RESUMEN
Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba+/+ and gba-/- immortalized neurons and primary human wild-type and type 2 GD fibroblasts.
Asunto(s)
Anticuerpos/química , Western Blotting , Fibroblastos/enzimología , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/metabolismo , Enfermedad de Parkinson/enzimología , Animales , Línea Celular Transformada , Fibroblastos/patología , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/patología , Glucosilceramidasa/genética , Humanos , Ratones , Ratones Noqueados , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patologíaRESUMEN
UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Enfermedad de Gaucher/patología , Glucosilceramidas/antagonistas & inhibidores , Glucolípidos/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Trastornos Parkinsonianos/patología , alfa-Sinucleína/metabolismo , Acetanilidas/farmacología , Benzamidas/farmacología , Catecolaminas/metabolismo , Diferenciación Celular/genética , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Glucosilceramidasa , Glucosilceramidas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Mutación/genética , Técnicas de Placa-Clamp , beta-Glucosidasa/genéticaRESUMEN
Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.
Asunto(s)
Enfermedad de Gaucher/tratamiento farmacológico , Glucosilceramidasa/efectos de los fármacos , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Humanos , Mutación , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Pliegue de Proteína/efectos de los fármacos , Sinucleínas/efectos de los fármacos , Sinucleínas/genéticaRESUMEN
Mutations in ATP13A2 (PARK9), encoding a lysosomal P-type ATPase, are associated with both Kufor-Rakeb syndrome (KRS) and neuronal ceroid lipofuscinosis (NCL). KRS has recently been classified as a rare genetic form of Parkinson's disease (PD), whereas NCL is a lysosomal storage disorder. Although the transport activity of ATP13A2 has not been defined, in vitro studies show that its loss compromises lysosomal function, which in turn is thought to cause neuronal degeneration. To understand the role of ATP13A2 dysfunction in disease, we disrupted its gene in mice. Atp13a2(-/-) and Atp13a2(+/+) mice were tested behaviorally to assess sensorimotor and cognitive function at multiple ages. In the brain, lipofuscin accumulation, α-synuclein aggregation and dopaminergic pathology were measured. Behaviorally, Atp13a2(-/-) mice displayed late-onset sensorimotor deficits. Accelerated deposition of autofluorescent storage material (lipofuscin) was observed in the cerebellum and in neurons of the hippocampus and the cortex of Atp13a2(-/-) mice. Immunoblot analysis showed increased insoluble α-synuclein in the hippocampus, but not in the cortex or cerebellum. There was no change in the number of dopaminergic neurons in the substantia nigra or in striatal dopamine levels in aged Atp13a2(-/-) mice. These results show that the loss of Atp13a2 causes sensorimotor impairments, α-synuclein accumulation as occurs in PD and related synucleinopathies, and accumulation of lipofuscin deposits characteristic of NCL, thus providing the first direct demonstration that null mutations in Atp13a2 can cause pathological features of both diseases in the same organism.
Asunto(s)
Adenosina Trifosfatasas , Envejecimiento/metabolismo , Encéfalo/metabolismo , Retroalimentación Sensorial , Proteínas de la Membrana , Lipofuscinosis Ceroideas Neuronales/enzimología , Trastornos Parkinsonianos/enzimología , alfa-Sinucleína/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Animales , Conducta Animal , Encéfalo/patología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Ratones , Ratones Mutantes , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/patología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , ATPasas de Translocación de Protón , alfa-Sinucleína/genéticaRESUMEN
The lysosomal enzyme glucocerebrosidase, encoded by the glucocerebrosidase gene, is involved in the breakdown of glucocerebroside into glucose and ceramide. Lysosomal build-up of the substrate glucocerebroside occurs in cells of the reticulo-endothelial system in patients with Gaucher disease, a rare lysosomal storage disorder caused by the recessively inherited deficiency of glucocerebrosidase. Gaucher disease has a broad clinical phenotypic spectrum, divided into non-neuronopathic and neuronopathic forms. Like many monogenic diseases, the correlation between clinical manifestations and molecular genotype is not straightforward. There is now a well-established clinical association between mutations in the glucocerebrosidase gene and the development of more prevalent multifactorial disorders including Parkinson's disease and other synucleinopathies. In this review we discuss recent studies advancing our understanding of the cellular relationship between glucocerebrosidase and α-synuclein, the potential impact of established and emerging therapeutics for Gaucher disease for the treatment of the synucleinopathies, and the role of lysosomal pathways in the pathogenesis of these neurodegenerative disorders.
