Clinical features and possible founder mutation of the 8bp duplication mutation in the SLC4A11 gene causing corneal dystrophy and perceptive deafness in three South American families

Background: Corneal Dystrophy and Perceptive Deafness (CDPD) or Harboyan syndrome is an autosomal recessive rare disorder, characterized by congenital corneal opacities and progressive sensorineural hearing loss, which usually begins after the second decades of life. This study reports the ophthalmic, audiological and genetic features, in five CDPD affected patients from three Chilean families.

Materials and Methods: Five individuals affected with CDPD from three unrelated Chilean families were clinically and genetically examined. To evaluate a putative founder mutation 7 SNPs were analyzed in the three families, an Argentinian patient (carrier of the same mutation previously reported) and 87 Chilean controls.

Results: The ophthalmic symptoms in the five patients were bilateral and symmetric, starting before one year of age, and visual acuity varied from 0.1 to 0.3. In all cases, hearing loss began over 8 years old. The sequence of the 19 exons of SLC4A11 gene of all the affected patients exhibited homozygous eight nucleotide sequence duplication (c.2233_2240dup TATGACAC, p.(Ile748Metfs*5)) at the end of exon 16. All the affected patients of the three families were homozygous for a haplotype composed of five SNPs and covering 4,1 Mb. The same haplotype was present in one allele of the heterozygous Argentinean patient and has a frequency of 2.76% in Chilean population.

Conclusions: The five CDPD patients were homozygous for the same mutation in the SLC4A11 gene. Haplotype analysis of all the affected, including the case reported from Argentina was in accordance with a founder mutation.     

Regioselectivity significantly impacts microsomal glucuronidation efficiency of R/S-6, 7-, and 8-hydroxywarfarin

1. Coumadin (R/S-warfarin) metabolism plays a critical role in patient response to anticoagulant therapy. Several cytochrome P450s oxidize warfarin into R/S-6-, 7-, 8-, 10, and 4′-hydroxywarfarin that can undergo subsequent glucuronidation by UDP-glucuronosyltransferases (UGTs); however, current studies on recombinant UGTs cannot be adequately extrapolated to microsomal glucuronidation capacities for the liver.

2. Herein, we estimated the capacity of the average human liver to glucuronidate hydroxywarfarin and identified UGTs responsible for those metabolic reactions through inhibitor phenotyping. There was no observable activity toward R/S-warfarin, R/S-10-hydroxywarfarin or R/S-4′-hydroxywarfarin.

3. The observed metabolic efficiencies (V_max/K_m) toward R/S-6-, 7-, and especially 8-hydroxywarfarin indicated a high glucuronidation capacity to metabolize these compounds.

4. UGTs demonstrated strong regioselectivity toward the hydroxywarfarins. UGT1A6 and UGT1A1 played a major role in R/S-6- and 7-hydroxywarfarin glucuronidation, respectively, whereas UGT1A9 accounted for almost all of the generation of the R/S-8-hydroxywarfarin glucuronide.

5. In summary, these studies expanded insights to glucuronidation of hydroxywarfarins by pooled human liver microsomes, novel roles for UGT1A6 and 1A9, and the overall degree of regioselectivity for the UGT reactions.

     

The Effect of PEI-Mediated E1A on the Radiosensitivity of Hepatic Carcinoma Cells

