Omics data integration facilitates target selection for new antiparasitic drugs against TriTryp infections.

Introduction: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., commonly referred to as TriTryps, are a group of protozoan parasites that cause important human diseases affecting millions of people belonging to the most vulnerable populations worldwide. Current treatments have limited efficiencies and can cause serious side effects, so there is an urgent need to develop new control strategies. Presently, the identification and prioritization of appropriate targets can be aided by integrative genomic and computational approaches. Methods: In this work, we conducted a genome-wide multidimensional data integration strategy to prioritize drug targets. We included genomic, transcriptomic, metabolic, and protein structural data sources, to delineate candidate proteins with relevant features for target selection in drug development. Results and Discussion: Our final ranked list includes proteins shared by TriTryps and covers a range of biological functions including essential proteins for parasite survival or growth, oxidative stress-related enzymes, virulence factors, and proteins that are exclusive to these parasites. Our strategy found previously described candidates, which validates our approach as well as new proteins that can be attractive targets to consider during the initial steps of drug discovery.

Facial Scanning and Additive Manufacturing Used in Production Nasal Prosthesis.

Maxillofacial prosthesis is an effective treatment for patients with facial sequelae, but it remains challenging for professionals due to its high esthetic complexity. This study describes a clinical case of successful nasal prosthetic rehabilitation using digital technology and additive manufacturing. Initially, the 76-year-old patient, with a facial defect in the nasal region, had her face scanned with 3-dimensional scanner for laboratory planning of the prototype of a nasal prosthesis. After approving the prototype image, working models in muffle shape were obtained in additive manufacture for the inclusion of the prosthesis. In the final session, the prosthesis was colored extrinsically and installed. The procedures digital in the manufacture of the facial prosthesis was applicable and agile, allowing the professional greater predictability regarding the shape of the rehabilitated organ, esthetic improvement in the mutilated area and patient satisfaction in relation to the speed, of the procedure and the quality of the prosthesis.

From Genome to Drugs: New Approaches in Antimicrobial Discovery.

Decades of successful use of antibiotics is currently challenged by the emergence of increasingly resistant bacterial strains. Novel drugs are urgently required but, in a scenario where private investment in the development of new antimicrobials is declining, efforts to combat drug-resistant infections become a worldwide public health problem. Reasons behind unsuccessful new antimicrobial development projects range from inadequate selection of the molecular targets to a lack of innovation. In this context, increasingly available omics data for multiple pathogens has created new drug discovery and development opportunities to fight infectious diseases. Identification of an appropriate molecular target is currently accepted as a critical step of the drug discovery process. Here, we review how diverse layers of multi-omics data in conjunction with structural/functional analysis and systems biology can be used to prioritize the best candidate proteins. Once the target is selected, virtual screening can be used as a robust methodology to explore molecular scaffolds that could act as inhibitors, guiding the development of new drug lead compounds. This review focuses on how the advent of omics and the development and application of bioinformatics strategies conduct a "big-data era" that improves target selection and lead compound identification in a cost-effective and shortened timeline.

African-Americans Have a Higher Propensity for Death from COVID-19: Rationale and Causation.

The purpose of this article is to provide an understanding about the mechanisms that contribute to the proliferation of COVID-19 morbidity and mortality among high-risk populations, and especially African-Americans. African-Americans are succumbing to novel SARS-CoV-2 (COVID-19) at an alarming rate. Current data indicate that while African-Americans represent less than 13.4% of the United States' population, they account for one-third of more than 4.77 million persons with verified COVID-19 infections. Currently, more than 50,258 African-Americans have succumbed to the disease. African-Americans are disproportionately impacted by COVID-19 to an extent unobserved in other racial/ethnic subgroups. In addition, this article describes the physiological event inflammation-mediation storming (cytokine storming). Social determinants of health such as income, education, and employment are hypothesized to impact cogent health care delivery for African-Americans. Included in this article are data on clinical outcomes that highlight the role of pre-existing (health disparities) conditions like diabetes, hypertension, cardiovascular disease, obesity, and lung disease, as barriers to optimal outcomes among African-Americans who are hospitalized with COVID-19. Also explored in this article is causation for vascular complications. A further aim of this article is to provide insight into cause and effect rationales for COVID-19 and health disparities, from both biosocial and health inequality perspectives. Linkages between these selected health disparities and COVID-19 are examined to determine possible deteriorating effects of COVID-19. Finally, techniques are offered to render culturally competent care to African-Americans diagnosed with COVID-19 who present concomitantly with health disparities.

