(+)-Borneol, the main component of "Natural Borneol" in the Chinese Pharmacopoeia, is a high-end spice and precious medicine. Plant extraction cannot meet the increasing demand for (+)-borneol, while microbial biosynthesis offers a sustainable supply route. However, its production was extremely low compared with other monoterpenes, even with extensively optimizing the mevalonate pathway. We found that the key challenge is the complex and unusual dephosphorylation reaction of bornyl diphosphate (BPP), which suffers the side-reaction and the competition from the cellular dephosphorylation process, especially lipid metabolism, thus limiting (+)-borneol synthesis. Here, we systematically optimized the dephosphorylation process by identifying, characterizing phosphatases, and balancing cellular dephosphorylation metabolism. For the first time, we identified two endogenous phosphatases and seven heterologous phosphatases, which significantly increased (+)-borneol production by up to 152%. By engineering BPP dephosphorylation and optimizing the MVA pathway, the production of (+)-borneol was increased by 33.8-fold, which enabled the production of 753 mg/L under fed-batch fermentation in shake flasks, so far the highest reported in the literature. This study showed that rewiring dephosphorylation metabolism was essential for high-level production of (+)-borneol in Saccharomyces cerevisiae, and balancing cellular dephosphorylation is also helpful for efficient biosynthesis of other terpenoids since all whose biosynthesis involves the dephosphorylation procedure.

Mechanical pain is one of the most common causes of clinical pain, but there remains a lack of effective treatment for debilitating mechanical and chronic forms of neuropathic pain. Recently, neurotoxin GsMTx4, a selective mechanosensitive (MS) channel inhibitor, has been found to be effective, while the underlying mechanism remains elusive. Here, with multiple rodent pain models, we demonstrated that a GsMTx4-based 17-residue peptide, which we call P10581, was able to reduce mechanical hyperalgesia and neuropathic pain. The analgesic effects of P10581 can be as strong as morphine but is not toxic in animal models. The anti-hyperalgesic effect of the peptide was resistant to naloxone (an μ-opioid receptor antagonist) and showed no side effects of morphine, including tolerance, motor impairment, and conditioned place preference. Pharmacological inhibition of TRPV4 by P10581 in a heterogeneous expression system, combined with the use of Trpv4 knockout mice indicates that TRPV4 channels may act as the potential target for the analgesic effect of P10581. Our study identified a potential drug for curing mechanical pain and exposed its mechanism.

Objective To observe the effects of electroacupuncture on intestinal flora and serum inflammatory factors in rabbit model with rheumatoid arthritis(RA);To explore the mechanism of its therapeutic effect on RA.Methods RA model rabbits were established by ovalbumin induction combined with Freund's complete adjuvant,and the rabbits after successful modeling were randomly divided into model group,probiotic group and electroacupuncture group,with 6 rabbits in each group.Another 6 rabbits were set as the normal group.The electroacupuncture group received electroacupuncture at the bilateral"Zusanli"and"Dubi"for 30 minutes,the probiotic group was given probiotic capsules solution(14.5 mg/kg)by gavage,once a day,for two consecutive weeks.The knee joint circumference and pain threshold of rabbits were measured,histopathological morphological changes of colonic tissue and synovial tissue ws observed by HE staining,16S rDNA sequencing was used to analyze structural changes of intestinal flora,the contents of TNF-α,IL-1β and IL-6 in serum were detected by ELISA.Results Compared with the normal group,the circumference of knee joint of rabbits in the model group increased significantly,the pain threshold was significantly decreased(P<0.01);the colonic mucosal damage was serious,the goblet cells were missing,a large number of inflammatory cells were infiltrate;the joint capsule synovial surface was rough,the synovial cell layer was hyperplasia and thickening,the synovial tissue inflammatory cell infiltration was obvious;the number and evenness of gut microbiota species decreased,while the relative abundance of Proteobacteria,Firmicutes,and Bacteroidetes decreased(P<0.05,P<0.01),the relative abundance of Desulfobacteria increased(P<0.01),while the relative abundance of Campylobacter,Lawsonella,and Pseudomonas increased(P<0.01),while the relative abundance of Heshanomonas and Herbaspirillum decreased(P<0.01);the contents of serum TNF-α,IL-1β and IL-6 increased(P<0.01).Compared with the model group,the knee joint circumference of the probiotic group and the electroacupuncture group decreased,the pain threshold increased(P<0.01,P<0.05);the degree of intestinal mucosal damage was reduced,the goblet cells were basically arranged neatly,the inflammatory cell infiltration was reduced;synovial cells proliferation and thickening decreased,with a small amount of inflammatory cell infiltration;the number and evenness of gut microbiota species increased,while the relative abundance of Proteobacteria,Firmicutes,and Bacteroidetes increased(P<0.05,P<0.01),the relative abundance of Desulfobacteria decreased(P<0.01),while the relative abundance of Campylobacter,Lawsonella and Pseudomonas decreased(P<0.01),the relative abundance of Heshanomonas and Herbaspirillum increased(P<0.05,P<0.01);the contents of serum TNF-α,IL-1β and IL-6 significantly decreased(P<0.01).Conclusion Electroacupuncture can significantly improve the symptoms of RA rabbits and reduce the inflammatory reaction in synovial tissue of joint,and the mechanism may be related to the regulation of intestinal flora structure.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Abstract Allergic diseases can occur in all systems of the body, covering the whole life cycle, from children to adults and to old age, can be lifelong onset and even fatal in severe cases. Children account for the largest proportion of the victims of allergic disease, Children s allergies start from scratch, ranging from mild to severe, from less to more, from single to multiple systems and systemic performance, so the prevention and treatment of allergic diseases in children is of great importance, which can not only prevent high risk allergic conditions from developing into allergic diseases, but also further block the process of allergy. At present, there is no consensus on the management system of allergic children in kindergartens and primary schools. The "Consensus on Allergy Management and Prevention in Kindergartens and Primary Schools", which includes the organizational structure, system construction and management of allergic children, provides evidence informed recommendations for the long term comprehensive management of allergic children in kindergartens and primary schools, and provides a basis for the establishment of the prevention system for allergic children.

