Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.Peer reviewe

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.EU/FP7/ESNATSDFGDoerenkamp-Zbinden Foundatio

Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells

Background & AimsThe differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories.MethodsGenome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified.ResultsGene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The “unwanted” intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC.ConclusionsThe present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation

Nucleic Acid Nanostructures - Design, Reconfigurability, and Applications

Self-assembly with near-atomic precision forms the basis for synthesizing biological structures found in living cells. These self-assembled structures retain the accuracy across sizes ranging from nanometers to macroscopic scale by exploiting the information encoded in the biomolecules. The fabrication of complex 3D synthetic molecular structures with such precision and controllability is cumbersome. Nucleic acid nanotechnology eases this process by enabling bottom-up construction of well-defined nanoscale objects through the predictability and programmability of interactions within DNA and RNA. The design, reconfigurability and a potential application of such nucleic acid nanostructures are presented in this thesis, which is based on three publications. Publication I presents a highly general and automated approach for the design and assembly of 3D RNA wireframe polyhedral nanostructures. Grounded on the principles of spanning tree-based strand routing, the method renders the target 3D wireframe structure as a single-stranded RNA. The output RNA sequence can be readily transcribed from the corresponding DNA template and assembled into the intended shape. As case examples, the design of three RNA polyhedral models: a tetrahedron, a triangular bipyramid, and a triangular prism were experimentally realized. The developed design pipeline is not restricted to polyhedral models and can be used to render arbitrary straight-line 3D meshes as RNA strands. Publication II and Publication III focus on the DNA origami approach to fabricate dynamic nanostructures. Publication II presents a reconfigurable chiral plasmonic molecule (CPM) that can be modulated remotely using visible light. The native CPMs are non-responsive to light but can change their spatial configuration by forming DNA triplex locks upon a decrease in the solution pH. To this end, a merocyanine-based photoacid was utilized as a photoresponsive medium that decreases the pH upon exposure to visible light. The chiral response from the CPMs was thus modulated by the intensity of the incident light, which alters the pH. By tuning the intensity, distinct steady out-of-equilibrium states were achieved, both in the pH and in the chiral response. In Publication III, the precise positioning capability and reconfigurability of the DNA origami technique were exploited. A DNA-origami-based device was fabricated to investigate DNA bending induced by TATA-binding protein. This bending was translated into an angular change in the DNA origami structures directly observable using a transmission electron microscope. Further, the role of transcription factors II A and II B in DNA bending was investigated. Our approach can be readily expanded to other DNA-distorting proteins with careful design considerations.Overall, this thesis contributes to various facets of nucleic acid nanotechnology from design to reconfigurability to applications. In essence, the findings presented in the individual sections illustrate that nucleic acid nanotechnology still has a large unexplored design space, novel switching mechanisms, and under-explored applications

Automated Detection of Seizure Types from the Higher-Order Moments of Maximal Overlap Wavelet Distribution

In this work, an attempt has been made to develop an automated system for detecting electroclinical seizures such as tonic-clonic seizures, complex partial seizures, and electrographic seizures (EGSZ) using higher-order moments of scalp electroencephalography (EEG). The scalp EEGs of the publicly available Temple University database are utilized in this study. The higher-order moments, namely skewness and kurtosis, are extracted from the temporal, spectral, and maximal overlap wavelet distributions of EEG. The features are computed from overlapping and non-overlapping moving windowing functions. The results show that the wavelet and spectral skewness of EEG is higher in EGSZ than in other types. All the extracted features are found to have significant differences (p < 0.05), except for temporal kurtosis and skewness. A support vector machine with a radial basis kernel designed using maximal overlap wavelet skewness yields a maximum accuracy of 87%. In order to improve the performance, the Bayesian optimization technique is utilized to determine the suitable kernel parameters. The optimized model achieves the highest accuracy of 96% and an MCC of 91% in three-class classification. The study is found to be promising, and it could facilitate the rapid identification process of life-threatening seizures