Asunto(s)
Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Mutación/genética , alfa-Sinucleína/genética , Enfermedad de Gaucher/clasificación , Enfermedad de Gaucher/fisiopatología , Estudios de Asociación Genética , Genotipo , Humanos , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/genética , FenotipoRESUMEN
Gaucher disease is an autosomal recessive disorder caused by deficiency of the enzyme glucocerebrosidase. Although it is a monogenic disease, there is vast phenotypic heterogeneity, even among patients with the same genotype. MicroRNAs (miRNAs) are small non-coding RNAs involved in many biological processes and diseases. To determine whether miRNAs can affect glucocerebrosidase activity, we performed a screen of 875 different miRNA mimics. The screen was performed using Gaucher fibroblasts, and glucocerebrosidase activity was used as the initial outcome parameter. We found several miRNAs that either up- or down-regulated glucocerebrosidase activity. In follow-up assays, we confirmed that one specific miRNA (miR-127-5p) down-regulated both glucocerebrosidase activity and protein levels by down-regulation of LIMP-2, the receptor involved in proper trafficking of glucocerebrosidase from the endoplasmic reticulum to the lysosome. A conditioned media assay demonstrated that cells treated with this miRNA secreted glucocerebrosidase into the extracellular environment, supporting impaired LIMP-2 function. Two other miRNAs, miR-16-5p and miR-195-5p, were found to up-regulate glucocerebrosidase activity by greater than 40% and to enhance expression and protein levels of the enzyme. In conclusion, we show that miRNAs can alter glucocerebrosidase activity in patient cells, indicating that miRNAs can potentially act as modifiers in Gaucher disease.
Asunto(s)
Fibroblastos/enzimología , Enfermedad de Gaucher/genética , Regulación Enzimológica de la Expresión Génica , Glucosilceramidasa/metabolismo , MicroARNs/genética , Western Blotting , Proliferación Celular , Células Cultivadas , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/genética , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Luciferasas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSC) migrate to primary tumours and drive tumour progression. This study aimed to identify the molecular mechanisms associated with these heterotypic cellular interactions and analyse their relevance in colorectal cancer (CRC). DESIGN: Paracrine interactions of BM-MSC with CRC cells were studied using collagen invasion assays, cell counts, flow cytometric cell-cycle analysis and tumour xenograft models. The role of neuregulin 1 (NRG1) and the human epidermal growth factor receptor (HER) family pathways were investigated using tyrosine kinase assays, mass spectrometry, pharmacological inhibition, antibody-mediated neutralisation and RNA interference. Transmembrane neuregulin 1 (tNRG1), HER2 and HER3 expression was analysed in primary CRC (n=54), adjacent normal colorectal tissues (n=4), liver metastases (n=3) and adjacent normal liver tissues (n=3) by immunohistochemistry. RESULTS: BM-MSC stimulate invasion, survival and tumorigenesis of CRC through the release of soluble NRG1, activating the HER2/HER3-dependent PI3K/AKT signalling cascade in CRC cells. Similarly, tumour-associated mesenchymal cells (T-MC) in CRC demonstrate high tNRG1 expression, which is significantly associated with advanced Union for International Cancer Control stage (p=0.005) and invasion depth (p=0.04) and decreased 5-year progression-free survival (p=0.01). HER2 and HER3 show membrane localisation in cancer cells of CRC tissue. CONCLUSION: Paracrine NRG1/HER3 signals initiated by BM-MSC and T-MC promote CRC cell progression, and high tNRG1 expression is associated with poor prognosis in CRC.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Células Madre Mesenquimatosas/patología , Neurregulina-1/metabolismo , Receptor ErbB-3/metabolismo , Análisis de Varianza , Western Blotting , Recuento de Células , Línea Celular Tumoral , Movimiento Celular/fisiología , Distribución de Chi-Cuadrado , Cromatografía Liquida , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Espectrometría de Masas , Comunicación Paracrina , Fosforilación , Interferencia de ARN , Receptor ErbB-2/metabolismo , Transducción de Señal , Estadísticas no ParamétricasRESUMEN
PURPOSE: Native Americans and Latinos have higher COVID-19 infection and mortality rates and may have limited access to diagnostic testing. Home-based testing may improve access to care in rural and underserved populations. This study tests the effect of community health worker (CHW) support on accessibility, feasibility, and completion of COVID-19 home testing among Native American and Latino adults living on the Flathead Reservation in Montana and in Yakima Valley, Washington. METHODS: A two-arm, multisite, pragmatic randomized controlled trial was conducted using block randomization stratified by site and participant age. Active arm participants received CHW assistance with online COVID-19 test kit registration and virtual swabbing support. The passive arm participants received standard-of-care support from the kit vendor. Logistic regression modeled the association between study arm and test completion (primary outcome) and between study arm and test completion with return of valid test results (secondary outcome). Responses to posttest surveys and interviews were summarized using deductive thematic analysis. FINDINGS: Overall, 63% of participants (n = 268) completed COVID-19 tests, and 50% completed tests yielding a valid result. Active arm participants had higher odds of test completion (odds ratio: 1.66, 95% confidence interval [1.01, 2.75]). Differences were most pronounced among adults ≥60 years. Participants cited ease of use and not having to leave home as positive aspects, and transportation and mailing issues as negative aspects of home-based testing. CONCLUSIONS: CHW support led to higher COVID-19 test completion rates, particularly among older adults. Significant testing barriers included language, educational level, rurality, and test kit issues.
