traditional autopsy in our center were selected and subjected to liver volume segmentation by 3D slicer method,Tada's formula method and literature method,and the data obtained from the traditional autopsy were compared and analyzed to obtain the accuracy rate.Results The 3D slicer method yielded higher consistency(95%confidence interval),lower volumetric variability(standard deviation),and a smaller region(variance)of uncertainty than the Tada formula method and the methods mentioned in the literature.Conclusion 3D slicer AI reconstruction based on virtual autopsy can visualize virtual anatomy,help increase the diagnostic accuracy of traditional autopsy,assist in pathological diagnosis,and provide new directions and tools for the development of imaging histology of virtual autopsy.

Objectives: Patients undergoing oral surgery exhibit high anxiety, which may elevate their cortisol levels and affect postoperative recovery. Overweight patients are often encountered in the dental clinic due to the increasing prevalence of overweight. We aimed to investigate the relationships between preoperatively assessed body mass index (BMI), serum cortisol and high-sensitivity C-reactive protein (hs-CRP) levels, and visual analog scale (VAS) scores and preoperative anxiety in patients undergoing mandibular third molar (MM3) extraction and to identify predictors of postoperative complications. Patients and Methods: We analyzed 43 patients (age, 20-42 years) undergoing MM3 extraction. At the first visit, patients completed the Modified Dental Anxiety Scale (MDAS) and Amsterdam Preoperative Anxiety and Information Scale (APAIS) questionnaires. Their BMI and VAS scores were also calculated. The participants underwent blood tests 1 hour before MM3 extraction. On the first postoperative day, the participants’ VAS scores and serum hs-CRP levels were reevaluated. Results: We found that BMI was significantly correlated with preoperative VAS scores. Further, BMI and preoperative hs-CRP levels were significantly correlated among women and patients undergoing extractions of fully impacted MM3s. No correlations were found between serum cortisol and other variables. The preoperative MDAS and VAS scores were significantly positively correlated, especially among patients undergoing extractions of fully impacted MM3s. Multiple linear regression showed that BMI and the eruption status of the MM3 were significant predictors of postoperative hsCRP levels and VAS scores, respectively. Conclusion In MM3 removals, patients with higher BMI showed elevated hs-CRP and higher VAS scores before surgery. Patients with higher anxiety among those undergoing extractions of fully impacted MM3s showed higher preoperative VAS scores. The two main predictors of postoperative complications were BMI and MM3 eruption status.

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Male infertility caused by idiopathic oligoasthenospermia (OAT) is known as idiopathic male infertility. Glutathione S-transferase (GST) and fluoride may play important roles in idiopathic male infertility, but their effects are still unknown. Our study examined the relationship between GST polymorphisms and fluoride-induced toxicity in idiopathic male infertility and determined the underlying mechanism. Sperm, blood, and urine samples were collected from 560 males. Fluoride levels were measured by a highly selective electrode method, and GST genotypes were identified using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Semen parameters, DNA fragmentation index (DFI), mitochondrial membrane potential (MMP), and oxidative stress (OS) biomarkers were statistically assessed at the P < 0.05 level. Compared with healthy fertile group, semen parameters, fluoride levels, OS biomarkers, sex hormone levels, and MMP and DFI levels were lower in the idiopathic male infertility group. For glutathione S-transferase M1 (GSTM1[-]) and glutathione S-transferase T1 (GSTT1[-]) or glutathione S-transferase P1 (GSTP1) mutant genotypes, levels of semen fluoride, OS, MMP, and DFI were considerably higher, and the mean levels of sperm parameters and testosterone were statistically significant in GSTM1(+), GSTT1(+), and GSTP1 wild-type genotypes. Both semen and blood fluoride levels were associated with oxidative stress in idiopathic male infertility patients. Elevated fluoride in semen with the genotypes listed above was linked to reproductive quality in idiopathic male infertility patients. In conclusion, GST polymorphisms and fluorine may have an indicative relationship between reproductive quality and sex hormone levels, and OS participates in the development of idiopathic male infertility.
Humans ; Male ; Fluorides/adverse effects* ; Semen ; Polymorphism, Genetic ; Glutathione Transferase/genetics* ; Glutathione S-Transferase pi/genetics* ; Infertility, Male/genetics* ; Genotype ; Biomarkers ; Genetic Predisposition to Disease ; Case-Control Studies