New Analogues of Amides and Quinolinones Produced by Streptomyces sp. YIM S01983.

Streptomide (1), a new amide analogue, streptomynone (2), a new quinolinone, and ten known compounds including three aliphatic acids (3-5), two amides (6-7), four cyclic dipeptides (8-11), and an adenosine (12) were isolated from the fermentation broth of Streptomyces sp. YIM S01983 isolated from a sediment sample collected in Bendong Village, Huadong Town, Chuxiong, China. Their structures were determined by analysis of the 1D/2D-NMR and HR-ESI-MS spectra. Compound 12 presented weak antimicrobial activities against Candida albicans and Aligenes faecalis (MIC=64 µg/mL). Compounds 7 and 12 showed weak cytotoxic activity against MHCC97H.

Case report of avascular necrosis of the glans penis after PAE embolization.

BACKGROUND: Prostate artery embolization (PAE) is a relatively safe and effective alternative method for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia. The adverse events caused by PAE are primarily mild, including urinary tract infection, acute urinary retention, dysuria, fever, etc. Severe complications, such as nontarget organ embolism syndrome or penile glans ischemic necrosis, are rare. Here, we report a case of severe ischemic necrosis of the glans penis after PAE and review the literature. CASE PRESENTATION: An 86-year-old male patient was admitted to the hospital due to progressive dysuria with gross hematuria. The patient underwent placement of a three-way urinary catheter to facilitate continuous bladder flushing, hemostasis, and rehydration. After admission, his hemoglobin decreased to 89 g/L. After an examination, the diagnosis was benign prostatic hyperplasia with bleeding. During communication with the patient regarding treatment, he requested prostate artery embolization due to his advanced age and concomitant disease status. He underwent bilateral prostate artery embolization under local anesthesia. His urine gradually turned clear. However, on the 6th day after embolization, the glans gradually showed ischemic changes. On the 10th day, there was partial necrosis and blackening of the glans. The glans completely healed, and the patient was able to urinate smoothly on the 60th day after local cleaning and debridement, the administration of pain relief, anti-inflammatory and anti-infection agents, and external application of burn ointment. CONCLUSION: Penile glans ischemic necrosis after PAE is rare. The symptoms include pain, congestion, swelling, and cyanosis in the glans.

Comparative Study on Growth Index and Nutritional Quality of Female Chinese Mitten Crab Eriocheir sinensis Selected at Different Growth Periods in Rice-Crab Culture Systems.

Research was conducted on the growth performance and nutritional quality of Chinese mitten crabs (Eriocheir sinensis) during a 62-day growing period in a symbiotic coculture comprising rice and crab. Culture experiments were conducted in three rice fields of equal size (996 m2). On days 0 (July 15, D0), 15 (July 30, D15), 31 (August 15, D31), 46 (August 30, D46), and 62 (September 2, D62), tissue samples of 50 female E. sinensis were collected randomly from each rice field. The results showed that the serum growth hormone (GH) content and muscle ecdysone receptor (EcR) mRNA expression levels were higher in the D31 and D46 groups; the content of serum 20-hydroxyecdysone (20-HE) and the mRNA expression levels of retinoid X receptor (RXR), insulin-like growth factor 2 (IGF2), and chitinase (CHI) reached the maximum in the D31 group. Muscle crude protein content gradually increased; hepatopancreas crude protein and crude lipid content began to decrease after reaching the maximum value in the D0 and D15 groups, respectively; the contents of crude protein and crude lipid in the ovary significantly increased in the D46 and D62 groups (P < 0.05). The content of muscle essential amino acids (EAA) reached the maximum in the D46 group; the hepatopancreas EAA content began to decrease significantly in the D31 group (P < 0.05); and the EAA content of the ovary decreased significantly after reaching the maximum value in the D46 group (P < 0.05). The muscle contents of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), polyunsaturated fatty acids (PUFA), and n-3 polyunsaturated fatty acids (n-3PUFA) and the ratio of n-3 polyunsaturated fatty acids/n-6 polyunsaturated fatty acids (n3/n6) decreased significantly in the D31 group (P < 0.05); the hepatopancreas contents of EPA, PUFA, n-3PUFA, and n-6 polyunsaturated fatty acids (n-6PUFA) and the ratio of n3/n6 began to decrease after reaching the maximum value in the D31 group, ethyl behenate (21:0), tetracosanoic acid (24:0), DPA, and DHA contents were detected for the first time in the D31 group; the ovary PUFA, n-3PUFA contents, and n3/n6 ratio of the D46 and D62 groups were significantly lower than those of the D31 group (P < 0.05). During the experimental conditions described here, female E. sinensis raised in rice fields reached rapid growth from August 15 to August 30. Additionally, the nutritional quality of the female E. sinensis edible tissues (muscle, hepatopancreas, and ovary) began to decline after August 15, when sufficient nutrients such as protein, lipid, EAA, and PUFA should be provided to the female E. sinensis.

