Hydroxyectoine, a vital compatible solute, is widely utilized in cosmetics, food, pharmaceutical industries, and biologics. However, the current microbial fermentation methods for hydroxyectoine production face challenges including insufficient precursor supply and low yields. Therefore, developing engineering microbial strains capable of efficiently synthesizing hydroxyectoine is of great significance. In this study, we first constructed a high-yield ectoine-producing strain ECT04 by multi-copy integration of the ectoine synthesis genes ectABC into the pseudogene loci of Escherichia coli MG1655(DE3), achieving an ectoine titer of 6.03 g/L. Subsequently, we employed plasmids with varying copy numbers to express ectD from Chromohalobacter salexigens to enable the conversion for hydroxyectoine production. We further investigated the effects of promoter, co-substrate ɑ-ketoglutarate, Fe²⁺ concentration, and dissolved oxygen on hydroxyectoine synthesis. Through fed-batch fermentation in a 7-L bioreactor, we significantly enhanced the hydroxyectoine production efficiency, attaining a final titer of 8.58 g/L and a productivity of 0.24 g/(L·h). This work successfully achieved the de novo synthesis of hydroxyectoine in E. coli, laying a foundation for the efficient bioproduction of this compound.
Escherichia coli/genetics* ; Fermentation ; Amino Acids, Diamino/biosynthesis* ; Bioreactors/microbiology* ; Metabolic Engineering/methods* ; Chromohalobacter/genetics* ; Plasmids/genetics*

Objective To investigate the morphological typing and clinical significance of the distal tibiofibular syndesmosis fibular notch based on CT images. Methods According to the inclusion and exclusion ceiteria‚ the imaging data of patients undergoing ankle joint CT examination were analyzed‚ and the inferior tibiofibular joint fibula notch was classified according to the morphological characteristics. The measurements included 8 distances. There were 123 males and 102 females‚ all of whom were Han nationality‚ aged 18-60 years old. Results Retrospectively analyzed the result of 225 patients from December 2013 to December 2022. The distal tibiofibular syndesmosis fibular notch was divided into four types according to morphological characteristics‚ C-shaped (50. 67%)‚ V-shaped (26. 67%)‚ flat-shaped (15. 11%) and L-shaped (7. 56%). The angle between the anterior and posterior facets of the flat shape (145. 56 ± 9. 25)° was the largest and the angle between the anterior and posterior facets of the L shape (125. 07 ± 13. 54)° was the smallest(P< 0. 05); the depth of the notch in the flat shape (3. 11 ± 0. 83) mm was the smallest and in the L shape (4. 47±1. 11) mm was the largest(P<0. 05);The posterior facet length (13. 06 ± 3. 56) mm and anterior tibiofibular gap (3. 83±1. 49) mm on left were larger than on the right side (P<0. 05); The posterior facet length (13. 36 ± 3. 46) mm‚ fibular notch depth (3. 93 ± 1. 10) mm and vertical distance of tibiofibular overlap (9. 10 ± 2. 55) mm larger in men than in women (P<0. 05). Conclusion In this study‚ the data related to the inferior tibiofibular syndesmosis notch were measured and divided into four types according to the shape. The flat inferior tibiofibular syndesmosis notch is more likely to have chronic ankle instability‚ and the fibula is more likely to move forward during anatomical reduction. The inferior tibiofibular syndesmosis of L-shaped and C-shaped notches is more prone to posterior displacement of fibula or poor rotation reduction during anatomical reduction.

Objective To investigate the effect of dexmedetomidine and small dose of ketamine on the hyperalgesia and brain-derived neurotrophic factor (BDNF) mRNA in the spinal cord of rats with neuropathic pain.Methods Forty-five male Sprague-Dawley rats,weighing 240-270 g,were randomly divided into 5 groups (n =9 each):sham operation group (group S),spinal nerve injury (SNI) group,ketamine group (group K),dexmedetomidine group (group D) and dexmedetomidine + ketamine group (group K+ D).The animals were anesthetized with intraperitoneal 10% chloral hydrate 400 mg/kg.SNI was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in groups SNI,K,D and K + D.In S group,the sciatic nerves and its branches were only exposed but not ligated.In groups D,K and K + D,dexmedetomidine 40 μg/kg (2 ml),ketamine 10 mg/kg (2 ml) and mixture of dexmedetomidine 20μg/kg + ketamine 5 mg/kg (2 ml) were injected intraperitoneally,respectively,once a day for 21 consecutive days starting from 24 h after successful SNI surgery.While the equal volume of normal saline 2 ml was injected instead in S and SNI groups.The paw withdrawal threshold to mechanical stimuli (PWT) was measured at 1 day before surgery,and 3,7,14 and 21 days after surgery.Three rats were sacrificed after measurement of pain threshold at 1 day before surgery,and 7 and 21 days after surgery and the lumbar segments (L4-6) of the spinal cord were removed for determination of BDNF mRNA expression in spinal dorsal horn by real time PCR.Results Compared with group S,the PWT was significantly decreased,and the expression of BDNF mRNA in spinal dorsal horn was up-regulated after surgery in groups SNI,K,D and K + D (P ＜ 0.05).The PWT was significantly higher,and the expression of BDNF mRNA in spinal dorsal horn was lower after surgery in groups K,D and K + D than in group SNI,and in group K + D than in groups K and D (P ＜ 0.05).Conclusion The combination of dexmedetomidine and small dose of ketamine produces a synergistic antinociception in rats with neuropathic pain,and direct or indirect inhibition of BDNF mRNA expression in spinal dorsal horn is involved in the underlying mechanism.

OBJECTIVETo explore the feasibility to construct genetic engineering human neural stem cells (hNSCs) mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI).

METHODSHuman neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3 (NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot.

RESULTSGenetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot.

CONCLUSIONSGenetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.

Animals ; Cell Differentiation ; Cells, Cultured ; Feasibility Studies ; Gene Expression ; Genetic Engineering ; methods ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Lentivirus ; genetics ; Microscopy, Fluorescence ; Neurons ; metabolism ; Rats ; Stem Cell Transplantation ; Stem Cells ; metabolism ; Transgenes

【Objective】To observe the effects of bamboo leaf extract(BLE)on cerebral ischemia.【Methods】 Effect of BLE(at the concentrations of 10,20 and 40 mg/kg respectively,iv)on rabbit carotid thrombus induced by cotton thread was observed.Meanwhile,the effects of BLE(at the concentrations of 15,30 and 60 mg/kg respectively,iv)on neurological deficit and cerebral infarct size in rats with middle cerebral arterial obstruction(MCAO),as well as the effects of BLE(at the concentrations of 22.5,45,and 90?mg/kg respectively,iv)on mice stroke index,grasping time of decapitated mice and survival time of mice subjecting to bilateral carotid ligation were also observed.【Results】BLE inhibited rabbit carotid thrombosis,improved neurological deficit and reduced cerebral infarct size in MCAO rats(P