[The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma].

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 µg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

[Application of next-generation sequencing technology in prenatal screening and diagnosis].

Developed rapidly for more than ten years, next-generation sequencing (NGS) has derived a series of new technologies which are widely used clinically, such as noninvasive prenatal testing(NIPT), noninvasive prenatal testing-plus(NIPT Plus), copy number variation sequencing(CNV-seq), and exome sequencing(ES), and plays an important role in birth defects prevention. Moreover, with in-deep development, the superiority of NGS is gradually recognized by clinicians, but there are still many challenges in practical application process. This study specifically elaborates on the development status, technological breakthroughs and future prospects of NGS in the field of prenatal screening and prenatal diagnosis at home and abroad.

[Polarization of bone marrow-derived macrophages induced by recombinant Trichinella spiralis cysteine protease inhibitors in vitro].

OBJECTIVE: To investigate the regulatory role of recombinant Trichinella spiralis cysteine protease inhibitors (rTs-Cys) in induction of polarization of bone marrow-derived macrophages (BMDMs) in vitro. METHODS: BMDMs were captured and cultured in conditioned medium for 7 days. Then, mature BMDMs were harvested and assigned into four groups. Cells in Group A (negative control) were given 10 ng/mL IFN-γ combined with 100 ng/mL LPS, cells in Group B (positive control) were treated with IL-4 and IL-10 (at 10 ng/mL both), and cells in Group C (recombinant protein alone) were stimulated with 1 µg/mL rTs-Cys, while cells in Group D (protein co-culture) were simultaneously treated with 1 µg/mL rTs-Cys, 10 ng/mL IFN-γ and 100 ng/mL LPS. Cells and culture supernatant were collected 24 hour post-treatment, and the proportions of F4/80+, CD11b+, CD206+ and CD11c+ cells were detected by flow cytometry. The levels of interleukin IL-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10 and transforming growth factor-ß (TGF-ß) in the cell culture supernatant were measured by ELISA and the CD86+ and CD206+ phenotypes were identified by immunofluorescent staining. RESULTS: Flow cytometry detected no significant difference in the proportion of F4/80+ CD11b+ CD11c+ cells among the four groups (F = 46.184, P < 0.001), and a lower proportion of F4/80+ CD11b+ CD11c+ cells was seen in groups C and D than in group A (all P values < 0.001). There was a significant difference in the proportion of F4/80+ CD11b+ CD206+ cells among the four groups (F = 11.032, P < 0.001), and a greater proportion of F4/80+ CD11b+ CD206+ cells was seen in groups C and D than in group A (all P values < 0.01). Immunofluorescent staining showed higher CD206+ expression and lower CD86+ expression in groups C and D than in Group A. There were significant differences in the IL-6 and (F = 3.950, P < 0.001) and TNF-α (F = 205.827, P < 0.001) levels in the cell culture supernatants among the four groups, and significantly lower IL-6 and TNF-α levels were measured in groups C and D than in Group A (both P < 0.05). There were significant differences in the IL-10 and (F = 8.274, P < 0.001) and TGF-ß (F = 13.559, P < 0.01) levels in the cell culture supernatants among the four groups, and greater IL-10 and TGF-ß levels were measured in Group C than in Group A (both P values < 0.01). In addition, the TGF-ß level was significantly higher in Group D than in Group A (P < 0.05); however, there was no significant difference in the IL-10 level between groups D and A (P > 0.05). CONCLUSIONS: rTs-Cys may induce the polarization of BMDMs to antiinflammatory M2 macrophages in vitro and inhibit the activation of M1 macrophages.

[Expression of RAGE in asthmatic rats and the intervention of Roxithromycin].