Objective To observe the effects of 4-week low intensity treadmill exercise on the learning and memory, amino acid levels and the protein expression of protein kinase A ( PKA) , cyclic adenosine monophosphate response element binding protein( CREB) and brain-derived neurotrophic factor(BDNF) in the prefrontal cortex (PFC) of the vascular dementia (VD) rats. Methods Thirty-nine SD rats were randomly allocated to 3 groups, sham group (sham, n= 13) , vascular dementia group (VD, n= 13) and vascular dementia treaded with exercise group (VD + EX, n= 13). Chronic cerebral ischemia model in VD group and VD+EX group rats were established by permanent ligation of bilateral, then VD+EX group rats were submitted to 4-week low intensity treadmill exercise. After exercise, spatial learning and memory ability were evaluated by Moms water maze test ( MWM ) , glutamic ( Glu ) and gamma-aminobutyric acid (GABA) levels in the PFC were measured by high performance liquid chromatography( HPLC) ; the protein expression of PKA, CREB and BDNF in the PFC of rats were detected by Western blotting. Results The result of the MWM showed the average escape latency of rats in the VD group on the 1 -5 days was significantly higer than sham group, the time to first find the original platform was significantly prolonged and the platform crossings decreased significantly ( P < 0 . 01 ) . The average escape latency of rats treated with four-week low intensity treadmill exercise on the 1-5 days was significantly lower than VD group, the time to first find the original platform was significantly shortened and the platform crossings increased significantly(P<0. Of). HPLC and Western blotting detection showed that the levels of Glu and GABA and the protein expression of PKA, CREB and BDNF in the PFC decreased significantly in VD group rats when compared with sham group ( P < 0.01 ) . Glu level and the protein expression of PKA, CREB and BDNF in the PFC of rats increased significantly ( P < 0 . 05) in VD + EX group rats when compared with VD group rats, but GABA level was not significant difference ( P > 0. 05 ) between the two groups. Conclusion Four-week low-intensity running exercise improves the learning and memory ability of VD rats through enhancing the Glu level and activating PKA-CREB-BDNF signaling in the PFC of rats.

Objective To examine the effect of estradiol (E2) treatment on ovariectomy (OVX) induced depressive-like behavior and possible mechanism by measuring inflammatory biomarkers levels oi interleukin-6 (IL-6) and tumor necrosis tactor-(x (TNF-ot) and brain-derived neurotrophic factor (BDNF) expression in amygdaia nucleus. Methods Thirty nine healthy aduit 缶male SD rats were randomly divided into three groups : sham operation group (SO), ovariectomized group (OVX) and ovariectomized estradiol treatment group (OVX + E2). After 6 weeks of E2 treatment, depressive like behavior was evaluated by opening field test (OFT) and sugar water preference test (SPT). The levels of inflammatory factors IL-6 and TNF-a in amygdala were measured by ELESA, and the expression of BDNF in rat amygdala was detected by immunohistochemical staining. Results The result of the SPT showed that OVX significantly decreased the sugar intake and sugar preference rate of rats, and E2 treatment significantly increased sugar water intake and sugar water preference rate of rats. The result of the OFT showed that OVX significantly decreased the numbers of crossing and rearing of rats, and reduced the time spent in the centre ; E2 treatment significantly increased the numbers of crossing and rearing of rats, and prolonged the time spent in the centre. ELESA and immunohistochemical analysis found the levels of IL-6 and TNF-a in amygdala increased significantly, while the average absorbance (AA) of BDNF in the amygdala reduced significantly (P<0.01 respectively) of rats in OVX group when compared with the SO group. And the levels of IL-6 and TNF-a in amygdala decreased significantly (P<0.01 respectively), while the A4 of BDNF increased significantly (P< 0.05) in the amygdala of rats in the OVX+EX group when compared with the OVX group. The difference was statistically significant. Conclusion E2 treatment improved depression-like behavior of OVX rats is partly due to increased antiinflammatory and activated the BDNF expression in amygdaloid nucleus, thus enhancing the neuroprotective effect of OVX rats.

