Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing thiC Riboswitch in Escherichia coli.

Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.

Monitoring RNA dynamics in native transcriptional complexes.

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition.

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Consensus statement on the treatment of massive irreparable rotator cuff tears: a Delphi approach by the Neer Circle of the American Shoulder and Elbow Surgeons.

BACKGROUND: Management of massive irreparable rotator cuff tears (MIRCTs) remains controversial owing to variability in patient features and outcomes contributing to a lack of unanimity in treatment recommendations. The purpose of this study was to implement the Delphi process using experts from the Neer Circle of the American Shoulder and Elbow Surgeons to determine areas of consensus regarding treatment options for a variety of MIRCTs. METHODS: A panel of 120 shoulder surgeons were sent a survey regarding MIRCT treatments including arthroscopic débridement and partial cuff repair, graft augmentation, reverse shoulder arthroplasty (RSA), superior capsular reconstruction (SCR), and tendon transfer. An iterative Delphi process was then conducted with a first-round questionnaire consisting of 13 patient factors with the option for open-ended responses to identify important features influencing the treatment of MIRCTs. The second-round survey sought to determine the importance of patient factors related to the 6 included treatment options. A third-round survey asked participants to classify treatment options for 60 MIRCT patient scenarios as either preferred treatment, acceptable treatment, not acceptable/contraindicated, or unsure/no opinion. Patient scenarios were declared to achieve consensus for the preferred and not acceptable/contraindicated categories when at least 80% of the survey respondents agreed on a response, and a 90% threshold was required for the acceptable treatment category, defined by an acceptable treatment or preferred treatment response. RESULTS: Seventy-two members agreed to participate and were deemed to have the requisite expertise to contribute based on their survey responses regarding clinical practice and patient volume. There were 20 clinical scenarios that reached 90% consensus as an acceptable treatment, with RSA selected for 18 scenarios and arthroscopic débridement and/or partial repair selected for 2. RSA was selected as the singular preferred treatment option in 8 scenarios. Not acceptable/contraindicated treatment options reached consensus in 8 scenarios, of which, 4 related to SCR, 3 related to RSA, and 1 related to partial repair with graft augmentation. CONCLUSION: This Delphi process exhibited significant consensus regarding RSA as a preferred treatment strategy in older patients with pseudoparesis, an irreparable subscapularis, and dynamic instability. In addition, the process identified certain unacceptable treatments for MIRCTs such as SCR in older patients with pseudoparesis and an irreparable subscapularis or RSA in young patients with an intact or reparable subscapularis without pseudoparesis or dynamic instability. The publication of these scenarios and areas of consensus may serve as a useful guide for practitioners in the management of MIRCTs.

A prospective study comparing tendon-to-bone interface healing using an interposition bioresorbable scaffold with a vented anchor for primary rotator cuff repair in sheep.

BACKGROUND: The purpose of this study was to evaluate the biomechanical and histologic properties of rotator cuff repairs using a vented anchor attached to a bioresorbable interpositional scaffold composed of aligned PLGA (poly(l-lactide-co-glycoside)) microfibers in an animal model compared to standard anchors in an ovine model. METHODS: Fifty-six (n = 56) skeletally mature sheep were randomly assigned to a repair of an acute infraspinatus tendon detachment using a innovative anchor-PLGA scaffold device (Treatment) or a similar anchor without the scaffold (Control). Animals were humanely euthanized at 7 and 12 weeks post repair. Histologic and biomechanical properties of the repairs were evaluated and compared. RESULTS: The Treatment group had a significantly higher fibroblast count at 7 weeks compared to the Control group. The tendon bone repair distance, percentage perpendicular fibers, new bone formation at the tendon-bone interface, and collagen type III deposition was significantly greater for the Treatment group compared with the Control group at 12 weeks (P ≤ .05). A positive correlation was identified in the Treatment group between increased failure loads at 12 weeks and the following parameters: tendon-bone integration, new bone formation, and collagen type III. No statistically significant differences in biomechanical properties were identified between Treatment and Control Groups (P > .05). CONCLUSIONS: Use of a vented anchor attached to a bioresorbable interpositional scaffold composed of aligned PLGA microfibers improves the histologic properties of rotator cuff repairs in a sheep model. Improved histology was correlated with improved final construct strength at the 12-week time point.

Decision making in treatment after a first-time anterior glenohumeral dislocation: A Delphi approach by the Neer Circle of the American Shoulder and Elbow Surgeons.

