Genome-wide identification of functional enhancers and their potential roles in pig breeding

Background:The pig is an economically important livestock species and is a widely applied large animal model in medical research.Enhancers are critical regulatory elements that have fundamental functions in evolution,development and disease.Genome-wide quantification of functional enhancers in the pig is needed.Results:We performed self-transcribing active regulatory region sequencing(STARR-seq)in the porcine kidney epithelial PK15 and testicular ST cell lines,and reliably identified 2576 functional enhancers.Most of these enhancers were located in repetitive sequences and were enriched within silent and lowly expressed genes.Enhancers poorly overlapped with chromatin accessibility regions and were highly enriched in chromatin with the repressive histone modification H3K9me3,which is different from predicted pig enhancers detected using ChIP-seq for H3K27ac or/and H3K4me1 modified histones.This suggests that most pig enhancers identified with STARR-seq are endogenously repressed at the chromatin level and may function during cell type-specific development or at specific developmental stages.Additionally,the PPP3CA gene is associated with the loin muscle area trait and the QKI gene is associated with alkaline phosphatase activity that may be regulated by distal functional enhancers.Conclusions:In summary,we generated the first functional enhancer map in PK15 and ST cells for the pig genome and highlight its potential roles in pig breeding.

The Effects of Some Penetration Enhancers on the Transdermal Iontophoretic Delivery of Insulin in Vitro *

The effects of some commonly used penetration enhancers such as laurocapram (AZ), oleic acid (OA), poloxamer (POL) and propylene glycol (PG) on the in vitro transdermal iontophoretic delivery of insulin through full-thickness mouse skin were investigated. The results showed that AZ had a synergistic effect on iontophoretic ability to enhance skin permeation of insulin, and PG could further increase this effect. 5% AZ / PG increased the iontophoretic steady state flux of insulin by a factor of 2.75 compared to that treated with iontophoresis alone. OA did not further enhance iontophoretic effect to increase skin permeation of insulin. The combination of iontophoresis and some enhancer provided a novel idea and possibility for transdermal delivery of insulin.

Enhancers and hindrances to doctor-nurse interdisciplinary collaborative practice in Nigeria

The purpose of this study was to identify factors that enhance and hinder interdisciplinary collaborative practice (ICP) among doctors and nurses at the Nnamdi Azikiwe teaching hospital, Nnewi, southeast Nigeria. The study was a cross-sectional descriptive survey and the quantitative method of data collection was employed. The population was all doctors irrespective of area of specialty and all nurses employed and working in the hospital as at the time of study. Proportionate stratified and convenience sampling methods were used to select study participants according to their categories. Using validated structured questionnaire, data were collected from 110 doctors and 95 nurses in the teaching hospital on their perception on ICP and factors that enhance/hinder ICP. Data were analyzed using both descriptive and inferential statistics. Specifically, frequencies, percentages, standard deviation and graphic presentation were used for descriptive analysis of scores while the unpaired t test of mean score using Graph Pad Prism, Version 5.30 was used to determine the influence of profession, gender, and years of experience on perception of ICP at 0.05 level of significance. The study found that both doctors and nurses have positive perception on ICP. Their years of experience have significant influence on their perception. Clear individual roles and good working relationships enhance ICP while giving priority to professional status rather than expertise was seen as a prominent hindrance to ICP. The study recommends collaborative continuing education for doctors and nurses to enhance ICP in patient care. In addition, the inclusion of interdisciplinary collaborative practice programmme into the curriculum of medical and nursing students (where it does not exist) would go a long way to strengthen ICP and decrease hindrances when they graduate.

