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This work deals with the optimization of an extracellular phospholipase C production by Bacillus cereus (PLCBc) using Response Surface Methodology (RMS) and Box-Behnken design. In fact, after optimization, a maximum phospholipase activity (51 U/ml) was obtained after 6 h of cultivation on tryptone (10 g/L), yeast extract (10 g/L), NaCl (8.125 g/L), pH 7.5 with initial OD (0.15). The PLCBc activity, esteemed by the model (51 U) was very approximate to activity gutted experimentally (50 U). The PLCBc can be considered as thermoactive phospholipase since it showed a maximal activity of 50 U/mL at 60 °C using egg yolk or egg phosphatidylcholine (PC) as substrate. In addition, the enzyme was active at pH 7 and is stable after incubation at 55 °C for 30 min. The application of B. cereus phospholipase C in soybean oil degumming was investigated. Our results showed that when using enzymatic degumming, the residual phosphorus decrease more than with water degumming, indeed, it passes from 718 ppm in soybean crude oil to 100 ppm and 52 ppm by degumming using water and enzymatic process, respectively. The diacylgycerol (DAG) yield showed an increase of 1.2% with enzymatic degumming compared to soybean crude oil. This makes our enzyme a potential candidate for food industrial applications such as enzymatic degumming of vegetable oils.
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Petróleo , Óleo de Soja , Fosfolipases Tipo C , Bacillus cereus , Fosfolipases , ÁguaRESUMO
A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72 h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54 kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 °C against 50 °C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 °C, r-AmyA reached 45 min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4 mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.
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Amilases/biossíntese , Amilases/isolamento & purificação , Aspergillus oryzae/enzimologia , Pichia/genética , Amilases/química , Amilases/metabolismo , Sítios de Ligação , Simulação por Computador , Estabilidade Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Metais/farmacologia , Modelos Moleculares , Oligopeptídeos/metabolismo , Proteínas Recombinantes/isolamento & purificação , TemperaturaRESUMO
BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an autosomal recessive metabolic disorder caused by inherited mutations in the AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT). PH1 is a clinically and genetically heterogeneous disorder. The aim of our study was to analyze and characterize the mutational spectrum of PH1 in Tunisian patients. MATERIALS AND METHODS: Molecular studies of 146 Tunisian patients suspected with PH were performed by PCR/Restriction fragment length polymorphism (RFLP) to detect seven mutations described as the most common. Direct sequencing for the 11 exons was performed in patients in whom any mutation was not identified. RESULTS: The genetic diagnosis of PH1 was confirmed in 62.3% of patients. The first molecular approach based on PCR/restriction enzyme test was positive in 37.6% of patients, whereas the second molecular approach based on whole gene sequencing was successful in 24% of cases. Twelve pathogenic mutations were detected in our cohort. Two mutations were novel, and five were detected for the first time in Tunisians. The three most frequent mutations were p.Ile244Thr, p.Gly190Arg, and c.33dupC, with a frequency of 43.4%, 21.4%, and 13.1%, respectively. CONCLUSION: The two novel mutations detected in our study extend the spectrum of known AGXT gene mutations. The screen for the mutations identified in this study can provide a useful, cost-effective, and first-line investigation in Tunisian PH1 patients.
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Hiperoxalúria Primária/genética , Transaminases/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Feminino , Frequência do Gene , Estudos de Associação Genética , Haplótipos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Adulto JovemRESUMO
BACKGROUND: Oral steroid treatment is the first line of therapy for childhood nephrotic syndrome (NS). However, resistance to this treatment has been observed in some patients. Here, we investigated the association of two steroid metabolism-related genes with susceptibility to childhood NS and the steroid response. METHODS: We genotyped the single nucleotide polymorphisms (SNP) of MDR-1 [C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642)] and the CYP3A5 gene (A6986G) in 63 NS patients and 110 age and gender matched controls by PCR-RFLP. RESULTS: Based on multivariate logistic regression analysis carrying the G2677A A allele seemed to multiply both the risk of NS and the risk of developing glucocorticoid (GC) resistance by three-fold (OR = 3.50, [1.37 - 7.06] , p < 0.001, OR = 3.07, [1.06 - 26.10], p = 0.048, respectively). When combined into haplotype, the TAT (1236_T, 2677_A, and 3435_T) haplotype conferred a two-fold NS risk (OR = 2.26, [1.11 - 4.58], p = 0.023) and almost three-fold risk to develop resistance to GC (OR = 2.69, [1.12 - 8.79], p = 0.044). However, TAT carriers seemed to have less risk to develop NS at late age (OR = 0.34, [0.12 - 0.92], p = 0.037). The C1236T (MDR-1) and the A6986G (CYP3A5) polymorphisms showed a trend of association to GC resistance but these associations did not reach the statistical significance (OR = 2.83, [0.54 - 14.67], p = 0.294), (OR = 2.11, [0.53 - 8.38], p = 0.28), respectively. CONCLUSIONS: Here we report that only the G2677A polymorphism was associated to NS susceptibility and steroid resistance. The TAT haplotype was associated with NS susceptibility especially at an early age and with steroid resistance.