RESUMEN
The secretory Rab27B small GTPase promotes invasive growth and metastasis in estrogen receptor (ER) α-positive breast cancer cells by orchestrating the peripheral targeting of vesicles secreting proinvasive growth regulators. Increased Rab27B expression is associated with poor prognosis in breast cancer patients. The molecular mechanisms of peripheral Rab27B secretory vesicle distribution are poorly understood. Mass spectrometry analysis on green fluorescent protein (GFP)-Rab27B vesicles prepared from GFP-Rab27B transfected MCF-7 human breast cancer cells detected eight subunits of the vacuolar H(+)-ATPase (V-ATPase) and the presence of V0a1 and V0d1 subunits was confirmed by Western blot analysis. Reversible inhibition of V-ATPase activity by bafilomycin A1 or transient silencing of V0a1 or V0d1 subunits demonstrated that V-ATPase controls peripheral localization and size of Rab27B vesicles. V-ATPase expression and activity further controls Rab27B-induced collagen type I invasion, cell-cycle progression and invasive growth in the chorioallantoic membrane assay. In agreement, Rab27B-dependent extracellular heat shock protein90α release and matrix metalloprotease-2 activation is markedly reduced by bafilomycin A1 and transient silencing of V0a1 and V0d1 subunits. Poor prognosis ERα-positive primary breast tumors expressing high levels of Rab27B also expressed multiple V-ATPase subunits and showed a strong cytoplasmic and peripheral V-ATPase V1E expression. In conclusion, inhibiting V-ATPase activity by interfering agents and drugs might be an effective strategy for blocking Rab27B-dependent proinvasive secretory vesicle trafficking in ERα-positive breast cancer patients.
Asunto(s)
Neoplasias de la Mama/enzimología , División Celular/fisiología , Metástasis de la Neoplasia , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas de Unión al GTP rab/fisiología , Animales , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/patología , Línea Celular Tumoral , Embrión de Pollo , Cartilla de ADN , Proteínas Fluorescentes Verdes/genética , Humanos , Inmunohistoquímica , Espectrometría de Masas , Microscopía Inmunoelectrónica , Proteínas de Unión al GTP rab/genéticaRESUMEN
BACKGROUND: Members of two seemingly unrelated kindreds of Arab Moslem origin presented with pronounced early onset spastic paraparesis of upper and lower limbs, mild intellectual disability, kyphosis, pectus carinatum and hypertrichosis. METHODS: The authors performed neurological and developmental examinations on the affected individuals. The authors conducted whole genome linkage and haplotype analyses, followed by sequencing of candidate genes; RNA and protein expression studies; and finally proof of principle investigations on knockdown morpholino oligonucleotide injected zebrafish. RESULTS: The authors characterise a novel form of autosomal recessive complex hereditary spastic paraparesis (CHSP). MRI studies of brain and spinal cord were normal. Within a single significantly linked locus the authors ultimately identified a homozygous missense mutation c.1146A>T (p.K382N) in the vacuolar protein sorting 37A (Vps37A) gene, fully penetrant and segregating with the disease in both families. Mobility was significantly reduced in Vps37A knockdown morpholino oligonucleotide injected zebrafish, supporting the causal relationship between mutations in this gene and the phenotype described in the patients of this study. CONCLUSIONS: The authors provide evidence for the involvement of Vps37A, a member of the endosomal sorting complex required for transport (ESCRT) system, in upper motor neuron disease. The ESCRT system has been shown to play a central role in intracellular trafficking, in the maturation of multivesicular bodies and the sorting of ubiquitinated membrane proteins into internal luminal vesicles. Further investigation of mechanisms by which dysfunction of this gene causes CHSP will contribute to the understanding of intracellular trafficking of vesicles by the ESCRT machinery and its relevance to CHSP.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Genes Recesivos , Mutación Missense , Paraplejía Espástica Hereditaria/genética , Animales , Encéfalo/metabolismo , Niño , Preescolar , Mapeo Cromosómico , Cromosomas Humanos Par 8/genética , Cromosomas Humanos Par 8/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Efecto Fundador , Técnicas de Silenciamiento del Gen , Ligamiento Genético , Haplotipos , Homocigoto , Humanos , Imagen por Resonancia Magnética , Masculino , Linaje , Fenotipo , Selección Genética , Paraplejía Espástica Hereditaria/fisiopatología , Pez CebraRESUMEN