Objective: To observe the expression of the Receptor of Advanced glycation end products (RAGE) in asthmatic rats, and explore the intervention of Roxithromycin. Methods: A total of 18 Specific Pathogen Free-class Brown Norway male rats were randomly divided into control group, asthma model group and Roxithromycin group, with 6 rats in each group. The asthmatic model was sensitized by intraperitoneal injection of Ovalbumin (OVA)+Al(OH)(3), and challenged with OVA. Rats in Roxithromycin group were given Roxithromycin 30 mg/kg 30 minutes before each challenge. Rats in control group and asthma model group were treated with equal volume of saline. The concentrations of RAGE and interleukin (IL)-4 in serum and bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent (ELISA); the pathological changes of lung tissues were observed by HE-staining; the thickness of airway wall and airway smooth muscle were measured by Image-Pro Plus; the relative expression of RAGE in lung tissues were detected by Western blot. Results: In asthma model group, the concentrations of RAGE and IL-4 in the serum and BALF were obviously higher than those in control group [(494±32) vs (327±45) ng/L; (32.4±5.8) vs (13.1±2.9) ng/L; (553±38) vs (399±56) ng/L; (37.8±3.4) vs (19.4±2.5) ng/L] (all P<0.01); in Roxithromycin group, the concentrations of RAGE and IL-4 in the serum and BALF were obviously lower than those in asthma model group [(438±18) vs (494±32) ng/L; (22.8±6.0) vs (32.4±5.8) ng/L; (444±42) vs (553±38) ng/L; (25.6±4.5) vs (37.8±3.4) ng/L] (all P<0.05). In asthma model group, the bronchial wall was thickened, the lumen was narrow, the mucosal wrinkles were significantly increased, edema appeared under the mucosa, and a large number of inflammatory cells infiltrated and aggregated in the bronchi, perivascular and alveolar spaces; the thickness of airway wall and airway smooth muscle were significantly increased than those in control group (P<0.01); in Roxithromycin group, airway inflammation and remodeling were alleviated compared with those in asthma model group (P<0.05). In asthma model group, the expression of RAGE in lung tissues were significantly increased than those in control group (P<0.01); in Roxithromycin group, the expression of RAGE were significantly decreased than those in asthma model group (P<0.01). There were positive correlations between the expression of RAGE and IL-4 in BALF and serum (r=0.782, 0.804, all P<0.01); there were positive correlations between RAGE and total white cell counts, eosinophil counts, smooth muscle thickness (r=0.897, 0.927, 0.860, all P<0.01). Conclusions: The increasing of RAGE in asthmatic rats are positively correlated with airway inflammation and airway remodeling. Roxithromycin may inhibit the development of asthma by reducing the expression of RAGE.

[Analysis of factors associated with failure of sequential mechanical ventilation in patients with acute exacerbation of chronic obstructive pulmonary disease complicated with respiratory failure].

Objective: By retrospectively analyzing the clinical data of patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with respiratory failure, to find the associated factors for failure of invasive-noninvasive sequential ventilation therapy. Methods: We conducted a cohort study of 64 patients with AECOPD complicated with respiratory failure, who were treated by invasive-noninvasive sequential ventilation. We took re-intubation, death or spontaneous discharge within 7 days following extubation as the endpoints. By comparing the APACHE Ⅱ score at admission into RICU, the ABCD grouping for COPD, the ratio of the diameter of the pulmonary artery to the diameter of the ascending aorta in chest CT(PA: A ratio), the levels of NT-proBNP, PaCO(2), PaO(2), the total number of leukocytes and the level of procalcitonin, we analyzed the differences between the success group(43 cases) and the failure group(21 cases). Results: The APACHE Ⅱ score at admission to RICU, the PA: A ratio, the level of NT-proBNP, the total leukocytes and the level of procalcitonin at admission to RICU showed significant differences in the univariate analysis(P<0.05). The average APACHE Ⅱ score was 23±4 in the success group and 27±6 in the failure group. The average PA: A ratio was 0.88±0.09 in the success group and 1.03±0.10 in the failure group. In the multivariate regression analysis, there were significant differences only in the APACHE Ⅱ score(P=0.02)and the PA: A ratio(P=0.012). The area under the ROC curve of the PA: A ratio for all patients was 0.894 and the cut-off value of the PA: A ratio was 0.98. Conclusion: The APACHE Ⅱ score and the PA: A ratio in CT are independent risk factors for failure of sequential ventilation in AECOPD patients complicated with respiratory failure. In particular, patients with a PA: A ratio greater than 0.98 have a higher risk of treatment failure.

[Clinicopathologic features of hepatocellular carcinoma patients surviving more than 10 years after radical hepatectomy].

