Construction of cell factories for production of valencene in Saccharomyces cerevisiae / 药学学报

Valencene, a kind of sesquiterpenoid with a citrus flavor, is mainly found in Valencia orange and is commonly used in cosmetics and food additives, as well as industrial synthetic nootkatone. In this study, synthetic biology was used to create a Saccharomyces cerevisiae cell factory to produce valencene. Fistly, valencene synthase gene (CnVS) from Callitropsis nootkatensis was inserted into the chromosome of the chassis strain YTT-T5. The resulting strain VAL-01 could produce 1.1 mg·L-1 valencene. Protein fusion technique was used, different valencene synthases were compared and the copy number of key genes was adjusted, yielding valencene to 436.4 mg·L-1. Then, knocking-out the transcription factor ROX1 resulted in valencene improvement by 17.4%. Moreover, the induction system of galactose was regulated, transcription factor PDR3 and INO2 were overexpressed. The engineered strain VAL-10 could produce 2 798.6 mg·L-1 valencene by high cell density fermentation method (nearly 2 500 times higher than VAL-01). This study provides a basis for green production of valencene.

Association of depressive symptoms, Internet addiction and insomnia among medical students in Anhui Province / 中国学校卫生

Objective@#To investigate the status of insomnia, Internet addiction, and depressive symptoms among medical students and to analyze the effect of Internet addiction on insomnia and the mediating role of depressive symptoms, in order to provide a basis for the development of targeted interventions and measurements for medical students.@*Methods@#A stratified whole group sampling method was used to select full-time college students from three medical universities in Anhui Province. The Chinese version of Insomnia Severity Index (ISI), Internet Addiction Test (IAT) scale and 9-item Patient Health Questionnaire (PHQ-9) were used to evaluate the symptoms of insomnia, Internet addiction and depressive in students. A multivariate Logistic regression analysis was used to explore the factors influencing insomnia among medical students and to analyze the relationship between insomnia with Internet addiction and depressive symptoms, respectively.@*Results@#The overall rate of Internet addiction was 49.5%, depressive symptoms was 39.5%, insomnia was 18.6%. High academic stress, and the presence of surrounding people diagnosed with COVID-19 were associated with a higher risk of insomnia ( P <0.05). The higher the level of Internet addiction (mild, OR =2.60; moderate/severe, OR =4.21) and depression. (mild, OR =6.35; moderate/severe, OR =19.32), the higher the risk of insomnia. Mediated effect analysis showed that Internet addiction had a direct predictive effect ( β =0.02, P <0.05) on insomnia and also indirectly affected insomnia through depression (indirect effect=0.07，95% CI =0.06-0.08).@*Conclusion@#The detected rates of insomnia, Internet addiction and depressive symptoms are high among medical students in Anhui Province, and Internet addiction and depressive symptoms are risk factors for insomnia, which should be given more attention and appropriate interventions when necessary to improve their physical and mental health.

Rectangular method: a modified technique for sampling the ischemic border zone in a rat model of transient middle cerebral artery occlusion

To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae / 中国中药杂志

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Depolymerized konjac glucomannan: preparation and application in health care / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Konjac glucomannan (KGM) is a water-soluble polysaccharide obtained from the roots and tubers of konjac plants. Recently, a degraded product of KGM, depolymerized KGM (DKGM), has attracted attention because of its low viscosity, improved hydrophily, and favorable physiological functions. In this review, we describe the preparation of DKGM and its prebiotic effects. Other health benefits of DKGM, covering antioxidant and immune activity, are also discussed, as well as its safety. DKGM could be a candidate for use as a tool for the treatment of various diseases, including intestinal flora imbalance, and oxidative- and immune-related disorders.

