CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

Extensive remodeling of sugar metabolism through gene loss and horizontal gene transfer in a eukaryotic lineage.

BACKGROUND: In yeasts belonging to the subphylum Saccharomycotina, genes encoding components of the main metabolic pathways, like alcoholic fermentation, are usually conserved. However, in fructophilic species belonging to the floral Wickerhamiella and Starmerella genera (W/S clade), alcoholic fermentation was uniquely shaped by events of gene loss and horizontal gene transfer (HGT). RESULTS: Because HGT and gene losses were first identified when only eight W/S-clade genomes were available, we collected publicly available genome data and sequenced the genomes of 36 additional species. A total of 63 genomes, representing most of the species described in the clade, were included in the analyses. Firstly, we inferred the phylogenomic tree of the clade and inspected the genomes for the presence of HGT-derived genes involved in fructophily and alcoholic fermentation. We predicted nine independent HGT events and several instances of secondary loss pertaining to both pathways. To investigate the possible links between gene loss and acquisition events and evolution of sugar metabolism, we conducted phenotypic characterization of 42 W/S-clade species including estimates of sugar consumption rates and fermentation byproduct formation. In some instances, the reconciliation of genotypes and phenotypes yielded unexpected results, such as the discovery of fructophily in the absence of the cornerstone gene (FFZ1) and robust alcoholic fermentation in the absence of the respective canonical pathway. CONCLUSIONS: These observations suggest that reinstatement of alcoholic fermentation in the W/S clade triggered a surge of innovation that goes beyond the utilization of xenologous enzymes, with fructose metabolism playing a key role.

A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast.

Microbe domestication has a major applied relevance but is still poorly understood from an evolutionary perspective. The yeast Torulaspora delbrueckii is gaining importance for biotechnology but little is known about its population structure, variation in gene content or possible domestication routes. Here, we show that T. delbrueckii is composed of five major clades. Among the three European clades, a lineage associated with the wild arboreal niche is sister to the two other lineages that are linked to anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix-Anthropic clade). Using 64 genomes we assembled the pangenome and the variable genome of T. delbrueckii. A comparison with Saccharomyces cerevisiae indicated that the weight of the variable genome in the pangenome of T. delbrueckii is considerably smaller. An association of gene content and ecology supported the hypothesis that the Mix-Anthropic clade has the most specialized genome and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. In contrast to S. cerevisiae, domestication in T. delbrueckii is not primarily driven by alcoholic fermentation but rather by adaptation to dairy and bread-production niches. This study expands our views on the processes of microbe domestication and on the trajectories leading to adaptation to anthropic niches.

Venous thromboembolism-related genetic determinant F11 rs4253417 is a potential prognostic factor in ischaemic stroke.

Ischaemic stroke (IS) and venous thromboembolism (VTE) are two forms of thromboembolism that, although distinct, seem to share numerous risk factors. Concerning genetic risk factors, while many VTE genetic markers have been reported, inclusively by genome-wide association studies (GWAS), the identification and validation of genetic determinants underlying IS pathogenesis have been challenging. Considering that IS and VTE shared biological pathways and aetiological factors, the severity of IS might be also influenced by VTE-related genetic variants. Thus, the present study was designed to analyse the impact of six VTE GWAS-identified genetic variants on the clinical outcome of 363 acute IS patients. Results revealed that the single-nucleotide polymorphism (SNP) F11 rs4253417 was an independent predictor of the 5-year risk of death among patients with total anterior circulation infarct (TACI). Namely, the ones carrying the SNP C allele presented a fourfold increase in the 5-year risk of death compared to TT genotype carriers (CC/CT vs. TT; adjusted HR, 4.240; 95% CI, 1.260-14.270; P = 0.020). This SNP is known to be associated with coagulation factor XI (FXI) levels, thus with implications in haemostasis and inflammation. As such, F11 rs4253417 might be a promising prognostic biomarker among TACI patients to aid in clinical decision-making. However, additional investigation is required to confirm the study's results and dissect the underlying mechanisms.

Physical Examination of Potential Deceased Organ and Tissue Donors: An Overview of the European Landscape.

