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Toxoplasmosis is a widespread zoonosis with an impact on immunocompromised people and critical in pregnant women because of its transmission to the fetus. Recombinant Toxoplasma gondii antigens produced in Escherichia coli are useful for antibody detection in patient’s blood. The aim of the study was to evaluate the feasibility of deriving chromosome-edited E. coli clones producing T. gondii antigens. Here, we present the CRISPR-Cas9 facilitated editing of the E. coli genome to produce SAG2 and GRA2 T. gondii antigens. Moreover, we have derived a clone that produces both proteins and an additional clone producing a novel fusion protein, SAG2-GRA2. These proteins, bearing His-tag, can be easily purified and are useful to detect anti-Toxoplasma antibodies in human blood. We conclude that it is feasible to edit the E. coli chromosome to produce T. gondii antigens that are bound by antibodies present in people affected by toxoplasmosis.

Gender discrimination (GD) has been frequently linked to mental health. The heterogeneity of GD definition has led to different assessment methodologies and variation around the analysis of GD. This can affect the study of the association between GD and health outcomes. The main goal of this systematic scoping review is the review of previous studies to operationalize the definition of the GD construct. Three search strategies were set in Pubmed, CINAHL and PsycINFO. 1st and 2nd search strategies included studies if their main focus was either, the analysis of discrimination perception, triggers of discrimination or the analysis of GD effects and associated factors to its perception. 3rd strategy was focused on the identification of GD questionnaires. The prevalence of GD, factors and consequences associated with GD perception and forms of discrimination were the principal variables collected. Risk of bias was assessed (PROSPERO: CRD42019120719). A total of 925 studies were obtained and 84 papers included. GD analysis environments were described. 60 questionnaires of discrimination were identified. Prevalence of GD varied between 3.4-67%. Female gender and a younger age were the factors most frequently related to GD. Poorer mental health was the most frequent consequence. Two components of the GD construct were identified: undervaluation (different recognition, opportunities in access, evaluation standards and expectations) and different treatment (verbal abuse and behavior). GD is measured in several environments and with different methodologies. The two component definition of GD can add order and precision to the measurement, increase response rates and reported GD. Key messages The heterogeneity in the conception of gender discrimination has led to different ways of assessment and to a great heterogeneity around the analysis of GD perception. This operative gender discrimination construct could add order and precision to its measurement, increase response rates and reported GD.

Therapeutic patient education (TPE) is effective and essential in the context of the growing prevalence of chronic diseases, in which tools are needed for planning structured programs. The objective of this project was to develop guidelines for designing and assessing a TPE program.1) We assembled a multidisciplinary group of 8 leaders in TPE, chronicity, quality and safety from the hospital and the university. 2) We conducted an exhaustive review of the scientific literature on the planning of TPE programs directed at chronically ill patients, their relatives and caregivers. 3) The final text underwent comments and suggestions by participants from the hospital and primary care centre during a course on information and TPE methodology. The recommendations were unanimously agreed upon by the writing group.We obtained a standardised work procedure targeted at professionals involved in planning TPE programs, based on international recommendations. The document is structured into sections: a) Definition of the health problem and analysis of the situation; b) Program structure (human resources and materials); objectives (health-related, behaviour-related and educational) and methodology; c) Path the patient and family/caregiver follows in the program; and d) Assessment and indicators. The assessment of the procedure, in the framework of the methodology courses, was favourable.The methodology provided by this document serves as an instrument for the standardised and systematic planning of educational programs and unifies the criteria in their drafting. However, the document needs to be adapted to the condition and population to which each program is directed.

In this prospective, multicentre cohort study, we analysed specific prognostic factors and the impact of timing of highly active antiretroviral therapy (HAART) on disease progression and death among 625 human immunodeficiency virus (HIV)-1-infected, treatment-naïve patients diagnosed with an AIDS-defining disease. HAART was classified as early (35 years, and low CD4+ count (

An eight-month-old Labrador retriever was presented with a grade IIIb open shearing injury of the left tarsus. Acute severe surgical site infection developed 2 days after pan-tarsal arthrodesis, resulting in wound dehiscence. Vacuum-assisted wound therapy was carried out for 12 days to treat an extensive full-thickness soft tissue defect with exposure of metal implants. New granulation tissue formation covering most of the defect was achieved by day 10 of this therapy. Epithelialization was achieved by second intention healing with open wound management. To the authors' knowledge, this is the first veterinary clinical case report documenting complete healing over exposed metal implants without any requirement for surgical revision.

