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.
Asians/genetics* ; China ; Female ; G(M1) Ganglioside ; Gangliosidosis, GM1/genetics* ; Humans ; Mutation ; beta-Galactosidase/genetics*

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.
Cloning, Molecular ; Escherichia coli/genetics* ; Genes, Reporter/genetics* ; Genetic Vectors/genetics* ; Lac Operon/genetics* ; Plasmids/genetics* ; beta-Galactosidase/genetics*

GM1 gangliosidosis is an autosomal recessive disorder caused by galactosidase beta1 (GLB1) gene variants which affect the activity of β-galactosidase (GLB). GLB dysfunction causes abnormalities in the degradation of GM1 and its accumulation in lysosome. This article reports the clinical and genetic features of a child with GM1 gangliosidosis. The girl, aged 2 years and 5 months, was referred to the hospital due to motor developmental regression for more than one year. Physical examination showed binocular deflection and horizontal nystagmus, but no abnormality was found on fundoscopy. The girl had increased muscular tone of the extremities, limitation of motion of the elbow, knee, and ankle joints, and hyperactive patellar tendon reflex. Blood biochemical examination showed a significant increase in aspartate aminotransferase. The 24-hour electroencephalographic monitoring detected frequent seizure attacks and diffuse θ wave activity, especially in the right hemisphere. Head magnetic resonance imaging showed thinner white matter in the periventricular region and diffuse high T2WI signal with unclear boundary. Three-dimensional reconstruction of white matter fiber tracts by diffusion tensor imaging showed smaller and thinner white matter fiber tracts, especially in the right hemisphere. Genetic analysis showed that the girl had compound heterozygous mutations of c.446C>T (p.Ser149Phe) and c.101T>C (p.Ile34Thr) in the GLB1 gene from her parents, among which c.101T>C (p.Ile34Thr) had not been reported in the literatures. The girl was finally diagnosed with GM1 gangliosidosis. Her conditions were not improved after antiepileptic treatment and rehabilitation training for 2 months.
Diffusion Tensor Imaging ; Female ; Gangliosidosis, GM1 ; genetics ; Humans ; Infant ; Mutation ; Virulence ; beta-Galactosidase ; genetics

OBJECTIVE: To explore the genetic cause for a child with growth retardation by next generation sequencing (NGS). METHODS: Clinical data of the patient was collected. Peripheral venous blood samples were taken from the neonate and his parents. Targeted capturing and NGS were carried out to detect mutations of genes associated with inborn errors of metabolism. Suspected mutations were validated by Sanger sequencing. RESULTS: The 15-month-old female patient was admitted to hospital for growth retardation for 4 months. Hypomyelination was found upon cranium MRI. Genetic testing revealed two novel insertional mutations in the GLB1 gene in the patient, namely c.2006-2007insT and c.475-476 insGGTCC. CONCLUSION The c.2006-2007insT and c.475-476 insGGTCC mutations of the GLB1 gene probably underlie the GM1 gangliosidosis resulting in the growth retardation in the child.
Female ; Gangliosidosis, GM1 ; genetics ; Humans ; Infant ; Infant, Newborn ; Mutation ; Pedigree ; beta-Galactosidase ; genetics

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*

The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Animals ; Bacteria/genetics* ; Bacterial Proteins/chemistry* ; Biofilms ; Cold Temperature ; Dairy Products ; Endopeptidases/chemistry* ; Enzyme Stability ; Food Microbiology ; Hot Temperature ; Lipase/chemistry* ; Milk/microbiology* ; Peptide Hydrolases/chemistry* ; Phospholipases/chemistry* ; RNA, Ribosomal, 16S/genetics* ; Raw Foods/microbiology* ; beta-Galactosidase/chemistry*

Probiotics that are able to provide beneficial effects on animal health have become important ingredients of dog foods. This study was conducted to characterize the probiotic potentials of two strains, Lactobacillus reuteri BCLR-42 and Lactobacillus plantarum BCLP-51, that were derived from feces of healthy dogs and evaluated based on tolerance to low pH and bile acid, antimicrobial activities, enzyme profiles, sensitivity to antibiotics, and innate immune enhancing potentials. Both strains showed survival of more than 90% at pH 3 and 0.2% bile acid and exhibited broad antimicrobial activities against indicator bacteria. Moreover, both strains showed high sensitivity to antibiotics, except vancomycin, metronidazole, and gentamicin. The alkaline phosphatase was negligible (score 0), whereas they showed strong beta galactosidase activity (score range 5 or 3, respectively). The phagocytosis and oxidative burst activities of canine granulocytes were significantly enhanced in response to both strains. These results show that both strains have the capability to act as probiotics and the potential for application as ingredients in dog foods.
Alkaline Phosphatase ; Animals ; Anti-Bacterial Agents ; Bacteria ; beta-Galactosidase ; Bile ; Dogs* ; Feces ; Gentamicins ; Granulocytes ; Hydrogen-Ion Concentration ; Lactobacillus plantarum* ; Lactobacillus reuteri* ; Lactobacillus* ; Metronidazole ; Phagocytosis ; Probiotics* ; Respiratory Burst ; Vancomycin