The immuneoreaction and antioxidant status of Chinese mitten crab (Eriocheir sinensis) involve protein metabolism and the response of mTOR signaling pathway to dietary methionine levels.

To study the effects of dietary methionine on growth performance, immunity, antioxidant capacity, protein metabolism, inflammatory response and apoptosis factors in Chinese mitten crabs (Eriocheir sinensis). Five diets with different methionine levels (0.63%, 0.85%, 1.06%, 1.25% and 1.47%) were fed to E. sinensis for 8 weeks. Results showed that in the 1.25% Met group, both growth performance and feed utilization were significantly increased. The crude protein content of crab muscle in the 1.06% and 1.25% Met groups was significantly higher than that in the control group. The immune and antioxidant enzyme activities, as well as gene expression levels of anti-lipopolysaccharide factor 1 (ALF1), Crustin-1, prophenoloxidase (proPO), cap 'n' collar isoform C (CncC) in 1.25% Met group were significantly higher than other groups. The activities of adenosine deaminase (ADA) and glutamate transaminase (GPT) in serum decreased first and then increased with the increase of methionine content, while the changes of ADA and GPT in hepatopancreas increased first and then decreased. 1.25% Met group exhibited significantly increased levels of GOT, GPT, and ADA compared to the control group. 1.25% Met diet group significantly up-regulated protein synthesis and anti-apoptotic factors, and significantly down-regulated inflammatory and pro-apoptotic factors in hepatopancreas. At 1.25% in the diet, methionine was found to boost E. sinensis growth, muscle protein deposition and immunity, as well as its antioxidant capacity. Combined with the above results, based on the expression of factors involved in the mammalian target of rapamycin (mTOR) signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway, it is proved that methionine can not only promote protein metabolism, improve feed utilization, but also alleviate the inflammatory response and apoptosis caused by oxidative stress in the body.

Oral Administration of Lactobacillus Casei Expressing Flagellin A Protein Confers Effective Protection against Aeromonas Veronii in Common Carp, Cyprinus Carpio.

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1ß, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.

IMPACT OF IMMUNE RESPONSE OF A PARASITIC BEETLE Dastarcus helophoroides ON ITS HOST BEETLE Monochamus alternatus.

Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one-seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses.

Nobiletin inhibited hypoxia-induced epithelial-mesenchymal transition of lung cancer cells by inactivating of Notch-1 signaling and switching on miR-200b.

Epithelial-mesenchymal transition (EMT) is an early step in the process of tumor metastasis. It is well known that tumor microenvironment affects malignancy in various carcinomas; in particular, that hypoxia induces EMT. Deregulated notch signaling also contributes a lot to the development of EMT in lung cancer. In this study, we investigated the use of Notch-1-inhibiting compound as novel therapeutic candidates to regulate hypoxia-induced EMT in lung cancer cells. According to previous screening, nobiletin was selected as a Notch-1 inhibitor. Hypoxia-induced EMT was characteristic of increased N-cadherin & vimentin expressions and decreased E-cadherin expressions. Treatment with nobiletin notably attenuated hypoxia-induced EMT, invasion and migration in H1299 cells, accompanied with reduced Notch-1, Jagged1/2 expressions and its downstream genes Hey-1 and Hes-1. Nobiletin treatment also promoted tumorsuppressive miR-200b level. Moreover, notch-1 siRNA prevented hypoxia-mediated cell migration and decreased Twist1, Snail1, and ZEB1/2 expressions, which are key EMT markers. Re-expression of miR-200b blocked hypoxia-induced EMT and cell invasion. Our findings suggest that downregulation of Notch-1 and reexpression of miR-200b by nobiletin might be a novel remedy for the therapy of lung cancer.

