The application value of prenatal ultrasound in conjoined twins.

OBJECTIVE: To evaluate the application and effectiveness of prenatal ultrasound in diagnosing and managing conjoined twins. METHODS: A retrospective analysis was conducted on 20 cases of conjoined twins diagnosed at our hospital between January 2016 and December 2022. The types of conjoined twins, ultrasonographic characteristics, and associated anomalies were assessed. RESULTS: The gestational age at diagnosis ranged from 10 to 35 weeks, with an average of 14.21 ± 5.69 weeks. Thirteen cases were detected in the first trimester, five in the early second trimester, one at 23 + 2 weeks, and one at 35 weeks. Thoracopagus was the most common type (11 cases, 55%), followed by omphalopagus (4 cases, 20%), cephalopagus (4 cases, 20%), and parapagus dicephalus (1 case, 5%). In the first trimester, the most common abnormalities observed included increased nuchal translucency (NT), cystic hygroma, hydrops fetalis, and generalized edema. Major birth defects identified in conjoined twins were omphalocele (3 cases), congenital heart malformations (3 cases), neural tube defects (2 cases), urachal cyst (1 case), and umbilical cyst (1 case). Pregnancy was terminated in 18 cases, one case resulted in spontaneous abortion during the second trimester, and one case was delivered by cesarean section at 37 weeks, with successful separation and recovery. CONCLUSION: Prenatal ultrasound is the primary diagnostic tool for conjoined twins. It effectively assesses the extent of twin fusion, provides critical information for clinical decision-making, and aids in the management of obstetric care.

Joint clustering multiple longitudinal features: A comparison of methods and software packages with practical guidance.

Clustering longitudinal features is a common goal in medical studies to identify distinct disease developmental trajectories. Compared to clustering a single longitudinal feature, integrating multiple longitudinal features allows additional information to be incorporated into the clustering process, which may reveal co-existing longitudinal patterns and generate deeper biological insight. Despite its increasing importance and popularity, there is limited practical guidance for implementing cluster analysis approaches for multiple longitudinal features and evaluating their comparative performance in medical datasets. In this paper, we provide an overview of several commonly used approaches to clustering multiple longitudinal features, with an emphasis on application and implementation through R software. These methods can be broadly categorized into two categories, namely model-based (including frequentist and Bayesian) approaches and algorithm-based approaches. To evaluate their performance, we compare these approaches using real-life and simulated datasets. These results provide practical guidance to applied researchers who are interested in applying these approaches for clustering multiple longitudinal features. Recommendations for applied researchers and suggestions for future research in this area are also discussed.

Effect of postoperative hypoalbuminemia and supplement of human serum albumin on the development of surgical site infection following spinal fusion surgery: a retrospective study.

PURPOSE: To determine the association between postoperative hypoalbuminemia and the development of surgical site infection (SSI) and evaluate whether the supplement of exogenous human serum albumin (HSA) in patients following spinal surgery would decrease the rate of postoperative SSI. METHODS: We performed a retrospective review of all patients who underwent lumbar spinal fusion surgery in our institution between January 2014 and December 2018. Patients with postoperative SSI were identified. We reviewed the demographic and clinical records of the patients and performed multiple logistic regression models to clarify the relevance between postoperative hypoalbuminemia, the supplement of HSA and SSI. Statistical adjustment for the potential confounders was also performed to exclude possible variation. RESULTS: Twenty-four of 602 patients developed SSI after lumbar spinal fusion surgery. No statistical significance was found between postoperative hypoalbuminemia and SSI rate (OR 0.74; 95% CI 0.22-2.48; P = 0.6199). However, the supplement of exogenous HSA was significantly associated with increased postoperative SSI rate (OR 1.21; 95% CI 1.05-1.41; P = 0.0094). Interestingly, stratified analyses showed supplement of HSA in patients without postoperative hypoalbuminemia increased the risk of SSI (OR 2.55; 95% CI 1.01-6.45; P = 0.0475), compared with patients with postoperative hypoalbuminemia (OR 1.17; 95% CI 1.00-1.36; P = 0.0434). CONCLUSIONS: The present study suggests that postoperative hypoalbuminemia is not associated with the development of SSI after spinal surgery. However, the supplement of HSA following spinal surgery will increase the rate of SSI. These slides can be retrieved under Electronic Supplementary Material.

Hypoxia reduces the osteogenic differentiation of peripheral blood mesenchymal stem cells by upregulating Notch-1 expression.