Galactosidase is generated from Aspergillus oryzae, which is widely used for antidiarrhea medicine to infants. Antibiotics and digestives were reported as a causative allergen inducing occupational asthma. Galatosidase-induced occupational asthma has not been reported yet. A forty-year-old female has suffered from rhinorrhea, sneezing, and nasal obstruction 1 year after handling galactosidase at obstetric and pediatric hospital, and then dyspnea appeared later. Skin prick test with inhalent allergens, beta-galactosidase, and Aspergillus oryzae showed strong positive reaction to beta-galactosidase only. Immunoinhibition test with beta-galactosidase and A. oryzae revealed inhibition to beta-galactosidase only. Bronchial provocation test with beta-galactosidase showed the dual asthmatic response. With these results, we confirmed that the patient has beta-galactosidase-induced occupational asthma and rhinitis.
Allergens ; Anti-Bacterial Agents ; Aspergillus oryzae ; Asthma, Occupational* ; beta-Galactosidase ; Bronchial Provocation Tests ; Dyspnea ; Female ; Galactosidases ; Hospitals, Pediatric ; Humans ; Infant ; Nasal Obstruction ; Oryza ; Rhinitis ; Skin ; Sneezing

OBJECTIVETo observe the time-effect relation of extracts from ginseng, notoginseng and chuanxiong on angiotensin II (Ang II)-induced senescence of vascular endothelial cells and explore the feature of Chinese medicine against vascular diseases.

METHODSHuman umbilical vein endothelial cells (HUVECs) cultured in vitro were stimulated with 10(-6) mol/L AngII to induce cell senescence, which were divided into 4 groups, the blank control group, the Ang II model group, the extracts group and the telmisartan group. The &beta;-gal was used to identify senescence of cells, the cell counting kit-8 method was applied to assess the cell viability, the cell function was examined with the level of endothelial nitric oxide synthase (eNOS) and the flow cytometry was used for analyzing the cell cycle changes.

RESULTSCompared with the control cells, the cells positive for &beta;-gal staining was significantly increased in the Ang II model group, and showed cell cycle arrest at G0/G1 phase with decreased S and G2/M phase cell percentage, eNOS expression and cell viability (P<0.05). The extracts and telmisartan treatment of Ang II-induced cells resulted in decreased &beta;-gal positive cells with a reduction in G0/G1 phase cells and an increasing in S, G2/M phase cells and eNOS expression (P<0.05). At 24 h, the extracts were more effective than telmisartan (P<0.05); while telmisartan was more effective at 48 h (P<0.05).

CONCLUSIONExtracts from ginseng, notoginseng and chuanxiong can delay Ang II-induced aging of HUVECs and may play an important role in early senescence.

Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cellular Senescence ; drug effects ; Down-Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Panax notoginseng ; chemistry ; Plant Extracts ; pharmacology ; Time Factors ; beta-Galactosidase ; metabolism

OBJECTIVES: This study investigated the question of whether adenoviral magnetofection can be a suitable method for increasing the efficacy of gene delivery into bone marrow stromal cell (BMSC) and for generation of a high level of bone morphogenic protein (BMP) secretion at a minimized viral titer. MATERIALS AND METHODS: Primary BMSCs were isolated from C57BL6 mice and transduced with adenoviral vectors encoding beta galactosidase or BMP2 and BMP7. The level of BMP secretion, activity of osteoblast differentiation, and cell viability of magnetofection were measured and compared with those of the control group. RESULTS: The expression level of beta galactosidase showed that the cell transduction efficiency of AdLacZ increased according to the increased amount of magnetic nanoparticles. No change in cell viability was observed after magnetofection with 2 microL of magnetic nanoparticle. Secretion of BMP2 or BMP7 was accelerated after transduction of AdBMP2 and 7 with magnetofection. AdBMP2 adenoviral magnetofection resulted in up to 7.2-fold higher secretion of BMP2, compared with conventional AdBMP2-transduced BMSCs. Magnetofection also induced a dramatic increase in secretion of BMP7 by up to 10-fold compared to the control. Use of only 1 multiplicity of infection (moi) of magnetofection with adenoviral transduction of AdBMP2 or AdBMP7 resulted in significantly higher transgene expression compared to 20 moi of conventional adenoviral transduction. CONCLUSION: Magnetic particle-mediated gene transudation is a highly efficient method of gene delivery to BMSCs. Magnetofection can lower the amount of viral particles while improving the efficacy of gene delivery.
Adenoviridae ; Animals ; beta-Galactosidase ; Bone Marrow ; Cell Survival ; Magnetics ; Magnets ; Mesenchymal Stromal Cells ; Mice ; Nanoparticles ; Osteoblasts ; Transgenes ; Virion