Physical examination (PE) of donors is essential to identify potential risks to the safety and efficacy of donated organs and tissues and is mandatory in the EU. However, no detailed guidance is available as to how PE should be performed. Health authorities (HA) and health professionals (HP) in member states of the European Committee on Organ Transplantation of the Council of Europe (CD-P-TO) and observer countries completed surveys relating to the regulatory requirements for PE and the professional practice of PE in their countries for organ and tissue donors. The HA survey addressed regulatory aspects, and the HP survey addressed professional practices, training, and respondents' opinions on the value of PE. These surveys revealed significant inter-country variation in the regulatory approach to PE and the performance of PE by professionals. Most respondents opined that PE was important and yielded valuable information in identifying contraindications to donation. There is no consensus at a regulatory or professional level as to how PE should be performed on organ and tissue donors. There is a requirement for agreed best practice guidelines in this area.

Manganese(I) tricarbonyl complexes as potential anticancer agents.

The antiproliferative activity of [Mn(CO)3(N^N)Br] (N^N = phendione 1, bipy 3) and of the two newly synthesized Mn complexes [Mn(CO)3(acridine)(phendione)]OTf (2) and [Mn(CO)3(di-triazole)Br] (4) has been evaluated by MTS against three tumor cell lines A2780 (ovarian carcinoma), HCT116 (colorectal carcinoma), HCT116doxR (colorectal carcinoma resistant to doxorubicin), and in human dermal fibroblasts. The antiproliferative assay showed a dose-dependent effect higher in complex 1 and 2 with a selectivity toward ovarian carcinoma cell line 21 times higher than in human fibroblasts. Exposure of A2780 cells to IC50 concentrations of complex 1 and 2 led to an increase of reactive oxygen species that led to the activation of cell death mechanisms, namely via intrinsic apoptosis for 2 and autophagy and extrinsic apoptosis for 1. Both complexes do not target DNA or interfere with cell cycle progression but are able to potentiate cell migration and neovascularization (for 2) an indicative that their application might be directed for initial tumor stages to avoid tumor invasion and metastization and opening a new avenue for complex 2 application in regenerative medicine. Interestingly, both complexes do not show toxicity in both in vivo models (CAM and zebrafish).

Acquired Hemophilia A associated with bullous pemphigoid: A rare combination.

Acquired Hemophilia A (AHA) is a rare autoimmune disorder, caused by the development of circulating autoantibodies against coagulation factor VIII (FVIII). AHA is associated with bullous pemphigoid in 2% of patients. We report a case of a 74-year-old man admitted with anemia and a tense subcutaneous and muscular hematoma in the right thigh. Blood analysis confirmed AHA. The patient had a recent diagnosis of bullous pemphigoid. Response to bypass agents and corticosteroids was good with bleeding control and normalization of FVIII and negative autoantibodies, respectively. In a 3-month follow-up period after tapering and stopping prednisolone, a relapse occurred, and immunosuppression was reinitiated. An early diagnosis and effective treatment in AHA are essential to reduce morbimortality. A careful tapering of immunosuppression is important to minimize FVIII inhibitor recurrence, as observed in this case.

Phaffia brasiliana sp. nov., a yeast species isolated from soil in a Cerrado-Atlantic Rain Forest ecotone site in Brazil.

During studies of yeasts associated with soil in a Cerrado-Atlantic Rain Forest ecotone site in Brazil, three orange-pigmented yeast strains were isolated from samples collected in Minas Gerais state, Brazil. Molecular analyses combining the 26S rRNA gene (D1/D2 domains) and the internal transcribed spacer (ITS) sequences as well as whole-genome sequence data showed that these strains could not be ascribed to any known species in the basidiomycetous genus Phaffia, and thus they are considered to represent a novel species for which the name Phaffia brasiliana sp. nov. is proposed. The holotype is CBS 16121T and the MycoBank number is MB 839315. The occurrence of P. brasiliana in a tropical region is unique for the genus, since all other species occur in temperate regions. Two factors appear to contribute to the distribution of the novel taxon: first, the region where it was found has relatively moderate temperature ranges and, second, an adaptation to grow or withstand temperatures higher than those of the other species in the genus seems to be in place.

ASP-Enzymosomes with Saccharomyces cerevisiae Asparaginase II Expressed in Pichia pastoris: Formulation Design and In Vitro Studies of a Potential Antileukemic Drug.