Nutritional status and consumption of inflammatory and anti-inflammatory foods by patients with inflammatory bowel diseases / Estado nutricional e consumo de alimentos inflamatórios e anti-inflamatórios por pacientes portadores de doenças inflamatórias intestinais

ABSTRACT Objective Evaluation of nutritional status and consumption frequency of inflammatory and anti-inflammatory food by patients with inflammatory bowel diseases. Methods An observational study of the patients assisted by the interdisciplinary inflammatory bowel diseases ambulatory of UNIVALI-SC. The nutritional status of patients was evaluated and each patient was categorized according to his/her body max index and also through a research questionnaire of the individual social-economy situation, life habits, and inflammatory and anti-inflammatory food consumption in a determinate period of time. Results Out of the 65 patients, 57% had Crohn's disease and 43% had ulcerative colitis. According to the disease activity, 71% were in remission and 29% in activity. Of the sample, 57% were classified as overweight. It was not possible to correlate nutritional status and type of inflammatory bowel diseases, nutritional status and income or nutritional status and level of education. The most inflammatory foods were beef (65%) and coffee (60%), while the anti-inflammatory ones were garlic (75%), olive oil (54%), and sweet potatoes (23%). There was no association between the most consumed inflammatory and anti-inflammatory food and body max index. Conclusion According to the results, most of the patients were overweight. The most commonly consumed inflammatory foods were beef and coffee and the anti-inflammatory ones were garlic, olive oil, and sweet potatoes.

RESUMO Objetivo Avaliar o estado nutricional e a frequência de consumo de alimentos inflamatórios e anti-inflamatórios por portadores de doenças inflamatórias intestinais. Metodologia Estudo transversal com indivíduos assistidos pelo ambulatório interdisciplinar de doenças inflamatórias intestinais da UNIVALI-SC. Avaliados por meio do estado nutricional e classificados de acordo com o índice de massa corporal, bem como através de um questionário contendo dados socioeconômicos, hábitos de vida e frequência do consumo de alimentos inflamatórios e anti-inflamatórios. Resultados Dos 65 pacientes, 57% eram portadores de doença de Crohn e 43% de retocolite ulcerativa. De acordo com a atividade da doença, 71% encontravam-se em remissão e 29% em atividade. Da amostra, 57% foram classificados como acima do peso. Não foi possível correlacionar estado nutricional e o tipo de doenças inflamatórias intestinais, estado nutricional e renda ou estado nutricional e escolaridade. Os alimentos inflamatórios mais consumidos foram carne de gado (65%) e café (60%), já os anti-inflamatórios foram alho (75%), azeite de oliva (54%) e batata doce (23%). Não houve associação entre os alimentos inflamatórios e anti-inflamatórios mais consumidos e o índice de massa corporal. Conclusão Segundo os resultados, observou-se que a maioria dos pacientes apresentava excesso de peso. Os alimentos inflamatórios mais consumidos foram carne de gado e café e os anti-inflamatórios foram alho, azeite de oliva e batata doce.

O ensino da prevenção nos projetos pedagógicos dos cursos de Medicina do estado de Santa Catarina / The teaching of prevention in the pedagogical projects of the Medicina courses of the state of Santa Catarina

A formação médica envolve o desenvolvimento de competências essenciais que incluem as ações preventivas, as quais apresentam-se em quatro níveis: primário, secundário, terciário e quaternário. O objetivo deste estudo é verificar de que forma os conceitos de prevenção estão presentes nos Projetos Pedagógicos dos cursos de medicina do Estado de Santa Catarina no ano de 2018. Trata-se de um estudo qualitativo e documental, de caráter exploratório, empregando análise temática. Entre 13 cursos identificados, obteve-se acesso a oito PP, dentre versões completas e resumidas. Na análise, foram verificadas cinco categorias distintas da prevenção em saúde: Prevenção influenciada pelas Diretrizes Curriculares Nacionais, Níveis de Prevenção Primária, Secundária e Terciária, e Estratégia de Prevenção Populacional. No exame não foram encontradas menções à P4, embora tenha sido enfatizada a importância da Medicina Baseada em Evidências. Evidencia-se que o destaque para a prevenção estabelecida nas Diretrizes Curriculares Nacionais é reproduzido nos Projetos Pedagógicos. Há, também, franca ênfase nas concepções de prevenção primária, secundária e terciária, em detrimento da quaternária. Ainda que a prevenção quaternária e a Prevenção Populacional possam estar mais presentes no cotidiano da formação médica, mas ausentes dos Projetos Pedagógicos examinados, a pouca visibilidade dessas categorias sugere que tal formação permanece centrada na abordagem de doenças no nível individual, reforçando o risco de medicalização da sociedade sem base em evidências científicas adequadas.

Medical training involves the development of essential skills, including preventive actions, which include the four levels of diseases prevention: primary, secondary, tertiary and quaternary. We verified how the concepts of prevention were presented in the Pedagogical Projects of the medical courses of the State of Santa Catarina in the year 2018. This was an exploratory, qualitative and documental study, using thematic analysis. Of the 13 courses contacted, we obtained access to eight Pedagogical Projects, either in complete or summarized versions. Five distinct categories of diseases prevention were identified: Prevention influenced by the National Curricular Guidelines, Primary, Secondary and Tertiary Prevention Levels, and Population Prevention Strategy. No mention of Quaternary Prevention was found, although the importance of Evidence Based Medicine has been emphasized. Prevention is highlighted in the National Curricular Guidelines and, therefore, in the Pedagogical Project. There is also a clear emphasis on the conceptions of primary, secondary and tertiary prevention, even so quaternary and Populational Prevention could be part of the medical education on a daily basis, but not highlighted on the Pedagogical Project. Such analysis suggests that medical education still lies upon approaches directed to disease in an individual basis, giving strength to medicalization in regard of a solid evidence based medicine practice.

Chloroquine and Hydroxychloroquine for the Treatment of COVID-19: Review and Appraisal of Current Evidence.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated coronavirus disease 19 (COVID-19) began ravaging most of the globe in November 2019. In the United States more than 25 million people have been infected with SARS-CoV-2. To date, COVID-19 has killed close to 400,000 U.S. citizens. In the face of limited pharmacotherapies, the current burden of SARS-CoV-2 and COVID-19 signals overwhelming sickness and trillions in healthcare costs ahead. The need to expeditiously identify safe and efficacious prophylaxis and treatment options is critical. Drug repositioning may be a promising strategy toward mitigating the impact of SARS-CoV-2 and COVID-19. This rapid review appraises available evidence on the viability of vintage antimalarial drugs chloroquine (CHQ) and its analog hydroxychloroquine (HCQ) repositioned for SARS-CoV-2 prophylaxis and COVID-19 treatment. Findings suggest neither the use of CHQ nor HCQ singularly, or concomitantly, with azithromycin and/or zinc provide definitive benefits for use against SARS-CoV-2 infection or COVID-19 illness. Moreover, administration of these medications was linked to significant and sometimes fatal complications.

Avaliação do conhecimento de Graduandos do curso de Odontologia sobre o uso de fluoretos em Odontopediatria: um estudo coorte / Evaluation of the knowledge of undergraduate dental students regarding the indications of fluoride for children: a cohort study

Objetivo: O objetivo do estudo foi avaliar o conhecimento de Graduandos da Faculdade de Odontologia da Universidade Federal de Minas Gerais (FAO-UFMG) quanto às indicações de uso de fluoretos em crianças. Métodos: Em 2015, dois grupos de estudantes foram convidados a participar do estudo: Grupo 1 (G1): 78 alunos do 1º período e Grupo 2 (G2): 72 alunos do 9º período (baseline). Em 2016, o G1 participou da reaplicação do mesmo questionário e a amostra de alunos recuperada foi de 66 graduandos (tempo 2 ­ T2). Foi realizada análise estatística dos dados usando os Testes Qui-quadrado de Pearson, Exato de Fisher e McNemar. Resultados: Comparando as respostas, houve diferenças de resposta em todas as questões entre G1 em baseline e G2 (p < 0,05). O conhecimento entre G1 no T2 e G2 foi semelhante (p > 0,05). As respostas foram diferentes entre G1 em baseline e G1 em T2 (p < 0,05), mostrando uma melhora do conhecimento para os mesmos alunos. Conclusão: No geral, pode-se dizer que ainda há uma defasagem no conhecimento, visto que muitas questões foram respondidas de maneira insatisfatória. (AU)

