Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Role of histone H1 in neural differentiation of embryonic stem cells

Linker histone H1 is a key structural protein facilitating the formation of higher order chromatin structures and regulates specific gene expression. In mammals, there exist 11 closely related H1 variants. Previous studies show that H1 depletion by 50% impairs specific gene regulation and differentiation of embryonic stem cells (ESCs). However, the mechanisms by which H1 and its variants regulate ESC differentiation remain elusive. Here, we demonstrate a dosage effect of H1 variants in mouse ESCs through severe H1 depletion and mutation analysis. We establish ultra-low H1 ESCs by sequential depletion of six somatic H1 variants. These cells exhibit normal ESC morphology and self-renewal. During neural differentiation, the total H1 level gradually increases, and H1 depletion reveals a dosage effect in neurite formation, induction of neural lineage-specific genes, and silencing of pluripotency-associated genes such as Oct4 and Nanog. In addition, severe H1 depletion causes reduced cell proliferation and cellular senescence in neural lineages. Significantly, Oct4 knockdown effectively restores neural differentiation and partially rescues the reduction in cell proliferation and cellular senescence. These results suggest that H1 is crucial for neural differentiation of ESCs and its regulation in the process acts in a dosage dependent, rather than a variant specific, manner. Another part of this thesis centers on analysis of H1 mutations frequently occurred in follicular lymphoma or transformed follicular lymphoma. These mutations in H1 are clustered in the globular and C-terminal domains directly involved in chromatin binding. By comparing the properties of wild-type human H1c (hH1c) and mutant hHcS102F expressed in H1c/H1d/H1e triple knockout mouse ESCs, we find that S102F mutation dramatically impairs the association of hH1c with chromatin. These results suggest that the identified H1 mutations in follicular lymphoma most likely result in a loss-of-function phenotype by reducing the binding affinity of H1 for chromatin, thus compromising chromatin compaction and the regulation of specific genes.Ph.D

Protection for Medical Workers Confronting the Challenges During the COVID-19 Outbreak-A Case of China

The COVID-19 outbreak is a major public health emergency initially occurred in Hubei, the epicenter in China, and then it rapidly turned into a global pandemic with much more than five million infected cases 28 May, 2020 [1]. Thanks to the joint efforts from all parties, especially the great contributions from Chinese medical workers, China’s domestic situation has improved significantly, while it remains severe in the rest of world.</p

Application of Transfer Learning for Object Detection on Manually Collected Data

This paper investigates the usage of pre-trained deep learning neural networks for object detection on a manually collected dataset for real-life indoor objects. Availability of object-specific datasets is a great challenge and the unavoidable task of collecting, processing and annotating ground truth data is laborious and time-consuming. In this paper, two famous models (AlexNet and Vgg16) have been evaluated as feature extractors in a Faster R-CNN network. Network models have been trained end-to-end on the collected dataset. The study highlights the poor performance of state of art systems when dealing with small size objects. Modifying the detector design by redesigning systems’ anchor boxes might help to tackle this problem. Detector results on the proposed dataset have been collected and compared. In addition, limitations and future work have been discussed

Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis

In atherosclerosis, plaques preferentially develop in arterial regions of disturbed blood flow (d-flow), which alters endothelial gene expression and function. Here, we determined that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase–dependent (DNMT-dependent) manner. Induction of d-flow by partial carotid ligation surgery in a murine model induced DNMT1 in arterial endothelium. In cultured endothelial cells, DNMT1 was enhanced by oscillatory shear stress (OS), and reduction of DNMT with either the inhibitor 5-aza-2′-deoxycytidine (5Aza) or siRNA markedly reduced OS-induced endothelial inflammation. Moreover, administration of 5Aza reduced lesion formation in 2 mouse models of atherosclerosis. Using both reduced representation bisulfite sequencing (RRBS) and microarray, we determined that d-flow in the carotid artery resulted in hyper-methylation within the promoters of 11 mechanosensitive genes and that 5Aza treatment restored normal methylation patterns. Of the identified genes, HoxA5 and Klf3 encode transcription factors that contain cAMP response elements, suggesting that the methylation status of these loci could serve as a mechanosensitive master switch in gene expression. Together, our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis

Tuning piezoelectricity via thermal annealing at a freestanding ferroelectric membrane

Tuning the ferroelectric domain structure by a combination of elastic and electrostatic engineering provides an effective route for enhanced piezoelectricity. However, for epitaxial thin films, the clamping effect imposed by the substrate does not allow aftergrowth tuning and also limits the electromechanical response. In contrast, freestanding membranes, which are free of substrate constraints, enable the tuning of a subtle balance between elastic and electrostatic energies, giving new platforms for enhanced and tunable functionalities. Here, highly tunable piezoelectricity is demonstrated in freestanding PbTiO3 membranes, by varying the ferroelectric domain structures from c-dominated to c/a and a domains via aftergrowth thermal treatment. Significantly, the piezoelectric coefficient of the c/a domain structure is enhanced by a factor of 2.5 compared with typical c domain PbTiO3. This work presents a new strategy to manipulate the piezoelectricity in ferroelectric membranes, highlighting their great potential for nano actuators, transducers, sensors and other NEMS device applications