Brain “inflammaging,” a low-grade and chronic inflammation, is a major hallmark for aging-related neurodegenerative diseases. Here, by profiling H3K27ac and gene expression patterns in human and ...mouse brains, we found that age-related up-regulated (Age-Up) and down-regulated (Age-Down) genes have distinct H3K27ac patterns. Although both groups show promoter H3K27ac, the Age-Up genes, enriched for inflammation-related functions, are additionally marked by broad H3K27ac distribution over their gene bodies, which is progressively reduced during aging. Age-related gene expression changes can be predicted by gene-body H3K27ac level. Contrary to the presumed transcription activation function of promoter H3K27ac, we found that broad gene-body hyper H3K27ac suppresses overexpression of inflammaging genes. Altogether, our findings revealed opposite regulations by H3K27ac of Age-Up and Age-Down genes and a mode of broad gene-body H3K27ac in repressing transcription.
Silver nanoplates are introduced as a new photoacoustic contrast agent that can be easily functionalized for molecular photoacoustic imaging in vivo. Methods are described for synthesis, ...functionalization, and stabilization of silver nanoplates using biocompatible (“green”) reagents. Directional antibody conjugation to the nanoplate surface is presented along with proof of molecular sensitivity in vitro with pancreatic cancer cells. Cell viability tests show the antibody-conjugated silver nanoplates to be nontoxic at concentrations up to 1 mg/mL. Furthermore, the silver nanoplates’ potential for in vivo application as a molecularly sensitive photoacoustic contrast agent is demonstrated using an orthotopic mouse model of pancreatic cancer. Results of these studies suggest that the synthesized silver nanoplates are well suited for a host of biomedical imaging and sensing applications.
Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease that can ultimately lead to cirrhosis and hepatocellular carcinoma. Although NASH is associated with excessive liver ...lipid accumulation, hepatocyte injury, inflammation, and fibrosis, its etiology remains incompletely understood. These can be characterized by determining transcriptional changes in specific genes previously found to be involved in these processes. As an inherently multifaceted disease, studies of NASH often require unbiased examination of major genes and pathways to identify the mechanisms involved in this disorder. To address this need, quantitative approaches such as mRNA-sequencing have been developed for the global assessment of gene expression. Here, we describe a protocol for bulk mRNA-sequencing that can be utilized for both liver samples and specific cell types isolated from the liver. This approach provides an important resource to further understand the molecular changes that occur during the development of NASH that can be utilized to design better therapeutic treatments.
Upscaling groundwater flow is a fundamental challenge in hydrogeology. This study proposed time‐fractional flow equations (t‐FFEs) for upscaling long‐term, transient groundwater flow and propagation ...of pressure heads in heterogeneous media. Monte Carlo simulations showed that, with increasing variance and correlation of the hydraulic conductivity (K), flow dynamics gradually deviated from Darcian flow and exhibit sub‐diffusive, time‐dependent evolution which can be separated into three major stages. At the early stage, the interconnected high‐K zones dominated flow, while at intermediate times, the transverse flow due to mixed high‐ and low‐K zones caused delayed rise of the piezometric head. At late times when flow in the relatively high‐K domains reached stability, cells with very low‐K continued to block the entry of water and generate “islands” with low piezometric head, significantly extending the temporal evolution of the piezometric head. The elongated water breakthrough curve cannot be quantified by the flow equation with an effective K, the space‐fractional flow equation, or the multi‐rate mass transfer (MRMT) flow model with a few rates, motivating the development of t‐FFEs assuming temporally non‐Darcian flow. Model applications showed that both the early and intermediate stages of flow dynamics can be captured by a single‐index t‐FFE (whose index is the exponent of the power‐law probability density function of the random operational time for water parcels), but the overall evolution of flow dynamics, especially the enhanced retention of flow at later times, required a distributed‐order t‐FFE with variable indexes for different flow phases that can dominate flow dynamics at different stages. Therefore, transient groundwater flow in aquifers with spatially stationary heterogeneity can be temporally non‐Darcian and non‐stationary, due to the time‐sensitive, combined effects of interconnected high‐K channels and isolated low‐K deposits on flow dynamics (which is the hydrogeological mechanism for the temporally non‐Darcian flow and sub‐diffusive pressure propagation), whose long‐term behavior can be quantified by multi‐index stochastic models.
Key Points
Transient groundwater flow can be temporally non‐Darcian and non‐stationary in porous media with stationary heterogeneity
Interconnected high‐K channels and isolated low‐K deposits caused temporally non‐Darcian flow in aquifers
The distributed time fractional flow equation can upscale the full‐range transient groundwater flow
Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 ...diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.
Early diagnosis of prostate cancer (PCa) is critical for the application of efficient treatment to PCa patients. However, the majority of PCas remains indolent from several months to several years ...before malignancy. Current diagnosis methods have limitations in their reliability and are inefficient in time cost. Thus, an efficient in vivo PCa cell xenograft model is highly desired for diagnostic studies in PCas. In the present study we present a standardized procedure to create a PCa cell xenograft model using zebrafish (Danio rerio) as the host. PC3-CTR cells, a cell line from adenocarcinoma with stable expression of calcitonin receptor (CRT), were subcutaneously injected into zebrafish larvae at 48 h post fertilization. The nursing conditions for the larvae were optimized with stable survival rates of post hatch and post PC3-CTR cell injection. In this system, the progression of PC3-CTR cells in vivo was evaluated by migration and proliferation of the cells. Massive migrations of PC3 cells in vivo were observed at post injection day (PID)3. The injected PC3-CTR cells eventually invaded the whole larval zebrafish at PID5. Quantification of PC3-CTR cell proliferation was done using quantitative PCR (qPCR) analysis targeting the expression profiles of two PCa housekeeping genes, TATA-binding protein (TBP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) encoding genes. The excessive proliferation of PC3 cells in vivo was detected with both qPCR assays. Expression levels of one non‑coding gene, prostate cancer associated 3 gene (pca3), and two other genes encoding transient receptor potential ion channel Melastatin 8 (trpm8) and prostate-specific membrane antigen (psma), showed a significantly enhanced aggressiveness of PC3-CTR cells in vivo. The model established in the present study provides an improved in vivo model for the diagnosis of PCas efficiently. This PCa cell xenograft model can also serve as a tool for high throughput anti-PCa drug screening in therapeutic treatments.
The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are ...negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.
DNA nanotechnology has proven to be a powerful strategy for the bottom-up preparation of colloidal nanoparticle (NP) superstructures, enabling the coordination of multiple NPs with orientation and ...separation approaching nanometer precision. To do this, NPs are often conjugated with chemically modified, single-stranded (ss) DNA that can recognize complementary ssDNA on the DNA nanostructure. The limitation is that many NPs cannot be easily conjugated with ssDNA, and other conjugation strategies are expensive, inefficient, or reduce the specificity and/or precision with which NPs can be placed. As an alternative, the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Here, we demonstrate a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. With this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement. Additionally, we investigated the effects of peptide-PNA charge on the efficacy of QD immobilization in suboptimal conditions. The results validate peptide-PNA as a viable alternative to ssDNA conjugation of NPs and warrant studies of other NP-binding peptides for peptide-PNA conjugation.
Abstract
Summary
Analysis of RNA sequencing (RNA-Seq) data have highlighted the fact that most genes undergo alternative splicing (AS) and that these patterns are tightly regulated. Many of these ...events are complex, resulting in numerous possible isoforms that quickly become difficult to visualize, interpret and experimentally validate. To address these challenges we developed MAJIQ-SPEL, a web-tool that takes as input local splicing variations (LSVs) quantified from RNA-Seq data and provides users with visualization and quantification of gene isoforms associated with those. Importantly, MAJIQ-SPEL is able to handle both classical (binary) and complex, non-binary, splicing variations. Using a matching primer design algorithm it also suggests to users possible primers for experimental validation by RT-PCR and displays those, along with the matching protein domains affected by the LSV, on UCSC Genome Browser for further downstream analysis.
Availability and implementation
Program and code will be available at http://majiq.biociphers.org/majiq-spel.
Supplementary information
Supplementary data are available at Bioinformatics online.
The physical and chemical properties of synthetic DNA have transformed this prototypical biopolymer into a versatile nanoscale building block material in the form of DNA nanotechnology. DNA ...nanotechnology is, in turn, providing unprecedented precision bioengineering for numerous biomedical applications at the nanoscale including next generation immune-modulatory materials, vectors for targeted delivery of nucleic acids, drugs, and contrast agents, intelligent sensors for diagnostics, and theranostics, which combines several of these functionalities into a single construct. Assembling a DNA nanostructure to be programmed with a specific number of targeting moieties on its surface to imbue it with concomitant cellular uptake and retention capabilities along with carrying a specific therapeutic dose is now eminently feasible due to the extraordinary self-assembling properties and high formation efficiency of these materials. However, what remains still only partially addressed is how exactly this class of materials is taken up into cells in both the native state and as targeted or chemically facilitated, along with how stable they are inside the cellular cytosol and other cellular organelles. In this minireview, we summarize what is currently reported in the literature about how (i) DNA nanostructures are taken up into cells along with (ii) what is understood about their subsequent stability in the complex multi-organelle environment of the cellular milieu along with biological fluids in general. This allows us to highlight the many challenges that still remain to overcome in understanding DNA nanostructure-cellular interactions in order to fully translate these exciting new materials.
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