Targeted chemotherapy overcomes drug resistance in melanoma.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Neuronal differentiation requires BRAT1 complex to remove REST from chromatin.

Repressor element-1 silencing transcription factor (REST) is required for the formation of mature neurons. REST dysregulation underlies a key mechanism of neurodegeneration associated with neurological disorders. However, the mechanisms leading to alterations of REST-mediated silencing of key neurogenesis genes are not known. Here, we show that BRCA1 Associated ATM Activator 1 (BRAT1), a gene linked to neurodegenerative diseases, is required for the activation of REST-responsive genes during neuronal differentiation. We find that INTS11 and INTS9 subunits of Integrator complex interact with BRAT1 as a distinct trimeric complex to activate critical neuronal genes during differentiation. BRAT1 depletion results in persistence of REST residence on critical neuronal genes disrupting the differentiation of NT2 cells into astrocytes and neuronal cells. We identified BRAT1 and INTS11 co-occupying the promoter region of these genes and pinpoint a role for BRAT1 in recruiting INTS11 to their promoters. Disease-causing mutations in BRAT1 diminish its association with INTS11/INTS9, linking the manifestation of disease phenotypes with a defect in transcriptional activation of key neuronal genes by BRAT1/INTS11/INTS9 complex. Finally, loss of Brat1 in mouse embryonic stem cells leads to a defect in neuronal differentiation assay. Importantly, while reconstitution with wild-type BRAT1 restores neuronal differentiation, the addition of a BRAT1 mutant is unable to associate with INTS11/INTS9 and fails to rescue the neuronal phenotype. Taken together, our study highlights the importance of BRAT1 association with INTS11 and INTS9 in the development of the nervous system.

Sex and body mass index impact on digit circumference for Leeds Dactylitis Index calculation.

OBJECTIVES: To estimate digit circumference and the impact of sex and body mass index (BMI) for the calculation of the Leeds Dactylitis Index (LDI) in psoriatic arthritis (PsA) patients with bilateral dactylitis. METHODS: Digit circumference of the hands and the foot were measured with a dactylometer and were studied according to sex and BMI (divided in 4 weight categories) in healthy Portuguese subjects, using Student's t-test and One-way ANOVA, respectively. The effect size of sex and BMI were calculated using Cohen's d test and Eta squared, respectively. Multiple linear regression was used to calculate the effect of sex and BMI, as well as their interaction, to create a formula to predict digit circumference. RESULTS: Fifty-nine participants (33 women, 26 men) with a mean BMI of 24.8 were included. Men's mean digit circumferences were statistically higher than those of women (p<0.001), with a large sex effect size in most of the digits. Differences in the mean circumference between the four BMI categories were statistically significant (p<0.05) for all digits, with a large BMI effect size. Sex and BMI were independent variables to predict mean digit circumference (p<0.001). A new tool (based on regression analysis) allowing to estimate the circumference of digits for males and females of different BMIs is presented. CONCLUSIONS: Our data allows the calculation of digit circumference for males and females of different BMIs in the Portuguese population; and shows that BMI influences digital circumference supporting BMI inclusion in LDI references tables.

Accuracy, potential, and limitations of probabilistic record linkage in identifying deaths by gender identity and sexual orientation in the state of Rio De Janeiro, Brazil.

BACKGROUND: Globally, the counting of deaths based on gender identity and sexual orientation has been a challenge for health systems. In most cases, non-governmental organizations have dedicated themselves to this work. Despite these efforts in generating information, the scarcity of official data presents significant limitations in policy formulation and actions guided by population needs. Therefore, this manuscript aims to evaluate the accuracy, potential, and limits of probabilistic data relationships to yield information on deaths according to gender identity and sexual orientation in the State of Rio de Janeiro. METHODS: This study evaluated the accuracy of the probabilistic record linkage to obtain information on deaths according to gender and sexual orientation. Data from two information systems were used from June 15, 2015 to December 31, 2020. We constructed nine probabilistic data relationship strategies and identified the performance and cutoff points of the best strategy. RESULTS: The best data blocking strategy was established through logical blocks with the first and last names, birthdate, and mother's name in the pairing strategy. With a population base of 80,178 records, 1556 deaths were retrieved. With an area under the curve of 0.979, this strategy presented 93.26% accuracy, 98.46% sensitivity, and 90.04% specificity for the cutoff point ≥ 17.9 of the data relationship score. The adoption of the cutoff point optimized the manual review phase, identifying 2259 (90.04%) of the 2509 false pairs and identifying 1532 (98.46%) of the 1556 true pairs. CONCLUSION: With the identification of possible strategies for determining probabilistic data relationships, the retrieval of information on mortality according to sexual and gender markers has become feasible. Based on information from the daily routine of health services, the formulation of public policies that consider the LGBTQ + population more closely reflects the reality experienced by these population groups.

