Optimization of newly isolated Bacillus cereus α-amylase production using orange peels and crab shells and application in wastewater treatment.

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.

Clinical and radiological findings of posterior reversible encephalopathy syndrome in children: About 16 children hospitalized in the pediatric department of a Tunisian tertiary care hospital.

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.

Optimization using response surface methodology of phospholipase C production from Bacillus cereus suitable for soybean oil degumming.

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.

Expression of a Copper Activated Xylanase in Yeast: Location of the His-Tag in the Protein Significantly Affects the Enzymatic Properties.

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.

Epidemiological, Bacteriological, and Evolutive Features of Children Hospitalized for Infective Endocarditis in a Tertiary Tunisian Pediatric Department.

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.

Molecular characterization of CTNS mutations in Tunisian patients with ocular cystinosis.

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.

Primary hyperoxaluria type 1 in developing countries: novel challenges in a new therapeutic era.

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.

Lupus Nephritis in Tunisian Children: Predictive Factors of Poor Outcomes.

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.

[Coronary artery aneurysm in Kawasaki disease and its risk factors : a retrospective study about 65 Tunisian children]. / Les complications coronaires de la maladie de Kawasaki et ses facteurs de risque : à propos de 65 cas d'enfants tunisien.

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.

Acute disseminated encephalomyelitis: A retrospective study of 20 children in a pediatrics department in Tunisia.

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.

The optimized production, purification, characterization, and application in the bread making industry of three acid-stable alpha-amylases isoforms from a new isolated Bacillus subtilis strain US586.

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.

Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties.

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.

Success of anti-CD20 monoclonal antibody treatment for severe autoimmune hemolytic anemia caused by warm-reactive immunoglobulin A, immunoglobulin G, and immunoglobulin M autoantibodies in a child: a case report.

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.

Pseudotumoral acute cerebellitis associated with mumps infection in a child.

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.

MDR-1 and CYP3A5 Polymorphisms in Pediatric Idiopathic Nephrotic Syndrome: Impact on Susceptibility and Response to Steroids (Preliminary Results).

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.

Clinical and imaging features of malignant infantile osteopetrosis.

Tfifha M, Gaha M, Gamaoun W, Chemli J, Mabrouk S, Hassayoun S, Zouari N, Jemni H, Abroug S. Clinical and imaging features of malignant infantile osteopetrosis. Turk J Pediatr 2017; 59: 452-457. Human osteopetrosis is a rare genetic disorder caused by osteoclast failure. It encompasses a group of highly heterogeneous forms, ranged widely in severity. Patients with autosomal recessive osteopetrosis are the most severely affected osteopetrotic patients. Here we describe Tunisian children with severe phenotype. They are native from the same geographic region, born to consanguineous parents. Clinical features were cranio-facial dysmorphy, macrocephaly, hepatosplenomegaly, severe anemia and thrombocytopenia with precocious onset of neuronopathic complications, blindness and deafness. Retinal atrophy, reported in a minority of forms is highlighted. Skeletal radiographs revealed generalized increase in bone density and abnormal metaphyseal remodeling, and superimposed rickets resulting from the defect in osteoclasts to provide a normal Ca/P balance. We report an exceptional association with congenital hypothyroidism. Multi-organ failure due to sepsis is one the most severe complications observed. The issue was fatal without hematopoietic stem cell transplantation.

Mutational Analysis of Agxt in Tunisian Population with Primary Hyperoxaluria Type 1.

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.

Ellis van Creveld syndrome in a Tunisian child revealed by an Eisenmenger syndrome.

Ellis-van Creveld syndrome (EvC) is an autosomal recessive inherited disease resulting from mutations in EVC1 or EVC2. Patients with this condition normally have chondrodysplasia, postaxial polydactyly, ectodermal dysplasia and congenital heart defects. We report the case of a 13-year-old Tunisian child who was admitted for cyanosis and acute heart failure. On clinical examination, he presented with typical features of EvC, cyanosis and dyspnea. EvC was confirmed by genetic tests, and echocardiography showed a partial atrioventricular canal defect with supra-systemic pulmonary artery pressure. The patient was treated; however, the evolution was fatal.

Improvement of Trichoderma reesei xylanase II thermal stability by serine to threonine surface mutations.

Three simple mutants, S80T, S146T, and S149T, and a double mutant, S80T-S149T, were constructed and expressed in Escherichia coli to replace Serine on the surface of the Trichoderma reesei xylanase protein with Threonine residues. While the Wild-type (WT) xylanase showed a half-life time (t1/2) of 20 min at 55 °C, the double mutant was more thermostable exhibiting a t1/2 value of 37 min, followed by the S80T and S149T mutants whose t1/2 values were 25 and 23 min, respectively. At 55 °C, the S146T mutant showed a decrease in thermostability with a t1/2 value of 3 min. While the WT enzyme retained only 32% of residual activity after incubation for 5 min at 60°C, the S80T, S149T, and the S80T-S149T mutant enzymes retained 45%, 41%, and 60%, respectively. Molecular modeling attributed the increase in the thermostability of the S80T and S149T mutants to a new hydrogen bond formation and a packing effect, respectively.