Purpose: Mesenchymal stem cells (MSCs) seeded on biocompatible scaffolds have therapeutic potential for bone defect repair. However, MSCs can be affected by hypoxia and nutritional deficiency due to a lack of blood vessels in the scaffolds. Here, we explored the effects of hypoxia on MSC differentiation to clarify these mechanisms. Methods: Peripheral blood mesenchymal stem cells (PBMSCs) were cultured in small individual chambers with oxygen concentrations of 1%, 9%, and 21%. Cell proliferation was evaluated by Cell Counting Kit 8 assays, and cell survival was determined using live/dead assays. Scratch assays were performed to evaluate cell migration. Ca2+ deposition/mineralization experiments, reverse transcription quantitative real-time polymerase chain reaction, and Western blotting were performed to assess the osteogenic differentiation of cells. Notch1 expression was downregulated by lentivirus-transfected PBMSCs to observe the effects of Notch1 knockdown on osteogenic gene and protein expression. Results: PBMSCs exposed to hypoxia (1% O2) demonstrated accelerated proliferation, increased migration, and reduced survival in the absence of serum. Although 9% oxygen promoted osteogenic differentiation, the osteogenic differentiation of PBMSCs was significantly reduced by 1% O2, and this effect was associated with increased Notch1 expression. Reducing Notch1 expression using small interfering RNA significantly restored the osteogenic differentiation of PBMSCs. Conclusions: Hypoxia accelerated proliferation, increased migration, and reduced PBMSC differentiation into osteoblasts by increasing Notch1 expression. These findings may contribute to the development of appropriate cell culture or in vivo transplantation conditions to maintain the full osteogenic potential of PBMSCs.

Effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of MC3T3-E1 cells.

In physiological and pathological environments, the concentration of oxygen around osteoblasts varies widely. No studies have systematically evaluated the effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of osteoblasts. In this study, we cultured the osteoblast precursor cell line MC3T3-E1 in small individual chambers with oxygen concentrations of 1%, 3%, 6%, 9%, and 21%. Cell proliferation was evaluated by the proliferation index test and EdU staining. To test cell survival, a live/dead assay was performed. A tablet scratch assay was performed to detect the migratory ability of the cells. Bone nodule formation experiments and immunofluorescence and Western blotting analyses of osteogenic-related proteins were performed to assess the osteogenic differentiation of the cells. We found that the proliferation and osteogenic differentiation ability of MC3T3-E1 cells in different oxygen concentrations were both approximately bell-shaped curves and that the optimal oxygen concentrations were approximately 6% and 9%, respectively. The live/dead assay showed that the survival of MC3T3-E1 cells in different oxygen concentrations was affected by the amount of serum. The tablet scratch experiment showed that there was greater cell migration with oxygen concentrations of 1%, 3%, and 21% than with oxygen concentrations of 6% and 9%. Our results have significant reference value for the intervention of the pathological processes involving osteoblasts, such as fracture, osteoporosis, and some vascular diseases. These results also have an important guiding role for the new scientific idea that osteoblasts can function as treatment cells to repair bone defects.

RNA sequencing and anthocyanin synthesis-related genes expression analyses in white-fruited Vaccinium uliginosum.

BACKGROUND: Vaccinium uliginosum (Ericaceae) is an important wild berry having high economic value. The white-fruited V. uliginosum variety found in the wild lacks anthocyanin and bears silvery white fruits. Hence, it is a good resource for investigating the mechanism of fruit color development. This study aimed to verify the differences in the expression levels of some structural genes and transcription factors affecting the anthocyanin biosynthesis pathway by conducting high-throughput transcriptome sequencing and real-time PCR analysis by using the ripening fruits of V. uliginosum and the white-fruited variety. RESULTS: We annotated 42,837 unigenes. Of the 325 differentially expressed genes, 41 were up-regulated and 284 were down-regulated. Further, 11 structural genes of the flavonoid pathway were up-regulated, whereas two were down-regulated. Of the seven genes encoding transcription factors, five were up-regulated and two were down-regulated. The structural genes VuCHS, VuF3'H, VuFHT, VuDFR, VuANS, VuANR, and VuUFGT and the transcription factors VubHLH92, VuMYB6, VuMYBPA1, VuMYB11, and VuMYB12 were significantly down-regulated. However, the expression of only VuMYB6 and VuMYBPA1 rapidly increased during the last two stages of V. uliginosum when the fruit was ripening, consistent with anthocyanin accumulation. CONCLUSIONS: VuMYB6 was annotated as MYB1 by the BLAST tool. Thus, the white fruit color in the V. uliginosum variant can be attributed to the down-regulation of transcription factors VuMYB1 and VuMYBPA1, which leads to the down-regulation of structural genes associated with the anthocyanin synthesis pathway.

