Translation regulation by a guanidine-II riboswitch is highly tunable in sensitivity, dynamic range, and apparent cooperativity.

Riboswitches function as important translational regulators in bacteria. Comprehensive mutational analysis of transcriptional riboswitches has been used to probe the energetic intricacies of interplay between the aptamer and expression platform, but translational riboswitches have been inaccessible to massively parallel techniques. The guanidine-II (gdm-II) riboswitch is an exclusively translational class. We have integrated RelE cleavage with next-generation sequencing to quantify ligand-dependent changes in translation initiation for all single and double mutations of the Pseudomonas aeruginosa gdm-II riboswitch, a total of more than 23,000 variants. This extensive mutational analysis is consistent with the prominent features of the bioinformatic consensus. These data indicate, unexpectedly, that direct sequestration of the Shine-Dalgarno sequence is dispensable for riboswitch function. Additionally, this comprehensive data set reveals important positions not identified in previous computational and crystallographic studies. Mutations in the variable linker region stabilize alternate conformations. The double mutant data reveal the functional importance of the previously modeled P0b helix formed by the 5' and 3' tails that serves as the basis for translational control. Additional mutations to GU wobble base pairs in both P1 and P2 reveal how the apparent cooperativity of the system involves an intricate network of communication between the two binding sites. This comprehensive examination of a translational riboswitch's expression platform illuminates how the riboswitch is precisely tuned and tunable with regard to ligand sensitivity, the amplitude of expression between ON and OFF states, and the cooperativity of ligand binding.

The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches.

Ligand specificity is an essential requirement for all riboswitches. Some variant riboswitches utilize a common structural motif, yet through subtle sequence differences, they are able to selectively respond to different small molecule ligands and regulate downstream gene expression. These variants discriminate between structurally and chemically similar ligands. Crystal structures provide insight into how specificity is achieved. However, ligand specificity cannot always be explained solely by nucleotides in direct contact with the ligand. The cyclic dinucleotide variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP riboswitches, that were distinguished based on the identity of a single nucleotide in contact with the ligand. Here we report a variant riboswitch with a mutation at a second ligand-contacting position that is promiscuous for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT, was used to quantitatively assess thousands of sites in the first- and second-shells of ligand contact for impacts on ligand specificity and promiscuity. In addition to nucleotides in direct ligand contact, nucleotides more distal from the binding site, within the J1/2 linker and the terminator helix, were identified that impact ligand specificity. These findings provide an example of how nucleotides outside the ligand binding pocket influence the riboswitch specificity. Moreover, these distal nucleotides could be used to predict promiscuous sequences.

The asymmetry and cooperativity of tandem glycine riboswitch aptamers.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

The Positively Charged Active Site of the Bacterial Toxin RelE Causes a Large Shift in the General Base pKa.

The bacterial toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted pKa. However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed pKa of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base pKa downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion.

Gene regulation by a glycine riboswitch singlet uses a finely tuned energetic landscape for helical switching.

Riboswitches contain structured aptamer domains that, upon ligand binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. Here, we report a sequencing-based high-throughput assay for monitoring in vitro transcription termination and use it to simultaneously characterize the functional effects of all 522 single point mutants of a glycine riboswitch type-1 singlet. Mutations throughout the riboswitch cause ligand-dependent defects, but only mutations within the terminator hairpin alter readthrough efficiencies in the absence of ligand. A comprehensive analysis of the expression platform reveals that ligand binding stabilizes the antiterminator by just 2-3 kcal/mol, indicating that the competing expression platform helices must be extremely close in energy to elicit a significant ligand-dependent response. These results demonstrate that gene regulation by this riboswitch is highly constrained by the energetics of ligand binding and conformational switching. These findings exemplify the energetic parameters of RNA conformational rearrangements driven by binding events.

Noninvasive detection of graft injury after heart transplant using donor-derived cell-free DNA: A prospective multicenter study.

