Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology

Metabolic and transcriptomic analysis of Huntington's disease model reveal changes in intracellular glucose levels and related genes.

Huntington's Disease (HD) is a neurodegenerative disorder caused by an expansion in a CAG-tri-nucleotide repeat that introduces a poly-glutamine stretch into the huntingtin protein (mHTT). Mutant huntingtin (mHTT) has been associated with several phenotypes including mood disorders and depression. Additionally, HD patients are known to be more susceptible to type II diabetes mellitus (T2DM), and HD mice model develops diabetes. However, the mechanism and pathways that link Huntington's disease and diabetes have not been well established. Understanding the underlying mechanisms can reveal potential targets for drug development in HD. In this study, we investigated the transcriptome of mHTT cell populations alongside intracellular glucose measurements using a functionalized nanopipette. Several genes related to glucose uptake and glucose homeostasis are affected. We observed changes in intracellular glucose concentrations and identified altered transcript levels of certain genes including Sorcs1, Hh-II and Vldlr. Our data suggest that these can be used as markers for HD progression. Sorcs1 may not only have a role in glucose metabolism and trafficking but also in glutamatergic pathways affecting trafficking of synaptic components

Adrenergic and mesenchymal signatures are identifiable in cell-free DNA and correlate with metastatic disease burden in children with neuroblastoma

Background: Cell-free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES-specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. Methods: We previously performed an integrative analysis to identify ADRN and MES-specific genes (n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples. Results: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p Conclusions: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.</p

Characterization of Huntington’s Disease Cell Model in Gene Expression, Intracellular Glucose Levels and Genetic Variation

An increased risk of diabetes has been observed among Huntington’s disease (HD) carri- ers, but there has not been follow-up investigation of the genetic nature of the risk. Here, we investigated diabetes phenotypes signatures in gene expression, DNA polymorphisms and intracellular glucose levels comparing rat and human data. Using a genome-wide association study (GWAS), RNA sequencing (RNA-Seq) analysis, and western blotting of Rattus norvegicus and human, we were able to identify a correlation between HD and DNA polymorphisms in the family of sortilins, tetraspanins and HLA/MHC proteins. We were also able to correlate SNP variants and gene expression with intracellular glu- cose levels using the nanopipette platform. Our results suggest that ST14A cells, from R. norvegicus, are a reliable model of HD, because they allowed confirmation of markers of diabetes, such as sortilins, glucose transporters and MHC proteins in our cell model. This model allowed experiments that highlight genes involved in mechanisms targeted by diabetes drugs, such as glucose transporters, and proteins controlling insulin release, related to mHTT. To the best of our knowledge, this is the first GWAS study using RNA-Seq data from both ST14A rat HD cell model and human HD. Similarly, for the first time, glucose levels were detected at the single-cell level in a HD model using nanopipettes

Characterization of Huntington’s Disease Cell Model in Gene Expression, Intracellular Glucose Levels and Genetic Variation

An increased risk of diabetes has been observed among Huntington’s disease (HD) carri- ers, but there has not been follow-up investigation of the genetic nature of the risk. Here, we investigated diabetes phenotypes signatures in gene expression, DNA polymorphisms and intracellular glucose levels comparing rat and human data. Using a genome-wide association study (GWAS), RNA sequencing (RNA-Seq) analysis, and western blotting of Rattus norvegicus and human, we were able to identify a correlation between HD and DNA polymorphisms in the family of sortilins, tetraspanins and HLA/MHC proteins. We were also able to correlate SNP variants and gene expression with intracellular glu- cose levels using the nanopipette platform. Our results suggest that ST14A cells, from R. norvegicus, are a reliable model of HD, because they allowed confirmation of markers of diabetes, such as sortilins, glucose transporters and MHC proteins in our cell model. This model allowed experiments that highlight genes involved in mechanisms targeted by diabetes drugs, such as glucose transporters, and proteins controlling insulin release, related to mHTT. To the best of our knowledge, this is the first GWAS study using RNA-Seq data from both ST14A rat HD cell model and human HD. Similarly, for the first time, glucose levels were detected at the single-cell level in a HD model using nanopipettes

Investigation of PfSR25, putative serpentine receptor of Plasmodium falciparum.

