A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed −1 ribosomal frameshifting (−1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine–Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA^Lys in the ribosomal peptidyl-tRNA–binding (P) site. We observed significantly slower elongation factor G (EF-G)–catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as −1PRF.The ribosome is the molecular machine that synthesizes proteins by translating messenger RNAs (mRNAs); each sequence of 3 nt, 1 codon, characterizes 1 aa (1–3). Failure to maintain frame during translation occurs with a low error of 10⁻⁵ (4); however, frameshifting with high efficiency (>10⁻²) is often programmed into many mRNAs to express two or more proteins from a single mRNA (5, 6). Many RNA viruses, including HIV-1, use programmed frameshifting to produce their vital proteins at a precise ratio (7, 8). The common −1 programmed ribosomal frameshifting (−1PRF) signals are a heptanucleotide slippery sequence (X XXY YYZ, underlining denotes the zero-frame) and a downstream stimulatory secondary structure such as a stem loop or a pseudoknot. Frameshifting that takes place on the slippery sequence results in minimal base pair mismatches. Prokaryotic systems have an additional stimulatory signal, an upstream, internal Shine–Dalgarno (SD) sequence (9). The dnaX gene of Escherichia coli has the three −1PRF signals; an SD sequence, an A AAA AAG slippery sequence, and a downstream stem loop (9–12). Highly efficient (50–80%) −1PRF during translation of the mRNA results in production of the γ DNA-polymerase subunit in the −1 frame and the τ DNA-polymerase subunit in the 0 frame (10).The −1PRF signals are spaced so that the slippery sequence is positioned within the ribosomal peptidyl-tRNA–binding (P) site and aminoacyl-tRNA–binding (A) site, whereas the downstream secondary structure is positioned at the ribosomal mRNA entry channel (Fig. 1) (5–8, 13). The upstream SD sequence base pairs with 16S ribosomal RNA (rRNA) near the ribosomal tRNA exit (E) site (Fig. 1) (9). Both the SD sequence and the downstream secondary structure can cause pausing during translation (14–19). However, frameshifting efficiency is not strictly related to the pausing extent (15, 17), and it is not proportional to the thermodynamic or mechanical stabilities of the secondary structures (7, 20). Nonetheless, it does correlate with the thermodynamic stability of the first 3–4 bp of the downstream secondary structure (21), and with the conformational plasticity of this structure (7, 20). However, a mechanism by which the stimulatory secondary structure promotes efficient frameshifitng has not emerged yet.Open in a separate windowFig. 1.A programmed −1 FSmRNA construct and a schematic drawing of a ribosomal complex translating the slippery sequence. FSmRNA contains three −1PRF signals from the dnaX gene in E. coli; an SD sequence, a slippery sequence, and a downstream stem loop. ΔSL mRNA has the same sequence as FSmRNA except with the stem loop (red box) deleted. Start and stop codons are highlighted in blue. Corresponding polypeptide sequences are shown below the mRNA. A schematic drawing of the POST-(Cy5)K₁ complex shows the 50S and 30S subunits in blue and purple rectangles, respectively. The L1 stalk in the small blue rectangle is labeled with Cy3. The ribosomal complex contains fMVK-(Cy5)tRNA^Lys in the P site, where the slippery sequence is being displayed. The upstream SD sequence forms base pairs with 16S rRNA and the downstream stem loop presents at the mRNA entry channel in the 30S subunit. The orange oval denotes the biotin on a DNA primer annealed to the 5′ end of the mRNA for immobilization.A translational elongation cycle starts with selecting a correct aminoacyl-tRNA in the A site via conformational changes of the posttranslocation (POST) ribosomal complex that are triggered upon binding an EF-Tu(GTP)⋅aminoacyl-tRNA ternary complex (TC) (1). Once peptidyl transfer takes place, the resulting pretranslocation (PRE) ribosomal complex undergoes large-scale conformational changes that facilitate translocation of the tRNAs from the P and A sites into the E and P sites, simultaneously advancing the ribosome along the mRNA by 3 nt (22). In the first step of translocation, the acceptor stems of the tRNAs are repositioned within the large ribosomal (50S, in prokaryotes) subunit to move the tRNAs from their classical (P/P, A/A) state to their hybrid (P/E, A/P) states, where X and Y in the X/Y notation refer to the position of the anticodon stem loop (ASL) of the tRNA in the small ribosomal (30S, in prokaryotes) subunit and the position of the acceptor stem of the tRNA in the 50S subunit, respectively. Hybrid state (H) formation is accompanied by rotation of the 30S subunit relative to the 50S subunit (23, 24) and a closure of the L1 stalk of the 50S subunit such that it forms a direct contact with the P/E hybrid tRNA (23–25), a global conformation of the PRE complex that we refer to as “global state 2” (25). Global state 1, in contrast, contains classical state (C) tRNAs, nonrotated subunits, and an open L1 stalk (25). Single-molecule fluorescence resonance energy transfer (smFRET) studies of this step of translocation have shown that the H state forms spontaneously upon peptidyl transfer and that, in the absence of an elongation factor-G (EF-G), the H state exists in a dynamic equilibrium with the C state (25–27). Translocation is completed by movement of the ASLs of the tRNAs and the mRNA in the 30S subunit. This step, which comprises the rate-limiting step for the overall process of translocation, requires unlocking of the PRE complex, a conformational change that is thought to involve swiveling of the head domain of the 30S subunit (28, 29) and that is catalyzed by EF-G (30). smFRET and structural studies suggest that the L1 stalk–P/E hybrid tRNA interaction that is established during the first step of translocation is preserved throughout the second step of translocation and is essential for guiding the translocation of the P/E hybrid tRNA into the E site (25, 31, 32).Here, we report an smFRET study of the dynamics of ribosomal complexes programmed with the −1PRF mRNA of the E. coli dnaX gene. We used a FRET pair composed of a Cy3-labeled L1 stalk [L1(Cy3)-stalk] and a Cy5-labeled P-site tRNA^Lys [(Cy5)tRNA^Lys] on the first lysine codon in the slippery sequence. As previously demonstrated (25), this FRET pair enabled us to monitor transitions of ribosomal complexes between C and H states and the subsequent release of the translocated (Cy5)tRNA^Lys from the E site, along one round of the translational elongation cycle. Two mRNA constructs, one containing the downstream stem loop and one lacking it, were compared to study the effect of the secondary structure on the dynamics and translocation of the ribosomal complexes. Our results show that the downstream stem loop changes the dynamics of the PRE ribosomal complexes and disturbs the translocation process. We propose that frameshifting is one of the favorable paths that the ribosome can adopt during the futile EF-G–driven translocation attempts from the H state.     