Objective: To clarify the clinicopathologic features of hepatocellular carcinoma (HCC) patients survived more than 10 years after radical hepatectomy. Methods: Two hundreds and fifty-two patients who underwent curative resection for HCC between January 1999 and March 2006 at Department of Hepatopancreatobiliary Surgery, Affiliated Hospital of Qingdao University were included.There were 217 male cases and 35 female cases aging from 17 to 82 years with median age of (53.8±10.5)years. Followed by March 31 2016, clinicopathologic factors in 10-year survivors and patients who died within 10 years were compared by χ(2) test, Kaplan-Meier survival analysis and Cox proportional hazards model and the prognostic factors affecting survival were identified. Results: All patients were followed-up for 4.0 to 205.7 months with median time of 53.4 months. The 10-year overall survival rate was 26%, there were 62 cases(26.2%) who survived for more than 10 years after initial hepatectomy. In survival >10-year group, the paitents with ALT<40 U/L, gamma-glutamyl transpeptidase<64 U/L, albumin≥35 g/L, without liver cirrhosis and portal hypertension, Child-Pugh grade A, no blood transfusion, AFP≤20 µg/L, tumor size ≤5.0 cm, single tumor, high differentiation, TNM stage Ⅰ and TACE negative after resection were more than the patients in survival <10-year group (P<0.05). In multivariate analysis, Child-Pugh grade A, the tumor size ≤5.0 cm and TACE negative after resection were favorable independent factors associated with 10-year survival (P<0.05). Conclusion: Based on the results of the study, Child-Pugh grade A, tumor size ≤5.0 cm and TACE negative after resection at initial hepatectomy might be biologically favorable conditions for patients surviving more than 10 years.

[Tissue heterogeneity analysis of CGG-repeat mutation in two fragile X affected male fetuses].

OBJECTIVE: To compare the CGG-repeat-length and its methylation status in fetal tissues and to explicate the heterogeneity of CGG repeats. METHOD: Multiple tissues from a full mutation(August 2013) and a mosaic aborted fetus of 23-week gestation(May 2012) were collected and genomic DNA from these tissues was extracted. The CGG-repeat-length and methylation status in fetal tissues were determined by a combined strategy of Southern blotting and GC-Rich PCR. FMR1 expression was measured by real time PCR and Western blotting. RESULT: CGG-repeat-length in different tissues of each fetus was similar.A major methylated band in the full mutation range (540 CGG repeats) was detected in the brain, skin, testis and kidney tissues of Case 1. An unmethylated premutation band with 160 CGG repeats, and another two bands with 470 and 1 100 CGG repeats in the full mutation range were shown in the brain, skin, testis, lung, stomach, gut, liver, kidney, heart and blood of Case 2. However, the methylation status of CGG repeats in the mosaic fetus was heterogeneous among different tissues. The lowest premutation ratio was in the brain of the mosaic fetus compared with other tissues, and correspondingly FMR1 expression in its brain was minimum. CONCLUSION: This study clarify the tissue heterogeneity of CGG repeats and provides information for the genetic counseling and clinical diagnosis in fragile X syndrome. Based on the fact that the mosaic fetus' mother is a carrier of full mutation, it is speculated that the maternal CGG repeat has contracted before the differentiation of trilaminar germ disc.

MicroRNA-92b promotes hepatocellular carcinoma progression by targeting Smad7 and is mediated by long non-coding RNA XIST.

MicroRNA (miRNA) and long non-coding RNA (lncRNA) have been demonstrated to participate in the progression of many cancers. Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumors worldwide, while the molecular mechanisms underlying HCC tumorigenesis are not completely clear. In this study, we showed that miR-92b was significantly upregulated in tumor tissue and plasma of HCC patients, and its expression level was highly correlated with gender and microvascular invasion. Functionally, miR-92b could promote cell proliferation and metastasis of HCC in vitro and in vivo. Mechanistic investigations suggested that Smad7, which exhibited an inverse relationship with miR-92b expression in HCC, was a direct target of miR-92b and could reverse its effects on HCC tumorigenesis. Furthermore, long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) and miR-92b could directly interact with and repress each other, and XIST could inhibit HCC cell proliferation and metastasis by targeting miR-92b. Taken together, our study not only revealed for the first time the importance of XIST/miR-92b/Smad7 signaling axis in HCC progression but also suggested the potential value of miR-92b as a biomarker in the clinical diagnosis and treatment of HCC.

Antisense expression of Gossypium hirsutum UDP-glucuronate decarboxylase in Arabidopsis leads to changes in cell wall components.

UDP-glucuronate decarboxylase (UDP-xylose synthase; UXS, EC 4.1.1.35) is an essential enzyme of the non-cellulosic polysaccharide biosynthetic pathway. In the present study, using transient expression of fluorescently labeled Gossypium hirsutum UXS (GhUXS3) protein in onion epidermal cells, we observed that this protein was distributed in the cytoplasm. The GhUXS3 cDNA of cotton was expressed in an antisense orientation in Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. Homozygous plants showing down-regulation of UXS were analyzed with northern blots. Compared to the untransformed control, transgenic plant showed shorter roots, earlier blossom formation, and delayed senescence. Biochemical analysis indicated that levels of rhamnose, mannose, galactose, glucose, xylose, and cellulose were reduced in some of the down-regulated antisense plants. These results suggest that GhUXS3 regulates the conversion of non-cellulosic polysaccharides and modulates their composition in plant cell walls. We also discuss a possible cellular function for GhUXS in determining the quality of cotton fibers.