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Citocromo P-450 CYP3A/genética , Síndrome Nefrótica/genética , Esteroides/uso terapêutico , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Criança , Genótipo , Haplótipos , Humanos , Síndrome Nefrótica/tratamento farmacológico , Polimorfismo GenéticoRESUMO
A newly isolated amylolytic strain was identified as Bacillus cereus spH1 based on 16S and 16-23S gene sequencing (Accession numbers OP811441.1 and OP819558, respectively), optimization strategies, using one variable at time (OVAT) and Plackett-Burman design, were employed to improve the alpha-amylase (α-amylase) production. Condition inferred revealed that the optimal physical parameters for maximum enzyme production were 30 °C, pH 7.5, and 12 h of incubation, using tryptone, malt extract, orange (Citrus sinensis) peels, crab (Portunus segnis) shells, calcium, and sodium chloride (NaCl) as culture medium. The full factorial design (FFD) model was observed to possess a predicted R2 and adjusted R2 values of 0.9788 and 0.9862, respectively, and it can effectively predict the response variables (p = 0). Following such efforts, α-amylase activity was increased 141.6-folds, ranging from 0.06 to 8.5 U/mL. The ideal temperature and pH for the crude enzyme activity were 65 °C and 7.5, respectively. The enzyme exhibited significant stability, with residual activity over 90% at 55 °C. The maltose was the only product generated during the starch hydrolysis. Moreover, the Bacillus cereus spH1 strain and its α-amylase were used in the treatment of effluents from the pasta industry. Germination index percentages of 143% and 139% were achieved when using the treated effluent with α-amylase and the strain, respectively. This work proposes the valorization of agro-industrial residues to improve enzyme production and to develop a green and sustainable approach that holds great promise for environmental and economic challenges.
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Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the K(m) values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward ß-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.
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Bacillus/enzimologia , Biocatálise , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Bacillus/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
A copper activated xylanase produced by E. coli BL21 was expressed in Pichia pastoris using the pGAPZαB expression vector. Two recombinant GH11 xylanase forms were obtained (N-His-rXAn11 and N-C-His-rXAn11). The findings revealed that the two recombinant xylanases displayed different behaviors toward the copper. In the presence of 3-mM Cu2+, the relative activity of the N-His-rXAn11 was enhanced by about 52%. However, the xylanase activity of the N- and C-terminal tagged one (N-C-His-rXAn11) was strongly inhibited by copper. In the presence of 3-mM Cu2+, the N-His-rXAn11 revealed to be thermostable at 60 °C with a half-life of 10 min. However, the N-C-His-rXAn11 was noted to be unstable since it was inactivated after 15 min of incubation at 55 °C. 3D models of the two recombinant forms showed that the created copper site in the N-His-rXAn11 was loosed in the C-terminal tagged protein. The C-terminal tag could trigger some structural changes with a notable displacement of secondary structures leading to great hindrance of the active site due to high fluctuations and probably new interactions among the N- and C-terminal amino acids.