Objective: The study was undertaken to investigate the effects of polyethyleneimine (PEI)-mediated adenovirus 5 early region 1A (E1A) on radiosensitivity of human hepatic carcinoma cell in vitro and to disclosure the underlying mechanism. Materials and Methods: Human hepatic carcinoma SMMC-7721 cell line was transfected with E1A gene using PEI vector. Untransfected cells (SMMC-7721 group), cells transfected with blank-vector (SMMC-7721-vect group), and cells transfected with E1A gene (SMMC-7721-E1A group) were treated with 6 MV X-ray irradiation at doses of 0, 1, 2, 4, 8 and Gy, respectively. Radiosensitivity was determined by MTT assay and quantified by calculating the cell survival rate. Cell-cycle distribution and apotosis rate were monitored by flow cytometry. Results: The survival rate of SMMC-7721-E1A was significantly lower than that of SMMC-7721 cell. Apoptosis rate of SMMC-7721-E1A group was significantly higher than that of SMMC-7721group (P<0.01).The ratio of S stage in cell cycle of SMMC-7721-E1A was significantly lower than that in SMMC-7721 cell. The ratio of G2/M stage in cell cycle of SMMC-7721-E1A was significantly higher than that in SMMC-7721 cell (P<0.01). Conclusion: PEI could transfect E1A gene into hepatic carcinoma cells PEI-mediated E1A could effectively enhance radiosensitivity of hepatic carcinoma cells which may be related to its effects on apoptosis promoting leading to S phase suppression and G2/M phase arrest.     

Cytoprotective autophagy induction by withaferin A in prostate cancer cells involves GABARAPL1

Withaferin A (WA) is a naturally occurring steroidal lactone with proven cancer chemopreventive activity in preclinical models of different cancers including prostate adenocarcinoma. Previously we compared the RNA-seq data from control and WA-treated 22Rv1 human prostate cancer cells to identify mechanistic targets of this phytochemical. The Gene Ontology pathway analysis of the RNA-seq data revealed significant upregulation of genes associated with autophagy upon WA treatment in 22Rv1 cells. In this study, we extended these findings to investigate the mechanism underlying WA-induced autophagy. Initially, we confirmed autophagy induction by WA treatment by transmission electron microscopy using three prostate cancer cell lines (LNCaP, 22Rv1, and PC-3). Fourteen common genes altered by 8- and 16-hour exposure to WA were identified from human autophagy PCR array and these results were consistent with the RNA-seq data. Two key autophagy markers (LC3BII and SQSTM1) were robustly increased in WA-exposed LNCaP, 22Rv1, and PC-3 cells as determined by immunoblotting, and this effect was elevated in the presence of autophagy inhibitor bafilomycin A1 (BafA1). BafA1 treatment augmented WA's cytotoxicity and subsequently its proapoptotic potential. WA treatment induced GABARAPL1 (ATG8L) protein expression in all three cell lines and its knockdown by RNA interference attenuated WA-mediated apoptosis. WA-induced autophagy was not affected in the presence of an antioxidant (EUK134). Taken together, the present study reveals that WA-mediated autophagy is cytoprotective and mediated by GABARAPL1.     

内镜活检胃癌组织中MUC3A,MUC13表达及与病理参数和预后的关系

目的:探讨内镜活检胃癌组织中黏蛋白3A(MUC3A)、黏蛋白13(MUC13)表达及与病理参数和预后的关系。方法:选取2013年1月至2014年12月南阳市中心医院肿瘤科收治的接受内镜活检的116例患者胃癌组织和癌旁正常组织进行分析,采用免疫组织化学法检测所有组织样本中MUC3A,MUC13表达,观察二者表达情况,并分析二者与临床病理参数及预后的关系。结果:胃癌患者组织MUC3A,MUC13阳性率分别为68.97%和66.38%,高于正常组织的32.76%和7.76%(P<0.05);胃癌组织中MUC3A,MUC13表达与年龄、性别、肿瘤大小无关(P>0.05),与组织分化、浸润深度、TNM分期、淋巴结转移有关(P<0.05);随访5年结果显示:胃癌组织中MUC3A阳性患者中位生存时间为(40.50±1.95)个月,低于阴性患者的(48.67±2.51)个月(P<0.05),MUC3A阳性患者预后5年生存率为32.50%,低于阴性患者的55.56%(P<0.05);MUC13阳性患者中位生存时间为(38.52±1.92)个月,低于阴性患者的(49.06±2.71)个月(P<0.05),MUC13阳性患者预后5年生存率为30.00%,低于阴性患者的61.11%(P<0.05);COX多因素回归分析显示:TNM分期,淋巴结转移,MUC3A,MUC13是影响胃癌预后的独立危险因素(P<0.05)。结论:MUC3A,MUC13在胃癌组织中呈高表达,且与临床病理参数及预后密切相关,二者均有望作为判定胃癌发生发展及预后评估的参考指标。     

