Genetic and clinical analysis of a novel GLB1 gene variant in a Chinese patient with GM1-gangliosidosis / 中华医学遗传学杂志

OBJECTIVE@#To explore the genotype-phenotype correlation of a case with GM1-gangliosidosis caused by compound heterogenic variants in GLB1.@*METHODS@#Genomic DNA was extracted from peripheral blood samples from the patient and her parents. Trio-based whole-exome sequencing (WES) was performed for the family and suspected mutation was verified by Sanger sequencing.@*RESULTS@#The proband, a 2-year-3-month old Chinese girl, presented with psychomotor deterioration, absent speech, intellectual disabilities and behavior problem. Trio-based WES has identified compound heterozygosity for 2 variants in the GLB1 gene: NM_000404.2:c.1343A>T, p.Asp448Val and c.1064A>C, p.Gln355Pro (GRCh37/hg19),which was inherited from the mother and father, respectively. Homozygous or compound heterozygous pathogenic variants in GLB1, encoding β-galactosidase, are responsible for GM1-gangliosidosis,an autosomal recessive lysosomal storage disorder characterized by variable degrees of neurodegeneration and skeletal abnormalities. The p.Asp448Val variant has been classified as pathogenic for GM1 gangliosidosis in medical literatures for the reason that functional studies demonstrated that expression of the p.Asp448Val variant in COS-1 cells resulted in no detectable β-galactosidase activity compared to wild type GLB1. The p.Gln355Pro variant has not been reported in literatures or database. The variant is highly conserved residue (PM1), and was not found in either the Genome Aggregation Database or the 1000 Genomes Project (PM2) and was predicted to have a deleterious effect on the gene product by multiple in silico prediction tools (PP3). Next, the β-galactosidase activity of the patient's peripheral blood leukocytes was determined by fluorescent method. The result was 0.0 nmol/mg. It showed that the p.Gln355Pro variant also resulted in loss of β-galactosidase activity, thus the variant was classified into clinical pathogenic variant.@*CONCLUSION@#Our study expands the mutational spectrum of the GLB1 gene and provides genetic counseling for the family.

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene / 生物工程学报

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Biochemical characterization of three Aspergillus niger ß-galactosidases

Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.

Nurses’ attitudes regarding the importance of families in pediatric nursing care / Actitudes de enfermeras sobre la importancia de las familias en los cuidados de enfermería en pediatría / Atitudes de enfermeiros em face da importância das famílias nos cuidados de enfermagem em pediatria

Affective, cognitive and behavioral components affect nurses´ attitudes to include families in the care processes. The purpose of this study was to investigate the attitudes of nurses about the importance of including families in nursing care. Data collection was performed in pediatric and maternal-child unit of a Brazilian university hospital. A sample of 50 nurses completed the Portuguese version of the instrument Families’Importance in Nursing Care-Nurses’ Attitudes (FINC-NA). The results indicated that nurses have supportive attitudes regarding families participation in nursing care. Attitudes of lower support for involving families in nursing care were found among nurses with older age, more time in the profession and who had no previous contact with contents related to Family Nursing. The application of the instrument in other contexts of assistance may help to illuminate important aspects of the challenges to implementing a family-centered approach in clinical practice.