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.
Cinnamomum camphora/enzymology* ; Alkyl and Aryl Transferases/chemistry*

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics* ; Child ; China/epidemiology* ; Cryptorchidism/genetics* ; Disorders of Sex Development/genetics* ; Female ; Genital Diseases, Male ; Genotype ; Humans ; Hypospadias/genetics* ; Male ; Membrane Proteins/genetics* ; Penis/abnormalities* ; Phenotype ; Retrospective Studies ; Steroid 21-Hydroxylase/genetics*

Objective: To establish a high glucose senescent model of human dermal fibroblasts (HDFs), and to investigate the effects of exosomes derived from human decidua mesenchymal stem cells (dMSCs) on the proliferation, migration, and apoptosis of senescent HDFs and possible mechanism. Methods: The experimental research method was used. From January to March 2021, discarded foreskin tissue was collected for isolation and culture of primary HDFs from 4 male phimosis patients (aged 18-22 years) admitted for circumcision in the Fourth Medical Center of the PLA General Hospital. The 6th passage of HDFs were taken and divided into low glucose group and high glucose group according to the random number table, and subsequently cultured in low-glucose complete medium and high-glucose complete medium, respectively, with medium changed every 72 h without subculturing. After 10 days of culture, the cells were taken and measured for cellular senescence using the β-galactosidase kit at 24 h after seeding; the expression of senescence-related proteins p16 and p53 was assessed by Western blotting at 48 h after seeding; cell proliferation was detected at 24, 48, and 72 h after seeding using the cell counting kit 8 (CCK-8) method; the cell proliferation was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) staining method, cell cycle and apoptosis were measured by flow cytometry after 48 h of seeding; Transwell experiment was used for the calculation of cell migration rate at 24 h after seeding. The human dMSCs were taken and cultured for 48-72 h from which the exosomes were extracted by differential high speed centrifugal method. The morphology of dMSC exosomes was observed by transmission electron microscopy, the particle size distribution of dMSC exosomes was measured by nanoparticle tracking analysis, and the expression of dMSC-exosomes marker proteins CD9 and tumor susceptibility gene101 (TSG101) were detected by Western blotting. The dMSC exosomes and high-glucose complete medium-induced senescent HDFs were co-cultured for 24 hours, then PKH67 kit was used to detect the uptake of exosomes by HDFs. High-glucose complete medium-induced senescent HDFs were taken and divided into high glucose alone group, high glucose+low concentration of exosomes group, and high glucose+high concentration of exosomes group according to the same method above. The high-glucose complete medium with equal volume of phosphate buffered saline, dMSC exosomes with final concentration of 50 μg/mL, and dMSC exosomes with final concentration of 100 μg/mL were added to the corresponding groups for conventional cell culture, respectively. After grouped, the cell proliferation, cell cycle and apoptosis as well as cell migration were detected by CCK-8 method and EdU staining method, flow cytometry, and Transwell experiment at the corresponding time points as before, respectively. Based on the previous results, high-glucose complete medium-induced senescent HDFs were taken and divided into high glucose alone group and high glucose+high concentration of exosomes group for the same treatment. After being grouped and cultured for 48 h, real-time fluorescent quantitative polymerase chain reaction was used to evaluate the mRNA expression of senescent-related microRNA (miR)-145-5p, miR-498, miR-503-5p, calcium/calmodulin dependent protein kinase 1D (CAMK1D), phosphates and tensin homologue deleted on chromosome ten (PTEN) gene, and Cyclin D1 in high glucose alone group and high glucose+high concentration of exosomes group. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference t test, and independent sample t test. Results: At 24 h after seeding, the rate of β-galactosidase-positive staining of HDF in high glucose group was (38.4±4.2)%, which was significantly higher than (16.5±2.2)% of low glucose group (t=4.65, P<0.01). At 48 h after seeding, the expression levels of senescence-related proteins p16 and p53 both were significantly higher in HDFs of high glucose group than those in low glucose group (with t values of 11.85 and 3.02, respectively, P<0.05 or P<0.01). At 0, 24, 48, and 72 h after seeding, the cell proliferation viability of HDFs in high glucose group was all significantly lower than in low glucose group (with t values of 4.13, 9.90, and 15.12, respectively, P<0.01). At 48 h after seeding, the rate of EdU-positive staining of HDFs in high glucose group was obviously lower than that of low glucose group (t=3.83, P<0.05). At 48 h after seeding, the percentage of G2/M+S subpopulations in three subpopulations (G0/G1, S, and G2/M) of HDF cycle was significantly lower in high glucose group than that in low glucose group (t=8.74, P<0.01). At 24 h after seeding, the number of HDFs migrated through the filter membrane to the lower chamber was 37±6 in high glucose group, which was significantly less than 74±7 in low glucose group (t=8.42, P<0.01). At 48 h after seeding, the HDF apoptosis rate was significantly higher in high glucose group than in low glucose group (t=8.48, P<0.01). The dMSC exosomes were cup-shaped or round vesicles with well-defined edges and uniform size distribution. The size of dMSC exosomes was basically in the range of 80-200 nm. Exosomal markers including CD9 and TSG101 were positively presented on the dMSC exosomes. After being co-cultured for 24 hours, the dMSC exosomes were taken up intracellularly by HDFs and mainly distributed around the nucleus of HDFs. After being grouped and cultured for 24, 48, and 72 h, the HDF proliferation viabilities in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly higher than in high glucose alone group (with t values of 6.36, 6.10, 7.76, 8.92, 12.17, and 10.74, respectively, P<0.01), the HDF proliferation viability in high glucose+high concentration of exosomes group was significantly higher than in high glucose+low concentration of exosomes group (with t values of 7.92, 4.82, and 4.72, respectively, P<0.01). After being grouped and cultured for 48 h, the percentages of EdU-positive HDFs in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly higher than in high glucose alone group (with t values of 5.32 and 9.88, respectively, P<0.01), the percentage of EdU-positive HDFs in high glucose+high concentration of exosomes group was notably higher than in high glucose+low concentration of exosomes group (t=5.27, P<0.01). After being grouped and cultured for 48 h, the proportion of G0/G1 subpopulation in both high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group was distinctly lower (with t values of 3.81 and 4.31, respectively, P<0.05), while the proportion of G2/M+S subpopulation was markedly higher (with t values of 3.81, 4.31, respectively, P<0.05) than in high glucose alone group. After being grouped and cultured for 24 h, the number of HDFs migrated through the filter membrane in both high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group was significantly higher than in high glucose alone group (with t values of 10.14 and 13.39, respectively, P<0.01), the number of HDFs migrated through the filter membrane in high glucose+high concentration of exosomes group was significantly increased than in high glucose+low concentration of exosomes group (t=6.27, P<0.01). After being grouped and cultured for 48 h, the HDF apoptosis rates in high glucose+low concentration of exosomes group and high glucose+high concentration of exosomes group were both significantly lower than in high glucose alone group (with t values of 3.72 and 5.53, respectively, P<0.05 or P<0.01). After being grouped and cultured for 48 h, compared with those in high glucose alone group, the mRNA expression levels of miR-145-5p and miR-498 were both obviously higher (with t values of 13.03 and 8.90, respectively, P<0.01), while the mRNA expression level of miR-503-5p was significantly lower (t=3.85, P<0.05) in high glucose+high concentration of exosomes group. After being grouped and cultured for 48 h, compared with those in high glucose alone group, the mRNA expression levels of CAMK1D and PTEN gene were both significantly lower (with t values of 8.83 and 5.97, respectively, P<0.01), while the mRNA expression level of Cyclin D1 was significantly higher in high glucose+high concentration of exosomes group (t=4.03, P<0.05). Conclusions: The dMSC exosomes are capable of improving cell proliferation and migration, and inhibiting cell apoptosis of high-glucose senescent HDFs. This may be related to the mechanism by which the increased expressions of intracellular miR-145-5p and miR-498 inhibit the expression of CAMK1D and PTEN gene, and the decreased expression of miR-503-5p promote the expression of Cyclin D1.
Adolescent ; Adult ; Cell Proliferation ; Decidua ; Exosomes ; Female ; Fibroblasts ; Glucose/pharmacology* ; Humans ; Male ; Mesenchymal Stem Cells ; MicroRNAs ; Young Adult