Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

Abstract Background The CHD7 (Chromodomain Helicase DNA binding protein 7) gene encodes a member of the chromodomain family of ATP-dependent chromatin remodeling enzymes. Mutations in the CHD7 gene are found in individuals with CHARGE, a syndrome characterized by multiple birth malformations in several tissues. CHD7 was identified as a binding partner of PBAF complex (Polybromo and BRG Associated Factor containing complex) playing a central role in the transcriptional reprogramming process associated to the formation of multipotent migratory neural crest, a transient cell population associated with the genesis of various tissues. CHD7 is a large gene containing 38 annotated exons and spanning 200 kb of genomic sequence. Although genes containing such number of exons are expected to have several alternative transcripts, there are very few evidences of alternative transcripts associated to CHD7 to date indicating that alternative splicing associated to this gene is poorly characterized. Findings Here, we report the cloning and characterization by experimental and computational studies of a novel alternative transcript of the human CHD7 (named CHD7 CRA_e), which lacks most of its coding exons. We confirmed by overexpression of CHD7 CRA_e alternative transcript that it is translated into a protein isoform lacking most of the domains displayed by the canonical isoform. Expression of the CHD7 CRA_e transcript was detected in normal liver, in addition to the DU145 human prostate carcinoma cell line from which it was originally isolated. Conclusions Our findings indicate that the splicing event associated to the CHD7 CRA_e alternative transcript is functional. The characterization of the CHD7 CRA_e novel isoform presented here not only sets the basis for more detailed functional studies of this isoform, but, also, contributes to the alternative splicing annotation of the CHD7 gene and the design of future functional studies aimed at the elucidation of the molecular functions of its gene products.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Abstract Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

Birth defects in newborns and stillborns: an example of the Brazilian reality

Abstract Background This study constitutes a clinical and genetic study of all newborn and stillborn infants with birth defects seen in a period of one year in a medical school hospital located in Brazil. The aims of this study were to estimate the incidence, causes and consequences of the defects. Methods For all infants we carried out physical assessment, photographic records, analysis of medical records and collection of additional information with the family, besides the karyotypic analysis or molecular tests in indicated cases. Result The incidence of birth defects was 2.8%. Among them, the etiology was identified in 73.6% (ci95%: 64.4-81.6%). Etiology involving the participation of genetic factors single or associated with environmental factors) was more frequent 94.5%, ci95%: 88.5-98.0%) than those caused exclusively by environmental factors (alcohol in and gestational diabetes mellitus). The conclusive or presumed diagnosis was possible in 85% of the cases. Among them, the isolated congenital heart disease (9.5%) and Down syndrome (9.5%) were the most common, followed by gastroschisis (8.4%), neural tube defects (7.4%) and clubfoot (5.3%). Maternal age, parental consanguinity, exposure to teratogenic agents and family susceptibility were some of the identified risk factors. The most common observed consequences were prolonged hospital stays and death. Conclusions The current incidence of birth defects among newborns and stillbirths of in our population is similar to those obtained by other studies performed in Brazil and in other underdeveloped countries. Birth defects are one of the major causes leading to lost years of potential life. The study of birth defects in underdeveloped countries should continue. The identification of incidence, risk factors and consequences are essential for planning preventive measures and effective treatments.

Electrophysiological study of hearing in full-term small-for-gestational-age newborns

PURPOSE: To describe the Brainstem Auditory Evoked Potential (BAEP) results of full-term small-for-gestational-age newborns, comparing them to the results of full-term appropriate-for-gestational-age newborns, in order to verify whether the small-for-gestational-age condition is a risk indicator for retrocochlear hearing impairment. METHODS: This multicentric prospective cross-sectional study assessed 86 full-term newborns - 47 small- (Study Group) and 39 appropriate-for-gestational-age (Control Group - of both genders, with ages between 2 and 12 days. Newborns with presence of transient evoked otoacoustic emissions and type A tympanometry were included in the study. Quantitative analysis was based on the mean and standard deviation of the absolute latencies of waves I, III and V and interpeak intervals I-III, III-V and I-V, for each group. For qualitative analysis, the BAEP results were classified as normal or altered by analyzing these data considering the age range of the newborn at the time of testing. RESULTS: In the Study Group, nine of the 18 (38%) subjects with altered BAEP results had the condition of small-for-gestational-age as the only risk factor for hearing impairments. In the Control Group, seven (18%) had altered results. Female subjects from the Study Group tended to present more central alterations. In the Control Group, the male group tended to have more alterations. CONCLUSION: Full-term children born small or appropriate for gestational age might present transitory or permanent central hearing impairments, regardless of the presence of risk indicators.

Hypersensitivity reactions to non beta-lactam antimicrobial agents, a statement of the WAO special committee on drug allergy

Antibiotics are used extensively in the treatment of various infections. Consequently, they can be considered among the most important agents involved in adverse reactions to drugs, including both allergic and non-allergic drug hypersensitivity [J Allergy Clin Immunol 113:832–836, 2004]. Most studies published to date deal mainly with reactions to the beta-lactam group, and information on hypersensitivity to each of the other antimicrobial agents is scarce. The present document has been produced by the Special Committee on Drug Allergy of the World Allergy Organization to present the most relevant information on the incidence, clinical manifestations, diagnosis, possible mechanisms, and management of hypersensitivity reactions to non beta-lactam antimicrobials for use by practitioners worldwide.

Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Measurement and model calculation of the temperature dependence of ligand-to-metal energy transfer rates in lanthanide complexes

Temperature dependent transient curves of excited levels of a model Eu3+ complex have been measured for the first time. A coincidence between the temperature dependent rise time of the 5D0 emitting level and decay time of the 5D1 excited level in the [Eu(tta)3(H2O)2] complex has been found, which unambiguously proves the T1→5D1→5D0 sensitization pathway. A theoretical approach for the temperature dependent energy transfer rates has been successfully applied to the rationalization of the experimental data.