BACKGROUND: The treatment of patients who sustain a first-time anterior glenohumeral dislocation (FTAGD) is controversial. The purpose of this study was to find consensus among experts using a validated iterative process in the treatment of patients after an FTAGD. METHODS: The Neer Circle is an organization of shoulder experts recognized for their service to the American Shoulder and Elbow Surgeons. Consensus among 72 identified experts from this group was sought with a series of surveys using the Delphi process. The first survey used open-ended questions designed to identify patient-related features that influence treatment decisions after an FTAGD. The second survey used a Likert scale to rank each feature's impact on treatment decisions. The third survey used highly impactful features to construct 162 clinical scenarios. For each scenario, experts recommended surgery or not and reported how strongly they made their recommendation. These data were analyzed to find clinical scenarios that had >90% consensus for recommending treatment. These data were also used in univariate and multivariate mixed-effects models to identify odds ratios (ORs) for different features and to assess how combining these features influenced the probability of surgery for specific populations. RESULTS: Of the 162 scenarios, 8 (5%) achieved >90% consensus for recommending surgery. All of these scenarios treated athletes with meaningful bone loss at the end of their season. In particular, for contact athletes aged > 14 years who were at the end of the season and had apprehension and meaningful bone loss, there was >90% consensus for recommending surgery after an FTAGD, with surgeons feeling very strongly about this recommendation. Of the scenarios, 22 (14%) reached >90% consensus for recommending nonoperative treatment. All of these scenarios lacked meaningful bone loss. In particular, surgeons felt very strongly about recommending nonoperative treatment after an FTAGD for non-athletes lacking apprehension without meaningful bone loss. The presence of meaningful bone loss (OR, 6.85; 95% confidence interval, 6.24-7.52) and apprehension (OR, 5.60; 95% confidence interval, 5.03-6.25) were the strongest predictors of surgery. When these 2 features were combined, profound effects increasing the probability of surgery for different populations (active-duty military, non-athletes, noncontact athletes, and contact athletes) were noted, particularly non-athletes. CONCLUSION: Consensus for recommending treatment of the FTAGD patient was not easily achieved. Certain combinations of patient-specific factors, such as the presence of meaningful bone loss and apprehension, increased the probability of surgery after an FTAGD in all populations. Over 90% of shoulder instability experts recommend surgery after an FTAGD for contact athletes aged > 14 years at the end of the season with both apprehension and meaningful bone loss. Over 90% of experts would not perform surgery after a first dislocation in patients who are not athletes and who lack apprehension without meaningful bone loss.

Dexmedetomidine Versus Remifentanil for Monitored Anesthesia Care During Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration: A Randomized Controlled Trial.

BACKGROUND: We hypothesized that, compared to remifentanil, dexmedetomidine used for endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) performed under monitored anesthesia care (MAC) in nonintubated patients would result in fewer episodes of major respiratory adverse events (number of episodes of bradypnea, apnea or desaturation) but no difference in satisfaction with perioperative conditions. METHODS: Sixty (American Society of Anesthesiologists physical status I-III) patients scheduled to undergo EBUS-TBNA under MAC were randomized to receive either remifentanil (0.5 µg/kg IV bolus) in 10 minutes, followed by 0.05-0.25 µg/kg/min, or dexmedetomidine (0.4 µg/kg IV bolus) in 10 minutes, followed by 0.5-1.0 µg/kg/h. The primary outcome was the number of major respiratory adverse events (bradypnea, apnea, or hypoxia). The secondary outcomes included hemodynamic variables, discharge time from the postanesthesia care unit, endotracheal lidocaine use, patient's sedation using the Observer Assessment of Alertness/Sedation Scale, operative conditions, operator and patient satisfaction, pain, coughing, vocal cord mobility, recall, and nausea/vomiting. RESULTS: Dexmedetomidine produced significantly fewer episodes of major respiratory events (bradypnea, apnea, or desaturation), with 0 [0-0.5] episodes versus 2 [0-5] (median [interquartile range]) (P = .001), than did remifentanil. Fewer episodes of bradypnea or apnea (dexmedetomidine: 0 [0-0] versus remifentanil: 0 [0-0.5]; P = .031), and fewer episodes of desaturation (dexmedetomidine: 0 [0-0.5] versus remifentanil: 1 [0-4]; P = .039) were recorded in the dexmedetomidine group. The time needed for patients to meet postanesthesia care unit discharge criteria (Aldrete score: 9) after EBUS-TBNA was longer in the dexmedetomidine group (10 [3-37.5] minutes) versus the remifentanil group (3 [3-5] minutes) (P < .001). No differences were observed in the 2 groups for sedation depth (Observer Assessment of Alertness/Sedation Scale), endotracheal lidocaine use, operative conditions, operator and patient satisfaction, pain, coughing, vocal cord mobility, recall, and nausea/vomiting episodes. CONCLUSIONS: Dexmedetomidine resulted in fewer respiratory adverse events during EBUS-TBNA under MAC, when compared to remifentanil, with no difference in overall operative conditions. However, dexmedetomidine use was associated with delayed postoperative discharge.