Standardized donor-derived cell-free DNA (dd-cfDNA) testing has been introduced into clinical use to monitor kidney transplant recipients for rejection. This report describes the performance of this dd-cfDNA assay to detect allograft rejection in samples from heart transplant (HT) recipients undergoing surveillance monitoring across the United States. Venous blood was longitudinally sampled from 740 HT recipients from 26 centers and in a single-center cohort of 33 patients at high risk for antibody-mediated rejection (AMR). Plasma dd-cfDNA was quantified by using targeted amplification and sequencing of a single nucleotide polymorphism panel. The dd-cfDNA levels were correlated to paired events of biopsy-based diagnosis of rejection. The median dd-cfDNA was 0.07% in reference HT recipients (2164 samples) and 0.17% in samples classified as acute rejection (35 samples; P = .005). At a 0.2% threshold, dd-cfDNA had a 44% sensitivity to detect rejection and a 97% negative predictive value. In the cohort at risk for AMR (11 samples), dd-cfDNA levels were elevated 3-fold in AMR compared with patients without AMR (99 samples, P = .004). The standardized dd-cfDNA test identified acute rejection in samples from a broad population of HT recipients. The reported test performance characteristics will guide the next stage of clinical utility studies of the dd-cfDNA assay.

Consequences of Increasing Complexity in Anorectal Surgery Performed at an Academic Center.

BACKGROUND: Anorectal surgery encompasses a wide range of procedures with varying complexity. The Accreditation Council for Graduate Medical Education Review Committee for Colon and Rectal Surgery recommends minimum case numbers (60) for 1-year specialty trainees in 6 categories of anorectal surgery, with definitions for procedural complexity. OBJECTIVE: The purpose of this study was to assess the scope of anorectal procedures and propose a stratification of procedures based on a consensus of levels of difficulty, as well as to identify a predictive charge cutoff suggestive of procedural complexity. DESIGN: This was a retrospective review. SETTINGS: The study was conducted at a tertiary academic center. PATIENTS: Patients undergoing anorectal procedures between January 2011 and December 2014 identified by Current Procedural Terminology coding were entered into 6 categories. Codes were stratified as routine or complex based on an assessment of perioperative care and technical expertise required. Patients with an abdominal portion to any procedure were excluded. MAIN OUTCOMES MEASURES: The study measured distribution of complexity in anorectal surgical procedures and procedural charge associated with differentiating routine from complex procedures. RESULTS: Seven colorectal surgeons performed 2483 anorectal procedures (mean = 620 per year). Mean age was 48 ± 16 years. Forty six (64%) of 71 procedures were classified as routine and 25 (36%) of 71 as complex. Most disease processes had subsets with routine or complex procedures, whereas all of the procedures performed for fecal incontinence or advanced anorectal techniques were considered complex. Fistula procedures and transanal excisions were most heterogeneous, with a high procedural complexity rate (37% and 50%). After a procedural complexity rating, intraclass correlation by 6 surgeons was 0.70, demonstrating good correlation. Receiver operating curve assessments of consensus categorization according to billing codes revealed $553 as the optimal cutoff between routine and complex procedures. LIMITATIONS: This was a single-institution retrospective review. CONCLUSIONS: Colorectal residents may benefit from anorectal case stratification, because it serves as a dialogue for those interested in complex anorectal surgery during training. Surgeon categorization of procedures correlates well with a charge-based model of complexity. See Video Abstract at http://links.lww.com/DCR/A806.

Mycofumigation through production of the volatile DNA-methylating agent N-methyl-N-nitrosoisobutyramide by fungi in the genus Muscodor.