It has been found that RNA can act not only as a genetic carrier, but also as a biological catalyst. The concept of a ribozyme, created in 1982, and the hypothesis of a RNA world, reflect this dual behaviour. This chapter is devoted to studying the mechanisms of several reactions catalysed by natural ribozymes with the aim of detecting the main catalytic factors that intervene in this kind of enzyme. It has been shown that the dications Mg2+, which help to neutralise the large negative charge of the main chain, not only play the structural role that they have in the ribosome, but can also play a direct role in the catalytic process in some cases. The molecular dynamics (MD) simulations are very important in order to clarify the catalytic mechanism of natural ribozymes and are becoming basic for achieving the rational design of low-cost artificial ribozymes in the near future.

This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (K(m) = 0.24 +/- 0.01 mM and V(max) of 8.5 +/- 0.12 pmol.microg protein(-1) min(-1)), pH-insensitive and largely Na(+)-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na(+). These results strongly implicate system y(+)L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y(+)LAT-1 but not y(+)LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y(+)LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Twenty three complexes of the type (1–23) have been synthesized by the three-component cycloaddition (3CC) of [M(CN)(CO)5]− (M  Cr, Mo, W) with various isocyanides CNR1 (R1  Me, Cy, tBu, p-Tol, CH2CO2Et and CH2SO2Tol-p) and carbonyl compounds R2R3CO (R2, R3 =for example, Me, Me; Et, Et; tBu, Me; H, iPr; CF3, CF3; [CH2]4; [CH2]5). Several other mono- and di-cyano complexes ([Mn2(CN)(CO)9]−, [Fe(CN)2Cp(CO)]−, [Fe(CN)Cp(dppe)], [Ru(CN)Cp(dppe)] and [Co(CN)2(dmgH)2]−) also undergo this obviously general reaction resulting in the 4-amino-oxazolin-2-ylidene complexes [M  Fe (34), Ru (35)] and (36). With amine hydrochloride as the fourth component, however, the 4CC products (29), [R1  Cy, R2 = Ph (37); p-Tol (38); R1  tBu, R2 = p-Tol (39)] have been obtained on using a molar ratio of cyano complex/organic reagents of 1: 2, afforded the bis(heterocyclic) complexes (40). To confirm the claimed nature of the products, X-ray structure analyses have been carried out on (27). Detachment of the carbenoid 4-amino-imidazoles from the complexes [M  W, R  Cy (46); M  Cr, R = tBu (47)] by oxidation with KmNO4/Fe(NO3)3 surprisingly gave the 4-amino-5-hydroxyimidazol-2-ones 48 and 49 which were characterized by X-ray analyses.

SummaryIn this case series we describe the diagnosis and surgical treatment of five cats affected by clinical cauda equina syndrome as a result of degenerative lumbosacral stenosis. Radiographic and magnetic resonance imaging findings confirmed the suspected diagnosis of disc-associated lumbosacral disease. Cauda equina decompression was achieved by dorsal laminectomy followed by dorsal annulectomy and nuclear extirpation. Dorsal stabilization was achieved using miniature positive-profile pins inserted into the vertebral body of L7 and the wings of S1 with the free ends of the pins being embedded in a bolus of gentamicin-impregnated polymethylmethacrylate. Reassessment two years postoperatively using a previously validated feline specific owner questionnaire indicated satisfactory outcome with complete return to normal activity and resolution of signs of pain in all cases.

To identify groups of patients receiving hospital-based educational programs, to determine whether the education was structured or ad hoc, and to analyse information on the structure, process and results of the programs.We performed a cross-sectional study using a translated and adapted version of the National Standards for Diabetes Self-Management Education. Information was collected on structure (resources, availability of a written program and professional training), the process followed by the patient (baseline assessment, methodology, training courses, support materials and record of activities), patient outcomes (post-intervention assessment, tools, record of evaluation and results) and program results (number of patients/relatives included, regular assessment).Patient groups receiving education were: patients with chronic diseases, patients with treatable psychiatric disorders and patients with oncological and haematological processes. Most educational activities involved informative activities and technical skills training, both on-demand and integrated in care activity. Structured therapeutic education programs were aimed at patients/relatives with: diabetes, obesity, musculoskeletal diseases, AIDS, splenectomy, chronic obstructive pulmonary disease, hypertension and urinary incontinence. Not all programs had written guidelines or defined parameters with respect to structure, process and results.The application of quality standards to hospital educational programs is useful in detecting: patients receiving education and the quality, type and weaknesses of the programs studied. Software based on these standards may provide information on trends in patient education, identify opportunities for improvement and aid the evaluation of the impact of each educational activity on the quality indicators associated with each program.