Editorial Commentary: Proficiency-Based Training: Are We Ready for a New Way to Train and Test Orthopaedic Surgeons?

Orthopaedic training has followed an apprentice/mentorship pathway of training throughout its history. This method of training is entrenched in Medicine and is the foundation for our current residency/fellowship method of attaining the appropriate training and credentialing of physicians and surgeons to safely care for our patients. Over the past 2 decades, the use of proficiency-based training has been shown to be effective in general surgery, cardiac surgery, vascular and plastic surgery, and now with the Copernicus project, in shoulder arthroscopy. Combining proficiency-based training with the rapid advances in virtual reality technology will allow surgeons to become competent with the surgical skills needed for surgery before ever touching a patient.

Low dose intravenous dexamethasone (4 mg and 10 mg) significantly prolongs the analgesic duration of single-shot interscalene block after arthroscopic shoulder surgery: a prospective randomized placebo-controlled study.

BACKGROUND: Although intravenous dexamethasone prolongs the analgesic duration of interscalene brachial plexus block, it is uncertain whether this effect can be observed using lower doses of dexamethasone. This study evaluated the impact of intravenous dexamethasone (4 mg and 10 mg) on the analgesic duration of single-shot interscalene block after arthroscopic shoulder surgery. We hypothesized that both doses would prolong the analgesic duration compared with placebo. METHODS: This was a prospective double-blind randomized placebo-controlled study in patients undergoing elective arthroscopic shoulder surgery under regional anesthesia with a single-shot interscalene block (0.5% ropivacaine 20 mL). Patients received dexamethasone 4 mg (D4), dexamethasone 10 mg (D10), or a placebo (normal saline [NS]) intravenously at the time of block completion. The primary outcome was the duration of analgesia, defined as the time from the onset of sensory blockade to the first analgesic request. The primary outcome was first analyzed with a Kruskal-Wallis test and then with a Mann-Whitney test for pairwise between-group comparison. RESULTS: Sixty-nine patients completed the study. The median [interquartile range] duration of analgesia was significantly different between the three groups (D4, 19.7 [16.9-23.3] hr; D10, 19.1 [11.5-22.8] hr; and NS, 11.8 [9.3-14.0] hr; P = 0.001). This difference was statistically significant for D4 and D10 compared with placebo (median difference [MD], 7.8 hr; 95% confidence interval [CI], 4.6 to 11.1 hr; P < 0.001; and MD, 7.4 hr; 95% CI, 4.2 to 10.5 hr; P = 0.001, respectively) but not for D4 compared with D10 (MD, 0.5 hr; 95% CI, -2.8 to 3.7 hr; P = 0.38). CONCLUSIONS: Low doses of intravenous dexamethasone (4 mg and 10 mg) significantly prolong the analgesic duration of interscalene block. This trial was registered at ClinicalTrials.gov (NCT02412657).

Fluorescence tools to investigate riboswitch structural dynamics.

Riboswitches are novel regulatory elements that respond to cellular metabolites to control gene expression. They are constituted of highly conserved domains that have evolved to recognize specific metabolites. Such domains, so-called aptamers, are folded into intricate structures to enable metabolite recognition. Over the years, the development of ensemble and single-molecule fluorescence techniques has allowed to probe most of the mechanistic aspects of aptamer folding and ligand binding. In this review, we summarize the current fluorescence toolkit available to study riboswitch structural dynamics. We fist describe those methods based on fluorescent nucleotide analogues, mostly 2-aminopurine (2AP), to investigate short-range conformational changes, including some key steady-state and time-resolved examples that exemplify the versatility of fluorescent analogues as structural probes. The study of long-range structural changes by Förster resonance energy transfer (FRET) is mostly discussed in the context of single-molecule studies, including some recent developments based on the combination of single-molecule FRET techniques with controlled chemical denaturation methods. This article is part of a Special Issue entitled: Riboswitches.