Synthesis and Characterization of Solution Processable Poly(pyrazolines)

Solution‐processable poly(dibenzalacetone) and poly(dibenzalcyclohexanone) are prepared by the condensation of 1,4‐dialkoxy‐bisbenzaldehyde and acetone/cyclohexanone using classic Claisen–Schmidt condensation. The prepared polymers are readily soluble in common organic solvents due to the presence of alkoxy chains present on the aromatic ring. The availability of α,β‐unsaturated ketone on the backbone is readily functionalized with hydrazine hydrate to obtain poly(pyrazoline acetate), a nonconjugated polymer on the backbone, which shows enhanced emission characteristics on comparison with the pristine polymer and small molecule analogues. The highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) of levels of poly(pyrazolines) has a band gap of 1.54 eV as calculated from cyclic voltammetry and UV–vis studies. Uniform thin film is obtained by spin casting as 1% solution on chlorobenzene and annealing under vacuum. Atomic force microscopy analysis shows fine morphology of the thin film. I–V characterization of the film shows low turn‐on voltage of 5.3 V. HOMO–LUMO is calculated by density functional theory and the result suggests that these molecules have great potential toward polymer organic light‐emitting diode applications.     

Early production of IL-17A by γδ T cells in the trachea promotes viral clearance during influenza infection in mice

The innate immune response generated against influenza infection is critical for the inhibition of viral dissemination. The trachea contains different types of innate immune cells that protect the respiratory tract from pathogen invasion. Among them, γδ T cells have the ability to rapidly generate large amounts of pro-inflammatory cytokines to preserve mucosal barrier homeostasis during infection. However, little is known about their role during the early phase of influenza infection in the airways. In this study, we found that, early after infection, γδ T cells are recruited and activated in the trachea and outnumber αβ T cells during the course of the influenza infection that follows. We also showed that the majority of the recruited γδ T cells express the Vγ4 TCR chain and infiltrate in a process that involves the chemokine receptor CXCR3. In addition, we demonstrated that γδ T cells promote the recruitment of protective neutrophils and NK cells to the tracheal mucosa. Altogether, our results highlight the importance of the immune responses mediated by γδ T cells.     

Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via c AMP-PKA signal pathway

AIM: To test our hypothesis that activation of protein kinase A(PKA) signal pathway by β-adrenergic agonist plays an important role in the protecting of cultured retinal pigment epithelial(RPE) cells against the hydroxychloroquine(HCQ) toxicity. METHODS: Cultured human RPE cells were treated with 1) HCQ, 2) HCQ with salbutamol(a β2-adrenergic receptor agonist), and 3) HCQ with salbutamol and a PKA inhibitor, and compared these to 4) untreated cells(controls). After treated for 24 h, cell vacuolation, cells viability, PKA and PKA kinase activity levels were determined by the measurement of the size of vacuoles using Image J software, the cell counting with a dye-exclusion testing, Western blot and PKA kinase detection, respectively. RESULTS: Cell vacuolation and cell death of cultured RPE cells were significantly increased by the treatment of HCQ. Salbutamol significantly elevated PKA and PKA activity levels and this was associated with the inhibition of the vacuolation and cell death. The PKA inhibitor significantly decreased the PKA levels and eliminated the protective effects of salbutamol on HCQ-treated RPE cells. CONCLUSION: The PKA pathway plays an important role in the protective effects of β2-adrenergic agonist on the RPE cells against HCQ toxicity. These findings reveal a novel potential strategy against HCQ retinopathy by treatment with PKA activating medications.