Design and Fabrication of High-Frequency Piezoelectric Micromachined Ultrasonic Transducer Based on an AlN Thin Film.

A piezoelectric micromachined ultrasonic transducer (PMUT) is a microelectromechanical system (MEMS) device that can transmit and receive ultrasonic waves. Given its advantages of high-frequency ultrasound with good directionality and high resolution, PMUT can be used in application scenarios with low power supply, such as fingerprint recognition, nondestructive testing, and medical diagnosis. Here, a PMUT based on an aluminum nitride thin-film material is designed and fabricated. First, the eigenfrequencies of the PMUT are studied with multiphysics coupling simulation software, and the relationship between eigenfrequencies and vibration layer parameters is determined. The transmission performance of the PMUT is obtained via simulation. The PMUT device is fabricated in accordance with the designed simple MEMS processing process. The topography of the PMUT vibration layer is determined via scanning electron microscopy, and the resonant frequency of the PMUT device is 7.43 MHz. The electromechanical coupling coefficient is 2.21% via an LCR tester.

Enhancement of the Transmission Performance of Piezoelectric Micromachined Ultrasound Transducers by Vibration Mode Optimization.

Ultrasound is widely used in industry and the agricultural, biomedical, military, and other fields. As key components in ultrasonic applications, the characteristic parameters of ultrasonic transducers fundamentally determine the performance of ultrasonic systems. High-frequency ultrasonic transducers are small in size and require high precision, which puts forward higher requirements for sensor design, material selection, and processing methods. In this paper, a three-dimensional model of a high-frequency piezoelectric micromachined ultrasonic transducer (PMUT) is established based on the finite element method (FEM). This 3D model consists of a substrate, a silicon device layer, and a molybdenum-aluminum nitride-molybdenum (Mo-AlN-Mo) sandwich piezoelectric layer. The effect of the shape of the transducer's vibrating membrane on the transmission performance was studied. Through a discussion of the parametric scanning of the key dimensions of the diaphragms of the three structures, it was concluded that the fundamental resonance frequency of the hexagonal diaphragm was higher than that of the circle and the square under the same size. Compared with the circular diaphragm, the sensitivity of the square diaphragm increased by 8.5%, and the sensitivity of the hexagonal diaphragm increased by 10.7%. The maximum emission sound-pressure level of the hexagonal diaphragm was 6.6 times higher than that of the circular diaphragm. The finite element results show that the hexagonal diaphragm design has great advantages for improving the transmission performance of the high-frequency PMUT.

An acetylation-enhanced interaction between transcription factor Sox2 and the steroid receptor coactivators facilitates Sox2 transcriptional activity and function.

SRY-box 2 (Sox2) is a transcription factor with critical roles in maintaining embryonic stem (ES) cell and adult stem cell functions and in tumorigenesis. However, how Sox2 exerts its transcriptional function remains unclear. Here, we used an in vitro protein-protein interaction assay to discover transcriptional regulators for ES cell core transcription factors (Oct4, Sox2, Klf4, and c-Myc) and identified members of the steroid receptor coactivators (SRCs) as Sox2-specific interacting proteins. The SRC family coactivators have broad roles in transcriptional regulation, but it is unknown whether they also serve as Sox2 coactivators. We demonstrated that these proteins facilitate Sox2 transcriptional activity and act synergistically with p300. Furthermore, we uncovered an acetylation-enhanced interaction between Sox2 and SRC-2/3, but not SRC-1, demonstrating it is Sox2 acetylation that promotes the interaction. We identified putative Sox2 acetylation sites required for acetylation-enhanced interaction between Sox2 and SRC-3 and demonstrated that acetylation on these sites contributes to Sox2 transcriptional activity and recruitment of SRC-3. We showed that activation domains 1 and 2 of SRC-3 both display a preferential binding to acetylated Sox2. Finally, functional analyses in mouse ES cells demonstrated that knockdown of SRC-2/3 but not SRC-1 in mouse ES cells significantly downregulates the transcriptional activities of various Sox2 target genes and impairs ES cell stemness. Taken together, we identify specific SRC family proteins as novel Sox2 coactivators and uncover the role of Sox2 acetylation in promoting coactivator recruitment and Sox2 transcriptional function.

Mutation-induced DNMT1 cleavage drives neurodegenerative disease.