Aim: This study s oughtto compare the knowledge of undergraduate students from the Dental School of Universidade Federal de Minas Gerais (FOUFMG) regarding the recommendations of fluoride use for children. Methods: In 2015, two groups of students took part in the study: Group 1 (G1): 78 students from the 1stsemester and Group 2 (G2): 72 students from the last semester (baseline). In 2016, G1 answered the same questionnaire, and the sample retrieved was of 66 students (time 2). Pearson's Chi-square, Fisher's exact, and McNemar tests were used for statistical analysis. Results: Comparing the responses, the answers were different between G1 at the baseline and G2 (p < 0.05). The answers of the questionnaire were similar between G1 at time 2 and G2 at the baseline (p > 0.05). The answers were different between G1 at the baseline and G1 at time 2 (p < 0.05), illustrating an improvement in knowledge. Conclusion:In conclusion, there is still a lack of knowledge, as many questions were not properly answered by the students. (AU)

Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders: evidence from SNP arrays.

Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.

Lyso-glycosphingolipid abnormalities in different murine models of lysosomal storage disorders.

In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC­MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed.

Globotriaosylceramide inhibits iNKT-cell activation in a CD1d-dependent manner.

Globotriaosylceramide (Gb3) is a glycosphingolipid present in cellular membranes that progressively accumulates in Fabry disease. Invariant Natural Killer T (iNKT) cells are a population of lipid-specific T cells that are phenotypically and functionally altered in Fabry disease. The mechanisms responsible for the iNKT-cell alterations in Fabry disease are not well understood. Here, we analyzed the effect of Gb3 on CD1d-mediated iNKT-cell activation in vitro using human cells and in vivo in the mouse model. We found that Gb3 competes with endogenous and exogenous antigens for CD1d binding, thereby reducing the activation of iNKT cells. This effect was exerted by a reduction in the amount of stimulatory CD1d:α-GalCer complexes in the presence of Gb3 as demonstrated by using an mAb specific for the complex. We also found that administration of Gb3 delivered to the same APC as α-GalCer, induces reduced iNKT-cell activation in vivo. This work highlights the complexity of iNKT-cell activation and the importance of nonantigenic glycosphingolipids in the modulation of this process.

Myelin Lipids Inhibit Axon Regeneration Following Spinal Cord Injury: a Novel Perspective for Therapy.

Lack of axon regeneration following spinal cord injury has been mainly ascribed to the inhibitory environment of the injury site, i.e., to chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs). Here, we used shiverer (shi) mice to assess axon regeneration following spinal cord injury in the presence of MAIs and CSPG but in the absence of compact myelin. Although in vitro shi neurons displayed a similar intrinsic neurite outgrowth to wild-type neurons, in vivo, shi fibers had increased regenerative capacity, suggesting that the wild-type spinal cord contains additional inhibitors besides MAIs and CSPG. Our data show that besides myelin protein, myelin lipids are highly inhibitory for neurite outgrowth and suggest that this inhibitory effect is released in the shi spinal cord given its decreased lipid content. Specifically, we identified cholesterol and sphingomyelin as novel myelin-associated inhibitors that operate through a Rho-dependent mechanism and have inhibitory activity in multiple neuron types. We further demonstrated the inhibitory action of myelin lipids in vivo, by showing that delivery of 2-hydroxypropyl-ß-cyclodextrin, a drug that reduces the levels of lipids specifically in the injury site, leads to increased axon regeneration of wild-type (WT) dorsal column axons following spinal cord injury. In summary, our work shows that myelin lipids are important modulators of axon regeneration that should be considered together with protein MAIs as critical targets in strategies aiming at improving axonal growth following injury.

Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis.

Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

Prevalence of Fabry's disease within hemodialysis patients in Spain.

AIMS: To identify patients with Fabry's disease (FD) within patients with kidney dysfunction, submitted to hemodialysis. SUBJECTS AND METHODS: Patients under hemodialysis were screened using a combined enzymatic α-Gal A assay and dried blood spot samples to determine GLA genotype. RESULTS: A total of 3,650 samples (18.5% of all patients under hemodialysis in Spain) were tested and 11 new unrelated FD patients (4 males and 7 females) were diagnosed. 66 relatives of 11 patients were tested and 23 new FD patients were identified. This study allowed the diagnosis of 34 FD patients. Among the 11 unrelated FD patients, 5 presented the same R118C mutation and one novel mutation was detected: D109G. CONCLUSIONS: This study reveals the need for screening for FD in all patients under hemodialysis for unknown causes and indicates that the incidence and prevalence of FD are underestimated so far.