Antagonistic microorganisms produce antimicrobials to inhibit the growth of competitors. Although water-soluble antimicrobials are limited to proximal interactions via aqueous diffusion, volatile antimicrobials are able to act at a distance and diffuse through heterogeneous environments. Here, we identify the mechanism of action of Muscodor albus, an endophytic fungus known for its volatile antimicrobial activity toward a wide range of human and plant pathogens and its potential use in mycofumigation. Proposed uses of the Muscodor species include protecting crops, produce, and building materials from undesired fungal or bacterial growth. By analyzing a collection of Muscodor isolates with varying toxicity, we demonstrate that the volatile mycotoxin, N-methyl-N-nitrosoisobutyramide, is the dominant factor in Muscodor toxicity and acts primarily through DNA methylation. Additionally, Muscodor isolates exhibit higher resistance to DNA methylation compared with other fungi. This work contributes to the evaluation of Muscodor isolates as potential mycofumigants, provides insight into chemical strategies that organisms use to manipulate their environment, and provokes questions regarding the mechanisms of resistance used to tolerate constitutive, long-term exposure to DNA methylation.

Cell-Free DNA and Active Rejection in Kidney Allografts.

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

A two-step chemical mechanism for ribosome-catalysed peptide bond formation.

The chemical step of natural protein synthesis, peptide bond formation, is catalysed by the large subunit of the ribosome. Crystal structures have shown that the active site for peptide bond formation is composed entirely of RNA. Recent work has focused on how an RNA active site is able to catalyse this fundamental biological reaction at a suitable rate for protein synthesis. On the basis of the absence of important ribosomal functional groups, lack of a dependence on pH, and the dominant contribution of entropy to catalysis, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2'-hydroxyl of the peptidyl-transfer RNA and a Brønsted coefficient near zero have been taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation, based on analysis of the kinetic isotope effect at five positions within the reaction centre of a peptidyl-transfer RNA mimic. Our results indicate that in contrast to the uncatalysed reaction, formation of the tetrahedral intermediate and proton transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike in previous proposals, the reaction is not fully concerted; instead, breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression.

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFß/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Depressive symptoms in older long-term colorectal cancer survivors: a population-based analysis using the SEER-Medicare healthcare outcomes survey.

PURPOSE: Colorectal cancer survivorship has improved significantly over the last 20 years; however, few studies have evaluated depression among older colorectal cancer survivors, especially using a population-based sample. The aim of this study was to identify correlates for positive depression screen among colorectal cancer survivors who underwent potentially curative surgery. METHODS: Using the 1998-2007 Surveillance, Epidemiology, and End-Result registry and the Medicare Health Outcome Survey linked dataset, we identified patients over 65 with pathology confirmed and resected colorectal cancer enrolled in Medicare. Using univariate and multiple variable analyses, we identified characteristics of patients with and without positive depression screen. RESULTS: Resected colorectal cancer patients (1785) (median age 77, 50.8 % female) were identified in the dataset with 278 (15.6 %) screening positive for symptoms of depression. Median time from diagnosis to survey was 62 months. On univariate analysis, larger tumor size, advanced cancer stage, and extent of resection were not correlates of depressive symptoms (all p > 0.05). After adjusting for confounders, income less than US$30,000 per year (OR 1.50, 1.02-2.22, 95 % CI, p = 0.042), non-white race (OR 1.51, 1.05-2.17, 95 % CI, p = 0.027), two or more comorbidities (OR 1.78, 1.25-2.52, 95 % CI, p = 0.001), and impairment in activities of daily living (OR 5.28, 3.67-7.60, 95 % CI, p < 0.001) were identified as independent correlates of depressive symptoms in colorectal cancer survivors. CONCLUSIONS: In the current study, socioeconomic status and features of physical health rather than tumor characteristics were associated with symptoms of depression among long-term colorectal cancer survivors.

Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE.

The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active site is atypical for acid-base catalysis, in that it is enriched with positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study, we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly dependent on pH, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pK(a) of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pK(a) back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pK(a) shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites.

Performance of gene-expression profiling test score variability to predict future clinical events in heart transplant recipients.

BACKGROUND: A single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0-40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events. METHODS: We defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group (death, re-transplantation or graft failure ≥315 days post-transplantation and within 3 years of the final GEP test) and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated. RESULTS: The estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8). CONCLUSION: In heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00761787.

Novel High-Frequency Peripheral Nerve Stimulator Treatment of Refractory Postherpetic Neuralgia: A Brief Technical Note.