A case of bilateral fibrotic contracture of the infraspinatus muscles in a five-year-old Belgian Shepherd dog is described. The dog was presented with progressive forelimb lameness with postural and gait abnormalities three months after an episode of overexertion. When walking, the lower part of both forelimbs swung in a lateral arc causing a circumduction movement and in the standing position, the dog showed elbow adduction with external rotation of the distal part of both front limbs. Orthopaedic examination revealed bilateral atrophy of both infraspinatus and supraspinatus muscles and restriction in the range of motion of both shoulders, especially when attempting abduction and flexion. No specific findings were observed in the shoulder or elbow radiographs but hyperechogenic areas were evident in the ultrasonographic examination of both infraspinatus muscles. A diagnosis of fibrotic contracture of both infraspinatus muscles was established and bilateral tenectomy of the insertion tendons of the infraspinatus muscles was performed. Complete recovery of the animal was achieved after the surgery, which was confirmed in a long-term follow-up (10 months). In conclusion, physical examination and ultrasonography allowed a proper diagnosis of the condition, and tenectomy of the infraspinatus muscles resulted in a complete recovery of the patient even with bilateral involvement.

Background: AEG1 is highly expressed in most solid tumors conferring multidrug resistance, increased proliferation, angiogenesis, metastatic potential and poor outcome. AEG1 activates multiple signal transduction pathways such as PIK3/AKT, NFKB, MAPK, WNT and is a rational target for therapeutic intervention in cancer. Our previous data in advanced EGFR mutated and EGFR wild type lung cancer patients confirmed that AEG1 overexpression is indicative of poor outcome to targeted and pleiotropic therapy. In addition, AEG1 functionality is instrumental in the natural history of brain tumors. Material and Methods: We designed an in silico model based on inhibition of the NFkB (p65) binding domain of AEG1, identifying a possible interacting interface between the two proteins that could be modeled as a 3-dimensional structure. This interface was reduced and transformed into a 3-point pharmacophore for virtual screeningcomparing both molecular interaction fields and the pharmacophore. A total of 217,000 chemical entities (CEs) were tested in this model. Each CE was scored using the Hercules affinity ranking. The top sixty CEs amongst the high scores were selected, and thus 4 compounds were proposed for in vitro screening. Twenty analogs were then synthesized, and PB03, a small molecular weight polyheterocyclic compound, was selected as the CE lead. Results: PB03 displays antitumor activity at low mM and nM concentrations. PB03-induced growth inhibition appears to be independent of EGFR, KRAS, BRAF, PIK3CA and p53 mutational status, Her2 amplification, and BRCA1 and AEG1 mRNA expression. PB03 clusters in vitro molar potency (MP) of Glial (Gliobastoma, GBM) and Neural Crest (Neuroblastoma (NBM) and Melanoma) -derived tumors as compared with PB03 profile in other solid tumors and in non-tumoral cells (p < 0.0001). A lack of impact of MGMT methylation status on PB03 (MP) sensitivity has been observed. PB03 is between 500 and 1000 fold more potent than temozolomide and MTIC in GBM and NBM (p < 0.0001). The mean PB03 IC50s in human non-tumoral cell lines is 2.08mM versus 1.22mM in the solid tumor panel vs 0.40mM in the Glial and Neural Crest-derived panel vs 0.11mM in GBM+NBM. Preliminary data in PC9 shows downregulation of AEG1 mRNA expression upon exposure to PB03. Expansion of the Glial and Neural Crest-derived tumor panel, cell kinetics, drug-induced cell death and in vivo/PK-PD studies in in vivo models is ongoing. PB03 has a negative score when screened in the SCIfinder chemical data base confirming PB03 as a first-in-class CE. Conclusions: This is the first successful attempt at the pharmacological intervention against the AEG1 pathway. The observed clustering of Glial and Neural Crest-derived malignancies is unique and a tool for rational development.

