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I have been unsuccessfully trying to buy a Colt King Cobra .22-caliber revolver (December 2023) for some time now. Colt has been advertising it for more than a year, but [...]

JTE-607 is an anticancer and anti-inflammatory compound and its active form, compound 2, directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-messenger RNA (pre-mRNA) 3′ processing. Surprisingly, compound 2-mediated inhibition of pre-mRNA cleavage is sequence specific and the drug sensitivity is predominantly determined by sequences flanking the cleavage site (CS). Using massively parallel in vitro assays, we identified key sequence features that determine drug sensitivity. We trained a machine learning model that can predict poly(A) site (PAS) relative sensitivity to compound 2 and provide the molecular basis for understanding the impact of JTE-607 on PAS selection and transcription termination genome wide. We propose that CPSF73 and associated factors bind to the CS region in a sequence-dependent manner and the interaction affinity determines compound 2 sensitivity. These results have not only elucidated the mechanism of action of JTE-607, but also unveiled an evolutionarily conserved sequence specificity of the mRNA 3′ processing machinery.

AbstractThe invasive tiger mosquito Aedes albopictusis a serious nuisance for people due to its biting and potential for disease transmission, even in Italian mountain regions. In this work, we examined its occurrence dynamic from the first invasion in two Alpine cities, Rovereto and Trento, only 25 km apart. Mosquito’s distribution was assessed using ovitraps, from mid-May to end-October, from its first reported occurrence, i.e. 2001 in Rovereto and 2010 in Trento. In both cities, ovitraps were located in seven habitat types: gathering places, residential areas, urban parks, car parks, near watercourses, industrial areas and croplands. The annual average temperature and precipitation were similar in the two cities from 2001 to 2020. We had hypothesized that the seasonal and annual differences in egg number between both cities would be limited. Significant linear and positive relationship was found between the average number of eggs and air temperature in both the cities, but with a steeper slope in Trento. In addition, the average number of eggs per ovitrap was higher in Trento than in Rovereto (average 50.3 vs 14.2). None of the considered risk factors (climate, habitat type) explained such difference in abundance between cities. Indeed, based on the temperature, the expectation was to find fewer eggs in Trento being cooler than Rovereto during 2010–2020 (the average in the period of activated traps was, respectively, 19.2 vs 19.7°C). Thus, we argue that other anthropogenic factors, such as different control activities (adulticide treatments were carried out solely in Rovereto), might explain the difference in egg abundance found between cities. A specific experimental design to evaluate treatment effect might validate our hypothesis (e.g. apply adulticides during the surveillance period, with treatments applied at regular distances from the ovitraps and with a specific periodicity to compare adult and egg abundance).

Supplemental Digital Content is available in the text.

RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome — the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.

We report a strategy for the orthogonal conjugation of the vinyl nucleosides, 5-vinyluridine (5-VU) and 2-vinyladenosine (2-VA), via selective reactivity with maleimide and tris(2-carboxyethyl)phosphine (TCEP), respectively. The orthogonality was investigated using density functional theory (DFT) and confirmed by reactions with vinyl nucleosides. Further, these chemistries were used to modify RNA for fluorescent cell imaging. These reactions allow for the expanded use of RNA metabolic labeling to study nascent RNA expression within different RNA populations.

When meconium (a mixture of bile salts, cholesterol, enzymes, and cell debris from the fetal intestinal tract) becomes lodged in the respiratory tract of infants, there is an increased risk for neonatal respiratory distress, a condition of serious impairment of breathing. Meconium is present in amniotic fluid (in which the fetus floats) in as many as 18 percent of all deliveries, and many infants inhale meconium during delivery. While much of the damage associated with meconium aspiration results from airway obstruction, it is possible that these substances also have chemical effects on the lung. In adult respiratory distress (RDS; acute respiratory failure), a frequently fatal condition, pulmonary surfactant may become inactivated. Surfactant is a substance that lines the lung and lowers its surface tension, preventing collapse of the organ. It is possible that meconium also affects surfactant adversely in the infant. This was evaluated in experiments that tested the effects of meconium obtained from healthy infants on lung surfactant from calves. Meconium was found to inhibit the ability of surfactant to lower the surface tension; at low concentrations of surfactant, even very small quantities of meconium led to considerable increases in surface tension. Above a certain concentration of surfactant, no inhibitory effect was seen, but that concentration was quite high. The results suggest that replacing surfactant may be an effective treatment for some cases of pulmonary disease involving meconium. (Consumer Summary produced by Reliance Medical Information, Inc.)

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10–23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10–23, overcomes the problem of product inhibition, which limited previous 10–23 designs, using molecular chemotypes with DNA, 2′-fluoroarabino nucleic acid and α-l-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10–23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.

In vivo RNA structure analysis has become a powerful tool in molecular biology, largely due to the coupling of an increasingly diverse set of chemical approaches with high-throughput sequencing. This has resulted in a transition from single target to transcriptome-wide approaches. However, these methods require sequencing depths that preclude studying low abundance targets, which are not sufficiently captured in transcriptome-wide approaches. Here we demonstrate that enrichment of low abundance targets before reverse transcription broadens the range of molecules analyzed and results in improved analysis for low abundance transcripts. In addition, this method is compatible with any choice of chemical adduct or read-out approach. We combine this method with inducible expression of an RBP of interest to study an autoregulated event in the pre-mRNA of the splicing factor, muscleblind-like splicing regulator 1 (MBNL1) in a cellular context.

Profiling RNA expression in a cell-specific manner continues to be a grand challenge in biochemical research. Bioorthogonal nucleosides can be utilized to track RNA expression; however, these methods currently have limitations due to background and incorporation of analogs into undesired cells. Herein, we design and demonstrate that uracil phosphoribosyltransferase can be engineered to match 5-vinyluracil for cell-specific metabolic labeling of RNA with exceptional specificity and stringency.

Tissues and organs are composed of diverse cell types, which poses a major challenge for cell-type-specific profiling of gene expression. Current metabolic labeling methods rely on exogenous pyrimidine analogs that are only incorporated into RNA in cells expressing an exogenous enzyme. This approach assumes that off-target cells cannot incorporate these analogs. We disprove this assumption and identify and characterize the enzymatic pathways responsible for high background incorporation. We demonstrate that mammalian cells can incorporate uracil analogs and characterize the enzymatic pathways responsible for high background incorporation. To overcome these limitations, we developed a new small molecule–enzyme pair consisting of uridine/cytidine kinase 2 and 2′-azidouridine. We demonstrate that 2′-azidouridine is only incorporated in cells expressing uridine/cytidine kinase 2 and characterize selectivity mechanisms using molecular dynamics and X-ray crystallography. Furthermore, this pair can be used to purify and track RNA from specific cellular populations, making it ideal for high-resolution cell-specific RNA labeling. Overall, these results reveal new aspects of mammalian salvage pathways and serve as a new benchmark for designing, characterizing and evaluating methodologies for cell-specific labeling of biomolecules.

Laboratory-evolved RNAs bind a wide variety of targets and serve highly diverse functions, including as diagnostic and therapeutic aptamers. The majority of aptamers have been identified using in vitroselection (SELEX), a molecular evolution technique based on selecting target-binding RNAs from highly diverse pools through serial rounds of enrichment and amplification. In vitroselection typically yields multiple distinct motifs of highly variable abundance and target-binding affinities. The discovery of new aptamers is often limited by the difficulty of characterizing the selected motifs, because testing of individual sequences tends to be a tedious process. To facilitate the discovery of new aptamers within in vitroselected pools, we developed Apta-Seq, a multiplex analysis based on quantitative, ligand-dependent 2′ acylation of solvent-accessible regions of the selected RNA pools, followed by reverse transcription (SHAPE) and deep sequencing. The method reveals, in a single sequencing experiment, the identity, structural features, and target dissociation constants for aptamers present in the selected pool. Application of Apta-Seq to a human genomic pool enriched for ATP-binding RNAs yielded three new aptamers, which together with previously identified human aptamers suggest that ligand-binding RNAs may be common in mammals.

Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond encoding proteins. Critical to RNA function is its transport to unique subcellular locations. Despite the importance of RNA localization, it is still very challenging to study in an unbiased manner. We recently described the ability to tag RNA molecules within subcellular locations through spatially restricted nucleobase oxidation. Herein, we describe a dramatic improvement of this protocol through the localized oxidation and tagging of proteins. Isolation of RNA–protein complexes enabled the enrichment of challenging RNA targets on chromatin and presented a considerably optimized protocol for the analysis of RNA subcellular localization within living cells.

New chemical probes have been designed to facilitate the identification of adenosine-to-inosine (A-to-I) edited RNAs. These reagents combine a conjugate acceptor for selective inosine covalent modification with functional groups for bioorthogonal biotinylation. The resulting biotinylated RNA was enriched and verified with RT-qPCR. This powerful chemical approach provides new opportunities to identify and quantify A-to-I editing sites.

Optimized and stringent chemical methods to profile nascent RNA expression are still in demand. Herein, we expand the toolkit for metabolic labeling of RNA through application of inverse electron demand Diels–Alder (IEDDA) chemistry. Structural examination of metabolic enzymes guided the design and synthesis of vinyl-modified nucleosides, which we systematically tested for their ability to be installed through cellular machinery. Further, we tested these nucleosides against a panel of tetrazines to identify those which are able to react with a terminal alkene, but are stable enough for selective conjugation. The selected pairings then facilitated RNA functionalization with biotin and fluorophores. We found that this chemistry not only is amenable to preserving RNA integrity but also endows the ability to both tag and image RNA in cells. These key findings represent a significant advancement in methods to profile the nascent transcriptome using chemical approaches.

DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2’-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons. The deposition of m6dA is associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with extinction-induced gene expression. The accumulation of m6dA is associated with transcriptional activation at the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnfexon IV messenger RNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as an epigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.

Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond encoding proteins. Critical to RNA function is its transport to unique subcellular locations, where it can be spatially regulated to perform its biological function. In this Perspective, we highlight the techniques developed to image and discover RNAs within their subcellular compartments and discuss the utility of these techniques in understanding RNA localization.

In response to the Letter to the Editor ('Response To Colt's Progress') in the April issue, we at Colt would like to reiterate we're very proud of the enormous progress [...]

The cellular RNA pool in animals arises from two separate genomes stored in the nucleus and multiple mitochondria. Chemical methods to track nascent RNA synthesis are unable to distinguish between these two with stringency. Herein, we report that spatially restricting bioorthogonal nucleoside biosynthesis enables, for the first time, selective metabolic labeling of the RNA transcribed in the mitochondria. We envision that this approach could open the door for heretofore-impossible analyses of mitochondrial RNA. Beyond our results revealed herein, our approach provides a roadmap for researchers to begin to design strategies to examine biomolecules within subcellular compartments.

The discovery of functional RNAs that are critical for normal and disease physiology continues to expand at a breakneck pace. Many RNA functions are controlled by the formation of specific structures, and an understanding of each structural component is necessary to elucidate its function. Measuring solvent accessibility intracellularly with experimental ease is an unmet need in the field. Here, we present a novel method for probing nucleobase solvent accessibility, Light Activated Structural Examination of RNA (LASER). LASER depends on light activation of a small molecule, nicotinoyl azide (NAz), to measure solvent accessibility of purine nucleobases. In vitro, this technique accurately monitors solvent accessibility and identifies rapid structural changes resulting from ligand binding in a metabolite-responsive RNA. LASER probing can further identify cellular RNA–protein interactions and unique intracellular RNA structures. Our photoactivation technique provides an adaptable framework to structurally characterize solvent accessibility of RNA in many environments.

We report herein a novel chemical-genetic method for assaying RNA localization within living cells. RNA localization is critical for normal physiology as well as the onset of cancer and neurodegenerative disorders. Despite its importance, there is a real lack of chemical methods to directly assay RNA localization with high resolution in living cells. Our novel approach relies on in situnucleobase oxidation by singlet oxygen generated from spatially confined fluorophores. We demonstrate that our novel method can identify RNA molecules localized within specific cellular compartments. We anticipate that this platform will provide the community with a much-needed methodology for tracking RNA localization within living cells, and set the stage for systematic large scale analysis of RNA localization in living systems.

Global inequalities in access to health care are reflected in differences in cancer survival. The CONCORD programme was designed to assess worldwide differences and trends in population-based cancer survival. In this population-based study, we aimed to estimate survival inequalities globally for several subtypes of childhood leukaemia.

RNA molecules depend on structural elements that are critical for cellular function. Chemical methods for probing RNA structure have emerged as a necessary component of characterizing RNA function. As such, understanding the limitations and idiosyncrasies of these methods is essential for their utility. Selective hydroxyl acylation has emerged as a common method for analyzing RNA structure. Ester products as a result of 2′-hydroxyl acylation can then be identified through reverse transcription or mutational enzyme profiling. The central aspect of selective hydroxyl acylation analyzed by primer extension (SHAPE) experiments is the fact that stable ester adducts are formed on the 2′-hydroxyl. Despite its importance, there has not been a direct comparison of SHAPE electrophiles for their ability to make stable RNA adducts. Herein, we conduct a systematic analysis of hydrolysis stability experiments to demonstrate that furoyl imidazole SHAPE reagents form stable ester adducts even at elevated temperatures. We also demonstrate that the acylation reaction with the furoyl acylimidaole SHAPE reagent can be controlled with dithiothreitol quenching, even in live cells. These results are important for our understanding of the biochemical details of the SHAPE experiment.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell–derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

In this Perspective, we expand the notion of temporal regulation of RNA in the brain and propose that the qualitative nature of RNA and its metabolism, together with RNA abundance, are essential for the molecular mechanisms underlying experience-dependent plasticity. We discuss emerging concepts in the newly burgeoning field of epitranscriptomics, which are predicted to be heavily involved in cognitive function. These include activity-induced RNA modifications, RNA editing, dynamic changes in the secondary structure of RNA, and RNA localization. Each is described with an emphasis on its role in regulating the function of both protein-coding genes, as well as various noncoding regulatory RNAs, and how each might influence learning and memory.

Proper control and maintenance of gene expression is critical for cellular identity and maintenance. Transcription of RNA from the genome is intimately controlled by post-translational chemical modification of histone tails and DNA. Recent studies have demonstrated that chromatin-remodeling complexes seek out their target genomic loci through the help of noncoding RNA molecules. Within this Review, we will outline how the use of biochemical techniques has shed light on the mechanisms employed by RNA to guide these complexes and therefore control gene expression.

Phobos, the innermost satellite of Mars, displays an extensive system of grooves that are mostly symmetric about its sub‐Mars point. Phobos is steadily spiraling inward due to the tides it raises on Mars lagging behind Phobos' orbital position and will suffer tidal disruption before colliding with Mars in a few tens of millions of years. We calculate the surface stress field of the deorbiting satellite and show that the first signs of tidal disruption are already present on its surface. Most of Phobos' prominent grooves have an excellent correlation with computed stress orientations. The model requires a weak interior that has very low rigidity on the tidal evolution time scale, overlain by an ~10–100 m exterior shell that has elastic properties similar to lunar regolith as described by Horvath et al. (1980). Tidal elongation from orbital decay produces surface stressesThese stresses align with the observed grooves on the surface, implying a connection to tidesOur model implies that Phobos' interior is weak with a stronger elastic outer layer

icSHAPE allows transcriptome-wide RNA structure profiling in living cells. It uses a clickable SHAPE reagent to enable biotin-based enrichment of SHAPE-modified RNA, reducing the necessary sequencing depth.

Many lakes in mountain regions have been used for hydropower generation since the 1950s. It has been estimated that as many as 79% of the rivers in the Alps have been affected by the presence of hydropower plants. In this context, the shutting down of hydropower plants on a group of Alpine lakes represented a good opportunity to study the ecological impact on them. We selected nine lakes that had been affected and nine that had not, and analysed the differences in environment, littoral diatoms and zoobenthos, phytoplankton, zooplankton, and fish. Results showed that benthic biota -diatoms and zoobenthos- were the most affected by water-level drawdown during winter months. Even six years after the end of hydroelectric operations, diatom species richness and diversity were lower in impacted lakes. Assemblage structure was different for both diatoms and zoobenthos. Phytoplankton and zooplankton were similar in impacted and unaffected lakes in terms of both species richness (and diversity) and assemblage structure. The degree of impact on fish was unclear because illegal stocking of lakes with allochthonous fish species had taken place. This study showed that compared to limnetic biota, littoral communities were the most affected by the decrease in water volume every winter. Six years after the end of hydroelectric operations, diatoms, and to lesser extent zoobenthos, were still different compared to those in natural (unaffected) lakes. Planktic communities seem to be either more resistant to the disturbances, or else able to recover more quickly to their former condition.

RNA–protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA–protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA–protein recognition across the diversity of human and pathogen RNAs.

RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base‐pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2′‐hydroxyl group in the ribose ring, and the reactivity of the 2′‐hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2′‐hydroxyl is reactive at single‐stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base‐pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2′ hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2′‐ hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome‐wide exploration of RNA structure and function. WIREs RNA2014, 5:867–881. doi: 10.1002/wrna.1253

Commercially pure magnesium was processed by ECAP from the as-cast condition and also processed by rolling followed by ECAP. The evolution of the microstructure was tracked in both processing routes and compared. The mechanical behavior was evaluated by compression and tensile tests. The results show that ECAP refines the grain structure of magnesium but leads to heterogeneous grain size distribution. The use of an intermediate step of rolling facilitates grain refinement and leads to homogeneous grain structure. Compression tests were carried out at three different orthogonal directions and the results show the development of anisotropy. Significant twinning activity is observed when the material is compressed perpendicular to the die channels. Tensile tests show that ECAP decreases the yield stress and increases the elongation of the material processed by rolling.

Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.

Commercially pure magnesium was processed by a single pass of Equal Channel Angular Pressing (ECAP) at 523K.The grain structure was evaluated at the region near the intersection between the channels of the die. Compression test workpieces were machined from the as-received material and the material processed by ECAP. These workpieces were tested at room temperature. The results show the grain structure is refined in the intersection between the channels. The ECAP processed material displays higher strength and reduced surface bulging due to the refined structure. The plastic flow during compression of the ECAP processing material concentrates along bands of refined grains.

This paper presents a general methodology for compensating LDO regulators which exploits a current amplifier to effectively multiply the Miller capacitor. After a general theoretical analysis that takes into account both the external and internal loop stability, it is examined the feasibility of applying this kind of compensation in different LDO applications, showing the role of the design parameters and proposing a specific design procedure for each practical cases which differ in terms of load capacitance and the adoption of a decoupling voltage buffer. Simulations and experimental results which validate the methodology are also included. [ABSTRACT FROM PUBLISHER]

AbstractEven though there is no reason to believe in a unique mechanism that would explain rarity, recognizing common patterns might aid in identifying effective conservation strategies. This study therefore approached the problem of rarity in spring habitats both at the level of multiple taxonomic groups (molluscs, oligochaeta, water mites, copepods, ostracods, chironomids, stoneflies, caddisflies, diatoms, and vascular plants) and, in more detail, at the level of a single group (bryophytes). The aim was to evaluate whether the proportion of rare species was associated with uncommon environmental conditions, while for the bryophytes an additional aim was to test whether common and rare species differed concerning their niche parameters (niche breadth and niche position), their biological traits (involved in dispersal processes), and their habitat preferences. Overall, 4 major results may be highlighted. 1) The significant concordance between the rarity of virtually all the taxonomic groups inhabiting spring habitats and their environmental conditions suggested that most of the rare species at community level might be explained by their uncommon resource requirements. 2) Rare bryophyte species not only had high niche positions, as did all the taxonomic groups, but also had narrow niche breadths. This result suggests an interesting distinction between resource use and resource availability. 3) Because the species traits linked to dispersal ability did not differ between common and rare species, the hypothesis of an important role being played by these traits in determining species distribution was not supported. 4) Among the rare bryophyte species, an undefined number were casual. Because they were less hygrophilous and less influenced by the environment, this result suggests that they might recruit essentially by chance from the surrounding habitats.

Cellular pathways must be synergized, controlled and organized to manage homeostasis. To achieve high selectivity within the crowded cellular milieu the cell utilizes scaffolding complexes whose role is to bring molecules in proximity thereby controlling and enhancing intermolecular interactions and signaling events. To date, scaffolds have been shown to be composed of proteinaceous units; however, recent evidence has supported the idea that non-coding RNAs may also play a similar role. In this point of view article we discuss recent data on ncRNA scaffolds, with particular focus on ncRNA HOTAIR. Using our current knowledge of signaling networks we discuss the role that RNA may play in writing and regulating histone modifications and the information needed for correct gene expression. Further, we speculate on additional, yet undiscovered roles that ncRNAs may be playing as molecular scaffolds.

It is thought that long-term monitoring is an essential tool through which conservationists and managers (i) are alerted when the system departs from the natural state, (ii) can check their environmental policy, and (iii) can detect disturbance effects. However, while long-term studies are growing in number, the lack of information on the background rates of natural changes could lead to a biased interpretation of results. In this study we analyzed the diatom composition of yearly samples (14-16 yrs) in four springs with the following goals: (1) to estimate the consequences of sampling-related processes on the species composition and relative abundance; (2) to determine the form of the assemblages' variation, evaluating whether the assemblages can be predicted by cyclic, directional, or stochastic changes; (3) to test if, and to what extent, the diatom variability (variation in species composition, relative abundance and diversity) on a temporal scale is driven by the environment; (4) to evaluate the degree of temporal concordance among the diatom assemblages inhabiting four springs. Results showed that because the effect of sampling was inversely proportional to the species abundances, the presence of the least abundant species over the years were mainly a result of chance. The analyses allowed us to discriminate between stochastic and directional patterns, revealing the ongoing changes in two out of four springs. Because the environmental variables did not explain a significant portion of this variability, other hypotheses are put forward. The assemblages' dynamics of species composition over time were significantly synchronous in two out of six couples of springs, and regardless of the environment. This result can suggest that internal, within springs, drivers may be more important than extrinsic forces operating over regional spatial scales. Overall, these results provide a benchmark of diatom variability over time and in natural conditions delimiting the "limits of acceptable changes".

Immunosuppressive treatment has changed the prognosis of Lupus nephritis over time, but improvement in prognosis is difficult to analyze in different historical periods, and should be better demonstrated in comparison with life expectancy of sex-and age-matched people. Long-term patient and renal survival of 90 patients diagnosed with Lupus nephritis at our center from 1968 to 2001 with a follow-up time of 14±8 years was retrospectively evaluated. Patient and kidney survival significantly increased over time. Multivariate analyses show that risks of patient and renal death decreased by 8% at each year of follow-up, and increased by more than 5 time in patients aged > 30 years at diagnosis. As only 14 patients were men, relative survival as compared to that of the sex- and age-matched general population of the Piedmont Region was calculated for the 76 women. Improvement in the survival of the cohort of women was seen at any time of follow-up: in particular, it was sharply lower in the first period (relative survival at 5,10 and 15 years = 0.784, 0.665, and 0.620, respectively) and increased in the second (relative survival at 5,10 and 15 years = 0.939, 0.921, and 0.850, respectively) nearly approaching that expected for the general population, i.e. 0.993, 0.983 and 0.967, respectively. Taken together, our data allow us to draw the conclusion that life expectancy in women with Lupus nephritis has improved over time, paralleling an improved awareness of the disease and a significant increase in steroid pulse therapy as induction/remission phase. Improvement in survival is for the first time demonstrated to cover the gap with life expectancy of the general population for women with Lupus nephritis.

This paper describes a model of a broad planetary ring whose sharp edge is confined by a satellite's mth Lindblad resonance (LR). This model uses the streamline formalism of Borderies et al. to calculate the ring's internal forces, namely, ring gravity, pressure, and viscosity. The model also allows for the possibility of a drag force that can affect small ring particles directly, and large ring particles indirectly via collisions with the small. The model calculates the streamlines' forced eccentricities e, their longitudes of periapse $\tilde{\omega }$, and the surface density s throughout the perturbed ring. This model is then applied to the outer edge of Saturn's B ring, which is maintained by an m = 2 inner LR with the satellite Mimas. A suite of ring models are used to illustrate how a ring's perturbed state depends on the ring's physical properties: its surface density, its viscosity, the ring particles' dispersion velocity, and the strength of the hypothetical drag force. A comparison of simulations with the outer B ring's observed properties suggests that the ring's surface density there is 10 [?] s [?] 280 gm cm-2 in the ring's outermost [?]40 km. The ring's sharp edge identifies the site where the ring's viscous torque precisely counterbalances the perturbing satellite's gravitational torque on the ring. However, an examination of several seemingly conventional viscous B ring models shows that they all fail, by wide margins, to balance these torques at the ring's outer edge. This is partly due to the ring's self-gravity, which tends to reduce forced eccentricities near the resonance. But this is also due to the fact that a viscous ring tends to be nearly peri-aligned with the satellite. Both effects conspire to reduce the satellite's torque on the ring, which in turn makes the ring's edge more difficult to maintain. Nonetheless, the following shows that a torque balance can still be achieved in a viscous B ring, but only in an extreme case where the ratio of the ring's bulk/shear viscosities satisfy n b /n s [?] 104. However, if the dissipation of the ring's forced motions is instead dominated by a weak drag force, then the satellite can exert a much stronger torque across a wider annulus in the ring, which can successfully counterbalance the ring's viscous torque there. We also show how this streamline model can be adapted to study other interesting ring phenomena, such as narrow eccentric ringlets and nonlinear spiral density waves.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivoexpression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.

Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N1-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT processivity and natural post-transcriptional modifications in RNA.

Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed “caged” nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.