Lubricant Biodegradation Enhancers: Designed Chemistry and Engineered Technology

In recent decades, a growing worldwide trend of developing the biodegradable lubricants has been prevailing to form a specific field of green chemistry and green engineering. Enhancement of biodegradability of unreadily biodegradable petroleum-based lubricants has as such become an urgent must. For over a decade the authors have been focusing on the improvement of biodegradability of unreadily biodegradable lubricants such as petroleum-based lubricating oils and greases. A new idea of lubricant biodegradation enhancer was put forward by the authors with the aim to stimulate the biodegradation of unreadily biodegradable lubricants by incorporating the enhancer into the lubricants in order to turn the lubricants into greener biodegradable ones and to help in situ bioremediation of lubricant-contaminated environment. This manuscript summarizes our recent efforts relating to the chemistry and technology of biodegradation enhancers for lubricants. Firstly, the chemistry of lubricant biodegradation enhancers was designed based on the principles of bioremediation for the treatment of hydrocarbon contaminated environment. Secondly, the ability of the designed biodegradation enhancers for increasing the biodegradability of unreadily biodegradable industrial lubricants was investigated through biodegradability evaluation tests, microbial population analysis, and biodegradation kinetics modeling. Finally, the impact of biodegradation enhancers on some crucial performance characteristics of lubricants such as lubricity and oxidation stability was tested via tribological evaluation and oxidation determinations. Our results have shown that the designed chemistry of nitrogenous and/or phosphorous compounds such as lauroyl glutamine, oleoyl glycine, oleic diethanolamide phosphate and lauric diethanolamide borate was outstanding in boosting biodegradation of petroleum-based lubricants which was ascribed to increase the microbial population and decrease the oil-water interfacial tension during the biodegradation process. Lubricants doped with the biodegradation enhancers exhibited much better biodegradability and higher biodegradation rate in the surrounding soils which could be well modeled by the exponential biodegradation kinetics. Furthermore, as lubricant dopants, the biodegradation enhancers also provided excellent capability in reducing friction and wear and in retarding oxidation of lubricants. In the nature of things, lubricant biodegradation enhancers, which are multi-functional not only in the improvement of biodegradability, but also in the fortification of lubricity and in the inhibition of oxidation of lubricants, are expected to be promising as a new category of lubricant additives.

Novel chemical permeation enhancers for transdermal drug delivery

Transdermal drug delivery has been accepted as a potential non-invasive route of drug administration,with advantages of prolonged therapeutic action,decreased side effect,easy use and better patient compliance.However,development of transdermal products is primarily hindered by the low permeability of the skin.To overcome this barrier effect,numerous new chemicals have been synthesized as potential permeation enhancers for transdermal drug delivery.In this review,we presented an overview of the investigations in this field,and further implications on selection or design of suitable permeation enhancers for transdermal drug delivery were also discussed.

The enhancing effect and promoting mechanisms of the stereoisomeric monoterpene alcohol esters as enhancers for drugs with different physicochemical properties

To explore the structure-activity connections of amphiphilic permeation enhancers containing the length of the hydrophobic chains as well as the properties of the polar head,O-acylgeraniol and O-acylnerol derivatives were synthesized from geraniol/nerol(cis-isomer of geraniol) and pharmaceutical excipient acids in this research. Their promotion of the percutaneous absorption of three drugs as the model, flurbiprofen(FP), isosorbide dinitrate(ISDN) and donepezil(DNP), which were selected based on their physicochemical properties,was tested by in vitro skin penetration and in vivo. Molecular simulation, ATR-FTIR, CLSM and histological observation were implement to evaluate the mode of action of the enhancers.The results indicated that(E)-3,7-dimethyl-2,6-octadien-1-yl tetradecanoate(GER-C14, trans-)achieved the highest enhancement ability for the three drugs;additionally, the in vivo results obtained were in good correlation with the in vitro data. Molecular docking results suggested that enhancers loosen the hydrogen bonds between ceramides, and the results of molecular simulation indicated that GER-C14, NER-C14 could insert into the middle of the lipid bilayer to form an independent phase. According to ATR-FTIR and histological evaluation, the enhancers extracted lipids and influenced the protein region, thereby disturbing the skin array. In addition, CLSM described the dynamic effects of enhancers on lipids between stratum corneum(SC) cells. In conclusion, GER-C14 had a better penetration promotion effect, which broadened our understanding of stereoisomeric penetration enhancers.