Renoprotection Provided by Dipeptidyl Peptidase-4 Inhibitors in Combination with Angiotensin Receptor Blockers in Patients with Type 2 Diabetic Nephropathy / 中华医学杂志(英文版)

Background@#Treatment with the dipeptidyl peptidase-4 inhibitors (DPP4i) and angiotensin receptor blockers (ARBs) in patients with type 2 diabetic nephropathy (DN) has not been well characterized. This study aimed to assess the renoprotection of this combined treatment in DN patients.@*Methods@#A total of 159 type 2 DN patients from 2013 to 2015 were enrolled retrospectively from a prospective DN cohort at the National Clinical Research Center of Kidney Diseases, Jinling Hospital (China). Fifty-seven patients received DPP4i and ARB treatment, and 102 patients were treated with ARBs alone. All patients were followed up for at least 12 months. Statistical analyses were performed using Stata version 12.0.@*Results@#There were no significant differences at baseline for age, sex, body mass index, duration of diabetes, fasting blood glucose (FBG), hemoglobin A1c (HbA1c), and estimated glomerular filtration rate (eGFR) between the two groups. Antihypertensive and antidiabetic medication use was similar in each group except calcium channel antagonists (P = 0.032). No significant changes in FBG and HbA1c were observed in the two groups after treatment. The eGFR decreased slower in the DPP4i + ARB group than in the ARB group at 12 months (Δ12 months: -2.48 ± 13.86 vs. -6.81 ± 12.52 ml·min·1.73m, P = 0.044). In addition, proteinuria was decreased further in the DPP4i + ARB group than in the ARB group after 24 months of treatment (Δ24 months: -0.18 [-1.00, 0.17] vs. 0.32 [-0.35, 0.88], P = 0.031). There were 36 patients with an eGFR decrease of more than 30% over 24 months. After adjusting for FBG, HbA1c, and other risk factors, DPP4i + ARB treatment was still associated with a reduced incidence of an eGFR decrease of 20% or 30%.@*Conclusions@#The combined treatment of DPP4i and ARBs is superior to ARBs alone, as evidenced by the greater proteinuria reduction and lower eGFR decline. In addition, the renoprotection of DPP4i combined with ARBs was independent of glycemic control.

Influence of amlodipine besylate combined metoprolol on serum inflammatory factor levels and cardiac function in aged patients with EH complicated CHF / 心血管康复医学杂志

Objective:To explore influence of amlodipine besylate tablet combined metoprolol on serum inflammatory factor levels and cardiac function in aged patients with essential hypertension(EH)complicated chronic heart failure(CHF).Methods:A total of 98 aged EH+CHF patients treated in our hospital from Jan 2014 to Oct 2015 were se-lected.Patients were randomly and equally divided into metoprolol group(received metoprolol based on routine treatment)and combined treatment group(received amlodipine besylate tablet based on metoprolol group),both groups were treated for six months.Serum levels of interleukin(IL)-1,IL-6 and tumor necrosis factor(TNF)-α, left ventricular end-diastolic dimension(LVEDd),left ventricular end-systolic dimension(LVESd)and left ventric-ular ejection fraction(LVEF)before and six months after treatment,and therapeutic effect were measured and compared between two groups.Results:Compared with before treatment,after six-month treatment,there were significant reductions in serum levels of IL-1,IL-6,TNF-α,LVEDd and LVESd,and significant rise in LVEF in two groups,P=0.001 all;compared with metoprolol group after six-month treatment,there were significant reduc-tions in serum levels of IL-1[(16.21 ± 1.45)ng/L vs.(12.34 ± 1.32)ng/L],IL-6[(46.21 ± 5.32)ng/L vs. (36.42 ± 4.51)ng/L],TNF-α[(23.12 ± 2.14)ng/L vs.(19.25 ± 2.41)ng/L],LVEDd[(51.68 ± 4.74)mm vs. (42.31 ± 4.25)mm]and LVESd[(46.27 ± 4.78)mm vs.(39.06 ± 4.35)mm],and significant rise in LVEF [(43.26 ± 4.37)% vs.(52.95 ± 5.18)%]in combined treatment group,P=0.001 all.Total effective rate of com-bined treatment group was significantly higher than that of metoprolol group(93.88% vs.79.59%),P= 0.037. There was no significant difference in incidence rate of adverse reactions between two groups,P=0.505. Conclu-sion:Amlodipine besylate combined metoprolol possesses significant therapeutic effect on aged EH + CHF patients, which may reduce serum inflammatory factor levels,relieve ventricular remodeling and improve cardiac function.

