Detection of occult atrial fibrillation by pacemaker interrogation in cryptogenic stroke

Purpose

Atrial fibrillation (AF) is a major cause of ischemic strokes, and it is assumed that occult intermittent episodes of AF are responsible for some of the seemingly cryptogenic strokes. Cardiac pacemakers feature rhythm diagnostic capabilities and data storage. We investigated whether pacemaker memory interrogation led to identification of undetected AF episodes prior to cryptogenic strokes.

Methods

The study enrolled all patients admitted between June 2010 and July 2013 for an acute cryptogenic stroke and who were implanted with a permanent pacemaker. Patients with a history of AF and a history of stroke with an identifiable origin were excluded. Pacemaker memories were interrogated to determine the presence of AF prior to the stroke and its temporal relationship with the stroke.

Results

Fourteen patients (nine men and five women) with a median (interquartile range) age of 84.5 (82.25–87.5) years were included. Median CHADS₂ and CHA₂DS₂-VASc scores were 2 (1–2.75) and 3.5 (3–4), respectively. Pacemaker memories were activated in 13 patients with atrial arrhythmia detection based on an atrial cutoff rate in 8 patients and on the detection of atrial rate acceleration in 5 patients. Electrograms were available for review in 10 patients. Unknown AF or atrial flutter was diagnosed previous to the stroke in six (43 %) patients. Four patients experienced more than one arrhythmia episode. The last episode occurred in the 48 h prior to stroke in three patients and in the previous 4 weeks in five patients. Anticoagulation was started after the stroke in all of these six patients.

Conclusions

Pacemaker interrogation has a high diagnostic yield in seemingly cryptogenic stroke, with frequent detection of occult AF. The causal link between AF and stroke is convincingly reinforced by their close temporal proximity, and anticoagulation is warranted in this clinical situation.     

Structural genomic variation in childhood epilepsies with complex phenotypes

A genetic contribution to a broad range of epilepsies has been postulated, and particularly copy number variations (CNVs) have emerged as significant genetic risk factors. However, the role of CNVs in patients with epilepsies with complex phenotypes is not known. Therefore, we investigated the role of CNVs in patients with unclassified epilepsies and complex phenotypes. A total of 222 patients from three European countries, including patients with structural lesions on magnetic resonance imaging (MRI), dysmorphic features, and multiple congenital anomalies, were clinically evaluated and screened for CNVs. MRI findings including acquired or developmental lesions and patient characteristics were subdivided and analyzed in subgroups. MRI data were available for 88.3% of patients, of whom 41.6% had abnormal MRI findings. Eighty-eight rare CNVs were discovered in 71 out of 222 patients (31.9%). Segregation of all identified variants could be assessed in 42 patients, 11 of which were de novo. The frequency of all structural variants and de novo variants was not statistically different between patients with or without MRI abnormalities or MRI subcategories. Patients with dysmorphic features were more likely to carry a rare CNV. Genome-wide screening methods for rare CNVs may provide clues for the genetic etiology in patients with a broader range of epilepsies than previously anticipated, including in patients with various brain anomalies detectable by MRI. Performing genome-wide screens for rare CNVs can be a valuable contribution to the routine diagnostic workup in patients with a broad range of childhood epilepsies.     

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.     

Angiotensin receptor blockade increases pancreatic insulin secretion and decreases glucose intolerance during glucose supplementation in a model of metabolic syndrome

Renin-angiotensin system blockade improves glucose intolerance and insulin resistance, which contribute to the development of metabolic syndrome. However, the contribution of impaired insulin secretion to the pathogenesis of metabolic syndrome is not well defined. To assess the contributions of angiotensin receptor type 1 (AT?) activation and high glucose intake on pancreatic function and their effects on insulin signaling in skeletal muscle and adipose tissue, an oral glucose tolerance test (oGTT) was performed in five groups (n = 10/group) of rats: 1) lean strain-control 2) obese Otsuka Long-Evans Tokushima Fatty (OLETF), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg/kg · d olmesartan for 6 wk; OLETF ARB), 4) OLETF + 5% glucose water (HG) for 6 wk (OLETF HG), and 5) OLETF + HG + ARB (OLETF HG/ARB). The glucose response to the oGTT increased 58% in OLETF compared with lean-strain control, whereas glucose supplementation increased it an additional 26%. Blockade of angiotensin receptor reduced the oGTT response 19% in the ARB-treated groups and increased pancreatic insulin secretion 64 and 113% in OLETF ARB and OLETF HG/ARB, respectively. ARB treatment in OLETF ARB and OLETF HG/ARB did not have an effect on insulin signaling proteins in skeletal muscle; however, it reduced pancreatic AT? protein expression 20 and 27%, increased pancreatic glucagon-like peptide-1 (GLP-1) receptor protein expression 41 and 88%, respectively, and increased fasting plasma GLP-1 approximately 2.5-fold in OLETF ARB. The results suggest that improvement of glucose intolerance is independent of an improvement in muscle insulin signaling, but rather by improved glucose-stimulated insulin secretion associated with decreased pancreatic AT? activation and increased GLP-1 signaling.     

Comparison of spatial and contrast resolution for cone-beam computed tomography scanners

OBJECTIVE: The purpose was to evaluate the perceived spatial and contrast resolution for a wide range of cone-beam computed tomography (CBCT) devices. STUDY DESIGN: A customized polymethyl methacrylate (PMMA) phantom was developed. Inserts containing a line-pair and rod pattern were used. The phantom was scanned with 13 CBCT devices and 1 multislice CT (MSCT) device using a variety of scanning protocols. The images were presented to 4 observers for scoring. RESULTS: The observer scores showed excellent agreement. A wide range was seen in image quality between CBCT exposure protocols. Compared with the average CBCT scores, the MSCT protocols scored lower for the line-pair insert but higher for the rod insert. CONCLUSIONS: CBCT devices are generally suitable for the visualization of high-contrast structures. Certain exposure protocols can be used for depicting low-contrast structures or fine details. The user should be able to select appropriate exposure protocols according to varying diagnostic requirements.     

A review of second primary malignancy in patients with relapsed or refractory multiple myeloma treated with lenalidomide

In a retrospective pooled analysis of 11 clinical trials of lenalidomide-based therapy for relapsed/refractory multiple myeloma (MM; N = 3846), the overall incidence rate (IR, events per 100 patient-years) of second primary malignancies (SPMs) was 3.62. IR of invasive (hematologic and solid tumor) SPMs was 2.08, consistent with the background incidence of developing cancer. In a separate analysis of pooled data from pivotal phase 3 trials of relapsed or refractory MM (N = 703), the overall IR of SPMs was 3.98 (95% confidence interval [CI], 2.51-6.31) with lenalidomide/dexamethasone and 1.38 (95% CI, 0.44-4.27) with placebo/dexamethasone; IRs of nonmelanoma skin cancers were 2.40 (95% CI, 1.33-4.33) and 0.91 (95% CI, 0.23-3.66), respectively; IRs of invasive SPMs were 1.71 (95% CI, 0.86-3.43) and 0.91 (95% CI, 0.23-3.66), respectively. The risk of SPMs must be taken into account before initiating lenalidomide treatment. In the context of the observed survival benefit in relapsed or refractory MM patients, the benefit/risk profile of lenalidomide/dexamethasone remains positive.