Identification of a novel mutation of the EDA gene in X-linked hypohidrotic ectodermal dysplasia.

This study aimed to identify the disease-causing mutation in the ectodysplasin A (EDA) gene in a Chinese family affected by X-linked hypohidrotic ectodermal dysplasia (XLHED). A family clinically diagnosed with XLHED was investigated. For mutation analysis, the coding region of EDA of 2 patients and 7 unaffected members of the family was sequenced. The detected mutation in EDA was investigated in 120 normal controls. A missense mutation (c.878T>G) in EDA was detected in 2 patients and 3 female carriers, but not in 4 unaffected members of the family. The mutation was not found in the 120 healthy controls and has not been reported previously. Our findings indicate that a novel mutation (c.878T>G) of EDA is associated with XLHED and adds to the repertoire of EDA mutations.

-173G/C polymorphism in the promoter of MIF is associated with hepatitis B virus infection in a Chinese Han population.

In addition to the host immune response, genetic and environmental factors play crucial roles in the manifestation of hepatitis B virus (HBV) infection. The macrophage migration inhibitory factor (MIF) -173G/C polymorphism (rs755622), located in the promoter region of MIF, may play integral roles in diverse processes, including the immune response. Thus, the MIF -173G/C polymorphism may influence the immune response to HBV during natural infection. We investigated whether the MIF -173G/C polymorphism was associated with susceptibility to HBV infection in a Chinese Han population. A total of 596 HBV infection cases and 612 age-matched controls were recruited for the study. Genotyping of the MIF -173G/C polymorphism was performed using the allele-specific polymerase chain reaction method. The frequencies of the alleles and genotypes in patients and controls were compared using the χ(2) test. Carriers of the variant C allele in MIF -173 G/C were at significantly higher risk of HBV infection than carriers of the wild-type allele (P = 0.032, odds ratio = 0.799, 95% confidence interval = 0.651-0.981). However, there was no significant difference in the distribution of MIF -173G/C genotypes between case and control groups in either population (P = 0.096, degrees of freedom = 2). Our findings indicate that the G to C base change in MIF -173 G/C confers an increased risk of development of HBV infection by altering the expression of MIF in our Chinese Han population.

Roxithromycin reduces the viability of cultured airway smooth muscle cells from a rat model of asthma.

OBJECTIVES: The purpose of this study was to investigate the effect of roxithromycin on apoptosis of airway smooth muscle cells (ASMCs) from a rat model of asthma and uncover signaling pathway underlying the cytotoxicity of roxithromycin. MATERIALS AND METHODS: ASMCs were isolated from a rat model of asthma and treated with or without roxithromycin for 48 h before parameter detection. Cell viability was assessed by WST-8 assay and flow cytometry after Annexin V/PI double staining. Changes in the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry using JC-1. Cytochrome C (Cyt c), cleaved Caspase-9/3 and P27 were evaluated by Western Blot. RESULTS: Incubation with roxithromycin reduced ASMCs proliferation and enhanced apoptosis in a dose-dependent manner. Flow cytometry revealed a loss of ΔΨm and Western Blot displayed Caspase-9/3 activation as well as Cyt c release from mitochondria to the the cytosol after the treatment of roxithromycin. In addition, P27 were more strongly expressed in AMSCs treated with roxithromycin compared with the control group. CONCLUSIONS: Roxithromycin induced apoptosis of ASMCs derived from a rat model of asthma in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway, involving the up-regulation of P27.

Morphology and innervation of the vestibular lagena in pigeons.

The morphological characteristics of the pigeon lagena were examined using histology, scanning electron microscopy, and biotinylated dextran amine (BDA) neural tracers. The lagena epithelium was observed to lie partially in a parasagittal plane, but was also U-shaped with orthogonal (lateral) directed tips. Hair cell planar polarities were oriented away from a central reversal line that ran nearly the length of the epithelium. Similar to the vertebrate utricle and saccule, three afferent classes were observed based upon their terminal innervation pattern, which include calyx, dimorph, and bouton fibers. Calyx and dimorph afferents innervated the striola region of the lagena, whereas bouton afferents innervated the extrastriola and a small region of the central striola known as the type II band. Calyx units had large calyceal terminal structures that innervated only type I hair cells. Dimorph afferents innervated both type I and II hair cells, with calyx and bouton terminals. Bouton afferents had the largest most complex innervation patterns and the greatest terminal areas contacting many hair cells.