Characteristics associated with poor COVID-19 outcomes in people with psoriasis, psoriatic arthritis and axial spondyloarthritis: data from the COVID-19 PsoProtect and Global Rheumatology Alliance physician-reported registries.

OBJECTIVES: To investigate factors associated with severe COVID-19 in people with psoriasis (PsO), psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA). METHODS: Demographic data, clinical characteristics and COVID-19 outcome severity of adults with PsO, PsA and axSpA were obtained from two international physician-reported registries. A three-point ordinal COVID-19 severity scale was defined: no hospitalisation, hospitalisation (and no death) and death. ORs were estimated using multivariable ordinal logistic regression. RESULTS: Of 5045 cases, 18.3% had PsO, 45.5% PsA and 36.3% axSpA. Most (83.6%) were not hospitalised, 14.6% were hospitalised and 1.8% died. Older age was non-linearly associated with COVID-19 severity. Male sex (OR 1.54, 95% CI 1.30 to 1.83), cardiovascular, respiratory, renal, metabolic and cancer comorbidities (ORs 1.25-2.89), moderate/high disease activity and/or glucocorticoid use (ORs 1.39-2.23, vs remission/low disease activity and no glucocorticoids) were associated with increased odds of severe COVID-19. Later pandemic time periods (ORs 0.42-0.52, vs until 15 June 2020), PsO (OR 0.49, 95% CI 0.37 to 0.65, vs PsA) and baseline exposure to TNFi, IL17i and IL-23i/IL-12+23i (OR 0.57, 95% CI 0.44 to 0.73; OR 0.62, 95% CI 0.45 to 0.87; OR 0.67, 95% CI 0.45 to 0.98; respectively; vs no disease-modifying antirheumatic drug) were associated with reduced odds of severe COVID-19. CONCLUSION: Older age, male sex, comorbidity burden, higher disease activity and glucocorticoid intake were associated with more severe COVID-19. Later pandemic time periods, PsO and exposure to TNFi, IL17i and IL-23i/IL-12+23i were associated with less severe COVID-19. These findings will enable risk stratification and inform management decisions for patients with PsO, PsA and axSpA during COVID-19 waves or similar future respiratory pandemics.

Determinants of health-related quality of life and global functioning and health in axSpA, pSpA, and PsA: results from the ASAS-PerSpA study.

OBJECTIVES: We aim to identify determinants of health-related quality of life (HRQoL) and global functioning and health (GH) in axial spondyloarthritis (axSpA), peripheral spondyloarthritis (pSpA), and psoriatic arthritis (PsA). METHODS: ASAS-perSpA study data were analyzed. Models for the three patient groups were performed separately to explore factors associated with HRQoL and GH, assessed by EQ-5D and ASAS-HI, respectively. RESULTS: The analyses included 4185 patients: 2719 with axSpA, 433 with pSpA, and 1033 with PsA.In axSpA, disease activity (DA) (ß=-0.061), physical function (ß=-0.041), female sex (ß=-0.019), and fibromyalgia (ß=-0.068) were associated with worse HRQoL; age (ß = 0.001) and university education (ß = 0.014) with better HRQoL. In pSpA, DA (ß=-0.04) and physical function (ß=-0.054) were associated with worse HRQoL. In PsA, DA (ß=-0.045), physical function (ß=-0.053), axial disease (ß=-0.041), and female sex (ß=-0.028) were associated with worse HRQoL.In axSpA, DA (ß = 0.889), physical function (ß = 0.887), peripheral disease (ß = 0.564), female sex (ß = 0.812) and fibromyalgia (ß = 1.639) were associated with worse GH; age (ß=-0.013) and university education (ß=-0.274) with better GH. In pSpA, physical function (ß = 1.142), and female sex (ß = 1.060) were associated with worse GH; university education (ß=-0.611) with better GH. In PsA, DA (ß = 0.703), physical function (ß = 1.025), axial involvement (ß = 0.659), female sex (ß = 0.924), and fibromyalgia (ß = 1.387) were associated with worse GH; age (ß=-0.024) and university education (ß=-0.856) with better GH. CONCLUSIONS: DA and physical function are major HRQoL and GH determinants across spondyloarthritis types, and clinical characteristics and sociodemographic factors play an important role, highlighting the importance of a holistic approach for individual patients.