Influence of chemical penetration enhancers on skin permeability of ellagic acid-loaded niosomes

This study aimed to develop niosomes of ellagic acid(EA),a potent antioxidant phytochemical substance,for dermal delivery and to investigate the influence of chemical penetration enhancers on the physicochemical properties of EA-loaded niosomes.The EA niosomes were prepared by reverse phase evaporation method using Span 60,Tween 60 and cholesterol as vesicle forming agents and Solulan C24 as a steric stabilizer.Polyethylene glycol 400(PEG)was used as a solubilizer while dimethylsulfoxide(DMSO)or Nmethyl-2-pyrrolidone(NMP)was used as a skin penetration enhancer.It was found that the mean particle sizes of EA-loaded niosomes were in the range of 312e402 nm with PI values of lower than 0.4.The niosomes were determined to be spherical multilamellar vesicles as observed by transmission electron microscope and optical microscopy.All niosomes were stable after 4 months storage at 4
C.In vitro skin permeation through human epidermis revealed that the skin enhancers affected the penetration of EA from the niosomes at 24 h.The DMSO niosomes showed the highest EA amount in epidermis;whereas the NMP niosomes had the highest EA amount in the acceptor medium.Concomitantly,the skin distribution by confocal laser scanning microscopy showed the high fluorescence intensity of the DMSO niosomes and NMP niosomes at a penetration depth of between 30e90 mm(the epidermis layer)and 90e120 mm(the dermis layer)under the skin,respectively.From the results,it can be concluded that the DMSO niosomes are suitable for epidermis delivery of EA while the NMP niosomes can be used for dermis delivery of EA.

The untold story between enhancers and skeletal muscle development

Currently,enhancers have key transcriptional regulatory roles in muscle development.Skeletal muscle formation involves various molecules,and in animals,enhancers are one of the main types of transcriptional regulatory regions that are of great importance to regulate myogenic gene expression.In muscle development,enhancers can generate enhancer RNAs(eRNAs)that are involved in the regulation of gene transcription.The regulation of gene expression by eRNAs offers great potential in improving animal production traits.Herein we comprehensively review the roles of enhancers in muscle formation and its potential function in skeletal muscle development.This review will describe the future application of enhancers in skeletal muscle development and discuss the prospects that enhancer studies offer for agriculture,biotechnology,and animal breeding.

Efficient and multiplex gene upregulation in plants through CRISPR-Cas-mediated knockin of enhancers

Geneupregulation through genome editing is important for plant research and breeding.Targeted insertion of short transcriptional enhancers(STEs)into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis.Here,we introduce an“in locus activation”technique in rice that leverages well-characterized STEs for refined,heritable,and multiplexed gene upregulation.To address the scarcity of potent enhancers,we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts.The in locus integration of these STEs into eight rice genes resulted in substantial tran-scriptional upregulation in the edited plants,with up to 869.1-fold increases in their transcript levels.Em-ploying a variety of STEs,we achieved delicate control of gene expression,enabling the fine-tuning of key phenotypic traits such as plant height.Our approach also enabled efficient multiplexed gene upregu-lation,with up to four genes activated simultaneously,significantly enhancing the nicotinamide mononucleotide metabolic pathway.Importantly,heritability studies from the To to T3 generations confirmed the stable and heritable nature of STE-driven gene activation.Collectively,our work demon-strates that coupled with STE mining,leveraging genome editing for in locus activation and gene upregu-lation holds great promise to be widely adopted in fundamental plant research and crop breeding.

