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PURPOSE: Biallelic INPP4A variants have recently been associated with severe neurodevelopmental disease in single case reports. Here, we expand and elucidate the clinical-genetic spectrum and provide a pathomechanistic explanation for genotype-phenotype correlations. METHODS: Clinical and genomic investigations of 30 individuals were undertaken alongside molecular and in silico modelling and translation reinitiation studies. RESULTS: We characterize a clinically variable disorder with cardinal features including global developmental delay, severe-profound intellectual disability, microcephaly, limb weakness, cerebellar signs and short stature. A more severe presentation associated with biallelic INPP4A variants downstream of exon 4 has additional features of (ponto)cerebellar hypoplasia, reduced cerebral volume, peripheral spasticity, contractures, intractable seizures and cortical visual impairment. Our studies identify the likely pathomechanism of this genotype-phenotype correlation entailing translational reinitiation in exon 4 resulting in an N-terminal truncated INPP4A protein retaining partial functionality, associated with less severe disease. We also identified identical reinitiation site conservation in Inpp4a-/- mouse models displaying similar genotype-phenotype correlation. Additionally, we show fibroblasts from a single affected individual exhibit disrupted endocytic trafficking pathways, indicating the potential biological basis of the condition. CONCLUSION: Our studies comprehensively characterise INPP4A-related neurodevelopmental disorder and suggest genotype-specific clinical assessment guidelines. We propose the potential mechanistic basis of observed genotype-phenotype correlations entails exon 4 translation reinitiation.
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Plaque-related diseases are amongst the most common ailments of the oral cavity. Streptococcus mutans is the causal agent of dental caries in animals and humans and is responsible for the formation and accumulation of plaques. This study aimed to identify and evaluate the role of the dental plaque isolates and its surrounding environment in plaque formation or inhibition. The study started with the identification of human dental plaque isolates from high caries index patients based on 16S rRNA and Mitis salivarius bacitracin agar (MSB) was used for S. mutans growing. Unexpectedly, the Streptococcus mutans was completely absent. The disc diffusion assay recorded that all the isolates had antimicrobial activity against the S. mutans growth. Enzymes assay revealed that the isolates produced dextransucrase, levansucrase and levanase activity with wide variation degrees. Also, the lactic acid production assay was done based in pH shift assessment. The highest pH shift and dextran yield were detected by the isolates Bacillus subtilis_AG1 and Bacillus mojavensis_AG3. The adherence test revealed that Lysinibacillus cresolivorans_W2 (MK411028) recorded the highest adhesion property (60%). Oligo- and polysaccharides were synthesized by the action of dextransucrase enzyme and their cytotoxicity tests were negative. Dextran with a molecular weight (117521â¯Da) recorded the highest antimicrobial efficacy against Bacillus subtilis_AG1 and Bacillusmojavensis_AG3 (65%, 63.5%) respectively. The results concluded that the dextran was the most important factor causing the dental plaque pathogenicity. Also, oral oligo- and polysaccharides might play a role in dental plaque control.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Placa Dentária/microbiologia , Streptococcus mutans/isolamento & purificação , Animais , Antibacterianos/farmacologia , Bacillaceae/isolamento & purificação , Bacillus/isolamento & purificação , Bacillus subtilis/isolamento & purificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Aderência Bacteriana , Linhagem Celular , Cárie Dentária/microbiologia , Dextranos/metabolismo , Glucosiltransferases , Hexosiltransferases , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Testes de Sensibilidade Microbiana , Boca/microbiologia , Filogenia , RNA Ribossômico 16S/genética , Streptococcus mutans/efeitos dos fármacosRESUMO
Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder caused by variants in ATM gene and characterized by progressive neurologic impairment, cerebellar ataxia, and oculo-cutaneous telangiectasia. Immunodeficiency with a recurrent sinopulmonary infections are observed in patients with A-T. Here, we report a novel stop codon variant, c.5944 C>T (p.Gln1982*), revealed by whole-exome sequencing in a 9-year old boy. He presented with recurrent upper respiratory tract infections, failure to thrive, developmental delay, ataxic gait, and bulbar telangiectasia.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Ataxia Telangiectasia/genética , Códon sem Sentido , Adulto , Ataxia Telangiectasia/patologia , Humanos , Masculino , Sequenciamento do ExomaRESUMO
Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ÏBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ÏC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Eritromicina/metabolismo , Plasmídeos/genética , Streptomyces/metabolismo , Proteínas de Bactérias/biossíntese , Cromatografia Líquida , Eritromicina/análogos & derivados , Eritromicina/biossíntese , Engenharia Genética , Glicosilação , Espectrometria de Massas , Complexos Multienzimáticos/metabolismo , Família Multigênica/genética , Streptomyces/genéticaRESUMO
This study aimed to investigate whether the gastroprotective effects of three types of bacterial levans are correlated with their prebiotic-associated anti-inflammatory/antioxidant potentials. Three levans designated as LevAE, LevP, and LevZ were prepared from bacterial honey isolates; purified, and characterized using TLC, NMR, and FTIR. The anti-inflammatory properties of levan preparations were assessed in LPS-stimulated RAW 264.7 cell lines, while their safety and gastroprotective potentials were assessed in Wistar rats. The three levans significantly reduced ulcer number (22.29-70.05 %) and severity (31.76-80.54 %) in the ethanol-induced gastric ulcer model compared to the control (P < 0.0001/each), with the highest effect observed in LevAE and levZ (200 mg/each) (P < 0.0001). LevZ produced the highest levels of glutathione; catalase activity, and the lowest MDA levels (P = 0.0001/each). The highest anti-inflammatory activity was observed in LevAE and levZ in terms of higher inhibitory effect on IL-1ß and TNF-α production (P < 0.0001 each); COX2, PGE2, and NF-κB gene expression. The three levan preparations also proved safe with no signs of toxicity, with anti-lipidemic properties as well as promising prebiotic activity that directly correlated with their antiulcer effect. This novel study highlights the implication of prebiotic-mediated systemic immunomodulation exhibited by bacterial levans that directly correlated with their gastroprotective activity.
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Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2â¯U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2â¯U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65⯰C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50-60⯰C for almost 90â¯min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.
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A novel synbiotic multiparticulate microparticle was produced in the current study to expand the synbiotic industrial applications. Initially, the inulin was fabricated into PLGA nanoparticles. After the inulin entrapment efficiency was boosted to reach 92.9⯱â¯8.4% by adjusting the formulation parameters, the developed particles were characterized by different techniques such as particle size analyzer, TEM, and TLC. The obtained data showed that the particle size was 115.8⯱â¯82.7â¯nm, the particles had smooth surface and round shape, and the fabrication procedure did not affect the integrity of the inulin. Later, the inulin loaded nanoparticles together with selected Bifidobacterium species were double coated with gum arabic and alginate. The maximum survivability of the encapsulated Bifidobacterium in the simulated gastric solution reached 88.29% of the initial population, which was significantly higher than the survivability of the free bacteria. Finally, the inulin release from the multiparticulate microparticles was studied and found to be sustained over three days.
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Bifidobacterium/isolamento & purificação , Trato Gastrointestinal/química , Inulina/química , Simbióticos , Cápsulas/química , Trato Gastrointestinal/microbiologia , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
A thermostable metallo-collagenase enzyme (150â¯kDa), recently identified in a newly isolated actinomycestes strain (Nocardiopsis dassonvillei NRC2aza), has been purified from natural source, characterized to have application in wound healing. A simple 3 step purification procedure gave an increase of purity by 6.23 fold with a specific activity of 387.2â¯Uâ¯mg-1. The enzyme activity showed stability across a range of pH (7.0-8.5) and temperature (40-55⯰C) with optima at pHâ¯8.0 and 60⯰C, respectively. Activators include Mg+2, Ca+2, Zn+2, Na+, K+ and Ba+2, while Mn+2, Co+2, Ni+2and Ag+ ions gave partial inhibition. Full inhibition was given by other tested ions and metalloproteinase inhibitors. Broad substrate specificity was demonstrated including activity against a native collagen. The Km and Vmax of the enzyme using azocollagen were 5.5â¯mg/ml and 1280â¯U, respectively. The purified collagenase enhanced wound closure in vitro and in vivo and the repair process was dose dependent. Topical application of the purified collagenase (either of 25 or 50â¯U) to cutaneous wounds significantly accelerated the rate of wound healing and the formation of granulation tissue. Hence, the purified collagenase has a great potential as a therapeutic agent in wound care and collagen related diseases.