Specific mutations within the replication foci targeting sequence (RFTS) domain of human DNMT1 are causative of two types of adult-onset neurodegenerative diseases, HSAN1E and ADCA-DN, but the underlying mechanisms are largely unknown. We generated Dnmt1-M1 and Dnmt1-M2 knock-in mouse models that are equivalent to Y495C and D490E-P491Y mutation in patients with HSAN1E, respectively. We found that both mutant heterozygous mice are viable, have reduced DNMT1 proteins, and exhibit neurodegenerative phenotypes including impaired learning and memory. The homozygous mutants die around embryonic day 10.5 and are apparently devoid of DNMT1 proteins. We present the evidence that the mutant DNMT1 proteins are unstable, most likely because of cleavage within RFTS domain by an unidentified proteinase. Moreover, we provide evidence that the RFTS mutation­induced cleavage of DNMT1, but not mutation itself, is responsible for functional defect of mutant DNMT1. Our study shed light on the mechanism of DNMT1 RFTS mutation causing neurodegenerative diseases.

Moderate DNA hypomethylation suppresses intestinal tumorigenesis by promoting caspase-3 expression and apoptosis.

Global DNA hypomethylation is a most common epigenetic alteration in human neoplasia. However, accumulative evidence shows that global DNA hypomethylation impacts tumorigenesis in a tissue-specific manner, promoting tumorigenesis in some but suppressing tumorigenesis in others including colorectal cancer. The underlying mechanisms, especially how DNA hypomethylation suppresses tumorigenesis, remain largely unknown. Here, we investigate how DNA hypomethylation affects intestinal tumorigenesis by using an Uhrf1 tandem tudor domain knockin mutant mouse model (Uhrf1ki/ki) that exhibits a moderate ~10% reduction of global DNA methylation. We found that both chemical-induced colorectal carcinogenesis and Apc loss of heterozygosity (LOH)-induced intestinal tumorigenesis are substantially suppressed in the Uhrf1 mutant mice. Furthermore, unlike Dnmt1 hypomorphic mice in which DNA hypomethylation suppresses the incidence of macroscopic intestinal tumors but promotes the formation of microadenoma in ApcMin/+ background, Uhrf1ki/ki/ApcMin/+ mice have markedly reduced incidence of both microadenoma and macroadenoma. DNA hypomethylation does not appear to affect Apc LOH, activation of the Wnt or Hippo pathway, or tumor cell proliferation, but acts cooperatively with activated Wnt pathway to enhance the caspase-3 gene expression, activation, and apoptosis. Furthermore, increased caspase-3 expression correlates with DNA hypomethylation within the caspase-3 enhancer regions. Taken together, we present a new mouse model for investigating the role of and the molecular mechanisms by which DNA hypomethylation suppresses intestinal tumorigenesis. Our finding that a moderate DNA hypomethylation is sufficient to suppress intestinal tumorigenesis by promoting caspase-3 expression and apoptosis sheds new light on DNA-methylation inhibitor-based colorectal cancer therapeutics.

Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1.

Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase1,2. Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions2-4. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility5-7, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF18,9, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.

The 5-Hydroxymethylcytosine (5hmC) Reader UHRF2 Is Required for Normal Levels of 5hmC in Mouse Adult Brain and Spatial Learning and Memory.

UHRF2 has been implicated as a novel regulator for both DNA methylation (5mC) and hydroxymethylation (5hmC), but its physiological function and role in DNA methylation/hydroxymethylation are unknown. Here we show that in mice, UHRF2 is more abundantly expressed in the brain and a few other tissues. Uhrf2 knock-out mice are viable and fertile and exhibit no gross defect. Although there is no significant change of DNA methylation, the Uhrf2 null mice exhibit a reduction of 5hmC in the brain, including the cortex and hippocampus. Furthermore, the Uhrf2 null mice exhibit a partial impairment in spatial memory acquisition and retention. Consistent with the phenotype, gene expression profiling uncovers a role for UHRF2 in regulating neuron-related gene expression. Finally, we provide evidence that UHRF2 binds 5hmC in cells but does not appear to affect the TET1 enzymatic activity. Together, our study supports UHRF2 as a bona fide 5hmC reader and further demonstrates a role for 5hmC in neuronal function.

Dissecting the precise role of H3K9 methylation in crosstalk with DNA maintenance methylation in mammals.

In mammals it is unclear if UHRF1-mediated DNA maintenance methylation by DNMT1 is strictly dependent on histone H3K9 methylation. Here we have generated an Uhrf1 knockin (KI) mouse model that specifically abolishes the H3K9me2/3-binding activity of Uhrf1. The homozygous Uhrf1 KI mice are viable and fertile, and exhibit ∼10% reduction of DNA methylation in various tissues. The reduced DNA methylation occurs globally in the genome and does not restrict only to the H3K9me2/3 enriched repetitive sequences. In vitro UHRF1 binds with higher affinity to reconstituted nucleosome with hemi-methylated CpGs than that with H3K9me2/3, although it binds cooperatively to nucleosome with both modifications. We also show that the nucleosome positioning affects the binding of methylated DNA by UHRF1. Thus, while our study supports a role for H3K9 methylation in promoting DNA methylation, it demonstrates for the first time that DNA maintenance methylation in mammals is largely independent of H3K9 methylation.