A novel single-base deletion mutation of the RUNX2 gene in a Chinese family with cleidocranial dysplasia.

We identified a disease-causing mutation of the RUNX2 gene in a four-generation Chinese family affected with cleidocranial dysplasia (CCD). For mutation analysis, the coding region of RUNX2 was sequenced with DNA from two patients and three unaffected family members. The RUNX2 mutation was investigated in 50 normal controls by denaturing high pressure liquid chromatography. A heterozygous single-base deletion (c.549delC) of RUNX2, which predicts a termination site at the 185th codon and leads to a stop in the runt domain of RUNX2 protein, was detected in both patients but not in the three unaffected members of the family. This mutation was also not found in 50 controls and has not been reported previously. We demonstrated that a novel mutation (c.549delC) of RUNX2 is associated with CCD in a Chinese family, adding to the repertoire of RUNX2 mutations related to CCD.

Two novel NPHS1 mutations in a Chinese family with congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (CNF) is a lethal, autosomal recessive disorder mainly caused by mutations in the NPHS1 gene; it is found at a relatively high frequency in Finns. We investigated the disease-causing mutations in a Chinese family with CNF and developed a prenatal genetic diagnosis for their latest pregnancy. Mutation analysis was made of all exons and exon/intron boundaries of NPHS1 in the fetus, parents and 50 unrelated controls using PCR and direct sequencing. A heterozygous nonsense mutation within exon 20 (c.2783C>A) and a missense mutation within exon 17 (c.2225T>C) in NPHS1 were detected in the proband's father and mother, respectively, but were not found in the fetus or in 50 unrelated controls. Two novel mutations of c.2783C>A and c.2225T>C in NPHS1 were found to be causative in this Chinese CNF family with no known Finnish ancestry. The most recent sibling did not inherit these two mutations and hence was unaffected with CNF. Determining the cumulative number and ethnic distribution of known mutations can help expedite further study of the pathogenesis of CNF.

New technique for mouse oocyte injection via a modified holding pipette.

To improve mouse oocyte survival from intracytoplasmic sperm injection, the sharp tip of the injection pipette has been modified to have a flat end. Here, for the same goal but for a more convenient manipulation, a sharp injection pipette was kept whereas the holding pipette was modified to have a trumpet-shaped opening, which allows deeper injection into the oocyte as it is held. Mouse oocyte injection with mouse and human spermatozoa was performed at 37°C. For the injection of mouse oocyte with mouse sperm head, a significantly higher survival rate (83%) was achieved by utilizing the modified holding pipette than the conventional one (21%; P<0.001) and the fertilization rates were normal and comparable for both methods (82% versus 81%). A superior survival rate (82%) and acceptable normal fertilization rate (71%) were also achieved by utilizing the modified holding pipette for interspecies ICSI (injecting mouse oocyte with human spermatozoon). Taken together, by utilizing a holding pipette with a trumpet-shaped opening, acceptable rates of mouse oocyte survival and fertilization can be achieved using a sharp injection pipette under conditions usual for human oocyte injection.

Mutation L437P in the 2B domain of keratin 1 causes diffuse palmoplantar keratoderma in a Chinese pedigree.

Diffuse palmoplantar keratoderma (DPPK) is an autosomal dominant genodermatosis characterized by uniform hyperkeratosis of the palm and sole epidermis. This disorder can be caused by mutations in the genes keratin 1, keratin 9, keratin 16, desmoglein 1 and plakoglobin. Here we present a DPPK Chinese pedigree and identify the aetiology as a novel missense mutation, L437P, located in a highly conserved helix motif in domain 2B of KRT1. Functional analysis shows that overexpression of the L437P mutant in cultured cells leads to abnormal intermediate filament networks and filament aggregation. This gain-of-function mutation highlights the role of domain 2B in mediating filament assembly.

Graft-versus-host disease after liver transplantation: successful treatment of a case.

Graft-versus-host disease (GVHD) is a rare, serious complication after liver transplantation. The incidence is about 1% abroad, but because of difficulties in diagnosis and treatment, the mortality rate is high (80%). Up to now, 78 patients have been reported to have this complication after liver transplantation worldwide; despite various therapeutic programs, the mortality of GVHD after orthotopic liver transplantation (OLT) is still high. Herein we have reported successful treatment of a case of GVHD after liver transplantation.

Transglutaminase crosslinked gelatin as a tissue engineering scaffold.

Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.