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Cobre , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Estabilidade Enzimática , Proteínas Recombinantes/química , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
BACKGROUND: Posterior reversible encephalopathy syndrome (PRES) is a clinico-radiological entity most frequently described in young- or middle-aged adults with a rare occurrence among children. AIM: To determine the clinical, radiological features and outcome of PRES in children admitted to a Tunisian tertiary care pediatric department. METHODS: we retrospectively reviewed records of all children under 18 years old diagnosed with PRES and admitted to the PICU of the Pediatric department of Sahloul University Hospital from January 2000 to August 2021. RESULTS: Sixteen patients were enrolled in this study. The mean age of the study population at PRES onset was 10 years (range: 4-14 years) and the male female ratio was 3. The most frequent neurological signs were seizures (n = 16 cases), headache (n = 8 cases), and impaired level of consciousness (7 cases). Visual disturbances were found in one patient. Arterial hypertension was the most underlying cause (16 cases). Brain MRI showed vasogenic edema, mostly localized in the parietal (13 cases) and occipital (11 cases) lobes. Moreover, cytotoxic edema (2 cases), pathologic contrast enhancement (1 case), and hemorrhage (3 cases) were isolated on MRI. The outcome after specific management was favorable after the first onset in 13 cases and death occurred in 3 patients. Relapses were observed in 4 patients. CONCLUSION: Clinical features presented by children with PRES are variable and non-specific. MRI typically shows reversible posterior cerebral edema. However, in some cases, atypical neuro-imaging findings, such as cytotoxic edema infarction, hemorrhage and contrast enhancement can be observed.
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Síndrome da Leucoencefalopatia Posterior , Adulto , Pessoa de Meia-Idade , Humanos , Masculino , Criança , Feminino , Adolescente , Pré-Escolar , Síndrome da Leucoencefalopatia Posterior/diagnóstico por imagem , Síndrome da Leucoencefalopatia Posterior/etiologia , Estudos Retrospectivos , Centros de Atenção Terciária , Imageamento por Ressonância Magnética , EdemaRESUMO
Background: Infective endocarditis is a rare condition in childhood, and there is limited data on this disease in Tunisia. Objective: This study aims to analyze the epidemiological profile, bacteriological data, and prognosis of infective endocarditis in children admitted to the pediatric department of a University Hospital in Tunisia. Methods: We conducted a comparative cross-sectional study in the pediatric department of Sahloul Teaching Hospital in Sousse, a tertiary referral hospital in Tunisia. The study included all children aged ≤ 18 years with infective endocarditis admitted to the tertiary referral center for pediatrics in Sahloul University Hospital from January 1994 to December 2022. The diagnosis of infective endocarditis was based on modified Duke's criteria. Results: Thirty-six patients met the diagnostic criteria for infective endocarditis, resulting in a proportion of 07 cases per 1000 hospital admissions. The mean age was 6 years (range: 40 days to 16 years). Congenital heart disease was identified as the underlying lesion in 23 cases (63.9 %). Blood cultures were positive in 20 patients (55.6 %), predominantly with Staphylococcus species (55 %). The most frequent complications involved the central nervous system (8 cases; 22.2 %). The mortality rate was 25 %, and factors predicting mortality included heart failure on admission or during the hospital stay, increased leukocyte count, and decreased prothrombin time. Conclusion: Our study reveals a shift in the prevalent underlying lesions, with rheumatic heart diseases no longer being the most common. Staphylococcus spp. emerged as the predominant organism in blood cultures. Notably, mortality predictors included heart failure, an elevated leukocyte count, and a decreased prothrombin time rate.
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Primary hyperoxaluria type 1 (PH1) is an autosomal recessive inborn error of metabolism characterized by marked hepatic overproduction of oxalate due to deficiency of hepatic peroxisomal alanine-glyoxylate aminotransferase caused by AGXT gene mutation. One major hallmark of PH1 in developed as well as developing countries (DC) is the diagnostic delay. Notably in DC, where the disease is most prevalent and probably underdiagnosed, there are many challenges in PH1 diagnosis and management, with economic constrains and ethical concerns. This has led to the existing gap in the management of PH1 between developed and DC, which is expected to further deepen with the advent of novel therapeutic agents unless appropriate actions are taken. Until recently, treatment possibilities were limited to supportive measures. Thanks to a better understanding of the molecular basis of the disease a number of new therapies are developed, or being developed, leading to profound changes in management strategies. In this review we discuss the current situation of PH1 in DC as well as the accessibility challenges and the advantages of using promising novel therapeutics to bridge the currently existing gap. We also provide an overview of an integrated approach to ensure equitable access of sustainable therapeutics to PH1 patients in DC. This is expected to reduce global PH1 healthcare disparities, improve its standard of care and reduce disability linked to extrarenal complications of PH1 by implementing personalized medicine.
