Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown

Developing classification criteria for skin-predominant dermatomyositis: the Delphi process

Background The European League Against Rheumatism/American College of Rheumatology classification criteria for inflammatory myopathies are able to classify patients with skin-predominant dermatomyositis (DM). However, approximately 25% of patients with skin-predominant DM do not meet two of the three hallmark skin signs and fail to meet the criteria. Objectives To develop a set of skin-focused classification criteria that will distinguish cutaneous DM from mimickers and allow a more inclusive definition of skin-predominant disease. Methods An extensive literature review was done to generate items for the Delphi process. Items were grouped into categories of distribution, morphology, symptoms, antibodies, histology and contextual factors. Using REDCap™, participants rated these items in terms of appropriateness and distinguishing ability from mimickers. The relevance score ranged from 1 to 100, and the median score determined a rank-ordered list. A prespecified median score cut-off was decided by the steering committee and the participants. There was a pre-Delphi and two rounds of actual Delphi. Results There were 50 participating dermatologists and rheumatologists from North America, South America, Europe and Asia. After a cut-off score of 70 during the first round, 37 of the initial 54 items were retained and carried over to the next round. The cut-off was raised to 80 during round two and a list of 25 items was generated. Conclusions This project is a key step in the development of prospectively validated classification criteria that will create a more inclusive population of patients with DM for clinical research

Definition of treatment goals for moderate to severe psoriasis: a European consensus

Patients with moderate to severe psoriasis are undertreated. To solve this persistent problem, the consensus programme was performed to define goals for treatment of plaque psoriasis with systemic therapy and to improve patient care. An expert consensus meeting and a collaborative Delphi procedure were carried out. Nineteen dermatologists from different European countries met for a face-to-face discussion and defined items through a four-round Delphi process. Severity of plaque psoriasis was graded into mild and moderate to severe disease. Mild disease was defined as body surface area (BSA) ≤10 and psoriasis area and severity index (PASI) ≤10 and dermatology life quality index (DLQI) ≤10 and moderate to severe psoriasis as (BSA > 10 or PASI > 10) and DLQI > 10. Special clinical situations may change mild psoriasis to moderate to severe including involvement of visible areas or severe nail involvement. For systemic therapy of plaque psoriasis two treatment phases were defined: (1) induction phase as the treatment period until week 16; however, depending on the type of drug and dose regimen used, this phase may be extended until week 24 and (2) maintenance phase for all drugs was defined as the treatment period after the induction phase. For the definition of treatment goals in plaque psoriasis, the change of PASI from baseline until the time of evaluation (ΔPASI) and the absolute DLQI were used. After induction and during maintenance therapy, treatment can be continued if reduction in PASI is ≥75%. The treatment regimen should be modified if improvement of PASI is <50%. In a situation where the therapeutic response improved ≥50% but <75%, as assessed by PASI, therapy should be modified if the DLQI is >5 but can be continued if the DLQI is ≤5. This programme defines the severity of plaque psoriasis for the first time using a formal consensus of 19 European experts. In addition, treatment goals for moderate to severe disease were established. Implementation of treatment goals in the daily management of psoriasis will improve patient care and mitigate the problem of undertreatment. It is planned to evaluate the implementation of these treatment goals in a subsequent programme involving patients and physicians

XAF1 as a modifier of p53 function and cancer susceptibility

Cancer risk is highly variable in carriers of the common TP53-R337H founder allele, possibly due to the influence of modifier genes. Whole-genome sequencing identified a variant in the tumor suppressor XAF1 (E134*/Glu134Ter/rs146752602) in a subset of R337H carriers. Haplotype-defining variants were verified in 203 patients with cancer, 582 relatives, and 42,438 newborns. The compound mutant haplotype was enriched in patients with cancer, conferring risk for sarcoma (P = 0.003) and subsequent malignancies (P = 0.006). Functional analyses demonstrated that wild-type XAF1 enhances transactivation of wild-type and hypomorphic TP53 variants, whereas XAF1-E134* is markedly attenuated in this activity. We propose that cosegregation of XAF1-E134* and TP53-R337H mutations leads to a more aggressive cancer phenotype than TP53-R337H alone, with implications for genetic counseling and clinical management of hypomorphic TP53 mutant carriers