Repalr of large-segmental femoral defects using lentivirus-mediated bone morphogenetic protein 2/bone marrow mesenchymal stem cells/demineralized bone matrix / 中国组织工程研究

BACKGROUND: Our research team has confirmed that compared to the adenoviral vector, transfection by lentiviral vector to rabbit bone marrow mesenchymal stem cells (BMSCs) is more effective that the expression of enhanced green fluorescent protein (EGFP)/bone morphogenetic protein 2 (BMP-2) can be persistent for a longer term. But further investigations are needed to explore whether BMSCs transfected with hBMP-2 through lentivirus combined with demineralized bone matrix (DBM) can promote bone defect repair. OBJECTIVE: To evaluate the effect of lentivirus-mediated hBMP-2/BMSCs/DBM (LV-hBMP-2/BMSCs/DBM) on the repair of large-segmental femoral defects and to explore the new treatments for large-segmental femoral defects. METHODS: Large-segmental bone defect models were made in the right femur of 48 New Zealand white rabbits by cutting the middle femoral bone and steel plate fixation. Then, animal models were randomly divided into four groups (n=12 per group) and implanted with nothing (control), DBM, hBMP-2/DBM, and LV-hBMP-2/BMSCs/DBM. Three rabbits from each group were sacrificed at 2, 4, 8 and 12 weeks after surgery to evaluate the repairing effect of femoral defects through hematoxylin-eosin staining, biomechanical analysis and radiological examination. RESULTS AND CONCLUSION: X-ray results revealed that osteotylus formed in all the four groups to different extents, and Lane - Sandhu X-ray scores were ranked as follows: control group < DBM group < hBMP-2/DBM group < LV-hBMP-2/BMSCs/DBM group (P < 0.05). Findings from the three-point bending test showed that the maximum load of the LV-hBMP-2/BMSCs/DBM group was significantly higher than that of the hBMP-2/DBM group, but is still lower than that of the normal femur at 8 and 12 weeks after modeling (P < 0.05). Hematoxylin-eosin staining results revealed that a few trabecular bones arranged disorderedly and a large amount of fibrous tissues in the control group; the DBM scaffold was basically degraded in the DBM group presenting with partially disordered trabecular bones and a large amount of fibrous tissues; the trabecular bones in the bone defect area were basically connected into line to start the shaping of the cortical bone, and recanalization of the medullary cavity was insignificant in the hBMP-2/DBM group; new cortical bone formed in the bone defect area and the medullary cavity was recanalized in the LV-hBMP-2/BMSCs/DBM group. These findings indicate that LV-hBMP-2/BMSCs/DBM can produce a large amount of calluses, promote formation of new cortical bone, and promote bone conduction, bone induction and osteogenesis after implantation into the bone defect; and this material has good repairing effect on large-segmental femoral defects of rabbits.

The Effects ofNesfatin-1 in the Paraventricular Nucleus on Gastric Motility and Its Potential Regulation by the Lateral Hypothalamic Area in Rats / 现代生物医学进展

Objective:The current study investigated the effects of nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) on gastric motility and the regulation of the lateral hypothalamic area (LHA).Methods:The projection of nerve ?ber and expression of nesfatin-1 were observed by retrograde tracing and fluo-immunohistochemistry staining;The nuclei microinjection and nuclei electrical stimulation,extracellular discharges of single unit neuron were used to observe the effects of nesfatin-1 on the GD neurons;Gastric motility recording in vivo were used to monitor the effects of nesfatin-1 on the amplitude of constriction and frequency of gastric motility in conscious rats.Results:Nesfatin-1 inhibited the majority of the GD-E neurons(1.97± 0.12 Hz vs.1.15± 0.07 Hz) and excited GD-I neurons (1.74± 0.10 Hz vs.3.04± 0.18 Hz) in the PVN,which were weakened by oxytocin receptor antagonist H4928 (GD-E:1.38± 0.08 Hz,P＜0.05 vs.nesfatin-1;GD-I:2.49± 0.15 Hz,P＜0.05 vs.nesfatin-1).Gastric motility experiments showed that administration ofnesfatin-1 in the PVN decreased gastric motility.Retrograde tracing and immunofluorescent staining showed that nucleobindin-2/nesfatin-1 and fluorogold double-labeled neurons were observed in the LHA.Electrical LHA stimulation excited the firing rate of GD-responsive neurons (GD-E:2.06± 0.12 Hz vs.4.23± 0.21 Hz,GD-I:1.61± 0.09 Hz vs.4.83± 0.25 Hz) in the PVN.Pre-administration of an antinucleobindin-2/nesfatin-1 antibody in the PVN strengthened gastric motility,decreased GD-E neurons (1.74± 0.10 Hz vs.3.04± 0.18 Hz) and excited the discharging of the GD-I neurons(4.15± 0.18 Hz vs.4.83± 0.25) induced by electrical stimulation of the LHA.Conclusion:Nesfatin-1 in the PVN could serve as an inhibitory factor to inhibit gastric motility,which might be regulated by the LHA.