The bacterial enzyme asparaginase is the main treatment option for acute lymphoblastic leukemia. However, it causes side effects, such as immunological reactions, and presents undesirable glutaminase activity. As an alternative, we have been studying asparaginase II from Saccharomyces cerevisiae, coded by ASP3 gene, which was cloned and expressed in Pichia pastoris. The recombinant asparaginase (ASP) presented antileukemic activity and a glutaminase activity 100 times lower in comparison to its asparaginase activity. In this work, we describe the development of a delivery system for ASP via its covalent attachment to functionalized polyethylene glycol (PEG) polymer chains in the outer surface of liposomes (ASP-enzymosomes). This new delivery system demonstrated antiproliferative activity against K562 (chronic myeloid leukemia) and Jurkat (acute lymphocytic leukemia) cell lines similar to that of ASP. The antiproliferative response of the ASP-enzymosomes against the Jurkat cells suggests equivalence to that of the free Escherichia coli commercial asparaginase (Aginasa®). Moreover, the ASP-enzymosomes were stable at 4 °C with no significant loss of activity within 4 days and retained 82% activity up to 37 days. Therefore, ASP-enzymosomes are a promising antileukemic drug.

Targeting Cancer Resistance via Multifunctional Gold Nanoparticles.

Resistance to chemotherapy is a major problem facing current cancer therapy, which is continuously aiming at the development of new compounds that are capable of tackling tumors that developed resistance toward common chemotherapeutic agents, such as doxorubicin (DOX). Alongside the development of new generations of compounds, nanotechnology-based delivery strategies can significantly improve the in vivo drug stability and target specificity for overcoming drug resistance. In this study, multifunctional gold nanoparticles (AuNP) have been used as a nanoplatform for the targeted delivery of an original anticancer agent, a Zn(II) coordination compound [Zn(DION)2]Cl2 (ZnD), toward better efficacy against DOX-resistant colorectal carcinoma cells (HCT116 DR). Selective delivery of the ZnD nanosystem to cancer cells was achieved by active targeting via cetuximab, NanoZnD, which significantly inhibited cell proliferation and triggered the death of resistant tumor cells, thus improving efficacy. In vivo studies in a colorectal DOX-resistant model corroborated the capability of NanoZnD for the selective targeting of cancer cells, leading to a reduction of tumor growth without systemic toxicity. This approach highlights the potential of gold nanoformulations for the targeting of drug-resistant cancer cells.

Detection of premature stop codons leading to truncated internalin A among food and clinical strains of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen responsible for outbreaks and sporadic cases of listeriosis, a severe invasive disease. Internalin A (InlA) a protein encoded by inlA has a key role in the mechanism of pathogenesis in L. monocytogenes infection, specifically in the invasion of human intestinal epithelial cells. Studies on inlA have shown that mutations leading to premature stop codons (PMSCs) occur naturally and are associated with impaired virulence of L. monocytogenes strains. Increasing evidence suggests that inlA PMSCs mutations are frequent in strains from foods, but rare among clinical isolates. In this study, 22 L. monocytogenes strains collected in Portugal from the processing environment of a bakery industry (n = 1), different food products (n = 10) and human clinical cases (n = 11) were analysed for mutations in inlA and invasion efficiency in Caco-2 cells. Sequencing revealed previously reported mutations types leading to PMSCs in three food and one clinical strain presenting different molecular serotypes (i.e., IIa, IIb and IIc). The remaining 18 isolates did not show PMSCs in inlA. The four strains with PMSCs in inlA presented lower invasiveness efficiencies in Caco-2 cells (below 8.9%) when compared to the control strain (full-length InlA). In addition, one clinical isolate showed reduced invasion efficiency but no PMSCs in inlA. This isolate showed increased inlA transcript levels to that obtained for the laboratory control strain. Our data support the hypothesis that L. monocytogenes isolated from food have attenuated invasion due to the presence of inlA PMSCs. This information would be critically needed for adequate risk-assessments of the foodborne illness burden associated with L. monocytogenes strains.

Guidelines for the Institutional Implementation of Developmental Neuroprotective Care in the NICU. Part B: Recommendations and Justification. A Joint Position Statement From the CANN, CAPWHN, NANN, and COINN.

The use of age-appropriate care as an organized framework for care delivery in the NICU is founded on the work of Heidelise Als, PhD, and her synactive theory of development. This theoretical construct has recently been advanced by the work of Gibbins and colleagues with the "universe of developmental care" conceptual model and developmental care core measures which were endorsed by the National Association of Neonatal Nurses in their age-appropriate care of premature infant guidelines as best-practice standards for the provision of high-quality care in the NICU. These guidelines were recently revised and expanded. In alignment with the Joint Commission's requirement for healthcare professionals to provide age-specific care across the lifespan, the core measures for developmental care suggest the necessary competencies for those caring for the premature and critically ill hospitalized infant. Further supported by the Primer Standards of Accreditation and Health Canada, the institutional implementation of these core measures require a strong framework for institutional operationalization presented in these guidelines. Part B will present the recommendations and justification of each steps behind the present guidelines to facilitate their implementation.