The presynaptic protein α-synuclein (α-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between α-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that α-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the α-syn C-terminal residues, 118-137. This α-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 µm in the presence of 25 to 100 mm NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for α-syn, as does the inhibitor conduritol-ß-epoxide-bound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the α-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.
Asunto(s)
Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/metabolismo , Lisosomas/metabolismo , Complejos Multiproteicos/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Enfermedad de Gaucher/genética , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/genética , Humanos , Concentración de Iones de Hidrógeno , Inositol/análogos & derivados , Inositol/farmacología , Lisosomas/genética , Complejos Multiproteicos/genética , Mutación Missense , Enfermedad de Parkinson/genética , alfa-Sinucleína/genéticaRESUMEN
Active learning in STEM education is essential for engaging the diverse pool of scholars needed to address pressing environmental and social challenges. However, active learning formats are difficult to scale and their incorporation into STEM teaching at U.S. universities varies widely. Here, we argue that urban agriculture as a theme can significantly increase active learning in undergraduate biology education by facilitating outdoor fieldwork and community-engaged education. We begin by reviewing benefits of field courses and community engagement activities for undergraduate biology and discuss constraints to their broader implementation. We then describe how urban agriculture can connect biology concepts to pressing global changes, provide field research opportunities, and connect students to communities. Next, we assess the extent to which urban agriculture and related themes have already been incorporated into biology-related programs in the United States using a review of major programs, reports on how campus gardens are used, and case studies from five higher education institutions (HEIs) engaging with this issue. We found that while field experiences are fairly common in major biology programs, community engagement opportunities are rare, and urban agriculture is almost nonexistent in course descriptions. We also found that many U.S. HEIs have campus gardens, but evidence suggests that they are rarely used in biology courses. Finally, case studies of five HEIs highlight innovative programming but also significant opportunities for further implementation. Together, our results suggest that urban agriculture is rarely incorporated into undergraduate biology in the United States, but there are significant prospects for doing so. We end with recommendations for integrating urban agriculture into undergraduate biology, including the development of campus gardens, research programs, community engagement partnerships, and collaborative networks. If done with care, this integration could help students make community contributions within required coursework, and help instructors feel a greater sense of accomplishment in an era of uncertainty.
RESUMEN
BACKGROUND: Home-based testing for COVID-19 has potential to reduce existing health care disparities among underserved populations in the United States. However, implementation of home-based tests in these communities may face significant barriers. This study evaluates the acceptability, feasibility, and success of home-based testing and the potential added benefit of active support from trusted community health workers for Native Americans and Hispanic/Latino adults living in rural Montana and Washington states. METHODS/DESIGN: The academic-community research team designed the trial to be responsive to community needs for understanding barriers and supports to home-based COVID-19 testing. The "Protecting Our Community" study is a two-arm pragmatic randomized controlled trial in which a total of 400 participants are randomized to active or passive arms. Participants of both study arms receive a commercially available home collection COVID-19 test kit, which is completed by mailing a self-collected nasal swab to a central laboratory. The primary study outcome is return of the kit to the central lab within 14 days. The cultural, social, behavioral, and economic barriers to home-based COVID-19 testing are also assessed by qualitative research methods. A survey and semi-structured interviews are conducted after the trial to evaluate perceptions and experience of home-based testing. DISCUSSION: Implementing home-based testing in underserved populations, including among Native American and Hispanic/Latino communities, may require additional support to be successful. The Protecting Our Community trial examines the effect of trusted community health workers on use of home-based testing, which may be adaptable for community-driven models of home-based testing in other underserved populations.
