DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.

Deciphering the multi-scale, quantitative cis-regulatory code.

Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.

mTORC1 promotes cell growth via m6A-dependent mRNA degradation.

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.

Mechanisms of Interplay between Transcription Factors and the 3D Genome.

Transcription factors (TFs) bind DNA in a sequence-specific manner and thereby serve as the protein anchors and determinants of 3D genome organization. Conversely, chromatin conformation shapes TF activity, for example, by looping TF-bound enhancers to distally located target genes. Despite considerable effort, our understanding of the mechanistic relation between TFs and 3D genome organization remains limited, in large part due to this interdependency. In this review, we summarize the evidence for the diverse mechanisms by which TFs and their activity shape the 3D genome and vice versa. We further highlight outstanding questions and potential approaches for untangling the complex relation between TF activity and the 3D genome.

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence.

Quiescence is a stress-resistant state in which cells reversibly exit the cell cycle and suspend most processes. Quiescence is essential for stem cell maintenance, and its misregulation is implicated in tumor formation. One of the hallmarks of quiescent cells is highly condensed chromatin. Because condensed chromatin often correlates with transcriptional silencing, it has been hypothesized that chromatin compaction represses transcription during quiescence. However, the technology to test this model by determining chromatin structure within cells at gene resolution has not previously been available. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide in quiescent Saccharomyces cerevisiae cells. We describe chromatin domains on the order of 10-60 kilobases that, only in quiescent cells, are formed by condensin-mediated loops. Condensin depletion prevents the compaction of chromatin within domains and leads to widespread transcriptional de-repression. Finally, we demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts.

An eQTL-based approach reveals candidate regulators of LINE-1 RNA levels in lymphoblastoid cells.

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition, the overwhelming majority of copies are degenerate and immobile. Nevertheless, both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability, inflammation, or cellular senescence) on their hosts, and L1's contributions to aging and aging diseases is an area of active research. However, because of the cell type-specific nature of transposon control, the catalogue of L1 regulators remains incomplete. Here, we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation, we experimentally modulate the levels of top candidates in vitro, including IL16, STARD5, HSD17B12, and RNF5, and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably, we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover, a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle, but widespread, upregulation of L1 subfamilies. Finally, we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data, we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels.

Temporal synchronization between two Q switched pulses in a 1 × 2 array distribution inside a single YAG/Yb:YAG/Cr:YAG laser resonator.

We propose for the first time, to the best of our knowledge, a coupling method of modes guided by gain waveguides to synchronize two Q switched pulses oscillating in a 1 × 2 array distribution inside a single YAG/Yb:YAG/Cr:YAG resonator. To analyze the temporal synchronization of spatially separated Q switched pulses, the buildup time interval, spatial distribution, and longitudinal modes distribution of the two pulse beams are investigated.

Recent Advances in Transducers for Intravascular Ultrasound (IVUS) Imaging.

As a well-known medical imaging methodology, intravascular ultrasound (IVUS) imaging plays a critical role in diagnosis, treatment guidance and post-treatment assessment of coronary artery diseases. By cannulating a miniature ultrasound transducer mounted catheter into an artery, the vessel lumen opening, vessel wall morphology and other associated blood and vessel properties can be precisely assessed in IVUS imaging. Ultrasound transducer, as the key component of an IVUS system, is critical in determining the IVUS imaging performance. In recent years, a wide range of achievements in ultrasound transducers have been reported for IVUS imaging applications. Herein, a comprehensive review is given on recent advances in ultrasound transducers for IVUS imaging. Firstly, a fundamental understanding of IVUS imaging principle, evaluation parameters and IVUS catheter are summarized. Secondly, three different types of ultrasound transducers (piezoelectric ultrasound transducer, piezoelectric micromachined ultrasound transducer and capacitive micromachined ultrasound transducer) for IVUS imaging are presented. Particularly, the recent advances in piezoelectric ultrasound transducer for IVUS imaging are extensively examined according to their different working mechanisms, configurations and materials adopted. Thirdly, IVUS-based multimodality intravascular imaging of atherosclerotic plaque is discussed. Finally, summary and perspectives on the future studies are highlighted for IVUS imaging applications.

Rational budgeting approach as a nutrient management tool for mixed crop-swine farms in Korea.

OBJECTIVE: Due to rapid economic return, mixed crop-swine farming systems in Korea have become more intensive. Intensive farming practices often cause nutrient surpluses and lead to environmental pollution. Nutrient budgets can be used to evaluate the environmental impact and as a regulatory policy instrument for nutrient management. This study was conducted to select a nutrient budgeting approach applicable to the mixed crop-swine farms in Korea and suggest an effective manure treatment method to reduce on-farm nutrient production. METHODS: In this study, we compared current and ideal gross nutrient balance (GNB) approaches of Organisation for Economic Co-operation and Development and soil system budget (SSB) approach with reference to on-farm manure treatment processes. Data obtained from farm census and published literature were used to develop the farm nutrient budgets. RESULTS: The average nitrogen (N) and phosphorus (P) surpluses were approximately 11 times and over 7 times respectively higher in the GNB approaches than the SSB. After solid-liquid separation of manure, during liquid composting a change in aeration method from intermittent to continuous reduced the N and P loading about 50% and 47%, respectively. Although changing in solid composting method from turning only to turning+aeration improved the N removal efficiency by 30.5%, not much improvement in P removal efficiency was observed. CONCLUSION: Although the GNB approaches depict the impact of nutrients produced in the mixed crop-swine farms on the overall agricultural environment, the SSB approach shows the partitioning among different nutrient loss pathways and storage of nutrients within the soil system; thus, can help design sustainable nutrient management plans for the mixed cropswine farms. The study also suggests that continuous aeration for liquid composting and turning+aeration for solid composting can reduce nutrient loading to the soil.

