Ambiviricota, a novel ribovirian phylum for viruses with viroid-like properties.

Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.

A comparative survey of the influence of small self-cleaving ribozymes on gene expression in human cell culture.

Self-cleaving ribozymes are versatile tools for synthetic biologists when it comes to controlling gene expression. Up to date, 12 different classes are known, and over the past decades more and more details about their structure, cleavage mechanisms and natural environments have been uncovered. However, when these motifs are applied to mammalian gene expression constructs, the outcome can often be unexpected. A variety of factors, such as surrounding sequences and positioning of the ribozyme influences the activity and hence performance of catalytic RNAs. While some information about the efficiency of individual ribozymes (each tested in specific contexts) is known, general trends obtained from standardized, comparable experiments are lacking, complicating decisions such as which ribozyme to choose and where to insert it into the target mRNA. In many cases, application-specific optimization is required, which can be very laborious. Here, we systematically compared different classes of ribozymes within the 3'-UTR of a given reporter gene. We then examined position-dependent effects of the best-performing ribozymes. Moreover, we tested additional variants of already widely used hammerhead ribozymes originating from various organisms. We were able to identify functional structures suited for aptazyme design and generated highly efficient hammerhead ribozyme variants originating from the human genome. The present dataset will aide decisions about how to apply ribozymes for affecting gene expression as well as for developing ribozyme-based switches for controlling gene expression in human cells.

Conformational Space of the Translocation Domain of Botulinum Toxin: Atomistic Modeling and Mesoscopic Description of the Coiled-Coil Helix Bundle.

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a ß hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin-membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water-ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed.

A scalable system for the fast production of RNA with homogeneous terminal ends.

In vitro transcription (IVT) using T7 RNA polymerase has become the most common method to synthesize RNAs for use in basic research and pharmaceutical applications. To solve the heterogeneity issue associated with the system, cis-acting ribozymes have been exploited to direct co-transcriptional processing to yield target RNAs with precisely defined ends. However, traditionally used ribozymes have many limitations, such as low efficiency and special sequence requirements of target RNAs. In addition, the introduction of ribozymes in IVT complicates the downstream purification of target RNAs. Here we describe a new cassette of engineered ribozymes (HHV-Kt and Twister-Kt) that can work in concert to produce RNA with defined 5' and 3' ends. The pair of ribozymes displayed reliably high activity when working with RNA of various lengths and structures. The engineered ribozymes also carry a K-turn RNA motif that enables fast post-IVT clearance of cleaved ribozymes and uncleaved precursors using K-turn affinity resins. Finally, we demonstrated the scalability of our system for the rapid production of large quantities of homogeneous RNA samples.

A clinical trial comparing BiVap (Richard Wolf®) saline vaporization of the prostate vs. TwisterTM Diode Laser System in the treatment of benign prostatic obstruction between prostate volume 90 to 150†ml.

INTRODUCTION: To evaluate the efficacy, safety and postoperative outcomes of the BiVap and Twister systems with benign prostatic obstruction (BPO) in prostate volüm between 90-150 ml. METHODS: In total, we included 131 patients treated with BiVap system (n = 68) and Twister system (n = 63). Postoperative complications including urinary tract infection, transient hematuria, severe dysuria and fever >38° C, urinary incontinence and urethral stricture were also noted. All patients were evaluated at the postoperative 1st, 3rd, 6th and 12th month and preoperative and postoperative values of IPSS score, QoL score, total PSA, IIEF 15, PVR, Qmax and Qave were compared. RESULTS: Preoperative demographic characteristics were similar in the 2 groups. There was observed significant improvement for IPSS, Qmax, Qave, PVR, and QoL score by the postoperative first month compared to the preoperative values in both groups. Maximum improvement in the IPSS, Qmax, Qave, QoL score and PVR were achieved at postoperative 6, 3, 12, 3 and 12th months respectively in group 1. In group 2 maximum improvement in the same parameters were achieved at postoperative 6, 3, 6, 6 and 12th months, respectively. CONCLUSIONS: BiVap and Twister systems are safe, effective, and useful technique, which can be used in the surgical treatment of BPO between 90-150 ml.

The 3'-untranslated region of mRNAs as a site for ribozyme cleavage-dependent processing and control in bacteria.

Besides its primary informational role, the sequence of the mRNA (mRNA) including its 5'- and 3'- untranslated regions (UTRs), contains important features that are relevant for post-transcriptional and translational regulation of gene expression. In this work a number of bacterial twister motifs are characterized both in vitro and in vivo. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. Our data show that the ribozyme-mediated cleavage of the bacterial 3'-UTR has major effects on gene expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they show dependence on motif-specific structural features and on mRNA stabilization properties of the secondary structures that remain on the 3'-UTR after ribozyme cleavage. Using these principles, novel artificial twister-based riboswitches are developed that exert their activity via ligand-dependent cleavage of the 3'-UTR and the removal of the protective intrinsic terminator. Our results provide insights into possible biological functions of these recently discovered and widespread catalytic RNA motifs and offer new tools for applications in biotechnology, synthetic biology and metabolic engineering.

Bilinguals' twisted tongues: Frequency lag or interference?