Exposure to herpes simplex virus,type 1 and reduced cognitive function

Herpes simplex virus, type 1 (HSV-1) causes cold sores, keratitis and rarely, fatal encephalitis. The infection is lifelong, with sensory ganglia serving as reservoirs of latent infection. Recently, exposure to HSV-1 has also been repeatedly associated with reduced cognitive function among healthy individuals without prior encephalitis. Though HSV-1 does not elevate risk for schizophrenia (SZ) per se, exposure is likewise associated with impaired cognitive functions among SZ patients. The range of cognitive changes observed in HSV-1 exposed persons has not been investigated systematically, nor is it known whether interaction between HSV-1 exposure and SZ related factors contributes to the impairment among SZ patients. Persons with or without schizophrenia/schizophreniform disorder (N = 298 total, DSM IV criteria) were assessed for HSV-1 exposure using serum HSV-1 antibody titers. The Penn Computerized Neurocognitive battery was used to assess eight cognitive domains with respect to accuracy and speed. There were no significant case–control differences in HSV-1 exposure. The SZ/schizophreniform disorder cases were significantly impaired in all cognitive domains compared with the controls. HSV-1 exposure was also associated with reduced cognitive function in the entire sample, but the magnitude of the effects and their patterns differed from the SZ related changes. Further, statistically significant interactions between HSV-1 exposure and SZ case status were not detected. HSV-1 exposure does not elevate risk for SZ, but it is associated with reduced function in specific cognitive domains regardless of SZ diagnostic status. An ‘epidiagnostic’ model for the association is proposed to explain the results.     

Comparison of the Heritability of Schizophrenia and Endophenotypes in the COGS-1 Family Study

Background:

Twin and multiplex family studies have established significant heritability for schizophrenia (SZ), often summarized as 81%. The Consortium on the Genetics of Schizophrenia (COGS-1) family study was designed to deconstruct the genetic architecture of SZ using neurocognitive and neurophysiological endophenotypes, for which heritability estimates ranged from 18% to 50% (mean = 30%). This study assessed the heritability of SZ in these families to determine whether there is a “heritability gap” between the diagnosis and related endophenotypes.

