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1.
Hum Mutat ; 43(1): 74-84, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34747535

RESUMO

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.


Assuntos
Neurilemoma , Neurofibromatoses , Cromossomos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neurilemoma/genética , Neurilemoma/patologia , Neurofibromatoses/genética , Proteína SMARCB1/genética , Fatores de Transcrição/genética
2.
J Biol Chem ; 296: 100034, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33148698

RESUMO

ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Pancreáticas/patologia , Sialiltransferases/fisiologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Pancreáticas/enzimologia , beta-D-Galactosídeo alfa 2-6-Sialiltransferase
3.
J Virol ; 95(21): e0135721, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34406867

RESUMO

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Assuntos
Evolução Molecular , Heparitina Sulfato/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Biológica , Animais , Sítios de Ligação , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar , Furina/metabolismo , Heparina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Ensaio de Placa Viral , Ligação Viral
4.
Curr Issues Mol Biol ; 43(2): 978-995, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34563039

RESUMO

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.


Assuntos
Bactérias/genética , Biologia Computacional , Microbioma Gastrointestinal/genética , Lytechinus/microbiologia , Metagenômica , Strongylocentrotus purpuratus/microbiologia , Animais , Bactérias/classificação , Bactérias/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA
5.
J Obstet Gynaecol Res ; 47(6): 2185-2195, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33754434

RESUMO

AIM: Next-generation sequencing (NGS) is able to describe the composition of human papillomaviruses (HPVs) as percent (%) reads rather than positive/negative results. Therefore, we used this unique approach to assess the prevalence of cervical HPVs of HIV infected (HIV+) in order to understand the determinants of being infected with higher % reads of high risk (HR)-HPVs and cervical abnormalities of atypical squamous cells of unknown significance or higher (ASCUS+). METHODS: Study included 66 women characterized for relevant risk factors/cytology. Receiver-operating curve curve was used to derive the optimal % read cut point to identify ASCUS+ in relation to any HR-HPV genotype or other specific HPV genotypes. The determinants of ASCUS+ and HR-HPVs were tested using logistic regression. RESULTS: Women with >20% reads of any HR-HPV or >12% any HR-HPV other than HPV 16/18 were 5.7 and 12.6 times more likely to be diagnosed with ASCUS+, respectively. Lower CD4 count was a significant determinant of >20% reads of HR-HPV (odds ratio [OR] = 4.1) or >12% any HR-HPV other than HPV 16/18 (OR = 4.5). CONCLUSION: We envision that the NGS-based HPV detection will be more accurate for screening and management of HIV+ at risk for developing cervical cancer (CC). We raise concerns regarding the limitations of 16/18-based HPV testing for triage and the efficacy of current HPV vaccines for preventing CC in HIV+.


Assuntos
Alphapapillomavirus , Infecções por HIV , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Genótipo , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Neoplasias do Colo do Útero/epidemiologia
6.
J Am Soc Nephrol ; 30(5): 767-781, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948627

RESUMO

BACKGROUND: Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients. METHODS: As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell-negative CD45+ innate immune cells in mouse, rat, pig, and human kidney tissue. RESULTS: We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species. CONCLUSIONS: Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Inata/genética , Molécula 1 de Adesão Intercelular/imunologia , Macrófagos/metabolismo , Análise de Variância , Animais , Biomarcadores/metabolismo , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Parabiose , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA , Especificidade da Espécie
7.
J Am Soc Nephrol ; 30(10): 1841-1856, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31337691

RESUMO

BACKGROUND: Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12-14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified. METHODS: We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation. RESULTS: Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11clo) to R2a (CD11chi) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease. CONCLUSIONS: These data uncover an important pathogenic role for resident macrophages during rapid cyst progression.