Auditor\ueda de gesti\uf3n para el \ue1rea administrativa del Centro Cl\uednico San Crist\uf3bal Hospital Privado, C.A. en la ciudad de San Crist\uf3bal del Estado T\ue1chira

La investigaci\uf3n estuvo dirigida a la realizaci\uf3n de la Auditor\ueda de Gesti\uf3n para el \uc1rea Administrativa del Centro Cl\uednico San Crist\uf3bal Hospital Privado, C.A. ubicado en la ciudad de San Crist\uf3bal del Estado T\ue1chira. La misma se clasific\uf3 dentro del tipo de investigaci\uf3n Explicativa-Casual, No Experimental. En este sentido, se realiz\uf3 una revisi\uf3n bibliogr\ue1fica especializada referida al tema objeto de estudio. Para recabar los datos se utilizaron los instrumentos de levantamiento de informaci\uf3n, tales como: observaci\uf3n directa, entrevista y cuestionario. Se enfoc\uf3 al \ue1rea administrativa, por lo que no se incluy\uf3 en otras \ue1reas operativas de la empresa. El trabajo contempla el cumplimiento de los siguientes objetivos: 1.- Determinar si el \ue1rea administrativa lleva a cabo una gesti\uf3n administrativa de acuerdo a los objetivos previamente definidos por la gerencia. 2.- Diagnosticar los controles internos existentes en dicha \ue1rea. 3.- Analizar las fortalezas y debilidades de la gesti\uf3n administrativa. 4.- Diagnosticar mecanismo de control que permitan al \ue1rea estudiada llevar una \uf3ptima gesti\uf3n administrativa. 5.- Evaluar las estrategias gerenciales aplicados al \ue1rea administrativa. Seg\ufan los resultados de la investigaci\uf3n, se determin\uf3 que la Junta Directiva de la empresa ocupa un punto importante dentro del \ue1rea administrativa, por cuanto es ella la que participa en la fijaci\uf3n de las metas generales y las metas especificas, as\ued como en la toma de decisiones, siendo un aspecto negativo para el \ue1rea administrativa. Se observaron deficiencias en los Controles Internos existentes. Las recomendaciones est\ue1n referidas a que cada unidad operativa debe intervenir tambi\ue9n en la fijaci\uf3n de las metas generales, las metas especificas y en la toma de decisiones. En cuanto a los Controles Internos existentes se sugiere realizar una revisi\uf3n a fin de actualizarlos y mejorarlos

European Guidelines (S1) on the use of high-dose intravenous immunoglobulin in dermatology

Background The treatment of severe dermatological autoimmune diseases and toxic epidermal necrolysis (TEN) with high-dose intravenous immunoglobulin (IVIg) is a well-established procedure in dermatology. As treatment with IVIg is usually considered for rare clinical entities or severe clinical cases, the use of immunoglobulin is not generally based on data from randomized controlled trials that are usually required for the practice of evidence-based medicine. Owing to the rarity of the indications for the use of IVIg, it is also unlikely that such studies will be available in the foreseeable future. Because the high costs of IVIg treatment also limit its first-line use, the first clinical guidelines on its use in dermatological conditions were established in 2008 and renewed in 2011. Materials and methods The European guidelines presented here were prepared by a panel of experts nominated by the EDF and the EADV. The guidelines were developed to update the indications for treatment currently considered as effective and to summarize the evidence base for the use of IVIg in dermatological autoimmune diseases and TEN. Results and conclusion The current guidelines represent consensual expert opinions and definitions on the use of IVIg reflecting current published evidence and are intended to serve as a decision-making tool for the use of IVIg in dermatological diseases