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Actinobacteria/enzimologia , Proteínas de Bactérias , Colagenases , Fibroblastos/metabolismo , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Células Cultivadas , Colagenases/química , Colagenases/isolamento & purificação , Colagenases/farmacologia , Fibroblastos/patologia , Humanos , Ratos , Ratos Wistar , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Enterococcus faecalis Esawy dextransucrase was immobilized in Fe(3+)-cross-linked alginate/carboxymethyl cellulose (AC) beads. The gel beads were modified with polyethylenimine (PEI) followed by glutaraldehyde (GA) to form Fe(3+) (ACPG) beads. Fe(3+) (ACPG) was characterized using FTIR and DSC techniques. GA activated beads showed new two peaks. The first was at 1,717 cm(-1) which refers to (CO) group of a free aldehyde end of glutaraldehyde, and another peak was at 1,660 cm(-1) referring to (CN) group. The immobilization process improved the optimum temperature from 35 to 45°C. The immobilized enzyme showed its optimum activity in wide pH range (4.5-5.4) compared to pH 5.4 in case of free form. Also, the immobilization process improved the thermal and pH enzyme stability to great extent. Reusability test proved that the enzyme activity retained 60% after 15 batch reactions. Immobilized enzyme was applied successfully in the synthesis of oligosaccharides and different molecular weights of dextran.
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Dextranos/química , Enterococcus faecalis/enzimologia , Enzimas Imobilizadas/química , Glucosiltransferases/química , Dextranos/síntese química , Enterococcus faecalis/isolamento & purificação , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glucosiltransferases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Microesferas , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , TermodinâmicaRESUMO
Dextrans enzymatic synthesis by immobilized Enterococcus faecalis Esawy dextransucrase was studied. Different parameters, such as: enzyme protein concentration (EPC), substrate concentration (SC), temperature and reaction time were evaluated. EPC played a fundamental role in controlling dextran molecular size with 0.1% dextran in reaction mixture. Dextran 38,397 and 125,471Da were yielded at EPC 4.78 and 5.78mg, respectively. Proper dextrans (73,378 and 117,521Da) demanded in pharmaceutical applications were achieved at 6% and 12% sucrose concentrations and at 4.78 and 5.78mg EPC, respectively. Optimum temperature for conversion of glucose to dextran was 30°C (73% and 80% at 5.78 and 4.78mg EPC, respectively). Varieties of maltooligosaccharides (MOS) were yielded by synergistic cooperation between sucrose and maltose. Six MOS and three dextrans samples in vitro have prebiotic effect on Lactobacillus casei with degree of variation. Two samples of MOS with different degree of polymerization (DP) and three samples of dextran with different molecular weight (MW) reported different fibrinolytic activity.
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Enterococcus faecalis/metabolismo , Glucosiltransferases/biossíntese , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Enterococcus faecalis/química , Glucosiltransferases/química , Lacticaseibacillus casei/química , Lacticaseibacillus casei/metabolismoRESUMO
The present experiments investigated the effect of water quality characteristics on the condition factor, the ovarian activity, cortisol level, and the immunoreactivity of stress-response cells (adrenocorticotropic hormone; ACTH- and melanin stimulating hormone; MSH- and somatolactin; SL- secreting cells) and gonadotropin (GTH)-secreting cells in the pituitary gland of Nile tilapia, Oreochromis niloticus. After 3 months of exposure to mixtures of water from different sources (Tap and Lake Manzalah waters), with high levels of minerals and heavy metals, water quality affected the number, size, and immunostaining of stress-response-immunoreactive (ir) cells and GTH-ir cells, which showed a dramatic decrease in their size. The integrated optical density (IOD) of immunoreactivity of MSH- and GTH- cells was significantly increased; however, it was significantly decreased for ACTH- and SL- cells. Also, high levels of cortisol were observed in females exposed to waters with high concentrations of minerals and heavy metals. In parallel, low values of gonadosomatic index (GSI%) and the ovarian histology revealed a decrease of maturing follicles concomitant with an increase of atretic follicles in females exposed to Lake Manzalah polluted water. Taken together, the increased activity of stress-response-ir pituitary cells, serum cortisol level and ovarian atretic follicles in response to elevated concentrations of minerals and heavy metals, supports the possible role of ACTH, MSH, and SL in the adaptive stress response of fish. Therefore, minerals and heavy metals must be considered when discussing tilapia aquaculture status.