Assuntos
Doenças Renais Císticas/etiologia , Macrófagos/fisiologia , Animais , Cílios/genética , Feminino , Rim/crescimento & desenvolvimento , Macrófagos/classificação , Masculino , Camundongos , Mutação
8.
Am J Physiol Renal Physiol ; 316(5): F1026-F1040, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30810063

RESUMO

Conditional gene targeting using Cre recombinase has offered a powerful tool to modify gene function precisely in defined cells/tissues and at specific times. However, in mammalian cells, Cre recombinase can be genotoxic. The importance of including Cre-expressing control mice to avoid misinterpretation and to maximize the validity of the experimental results has been increasingly recognized. While studying the role of podocytes in the pathogenesis of glomerular basement membrane (GBM) thickening, we used Cre recombinase driven by the podocyte-specific podocin promoter (NPHS2-Cre) to generate a conditional knockout. By conventional structural and functional measures (histology by periodic acid-Schiff staining, albuminuria, and plasma creatinine), we did not detect significant differences between NPHS2-Cre transgenic and wild-type control mice. However, surprisingly, the group that expressed Cre transgene alone developed signs of podocyte toxicity, including marked GBM thickening, loss of normal foot process morphology, and reduced Wilms tumor 1 expression. GBM thickening was characterized by altered expression of core structural protein laminin isoform α5ß2γ1. RNA sequencing analysis of extracted glomeruli identified 230 genes that were significant and differentially expressed (applying a q < 0.05-fold change ≥ ±2 cutoff) in NPHS2-Cre mice compared with wild-type control mice. Many biological processes were reflected in the RNA sequencing data, including regulation of the extracellular matrix and pathways related to apoptosis and cell death. This study highlights the importance of including the appropriate controls for potential Cre-mediated toxicity in conditional gene-targeting experiments. Indeed, omitting the Cre transgene control can result in critical errors during interpretation of experimental data.


Assuntos
Marcação de Genes/efeitos adversos , Membrana Basal Glomerular/enzimologia , Integrases/metabolismo , Podócitos/enzimologia , Animais , Regulação da Expressão Gênica , Membrana Basal Glomerular/ultraestrutura , Integrases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Camundongos Transgênicos , Podócitos/ultraestrutura , Regiões Promotoras Genéticas , Fatores de Tempo , Proteínas WT1/genética , Proteínas WT1/metabolismo
9.
J Virol ; 92(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29167335

RESUMO

Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, ß-d-N4-hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections.IMPORTANCE Venezuelan equine encephalitis virus (VEEV) can cause widespread epidemics among humans and domestic animals. VEEV infections result in severe meningoencephalitis and long-term sequelae. No approved therapeutics exist for treatment of VEEV infections. Our study demonstrates that ß-d-N4-hydroxycytidine (NHC) is a very potent anti-VEEV compound, with the 50% effective concentration being below 1 µM. The mechanism of NHC antiviral activity is based on induction of high mutation rates in the viral genome. Accordingly, NHC treatment affects both the rates of particle release and the particle infectivity. Most importantly, in contrast to most of the anti-alphavirus drugs that are under development, resistance of VEEV to NHC develops very inefficiently. Even low levels of resistance require acquisition of multiple mutations in the gene of the VEEV-specific RNA-dependent RNA polymerase nsP4.


Assuntos
Alphavirus/patogenicidade , Antivirais/farmacologia , Citidina/análogos & derivados , Mutação , Alphavirus/efeitos dos fármacos , Alphavirus/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Citidina/farmacologia , Genoma Viral/efeitos dos fármacos , Humanos , Ribavirina/farmacologia , Células Vero , Carga Viral , Proteínas não Estruturais Virais/genética
10.
Pediatr Transplant ; 23(5): e13494, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31124575

RESUMO

BACKGROUND: Optimal management of immunosuppression in kidney transplantation requires a delicate balance of efficacy and toxicity. Tacrolimus (TAC) dose requirements are significantly impacted by genetic variation in CYP3A5 polymorphisms, however the impact that genotype has on clinical outcomes in the pediatric kidney transplant population remains unclear. METHODS: We evaluated a retrospective cohort of 98 pediatric kidney transplant recipients. The primary exposure was CYP3A5 genotype, which classified each recipient into the expresser (at least one CYP3A5*1 allele) or non-expresser group (only CYP3A5*3 alleles). The primary outcome was time to achieve a steady therapeutic TAC concentration. Secondary outcomes include incidence of early allograft rejection and calcineurin inhibitor (CNI) nephrotoxicity during the first year post-transplant. RESULTS: The study cohort included 55 (56%) expressers and 43 (44%) non-expressers of the CYP3A5*1 allele. Expressers had a significantly longer time to achieve a steady therapeutic TAC concentration than non-expressers (log rank, P = 0.03). Expressers had a trend for higher incidence of early allograft rejection (29.1% vs 16.3%, log rank, P = 0.16). Early biopsy-proven CNI nephrotoxicity was seen in 60% of recipients, with no differences in the rate between expressers and non-expressers. CONCLUSIONS: Pediatric kidney transplant recipients with the CYP3A5*1 allele (expressers) take a longer time to achieve therapeutic TAC levels than those with the CYP3A5*3 allele (non-expressers). However, we observed no significant differences in acute rejection or CNI nephrotoxicity based on CYP3A5 genotype. Thus CYP3A5 genotype was not observed to have an immediate impact on early transplant outcomes.