Interplay between mesenchymal stem cells and macrophages: Promoting bone tissue repair.

The repair of bone tissue damage is a complex process that is well-orchestrated in time and space, a focus and difficulty in orthopedic treatment. In recent years, the success of mesenchymal stem cells (MSCs)-mediated bone repair in clinical trials of large-area bone defects and bone necrosis has made it a candidate in bone tissue repair engineering and regenerative medicine. MSCs are closely related to macrophages. On one hand, MSCs regulate the immune regulatory function by influencing macrophages proliferation, infiltration, and phenotype polarization, while also affecting the osteoclasts differentiation of macrophages. On the other hand, macrophages activate MSCs and mediate the multilineage differentiation of MSCs by regulating the immune microenvironment. The cross-talk between MSCs and macrophages plays a crucial role in regulating the immune system and in promoting tissue regeneration. Making full use of the relationship between MSCs and macrophages will enhance the efficacy of MSCs therapy in bone tissue repair, and will also provide a reference for further application of MSCs in other diseases.

[Genetic analysis and prenatal diagnosis for non-syndromic hearing impairment].

OBJECTIVE: To detect genetic mutations underlying non-syndromic hearing impairment (NSHI) and establish a method for prenatal diagnosis. METHODS: Sixty six NSHI patients were included in this study. DNA was extracted from peripheral blood. Genetic mutations were detected by gene chip analysis and direct sequencing of GJB2 gene. For 7 pregnant women at high risk, prenatal genetic diagnosis was provided. RESULTS: Fourteen cases (21.21%) were found to have GJB2 mutations by both methods (homozygous 235delC mutation in 3 cases, homozygous 176del16 mutation in 2 cases, 235delC and 299delAT compound heterozygous mutation in 2 cases, 299delAT and 176del16 compound heterozygous mutation in 1 case, c.339T > G and 313del12bp compound heterozygous mutation 1 case, and 235delC heterozygous mutation in 5 cases). 13 (19.70%) had SLC26A4 mutations (IVS7-2 A >G homozygous mutation in 2 cases, IVS7-2 A > G homozygous mutation in 2 cases, IVS7-2 A > G and 2168A > G compound heterozygous mutation in 3 cases, 2168A>G heterozygous mutation in 3 cases, and IVS7-2 heterozygous mutation in 3 cases); and 3 had mtDNA12S rRNA mutation (1555A > G mutation in 2 cases, 1494C > T mutation in 1 case). Prenatal diagnosis suggested that 3 fetuses have carried a heterozygous mutation. Two fetuses were detected as normal and confirmed to have normal hearing after birth. Two fetuses were found to have carried compound mutations of GJB2. CONCLUSION: Gene chip combined with GJB2 gene analysis is an accurate and effective method for the diagnosis of NSHI. The results can facilitate accurate prenatal diagnosis.

Retroperitoneal tumor finally diagnosed as a bronchogenic cyst: A case report and review of literature.

