21419335
TRBP alters human precursor microRNA processing in vitro.
MicroRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms that can vary in length at their 5' or 3' ends, but the biogenesis and functional significance of these RNAs are largely unknown. We show here that the human trans-activation response (TAR) RNA binding protein (TRBP), a known molecular partner of the miRNA processing enzyme Dicer, changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. Furthermore, TRBP can trigger the generation of iso-miRNAs (isomiRs) that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. These results implicate TRBP as a key regulator of miRNA processing and targeting in humans.
2859155
Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli
Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.
24037980
RNA–protein analysis using a conditional CRISPR nuclease
RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA–protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.
19782986
Reconstitution and characterization of a new desosaminyl transferase, EryCIII, from the erythromycin biosynthetic pathway.
EryCIII converts alpha-mycarosyl erythronolide B into erythromycin D using TDP-d-desosamine as the glycosyl donor. We report the heterologous expression, purification, in vitro reconstitution, and preliminary characterization of EryCIII. Coexpression of EryCIII with the GroEL/ES chaperone complex was found to enhance greatly the expression of soluble EryCIII protein. The enzyme was found to be highly active with a kcat greater than 100 min-1. EryCIII was quite selective for the natural nucleotide sugar donor and macrolide acceptor substrates, unlike several other antibiotic glycosyl transferases with broad specificity such as desVII, oleG2, and UrdGT2. Within detectable limits, neither 6-deoxyerythronolide B nor 10-deoxymethynolide were found to be glycosylated by EryCIII. Furthermore, TDP-d-mycaminose, which only differs from TDP-d-desosamine at the C4 position, could not be transferred to alphaMEB. These studies lay the groundwork for detailed structural and mechanistic analysis of an important member of the desosaminyl transferase family of enzymes.
1465194
Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing
During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer–TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer–dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.
8585824
Improved Precursor Directed Biosynthesis in E. coli via Directed Evolution
Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor-directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure–function relationships in the bacterial ribosome.
5387554
Amicrobial Pustulosis-Like Rash in a Patient with Crohn’s Disease under Anti-TNF-Alpha Blocker
Amicrobial pustulosis of the folds (APF) is a recently described entity characterized by relapsing pustular lesions predominantly involving the cutaneous flexures and scalp. This disease typically occurs in association with systemic lupus erythematosus and a variety of other autoimmune diseases. We here describe an APF-like pustular eruption predominantly affecting the scalp, face and trunk, occurring during long-term infliximab treatment for Crohn’s disease. Immunohistochemical staining of skin biopsy specimens for myxovirus resistance protein A, a marker for type 1 interferon-inducible proteins, showed increased staining in the epidermis and dermal mononuclear inflammatory infiltrate. Our observation further extends the spectrum of cutaneous adverse reactions potentially related to anti-tumor necrosis factor-α, the clinical context in which APF can occur as well as its clinical presentations.
6882949
Hydrocortisone for refractory hypotension of very low birth weight infant with patent ductus arteriosus: a case report
To the Editor: A male infant weighing 1,090 g, 28 weeks of gestation, was scheduled to take a ligation of a patent ductus arteriosus (PDA). Eight days after birth, mean arterial pressure (MAP) has fall to \30 mmHg. We started with dopamine (5–10 lg/kg/min), to maintain the blood pressure, but this was not successful. Although we did not confirm an adrenal insufficiency (AI), we decided to start medication of hydrocortisone, 3 mg/kg/day, which resulted in over 50/35 mmHg. The hydrocortisone was tapered for 3 days because MBP was maintained at[40 mmHg, and 5 lg/kg/min of dopamine was maintained until the day of surgery. At 31 days after birth, we induced anesthesia with 2 mg/kg of ketamine, 20 lg/kg of fentanyl, and 0.9 mg/kg of rocuronium without premedication. Anesthesia was maintained by a continuous infusion of ketamine (1 mg/kg/h) and intermittent rocuronium in 50% O2 with air. After ligation of a PDA, MAP was decreased to 30 mmHg by degrees which required an increase to 10 lg/kg/ min of dopamine and the addition of 5 lg/kg/min of dobutamine to maintain over 30 mmHg. An echocardiogram showed globally normal contractility without a PDA. In spite of our efforts, mean blood pressure had fallen to 24 mmHg at 6 h after ligation. The patient was commenced on intravenous hydrocortisone (3 mg/kg/day), which increased MAP to above 30 mmHg and heart rate to 172 bpm after 90 min. After 4 h, MAP was maintained over 50 mmHg and heart rate was 190 bpm. Two days after surgery, the dopamine, dobutamine, and hydrocortisone were tapered and the blood pressure was maintained until discharge. Hypotension of very low birth weight (VLBW) premature infants is a major factor of morbidity, and mortality. Inotropes such as dopamine and dobutamine are widely used to normalize blood pressure but more than 50% of hypotensive VLBW infants requiring dopamine at doses[10 mg/kg/min develop refractory hypotension (RH) that is resistant to vasopressor [1]. Hydrocortisone is beneficial in treatment of RH, but the mechanisms are not completely understood. Down-regulation of cardiovascular adrenergic receptors and the development of relative adrenal insufficiency are thought to be the factors that may explain the corticosteroid responsiveness of RH [2, 3]. Cardiovascular response to catechol amines is attenuated due to down-regulation of the cardiovascular adrenergic receptors in VLBW infants with critical illness [2]. Moreover, VLBW infants have low adrenal reserves and reach a relatively adrenal insufficiency (AI) state in stressed situations [4]. Watterberg et al. [5] has reported hydrocortisone (1 mg/kg every 8 h) was effective for VLBW infants with AI. In this case, the infant had RH in spite of the treatment with inotropes after birth and PDA ligation, and we successfully treated him with hydrocortisone. Thus, we recommend that early hydrocortisone therapy be administered to VLBW premature infants for perioperative RH resistant to inotropes..
44677500
Interaction of pitch and duration processing by non-musicians in reading musical notation.
To study the relation of pitch and duration processing in reading musical notation, a Stroop-like task was used by 20 non-musicians. A probe display was presented before each target note. Participants were required to process the tonal and metric information of the probe and then to make a match or mismatch decision between probe and target. The target's color informed participants which dimension (pitch or duration) required analysis. The congruity of the irrelevant dimension of the target was manipulated to examine the effect on the relevant dimension. The interference effect of the irrelevant dimension on the relevant dimension was obvious for number of errors and reaction times. This result is consistent with pitch and duration being processed interdependently and reconciles with the theory of dynamic attention.
8896689
Secretory Low Molecular Weight Phospholipase A2 Plays Important Roles in Cell Elongation and Shoot Gravitropism in Arabidopsis Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.014423.
To elucidate the cellular functions of phospholipase A2 in plants, an Arabidopsis cDNA encoding a secretory low molecular weight phospholipase A2 (AtsPLA2β) was isolated. Phenotype analyses of transgenic plants showed that overexpression of AtsPLA2β promotes cell elongation, resulting in prolonged leaf petioles and inflorescence stems, whereas RNA interference–mediated silencing of AtsPLA2β expression retards cell elongation, resulting in shortened leaf petioles and stems. AtsPLA2β is expressed in the cortical, vascular, and endodermal cells of the actively growing tissues of inflorescence stems and hypocotyls. AtsPLA2β then is secreted into the extracellular spaces, where signaling for cell wall acidification is thought to occur. AtsPLA2β-overexpressing or -silenced transgenic plants showed altered gravitropism in inflorescence stems and hypocotyls. AtsPLA2β expression is induced rapidly by auxin treatment and in the curving regions of inflorescence stems undergoing the gravitropic response. These results suggest that AtsPLA2β regulates the process of cell elongation and plays important roles in shoot gravitropism by mediating auxin-induced cell elongation.
6648314
No Association between Serotonin Receptor 2C-759C/T Polymorphism and Weight Change or Treatment Response to Mirtazapine in Korean Depressive Patients
Objective Activation of one or more serotonin (5-HT) receptors may play a role in mediating the antidepressant effects of serotonergic antidepressants. The serotonin 2C (5HT 2C) receptor is known to be associated with antidepressant action and weight gain. We sought to determine whether the 5-HTR 2C receptor -759C/T polymorphism was associated with weight gain and treatment response to mirtazapine in major depressive disorder (MDD) patients. Methods The 5-HT 2C receptor -759C/T polymorphism was analyzed in 323 MDD patients. All patients were evaluated using the 21-item Hamilton Depression Rating Scale at the beginning of the study and at 1, 2, 4, and 8 weeks of mirtazapine treatment. Results There was no significant difference in the 5-HT 2C receptor -759C/T genotype distribution between responder and non-responder groups. The 5-HT 2C receptor -759C/T polymorphism was not associated with weight change over time after mirtazapine administration. Conclusion The 5-HT 2C receptor -759C/T polymorphism does not appear to be a predictor of treatment response to mirtazapine. This polymorphism was not associated with weight change after 8 weeks of mirtazapine treatment. Further investigation on other polymorphisms of the 5-HT 2C gene is required to determine whether the 5-HT 2C gene influences treatment response and weight change after mirtazapine administration in patients with major depressive disorder.