Assuntos
Citocromo P-450 CYP3A/genética , Imunossupressores/uso terapêutico , Transplante de Rim , Tacrolimo/uso terapêutico , Adolescente , Criança , Relação Dose-Resposta a Droga , Feminino , Variação Genética , Genótipo , Rejeição de Enxerto , Humanos , Masculino , Estudos Retrospectivos , Fatores de Tempo
11.
Dev Biol ; 426(2): 429-441, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27209239

RESUMO

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation.


Assuntos
Padronização Corporal/genética , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Análise de Sequência de RNA , Xenopus/genética , Ativinas/fisiologia , Animais , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas da Matriz Extracelular/fisiologia , Gástrula/ultraestrutura , Camadas Germinativas/metabolismo , Morfogênese/genética , Organizadores Embrionários , Proteínas Tirosina Quinases/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Xenopus/embriologia , Proteínas de Xenopus/fisiologia
12.
Hum Mol Genet ; 25(8): 1619-36, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26908599

RESUMO

Identifying molecular drivers of pathology provides potential therapeutic targets. Differentiating between drivers and coincidental molecular alterations presents a major challenge. Variation unrelated to pathology further complicates transcriptomic, proteomic and metabolomic studies which measure large numbers of individual molecules. To overcome these challenges towards the goal of determining drivers of Huntington's disease (HD), we generated an allelic series of HD knock-in mice with graded levels of phenotypic severity for comparison with molecular alterations. RNA-sequencing analysis of this series reveals high numbers of transcripts with level alterations that do not correlate with phenotypic severity. These discorrelated molecular changes are unlikely to be drivers of pathology allowing an exclusion-based strategy to provide a short list of driver candidates. Further analysis of the data shows that a majority of transcript level changes in HD knock-in mice involve alteration of the rate of mRNA processing and/or degradation rather than solely being due to alteration of transcription rate. The overall strategy described can be applied to assess the influence of any molecular change on pathology for diseases where different mutations cause graded phenotypic severity.


Assuntos
Regulação da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Doença de Huntington/patologia , RNA Mensageiro/metabolismo , Alelos , Animais , Modelos Animais de Doenças , Humanos , Doença de Huntington/genética , Camundongos , Fenótipo , Análise de Sequência de RNA
13.
J Am Soc Nephrol ; 27(11): 3278-3284, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26966014

RESUMO

Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, Y1CS3, in the complementarity-determining region 3 of the heavy chain variable region compared with a Y1CA3 sequence in similar isotype-matched IgG from healthy controls. We previously found that the S3 residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the Y1C(A/V)3 amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S3 hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent ß-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.


Assuntos
Autoanticorpos/genética , Galactose/deficiência , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/imunologia , Imunoglobulina A , Mutação , Glomerulonefrite por IGA/enzimologia , Humanos
14.
BMC Microbiol ; 16: 5, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26758906

RESUMO

BACKGROUND: Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient's gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice. RESULTS: A total of nine samples from three human donors, recipient's pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01%). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8%, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55%, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2-4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7% of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10%. CONCLUSION: We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.


Assuntos
Bactérias/crescimento & desenvolvimento , Clostridioides difficile/fisiologia , Infecções por Clostridium/terapia , Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Infecções por Clostridium/microbiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
Hum Mutat ; 36(11): 1088-99, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26219265

RESUMO

Somatic mosaicism for DNA copy-number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array-based comparative genomic hybridization showed 10% (6/59) of patients harbored one to 359 large SMC-CNAs (mean: 1,328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro), and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients.