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INTRODUCTION: Kawasaki syndrome (KS) is a systemic vasculitis of unknown etiology that affects medium and small blood vessels. The aim of our study is to analyze coronary artery lesions in children with KS and their risk factors. MATERIAL AND METHODS: All children under the age of 15 years-old presenting KS and admitted in the pediatric department of three university hospital (Sahloul hospital, and Farhat Hached hospital of Sousse, Ibn El Jazzar hospital of Kairoun) from January 2000 to December 2018 were included. RESULTS: Sixty-five patients were included in our study. The mean age at diagnosis was of 29.9 months [2-120 months] and the sex ratio was of 1.7. Echocardiography was performed in all patients. It showed coronary dilation in 37% of patients with coronary artery diameter of 4.2 mm on average [3.2-7mm]. The coronary aneurysm was small in 19 cases and medium in 5 cases. No giant aneurysm has been identified. In univariate analysis, the predictors of coronary artery lesions were male sex, atypical form, fever duration more than 10 days, hepatic cytolysis, thrombocytosis and anemia. In multivariate analysis, only the last four parameters were the predictive factors of the coronary artery involvement. CONCLUSION: Several risk factors can be used to determine which children are predisposed to develop coronary dilations. In case of patient with risk factors, intravenous immunoglobulins should be initiated early to avoid these serious complications.
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Aneurisma Coronário , Doença da Artéria Coronariana , Síndrome de Linfonodos Mucocutâneos , Adolescente , Criança , Aneurisma Coronário/diagnóstico por imagem , Aneurisma Coronário/epidemiologia , Aneurisma Coronário/etiologia , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/etiologia , Vasos Coronários , Feminino , Humanos , Lactente , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/diagnóstico , Estudos Retrospectivos , Fatores de RiscoRESUMO
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder of unknown etiology. Lupus nephritis (LN) is one of the most severe clinical manifestations observed in patients with SLE; it is more frequent and more severe in children than in adults. The aim of our study was to assess the predictive factors of poor outcomes in Tunisian children with LN. This was a multicenter retrospective observational study on 40 pediatric patients with biopsy-proven LN from five nephrology departments in Tunisia. The patients were 12.33 ± 3.3 years of age at the time of their kidney biopsy. Eleven patients developed end-stage renal disease (ESRD) (27.5%), and seven patients died. Overall, 18 (45%) patients reached our composite endpoint (ESRD or death). An age at diagnosis of more than 14 years, elevated serum creatinine at the time of the kidney biopsy, the existence of wire loops, thromboembolic complications as well as infectious complications are the most important clinical features associated with an increased risk of ESRD. Predictive factors of death were a baseline creatinine level of more than 2.26 mg/dL, a high proteinuria at baseline, fibrous crescents determined by renal biopsy, thromboembolic complications, infectious compli-cations, and ESRD. In summary, our results suggest that early and appropriate management is the best guarantee of a good renal outcome in children with LN.
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Falência Renal Crônica , Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Adulto , Humanos , Criança , Adolescente , Nefrite Lúpica/complicações , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/epidemiologia , Rim/patologia , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/etiologia , Lúpus Eritematoso Sistêmico/complicações , Biópsia , Estudos RetrospectivosRESUMO
BACKGROUND: Ocular cystinosis is a rare autosomal recessive disorder characterized by intralysosomal cystine accumulation in renal, ophthalmic (cornea, conjunctiva), and other organ abnormalities. Patients with ocular cystinosis are mostly asymptomatic and typically experience mild photophobia due to cystine crystals in the cornea observed accidently during a routine ocular examination. The ocular cystinosis is associated with different mutations in CTNS gene. Cysteamine therapy mostly corrects the organ abnormalities. METHODS: This study was performed in collaboration with the department of ophthalmology of Farhat Hached Hospital. The Optical Coherence Tomography (OCT) of the cornea and retinal photography were used to search cystine crystals within the corneas and conjunctiva in eight Tunisian patients. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the entire CTNS gene. RESULTS: The studied patients were found to have cystine crystal limited anterior corneal stroma and the conjunctiva associated with retinal crystals accumulation. CTNS gene sequencing disclosed 7 mutations: three missense mutations (G308R, p.Q88K, and p.S139Y); one duplication (C.829dup), one framshift mutation (p.G258f), one splice site mutation (c.681 + 7delC) and a large deletion (20,327-bp deletion). Crystallographic structure analysis suggests that the novel mutation p.S139Y is buried in a first transmembrane helix closed to the lipid bilayer polar region, introducing a difference in hydrophobicity which could affect the hydrophobic interactions with the membrane lipids. The second novel mutation p.Q88K which is located in the lysosomal lumen close to the lipid membrane polar head region, introduced a basic amino acid in a region which tolerate only uncharged residue. The third missense mutation introduces a positive change in nonpolar tail region of the phospholipid bilayer membrane affecting the folding and stability of the protein in the lipid bilayer. CONCLUSIONS: Our data demonstrate that impaired transport of cystine out of lysosomes is the most common, which is obviously associated with the mutations of transmembrane domains of cystinosine resulting from a total loss of its activity.