Methods:

Nuclear families (N = 296) with a SZ proband, an unaffected sibling, and both parents (n = 1366 subjects; mean family size = 4.6) underwent comprehensive endophenotype and clinical characterization. The Family Interview for Genetic Studies was administered to all participants and used to obtain convergent psychiatric symptom information for additional first-degree relatives of interviewed subjects (N = 3304 subjects; mean family size = 11.2). Heritability estimates of psychotic disorders were computed for both nuclear and extended families.

Results:

The heritability of SZ was 31% and 44% for nuclear and extended families. The inclusion of bipolar disorder increased the heritability to 37% for the nuclear families. When major depression was added, heritability estimates dropped to 34% and 20% for nuclear and extended families, respectively.

Conclusions:

Endophenotypes and psychotic disorders exhibit comparable levels of heritability in the COGS-1 family sample. The ascertainment of families with discordant sibpairs to increase endophenotypic contrast may underestimate diagnostic heritability relative to other studies. However, population-based studies also report significantly lower heritability estimates for SZ. Collectively, these findings support the importance of endophenotype-based strategies and the dimensional view of psychosis.Key words: schizophrenia, psychosis, endophenotypes, cognition, biomarkers, heritability     

Localized transmeningeal muscimol prevents neocortical seizures in rats and nonhuman primates: Therapeutic implications

Purpose:   To determine whether muscimol delivered epidurally or into the subarachnoid space can prevent and/or terminate acetylcholine (Ach)–induced focal neocortical seizures at concentrations not affecting behavior and background electroencephalography (EEG) activity.
Methods:   Rats (n = 12) and squirrel monkeys (n = 3) were chronically implanted with an epidural or subarachnoid drug delivery device, respectively, over the right frontal/parietal cortex, with adjacent EEG electrodes. Recordings were performed in behaving rats and chaired monkeys. Via the implants, either a control solution (artificial cerebrospinal fluid, ACSF) or muscimol (0.25–12.5 m m ) was delivered locally as a "pretreatment," followed by the similar delivery of a seizure-inducing concentration of Ach. In five additional rats, the quantities of food-pellets consumed during epidural ACSF and muscimol (2.5 m m ) exposures were measured. In a last group of four rats, muscimol (0.8–2.5 m m ) was delivered epidurally during the ongoing, Ach-induced EEG seizure.
Results:   In contrast to ACSF pretreatments, epidural muscimol pretreatment in rats completely prevented the seizures at and above 2.5 m m . In the monkeys, subarachnoid muscimol pretreatments at 2.5 m m completely prevented the focal-seizure–inducing effect of Ach, whereas similar deliveries of ACSF did not affect the seizures. Furthermore, 2.5 m m epidural muscimol left the eating behavior of rats intact and caused only slight changes in the EEG power spectra. Finally, muscimol delivery during Ach-induced EEG seizures terminated the seizure activity within 1–3 min.
Conclusions:   The results of this study suggest that muscimol is a viable candidate for the transmeningeal pharmacotherapy of intractable focal epilepsy.     

Permanent Deformation and Stiffness Degradation of Open Hole Glass/PA6 UD Thermoplastic Composite in Tension and Compression

UD glass/PA6 coupons with an open hole are subjected to tensile and compressive loading. Three layups: [0/90]_5s, [+45/−45]_5s and [+45/0/−45/90]_3s with a shape based on ASTM D5766 were tested. Both monotonic loading as well as loading–unloading–reloading tests were executed. The strain field on the sample surface was measured with digital image correlation. This allowed identifying the distribution of the strain field during loading, permanent deformation and the evolution of the sample elastic modulus. This information is not frequently measured. Yet, it is vital for the development and validation of advanced failure models. The results indicate that the thermoplastic matrix allows large plastic deformation under tensile loading for the specimens with layup [+45/−45]_5s. In addition, the specimen elastic modulus reduces by about 70%. The other layups show minor permanent deformation, while the elastic modulus reduces by up to 15%. Furthermore, the quasi-isotropic laminate shows a significant post-failure load-bearing capacity under compression loading. The results are complemented with post-mortem damage and fracture observations using optical microscopy and ultrasound inspection.     