Characterization of HSP90 expression and function following CNS injury.

Spinal cord injury induces significant cellular stress responses. The Heat Shock Protein 90 (HSP90) plays a pivotal role as a molecular chaperone and is crucial for protein folding, stabilization, and cellular signaling pathways. Despite its important function in stress adaptation, the specific expression patterns and functional roles of HSP90 after nerve injury remain unclear. This study aimed to elucidate the expression dynamics and functional implications of HSP90 following central nervous system (CNS) injury. Using western blotting and immunohistochemical analyses, we observed upregulation of HSP90 expression in spinal cord tissues and within injured neurons in a spinal cord contusion injury model. Additionally, HSP90 was found to enhance neurite outgrowth in primary cortical neurons cultured in vitro. Furthermore, in a glutamate-induced neuronal injury model, the expression of HSP90 was up-regulated, and overexpression of HSP90 promoted neurite re-growth in damaged neurons. Overall, our findings highlight the critical involvement of HSP90 in the neural response to injury and offer valuable insights into potential therapeutic strategies for CNS repair.

Stimulation of oxalate root exudate in arsenic speciation and fluctuation with phosphate and iron in anoxic mangrove sediment.

Mutual transformations of rhizospheric arsenic (As) in pollution-prone mangrove sediments affected by root exudate oxalate were simulated. This study focuses on the effect of oxalate on As release, mobilization, and phase speciation associated with P and Fe was examined under anoxic conditions in time-dependent changes. Results showed that oxalate addition significantly facilitated As-Fe-P release from As-contaminated mangrove sediments. Sediment As formed the adsorptive and the carbonate-binding fractionations, facilitating the re-adsorption processes. Solution As and As5+ correlated with NaOH-P positively but with NaHCO3-P and HCl-P negatively. Dominant Fe3+ (>84 %) from the amorphous Fe regulated suspension changes and then time-dependent co-precipitation with As and P. Sediment P formed strong complexes with Fe oxides and could be substituted for As via STEM analysis. Oxalate ligand exchange, competitive adsorption of oxalate, and Fe-reduced dissolution are confirmed to involve, allowing for an insight As/P/Fe mobilization and fate in mangrove wetland.

A systematic review and meta-analysis of acupuncture combined with Tuina in the treatment of insomnia.

BACKGROUND: Insomnia is a sleep disorder with insufficient sleep time or/and poor sleep quality. Relevant epidemiological studies have shown that insomnia symptoms occur in about 35% to 50% of the adult population, and it is one of the most common diseases in the elderly. Patients who often suffer from insomnia are prone to symptoms such as fatigue, weakened cognitive function, depression, and even mental illness, which bring serious physical and mental damage to individuals and a heavy economic burden to social medical care and families. Traditional Chinese medicine and modern medicine have their own advantages in the treatment of insomnia, and there is currently a lack of reports on the comparison of acupuncture combined with massage and conventional medicine. OBJECTIVE: To evaluate the clinical efficacy of acupuncture combined with Tuina in the treatment of insomnia. METHODS: Search for clinical randomized controlled trials (RCTs) of acupuncture combined with Tuina in the treatment of insomnia from PubMed, Cochrane Library, Web of Science, China National Knowledge Infrastructure, Wan Fang Database, and China Science and Technology Journal Database. The RevMan5.4 software was used for Meta- analysis after literature screening, data extraction and quality evaluation. RESULTS: A total of 29 studies were included with a total of 2688 cases. Compared with drugs or acupuncture alone, acupuncture combined with Tuina has advantages in the total clinical effectiveness, as well as the Pittsburgh Sleep Quality Index (PSQI) and Statistical Self-Rating Anxiety Scale score (SAS) (OR = 3.59, 95% confidence interval [CI] [2.77, 4.66], Z = 9.62 [P < .00001]) (MD = -2.44, 95% CI [-2.93, -1.95], Z = 9.72 [P < .00001]) (MD = -8.42, 95% CI [-10.23, -6.61], Z = 9.09 [P < .00001]). There was no statistically significant difference in Statistical Self-rating Depression Scale score (SDS) (MD = -5.26, 95% CI [-11.29, 0.78], Z = 1.71 [P > .05]). CONCLUSION: Acupuncture combined with Tuina has obvious clinical advantages in the treatment of insomnia. This result is expected to provide a reference for the clinical treatment of insomnia, but the long-term effect of clinical efficacy still needs further study.