Health-related quality of life among spondyloarthritis and chronic low back pain patients: results from a nationwide population-based survey.

PURPOSE: Both spondyloarthritis and chronic low back pain (CLBP) significantly impact health-related quality of life (HRQoL). It is important to clarify whether these disorders have different impacts on the several domains of HRQoL as different mechanisms may necessitate different treatment interventions. Moreover, the factors associated with HRQoL can inform more targeted group interventions to promote HRQoL. METHODS: We used data from EpiReumaPt, a population-based survey conducted from September 2011 to December 2013. HRQoL was assessed with EuroQoL-5-Dimensions (EQ-5D). Spondyloarthritis was diagnosed by expert opinion (rheumatologist) and predefined criteria. CLBP was diagnosed if low back pain was present on the day of the interview and persisted for > 90 days. Univariable and multivariable linear regression analyses compared HRQoL among subjects with spondyloarthritis, CLBP, and no rheumatic diseases. Multivariable linear regression analyses evaluated HRQoL factors in spondyloarthritis and CLBP subjects. RESULTS: We included 92 spondyloarthritis patients, 1376 CLBP patients, and 679 subjects without rheumatic diseases. HRQoL was similarly affected in spondyloarthritis and CLBP (ß = - 0.03, 95% CI [- 0.08; 0.03]) in all EQ5D dimensions. A much lower HRQoL was found in spondyloarthritis and CLBP patients compared with subjects without rheumatic diseases (ß = - 0.14, 95% CI [- 0.19; - 0.10]; ß = - 0.12, 95% CI [- 0.14; - 0.09], respectively). In spondyloarthritis subjects, multimorbidity and active disease were associated with worse HRQoL (ß = - 0.18; 95% CI [- 0.24; 0.03]; ß = - 0.13; 95% CI [- 0.29; - 0.05], respectively), and regular physical exercise was associated with better HRQoL (ß = 0.18; 95% CI [0.10; 0.30]). In CLBP subjects, multimorbidity (ß = - 0.11; 95% CI [- 0.14; - 0.08]), obesity (ß = - 0.04; 95% CI [- 0.08; - 0.01]), and low back pain intensity (ß = - 0.02; 95% CI [- 0.03; - 0.02]) were associated with worse HRQoL, and regular physical exercise (ß = 0.08; 95% CI [0.05; 0.11]) was significantly associated with better HRQoL. CONCLUSION: Spondyloarthritis and CLBP subjects reported similar levels of impairment in the mental, physical, and social domains of HRQoL. Future health plans should address modifiable factors associated with HRQoL in these conditions to achieve better outcomes.

Record linkage under suboptimal conditions for data-intensive evaluation of primary care in Rio de Janeiro, Brazil.

BACKGROUND: Linking Brazilian databases demands the development of algorithms and processes to deal with various challenges including the large size of the databases, the low number and poor quality of personal identifiers available to be compared (national security number not mandatory), and some characteristics of Brazilian names that make the linkage process prone to errors. This study aims to describe and evaluate the quality of the processes used to create an individual-linked database for data-intensive research on the impacts on health indicators of the expansion of primary care in Rio de Janeiro City, Brazil. METHODS: We created an individual-level dataset linking social benefits recipients, primary health care, hospital admission and mortality data. The databases were pre-processed, and we adopted a multiple approach strategy combining deterministic and probabilistic record linkage techniques, and an extensive clerical review of the potential matches. Relying on manual review as the gold standard, we estimated the false match (false-positive) proportion of each approach (deterministic, probabilistic, clerical review) and the missed match proportion (false-negative) of the clerical review approach. To assess the sensitivity (recall) to identifying social benefits recipients' deaths, we used their vital status registered on the primary care database as the gold standard. RESULTS: In all linkage processes, the deterministic approach identified most of the matches. However, the proportion of matches identified in each approach varied. The false match proportion was around 1% or less in almost all approaches. The missed match proportion in the clerical review approach of all linkage processes were under 3%. We estimated a recall of 93.6% (95% CI 92.8-94.3) for the linkage between social benefits recipients and mortality data. CONCLUSION: The adoption of a linkage strategy combining pre-processing routines, deterministic, and probabilistic strategies, as well as an extensive clerical review approach minimized linkage errors in the context of suboptimal data quality.