A DNA origami-based device for investigating DNA bending proteins by transmission electron microscopy

The DNA origami technique offers precise positioning of nanoscale objects with high accuracy. This has facilitated the development of DNA origami-based functional nanomechanical devices that enable the investigation of DNA–protein interactions at the single particle level. Herein, we used the DNA origami technique to fabricate a nanoscale device for studying DNA bending proteins. For a proof of concept, we used TATA-box binding protein (TBP) to evaluate our approach. Upon binding to the TATA box, TBP causes a bend to DNA of ∼90°. Our device translates this bending into an angular change that is readily observable with a conventional transmission electron microscope (TEM). Furthermore, we investigated the roles of transcription factor II A (TF(II)A) and transcription factor II B (TF(II)B). Our results indicate that TF(II)A introduces additional bending, whereas TF(II)B does not significantly alter the TBP–DNA structure. Our approach can be readily adopted to a wide range of DNA-bending proteins and will aid the development of DNA-origami-based devices tailored for the investigation of DNA–protein interactions.Peer reviewe

Signaling molecules, transcription growth factors and other regulators revealed from in-vivo and in-vitro models for the regulation of cardiac development

Several in-vivo heart developmental models have been applied to decipher the cardiac developmental patterning encompassing early, dorsal, cardiac and visceral mesoderm as well as various transcription factors such as Gata, Hand, Tin, Dpp, Pnr. The expression of cardiac specific transcription factors, such as Gata4, Tbx5, Tbx20, Tbx2, Tbx3, Mef2c, Hey1 and Hand1 are of fundamental significance for the in-vivo cardiac development. Not only the transcription factors, but also the signaling molecules involved in cardiac development were conserved among various species. Enrichment of the bone morphogenic proteins (BMPs) in the anterior lateral plate mesoderm is essential for the initiation of myocardial differentiation and the cardiac developmental process. Moreover, the expression of a number of cardiac transcription factors and structural genes initiate cardiac differentiation in the medial mesoderm. Other signaling molecules such as TGF-beta, IGF-1/2 and the fibroblast growth factor (FGF) play a significant role in cardiac repair/regeneration, ventricular heart development and specification of early cardiac mesoderm, respectively. The role of the Wnt signaling in cardiac development is still controversial discussed, as in-vitro results differ dramatically in relation to the animal models. Embryonic stem cells (ESC) were utilized as an important in-vitro model for the elucidation of the cardiac developmental processes since they can be easily manipulated by numerous signaling molecules, growth factors, small molecules and genetic manipulation. Finally, in the present review the dynamic role of the long noncoding RNA and miRNAs in the regulation of cardiac development are summarized and discussed. (C) 2015 Elsevier Ireland Ltd. All rights reserved

Standardization, Calibration, and Evaluation of Tantalum-Nano rGO-SnO2 Composite as a Possible Candidate Material in Humidity Sensors

The present study focuses the development and the evaluation of humidity sensors based on reduced graphene oxide—tin oxide (rGO-SnO2) nanocomposites, synthesized by a simple redox reaction between GO and SnCl2. The physico-chemical characteristics of the nanocomposites were analyzed by XRD, TEM, FTIR, and Raman spectroscopy. The formation of SnO2 crystal phase was observed through XRD. The SnO2 crystal phase anchoring to the graphene sheet was confirmed through TEM images. For the preparation of the sensors, tantalum substrates were coated with the sensing material. The sensitivity of the fabricated sensor was studied by varying the relative humidity (RH) from 11% to 95% over a period of 30 days. The dependence of the impedance and of the capacitance with RH of the sensor was measured with varying frequency ranging from 1 kHz to 100 Hz. The long-term stability of the sensor was measured at 95% RH over a period of 30 days. The results proved that rGO-SnO2 nanocomposites are an ideal conducting material for humidity sensors due to their high sensitivity, rapid response and recovery times, as well as their good long-term stability