A Prospective Study of the Surgical Outcome of Simple Uvulopalatopharyngoplasty (UPPP), UPPP Combined With Genioglossus Advancement or Tongue Base Advancement for Obstructive Sleep Apnea Hypopnea Syndrome Patients With Multilevel Obstruction

The editorial board and publication ethics committee of the CEO decided to retract this paper from our journal.

Clinical analysis on gastrointestinal stromal tumor patients with hepatic metastases: 24 cases report / 肿瘤研究与临床

Objective To analyze the clinical characters,treatment effects and influential factors of gastrointestinal stromal tumor patients with hepatic metastases,and to explore the reasonable clinical treatment protocols.Methods The clinical data of 24 gastrointestinal stromal tumor patients with hepatic metastases who were admitted to Peoples Liberation Army Hospital from April 2007 to April 2010 were retrospectively analyzed.Results The 1-,2-,3-and 4-year cumulative survival rates of hepatic metastases patients with gastrointestinal stromal tumors were 91％,72 ％,22 ％ and 6 ％,respectively.The 1-,2-and 3-year survival rates of the patients treated with imatinib after operation were 91％,70 ％,28 ％,respectively,the median survival time was 26 months,the mortality rate was 27.3 ％ (3/11),and 9 cases got the tumor complete elimination.The 1-,2-and 3-year survival rates of the patients treated without imatinib after operation were 90 ％,68 ％,18 ％,respectively,the median survival time was 24 months,the mortality rate was 14.3 ％ (2/13),and 12 cases got the tumor complete elimination.The recurrence rate after complete resection was 28.6 ％(4/14),and the mortality rate was 7.1％ (1/14).The mortality rate after incomplete resection was 40 ％ (4/10).Conclusion Surgical treatment of eliminating the primary and metastatic lesion,and adjuvant therapy of imtinib could prolong the survival time,reduce postoperative recurrence rate and improve the quality of life,which are the effective methods for the treatment of the gastrointestinal stromal tumor patients with hepatic metastases.

A Prospective Study of the Surgical Outcome of Simple Uvulopalatopharyngoplasty (UPPP), UPPP Combined With Genioglossus Advancement or Tongue Base Advancement for Obstructive Sleep Apnea Hypopnea Syndrome Patients With Multilevel Obstruction

OBJECTIVES: To investigate the surgical outcomes of different uvulopalatopharyngoplasty (UPPP). METHODS: All subjects underwent overnight polysomnography and were evaluated using the Epworth sleepiness scale (ESS), the Quebec sleep questionnaire and the snoring scale at the baseline and 3 and 12 months following operation. The primary endpoint was the overall effective rate representing the sum of the surgical success rate and effective rate. RESULTS: The overall effective rate at 12 months post surgery was 55.6% for simple UPPP, 95.8% for UPPP+GA, and 92.3% for UPPP+TBA. The surgical success rate at 3 and 12 months postoperation for UPPP+GA or UPPP+TBA was significantly higher than simple UPPP (P<0.05). Marked improvement was observed in all patients in the snoring scale score and the ESS score 3 and 12 months following surgery compared to the baseline (P<0.05 in all). CONCLUSION: UPPP, UPPP+GA, and UPPP+TBA are all effective in improving the surgical outcome of obstructive sleep apnea hypopnea syndrome (OSAHS) patients with multilevel obstruction. UPPP+TBA appears to be the most effective in treating OSAHS patients.