Single-molecule chemical denaturation of riboswitches.

To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg(2+) ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods.

Randomized prospective trial comparing two supraglottic airway devices: i-gel™ and LMA-Supreme™ in paralyzed patients.

PURPOSE: Many features can influence the choice of a supraglottic airway device (SAD), including ease of insertion, adequate ventilation pressures and lack of adverse effects. The goal of this randomized prospective trial was to compare the performance of the i-gel™ with that of the LMA-Supreme™. METHODS: One hundred adult patients (American Society of Anesthesiologists I-III) scheduled to undergo elective surgery under general anesthesia were randomized to either an i-gel (n = 50) or an LMA-Supreme (n = 50). The primary objective was to compare ventilation pressures. Secondary objectives included time and number of attempts needed to introduce the device, adverse effects, and repositioning. The endoscopic view of the glottic aperture and the position of the drain tubes in relation to the esophagus were also evaluated. RESULTS: The devices were inserted successfully in 46 (92%) patients in both groups. There was no significant difference in the [mean (SD)] leak pressure [i-gel: 23 (7) cm H2O vs LMA-Supreme: 21 (8) cm H2O; P = 0.14] or peak inspiratory pressure between both devices. Insertion time was shorter with the i-gel than with the LMA-Supreme [19 (7) sec vs 27 (17) sec, respectively; P = 0.003]. The vocal cords were completely visualized more often through the i-gel (70%) than through the LMA-Supreme (50%) (P = 0.007). Esophageal mucosa was easily visualized through the drain port in all but four patients, two patients in each group. There was no difference between groups regarding preoperative or postoperative complications. Postoperative patient discomfort was generally mild and comparable between both devices. CONCLUSION: Both the LMA-Supreme and the i-gel offer similar performance for positive pressure ventilation in paralyzed patients during general anesthesia. The i-gel was associated with a slightly faster insertion time and better fibrescopic visualization of the glottis. This trial was registered at Clinicaltrials.gov: NCT01001078.

Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms.

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

When is a Reverse Shoulder Arthroplasty Indicated for a Rotator Cuff Tear?

Rotator cuff tears are the most frequent shoulder injury in patients over 50 years old. Treatment of rotator cuff tears is individualized based on the age, demands, and symptoms of the patient. Nonoperative treatment may often be effective, especially in lower-demand or elderly patients. Surgical repair is indicated after failure of nonoperative treatment or in patients whose high-demand lifestyle requires full strength and function of the shoulder. In older patients, larger tears are less likely to heal and rotator cuff repair may not reliable. In cases of failed previous rotator cuff repair, there may not be enough viable tissue for complete repair. If there is underlying arthritis, a rotator cuff repair is unlikely to resolve all symptoms, and treatment to care for the arthritis and rotator cuff tear is indicated. Reverse shoulder arthroplasty provides the surgical answer for these scenarios and is now being used for numerous indications of shoulder pathology.

A double-blind randomized controlled trial comparing the effects of subacromial injection with corticosteroid versus NSAID in patients with shoulder impingement syndrome.

HYPOTHESIS: The objective of this study was to compare the efficacy of subacromial injection of triamcinolone compared to injection of ketorolac in the treatment of external shoulder impingement syndrome. METHODS: Thirty-two patients diagnosed with external shoulder impingement syndrome were included in this double-blinded randomized controlled clinical trial. Each patient was randomized into the steroid group or nonsteroidal anti-inflammatory drugs (NSAID) group. The steroid syringe contained 40 mg triamcinolone; and the NSAID syringe contained 60 mg ketorolac. Each patient was evaluated in terms of arc of motion, visual analog scale (VAS) for evaluating pain, and the UCLA (The University of California at Los Angeles) shoulder rating scale. RESULTS: At 1 month follow-up, both treatment arms resulted in increased range of motion and decreased pain. The steroid group decreased in active abduction while the NSAID group increased (steroid: 134°, NSAID: 151°, P = .03). The mean improvement in the UCLA shoulder rating scale at 4 weeks was 7.15 for the NSAID group and 2.13 for the steroid group (P = .03). Subgroup analysis of the UCLA scale demonstrated an increase in both forward flexion strength (P = .04) and patient satisfaction (P = .03) in the NSAID group. No significant difference could be seen in all other outcome measures. CONCLUSION: In this study, an injection of ketorolac resulted in greater improvements in the UCLA shoulder rating scale than an injection of triamcinolone at 4 weeks follow-up. While both triamcinolone and ketorolac are effective in the treatment of isolated subacromial impingement, ketorolac appears to have equivalent if not superior efficacy; all the while decreasing patient exposure to the potential side-effects of corticosteroids.