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Gonadotropinas/análise , Hidrocortisona/sangue , Metais Pesados/toxicidade , Folículo Ovariano/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Qualidade da Água , Animais , Ciclídeos/sangue , Ciclídeos/metabolismo , Feminino , Gonadotropinas/imunologia , Folículo Ovariano/química , Hipófise/química , Hipófise/imunologia , Estresse FisiológicoRESUMO
BACKGROUND: Although the role of folic acid (FA) in preventing neural tube defects (NTDs) is well documented, its optimal intake in pregnant women is still low in many countries. Here, we prospectively studied the prevalence of NTDs in the newborns and the patterns of FA intake in pregnant Saudi mothers. METHODS: This case-control study was nested within a 3-year project (July 2010 to June 2013) to study the patterns of birth defects in the offspring of Saudi women who received their antenatal care and delivered at Prince Sultan Military Medical City, Riyadh-Saudi Arabia. Enrolled mothers were divided into 4 groups: group 1 (FA taken before pregnancy and continued regularly after conception), group 2 (FA taken post-conception), group 3 (no FA intake), and group 4 (did not remember or were unsure of taking FA). Control mothers were randomly selected from those with normal first obstetrical ultrasound scan at 18-22 weeks of gestation. RESULTS: The cohort included 30,531 mothers giving birth to 28,646 infants. We studied 1179 mothers of babies with birth defects (BDs) and 1262 control mothers. There were 237 (9.7%) mothers in-group 1; 2001 (82%) in-group 2; 154 (6.3%) in-group 3; and 49 (2%) in-group 4. There were 49 babies with NTDs, a prevalence of 1.7/1000 total births. Among the studied mothers 2274 (93%) took FA either full or partial course. CONCLUSION: The high prevalence of NTDs and the low optimal FA intake highlight the need for a strict implementation of staple food fortification and health education program for Saudi women.
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Bacillus subtilis NRC1aza produced levansucrase under solid state fermentation using starch as support. A sequential optimization strategy, based on statistical experimental designs is employed to enhance enzyme productivity. First, a 2-level Plackett-Burman design was applied for bioprocess parameters screen that significantly increase levansucrase production. Second optimization step was performed using fractional factorial design in order to optimize the amounts of highest positive variables that had significant effect on levansucrase productivity. Maximal enzyme productivity of 170 U/gds was achieved in presence of glucose, yeast extract, and pH 8. In vitro, experiments confirmed that LevCR and LevQT had an antitumor activity against different animal and human cancer cell lines by demonstrating inhibitory effects on growth of Ehrlich ascites carcinoma cell line, human MCF-7 breast and liver HepG2 cancer cell lines, in particular LevQT was found to be efficacious compared to anticancer drug, cisplatin. Result focused in LevCR as strong fibrinolytic agent.
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Bacillus subtilis/enzimologia , Hexosiltransferases/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Carcinoma de Ehrlich/tratamento farmacológico , Feminino , Fermentação , Frutanos/farmacologia , Frutanos/uso terapêutico , Células Hep G2 , Hexosiltransferases/farmacologia , Hexosiltransferases/uso terapêutico , Humanos , Células MCF-7 , CamundongosRESUMO
The present experiments were designed to determine the effect of different stress factors; handling, seawater acclimation, confinement, and induced spawning on plasma cortisol, hydro mineral balance as well as changes in size, number and integrated intensity of somatolactin (SL)-expressing cells in Liza ramada mature females confined to fresh water ponds. The plasma levels of cortisol, PO(4)(3-), Na(+), and K(+) were higher, while Ca(2+) and Mg(2+) were lower than controls during transportation without anesthesia. By using clove oil (5 mg L(-1)) as an anesthetic during transportation, the plasma cortisol, PO(4) (3-), Na(+), and K(+) were similar to controls, while Ca(2+) and Mg(2+) were higher. During seawater acclimation, the plasma cortisol and minerals were significantly higher except Na(+) which was lower than controls. In addition, during induction of spawning, the plasma levels of cortisol, PO(4)(3-), Na(+), K(+), and Mg(2+) were significantly higher than controls. The SL-producing cells are located in the pars intermedia (PI) bordering the neurohypophysis. The stress affected the number, size, and immunostaining of SL-expressing cells. During seawater acclimation, the size and the integrated intensity of SL immunoreactivity were lower, but the number of these cells was higher than controls. Furthermore, the number, size, and the integrated intensity of SL immunoreactivity were significantly lower than controls during handling and after spawning, which was opposite to confinement. The response of SL-expressing cells in PI in parallel with changes in cortisol and hydro mineral balance induced by stress support the possible role of SL in the adaptive response of fish to stress.