Risks and Recommendations in Prenatally Detected De Novo Balanced Chromosomal Rearrangements from Assessment of Long-Term Outcomes.

The 6%-9% risk of an untoward outcome previously established by Warburton for prenatally detected de novo balanced chromosomal rearrangements (BCRs) does not account for long-term morbidity. We performed long-term follow-up (mean 17 years) of a registry-based nationwide cohort of 41 individuals carrying a prenatally detected de novo BCR with normal first trimester screening/ultrasound scan. We observed a significantly higher frequency of neurodevelopmental and/or neuropsychiatric disorders than in a matched control group (19.5% versus 8.3%, p = 0.04), which was increased to 26.8% upon clinical follow-up. Chromosomal microarray of 32 carriers revealed no pathogenic imbalances, illustrating a low prognostic value when fetal ultrasound scan is normal. In contrast, mate-pair sequencing revealed disrupted genes (ARID1B, NPAS3, CELF4), regulatory domains of known developmental genes (ZEB2, HOXC), and complex BCRs associated with adverse outcomes. Seven unmappable autosomal-autosomal BCRs with breakpoints involving pericentromeric/heterochromatic regions may represent a low-risk group. We performed independent phenotype-aware and blinded interpretation, which accurately predicted benign outcomes (specificity = 100%) but demonstrated relatively low sensitivity for prediction of the clinical outcome in affected carriers (sensitivity = 45%-55%). This sensitivity emphasizes the challenges associated with prenatal risk prediction for long-term morbidity in the absence of phenotypic data given the still immature annotation of the morbidity genome and poorly understood long-range regulatory mechanisms. In conclusion, we upwardly revise the previous estimates of Warburton to a morbidity risk of 27% and recommend sequencing of the chromosomal breakpoints as the first-tier diagnostic test in pregnancies with a de novo BCR.

Different expression levels of glycans on leukemic cells-a novel screening method with molecularly imprinted polymers (MIP) targeting sialic acid.

Sialic acid (SA) is normally expressed on the cell membranes and is located at the terminal position of the sugar chains. SA plays an important role for regulation of the innate immunity, function as markers of the cells and can be recognized by a variety of receptors. Interestingly, the level of SA expression is increased on metastatic cancer cells. The availability of specific antibodies against SA is limited and, therefore, biomarker tools for detection of SA are lacking. We have recently presented a novel method for specific fluorescence labeling of SA molecular imprinted polymers (MIP). Here, we have performed an extended screening of SA expression by using SA-MIP and included four different chronic lymphocytic leukemia (CLL) cell lines, conveniently analyzed by flow cytometry and fluorescence microscopy. SA expression was detected in four cell lines at different levels, and the SA expression were verified with lectin-FITC. These results show that SA-MIP can be used as a plastic antibody for detection of SA using both flow cytometry and fluorescence microscopy. We suggest that SA-MIP can be used for screening of different tumor cells of various stages, including CLL cells.

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds.

Sialic Acid-Imprinted Fluorescent Core-Shell Particles for Selective Labeling of Cell Surface Glycans.

The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid-imprinted core-shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities, and the use of an NBD-appended urea-monomer as a binary hydrogen-bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT-modified silica core particles using ethylene glycol dimethacrylate (EGDMA) as cross-linker resulting in a shell thickness of ca. 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 × 10(5) M(-1) in 2% water, 5.9 × 10(3) M(-1) in 98% water, B(max) ≈ 10 µmol g(-1)), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 10(3) M(-1) in 98% water). In cell imaging experiments, the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin.

Influence of substratum hydrophobicity on salivary pellicles: organization or composition?

Different physico-chemical properties (eg adsorption kinetics, thickness, viscoelasticity, and mechanical stability) of adsorbed salivary pellicles depend on different factors, including the properties (eg charge, roughness, wettability, and surface chemistry) of the substratum. Whether these differences in the physico-chemical properties are a result of differences in the composition or in the organization of the pellicles is not known. In this work, the influence of substratum wettability on the composition of the pellicle was studied. For this purpose, pellicles eluted from substrata of different but well-characterized wettabilities were examined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that substratum hydrophobicity did not have a major impact on pellicle composition. In all substrata, the major pellicle components were found to be cystatins, amylases and large glycoproteins, presumably mucins. In turn, interpretation of previously reported data based on the present results suggests that variations in substratum wettability mostly affect the organization of the pellicle components.

Sequence and expression analysis of gaps in human chromosome 20.