Este trabalho teve por objetivo dissecar o papel potencial de fosforilação/desfosforilação como efeitos da ativação de PfSR25, um candidato a receptor serpentina de P. falciparum. Como a sinalização é um evento celular complexo, não excluímos a possibilidade de que outros mecanismos moleculares ocorram além dos aqui descritos. Nossas conclusões são que potássio modula PfSR25, ativando quinases/fosfatases, levando à ativação de moléculas efetoras. Encontramos MSP1, proteína já caracterizada e Pf4-4-13 e o fator básico de transcrição 3B (PfBTF3B), que ainda não foram caracterizados em P. falciparum, como efetores. Estes dados sugerem que pelo menos em parte, o mecanismo pelo qual PfSR25 exerce seu papel no desenvolvimento de P. falciparum seja através da ativação de quinases/fosfatases. Isto não é surpreendente, pois a sinalização de PfSR25 ocorre através de K+/cálcio e o segundo mensageiro é um modulador destas classes de proteínas. No entanto, deve ser investigado se cálcio tem algum efeito direto sobre o processamento/ativação dos efetores aqui identificados.This work aimed at dissecting the potential role of phosphorylation/dephosphorylation as downstream effect of PfSR25 activation. PfSR25 is a serpentine receptor candidate from P. falciparum. As signaling is a quite complex cellular event, we do not exclude the possibility that other molecular mechanisms, take place additionally to those here described. Our conclusions are that potassium modulates PfSR25 by activation of kinases/phosphatases, leading to activation of effector molecules. We found MSP1 already characterized protein and Pf4.4.13 and the basic transcription factor 3B (PfBTF3B), which have not yet been characterized in P. falciparum as effectors. These data suggest that, at least in part, the mechanism by which PfSR25 exerts its role in the P. falciparum development is through the activation of kinase/phosphatase. This is not surprising, since PfSR25 signaling occurs through K+/Calcium and the second messenger is a modulator of these classes of proteins. However, remains to be investigated rather calcium has a direct effect on processing/activation the effectors here identified

Mutant Huntingtin Affects Diabetes and Alzheimer’s Markers in Human and Cell Models of Huntington’s Disease

A higher incidence of diabetes was observed among family members of individuals affected by Huntington&rsquo;s Disease with no follow-up studies investigating the genetic nature of the observation. Using a genome-wide association study (GWAS), RNA sequencing (RNA-Seq) analysis and western blotting of Rattus norvegicus and human, we were able to identify that the gene family of sortilin receptors was affected in Huntington&rsquo;s Disease patients. We observed that less than 5% of SNPs were of statistical significance and that sortilins and HLA/MHC gene expression or SNPs were associated with mutant huntingtin (mHTT). These results suggest that ST14A cells derived from R. norvegicus are a reliable model of HD, since sortilins were identified through analysis of the transcriptome in these cells. These findings help highlight the genes involved in mechanisms targeted by diabetes drugs, such as glucose transporters as well as proteins controlling insulin release related to mHTT. To the best of our knowledge, this is the first GWAS using RNA-Seq data from both ST14A rat HD cell model and human Huntington&rsquo;s Disease

Mutant Huntingtin Affects Diabetes and Alzheimer's Markers in Human and Cell Models of Huntington's Disease.

A higher incidence of diabetes was observed among family members of individuals affected by Huntington's Disease with no follow-up studies investigating the genetic nature of the observation. Using a genome-wide association study (GWAS), RNA sequencing (RNA-Seq) analysis and western blotting of Rattus norvegicus and human, we were able to identify that the gene family of sortilin receptors was affected in Huntington's Disease patients. We observed that less than 5% of SNPs were of statistical significance and that sortilins and HLA/MHC gene expression or SNPs were associated with mutant huntingtin (mHTT). These results suggest that ST14A cells derived from R. norvegicus are a reliable model of HD, since sortilins were identified through analysis of the transcriptome in these cells. These findings help highlight the genes involved in mechanisms targeted by diabetes drugs, such as glucose transporters as well as proteins controlling insulin release related to mHTT. To the best of our knowledge, this is the first GWAS using RNA-Seq data from both ST14A rat HD cell model and human Huntington's Disease

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology