m6A enhances the phase separation potential of mRNA.

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.

Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag.

Fluorogenic RNA aptamers are used to genetically encode fluorescent RNA and to construct RNA-based metabolite sensors. Unlike naturally occurring aptamers that efficiently fold and undergo metabolite-induced conformational changes, fluorogenic aptamers can exhibit poor folding, which limits their cellular fluorescence. To overcome this, we evolved a naturally occurring well-folded adenine riboswitch into a fluorogenic aptamer. We generated a library of roughly 1015 adenine aptamer-like RNAs in which the adenine-binding pocket was randomized for both size and sequence, and selected Squash, which binds and activates the fluorescence of green fluorescent protein-like fluorophores. Squash exhibits markedly improved in-cell folding and highly efficient metabolite-dependent folding when fused to a S-adenosylmethionine (SAM)-binding aptamer. A Squash-based ratiometric sensor achieved quantitative SAM measurements, revealed cell-to-cell heterogeneity in SAM levels and revealed metabolic origins of SAM. These studies show that the efficient folding of naturally occurring aptamers can be exploited to engineer well-folded cell-compatible fluorogenic aptamers and devices.

Effect of neighborhood deprivation index on breast cancer survival in the United States.

PURPOSE: To analyze the association between the Neighborhood Deprivation Index (NDI) and clinical outcomes of locoregional breast cancer (BC). METHODS: Surveillance, Epidemiology and End Results (SEER) database is queried to evaluate overall survival (OS) and disease-specific survival (DSS) of early- stage BC patients diagnosed between 2010 and 2016. Cox multivariate regression was performed to measure the association between NDI (Quintiles corresponding to most deprivation (Q1), above average deprivation (Q2), average deprivation (Q3), below average deprivation (Q4), least deprivation (Q5)) and OS/DSS. RESULTS: Of the 88,572 locoregional BC patients, 27.4% (n = 24,307) were in the Q1 quintile, 26.5% (n = 23,447) were in the Q3 quintile, 17% (n = 15,035) were in the Q2 quintile, 13.5% (n = 11,945) were in the Q4 quintile, and 15.6% (n = 13,838) were in the Q5 quintile. There was a predominance of racial minorities in the Q1 and Q2 quintiles with Black women being 13-15% and Hispanic women being 15% compared to only 8% Black women and 6% Hispanic women in the Q5 quintile (p < 0.001). In multivariate analysis, in the overall cohort, those who live in Q2 and Q1 quintile have inferior OS and DSS compared to those who live in Q5 quintile (OS:- Q2: Hazard Ratio (HR) 1.28, Q1: HR 1.2; DSS:- Q2: HR 1.33, Q1: HR 1.25, all p < 0.001). CONCLUSION: Locoregional BC patients from areas with worse NDI have poor OS and DSS. Investments to improve the socioeconomic status of areas with high deprivation may help to reduce healthcare disparities and improve breast cancer outcomes.

Clinicopathologic Predictors of Clinical Outcomes in Mammary Adenoid Cystic Carcinoma: A Multi-institutional Study.

Adenoid cystic carcinoma (AdCC) is an uncommon type of invasive breast carcinoma with a favorable prognosis. However, some cases are aggressive. The study aims to define the clinicopathologic predictors of outcome. Clinical, radiological, and pathologic variables were recorded for 76 AdCC cases from 11 institutions. The following histologic characteristics were evaluated by the breast pathologist in each respective institution, including Nottingham grade (NG), percentages of various growth patterns (solid, cribriform, trabecular-tubular), percentage of basaloid component, tumor borders (pushing, infiltrative), perineural invasion, lymphovascular invasion, necrosis, and distance from the closest margin. Various grading systems were evaluated, including NG, salivary gland-type grading systems, and a new proposed grading system. The new grading system incorporated the growth pattern (percent solid, percent cribriform), percent basaloid morphology, and mitotic count using the Youden index criterion. All variables were correlated with recurrence-free survival. Nineteen (25%) women developed local and/or distant recurrence. Basaloid morphology (≥25% of the tumor) was identified in 20 (26.3%) cases and a solid growth pattern (using ≥60% cutoff) in 22 (28.9%) cases. In the univariate analysis, the following variables were significantly correlated with worse recurrence-free survival: solid growth pattern, basaloid morphology, lymphovascular invasion, necrosis, perineural invasion, and pN-stage. In the multivariate analysis including basaloid morphology, pN-stage, lymphovascular invasion, and perineural invasion, basaloid morphology was statistically significant, with a hazard ratio of 3.872 (95% CI, 1.077; 13.924; P =.038). The NG and the new grading system both correlated with recurrence-free survival. However, grade 2 had a similar risk as grade 3 in the NG system and a similar risk as grade 1 in the new grading system. For solid growth patterns and basaloid morphology, using a 2-tier system with 1 cutoff was better than a 3-tier system with 2 cutoffs. Basaloid morphology and solid growth pattern have prognostic values for AdCC, with a 2-tier grading system performing better than a 3-tier system.

ABC Transporters 45 Years On.

ABC transporters constitute one of the largest gene families among all species [...].

The Switch and Reciprocating Models for the Function of ABC Multidrug Exporters: Perspectives on Recent Research.

ATP-binding cassette (ABC) transporters comprise a large superfamily of primary active transporters, which are integral membrane proteins that couple energy to the uphill vectorial transport of substrates across cellular membranes, with concomitant hydrolysis of ATP. ABC transporters are found in all living organisms, coordinating mostly import in prokaryotes and export in eukaryotes. Unlike the highly conserved nucleotide binding domains (NBDs), sequence conservation in the transmembrane domains (TMDs) is low, with their divergent nature likely reflecting a need to accommodate a wide range of substrate types in terms of mass and polarity. An explosion in high resolution structural analysis over the past decade and a half has produced a wealth of structural information for ABCs. Based on the structures, a general mechanism for ABC transporters has been proposed, known as the Switch or Alternating Access Model, which holds that the NBDs are widely separated, with the TMDs and NBDs together forming an intracellular-facing inverted "V" shape. Binding of two ATPs and the substrate to the inward-facing conformation induces a transition to an outward conformation. Despite this apparent progress, certainty around the transport mechanism for any given ABC remains elusive. How substrate binding and transport is coupled to ATP binding and hydrolysis is not known, and there is a large body of biochemical and biophysical data that is at odds with the widely separated NBDs being a functional physiological state. An alternative Constant Contact model has been proposed in which the two NBSs operate 180 degrees out of phase with respect to ATP hydrolysis, with the NBDs remaining in close proximity throughout the transport cycle and operating in an asymmetric allosteric manner. The two models are discussed in the light of recent nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses of three ABC exporters.

Asbestos and Zeolites: from A to Z via a Common Ion.

Asbestos and zeolites are silicate-based minerals, linked inextricably via paradoxical similarities and differences which have emanated from different geological epochs. Both have been employed in the service of humanity through millennia: asbestos, for its "inextinguishable" quality of being an insulator against heat and fire; zeolite, a "boiling stone" with its volcanic and marine sedimentary rock origins, for its propensity to adsorb water and remove metals and toxins. Serious adverse health effects observed in asbestos miners as long ago as the 1st Century AD did not halt the rising popularity of asbestos. As the miracle material of the 1900s, asbestos production and consumption exploded, culminating in its ubiquity in ships, vehicles, homes, commercial buildings, and over 3000 different industrial and household products. Through the 1940s and 1950s, epidemiological studies concluded that asbestos was a likely cause of asbestosis, lung cancer, and malignant mesothelioma, and it is now banned in many but far from all countries. The long latency between exposure to asbestos and the occurrence of cancer has obscured the deadly consequences of asbestos exposure for centuries. Even today, a considerable part of the world population is insufficiently aware of the dangers of asbestos, and millions of tons of this carcinogen continue to be mined and used worldwide. Zeolites, both natural and synthetic, are microporous aluminosilicate minerals commonly used in a myriad of processes, in the petrochemical industry, in domestic appliances and cleaning agents, as commercial adsorbents and exchangers for toxins and pollutants, and as catalysts. Zeolites are found in agriculture, veterinary science, and human health. More recently, new materials such as carbon nanotubes are being employed in materials requiring durability and thermal and electrical conductivity, yet nanotubes are now joining the ranks of more established particulates such as asbestos and silica, in causing human disease. In this review, we compare and contrast the similarities and differences of these two groups of silicate minerals and their waxing and waning use in the employ of humanity.

Detection of circulating tumor cells and circulating tumor DNA before and after mammographic breast compression in a cohort of breast cancer patients scheduled for neoadjuvant treatment.

PURPOSE: It is not known if mammographic breast compression of a primary tumor causes shedding of tumor cells into the circulatory system. Little is known about how the detection of circulating biomarkers such as circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) is affected by breast compression intervention. METHODS: CTCs and ctDNA were analyzed in blood samples collected before and after breast compression in 31 patients with primary breast cancer scheduled for neoadjuvant therapy. All patients had a central venous access to allow administration of intravenous neoadjuvant chemotherapy, which enabled blood collection from superior vena cava, draining the breasts, in addition to sampling from a peripheral vein. RESULTS: CTC and ctDNA positivity was seen in 26% and 65% of the patients, respectively. There was a significant increase of ctDNA after breast compression in central blood (p = 0.01), not observed in peripheral testing. No increase related with breast compression was observed for CTC. ctDNA positivity was associated with older age (p = 0.05), and ctDNA increase after breast compression was associated with high Ki67 proliferating tumors (p = 0.04). CTCs were more abundant in central compared to peripheral blood samples (p = 0.04). CONCLUSIONS: There was no significant release of CTCs after mammographic breast compression but more CTCs were present in central compared to peripheral blood. No significant difference between central and peripheral levels of ctDNA was observed. The small average increase in ctDNA after breast compression is unlikely to be clinically relevant. The results give support for mammography as a safe procedure from the point of view of CTC and ctDNA shedding to the blood circulation. The results may have implications for the standardization of sampling procedures for circulating tumor markers.

Pre-operative plasma cell-free circulating tumor DNA and serum protein tumor markers as predictors of lung adenocarcinoma recurrence.

Background: Lung cancer patients have a risk of recurrence even after curatively intended surgery. Cell-free circulating tumor DNA (ctDNA) and circulating tumor marker measurements are easily accessible through peripheral blood and could potentially identify patients with worse prognosis. The aim of this study was to examine ctDNA in pre-operative plasma and the role of tumor markers in pre-operative serum for their predictive potential on risk of tumor recurrence. Methods: Mutation analysis by 26-gene targeted sequencing was performed on 157 lung adenocarcinomas (ACs) from patients surgically treated at the Lund University Hospital 2005-2014. Of these, 58 tumors from patients in stages I-IIIA (34 stage I, 14 stage II and 10 stage III) with mutation(s) in EGFR, BRAF or KRAS were included. ctDNA from corresponding plasma (median 1.5 ml, range 1-1.6) was analyzed for one tumor-specific mutation in either of these three oncogenes using ultrasensitive IBSAFE droplet digital PCR (ddPCR). The tumor markers cancer antigen 125 (CA 125) and carbohydrate antigen 19-9 (CA 19-9) were analyzed in corresponding serum with electrochemiluminiscence immunoassay. Results: 6/7 patients with ctDNA and 19/51 without detected ctDNA were diagnosed with recurrence (log-rank test p = .001). 8/10 patients with positive serum tumor markers and 17/47 without tumor markers were diagnosed with recurrence (log-rank test, p = .0002). Fifteen patients had positive ctDNA and/or tumor markers, 12 of these had recurrence (log-rank test, p < .0001). Conclusion: A combination of tumor markers and ctDNA single mutation detection in low-volume pre-operative blood samples is a promising prognostic test. Prediction of recurrent disease in surgically treated early stage lung cancer can likely be further improved by using larger volumes of blood.

Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.

N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100-200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs).

MetaPlotR: a Perl/R pipeline for plotting metagenes of nucleotide modifications and other transcriptomic sites.

SUMMARY: An increasing number of studies are mapping protein binding and nucleotide modifications sites throughout the transcriptome. Often, these sites cluster in certain regions of the transcript, giving clues to their function. Hence, it is informative to summarize where in the transcript these sites occur. A metagene is a simple and effective tool for visualizing the distribution of sites along a simplified transcript model. In this work, we introduce MetaPlotR, a Perl/R pipeline for creating metagene plots. AVAILABILITY AND IMPLEMENTATION: The code and associated tutorial are available at https://github.com/olarerin/metaPlotR . CONTACT: srj2003@med.cornell.edu.

Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive.

Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as ≥100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. This was unexpected given 37% of contaminated series used poly(A)-selection for mRNA enrichment. Lastly, we examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality.

Intrathoracic tuberculous lymphadenopathy in children: a guide to chest radiography.

Making the diagnosis of pulmonary tuberculosis in children can be difficult because microbiological confirmation is not often achieved. Diagnosis is therefore often based on clinical features in combination with chest radiograph findings. Chest radiographs can demonstrate lymphadenopathy of the hilar and para-tracheal regions on the anteroposterior view, and subcarinal lymphadenopathy on the lateral view. However poor interobserver agreement has been reported for radiologist and clinician assessment of lymphadenopathy. This might reflect the lack of standardised imaging criteria for diagnosis as well as radiologists' objectives for achieving sensitivity rather than specificity. In this paper the authors provide a pictorial aid of chest radiographs in children with culture-confirmed tuberculosis to help clinicians identify lymph node enlargement in primary pulmonary tuberculosis. This collection of images comprises chest radiographs accompanied by schematics and either CT or MRI scan confirmation of pathological lymph node enlargement at the positions commonly affected in tuberculosis.

Preparation of balanced trichromatic white phosphors for solid-state white lighting.

High quality white light-emitting diodes (LEDs) employ multi-component phosphor mixtures to generate light of a high color rendering index (CRI). The number of distinct components in a typical phosphor mix usually ranges from two to four. Here we describe a systematic experimental technique for starting with phosphors of known chromatic properties and arriving at their respective proportions for creating a blended phosphor to produce light of the desired chromaticity. This method is applicable to both LED pumped and laser diode (LD) pumped white light sources. In this approach, the radiometric power in the down-converted luminescence of each phosphor is determined and that information is used to estimate the CIE chromaticity coordinate of light generated from the mixed phosphor. A suitable method for mixing multi-component phosphors is also described. This paper also examines the effect of light scattering particles in phosphors and their use for altering the spectral characteristics of LD- and LED-generated light. This is the only approach available for making high efficiency phosphor-converted single-color LEDs that emit light of wide spectral width.

A reciprocating twin-channel model for ABC transporters.

ABC transporters comprise a large, diverse, and ubiquitous superfamily of membrane active transporters. Their core architecture is a dimer of dimers, comprising two transmembrane (TM) domains that bind substrate, and two ATP-binding cassettes, which use the cell's energy currency to couple substrate translocation to ATP hydrolysis. Despite the availability of over a dozen resolved structures and a wealth of biochemical and biophysical data, this field is bedeviled by controversy and long-standing mechanistic questions remain unresolved. The prevailing paradigm for the ABC transport mechanism is the Switch Model, in which the ATP-binding cassettes dimerize upon binding two ATP molecules, and thence dissociate upon sequential ATP hydrolysis. This cycle of nucleotide-binding domain (NBD) dimerization and dissociation is coupled to a switch between inward- or outward facing conformations of a single TM channel; this alternating access enables substrate binding on one face of the membrane and its release at the other. Notwithstanding widespread acceptance of the Switch Model, there is substantial evidence that the NBDs do not separate very much, if at all, and thus physical separation of the ATP cassettes observed in crystallographic structures may be an artefact. An alternative Constant Contact Model has been proposed, in which ATP hydrolysis occurs alternately at the two ATP-binding sites, with one of the sites remaining closed and containing occluded nucleotide at all times. In this model, the cassettes remain in contact and the active sites swing open in an alternately seesawing motion. Whilst the concept of NBD association/dissociation in the Switch Model is naturally compatible with a single alternating-access channel, the asymmetric functioning proposed by the Constant Contact model suggests an alternating or reciprocating function in the TMDs. Here, a new model for the function of ABC transporters is proposed in which the sequence of ATP binding, hydrolysis, and product release in each active site is directly coupled to the analogous sequence of substrate binding, translocation and release in one of two functionally separate substrate translocation pathways. Each translocation pathway functions 180° out of phase. A wide and diverse selection of data for both ABC importers and exporters is examined, and the ability of the Switch and Reciprocating Models to explain the data is compared and contrasted. This analysis shows that not only can the Reciprocating Model readily explain the data; it also suggests straightforward explanations for the function of a number of atypical ABC transporters. This study represents the most coherent and complete attempt at an all-encompassing scheme to explain how these important proteins work, one that is consistent with sound biochemical and biophysical evidence.

Cholesterol sensing by the ABCG1 lipid transporter: Requirement of a CRAC motif in the final transmembrane domain.

The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate-transporter interaction of an ABC lipid exporter.

Laser-driven phosphor-converted white light source for solid-state illumination.

Energy efficiency and lighting quality considerations are driving research into laser-pumped white light sources. Laser diodes as pump sources for downconversion phosphors promise freedom from "droop" that adversely affects the efficiency of light-emitting diodes (LEDs). High-intensity laser diode-pumped light sources for applications such as search lights and automobile headlights have been demonstrated recently. Our paper describes the design and construction of a domestic/office-type solid-state luminaire driven by light from an integrated violet laser-diode module. A trichromatic phosphor made from a blend of separate europium-containing rare-earth phosphors was used as the downconversion medium. Mechanical and optical design of the reflector and the phosphor plate are described. Characteristics of both the pump light and the downconverted light are also described. Our studies also looked at the variation of chromaticity coordinates with variation in pump power and the effect of laser speckle on the lamp's light output. Finally, there is a brief discussion of energy conversion efficiency and longevity considerations, comparing pumping with LEDs versus pumping with laser diodes.

Mechanism of the ABC transporter ATPase domains: catalytic models and the biochemical and biophysical record.

ABC transporters comprise a large, diverse, and ubiquitous superfamily of membrane active transporters. Their core architecture is a dimer of dimers, comprising two transmembrane domains that bind substrate and form the channel, and two ATP-binding cassettes, which bind and hydrolyze ATP to energize the translocase function. The prevailing paradigm for the ABC transport mechanism is the Switch Model, in which the nucleotide binding domains are proposed to dimerise upon binding of two ATP molecules, and thence dissociate upon sequential hydrolysis of the ATP. This idea appears consistent with crystal structures of both isolated subunits and whole transporters, as well as with a significant body of biochemical data. Nonetheless, an alternative Constant Contact Model has been proposed, in which the nucleotide binding domains do not fully dissociate, and ATP hydrolysis occurs alternately at each of the two active sites. Here, we review the biochemical and biophysical data relating to the ABC catalytic mechanism, to show how they may be construed as consistent with a Constant Contact Model, and to assess to what extent they support the Switch Model.