Aim: In the SLCG large-scale screening, 16.6% of EGFR mutations were found, median PFS and OS for pts receiving erlotinib was 14 mos and 27 mos, respectively. The HR for the duration of PFS was 1.68 for the presence of L858R mutation in paired serum DNA (P = 0.02) (NEJM 2009; 361:958-67). Mutant EGFR NSCLC selectively activates STAT3 signaling which promotes cell survival and single EGFR inhibition can even enhance STAT3 activation. Growing evidence shows that targeting STAT3 or upstream or downstream components could improve the outcome. Hence, the resistance mechanisms should be revised, T790M is detected in 60% before therapy (reported by SLCG) and MET signaling can cause inter-receptor crosstalk with AXL, EPHA2, JAK1 and CDCP1. BIM mRNA expression is an independent predictive marker of response to erlotinib (reported by SLCG). High BIM can predict response and early adaptive resistance via IL6-STAT3-Bcl2. In those patients with low BIM, STAT3 can be activated through over-expression of AXL and EPHA2. Crosstalk between TGFb and NOTCH signaling can also be involved. Methods: Transcripts involving STAT3 signaling were examined from 80 EGFR mutant NSCLC pts, including BIM, STAT3 and SHP2. Also MET, AXL, Mer, EphA2 and Gas6 were analyzed. Together with TGFb and NOTCH constituents: ADAM17, NUMB, HES1, DUSP1, RBJ, LncRNA-ATB, SLUG, GLI and PKCi. Repurposing drugs, in combination with gefitinib, erlotinib and afatinib, were examined in cell cultures and in subcutaneous and orthotopic xenograft mice models. Results: Data will be presented on the correlation of BIM and SHP2 with regard to the mechanism of activation of STAT3 and the influence in response, PFS and OS in the 80 erlotinib-treated EGFR mutant NSCLC pts, and the influence of the above mentioned signaling constituents. Early data shows synergism of gefitinib with AXL inhibitor in the PC9-R cells. Also, activity of niclosamide and paclitaxel. Conclusions: Based on the results, the SLCG-GFCP will carry out a trial with erlotinib in combination with the most active repurposing drugs found, customized by BIM expression. Disclosure: All authors have declared no conflicts of interest.

The intramolecular proton transfer in cationized glycine and chlorine substituted derivatives with M = Na+, Mg2+, Ni+, Cu+, and Cu2+ has been studied with the three parameter B3LYP density functional method. The coordination of metal cations to the oxygens of the carboxylic group of glycine stabilizes the zwitterionic structure. For all monocations the intramolecular proton transfer occurs readily with small energy barriers (1-2 kcalmol(-1)). For the dication Mg2+ and Cu2+ systems, the zwitterionic structure becomes very stable. However, whereas for Mg2+, the proton transfer process takes place spontaneously, for Cu2+ the reaction occurs with an important energy barrier. The substitution of the hydrogens of the amino group by chlorine atoms decreases the basicity of nitrogen, which destabilizes the zwitterionic structure. For monosubstituted glycine complexed with Na+, the zwitterionic structure still exists as a minimum, but for disubstituted glycine no minimum appears for this structure. In contrast, for Mg2+ complexed to mono- and disubstituted glycine, the zwitterionic structure remains the only minimum, since the enhanced electrostatic interaction with the dication overcomes the destabilizing effect of the chlorine atoms.

to assess the relations between cardiovascular risk factors and psychological or socio-economic status.We used a self-questionnaire including data upon usual risk factors, anxiety, depression, behavioural pattern, somatic manifestations, life style, incomes and work-related stress. We gathered 955 people, mainly attending an occupational health examination, filling anonymously the same restricted questionnaire (83 questions).Non-working people represented only 4.4 per cent of the study group and included mainly retired people. They differed significantly from wage earners by a higher level of cardiovascular risk and depression, respectively 15.8 (s.d. 11.3) versus 7.8 (6.7) and 5.6 (3.5) versus 3.9 (2.8). These differences can be related to older age of this group. Among working people, 30 per cent felt job-insecurity; they differed significantly from working people by a higher level of anxiety, 8.9 (3.7) versus 7.5 (3.3). There was no difference concerning the absolute cardiovascular risk or the behavioural pattern. A comprehensive self questionnaire is proposed.