Guidelines for the Institutional Implementation of Developmental Neuroprotective Care in the Neonatal Intensive Care Unit. Part A: Background and Rationale. A Joint Position Statement From the CANN, CAPWHN, NANN, and COINN.

The use of age-appropriate care as an organized framework for care delivery in the neonatal intensive care unit is founded on the work of Heidelise Als, PhD, and her synactive theory of development. This theoretical construct has recently been advanced by the work of Gibbins and colleagues with the "universe of developmental care" conceptual model and developmental care core measures which were endorsed by the National Association of Neonatal Nurses in their age-appropriate care of premature infant guidelines as best-practice standards for the provision of high-quality care in the neonatal intensive care unit. These guidelines were recently revised and expanded. In alignment with the Joint Commission's requirement for health-care professionals to provide age-specific care across the lifespan, the core measures for developmental care suggest the necessary competencies for those caring for the premature and critically ill hospitalized infant. Further supported by the Primer Standards of Accreditation and Health Canada, the institutional implementation of theses core measures requires a strong framework for institutional operationalization, presented in these guidelines. Part A of this article will present the background and rationale behind the present guidelines and their condensed table of recommendations.

Assessment of discomfort in patients with cognitive failure in palliative care.

OBJECTIVE: When cognitive impairment precludes patients' report of symptoms, it becomes necessary to use other means. The purpose of our study was to evaluate the validity of the method currently in use on our service. METHOD: Two members of the team simultaneously assessed the patient and independently recorded whether the patient showed signs of discomfort, and a third questioned patients with cognitive failure who maintained some ability to respond if something was bothering them. RESULTS: Some 200 assessments were made of 116 patients. The kappa coefficient of agreement was 0.615. The sensitivity was 17% and specificity 99%. The positive predictive value was 88%, and the negative predictive value was 73%. SIGNIFICANCE OF RESULTS: Due to the low sensitivity of this method, it cannot be recommended as a screening tool.

Can the previous therapeutic control of the main risk factors of cerebrovascular disease influence the acetylsalicylic Acid-nonresponsive status in acute ischemic stroke patients? Results from a portuguese prospective cohort study.

BACKGROUND: Acute ischemic stroke (AIS) is a complex disease, and the therapeutic control of its risk factors may influence the efficacy of acetylsalicylic acid (ASA) and the occurrence of new vascular events. The aim of this study was to investigate the potential in vivo properties of a previous treatment controlling the main risk factors of cerebrovascular disease, in the ASA-nonresponsive status in a Portuguese population. METHODS: We conducted a prospective cohort study with the recruitment of 90 patients diagnosed with AIS and a follow-up protocol was set up with recurrent stroke as the main clinical end point under evaluation. At admission, PFA-100 (platelet function analyzer) test was evaluated in blood samples from AIS patients treated with 100 mg/day of ASA and previously treated with antihypertensive, antidiabetic, or statin drugs. RESULTS: We observed that 30% of patients were ASA nonresponders. Multivariate regression analysis indicated that the previous treatment with antihypertensive drugs emerged with a significant risk reduction of an ASA-nonresponsive status (odds ratio, .119; 95% confidence interval, .026-.538; P = .006). Furthermore, our results indicated an influence of ASA-nonresponsive status in a decreased period to a new recurrent stroke event in the time frame of 24 months (P = .005, log-rank test). CONCLUSIONS: ASA is an important part of treatment of AIS, and its efficacy may be improved in previous high-risk cerebrovascular patients, particularly with antihypertensive therapeutic control.

Abnormal lung function in adults with congenital heart disease: prevalence, relation to cardiac anatomy, and association with survival.