Long Noncoding RNA SNHG1 Knockdown Ameliorates Apoptosis, Oxidative Stress and Inflammation in Models of Parkinson's Disease by Inhibiting the miR-125b-5p/MAPK1 Axis.

BACKGROUND: Parkinson's disease (PD) is a prevalent neurodegenerative disease. Long noncoding RNA small molecule RNA host gene 1 (SNHG1) has been reported to play critical roles in Parkinson's disease (PD) progression. The study aimed to further elucidate the mechanism of SNHG1 in PD pathogenesis. METHODS: The levels of SNHG1, miR-125b-5p and mitogen-activated protein kinase 1 (MAPK1) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell viability and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The activity of Caspase-3 or Caspase-9 was measured using a Caspase-3 or Caspase-9 Assay Kit. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, lactic dehydrogenase (LDH) activity, reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were gauged by enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter assay was performed to identify the relationship between miR-125b-5p and SNHG1 or MAPK1. The MPTP-induced PD mouse was used as an in vivo model of PD and MPP+-treated SK-N-SH and MN9D cells were used as in vitro models of PD. RESULTS: SNHG1 and MAPK1 were significantly up-regulated while miR-125b-5p was down-regulated in the MPTP-induced PD mouse model and MPP+-induced PD cell models. SNHG1 silence or miR-125b-5p overexpression protected against MPP+-evoked apoptosis, oxidative stress and inflammation in SK-N-SH and MN9D cells. Moreover, SNHG1 acted as a molecular sponge of miR-125b-5p, and the protective impact of SNHG1 silence on MPP+-evoked cell damage was reversed by miR-125b-5p inhibition. Furthermore, MAPK1 was a functional target of miR-125b-5p and its overexpression attenuated the effects of miR-125b-5p restoration in MPP+-triggered cell injury. In addition, the behavioral changes in MPTP-induced PD mouse in vivo model were relieved by SNHG1 silence. CONCLUSION: SNHG1 knockdown exerted neuroprotective effects in MPP+-evoked cytotoxicity through regulating the miR-125b-5p/MAPK1 axis both in human and mouse PD cell models, highlighting a possible target for PD therapy.

The Regulatory Effect of VEGF-Ax on Rat Bone Marrow Mesenchymal Stem Cells' Angioblastic Differentiation and Its Proangiogenic Ability.

Vascular endothelial growth factor A (VEGFA), which plays a key role in angiogenesis, is composed of many isoforms. Distinct VEGFA isoforms are generated by alternative splicing of VEGFA mRNA and named as VEGFxxx, where xxx represents the number of amino acids present in the final protein sequence. These isoforms have opponent pro- and antiangiogenic effects. VEGF-Ax, an additional isoform containing a 22-amino-acid extension in the COOH terminus, arising from VEGFA mRNA, programmed translational readthrough. The function of VEGF-Ax is not clear, especially the conclusion that VEGF-Ax regulates angiogenesis is contradictory. Thus, we investigated the effect of VEGF-Ax on differentiation and angiogenesis of rat bone marrow mesenchymal stem cells (BMMSCs). The results showed that VEGF-Ax could promote the proliferation and migration of BMMSCs, stimulate the differentiation of BMMSCs into endothelial cell-like cells, and protect BMMSCs from endoplasmic reticulum stress-induced apoptosis.

PC-3-Derived Exosomes Inhibit Osteoclast Differentiation by Downregulating miR-214 and Blocking NF-κB Signaling Pathway.

Prostate cancer is a serious disease that can invade bone tissues. These bone metastases can greatly decrease a patient's quality of life, pose a financial burden, and even result in death. In recent years, tumor cell-secreted microvesicles have been identified and proposed to be a key factor in cell interaction. However, the impact of cancer-derived exosomes on bone cells remains unclear. Herein, we isolated exosomes from prostate cancer cell line PC-3 and investigated their effects on human osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining. The potential mechanism was evaluated by qRT-PCR, western blotting, and microRNA transfection experiments. The results showed that PC-3-derived exosomes dramatically inhibited osteoclast differentiation. Marker genes of mature osteoclasts, including CTSK, NFATc1, ACP5, and miR-214, were all downregulated in the presence of PC-3 exosomes. Furthermore, transfection experiments showed that miR-214 downregulation severely impaired osteoclast differentiation, whereas overexpression of miR-214 promoted differentiation. Furthermore, we demonstrated that PC-3-derived exosomes block the NF-κB signaling pathway. Our study suggested that PC-3-derived exosomes inhibit osteoclast differentiation by downregulating miR-214 and blocking the NF-κB signaling pathway. Therefore, elevating miR-214 levels in the bone metastatic site may attenuate the invasion of prostate cancer.