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Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Sistemas de Transporte de Aminoácidos Neutros/genética , Cistina/genética , Cistina/metabolismo , Cistinose/genética , Cistinose/metabolismo , Humanos , Bicamadas Lipídicas , Mutação , TunísiaRESUMO
BACKGROUND: Acute disseminated encephalomyelitis (ADEM) is an inflammatory demyelinating disorder of the central nervous system. Little information is available about the clinical and neuroradiological profile or the follow-up of this disease in Tunisian children. AIM: To determine the clinical, laboratory, and radiological features and the outcome of ADEM in children admitted to the pediatrics department of a university hospital in Tunisia. METHODS: All children ≤ 18 years old presenting with ADEM and admitted to the tertiary referral center for pediatrics at Sahloul University Hospital from January 2000 to December 2020 were included in the study. The diagnosis of ADEM was confirmed according to the international pediatric multiple sclerosis study group criteria. RESULTS: A total of 20 patients (13 girls and 7 boys) fulfilled the diagnostic criteria for ADEM. The mean age at diagnosis was 5.6 years. The clinical presentation included polyfocal neurological signs such as cranial hypertension (45%), seizures (35%), and motor weaknesses (55%). Pyramidal tract signs and cranial nerve palsies were noted in 55% of cases. Brain magnetic resonance imaging showed particular features, namely, a relapsing tumor-like form in one case, and optic neuritis and demyelinating lesions of the white matter in the brain and the spinal cord with gadolinium cerebral ring-like enhancement in another case. The treatment consisted of intravenous immunoglobulin in 16 cases (80%) and corticosteroid in 19 cases (95%). Plasmapheresis was used for one patient. Complete recovery was observed in 12 patients (60%); 19 patients (95%) had a monophasic course of the disease while only one patient developed multiphasic ADEM. CONCLUSIONS: ADEM remains a difficult diagnosis in children. Nevertheless, after prompt diagnosis and adequate treatment, most children with ADEM have a favorable outcome with restitutio ad integrum.
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Encefalomielite Aguda Disseminada/diagnóstico , Adolescente , Corticosteroides/uso terapêutico , Criança , Pré-Escolar , Encefalomielite Aguda Disseminada/classificação , Encefalomielite Aguda Disseminada/epidemiologia , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , Masculino , Pediatria/métodos , Pediatria/estatística & dados numéricos , Estudos Retrospectivos , Tunísia/epidemiologiaRESUMO
A new alpha-amylase-producing strain was assigned as Bacillus subtilis US586. The used statistical methodology indicated that amylase production was enhanced by 5.3 folds. The crude enzyme analysis proved the presence of three amylases isoforms Amy1, Amy2, and Amy3 called Amy586. The purified amylases had molecular masses of 48, 52, and 68 kDa with a total specific activity of 2,133 U/mg. Amy586 generated maltose, maltotriose, and maltopentaose as main final products after starch hydrolysis. It exhibited a large 4-6 optimal pH, a 60°C temperature activity, and a moderate thermostability. Amy586 displayed a high pH stability ranging from 3.5 to 6. The addition of Amy586 to weak wheat flour decreased its P/L ratio from 1.9 to 1.2 and increased its dough baking strength (W) from 138 × 10-4 to 172 × 10-4 J. Amy586 also improved the bread texture parameters by reducing its firmness and boosting the cohesion and elasticity values. PRACTICAL APPLICATIONS: Bacterial alpha-amylases with novel properties have been the major extent of recent research. In this paper, we managed to demonstrate that the addition of a purified amylolytic extract from the new isolated Bacillus subtilis strain US586 to weak local flour improves dough rheological proprieties and bread quality. Therefore, Amy586 can be considered as a bread making improver.