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.

Methods for detecting residual malignant cells in patients suffering from lymphoid malignancies have neither been sufficiently sensitive nor easy to routinize, hampering a potential prediction of disease outcome. Taking advantage of clone-specific DNA sequences, generated during lymphocyte differentiation and the polymerase chain reaction, some strategies have been developed for several groups. Up to now the most specific and sensitive methodology, which consists of designing leukemia-specific oligonucleotides, requires sequencing of the complementary determining region III-DNA for each particular patient and is too laborious to be applied to each case for routine monitoring in most hospital laboratories. In an attempt to achieve an easy way to detect residual malignant cells in B lymphoproliferative diseases, we have used a new PCR-based approach, named here as PCR-nuclease protection assay, consisting of: (i) amplification of DNA segments corresponding to the complementarity determining region III of the immunoglobulin heavy chain genes from samples at disease diagnosis; (ii) isolation of the disease-specific single-stranded DNA; (iii) labeling of the single-stranded DNA to generate specific probes; (iv) hybridization to amplified DNA from samples corresponding to different disease phases; and (v) digestion with S1-nuclease. Using this approach, we could detect one malignant cell in a background of 10(5) healthy cells. The sensitivity and specificity of this approach compares with those of the above mentioned specific oligonucleotide strategy in detecting residual malignant B cells. Moreover, this strategy is much less tedious and could be used by most hospital laboratories.

Nucleophile-electrophile combination reactions, hydrolytic reactions, intramolecular hydrogen shifts and some reactions in which the solvent relaxation plays an important role are studied in order to demonstrate the active role of the solvent. The equilibrium or nonequilibrium of transition state solvation are examined in light of the solvent's active role.

By means of the MINDO/3 method the potential surfaces for the method of attack of hydroxide ion on methyl formate and methyl oxalate anion have been studied. In the case of the neutral ester three competitive mechanisms have been found, in good agreemen with experimental results in the gas phase.

Semiempirical MINDO/3 method is used to examine the experimental controversy about the spontaneous migration of a water molecule from the nucleophile to the leaving group in the Walden inversion reaction, HCOOCH 3 + OH − → HCOO − + CH 3 OH, by solvating the nucleophile with different numbers of water molecules.

The semiempirical MINDO/3 method is used to study the kinetic stability of the intermediate that results from the addition of a hydroxide ion to the semiester methyl oxalate anion. The influence of the methyl group orientation is discussed.

En suivant la methode dite du «cycle de Forster», on determine au moyen des methodes de la Chimie Quantique les valeurs des PK des etats excites singulet et triplet de la β-naphtylamine et du β-naphtol. Dans le cas de cette derniere molecule, on propose un modele pour decrire l'ion β-naphtolate intervenant dans les equilibres classiques etudies.

On montre a partir de considerations theoriques qu'une molecule conjugee dont un substituant donneur d'electrons est le centre d'acidite doit devenir plus acide quand eile passe de l'etat fondamental au premier etat electronique excite. Le contraire doit se produire lorsque le substituant est accepteur d'electrons. Enfin une molecule conjugee contenant un heteroatome centre de basicite doit devenir plus basique au cours de la mEme transition. On suggere, par ailleurs, une interpretation du fait que dans cette serie de molecules le premier etat excite triplet possede une force acido-basique tres differente de celle du premier etat excite singlet.

An attempt is made to develop a unified theoretical treatment of the transition state in the reactions of unsaturated molecules. By careful choice of parameters, this treatment is shown to encompass both the Wheland and the delocalized models of the transition state.

The pairing and stacking energies of nucleic acid bases have been computed by the perturbation formulas from reliable eigenvalues and wavefunctions involving all valence electrons for the bases and for the base pairs. The position of minimal energies for the electrostatic components, `corresponding to a free rotation of one base pair around the helix axis to a fixed vertical distance between two monomers equal to 3·36 A, will reach angles around 45 °, whilst the minima of dispersion and exchange components will not reach angles higher than 18 °. Consequently, the angle for the minima of total energy, although not directly computable, is reasonably expected to be around 36 °.

The semiempirical MINDO/3 method is used to study the kinetic stability of the intermediate that results from the addition of a hydroxide ion to the semiester methyl oxalate anion. The influence of the methyl group orientation is discussed.