Correlation between circulating tumor cells and D-D and platelet in patients with pulmonary malignancies.

The aim of the present study was to investigate the correlation between circulating tumor cells (CTC) and D-dimer (D-D) and platelet (PLT) in patients with pulmonary malignancies. A total of 98 patients with lung cancer admitted to West China Hospital, Sichuan University, from June 2016 to February 2017 were enrolled in the present study. D-D and PLT levels were measured in the fasting elbow vein of the patients. The expression of CTC in peripheral blood was detected by negative separation using immunomagnetic beads and immunocytochemical staining. The correlation between CTC and D-D and PLT in patients with lung cancer was analyzed. The mean level of D-D in the peripheral blood of 98 patients was 1.80±1.63 µg/l, and the level of D-D was correlated with distant metastasis (P<0.05). The mean level of PLT in peripheral blood was 305.53±141.22×109/l in 98 patients, and the level of PLT was correlated with patient age, clinical stage and distant metastasis (P<0.05). The levels of D-D, PLT and distant metastasis were significantly higher in CTC-positive than in CTC-negative patients (P<0.05). Therefore, CTC can predict the distant metastasis of lung cancer, and the incidence of distant metastasis is high in patients with hypercoagulable state.

Caudatin targets TNFAIP1/NF-κB and cytochrome c/caspase signaling to suppress tumor progression in human uterine cancer.

Caudatin, a C-21 steroidal glyco-side isolated from Chinese herbs, has a long history of use for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of caudatin in human uterine cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which caudatin inhibits cell growth in human cervical carcinoma cell line (HeLa) and endometrial carcinoma cell line (HEC-1A). Treatment with caudatin promoted cell morphology change, inhibited cell proliferation, colony formation, migration and spheroid formation, and induced cell apoptosis. Our results showed that the expression of tumor necrosis factor; α-induced protein 1 (TNFAIP1) was downregulated in uterine cancer cells and tissues compared to paired adjacent non-tumor uterine tissues. Further molecular mechanism study showed that caudatin can directly regulate TNFAIP1 expression in a concentration-dependent manner and also associated with the downregulation of NF-κB and upregulation of BAX/BcL-2 ratio and caspase-3. Moreover, we found that overexpression of TNFAIP1 inhibits the growth and invasion, and induces apoptosis in uterine cancer cells through inhibition of the NF-κB pathway, suggesting that TNFAIP1 may act as a potential therapeutic target for the treatment of cancer. We found that caudatin inhibited tumorigenicity and upregulated TNFAIP1 in vivo. Taken together, caudatin impacts on cell proliferation, migration and apoptosis of uterine cancer cells by regulating several carcinogenesis-related processes, including a novel mechanism involving the targeting of TNFAIP1/NF-κB signaling. Our findings provide new insights into understanding the anticancer mechanisms of caudatin in human uterine cancer therapy.

microRNA-372 maintains oncogene characteristics by targeting TNFAIP1 and affects NFκB signaling in human gastric carcinoma cells.

Aberrant microRNA (miRNA) expression has been investigated in gastric cancer, which is one of the most common malignancies. However, the roles of miRNAs in gastric cancer remain largely unknown. In the present study, we found that microRNA-372 (miR-372) directly targets tumor necrosis factor, α-induced protein 1 (TNFAIP1), and is involved in the regulation of the NFκB signaling pathway. Furthermore, overexpression of TNFAIP1 induced changes in AGS cells similar to those in AGS cells treated with miR-372-ASO. Collectively, these findings demonstrate an oncogenic role for miR-372 in controlling cell growth and apoptosis through downregulation of TNFAIP1. This novel molecular basis provides new insights into the etiology of gastric cancer.

MicroRNA-373 is upregulated and targets TNFAIP1 in human gastric cancer, contributing to tumorigenesis.