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Bacillus subtilis/enzimologia , Amido/metabolismo , Triticum/química , alfa-Amilases/metabolismo , Pão , Farinha , Hidrólise , Isoenzimas , Maltose/metabolismo , Peso Molecular , Oligossacarídeos/metabolismo , Temperatura , Trissacarídeos/metabolismo , alfa-Amilases/isolamento & purificaçãoRESUMO
The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the beta-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37 degrees C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19 degrees C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed V(max) and K(m) values of 253 +/- 36 mumol of beta-cyclodextrin/mg/min and 0.36 +/- 0.18 g/L, respectively.
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Bacillus/classificação , Bacillus/enzimologia , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Engenharia de Proteínas/métodos , Bacillus/genética , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/fisiologia , Glucosiltransferases/genética , Glucosiltransferases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases. In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum activity was obtained at 40 degrees C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from beta-cyclodextrin, and panose from pullulan.
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Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Proteínas de Bactérias/genética , Cromatografia em Camada Fina , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Maltose/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
BACKGROUND: Autoimmune hemolytic anemia is rare in children. First-line therapies for this disease consist of corticosteroids and intravenously administered immunoglobulin that are effective in most patients. However, a small proportion of cases (5 to 10%) is refractory to these therapies and may represent a medical emergency, especially when hemolysis is due to warm immunoglobulin M. Recently, reports of the use of rituximab in adult autoimmune diseases have shown promising results. In children, there are few studies on the use of rituximab in the treatment for autoimmune hemolytic anemia, especially on its long-term efficacy and adverse effects. CASE PRESENTATION: Here, we report the case of a 10-year-old Tunisian girl with refractory acute autoimmune hemolytic anemia caused by warm-reactive immunoglobulin A, immunoglobulin G, immunoglobulin M, and C3d autoantibodies. First-line treatments using corticosteroids and intravenously administered immunoglobulin were ineffective in controlling her severe disease. On the other hand, she was successfully treated with rituximab. In fact, her hemolytic anemia improved rapidly and no adverse effects were observed. CONCLUSIONS: The case that we report in this paper shows that rituximab could be an alternative therapeutic option in severe acute autoimmune hemolytic anemia with profound hemolysis refractory to conventional treatment. Moreover, it may preclude the use of plasmapheresis in such an urgent situation with a sustained remission.
Assuntos
Anemia Hemolítica Autoimune/induzido quimicamente , Anemia Hemolítica Autoimune/tratamento farmacológico , Imunoglobulinas Intravenosas/efeitos adversos , Fatores Imunológicos/administração & dosagem , Rituximab/administração & dosagem , Anemia Hemolítica Autoimune/diagnóstico , Antígenos CD20 , Transfusão de Sangue , Criança , Feminino , Humanos , Imunoglobulina A/administração & dosagem , Imunoglobulina A/efeitos adversos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/efeitos adversos , Imunoglobulina M/administração & dosagem , Imunoglobulina M/efeitos adversos , Imunoglobulinas Intravenosas/administração & dosagem , Fatores Imunológicos/efeitos adversos , Contagem de Leucócitos , Indução de Remissão , Rituximab/efeitos adversos , Resultado do TratamentoRESUMO
Pseudotumoral cerebellitis in childhood is an uncommon presentation of cerebellitis mimicking a brain tumor. It often follows an inflammatory or infectious event, particularly due to varicella virus. Patients could have a wide clinical spectrum on presentation. Some patients may be asymptomatic or present at most with mild cerebellar signs, whereas others may suffer severe forms with brainstem involvement and severe intracranial hypertension mimicking tumor warranting surgical intervention. Imaging techniques especially multimodal magnetic resonance imaging represent an interesting tool to differentiate between posterior fossa tumors and acute cerebellitis. We describe a case of pseudotumoral cerebellitis in a 6-year-old girl consequent to mumps infection and review the literature on this rare association.