Cause of death related to medical disputes in Yancheng area: a study of 60 autopsy cases / 法医学杂志

OBJECTIVE@#To summarize the pattern and main characteristics of fatal cases related to medical disputes in Yancheng area.@*METHODS@#Sixty fatal cases of medical disputes were retrospectively analyzed to elucidate the annual incidence, characters of distribution of hospitals, gender and age of the decedents, types of diseases, and cause of death.@*RESULTS@#Among 60 fatal cases, most cases happened in health clinics of county, township and village. There were more males than females. The major medical specialties involved included internal medicine, surgery, gynecology and pediatrics, with the internal medicine specialty having the highest incidence.@*CONCLUSION@#Police institutions have advantages in investigation of these cases in their jurisdictions, which could enhance the ability of local medicolegal examination.

Surgical treatment of iatrogenic traumatic biliary stricture / 中华消化外科杂志

Iatrogenic traumatic biliary stricture is one of the difficult points in the biliary surgery,and operation is the only definitive treatment. The operative opportunity,surgical procedure and techniques are important for the prognosis.From January 1998 to December 2011,173 patients with iatrogenic traumatic stricture were admitted to the Eastern Hepatobiliary Surgery Hospital. According to the Bismuth classification of traumatic biliary stricture,10 patients were in type Ⅰ,22 in type Ⅱ,87 in type Ⅲ,38 in type Ⅳ and 16 in type Ⅴ.Excision of the traumatic stricture with end-to-end anastomosis was performed on 19 patients.Of the 173 patients,154 were treated by Roux-en-Y duodenojejunostomy, and 8 of them received additional hemihepatectomy or partial hepatectomy.A total of 155patients were followed up,with a median time of 74 months,the total excellent and good rate was 94.8％ ( 147/155 ).Surgery is the most effective therapy for iatrogenic traumatic biliary stricture. Optimal timing, reasonable surgical methods, strictly following the principle of biliary surgery and perfect operative skills are key points for a better prognosis.

Proliferation of bone marrow mesenchymal stem cells induced by cyclic mechanical stretch mediated with ERK signal molecules / 医用生物力学

Objective To investigate effects of the cyclic stretch on rat bone marrow mesenchymal stem cells (rBMSCs) proliferation and the related signal molecules. Methods The cyclic mechanical stretching apparatus was used to study the effects of the cyclic stretch on the growth of rBMSCs with different parameters. The expression of c fos was measured by RT PCR at mRNA level and the levels of phosphorylated extracellular signal regulated kinase 1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2) and c-fos proteins were detected by Western blotting. Results The proliferation of rBMSCs could be stimulated by the cyclic stretch, and the number of cells was increased to 1.4 times compared with that of the control group at 1 Hz, 10% strain for 15 min and then being static for 6 h. Moreover, the expressions of c-fos mRNA and protein levels were increased significantly after the stimulation of the stretch. On the other hand, it was found that the cyclic stretch could remarkably enhance the phosphorylation of ERK1/2, but did not influence the level of t-ERK1/2. After treated with PD98059, the proliferation of rBMSCs stimulated by the cyclic stretch was inhibited prominently in a dose dependent manner and the expressions of p-ERK1/2, as well as c-fos at gene and protein levels were sharply down regulated. Conclusions ERK1/2 signal molecules play an important role in regulating the cyclic stretch on the proliferation of rBMSCs and transcription factors may take part in the regulations during this progress.

Shikani™ Seeing Optical Stylet-aided tracheal intubation in patients with a large epiglottic cyst / 中华医学杂志(英文版)

Large epiglottic cysts can block the glottis, leading to serious consequences. This condition presents a challenge in terms of airway management for anesthesiologists during induction of anesthesia. We report the use of a Shikani™ Seeing Optical Stylet combined with a Macintosh laryngoscope to aid tracheal intubation in seven patients with large epiglottic cysts. Use of this technique can avoid cyst rupture and allow smooth, safe intubation.