BACKGROUND: Retroperitoneal bronchogenic cyst (RBC) is an extremely rare developmental abnormality. Most are benign tumors but malignant transformation is possible. Because of their anatomical position, RBCs are easily misdiagnosed as adrenal or pancreatic solid tumors on radiological evaluation. Here, we report a case of RBC, review the literature, and summarize some important features. CASE SUMMARY: A 49-year-old woman was incidentally found to have a retroperitoneal tumor during a physical examination. Enhanced computed tomography and laboratory evaluations, including routine blood examination, blood biochemistry, 24-h urine 17 ketones, 17 hydroxyls, adrenocortical hormone, serum potassium concentration, serum amylase, lipase, and epithelial tumor markers, revealed a moderate density, 54 mm × 40 mm mass with a clear boundary near the left adrenal gland. The were no abnormalities in the blood and urine values. Because the patient had a history of hypertension and the location of the mass was adjacent to the adrenal gland, it was initially diagnosed as a left adrenal tumor and was resected by retroperitoneal laparoscopy. However, the pathological examination after surgery confirmed it to be a bronchogenic cyst. CONCLUSION: Retroperitoneal laparoscopic surgery can be prioritized for symptomatic RBC patients. Conservative treatment is feasible for selected patients.

Construction of PD1/CD28 chimeric-switch receptor enhances anti-tumor ability of c-Met CAR-T in gastric cancer.

Chimeric antigen receptor (CAR) T cell is a promising method in cancer immunotherapy but faces many challenges in solid tumors. One of the major problems was immunosuppression caused by PD-1. In our study, the expression of c-Met in GC was analyzed from TCGA datasets, GC tissues, and cell lines. The c-Met CAR was a second-generation CAR with 4-1BB, cMet-PD1/CD28 CAR was c-Met CAR adding PD1/CD28 chimeric-switch receptor (CSR). In vitro, we measured the changes of different subgroups, phenotypes and PD-1 expression in CAR-T cells. We detected the secretion levels of different cytokines and the killing ability of CAR-Ts. In vivo, we established a xenograft GC model and observed the anti-tumor effect and off-target toxicity of different CAR-Ts. We find that the expression of c-Met was increased in GC. CD3+CD8+ T cells and CD62L+CCR7+ central memory T cells (TCM) were increased in two CAR-Ts. The stimulation of target cells could promote the expression of PD-1 in c-Met CAR-T. Compared with Mock T, the secretion of cytokines as IFN-γ, TNF-α, IL-6, IL-10 secreted by two CAR-Ts was increased, and the killing ability to c-Met positive GC cells was enhanced. The PD1/CD28 CSR could further enhance the killing ability, especially the long-term anti-tumor effect of c-Met CAR-T, and reduce the release level of IL-6. CAR-Ts target c-Met had no obvious off-target toxicity to normal organs. Thus, the PD1/CD28 CSR could further enhance the anti-tumor ability of c-Met CAR-T, and provides a promising design strategy to improve the efficacy of CAR-T in GC.

The relationship between the extracellular matrix and the angiogenesis and metastasis of laryngeal carcinoma.

AIMS: We studied the relationships between the growth, metastasis and angiogenesis of laryngeal carcinoma and extracellular matrix protein-1 (ECM1) and hyaluronan (HA). METHODS: Thirty-three cases of benign and malignant laryngeal tumors were included. Using immunohistochemical staining, we examined the expression of ECM1 and HA in tumors and metastatic lymph nodes and correlated it with counts of microvessel density. RESULTS: The expression of ECM1 (p = 0.004) and HA (p = 0.036) was significantly different between benign and malignant tumors. The expression of ECM1 was strongly associated with microvessel density (Spearman's rho = 0.513, p = 0.017), with the strongest expression present within caner nests. With respect to HA, expression was significantly associated with more advanced clinical TNM stage (Spearman's rho = 0.521, p = 0.015). The link was stronger in cases where metastasis or recurrence occurred than in those without complication (p = 0.014). No significant relationship was found between ECM1 and HA expression among malignant samples (Spearman's rho = 0.383, p = 0.087). CONCLUSIONS: ECM1 has a high association with the growth, metastasis and angiogenesis of laryngeal carcinoma. An increased level of HA expression was related to the process of laryngeal carcinogenesis. Specifically, carcinoma cells with high levels of HA secretion had a higher potential for metastasis. No significant relative expression of ECM1 and HA was found.