The role of microRNAs (miRNAs) in regulating gene expression is currently an area of intense interest. Previous studies have shown that miRNA-372 plays crucial roles in gastric tumorigenesis by targeting the mRNA of tumor necrosis factor, α-induced protein 1 (TNFAIP1). The present study showed that miR-373 is upregulated in gastric adenocarcinoma tissue and gastric carcinoma cell lines when compared to normal gastric tissues. The overexpression of miR-373 in the gastric cancer cells increased cell proliferation. A bioinformatics search revealed a conserved target site within the 3' untranslated region (UTR) of TNFAIP1, an immediate-early response gene of the endothelium induced by TNF-α. The overexpression of miR-373 caused the suppression of a luciferase reporter containing the TNFAIP1 3'UTR in the HEK293 cells and reduced the levels of TNFAIP1 protein in the AGS cells. The mRNA levels of TNFAIP1 in the gastric cancer and normal gastric tissues were negatively correlated with the expression levels of miR-373 in these tissues. Moreover, the knockdown of TNFAIP1 had a similar effect to the overexpression of miR-373. The overexpression of TNFAIP1 may partly rescue the inhibition of proliferation caused by the inhibitor, miR-373-ASO. Taken together, these findings demonstrate an oncogenic role for miR-373, similar to that of miR-372, in controlling cell growth through the downregulation of TNFAIP1.

Caudatin induces cell apoptosis in gastric cancer cells through modulation of Wnt/ß-catenin signaling.

Caudatin has been reported to trigger apoptosis in several types of cancer cell lines. In the present study, we investigated whether caudatin has therapeutic value in gastric cancer and examined the effects of caudatin on the expression of ß-catenin in human gastric carcinoma cell lines. Here, we showed that caudatin treatment resulted in a dose- and time­dependent inhibition of proliferation of the gastric carcinoma cell lines. Cell cycle analysis demonstrated that caudatin induced G0/G1 arrest and downregulated CDK2 levels. In contrast, the expression of the p21 protein was increased. AGS cells treated with caudatin exhibited typical characteristics of apoptosis, which were accompanied by activation of caspase­3, ­8, ­9 and PARP. Western blot analysis and immunocytochemical staining showed that caudatin induced a reduction in ß­catenin expression and this reduction was due to proteosome-mediated degradation. This reduction in ß­catenin activation was due to the downregulation of its downstream targets cyclinD1 and c­MYC in all gastric carcinoma cell lines. Furthermore, we demonstrated that gastric adenocarcinoma tissues and AGS cells exhibited abnormal expression of miR­372. Additionally, caudatin downregulated the expression of oncomir miR­372 and miR­21, and upregulated tumor suppressor let­7a miRNA expression. These data revealed that inhibition of Wnt/ß­catenin signaling is a novel mechanism of action for caudatin during therapeutic intervention in gastric cancers.

[Expression, purification and enzymatic characterization of adenylate kinase of Thermus thermophilus HB27 in Escherichia coli].

OBJECTIVE: To clone the gene encoding adenylate kinase of Thermus thermophilus HB27, an extremely thermophilic bacterium, express the protein in Escherichia coil and study the enzymatic characterization. METHODS: The DNA fragment encoding adenylate kinase was obtained by PCR from the total DNA of Thermus thermophilus HB27 and cloned into the vector pET-28a. The recombinant plasmid was identified by PCR, restriction endonuclease digestion and sequence analysis. Enzymatic characterization of the expressed protein was carried out using spectrophotometric assays. RESULTS: The gene coding for adenylate kinase from Thermus thermophilus HB27 was cloned and the protein was overexpressed in Escherichia coli BL21(DE3). The optimum reactive pH and temperature for the enzyme were 8.5 and 90 degrees celsius;, respectively. The Km of the recombinant adenylate kinase for ADP was 68.6 micromol/L, with an V(max)ADP of 0.294 mmol/(L.min). Under the condition of environmental temperature at 70, 80, 90, or 100 degrees celsius; for 7 h, the recombinant adenylate kinase still retained the enzymatic activity with high thermostability. AP5A, a specific adenylate kinase inhibitor, inhibited the enzymatic activity of the protein by 70% at the concentration of 2.0 mmol/L, with a Ki value of 46.39 micromol/L for ADP. CONCLUSION: The gene coding for adenylate kinase of Thermus thermophilus HB27 has been successfully cloned and expressed in Escherichia coil, which provides the basis for potential use of the highly thermostable recombinant HB27 adenylate kinase.