Folding of the SAM-I riboswitch: a tale with a twist.

Riboswitches are ligand-dependent RNA genetic regulators that control gene expression by altering their structures. The elucidation of riboswitch conformational changes before and after ligand recognition is crucial to understand how riboswitches can achieve high ligand binding affinity and discrimination against cellular analogs. The detailed characterization of riboswitch folding pathways suggest that they may use their intrinsic conformational dynamics to sample a large array of structures, some of which being nearly identical to ligand-bound molecules. Some of these structural conformers can be "captured" upon ligand binding, which is crucial for the outcome of gene regulation. Recent studies about the SAM-I riboswitch have revealed unexpected and previously unknown RNA folding mechanisms. For instance, the observed helical twist of the P1 stem upon ligand binding to the SAM-I aptamer adds a new element in the repertoire of RNA strategies for recognition of small metabolites. From an RNA folding perspective, these findings also strongly indicate that the SAM-I riboswitch could achieve ligand recognition by using an optimized combination of conformational capture and induced-fit approaches, a feature that may be shared by other RNA regulatory sequences.

In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection.

BACKGROUND: It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference machinery. RESULTS: The RNA isolated from an infected tree have been used to sequence in parallel viroid species and small non-coding RNA species. Specifically, PLMVd RNAs were amplified, cloned and sequenced according to a conventional approach, while small non-coding RNAs were determined by high-throughput sequencing. The first led to the typing of 18 novel PLMVd variants. The second provided a library of small RNAs including 880 000 sequences corresponding to PLMVd-derived siRNAs, which makes up 11.2% of the sequences of the infected library. These siRNAs contain mainly 21-22 nucleotide RNAs and are equivalently distributed between the plus and the minus polarities of the viroid. They cover the complete viroid genome, although the amount varies depending on the regions. These regions do not necessarily correlate with the double-stranded requirement to be a substrate for Dicer-like enzymes. We noted that some sequences encompass the hammerhead self-cleavage site, indicating that the circular conformers could be processed by the RNA-silencing machinery. Finally, a bias in the relative abundance of the nature of the 5' nucleotides was observed (A, U >> G, C). CONCLUSIONS: The approach used provided us a quantitative representation of the PLMVd-derived siRNAs retrieved from infected peach trees. These siRNAs account for a relatively large proportion of the small non-coding RNAs. Surprisingly, the siRNAs from some regions of the PLMVd genome appear over-represented, although these regions are not necessarily forming sufficiently long double-stranded structures to satisfy Dicer-like criteria for substrate specificity. Importantly, this large library of siRNAs gave several hints as to the components of the involved silencing machinery.

Risk factors associated with noncontact injury of the anterior cruciate ligament: a prospective four-year evaluation of 859 West Point cadets.

BACKGROUND: The causes of noncontact anterior cruciate ligament injury remain an enigma. PURPOSE: To prospectively evaluate risk factors for noncontact anterior cruciate ligament injuries in a large population of young athletic people. STUDY DESIGN: Prospective cohort study. METHODS: In 1995, 1198 new United States Military Academy cadets underwent detailed testing and many parameters were documented. During their 4-year tenure, all anterior cruciate ligament injuries that occurred were identified. Statistical analyses were used to identify the factors that may have predisposed the cadets to noncontact anterior cruciate ligament injuries. RESULTS: Among the 895 cadets who completed the entire 4-year study, there were 24 noncontact anterior cruciate ligament tears (16 in men, 8 in women). Significant risk factors included small femoral notch width, generalized joint laxity, and, in women, higher than normal body mass index and KT-2000 arthrometer values that were 1 standard deviation or more above the mean. The presence of more than one of these risk factors greatly increased the relative risk of injury. All female cadets who had some combination of risk factors sustained noncontact anterior cruciate ligament injuries, indicating that some combinations of factors are especially perilous to the female knee. CONCLUSION: Several risk factors may predispose young athletes to noncontact anterior cruciate ligament injury.