Auricle reconstruction with a nickel-titanium shape memory alloy as the framework.

OBJECTIVE: The objective of this study is to explore the biocompatibility and implantability of a nickel-titanium (NiTi) alloy in auricle reconstruction. METHODS AND MATERIALS: Twelve New Zealand rabbits underwent subcutaneous implantation with a NiTi alloy framework shaped like the human auricle under general anesthesia. The implant was inserted after skin expansion. Implant vascularization was evaluated at months 1, 3, 6, 9, and 12 after implantation by histologic analysis. Immunohistochemical methods were used to examine expression of vascular endothelial growth factor in tissue around the implant. The fibrovascular ingrowth rate of implants was determined by bone scanning using (99m)Tc-PYP. The surface of the NiTi alloy implant was examined microscopically with scanning electron microscopy. RESULT: The implant harvested showed only partial vascularization at 1 month and completely vascularized at 3 months. The amount of vascular endothelial growth factor-positive cells was markedly increased at 6 months and reached the highest number at 3 months. The fibrovascular ingrowth rate of implant was assessed by (99m)Tc-PYP bone scan using ratios of (99m)Tc-PYP activity in placement regions versus the contralateral normal region. One rabbit had exposure of the NiTi alloy framework as a result of overlying skin flap necrosis. It was rescued with animal skin without the complete removal of the framework. All the other rabbits tolerated the implant well, and there were no complications. CONCLUSION: The NiTi alloy implant represents an alternative implant for auricular reconstruction.

[Genotype and phenotype analyses of three families with autosomal recessive juvenile parkinsonism].

OBJECTIVE: To investigate the gene mutations and the clinical features of Chinese patients with autosomal recessive juvenile parkinsonism(AR-JP). METHODS: the polymerase chain reaction (PCR), DNA sequence analysis, and restriction enzyme digestion analysis were applied to check parkin gene mutations of 15 index patients from 15 families with AR-JP. RESULTS: Three families were detected to have parkin mutations. Two of them had heterozygous deletion mutations (202-203 del AG in exon 2, 1069-1074 del GTGTCC in exon 9) and another of them carried a heterozygous missense mutation [1422(T-->C) in exon 12]. Two of the mutations [1069-1074delGTGTCC and 1422(T-->C)] were not reported previously. There were six patients in the three families. Mean age at onset was 25.2+/-5.7 years, ranging from 18 to 31 years. The symptoms were under slow progression, diurnal fluctuation with sleep benefit, and hyperreflexia were relatively prominent. Response to levodopa was satisfactory. CONCLUSION: There are parkin mutations happened in Chinese patients with AR-JP. Patients with parkin mutations have distinct clinical features besides the common clinical features of Parkinson's disease.

Functional remobilization evaluation of the paralyzed vocal cord by end-to-side neurorrhaphy in rats.

OBJECTIVE: To investigate the value of end-to-side neurorrhaphy to treat vocal cord paralysis. STUDY DESIGN: A prospective study evaluating the effects of end-to-side neurorrhaphy to treat vocal cord paralysis by means of fiberoptic laryngoscopy and nerve electromyography. METHODS: Thirty Sprague-Dawley rats were divided into experimental group 1, experimental group 2, and a control group randomly. Right recurrent laryngeal nerve (RLN) was incised, and the distal end of the RLN was anastomosed to the right phrenic nerve by end-to-side neurorrhaphy in experimental group 1 or by end-to-end nerve anastomosis in experimental group 2, respectively. The adductor nerve branch of the right RLN was incised and anastomosed to the proximal end of the right ansa cervicalis nerve by end-to-end nerve anastomosis. Fiberoptic laryngoscopy and nerve electromyography were used to examine the vocal cord movement and nerve regeneration. RESULTS: Three months after operation, this effect of end-to-side neurorrhaphy created a significant difference compared with the end-to-end nerve anastomosis (P < .05). The end-to-side neurorrhaphy did not lead to vocal cord movement compared with end-to-end nerve anastomosis. CONCLUSION: Vocal cord paralysis cannot be treated by this microsurgical technique.