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El propósito de este estudio fue identificar las actitudes de los enfermeros sobre la importancia de incluir a las familias en el cuidado de enfermería. La recolección de datos se llevó a cabo en las unidades de pediatría y materno-infantil de un hospital universitario brasileño. Una muestra de 50 enfermeras completó la versión en portugués del el instrumento Families’ Importance in Nursing Care-Nurses’ Attitudes (FINC-NA). Los resultados indicaron las puntuaciones más altas en dimensiones indicativas de actitudes de apoyo a la participación de las familias en el cuidado. Enfermeras con más tiempo en la profesión y que no tenían conocimiento previo de enfermería de familia tuvieron puntuaciones que indican actitudes de menor apoyo para involucrar a las familias en el cuidado de enfermería. La aplicación de este instrumento en otro tipo de asistencia contextos puede ayudar a iluminar aspectos importantes de los desafíos para la implementación de un enfoque centrado en la familia, en la práctica clínica.
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Objetivo Identificar as atitudes dos enfermeiros sobre a importância de incluir as famílias nos cuidados de enfermagem. Método Estudo de abordagem quantitativa descritiva, cuja coleta de dados foi realizada em unidades de pediatria e materno-infantil de um hospital universitário brasileiro. Uma amostra de 50 enfermeiros completou a versão em português da escala Families Importance in Nursing Care-Nurses Attitudes (FINC-NA). Resultados Indicaram escores mais elevados em dimensões indicativas de atitudes de apoio sobre a participação das famílias no cuidado de enfermagem. Enfermeiros com mais tempo na profissão e que não tiveram conhecimento prévio de enfermagem da família apresentaram escores indicativos de atitudes de menor apoio para envolver as famílias no cuidado de enfermagem. Conclusão A aplicação desse instrumento em outros contextos de assistência poderá contribuir para iluminar importantes aspectos relacionados aos desafios para a implementação de uma abordagem centrada na família na prática clínica e subsidiar o desenvolvimento de pesquisas mais amplas.
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Gangliosidosis generalizada tipo 1 / Generalized gangliosidosis type 1

La gangliosidosis generalizada tipo 1 es una enfermedad de acúmulo lisosomal producida por mutaciones en el gen de la enzima b-galactosidasa, caracterizada fundamentalmente por toma del sistema nervioso central, la visceromegalia, disostosis ósea y dimorfismo facial. Se presenta el caso de un lactante varón, hijo de padres no consanguíneos, de 5 meses de edad, Apgar 6/8 debido a hipoxia neonatal, con historia de múltiples ingresos por enfermedad diarreica e infecciones respiratorias. Es remitido a la Consulta de Genética Clínica por retardo del desarrollo psicomotor, macrocráneo y hepatomegalia, además de máculas hipercrómicas en piel. En el examen físico se encontraron evidencias de una posible afectación por enfermedad metabólica lisosomal. Entre las enfermedades a descartar estaban la galactosialidosis, de características clínicas similares, y la enfermedad de Morquio, con diferente presentación clínica pero idéntico defecto enzimático

Generalized or GM 1 gangliosidosis is a lysosomal storage disease caused by mutations in the enzyme b-galactosidase gene, mainly characterized by affecting the central nervous system, visceromegalia, osseous dysostosis and facial dimorphism. This is the case of a male nursling born to non-consanguineous parents, 5 months of age, Apgar index of 6/8 due to neonatal hypoxia, with a history of several admissions to hospital because of diarrheal disease and respiratory infections. He was referred to the clinical genetic service since he presented with retarded psychomotor development, macrocrania and hepatomegalia, in addition to hyperchromic skin spots. The physical exam found evidence of possible effects by lysosomal metabolic disease. Among the diseases to be ruled out for the diagnosis were galactosialidosis of similar clinical characteristics and Morquio B disease with different clinical presentation but identical enzymatic deficiency

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAitransfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated beta-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.

Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

HilA gene expression in SCFAs adapted and inorganic acid challenged Salmonella enterica serovar typhimurium.