The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and/or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ∼ 99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum. One of these CpG islands was differentially methylated and paternally hypermethylated. We found all chr 20 gaps to comprise structured non-coding RNAs (ncRNAs) and to be conserved in primates. We verified expression for 13 candidate ncRNAs, some of which showed tissue specificity. Four ncRNAs expressed within the gap at DLGAP4 show elevated expression in the human brain. Our data suggest that unfinished human genome gaps are likely to comprise numerous functional elements.

Gene-Mutation-Based Algorithm for Prediction of Treatment Response in Colorectal Cancer Patients.

Purpose: Despite the high mortality of metastatic colorectal cancer (mCRC), no new biomarker tools are available for predicting treatment response. We developed gene-mutation-based algorithms as a biomarker classifier to predict treatment response with better precision than the current predictive factors. Methods: Random forest machine learning (ML) was applied to identify the candidate algorithms using the MSK Cohort (n = 471) as a training set and validated in the TCGA Cohort (n = 221). Logistic regression, progression-free survival (PFS), and univariate/multivariate Cox proportional hazard analyses were performed and the performance of the candidate algorithms was compared with the established risk parameters. Results: A novel 7-Gene Algorithm based on mutation profiles of seven KRAS-associated genes was identified. The algorithm was able to distinguish non-progressed (responder) vs. progressed (non-responder) patients with AUC of 0.97 and had predictive power for PFS with a hazard ratio (HR) of 16.9 (p < 0.001) in the MSK cohort. The predictive power of this algorithm for PFS was more pronounced in mCRC (HR = 16.9, p < 0.001, n = 388). Similarly, in the TCGA validation cohort, the algorithm had AUC of 0.98 and a significant predictive power for PFS (p < 0.001). Conclusion: The novel 7-Gene Algorithm can be further developed as a biomarker model for prediction of treatment response in mCRC patients to improve personalized therapies.

Reversible Self-Assembled Monolayers with Tunable Surface Dynamics for Controlling Cell Adhesion Behavior.

Cells adhering onto surfaces sense and respond to chemical and physical surface features. The control over cell adhesion behavior influences cell migration, proliferation, and differentiation, which are important considerations in biomaterial design for cell culture, tissue engineering, and regenerative medicine. Here, we report on a supramolecular-based approach to prepare reversible self-assembled monolayers (rSAMs) with tunable lateral mobility and dynamic control over surface composition to regulate cell adhesion behavior. These layers were prepared by incubating oxoacid-terminated thiol SAMs on gold in a pH 8 HEPES buffer solution containing different mole fractions of ω-(ethylene glycol)2-4- and ω-(GRGDS)-, α-benzamidino bolaamphiphiles. Cell shape and morphology were influenced by the strength of the interactions between the amidine-functionalized amphiphiles and the oxoacid of the underlying SAMs. Dynamic control over surface composition, achieved by the addition of inert filler amphiphiles to the RGD-functionalized rSAMs, reversed the cell adhesion process. In summary, rSAMs featuring mobile bioactive ligands offer unique capabilities to influence and control cell adhesion behavior, suggesting a broad use in biomaterial design, tissue engineering, and regenerative medicine.

Fluorescent Molecularly Imprinted Polymer Layers against Sialic Acid on Silica-Coated Polystyrene Cores-Assessment of the Binding Behavior to Cancer Cells.

Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells.

PIP5K1α is Required for Promoting Tumor Progression in Castration-Resistant Prostate Cancer.

PIP5K1α has emerged as a promising drug target for the treatment of castration-resistant prostate cancer (CRPC), as it acts upstream of the PI3K/AKT signaling pathway to promote prostate cancer (PCa) growth, survival and invasion. However, little is known of the molecular actions of PIP5K1α in this process. Here, we show that siRNA-mediated knockdown of PIP5K1α and blockade of PIP5K1α action using its small molecule inhibitor ISA-2011B suppress growth and invasion of CRPC cells. We demonstrate that targeted deletion of the N-terminal domain of PIP5K1α in CRPC cells results in reduced growth and migratory ability of cancer cells. Further, the xenograft tumors lacking the N-terminal domain of PIP5K1α exhibited reduced tumor growth and aggressiveness in xenograft mice as compared to that of controls. The N-terminal domain of PIP5K1α is required for regulation of mRNA expression and protein stability of PIP5K1α. This suggests that the expression and oncogenic activity of PIP5K1α are in part dependent on its N-terminal domain. We further show that PIP5K1α acts as an upstream regulator of the androgen receptor (AR) and AR target genes including CDK1 and MMP9 that are key factors promoting growth, survival and invasion of PCa cells. ISA-2011B exhibited a significant inhibitory effect on AR target genes including CDK1 and MMP9 in CRPC cells with wild-type PIP5K1α and in CRPC cells lacking the N-terminal domain of PIP5K1α. These results indicate that the growth of PIP5K1α-dependent tumors is in part dependent on the integrity of the N-terminal sequence of this kinase. Our study identifies a novel functional mechanism involving PIP5K1α, confirming that PIP5K1α is an intriguing target for cancer treatment, especially for treatment of CRPC.