TGF-ß1-509C/T polymorphism and the risk of ESCC in a Chinese Han population.

BACKGROUND: The studies investigating whether transforming growth factor (TGF)-ß1-509C/T polymorphism is associated with the risk of ESCC is inconsistent. METHODS: The TGF-ß1-509C/T genotypes were determined by using a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay and DNA sequencing analysis. The differences in demographic variables and genotype distributions of TGF-ß1-509C/T polymorphism between ESCC patients and controls were calculated by Pearson's Chi square test. Associations between TGF-ß1-509C/T polymorphism genotypes and ESCC risk were estimated by OR and their 95% CIs computed using unconditional logistic regression model. RESULTS: There was a significant difference of TGF-ß1-509C/T polymorphism genotype distribution between ESCC group and control group (P<0.001). With the CC genotype as reference, the adjusted OR for CT genotype reached to 0.78 (95% CI: 0.65-0.89; P=0.041), and the adjusted OR for TT homozygous carriers was 0.52 (95% CI: 0.33-0.78; P=0.017). The T allele carriage also presented a lower risk for ESCC (adjusted OR=0.43; 95% CI, 0.29-0.71; P=0.009). CONCLUSION: TGF-ß1-509C/T polymorphism may contributes to ESCC susceptibility in Chinese population.

[Improved three-dimension reconstruction of serial frozen tissue sections of the temporal bone of guinea pigs].

OBJECTIVE: To improve the serial frozen tissue sections of the temporal bone of guinea pigs for better three-dimension reconstruction of the computer images. METHODS: The temporal bone of the guinea pigs was first decalcified by formate before embedding with lines marking the position of the specimen in glutin at low temperature. RESULT AND CONCLUSION: Thinner slices, shorter period and more accurate orientation were achieved to highlight the spatial features of the temporal bone, thus facilitating the anatomical study of the temporal bone.

Inactivation of recombinant human brain-type creatine kinase during denaturation by guanidine hydrochloride in a macromolecular crowding system.

In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k (2) value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8 × 10(-5) s(-1). The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment.

Optimized transformation of Streptomyces sp. ATCC 39366 producing leptomycin by electroporation.

Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam (-) ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×10(2) CFU/µg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-µF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives.

[Auricular reconstruction for microtia with modified Nagata method].

OBJECTIVE: To investigate a method for total reconstruction of auricle. METHODS: 90 patients (101 ears) with congenital microtia underwent two-stage operations for auricular reconstruction. The first stage involved fabrication and grafting of autologous costal cartilage, removing the remnant ear cartilage, embedding the framework into local flap of the mastoid region, transferring the remnant ear lobule flap to link to the inferior framework. The second stage was creating an auriculocephalic sulcus. The reconstruction was performed 4 - 12 months after the first surgery. Skin incision was made 5 mm lateral side of the posterior margin of the auricle. The ear framework carrying a thick ear fascia was separated from the side of the head, the frames of the costal cartilage banked at the first operation were harvested, shaved and transplanted to the posterior wall of the concha with sutures; adjust stand position and angle, so that made the ear shape, position, axis, close to the healthy ear, and auriculocephalic angle was slightly larger than the contralateral ear. Two random flap was designed with superior on the root of the helix and in the inferior-posterior direction of the inferior mastoid area, two flapes were elevated and transplanted to posterior auricular sulcus to cover the grafted cartilage. Skin graft was performed in the remaining raw surface. RESULTS: A total of 90 patients were operated, all of 101 constructed ears achieved satisfied or near satisfied shapes. Five cases of partial skin flap necrosis were caused by pedicle impairment. Exposure of cartilage framework happened in two cases. The auriculocephalic sulcus of four cases diminished after the second stage operation. Three month to two-year follow-up of 67 patients showed that the reconstructed ears were satisfied with the results, including good shapes and steady auriculocephalic angles. CONCLUSIONS: The method is a simple, safe and reliable method for total aural reconstruction.