Clinical and Multimodal Imaging Findings and Risk Factors for Ocular Involvement in a Presumed Waterborne Toxoplasmosis Outbreak, Brazil1.

In 2015, an outbreak of presumed waterborne toxoplasmosis occurred in Gouveia, Brazil. We conducted a 3-year prospective study on a cohort of 52 patients from this outbreak, collected clinical and multimodal imaging findings, and determined risk factors for ocular involvement. At baseline examination, 12 (23%) patients had retinochoroiditis; 4 patients had bilateral and 2 had macular lesions. Multimodal imaging revealed 2 distinct retinochoroiditis patterns: necrotizing focal retinochoroiditis and punctate retinochoroiditis. Older age, worse visual acuity, self-reported recent reduction of visual acuity, and presence of floaters were associated with retinochoroiditis. Among patients, persons >40 years of age had 5 times the risk for ocular involvement. Five patients had recurrences during follow-up, a rate of 22% per person-year. Recurrences were associated with binocular involvement. Two patients had late ocular involvement that occurred >34 months after initial diagnosis. Patients with acquired toxoplasmosis should have long-term ophthalmic follow-up, regardless of initial ocular involvement.

Trehalose-6-phosphate-mediated phenotypic change in Acinetobacter baumannii.

The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD-glucose and glucose-6-phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose-6-phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose-6-phosphate phosphatase activity accumulated trehalose-6-phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose-6-phosphate. Our results demonstrate that trehalose-6-phosphate affects multiple physiological activities in A. baumannii ATCC 19606.

Predicted dynamical couplings of protein residues characterize catalysis, transport and allostery.

MOTIVATION: Protein function is intrinsically linked to native dynamics, but the systematic characterization of functionally relevant dynamics remains elusive besides specific examples. Here we exhaustively characterize three types of dynamical couplings between protein residues: co-directionality (moving along collinear directions), coordination (small fluctuations of the interatomic distance) and deformation (the extent by which perturbations applied at one residue modify the local structure of the other one), which we analytically compute through the torsional network model. RESULTS: We find that ligand binding sites are characterized by large within-site coordination and co-directionality, much larger than expected for generic sets of residues with equivalent sequence distances. In addition, catalytic sites are characterized by high coordination couplings with other residues in the protein, supporting the view that the overall protein structure facilitates the catalytic dynamics. The binding sites of allosteric effectors are characterized by comparably smaller coordination and higher within-site deformation than other ligands, which supports their dynamic nature. Allosteric inhibitors are coupled to the active site more frequently through deformation than through coordination, while the contrary holds for activators. We characterize the dynamical couplings of the sodium-dependent Leucine transporter protein (LeuT). The couplings between and within sites progress consistently along the transport cycle, providing a mechanistic description of the coupling between the uptake and release of ions and substrate, and they highlight qualitative differences between the wild-type and a mutant for which chloride is necessary for transport. AVAILABILITY AND IMPLEMENTATION: The program tnm is freely available at https://github.com/ugobas/tnm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GO-DACT: a phase 3b randomised, double-blind, placebo-controlled trial of GOlimumab plus methotrexate (MTX) versus placebo plus MTX in improving DACTylitis in MTX-naive patients with psoriatic arthritis.