Salmonella enterica serovar typhimurium (S. typhimurium) encounters short chain fatty acids (inorganic acids containing propionate, butyrate and acetate) in the intestine as well as in food preservatives. Short chain fatty acids (SCFAs) exposed organisms have been reported to offer resistance to organic acid resulting into enhanced virulence. However, the role of hilA (hyper invasive loci) gene expression has not been assessed in this context. In the present study, S. typhimurium was grown under SCFAs stress condition simulating the in vivo environment and hilA gene expression was evaluated. The gene expression was measured by beta-galactosidase (beta-gal) assay using a hilA-lacZY fusion strain and calculated as Miller units. hilA gene expression was found to be significantly higher in the SCFAs exposed cells than the unexposed ones, after 2 hrs and 4 hrs of exposure. However, no significant difference was observed between the activities at 2 hrs and 4 hrs. It indicates that hilA gene gets expressed by 2 hrs and persists till 4 hrs at least. The beta-gal activity was also measured in the unadapted / SCFAs adapted organisms followed by acid shock for 1 hr. The gene expression was also found to be higher in the SCFAs adapted--acid (pH 3) challenged as compared to the unadapated acid challenged organisms suggesting that SCFAs adaptation may induce organic acid tolerance by modulating the hilA response. This observation indicates that hilA may be the additional gene contributing to acid resistance and thereby increasing virulence of the organism after SCFAs adaptation.

Effectiveness of liposomes to transfect livestock fibroblasts

The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.

Catheter-based adenovirus-mediated local intravascular gene delivery of a soluble TGF-beta type II receptor using an Infiltrator in porcine coronary arteries: efficacy and complications

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor b1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdT beta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdT beta-ExR or adenovirus expressing b-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RTPCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify b-galactosidase. Both soluble TGF-beta receptor and b-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdT beta-ExR of 5x10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.

Catheter-based adenovirus-mediated local intravascular gene delivery of a soluble TGF-beta type II receptor using an Infiltrator in porcine coronary arteries: efficacy and complications

Enhanced extracellular matrix (ECM) accumulation is an important finding in human restenotic arterial neointima after angioplasty. Transforming growth factor b1(TGF-beta1) is known to regulate the synthesis and turnover of a variety of ECM components, and may play an important role in restenosis. Recombinant adenoviral vector expressing an ectodomain of the TGF-beta type II receptor fused to the human immunoglobulin Fc portion (AdT beta-ExR) inhibits the action of TGF-beta probably either by adsorbing TGF-beta or by acting as a dominant negative receptor. We carried out a catheter-based local adenovirus mediated gene delivery using an Infiltrator in porcine coronary arteries to know the pattern of gene expression, efficacy and procedural complications. Twenty four coronary arteries in 13 pigs were used for intravascular gene delivery by intramural injection with either AdT beta-ExR or adenovirus expressing b-galactosidase (AdCALacZ). Direct immunofluorescent staining and reverse transcription polymerase chain reaction (RTPCR) were used for detection of type II TGF-beta receptor and its mRNA respectively. X-Gal histochemistry was performed to identify b-galactosidase. Both soluble TGF-beta receptor and b-galactosidase were expressed locally in the media and adventita at injected arterial segments without any significant dissemination to remote area. Intravascular gene transfection performed with various titer of each adenoviral vector showed that AdT beta-ExR of 5x10(8) pfu and AdCALacZ of 2.5 x 10(8) pfu were the minimum titer for the expression of each transgene. Infiltration of CD3 positive T cells was detected by immunohistochemical staining in the area of each transgene expression, and tends to decrease over time after gene delivery. Pathological study of 24 treated arteries showed complications such as disruption of external elastic lamina with hemorrhage (n = 4), minimal disruption of internal elastic lamina and endothelial layer, and medial thickening. In conclusion, catheter-based local intravascular gene delivery of adenoviral vector is feasible and effective in a selected artery, but must be undertaken with caution due to possible lethal complications. Local delivery of soluble TGF-beta type II receptor in this way may provide an effective intravascular gene therapy to inhibit TGF-beta signal pathway without any significant systemic side effect.

Biolistic-mediated gene expression in guinea pigs and cattle tissues in vivo

Foreign genes were introduced and expressed in vivo in guinea pigs and cattle utilizing a new hand-held device based on high-pressure helium gas to accelerate DNA-coated microparticles. Guinea pigs were used to evaluate the physical parameters to introduce and express the exogenous DNA. The best conditions were applied to conduct bombardments in cattle. The results showed a high frequency of gene expression in all the bombarded cattle. This procedure could be used to study the immune responses in cattle and in a wide variety of animals through genetic immunization.