OBJECTIVES: The study aims to describe an ultrasound (US)-guided peripheral nerve stimulation implant technique and describe the effect of high-frequency peripheral nerve stimulation on refractory postherpetic neuralgia. MATERIALS AND METHODS: Following a cadaver pilot trial using US and confirmatory fluoroscopic guidance, a 52-year-old man with refractory left supraorbital neuralgia underwent combined US and fluoroscopic-guided supraorbital peripheral nerve stimulator trial. The patient was subsequently implanted with a percutaneous lead over the left supraorbital and supratrochlear nerve utilizing a high-frequency stimulation paradigm. RESULTS: At 9 months follow-up, the pain intensity had declined from a weekly average of 8/10 to 1/10 on the pain visual analog scale (VAS). After implant, both nerve conduction and blink reflex studies were performed, which demonstrated herpetic nerve damage and frequency-specific peripheral nerve stimulation effects. The patient preferred analgesia in the supraorbital nerve distribution accomplished with high-frequency paresthesia-free stimulation (HFS) at an amplitude of 6.2 mA, a frequency of 100-1200 Hz, and a pulse width of 130 µsec, to paresthesia-mediated pain relief associated with low-frequency stimulation. CONCLUSION: We report the implant of a supraorbital peripheral nerve stimulating electrode that utilizes a high-frequency program resulting in sustained suppression of intractable postherpetic neuralgia.

p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation.

Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.

Epinephrine induces rapid deterioration in pulmonary oxygen exchange in intact, anesthetized rats: a flow and pulmonary capillary pressure-dependent phenomenon.

BACKGROUND: Previous studies indicate epinephrine adversely affects arterial oxygenation when administered in a rat model of local anesthetic overdose. The authors tested whether epinephrine alone exerts similar effects in the intact animal. METHODS: Anesthetized rats received a single intravenous injection of epinephrine (25, 50, or 100 mcg/kg); matched cohorts were pretreated with phentolamine (100 mcg/kg); n = 5 for each of the six treatment groups. Arterial pressure and blood gases were measured at baseline, 1 and 10 min after epinephrine administration. Pulmonary capillary pressures during epinephrine infusion with normal and increased flows were measured in an isolated lung preparation. RESULTS: Epinephrine injection in the intact animal caused hypoxemia, hypercapnia, and acidosis at all doses. Arterial oxygen tension was reduced within 1 min of injection. Hyperlactatemia occurred by 10 min after 50 and 100 mcg/kg. Rate pressure product was decreased by 10 min after 100 mcg/kg epinephrine. Pretreatment with phentolamine attenuated these effects except at 100 mcg/kg epinephrine. In the isolated lung preparation, epinephrine in combination with increased pulmonary flow increased pulmonary capillary pressure and lung water. CONCLUSIONS: Bolus injection of epinephrine in the intact, anesthetized rat impairs pulmonary oxygen exchange within 1 min of treatment. Effects were blunted by α-adrenergic receptor blockade. Edema occurred in the isolated lung above a threshold pulmonary capillary pressure when epinephrine treatment was coupled with an increase in pulmonary flow. These results potentially argue against using traditional doses of epinephrine for resuscitation, particularly in the anesthetized patient.

How Do Bacteria "See" Molecules Inside Themselves?

RNA, like its close cousin DNA, is used to store information in the cell. Unlike DNA, it is really good at folding up into interesting shapes, which makes it good at lots of other important jobs. Some kinds of RNA, called riboswitches, can sense what is going on inside a cell. Each riboswitch fits a specific small molecule. When the riboswitch and small molecule interact it changes what the cell does. For example, if the small molecule is harmful the cell might start making a protein that will get rid of it. Recently, scientists discovered some riboswitches that look very similar to each other but recognize very different small molecules. We used X-ray crystallography to get pictures of these riboswitches. We saw how changing just one piece of the riboswitch changed which small molecule it recognized. This shows us how RNA can gain new functions as an organism evolves.