BACKGROUND: Restrictive lung defects are associated with higher mortality in patients with acquired chronic heart failure. We investigated the prevalence of abnormal lung function, its relation to severity of underlying cardiac defect, its surgical history, and its impact on outcome across the spectrum of adult congenital heart disease. METHODS AND RESULTS: A total of 1188 patients with adult congenital heart disease (age, 33.1±13.1 years) undergoing lung function testing between 2000 and 2009 were included. Patients were classified according to the severity of lung dysfunction based on predicted values of forced vital capacity. Lung function was normal in 53% of patients with adult congenital heart disease, mildly impaired in 17%, and moderately to severely impaired in the remainder (30%). Moderate to severe impairment of lung function related to complexity of underlying cardiac defect, enlarged cardiothoracic ratio, previous thoracotomy/ies, body mass index, scoliosis, and diaphragm palsy. Over a median follow-up period of 6.7 years, 106 patients died. Moderate to severe impairment of lung function was an independent predictor of survival in this cohort. Patients with reduced force vital capacity of at least moderate severity had a 1.6-fold increased risk of death compared with patients with normal lung function (P=0.04). CONCLUSIONS: A reduced forced vital capacity is prevalent in patients with adult congenital heart disease; its severity relates to the complexity of the underlying heart defect, surgical history, and scoliosis. Moderate to severe impairment of lung function is an independent predictor of mortality in contemporary patients with adult congenital heart disease.

Valproyl-CoA inhibits the activity of ATP- and GTP-dependent succinate:CoA ligases.

BACKGROUND: Valproic acid (VPA) is an effective antiepileptic drug that may induce progressive microvesicular steatosis. The impairment of mitochondrial function may be an important metabolic effect of VPA treatment with potential adverse consequences. OBJECTIVE: To investigate the influence of VPA on the activity of GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL). METHODS: The GTP- and ATP-specific SUCL activities were measured in human fibroblasts in the reverse direction, i.e. the formation of succinyl-CoA. These were assessed at different concentrations of succinate in the presence of VPA, valproyl-CoA and zinc chloride, an established inhibitor of the enzymes. Activities were measured using an optimized HPLC procedure. RESULTS: Valproyl-CoA (1 mM) inhibited the activity of A-SUCL and G-SUCL by 45-55% and 25-50%, respectively. VPA (1 mM) had no influence on the activity of the two enzymes. DISCUSSION: Valproyl-CoA appears to affect the activity of SUCL, especially with the ATP-specific enzyme. Considering the key role of SUCL in the Krebs cycle, interference with its activity might impair the cellular energy status. Moreover, A-SUCL is bound to the nucleoside diphosphate kinase (NDPK), which is responsible for the mitochondrial (deoxy)nucleotide synthesis. An inhibition of A-SUCL might influence the activity of NDPK inducing an imbalance of nucleotides in the mitochondria and eventually mitochondrial DNA depletion. This may account for the potential liver failure associated with valproate therapy, reported in patients with deficiencies within the mitochondrial DNA replicase system such as polymerase gamma 1.

Comparison of ambient solvent extraction methods for the analysis of fatty acids in non-starch lipids of flour and starch.

BACKGROUND: Lipids are minor components of flours, but are major determinants of baking properties and end-product quality. To the best of our knowledge, there is no single solvent system currently known that efficiently extracts all non-starch lipids from all flours without the risk of chemical, mechanical or thermal damage. This paper compares nine ambient solvent systems (monophasic and biphasic) with varying polarities: Bligh and Dyer (BD); modified Bligh and Dyer using HCl (BDHCL); modified BD using NaCl (BDNaCl); methanol-chloroform-hexane (3:2:1, v/v); Hara and Radin (hexane-isopropanol, 3:2, v/v); water-saturated n-butanol; chloroform; methanol and hexane for their ability to extract total non-starch lipids (separated by lipid classes) from wheat flour (Triticum aestivum L.). Seven ambient extraction protocols were further compared for their ability to extract total non-starch lipids from three alternative samples: barley flour (Hordeum vulgare L.), maize starch (Zea mays L.) and tapioca starch (Manihot esculenta Crantz). RESULTS: For wheat flour the original BD method and those containing HCl or NaCl tended to extract the maximum lipid and a significant correlation between lipid extraction yield (especially the glycolipids and phospholipids) and the polarity of the solvent was observed. For the wider range of samples BD and BD HCl repeatedly offered the maximum extraction yield and using pooled standardized (by sample) data from all flours, total non-starch lipid extraction yield was positively correlated with solvent polarity (r = 0.5682, P < 0.05) and water ratio in the solvent mixture (r = 0.5299, P < 0.05). CONCLUSION: In general, BD-based methods showed better extraction yields compared to methods without the addition of water and, most interestingly, there was much greater method dependence of lipid yields in the starches when compared to the flour samples, which is due to the differences in lipid profiles between the two sample types (flours and starches).