Status of iron metabolism and erythropoietic proliferation in children with various genotypes of thalassemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the status of iron metabolism and erythropoietic proliferation in children with various genotypes of thalassemia.</p><p><b>METHODS</b>Serum concentrations of ferritin (SF), transferrin receptor (sTfR) and erythropoietin (EPO) were measured in 158 children with thalassemia. The differences in the concentrations of the three indices among children with different genotypes of thalassemia were compared. The correlations of the hemoglobin level with sereum SF, sTfR and EPO levels were assessed.</p><p><b>RESULTS</b>Among the 158 children with thalassemia, 52(32.9%) were diagnosed with alpha-thalassemia minor, 27(17.1%) with HbH disease, 59(37.4%) with beta-thalassemia minor, 13(8.2%) with beta-thalassemia major, and 7(4.4%) with combining alpha beta thalassemia. The SF levels in children with HbH disease or beta-thalassemia major were significantly higher than those in the other thalassemia groups (P<0.01). The sTfR levels in children with beta-thalassemia major were the highest when compared with those in the other thalassemia groups (P<0.05). The EPO levels in children with beta-thalassemia major were also the highest when compared with those in the other thalassemia groups (P<0.01). There was a negative correlation between hemoglobin and EPO levels in children with HbH disease (r=-0.656, P<0.01) and beta-thalassemia major (r=-0.641; P<0.05).</p><p><b>CONCLUSIONS</b>The status of iron metabolism and erythropoietic proliferation is different in children with different genotypes of thalassemia. A combined measurement of SF, sTfR and EPO may reflect the status of erythropoietic proliferation.</p>

Cytotoxicity of human doppel protein and Dpl-like protein PrPΔ32-121 to SH-SY5Y cells / 西安交通大学学报(医学版)

Objective To observe the biological activities of human doppel (Dpl) protein transiently expressed and Dpl-like protein PrPΔ32-121 on a human neuroblastoma cell line SH-SY5Y. Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrPΔ32-121 fragment were generated by PCR. The expression and location of Dpl and PrPΔ32-121 post-transfection were observed by IFA. The cytotoxicity was measured by MTT analysis. Cellular apoptosis was investigated by flow cytometry and Western blot. Results Both Dpl and PrPΔ32-121 protein were expressed and mainly located on the cell membrane. Remarkable cytotoxicity was detected on SH-SY5Y cells after 24 h transfection. Meanwhile, more Annexin V/PI positively-stained cells as well as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrPΔ32-121 expressing plasmids. Conclusion Dpl protein transiently expressed and PrPΔ32-121 can lead to the similar neural cytotoxicity, probably triggering the cell apoptosis program.

Studies on the mutation and polymorphism of the TPMT gene in Chinese children with acute leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the allelic frequencies and distribution of single-nucleotide polymorphisms within the coding region (cSNPs) of thiopurine S-methyltransferase gene (TPMT) in Chinese children with acute leukemia (AL) and healthy controls, in order to provide genetic references for individual chemotherapy for AL patients by studying the relationship between the cSNP in human TPMT and chemotherapeutic effect of thiopurine drugs.</p><p><b>METHODS</b>The bone marrow samples from 53 children with AL and peripheral blood samples from 115 healthy children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in the TPMT gene by reverse transcriptase-polymerase chain reaction (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. The distribution of each genotype was evaluated.</p><p><b>RESULTS</b>Two novel heterozygote mutations, 210C>T (C70C, silent) and 622T>C (F208L), were identified in the coding region of the TPMT in a single sample, respectively. The mother of the child with mutation 622T>C was confirmed as the same genotype by DGGE and sequencing (NCBI_ss accession numbers 107796292 and 107795933). Two known polymorphisms, 474T>C (silent) and 719A>G (T240C), were identified. The allelic frequencies were 14.2%, 2.83% and 17.0%, 3.04% in the AL children and control children respectively, with the total allelic frequencies of 16.2% (first reported in the Chinese Han population) and 2.99% respectively. No association with susceptibility to disease was observed.</p><p><b>CONCLUSION</b>Two novel mutations and two known polymorphisms were identified in Chinese children by RT-PCR-DGGE combined with DNA sequencing, which provides the first step to identify genetic markers for predicting variability in response to and toxicity of thiopurine drugs.</p>