Digital holographic microscopy: a noninvasive method to analyze the formation of spheroids.

Digital holographic (DH) microscopy is a unique noninvasive method to analyze living cells. With DH microscopy, in vitro cell cultures can be imaged in 2D and pseudo-3D and measurements of size and morphology of the cells are provided. Here, a description of a novel methodology utilizing DH microscopy for the analysis of spheroids is presented. A cell culture protocol is introduced and morphological parameters of cell spheroids as measured by DH microscopy are presented. The study confirms the use of DH microscopy for the analysis of cell spheroids. In the future, organoids can be analyzed with DH microscopy, and it can also be used for drug response and cell death analyses.

Molecularly imprinted polymers in biological applications.

Molecularly imprinted polymers (MIPs) are currently widely used and further developed for biological applications. The MIP synthesis procedure is a key process, and a wide variety of protocols exist. The templates that are used for imprinting vary from the smallest glycosylated glycan structures or even amino acids to whole proteins or bacteria. The low cost, quick preparation, stability and reproducibility have been highlighted as advantages of MIPs. The biological applications utilizing MIPs discussed here include enzyme-linked assays, sensors, in vivo applications, drug delivery, cancer diagnostics and more. Indeed, there are numerous examples of how MIPs can be used as recognition elements similar to natural antibodies.

Supervised classification of etoposide-treated in vitro adherent cells based on noninvasive imaging morphology.

Single-cell studies using noninvasive imaging is a challenging, yet appealing way to study cellular characteristics over extended periods of time, for instance to follow cell interactions and the behavior of different cell types within the same sample. In some cases, e.g., transplantation culturing, real-time cellular monitoring, stem cell studies, in vivo studies, and embryo growth studies, it is also crucial to keep the sample intact and invasive imaging using fluorophores or dyes is not an option. Computerized methods are needed to improve throughput of image-based analysis and for use with noninvasive microscopy such methods are poorly developed. By combining a set of well-documented image analysis and classification tools with noninvasive microscopy, we demonstrate the ability for long-term image-based analysis of morphological changes in single cells as induced by a toxin, and show how these changes can be used to indicate changes in biological function. In this study, adherent cell cultures of DU-145 treated with low-concentration (LC) etoposide were imaged during 3 days. Single cells were identified by image segmentation and subsequently classified on image features, extracted for each cell. In parallel with image analysis, an MTS assay was performed to allow comparison between metabolic activity and morphological changes after long-term low-level drug response. Results show a decrease in proliferation rate for LC etoposide, accompanied by changes in cell morphology, primarily leading to an increase in cell area and textural changes. It is shown that changes detected by image analysis are already visible on day 1 for [Formula: see text] etoposide, whereas effects on MTS and viability are detected only on day 3 for [Formula: see text] etoposide concentration, leading to the conclusion that the morphological changes observed occur before and at lower concentrations than a reduction in cell metabolic activity or viability. Three classifiers are compared and we report a best case sensitivity of 88% and specificity of 94% for classification of cells as treated/untreated.

Interfacing antibody-based microarrays and digital holography enables label-free detection for loss of cell volume.

BACKGROUND: We introduce the combination of digital holographic microscopy (DHM) and antibody microarrays as a powerful tool to measure morphological changes in specifically antibody-captured cells. The aim of the study was to develop DHM for analysis of cell death of etoposide-treated suspension cells. RESULTS/METHODOLOGY: We demonstrate that the cell number, mean area, thickness and volume were noninvasively measured by using DHM. The cell number was stable over time, but the two cell lines showed changes of cell area and cell irregularity after treatment. The cell volume in etoposide-treated cells was decreased, whereas untreated cells showed stable volume. CONCLUSION: Our results provide proof of concept for using DHM combined with antibody-based microarray technology for detecting morphological changes in captured cells.