Asunto(s)
COVID-19 , Prueba de COVID-19 , Hispánicos o Latinos , Humanos , Ensayos Clínicos Pragmáticos como Asunto , Ensayos Clínicos Controlados Aleatorios como Asunto , SARS-CoV-2 , Estados Unidos , Indio Americano o Nativo de AlaskaRESUMEN
Lysosomal integral membrane protein type 2 (LIMP-2) is responsible for proper sorting and lysosomal targeting of glucocerebrosidase, the enzyme deficient in Gaucher disease (GD). Mutations in the gene for LIMP-2, SCARB2, are implicated in inherited forms of myoclonic epilepsy, and myoclonic epilepsy is part of the phenotypic spectrum associated with GD. We investigated whether SCARB2 mutations impact the Gaucher phenotype focusing on patients with myoclonic epilepsy, including a pair of siblings with GD who were discordant for myoclonic seizures. Sequencing of SCARB2 genomic and cDNA identified a heterozygous, maternally inherited novel mutation, c.1412A>G (p.Glu471Gly), in the brother with GD and myoclonic epilepsy, absent from his sibling and controls. Glucocerebrosidase activity, Western blots, real-time PCR, and immunofluorescence studies demonstrated markedly decreased LIMP-2 and glucocerebrosidase in cells from the sibling with (p.Glu471Gly) LIMP-2, and diminished glucocerebrosidase in lysosomes. The cells secreted highly glycosylated enzyme and showed mistrafficking of glucocerebrosidase. Sequencing of SCARB2 in 13 other subjects with GD and myoclonic epilepsy and 40 controls failed to identify additional mutations. The study provides further evidence for the association of LIMP-2 and myoclonic epilepsy, explains the drastically different phenotypes encountered in the siblings, and demonstrates that LIMP-2 can serve as a modifier in GD.
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Enfermedad de Gaucher/genética , Proteínas de Membrana de los Lisosomas/genética , Mutación , Receptores Depuradores/genética , Adulto , Epilepsias Mioclónicas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Griscelli syndrome (GS), a rare autosomal recessive disorder characterized by partial albinism and immunological impairment and/or severe neurological impairment, results from mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes. We identified a Hispanic patient born of a consanguineous union who presented with immunodeficiency, partial albinism, hepatic dysfunction, hemophagocytosis, neurological impairment, nystagmus, and silvery hair indicative of Griscelli Syndrome Type 2 (GS2). We screened for point mutations, but only exons 2-6 of the patient's DNA could be PCR-amplified. Whole genome analysis using the Illumina 1M-Duo DNA Analysis BeadChip identified a homozygous deletion in the patient's DNA. The exact breakpoints of the 47.5-kb deletion were identified as chr15q15-q21.1: g.53332432_53379990del (NCBI Build 37.1); the patient lacks the promoter and 5'UTR regions of RAB27A, thus confirming the diagnosis of GS2.
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Eliminación de Secuencia/genética , Proteínas de Unión al GTP rab/genética , Secuencia de Bases , Humanos , Síndromes de Inmunodeficiencia/genética , Linfohistiocitosis Hemofagocítica , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Linaje , Piebaldismo/genética , Enfermedades de Inmunodeficiencia Primaria , Proteínas rab27 de Unión a GTPRESUMEN
Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by variable oculocutaneous albinism, immunodeficiency, mild bleeding diathesis, and an accelerated lymphoproliferative state. Abnormal lysosome-related organelle membrane function leads to the accumulation of large intracellular vesicles in several cell types, including granulocytes, melanocytes, and platelets. This report describes a severe case of CHS resulting from paternal heterodisomy of chromosome 1, causing homozygosity for the most distal nonsense mutation (p.E3668X, exon 50) reported to date in the LYST/CHS1 gene. The mutation is located in the WD40 region of the CHS1 protein. The patient's fibroblasts expressed no detectable CHS1. Besides manifesting the classical CHS findings, the patient exhibited hypotonia and global developmental delays, raising concerns about other effects of heterodisomy. An interstitial 747 kb duplication on 6q14.2-6q14.3 was identified in the propositus and paternal samples by comparative genomic hybridization. SNP genotyping revealed no additional whole chromosome or segmental isodisomic regions or other dosage variations near the crossover breakpoints on chromosome 1. Unmasking of a separate autosomal recessive cause of developmental delay, or an additive effect of the paternal heterodisomy, could underlie the severity of the phenotype in this patient.