Air pollution and heart failure: Relationship with the ejection fraction

AIM: To study whether the concentrations of particulate matter in ambient air are associated with hospitaladmission due to heart failure in patients with heart failure with preserved ejection fraction and reduced ejection fraction. METHODS: We studied 353 consecutive patients admitted into a tertiary care hospital with a diagnosis of heart failure. Patients with ejection fraction of ≥ 45% were classified as having heart failure with preserved ejection fraction and those with an ejection fraction of < 45% were classified as having heart failure with reduced ejection fraction. We determined the average concentrations of different sizes of particulate matter (< 10, < 2.5, and < 1 μm) and the concentrations of gaseous pollutants (carbon monoxide, sulphur dioxide, nitrogen dioxide and ozone) from 1 d up to 7 d prior to admission. RESULTS: The heart failure with preserved ejection fraction population was exposed to higher nitrogen dioxide concentrations compared to the heart failure with reduced ejection fraction population (12.95 ± 8.22 μg/m 3 vs 4.50 ± 2.34 μg/m 3 , P < 0.0001). Multivariate analysis showed that nitrogen dioxide was a significant predictor of heart failure with preserved ejection fraction (odds ratio ranging from (1.403, 95%CI: 1.003-2.007, P = 0.04) to (1.669, 95%CI: 1.043-2.671, P = 0.03). CONCLUSION: This study demonstrates that shortterm nitrogen dioxide exposure is independently associated with admission in the heart failure with preserved ejection fraction population.     

Treatment of pruritus of primary biliary cirrhosis with rifampin

Pruritus can be a debilitating symptom in patients with chronic cholestasis. Based on previous reports of its efficacy, we evaluated the impact of rifampin on the pruritus associated with primary biliary cirrhosis. Fourteen patients were included in a randomized, crossover study. After a 15-day washout period, subjects were followed for three weeks. During the first and third week, patients received 600 mg of rifampin or placebo; no treatment was administered during the second week. Pruritus was subjectively scored on a scale from 0 to 100. With rifampin, pruritus disappeared in 11 patients and partially improved in three; with placebo, only two had a partial response (P<0.001). Six patients with a prior poor or no response to cholestyramine improved with rifampin. No changes in biochemical tests or side effects were observed during this period. We conclude that short-term administration of rifampin relieves pruritus in primary biliary cirrhosis. When administered over a period of eight months in an open study, the relief of pruritus was maintained, while one individual developed an allergic reaction. Rifampin appears to be a safe drug in the management of the pruritus of primary biliary cirrhosis.     

Inhibition of poly(ADP-ribose) polymerase attenuates the severity of acute pancreatitis and associated lung injury

The severity of acute pancreatitis results from the transmigration and activation of leukocytes within the pancreas and the local synthesis and release of proinflammatory-soluble mediators that transform a local injury into a systemic inflammatory response. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether the genetic deletion of PARP-1 or PARP-2 (a new member of the PARP family) genes, or pharmacological inhibition of PARP activity might affect the development and severity of acute pancreatitis and pancreatitis-associated lung injury. Secretagogue-induced acute pancreatitis was achieved by 12 hourly intraperitoneal injections of cerulein in mice deficient in PARP-1 or PARP-2 genes, and wild-type (WT) littermate mice untreated or treated with PARP activity inhibitors. The severity of pancreatitis was assessed by measurements of serum amylase, lipase, interleukin-1beta and IL-6, pancreatic water content, histologic grading and pancreas myeloperoxidase (MPO) activity. Lung injury was evaluated by quantifying MPO activity and morphological changes. We found that the severity of acute pancreatitis and pancreatitis-associated lung injury was significantly attenuated in mice lacking PARP-1, but not PARP-2, compared with WT mice. Interestingly, administration of PARP inhibitors, 3-aminobenzamide or PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyacetamide HCl), in WT mice markedly decreased acute pancreatitis severity and pulmonary-associated injury in a larger extension than genetic deletion of PARP-1. Our results support the potential therapeutic application of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury.     

Role of neuropsychologists in the evaluation and management of sport-related concussion: an inter-organization position statement

Over the past 20 years, clinical neuropsychologists have been at the forefront of both scientific and clinical initiatives aimed at developing evidence-based approaches to the evaluation and management of sport-related concussion (SRC). These efforts have directly impacted current policy on strategies for injury assessment and return-to-play by athletes after concussion. Many states are considering legislation requiring (a) education of athletes, parents, coaches, and school/organization officials on the recognition, evaluation, and management of SRCs; (b) removal from play of any youth athlete that is suspected of having sustained a concussion; and (c) not allowing the student to return to participation until the student is evaluated and cleared for return to participation in writing by an appropriate healthcare professional. It is the official position of the American Academy of Clinical Neuropsychology (AACN), American Board of Professional Neuropsychology (ABN), Division 40 (Neuropsychology) of the American Psychological Association (APA), and the National Academy of Neuropsychology (NAN) that neuropsychologists should be included among the licensed healthcare professionals authorized to evaluate, clinically manage, and provide return to play clearance for athletes who sustain a SRC.