Identification of Highly Repetitive Enhancers with Long-range Regulation Potential in Barley via STARR-seq

Enhancers are DNA sequences that can strengthen transcription initiation.However,the global identification of plant enhancers is complicated due to uncertainty in the distance and orientation of enhancers,especially in species with large genomes.In this study,we performed self-transcribing active regulatory region sequencing(STARR-seq)for the first time to identify enhancers across the barley genome.A total of 7323 enhancers were successfully identified,and among 45 randomly selected enhancers,over 75%were effective as validated by a dual-luciferase reporter assay system in the lower epidermis of tobacco leaves.Interestingly,up to 53.5%of the barley enhancers were repetitive sequences,especially transposable elements(TEs),thus reinforcing the vital role of repetitive enhancers in gene expression.Both the common active mark H3K4me3 and repressive mark H3K27me3 were abundant among the barley STARR-seq enhancers.In addition,the functional range of barley STARR-seq enhancers seemed much broader than that of rice or maize and extended to±100 kb of the gene body,and this finding was consistent with the high expression levels of genes in the genome.This study specifically depicts the unique features of barley enhancers and provides available barley enhancers for further utilization.

Steering Against Wind： A New Network of NamiRNAs and Enhancers

MicroRNAs （miRNAs） are a class of endogenous non-coding RNAs with regulatory functions. Traditionally, miRNAs are thought to play a negative regulatory role in the cytoplasm by binding to the YUTR of target genes to degrade mRNA or inhibit translation. However, it remains a challenge to interpret the potential function of many miRNAs located in the nucleus. Recently, we reported a new type of miRNAs present in the nucleus, which can activate gene expres- sion by binding to the enhancer, and named them nuclear activating miRNAs （NamiRNAs）. The discovery of NamiRNAs showcases a complementary regulatory mechanism of miRNA, demon- strating their differential roles in the nucleus and cytoplasm. Here, we reviewed miRNAs in nucleus to better understand the function of NamiRNAs in their interactions with the enhancers. Accord- ingly, we propose a NamiRNA--enhancer-target gene activation network model to better under- stand the crosstalk between NamiRNAs and enhancers in regulating gene transcription. Moreover, we hypothesize that NamiRNAs may be involved in cell identity or cell fate determina- tion during development, although further study is needed to elucidate the underlying mechanisms in detail.

Effect of paracellular permeation enhancers on intestinal permeability of two peptide drugs,enalaprilat and hexarelin, in rats

Transcellular permeation enhancers are known to increase the intestinal permeability of enalaprilat,a 349 Da peptide,but not hexarelin(887 Da).The primary aim of this paper was to investigate if paracellular permeability enhancers affected the intestinal permeation of the two peptides.This was investigated using the rat single-pass intestinal perfusion model with concomitant blood sampling.These luminal compositions included two paracellular permeation enhancers,chitosan(5 mg/mL) and ethylenediaminetetraacetate(EDTA,1 and 5 mg/mL),as well as low luminal tonicity(100 mOsm) with or without lidocaine.Effects were evaluated by the change in lumen-to-blood permeability of hexarelin and enalaprilat,and the blood-to-lumen clearance of ^(51)chromium-labeled EDTA(CL_(Cr-EDTA)),a clinical marker for mucosal barrier integrity.The two paracellular permeation enhancers increased the mucosal permeability of both peptide drugs to a similar extent.The data in this study suggests that the potential for paracellular permeability enhancers to increase intestinal absorption of hydrophilic peptides with low molecular mass is greater than for those with transcellular mechanism-of-action.Further,the mucosal blood-to-lumen flux of ^(51)Cr-EDTA was increased by the two paracellular permeation enhancers and by luminal hypotonicity.In contrast,luminal hypotonicity did not affect the lumen-to-blood transport of enalaprilat and hexarelin.This suggests that hypotonicity affects paracellular solute transport primarily in the mucosal crypt region,as this area is protected from luminal contents by a constant water flow from the crypts.