OBJECTIVES: To assess the efficacy of golimumab in combination with methotrexate (MTX) versus MTX monotherapy in psoriatic arthritis (PsA) dactylitis. METHODS: Multicentre, investigator-initiated, randomised, double-blind, placebo-controlled, parallel-design phase 3b trial in 11 Portuguese rheumatology centres. Patients with PsA along with active dactylitis and naive to MTX and biologic disease-modifying antirheumatic drugs (bDMARDs) were randomly assigned to golimumab or placebo, both in combination with MTX. The primary endpoint was Dactylitis Severity Score (DSS) change from baseline to week 24. Key secondary endpoints included DSS and Leeds Dactylitis Index (LDI) response, and changes from baseline in the LDI and MRI dactylitis score. Analysis was by intention-to-treat for the primary endpoint. RESULTS: Twenty-one patients received golimumab plus MTX and 23 MTX monotherapy for 24 weeks. One patient from each arm discontinued. Patient inclusion was halted at 50% planned recruitment due to a favourable interim analysis. Median baseline DSS was 6 in both arms. By week 24, patients treated with golimumab plus MTX exhibited significantly greater improvements in DSS relative to MTX monotherapy (median change of 5 vs 2 points, respectively; p=0.026). In the golimumab plus MTX arm, significantly higher proportions of patients achieved at least 50% or 70% improvement in DSS and 20%, 50% or 70% improvement in LDI in comparison to MTX monotherapy. CONCLUSIONS: The combination of golimumab and MTX as first-line bDMARD therapy is superior to MTX monotherapy for the treatment of PsA dactylitis. TRIAL REGISTRATION NUMBER: NCT02065713.

Analysing ambiguities in trypanosomatids taxonomy by barcoding.

BACKGROUND: Biodiversity screens and phylogenetic studies are dependent on reliable DNA sequences in public databases. Biological collections possess vouchered specimens with a traceable history. Therefore, DNA sequencing of samples available at institutional collections can greatly contribute to taxonomy, and studies on evolution and biodiversity. METHODS: We sequenced part of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and the SSU rRNA (V7/V8) genes from 102 trypanosomatid cultures, which are available on request at www.colprot.fiocruz.br. OBJECTIVE: The main objective of this work was to use phylogenetic inferences, using the obtained DNA sequences and those from representatives of all Trypanosomatidae genera, to generate phylogenetic trees that can simplify new isolates screenings. FINDINGS: A DNA sequence is provided for the first time for several isolates, the phylogenetic analysis allowed the classification or reclassification of several specimens, identification of candidates for new genera and species, as well as the taxonomic validation of several deposits. MAIN CONCLUSIONS: This survey aimed at presenting a list of validated species and their associated DNA sequences combined with a short historical overview of each isolate, which can support taxonomic and biodiversity research and promote culture collections.

Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants.

Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.

Combined transcriptomics-metabolomics profiling of the heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a remarkable archaeon able to grow at temperatures around 100 °C. To gain insight into how this model hyperthermophile copes with heat stress, we compared transcriptomic and metabolomic data of cells subjected to a temperature shift from 90 °C to 97 °C. In this study, we used RNA-sequencing to characterize the global variation in gene expression levels, while nuclear magnetic resonance (NMR) and targeted ion exchange liquid chromatography-mass spectrometry (LC-MS) were used to determine changes in metabolite levels. Of the 552 differentially expressed genes in response to heat shock conditions, 257 were upregulated and 295 were downregulated. In particular, there was a significant downregulation of genes for synthesis and transport of amino acids. At the metabolite level, 37 compounds were quantified. The level of di-myo-inositol phosphate, a canonical heat stress solute among marine hyperthermophiles, increased considerably (5.4-fold) at elevated temperature. Also, the levels of mannosylglycerate, UDP-N-acetylglucosamine (UDPGlcNac) and UDP-N-acetylgalactosamine were enhanced. The increase in the pool of UDPGlcNac was concurrent with an increase in the transcript levels of the respective biosynthetic genes. This work provides the first metabolomic analysis of the heat shock response of a hyperthermophile.

Characterisation of ocular involvement in an experimental model of neuroschistosomiasis mansoni.

The Global Burden of Disease Study 2010 listed schistosomiasis among the leading 100 causes of death in Brazil, responsible for 3.6% of the estimated total of deaths globally. Eye and adnexa are very rarely affected by schistosomiasis mansoni, with limited documentation of ocular pathology in this setting. This short communication reports ocular histolopathological findings in a murine model of neuroschistosomiasis mansoni. Lesions were found in the bulbar conjunctiva, lacrimal gland, choroid and corneoscleral limbus.

Development of a Measurement Tool to Assess Students' Knowledge and Perceptions About Cancer (SKPaC).

Cancer literacy is currently one of the most important dimensions of cancer continuum. Objective assessment of cancer knowledge in populations remains a challenging field to public health entities. Different evaluation tools are currently available; still, some groups remain disregarded due to the absence of validated instruments. Cancer literacy in adolescents and young adults has been clearly overlooked being a subject that requires new tools to be properly studied. To address this topic, we developed a new instrument and field tested it in a classroom environment for internal reliability, construct, and face validity. "Students Knowledge and Perceptions about Cancer questionnaire" was designed in Portuguese language and adapted to the Portuguese context by a multidisciplinary team. The final version of the questionnaire includes 35 items organized in three sections, encompassing knowledge and perceptions about cancer and socio-biographic data. Cancer experts ensured content validity, while tailoring of contents was refined with high school teachers. Test and retest of the instrument showed a good reliability of the scale and construct validity. Also, the clarity of the questionnaire and suitability to proper evaluate cancer knowledge was consistent between test and retest. The Students' Knowledge and Perceptions About Cancer Questionnaire (SKPaC) showed to be a valid tool to assess adolescents' knowledge and perceptions about cancer that can be used in the educational context.

Classical fragile-X phenotype in a female infant disclosed by comprehensive genomic studies.

BACKGROUND: We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities. CASE PRESENTATION: We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation. CONCLUSIONS: Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.

Production of mannosylglycerate in Saccharomyces cerevisiae by metabolic engineering and bioprocess optimization.

BACKGROUND: Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075-4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production. RESULTS: To drive MG production, the pathway for the synthesis of GDP-mannose, one of the MG biosynthetic precursors, was overexpressed in S. cerevisiae along with the MG biosynthetic pathway. MG production was evaluated under different cultivation modes, i.e., flask bottle, batch, and continuous mode with different dilution rates. The genes encoding mannose-6-phosphate isomerase (PMI40) and GDP-mannose pyrophosphorylase (PSA1) were introduced into strain MG01, hosting a plasmid encoding the MG biosynthetic machinery. The resulting engineered strain (MG02) showed around a twofold increase in the activity of PMI40 and PSA1 in comparison to the wild-type. In batch mode, strain MG02 accumulated 15.86 mgMG g DCW -1 , representing a 2.2-fold increase relative to the reference strain (MG01). In continuous culture, at a dilution rate of 0.15 h-1, there was a 1.5-fold improvement in productivity. CONCLUSION: In the present study, the yield and productivity of MG were increased by overexpression of the GDP-mannose pathway and optimization of the mode of cultivation. A maximum of 15.86 mgMG g DCW -1 was achieved in batch cultivation and maximal productivity of 1.79 mgMG g DCW -1  h-1 in continuous mode. Additionally, a positive correlation between MG productivity and growth rate/dilution rate was established, although this correlation is not observed for MG yield.

The Nuanced Interplay of Intrinsic Disorder and Other Structural Properties Driving Protein Evolution.

Protein evolution often occurs at unequal rates in different sites along an amino acid chain. Site-specific evolutionary rates have been linked to several structural and functional properties of proteins. Previous analyses of this phenomenon have involved relatively small datasets and, in some cases, the interaction among multiple structural factors is not evaluated. Here, we present the results of a large-scale phylogenetic and statistical analysis, testing the effects and interactions of three structural properties on amino acid replacement rates. We used sequence-based computational methods to predict (i) intrinsic disorder propensity, (ii) secondary structure, and (iii) functional domain involvement across millions of amino acid sites in thousands of sequence alignments of metazoan proteins. Our results somewhat corroborate earlier findings that intrinsically disordered sites tend to be more variable than ordered sites, but there is considerable overlap among their rate distributions, and a significant confounding interaction exists between intrinsic disorder and secondary structure. Notably, protein sites that are consistently predicted to be both intrinsically disordered and involved in secondary structures tend to be the most conserved at the amino acid level, suggesting that they are highly constrained and functionally important. In addition, a significant interaction exists between functional domain involvement and secondary structure. These findings suggest that multiple structural drivers of protein evolution should be evaluated simultaneously in order to get a clear picture of their individual effects as well as any confounding interactions among them.