Electrochemical Sensing with Spatially Patterned Pt Octahedra Electrodes

Locally controlling the position of electrodes in 3D can open new avenues to collect electrochemical signals in complex sensing environments. Implementing such electrodes via an electrical network requires advanced fabrication approaches. This work uses corner lithography and Pt ALD to produce electrochemical 3D electrodes. The approach allows the fabrication of (sub)micrometer size Pt octahedra electrodes spatially supported over 3D fractal-like structures. As a proof of concept, electrochemical sensing of ferrocyanide in biofouling environments, e.g., bovine serum albumin (BSA) and Pseudomonas aeruginosa (P. aeruginosa), is assessed. Differences between before and after BSA addition show a reduction in the active electrode surface area (ΔAeff) ≈49% ± 7% for the flat electrode. In comparison, a ΔAeff reduction of 25% ± 2% for the 3D electrode has been found. The results are accompanied by a 24% ± 16% decrease in peak current for the flat Pt substrate and a 14% ± 5% decrease in peak current for the 3D electrode 24 h after adding BSA. In the case of P. aeruginosa, the 3D electrode retains electrochemical signals, while the flat electrode does not. The results demonstrate that the 3D Pt electrodes are more stable than their flat counterparts under biofouling conditions.</p

Somatic tumor mutations detected by targeted next generation sequencing in minute amounts of serum-derived cell-free DNA

The use of blood-circulating cell-free DNA (cfDNA) as 'liquid-biopsy' is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens

Molecular alterations in dog pheochromocytomas and paragangliomas

Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL; together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases; SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways

Fractals in tissue engineering

The disclosure relates to a method for producing three-dimensional cell cluster on an inorganic cell culture platform comprising three-dimensional structures, preferably fractal structures. Such three-dimensional structures are useful for culturing cells and tissues, preferably in three dimensions. Such three-dimensional structures are useful for inducing differentiation, preferably of non-embryonic stem cells. In particular, such three-dimensional (3D) structures are useful for culturing primary tissue cells

Fractals in tissue engineering

The disclosure relates to a method for producing three-dimensional cell cluster on an inorganic cell culture platform comprising three-dimensional structures, preferably fractal structures. Such three-dimensional structures are useful for culturing cells and tissues, preferably in three dimensions. Such three-dimensional structures are useful for inducing differentiation, preferably of non-embryonic stem cells. In particular, such three-dimensional (3D) structures are useful for culturing primary tissue cells

Complex tumor spheroid formation and one‐step cancer‐ associated fibroblasts purification from hepatocellular carcinoma tissue promoted by inorganic surface topography

In vitro cell models play important roles as testbeds for toxicity studies, drug development, or as replacements in animal experiments. In particular, complex tumor models such as hepatocellular carcinoma (HCC) are needed to predict drug efficacy and facilitate translation into clinical practice. In this work, topographical features of amorphous silicon dioxide (SiO2) are fabricated and tested for cell culture of primary HCC cells and cell lines. The topographies vary from pyramids to octahedrons to structures named fractals, with increased hierarchy and organized in periodic arrays (square or Hexagonal). The pyramids were found to promote complex 2D/3D tissue formation from primary HCC cells. It was found that the 2D layer was mainly composed of cancer‐associated fibroblasts (CAFs), while the 3D spheroids were composed of tumor cells enwrapped by a CAF layer. Compared with conventional protocols for 3D cultures, this novel approach mimics the 2D/3D complexity of the original tumor by invading CAFs and a microtumor. Topographies such as octahedrons and fractals exclude tumor cells and allow one‐step isolation of CAFs even directly from tumor tissue of patients as the CAFs migrate into the structured substrate. Cell lines form spheroids within a short time. The presented inorganic topographical surfaces stimulate complex spheroid formation while avoiding additional biological scaffolds and allowing direct visualization on the substrate

Toward an improved definition of the genetic and tumor spectrum associated with SDH germ-line mutations

The tricarboxylic acid, or Krebs, cycle is central to the cellular metabolism of sugars, lipids, and amino acids; it fuels the mitochondrial respiratory chain for energy generation. In the past decade, mutations in the Krebs-cycle enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase have been documented to be causally involved in carcinogenesis. This review is focused on the relationship between SDH mutations and the carcinogenic phenotype. The succinate dehydrogenase complex catalyzes the oxidation of succinate to fumarate; mutations in its subunits SDHA, SDHB, SDHC, and SDHD, and in the assembly factor SDHAF2, result in syndromes with distinct tumor types, including pheochromocytoma/paraganglioma, gastrointestinal stromal tumor, and, less often, renal-cell carcinoma and pituitary adenoma. In this study we collected all previously reported SDH mutations with the aim of defining their nature and tumor spectrum. In addition, genotype-phenotype correlations as well as mechanisms of biallelic inactivation were analyzed in the SDH-deficient setting. Finally, we performed bioinformatics analysis using SIFT, Polyphen2, and Mutation Assessor to predict the functional impact of nonsynonymous mutations. The prediction of the latter was further compared with available SDHA and/or SDHB immunohistochemistry data