Assuntos
Neoplasias da Mama/genética , Variações do Número de Cópias de DNA , Instabilidade Genômica , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Feminino , Genes BRCA1 , Genes BRCA2 , Estudos de Associação Genética , Loci Gênicos , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Reprodutibilidade dos Testes , Carga Tumoral
16.
Am J Kidney Dis ; 65(5): 701-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25468385

RESUMO

BACKGROUND: Anticoagulation management is difficult in chronic kidney disease, with frequent supratherapeutic international normalized ratios (INRs ≥ 4) increasing hemorrhagic risk. We evaluated whether the interaction of INR and lower estimated glomerular filtration rate (eGFR) increases hemorrhage risk and whether patients with lower eGFRs experience slower anticoagulation reversal. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: Warfarin pharmacogenetics cohort (1,273 long-term warfarin users); warfarin reversal cohort (74 warfarin users admitted with INRs ≥ 4). PREDICTOR: eGFR, INR as time-dependent covariate, and their interaction in the pharmacogenetics cohort; eGFR in the reversal cohort. OUTCOMES & MEASUREMENTS: In the pharmacogenetics cohort, hemorrhagic (serious, life-threatening, and fatal bleeding) risk was assessed using proportional hazards regression. In the reversal cohort, anticoagulation reversal was assessed from changes in INR, warfarin and metabolite concentrations, clotting factors (II, VII, IX, and X), and PIVKA-II (protein induced by vitamin K absence or antagonist II) levels at presentation and after reversal, using linear regression and path analysis. RESULTS: In the pharmacogenetics cohort, 454 (35.7%) had eGFRs < 60 mL/min/1.73 m(2). There were 137 hemorrhages in 119 patients over 1,802 person-years of follow-up (incidence rate, 7.6 [95% CI, 6.4-8.9]/100 person-years). Patients with lower eGFRs had a higher frequency of INR ≥ 4 (P<0.001). Risk of hemorrhage was affected significantly by eGFR-INR interaction. At INR<4, there was no difference in hemorrhage risk by eGFR (all P ≥ 0.4). At INR≥4, patients with eGFRs of 30 to 44 and < 30 mL/min/1.73 m(2) had 2.2-fold (95% CI, 0.8-6.1; P=0.1) and 5.8-fold (95% CI, 2.9-11.4; P<0.001) higher hemorrhage risks, respectively, versus those with eGFRs ≥ 60 mL/min/1.73 m(2). In the reversal cohort, 35 (47%) had eGFRs < 45 mL/min/1.73 m(2). Patients with eGFRs < 45 mL/min/1.73 m(2) experienced slower anticoagulation reversal as assessed by INR (P=0.04) and PIVKA-II level (P=0.008) than those with eGFRs ≥ 45 mL/min/1.73 m(2). LIMITATIONS: Limited sample size in the reversal cohort, unavailability of antibiotic use and urine albumin data. CONCLUSIONS: Patients with lower eGFRs have differentially higher hemorrhage risk at INR ≥ 4. Moreover, because the INR reversal rate is slower, hemorrhage risk is prolonged.


Assuntos
Anticoagulantes/efeitos adversos , Hemorragia/induzido quimicamente , Hemorragia/fisiopatologia , Coeficiente Internacional Normatizado , Varfarina/efeitos adversos , Idoso , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Medição de Risco
17.
PLoS One ; 19(5): e0303257, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753830

RESUMO

Patterns of single nucleotide polymorphisms (SNPs) in eukaryotic DNA are traditionally attributed to selective pressure, drift, identity descent, or related factors-without accounting for ways in which bias during de novo SNP formation, itself, might contribute. A functional and phenotypic analysis based on evolutionary resilience of DNA points to decreased numbers of non-synonymous SNPs in human and other genomes, with a predominant component of SNP depletion in the human gene pool caused by robust preferences during de novo SNP formation (rather than selective constraint). Ramifications of these findings are broad, belie a number of concepts regarding human evolution, and point to a novel interpretation of evolving DNA across diverse species.


Assuntos
Evolução Molecular , Polimorfismo de Nucleotídeo Único , Humanos , Genoma Humano , Animais , Genoma/genética , Genômica/métodos
18.
Neurosurgery ; 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38747605

RESUMO

BACKGROUND AND OBJECTIVES: Vein of Galen malformation (VOGM), the result of arteriovenous shunting between choroidal and/or subependymal arteries and the embryologic prosencephalic vein, is among the most severe cerebrovascular disorders of childhood. We hypothesized that in situ analysis of the VOGM lesion using endoluminal tissue sampling (ETS) is feasible and may advance our understanding of VOGM genetics, pathogenesis, and maintenance. METHODS: We collected germline DNA (cheek swab) from patients and their families for genetic analysis. In situ VOGM "endothelial" cells (ECs), defined as CD31+ and CD45-, were obtained from coils through ETS during routine endovascular treatment. Autologous peripheral femoral ECs were also collected from the access sheath. Single-cell RNA sequencing of both VOGM and peripheral ECs was performed to demonstrate feasibility to define the transcriptional architecture. Comparison was also made with a published normative cerebrovascular transcriptome atlas. A subset of VOGM ECs was reserved for future DNA sequencing to assess for somatic and second-hit mutations. RESULTS: Our cohort contains 6 patients who underwent 10 ETS procedures from arterial and/or venous access during routine VOGM treatment (aged 12 days to ∼6 years). No periprocedural complications attributable to ETS occurred. Six unique coil types were used. ETS captured 98 ± 88 (mean ± SD; range 17-256) experimental ECs (CD31+ and CD45-). There was no discernible correlation between cell yield and coil type or route of access. Single-cell RNA sequencing demonstrated hierarchical clustering and unique cell populations within the VOGM EC compartment compared with peripheral EC controls when annotated using a publicly available cerebrovascular cell atlas. CONCLUSION: ETS may supplement investigations aimed at development of a molecular-genetic taxonomic classification scheme for VOGM. Moreover, results may eventually inform the selection of personalized pharmacologic or genetic therapies for VOGM and cerebrovascular disorders more broadly.

19.
Sci Rep ; 13(1): 2137, 2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36747074

RESUMO

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is a frequent cause of infections, including bacteraemia and other acute diseases in adults and immunocompromised individuals. We developed a novel system to study GBS within human monocytes to define the co-transcriptome of intracellular GBS (iGBS) and host cells simultaneously using dual RNA-sequencing (RNA-seq) to better define how this pathogen responds to host cells. Using human U937 monocytes and genome-sequenced GBS reference strain 874,391 in antibiotic protection assays we validated a system for dual-RNA seq based on measures of GBS and monocyte viability to ensure that the bacterial and host cell co-transcriptome reflected mainly intracellular (iGBS) rather than extracellular GBS. Elucidation of the co-transcriptome revealed 1119 dysregulated transcripts in iGBS with most genes, including several that encode virulence factors (e.g., scpB, hvgA, ribD, pil2b) exhibiting activation by upregulated expression. Infection with iGBS resulted in significant remodelling of the monocyte transcriptome, with 7587 transcripts differentially expressed including 7040 up-regulated and 547 down-regulated. qPCR confirmed that the most strongly activated genes included sht, encoding Streptococcal Histidine Triad Protein. An isogenic GBS mutant strain deficient in sht revealed a significant effect of this gene on phagocytosis of GBS and survival of the bacteria during systemic infection in mice. Identification of a novel contribution of sht to GBS virulence shows the co-transcriptome responses elucidated in GBS-infected monocytes help to shape the host-pathogen interaction and establish a role for sht in the response of the bacteria to phagocytic uptake. This study provides comprehension of concurrent transcriptional responses that occur in GBS and human monocytes that shape the host-pathogen interaction.


Assuntos
Monócitos , Infecções Estreptocócicas , Adulto , Humanos , Camundongos , Animais , Monócitos/metabolismo , Streptococcus agalactiae , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , RNA-Seq , Fagocitose/genética , Interações Hospedeiro-Patógeno/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
20.
JCI Insight ; 8(19)2023 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-37643018

RESUMO

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Neoplasias Pancreáticas/patologia , Pâncreas/patologia , Carcinoma Ductal Pancreático/patologia , Receptores ErbB/genética , Metaplasia/patologia , Sialiltransferases/genética , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , Antígenos CD
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