A cargo-centered perspective on the PEX5 receptor-mediated peroxisomal protein import pathway.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.

Invariant natural killer T cells are phenotypically and functionally altered in Fabry disease.

Fabry disease is a lysosomal storage disease belonging to the group of sphingolipidoses. In Fabry disease there is accumulation of mainly globotriaosylceramide due to deficiency of the lysosomal enzyme α-galactosidase A. The lysosome is an important compartment for the activity of invariant natural killer T (iNKT) cells. iNKT cells are lipid-specific T cells that were shown to be important in infection, autoimmunity and tumor surveillance. In several mouse models of lysosomal storage disorders there is a decrease in iNKT cell numbers. Furthermore, alterations on iNKT cell subsets have been recently described in the Fabry disease mouse model. Herein, we analyzed iNKT cells and their subsets in Fabry disease patients. Although there were no differences in the percentage of iNKT cells between Fabry disease patients and control subjects, Fabry disease patients presented a reduction in the iNKT CD4(+) cells accompanied by an increase in the iNKT DN cells. Since iNKT cell subsets produce different quantities of pro-inflammatory and anti-inflammatory cytokines, we analyzed IFN-γ and IL-4 production by iNKT cells of Fabry disease patients and mice. We found a significant reduction in the production of IL-4 by mice splenic iNKT cells and human iNKT cell subsets, but no significant alterations in the production of IFN-γ. Altogether, our results suggest a bias towards a pro-inflammatory phenotype in Fabry disease iNKT cells.

Heat shock induces a massive but differential inactivation of SUMO-specific proteases.

Covalent conjugation of the small ubiquitin-like modifier (SUMO) to proteins is a highly dynamic and reversible process. Cells maintain a fine-tuned balance between SUMO conjugation and deconjugation. In response to stress stimuli such as heat shock, this balance is altered resulting in a dramatic increase in the levels of SUMO conjugates. Whether this reflects an activation of the conjugation cascade, a decrease in the activity of SUMO-specific proteases (SENPs), or both, remains unknown. Here, we show that from the five human SENPs detected in HeLa cells (SENP1/2/3/6/7) the activities of all but one (SENP6) were largely diminished after 30min of heat shock. The decreased activity is not due to changes in their steady-state levels. Rather, in vitro experiments suggest that these SENPs are intrinsically heat-sensitive, a property most likely emerging from their catalytic domains. Heat shock inactivation seems to be a specific property of SENPs because numerous members of the related deubiquitinase family of cysteine proteases are not affected by this stress condition. Overall, our results suggest that SENPs are particularly sensitive to heat shock, a property that may be important for the adaptation of cells to this stress condition.

Mapping the genetic and clinical characteristics of Gaucher disease in the Iberian Peninsula.

BACKGROUND: Gaucher disease (GD) is due to deficiency of the glucocerebrosidase enzyme. It is panethnic, but its presentation reveals ethnicity-specific characteristics. METHODS: We evaluated the distribution, and clinical and genetic characteristics of GD patients in the Iberian Peninsula (IP). We analysed geographical distribution, demographic, genetic and clinical data, age at diagnosis, type, and years of therapy in 436 GD patients from the IP. RESULTS: The prevalence of GD was 1/149,000 inhabitants; 88.3% were type 1, 6.7% type 2, and 5.0% type 3. The mean age at diagnosis in type 1 was 28.7 years. A total of 72.7% were classified as having mild forms, 25.5% moderate, and 1.7% severe. Anemia and thrombocytopenia were present in 56% and 55%, respectively. Bone disease and hepatomegaly were reported in 62% and 68%, respectively, and were more likely in asplenic than in non-splenectomized patients. Sixty-nine mutant alleles were identified, and five mutations accounted for 75% of the GBA alleles. Several patients described in our series had interesting phenotypes. A total of 58.7% of patients had received enzyme replacement therapy and 12.6% were treated with miglustat. CONCLUSIONS: A broad spectrum of GBA mutations is present in the IP, with 98.2% of type 1 GD being mild and 23.0% never treated. These data highlight genetic and phenotypic heterogeneities among geographic populations.

Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate.

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1.

Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.