MnO2 Nanowire/Biomass-Derived Carbon from Hemp Stem for High-Performance Supercapacitors.

Hierarchical 3D nanostructures based on waste biomass are being offered as promising materials for energy storage due to their processabilities, multifunctionalities, environmental benignities, and low cost. Here we report a facile, inexpensive, and scalable strategy for the fabrication of hierarchical porous 3D structure as electrode materials for supercapacitors based on MnO2 nanowires and hemp-derived activated carbon (HC). Vertical MnO2 wires are uniformly deposited onto the surface of HC using a one-step hydrothermal method to produce hierarchical porous structures with conductive interconnected 3D networks. HC acts as a near-ideal 3D current collector and anchors electroactive materials, and this confers a specific capacitance of 340 F g-1 at 1 A g-1 with a high rate capability (88% retention) of the 3D MnO2/HC composite because of its open-pore system, which facilitates ion and electron transports and synergistic contribution of two energy-storage materials. Moreover, asymmetric supercapacitors fabricated using 3D HC as the anode and 3D MnO2/HC as the cathode are able to store 33.3 Wh kg-1 of energy and have a power delivery of 14.8 kW kg-1.

Alternating antibiotic treatments constrain evolutionary paths to multidrug resistance.

Alternating antibiotic therapy, in which pairs of drugs are cycled during treatment, has been suggested as a means to inhibit the evolution of de novo resistance while avoiding the toxicity associated with more traditional combination therapy. However, it remains unclear under which conditions and by what means such alternating treatments impede the evolution of resistance. Here, we tracked multistep evolution of resistance in replicate populations of Staphylococcus aureus during 22 d of continuously increasing single-, mixed-, and alternating-drug treatment. In all three tested drug pairs, the alternating treatment reduced the overall rate of resistance by slowing the acquisition of resistance to one of the two component drugs, sometimes as effectively as mixed treatment. This slower rate of evolution is reflected in the genome-wide mutational profiles; under alternating treatments, bacteria acquire mutations in different genes than under corresponding single-drug treatments. To test whether this observed constraint on adaptive paths reflects trade-offs in which resistance to one drug is accompanied by sensitivity to a second drug, we profiled many single-step mutants for cross-resistance. Indeed, the average cross-resistance of single-step mutants can help predict whether or not evolution was slower in alternating drugs. Together, these results show that despite the complex evolutionary landscape of multidrug resistance, alternating-drug therapy can slow evolution by constraining the mutational paths toward resistance.

Whole-transcriptome analysis of mouse adipose tissue in response to short-term caloric restriction.

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptome analysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-ß, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan.

Transfer learning reveals sequence determinants of the quantitative response to transcription factor dosage.

Deep learning approaches have made significant advances in predicting cell type-specific chromatin patterns from the identity and arrangement of transcription factor (TF) binding motifs. However, most models have been applied in unperturbed contexts, precluding a predictive understanding of how chromatin state responds to TF perturbation. Here, we used transfer learning to train and interpret deep learning models that use DNA sequence to predict, with accuracy approaching experimental reproducibility, how the concentration of two dosage-sensitive TFs (TWIST1, SOX9) affects regulatory element (RE) chromatin accessibility in facial progenitor cells. High-affinity motifs that allow for heterotypic TF co-binding and are concentrated at the center of REs buffer against quantitative changes in TF dosage and strongly predict unperturbed accessibility. In contrast, motifs with low-affinity or homotypic binding distributed throughout REs lead to sensitive responses with minimal contributions to unperturbed accessibility. Both buffering and sensitizing features show signatures of purifying selection. We validated these predictive sequence features using reporter assays and showed that a biophysical model of TF-nucleosome competition can explain the sensitizing effect of low-affinity motifs. Our approach of combining transfer learning and quantitative measurements of the chromatin response to TF dosage therefore represents a powerful method to reveal additional layers of the cis-regulatory code.

In vitro heterochronic parabiosis identifies pigment epithelium-derived factor as a systemic mediator of rejuvenation by young blood.

