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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[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.

[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.

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.

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.

Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems.

The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100 g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8 M GdnHCl and aid in the refolding of denatured-rHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment.

[Reconstruction of segmental ear helix defect with post-auricular skin flap].

OBJECTIVE: To explore the method of repairing segmental ear helix defect. METHODS: Twenty-one patients with segmental ear helix defect were repaired with post-auricular skin flap. In the first stage operation, ear helix defect was assessed, including the anterior and posterior area defect. According to the defect, post-auricular skin flap was designed and transplanted to repair the defect. Six weeks later, the pedicle of the post-auricular skin flap was cut off, elevated, and folded to form the helix. The secondary defect was directly sutured or repaired with skin graft. RESULTS: Twenty-one patients were treated with this method. In two to 12 months follow-up, all flaps survived and reconstructed ear helices were in good shape. The reconstructed ears were in symmetry to the healthy ones. CONCLUSION: The method is safe and effective for the correction of segmental ear helix defect.

Effects of macromolecular crowding on refolding of recombinant human brain-type creatine kinase.

In this study, we quantitatively measured the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000), dextran 70, and calf thymus DNA (CT DNA), on the refolding and aggregation of recombinant human brain-type creatine kinase (rHBCK) denatured by guanidine hydrochloride (GdnHCl). The results showed that there is more aggregation in the presence of either a single crowding agent or in a mixture of crowding agents than in the absence of crowding agents, especially in the presence of a mixture containing CT DNA and PEG 2000 (or dextran 70). In the presence of high concentrations of PEG 2000 (100 g/L), dextran 70 (100 g/L), and CT DNA (15 g/L), the refolding yield remarkably decreased from 70% to 20%, 52% and 57%, respectively. A remarkable decrease in the refolding yield and rate with mixed crowding agent containing CT DNA and PEG 2000 (or dextran 70) was also observed. In comparison to refolding in the presence of 100 g/L PEG 2000, the refolding yields and rates improved in the presence of a mixture of PEG 2000 and dextran 70. We speculate that the crowding agents can favor both correct folding and misfolding/aggregation of denatured-rHBCK. Though it is not known what combination of crowding agents most accurately reflects the physiological environment within a cell, we believe our study could contribute to the understanding of protein folding and the factors that contribute to proper conformation and function in the intracellular environment.

The Effect of D-(-)-arabinose on Tyrosinase: An Integrated Study Using Computational Simulation and Inhibition Kinetics.

Tyrosinase is a ubiquitous enzyme with diverse physiologic roles related to pigment production. Tyrosinase inhibition has been well studied for cosmetic, medicinal, and agricultural purposes. We simulated the docking of tyrosinase and D-(-)-arabinose and found a binding energy of -4.5 kcal/mol for theup-formof D-(-)-arabinose and -4.4 kcal/mol for thedown-form of D-(-)-arabinose. The results of molecular dynamics simulation suggested that D-(-)-arabinose interacts mostly with HIS85, HIS259, and HIS263, which are believed to be in the active site. Our kinetic study showed that D-(-)-arabinose is a reversible, mixed-type inhibitor of tyrosinase (α-value = 6.11 ± 0.98, K(i) = 0.21 ± 0.19 M). Measurements of intrinsic fluorescence showed that D-(-)-arabinose induced obvious tertiary changes to tyrosinase (binding constant K = 1.58 ± 0.02 M(-1), binding number n = 1.49 ± 0.06). This strategy of predicting tyrosinase inhibition based on specific interactions of aldehyde and hydroxyl groups with the enzyme may prove useful for screening potential tyrosinase inhibitors.

Analysis of long-term outcomes of double-strut bone graft for osteonecrosis of the femoral head.

OBJECTIVE: To analyze the long-term effect of double-strut bone graft for osteonecrosis of the femoral head (ONFH). METHODS: A total of 366 adult patients with ONFH in 466 hips underwent double-strut bone graft from March 1988 to January 1999. Of them, 186 patients with 206 hips and an average age of 32.2 years (range, 20-60 years) were followed up for more than five years, up to January 2006. Based on the Association Research Circulation Osseous (ARCO) classification, there were 36 hips in stage IIB, and 30, 40, 40, 32 and 28 in stage IIC, IIIA, IIIB, IIIC and IV, respectively. The functional results of affected hips were evaluated by the hundred forked method. RESULTS: Hip pain in all patients disappeared or alleviated greatly after the operation. The height of the femoral head improved to various extents, and the range of motion of the hip joint increased. The patients were followed up for 5-16 years, (average 10.5 years). The total scores increased significantly postoperatively (P < 0.01). The rate of excellent and good results was 83.3%, 80.0%, 75.0%, 65.0%, 40.6% and 28.6% in stage IIB, IIC, IIIA, IIIB, IIIC and IV, respectively (63.6% for the whole group). CONCLUSION: The long-term effect of double-strut bone graft for ONFH is satisfactory in relation to staging of ONFH. Favorable results can be expected in young ONFH patients in stage IIB, IIC, and IIIA, IIIB.