Radiomics and molecular analysis:Bridging the gap for predicting hepatocellular carcinoma prognosis

This editorial examines a recent study that used radiomics based on computed tomography(CT)to predict the expression of the fibroblast-related gene enhancer of zeste homolog 2(EZH2)and its correlation with the survival of patients with hepatocellular carcinoma(HCC).By integrating radiomics with molecular analysis,the study presented a strategy for accurately predicting the expression of EZH2 from CT scans.The findings demonstrated a strong link between the radiomics model,EZH2 expression,and patient prognosis.This noninvasive approach provides valuable insights into the therapeutic management of HCC.

Crosstalk among canonical Wnt and Hippo pathway members in skeletal muscle and at the neuromuscular junction

Skeletal muscles are essential for locomotion,posture,and metabolic regulation.To understand physiological processes,exercise adaptation,and muscle-related disorders,it is critical to understand the molecular pathways that underlie skeletal muscle function.The process of muscle contra ction,orchestrated by a complex interplay of molecular events,is at the core of skeletal muscle function.Muscle contraction is initiated by an action potential and neuromuscular transmission requiring a neuromuscular junction.Within muscle fibers,calcium ions play a critical role in mediating the interaction between actin and myosin filaments that generate force.Regulation of calcium release from the sarcoplasmic reticulum plays a key role in excitation-contraction coupling.The development and growth of skeletal muscle are regulated by a network of molecular pathways collectively known as myogenesis.Myogenic regulators coordinate the diffe rentiation of myoblasts into mature muscle fibers.Signaling pathways regulate muscle protein synthesis and hypertrophy in response to mechanical stimuli and nutrient availability.Seve ral muscle-related diseases,including congenital myasthenic disorders,sarcopenia,muscular dystrophies,and metabolic myopathies,are underpinned by dys regulated molecular pathways in skeletal muscle.Therapeutic interventions aimed at preserving muscle mass and function,enhancing regeneration,and improving metabolic health hold promise by targeting specific molecular pathways.Other molecular signaling pathways in skeletal muscle include the canonical Wnt signaling pathway,a critical regulator of myogenesis,muscle regeneration,and metabolic function,and the Hippo signaling pathway.In recent years,more details have been uncovered about the role of these two pathways during myogenesis and in developing and adult skeletal muscle fibers,and at the neuromuscular junction.In fact,research in the last few years now suggests that these two signaling pathways are interconnected and that they jointly control physiological and pathophysiological processes in muscle fibers.In this review,we will summarize and discuss the data on these two pathways,focusing on their concerted action next to their contribution to skeletal muscle biology.However,an in-depth discussion of the noncanonical Wnt pathway,the fibro/a dipogenic precursors,or the mechanosensory aspects of these pathways is not the focus of this review.

Potential of Essential Oils as Alternative Permeation Enhancers for Transdermal Delivery

Transdermal drug delivery plays a significant part in the drug delivery system when compared to other routes of drug administration.The function of the stratum corneum(SC)is a barrier.Recently,numerous methods have been thrived to improve the perforation of drugs across the skin.The most effective method is to use enhancers since these agents enhance skin permeability.Natural penetration enhancers like essential oils demonstrate higher enhancement activity and are more widely accepted than synthetic penetration enhancers.High potential in the expansion and interaction with the SC intercellular lipids has led to an increasing interest in these oils as penetration enhancers.This article gives an overview of a few essential oils,including their mode of action and important parameters for permeation improvement.The present work can provide essential oils as alternative enhancers,and this could be useful in transdermal administration.