Several decades of heterochronic parabiosis (HCPB) studies have demonstrated the restorative impact of young blood, and deleterious influence of aged blood, on physiological function and homeostasis across tissues, although few of the factors responsible for these observations have been identified. Here we develop an in vitro HCPB system to identify these circulating factors, using replicative lifespan (RLS) of primary human fibroblasts as an endpoint of cellular health. We find that RLS is inversely correlated with serum donor age and sensitive to the presence or absence of specific serum components. Through in vitro HCPB, we identify the secreted protein pigment epithelium-derived factor (PEDF) as a circulating factor that extends RLS of primary human fibroblasts and declines with age in mammals. Systemic administration of PEDF to aged mice reverses age-related functional decline and pathology across several tissues, improving cognitive function and reducing hepatic fibrosis and renal lipid accumulation. Together, our data supports PEDF as a systemic mediator of the effect of young blood on organismal health and homeostasis and establishes our in vitro HCPB system as a valuable screening platform for the identification of candidate circulating factors involved in aging and rejuvenation.

The retrotransposon - derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag -like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag -like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

Pulmonary actinomycosis during the first decade of 21st century: cases of 94 patients.

BACKGROUND: Pulmonary actinomycosis is a chronic pulmonary infection caused by Actinomyces. Both improving oral hygiene and early application of antibiotics to the case of suspicious pulmonary infections result in changes in incidences and presentations of pulmonary actinomycosis. However, there are little reports dealt with the recent clinical characteristics of pulmonary actinomycosis. This study aimed to review the characteristics of pulmonary actinomycosis occurred during the first decade of 21st century. METHODS: This retrospective study was performed on 94 subjects with pulmonary actinomycosis diagnosed pathologically from January 2000 to December 2010 in 13 hospitals in Korea. RESULTS: The data of the study showed that pulmonary actinomycosis occurs frequently in middle to old-aged males (mean age; 57.7 years old) and that the most common symptoms are cough, hemoptysis, and sputum production. Various radiologic features such as the consolidation with central low attenuation (74.5%) and no regional predominance were also observed. Most of patients recovered completely with medical and/or surgical treatment, reaching approximately 98% cure rate. CONCLUSIONS: The results demonstrate that pulmonary actinomycosis is one of the cautious pulmonary diseases. More importantly, in cases of persistent hemoptysis or for differential diagnosis from lung malignancy, our data have revealed that surgical resection appears to be a useful intervention and that radiologic diagnosis may not provide decisive information. These findings indicate that it is important for the clinicians to include pulmonary actinomycosis as one of differential diagnoses for refractory pulmonary abnormal lesions to the current usual management.

Clinical use of alumina-toughened zirconia abutments for implant-supported restoration: prospective cohort study of survival analysis.

OBJECTIVES: The aim of this prospective cohort study was to compute the long-term clinical survival and complication rates of alumina-toughened zirconia abutments used for implant-supported restorations and to evaluate the effects of several clinical variables on these rates. MATERIAL AND METHODS: From May 1998 to September 2010, 213 patients aged 18 years or older were recruited. The patients received 611 external hex implants and 328 implant-supported fixed restorations using alumina-toughened zirconia abutments. During the follow-up, each restoration was coded as a dental event, which included loosening or fracture of abutment screws, and abutment fracture. From the coded data, the effects of the investigated clinical variables-restored area (anterior/posterior), number of prosthodontic units (one/two units or over), prosthesis type (single-unit/multiunit without pontic/multiunit with pontic), implant system, and patient gender-on the survival of the abutments were evaluated. Survival analysis using Kaplan-Meier method and Cox proportional hazard model was applied. The 5-year survival and complication rates of the abutments were assessed. RESULTS: The number of prosthodontic units and the type of prosthesis had a significant association with complication rates (P < 0.05). Kaplan-Meier survival analysis estimated that the cumulative 5-year complication rate of the abutments used in single restorations was 19.7%. Multiunit-fixed dental prostheses without and with pontics had complication rates of 3.9% and 3.8%, respectively. The 5-year survival rate of the abutments was more than 95%, regardless of the type of prosthesis. CONCLUSIONS: Alumina-toughened zirconia abutments are likely to exhibit excellent long-term survival in clinical use for fixed restorations. Single tooth replacement with the abutment at the molar region may require special care and extra attention.

Assessment of a Novel Real-Time Bio-Liquor Circulation System for Manure Management and Mitigation of Odor Potential in Swine Farming.

Recently, circulating biologically treated manure in slurry pits has been used as an odor reduction technology, but few successful results have been reported, due to the lack of proper control strategies for bioreactors. This study was conducted to investigate the performance of the developed real-time controlled bio-liquor circulation system (BCS) at farm scale. The BCS was operated sequentially as per swine manure inflow (anoxic, aerobic, and settling) circulation to the slurry pit. Each operational phase was self-adjusted in real-time using a novel algorithm for detecting the control point on the oxidation reduction potential (ORP) and pH (mV)-time profiles, the nitrogen break point (NBP), and the nitrate knee point (NKP) in the aerobic and anoxic phases, respectively. The NH4-N in the slurry manure was thoroughly removed (100%) in the bioreactor, optimizing the duration of each operational phase by accurately detecting real-time control points. The newly developed real-time BCS decreased the nitrogen and organic matter in the slurry pit by >70%, and the potential ammonia and methane emissions by 75% and 95%, respectively. This study highlights that improved BCS that utilizes ORP tracking and pH (mV)-time profiles can effectively optimize BCS operation, and thereby reduce malodor and GHG emissions from swine farms.