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Aneuploidia , Síndrome de Chediak-Higashi/genética , Cromosomas Humanos Par 1/genética , Síndrome de Chediak-Higashi/patología , Codón sin Sentido , Exones/genética , Fibroblastos/patología , Humanos , Lactante , Lisosomas/patología , Retina/patología , Análisis de Secuencia de ADNRESUMEN
HPS is an autosomal recessive disorder characterized by oculocutaneous albinism and prolonged bleeding. Eight human genes are described resulting in the HPS subtypes 1-8. Certain HPS proteins combine to form Biogenesis of Lysosome-related Organelles Complexes (BLOCs), thought to function in the formation of intracellular vesicles such as melanosomes, platelet dense bodies, and lytic granules. Specifically, BLOC-2 contains the HPS3, HPS5 and HPS6 proteins. We used phylogenetic footprinting to identify conserved regions in the upstream sequences of HPS3, HPS5 and HPS6. These conserved regions were verified to have in vitro transcription activation activity using luciferase reporter assays. Transcription factor binding site analyses of the regions identified 52 putative sites shared by all three genes. When analysis was limited to the conserved footprints, seven binding sites were found shared among all three genes: Pax-5, AIRE, CACD, ZF5, Zic1, E2F and Churchill. The HPS3 conserved upstream region was sequenced in four patients with decreased fibroblast HPS3 RNA levels and only one HPS3 mutation in the coding exons and surrounding exon/intron boundaries; no mutation was found. These findings illustrate the power of phylogenetic footprinting for identifying potential regulatory regions in non-coding sequences and define the first putative promoter elements for any HPS genes.
Asunto(s)
Proteínas Portadoras/genética , Síndrome de Hermanski-Pudlak/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Regiones Promotoras Genéticas , Huella de ADN , Genes Reporteros , HumanosRESUMEN
The autosomal recessive Griscelli syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking steps within cells. In melanocytes, the GTP-bound form of Rab27A associates with the membranes of mature fully-pigmented melanosomes through its geranylgeranyl group. Once attached, Rab27A recruits the downstream effector Melanophilin (Mlph) and the actin-dependent motor protein Myosin Va (MyoVa). The molecular Rab27A/Mlph/MyoVA tripartite complex, which links melanosomes to the peripheral actin network, is required to achieve melanosome transfer to surrounding keratinocytes in the epidermis. Here we report a novel homozygous missense mutation c.127G>A, p.G43S in exon 2 of the RAB27A gene of an Afghani GSII patient. Laser scanning confocal microscopy showed that the G43S mutation, which is located in the highly conserved switch I region of Rab27A, induces perinuclear localization of melanosomes in normal melanocytes, and fails to restore melanosomes to the actin-rich periphery in GSII melanocytes. Co-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins.
Asunto(s)
Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Mutación Missense , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Afganistán , Secuencias de Aminoácidos , Núcleo Celular/química , Núcleo Celular/genética , Niño , Preescolar , Femenino , Humanos , Masculino , Melanocitos/química , Melanocitos/metabolismo , Melanosomas/química , Melanosomas/metabolismo , Errores Innatos del Metabolismo/diagnóstico , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Síndrome , Proteínas de Unión al GTP rab/química , Proteínas rab27 de Unión a GTPRESUMEN
Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.
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Neoplasias del Colon/patología , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/fisiología , Factor de Crecimiento Transformador beta/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Actinas/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Colágeno Tipo I/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Invasividad Neoplásica , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The discovery of a link between mutations in GBA1, encoding the lysosomal enzyme glucocerebrosidase, and the synucleinopathies directly resulted from the clinical recognition of patients with Gaucher disease with parkinsonism. Mutations in GBA1 are now the most common known genetic risk factor for several Lewy body disorders, and an inverse relationship exists between levels of glucocerebrosidase and oligomeric α-synuclein. While the underlying mechanisms are still debated, this complicated association is shedding light on the role of lysosomes in neurodegenerative disorders, demonstrating how insights from a rare disorder can direct research into the pathogenesis and therapy of seemingly unrelated common diseases.