Identification and characteristic analysis of enhancers across 13 major cancer types

Enhancers are often mutated and dysregulated in various diseases such as cancer.By integrating the function annotation of the mammalian genome(FANTOM)enhancers expression profiles and RNA-seq data from The Cancer Genome Atlas(TCGA)of 13 cancers and their corresponding para-cancerous tissues,we systematically identified a total of 4702 significantly differentially expressed(DE)enhancers.Furthermore,a total of 1036 DE genes regulated by DE enhancerswere identified.Itwas found that in these 13 cancers,most(61.13%)enhancers were ubiquitously expressed,whereas DE enhancers were more likely to be tissue-specific expressed,and the DE genes regulated by DE enhancers were significantly enriched in cancer-related pathways.Finally,it was manifested that 74 single nucleotide polymorphisms(SNPs)were located in 37 DE enhancers,and these SNPs affected the gain and loss of functional transcription factor binding sites of 758 transcription factors,which were shown to be highly correlated with tumorigenesis and development.

Global Quantitative Mapping of Enhancers in Rice by STARR-seq

Enhancers activate transcription in a distance-,orientation-,and position-independent manner,which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes,especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40%of identified enhancers car-ried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks,H3K4me3 and/or H3K27ac,which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes,which overlap poorly with STARR-seq enhancers. In summary,we quantitatively identified enhancers by functional analysis in the genome of rice,an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.

Three-dimensional genome architectural CCCTC-binding factor makes choice in duplicated enhancers at Pcdhα locus

During development, gene expression is spatiotemporally regulated by long-distance chromatin interactions between distal enhancers and target promoters. However, how specificity of the interactions between enhancers and promoters is achieved remains largely unknown. As there are far more enhancers than promoters in mammalian genomes, the complexities of enhancer choice during gene regulation remain obscure. CTCF, the CCCTC-binding factor that directionally binds to a vast range of genomic sites known as CBSs(CTCF-binding sites), mediates oriented chromatin looping between a substantial set of distal enhancers and target promoters. To investigate mechanisms by which CTCF engages in enhancer choice, we used CRISPR/Cas9-based DNA-fragment editing to duplicate CBS-containing enhancers and promoters in the native genomic locus of the clustered Pcdhα genes. We found that the promoter is regulated by the proximal one among duplicated enhancers and that this choice is dependent on CTCF-mediated directional enhancer-promoter looping. In addition, gene expression is unaltered upon the switch of enhancers. Moreover, after promoter duplication, only the proximal promoter is chosen by CTCF-mediated directional chromatin looping to contact with the distal enhancer. Finally, we demonstrated that both enhancer activation and chromatin looping with the promoter are essential for gene expression. These findings have important implications regarding the role of CTCF in specific interactions between enhancers and promoters as well as developmental regulation of gene expression by enhancer switching.

Non-coding Transcripts from Enhancers:New Insights into Enhancer Activity and Gene Expression Regulation

Long non-coding RNAs （lncRNAs） have gained widespread interest in the past decade owing to their enormous amount and surprising functions implicated in a variety of biological pro- cesses. Some lncRNAs exert function as enhancers, i.e., activating gene transcription by serving as the cis-regulatory molecules. Furthermore, recent studies have demonstrated that many enhancer elements can be transcribed and produce RNA molecules, which are termed as enhancer RNAs （eRNAs）. The eRNAs are not merely the by-product of the enhancer transcription. In fact, many of them directly exert or regulate enhancer activity in gene activation through diverse mechanisms. Here, we provide an overview of enhancer activity, transcription of enhancer itself, characteristics of eRNAs, as well as their roles in regulating enhancer activity and gene expression.

Super enhancers:Pathogenic roles and potential therapeutic targets for acute myeloid leukemia(AML)

Acute myeloid leukemia(AML)is a malignant hematological tumor with disordered oncogenes/tumor suppressor genes and limited treatments.The potent anti-cancer effects of bromodomain and extra-terminal domain(BET)inhibitors,targeting the key component of super enhancers,in early clinical trials on AML patients,implies the critical role of super enhancers in AML.Here,we review the concept and characteristic of super enhancer,and then summarize the current researches about super enhancers in AML pathogenesis,diagnosis and classification,followed by illustrate the potential super enhancer-related targets and drugs,and propose the future directions of super enhancers in AML.This information provides integrated insight into the roles of super enhancers in this disease.