Though bilinguals know many more words than monolinguals, within each language bilinguals exhibit some processing disadvantages, extending to sublexical processes specifying the sound structure of words (Gollan & Goldrick, Cognition, 125(3), 491-497, 2012). This study investigated the source of this bilingual disadvantage. Spanish-English bilinguals, Mandarin-English bilinguals, and English monolinguals repeated tongue twisters composed of English nonwords. Twister materials were made up of sound sequences that are unique to the English language (nonoverlapping) or sound sequences that are highly similar-yet phonetically distinct-in the two languages for the bilingual groups (overlapping). If bilingual disadvantages in tongue-twister production result from competition between phonetic representations in their two languages, bilinguals should have more difficulty selecting an intended target when similar sounds are activated in the overlapping sound sequences. Alternatively, if bilingual disadvantages reflect the relatively reduced frequency of use of sound sequences, bilinguals should have greater difficulty in the nonoverlapping condition (as the elements of such sound sequences are limited to a single language). Consistent with the frequency-lag account, but not the competition account, both Spanish-English and Mandarin-English bilinguals were disadvantaged in tongue-twister production only when producing twisters with nonoverlapping sound sequences. Thus, the bilingual disadvantage in tongue-twister production likely reflects reduced frequency of use of sound sequences specific to each language.

Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes.

Fourteen well-defined ribozyme classes have been identified to date, among which nine are site-specific self-cleaving ribozymes. Very recently, small self-cleaving ribozymes have attracted renewed interest in their structure, biochemistry, and biological function since the discovery, during the last three years, of four novel ribozymes, termed twister, twister sister, pistol, and hatchet. In this review, we mainly address the structure, biochemistry, and catalytic mechanism of the novel ribozymes. They are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of the ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site. It is most likely that a guanine and adenine in close proximity of the cleavage site are involved in general acid-base catalysis. In addition, metal ions appear to play a structural rather than catalytic role although some of their crystal structures have shown a direct metal ion coordination to a non-bridging phosphate oxygen at the cleavage site. Collectively, the structural and biochemical data of the four newest ribozymes could contribute to advance our mechanistic understanding of how self-cleaving ribozymes accomplish their efficient site-specific RNA cleavages.

Development of a new stir bar sorptive extraction method for the determination of medium-level volatile thiols in wine.

A fast, simple, and reliable analytical method for the determination of medium-level volatile thiols in wines is presented. Stir bar sorptive extraction using ethylene glycol-silicone coated stir bars has been used in combination with thermal desorption gas chromatography with mass spectrometry for the analysis of 4-mercapto-4-methylpentan-2-one, 2-furanmethanethiol, 3-mercaptohexyl acetate, and 3-mercaptohexanol in wine. Optimization of the extraction technique was performed using a two-level fractional factorial design. For the extraction step, the optimum conditions were: Ethylene glycol and silicone coated stir bars, pH at 3.5, sample volume of 25 mL, extraction time of 90 min, NaCl content 4.0 g, and stirring speed at 500 rpm. The optimized method achieved good linearity for all studied compounds (r(2) > 0.995) and it provided detection limits of 21.52, 0.36, 0.73, and 2.55 µg/L for 4-mercapto-4-methylpentan-2-one, 2-furanmethanethiol, 3-mercaptohexyl acetate, and 3-mercaptohexanol, respectively. It was repeatable, with precisions lower than 18% relative standard deviation for both intraday and interday repeatability. The developed procedure is suitable for the determination of these kinds of compounds when they are present at medium concentration levels. It was finally applied to real wine samples with negative aroma derived from the high concentration levels of these compounds.

Production of tongue twisters by speakers with partial glossectomy.

Abstract A partial glossectomy can affect speech production. The goal of this study was to investigate the effect of the presence of a tumour as well as the glossectomy surgery on the patients' production of tongue twisters with the sounds [t] and [k]. Fifteen patients with tongue cancer and 10 healthy controls took part in the study. The outcome measures were the patients' speech acceptability, rate of errors, the time needed to produce the tongue twisters, pause duration between item repetitions and the tongue shape during the production of the consonants [t] and [k] before and after surgery. The patients' speech acceptability deteriorated after the surgery. Compared to controls, the patients' productions of the tongue twisters were slower but not more errorful. Following the surgery, their speed of production did not change, but the rate of errors was higher. Pause duration between items was longer in the patients than in the controls but did not increase from before to after surgery. Analysis of the patients' tongue shapes for the productions of [t] and [k] indicated a higher elevation following the surgery for the patients with flap reconstructions. The results demonstrated that the surgical resection of the tongue changed the error rate but not the speed of production for the patient. The differences in pause duration also indicate that the tumour and the surgical resection of the tongue may impact the phonological planning of the tongue twister.

Concurrent waterlogging and anthracnose-twister disease in rainy-season onions (Allium cepa): Impact and management.

Waterlogging and anthracnose-twister disease are significant obstacles in rainy-season onion cultivation. As a shallow-rooted crop, onions are highly sensitive to waterlogging. Wherever rainy-season onion cultivation has been undertaken, the anthracnose-twister disease complex is also widespread across the world in addition to waterlogging. Waterlogging is the major predisposing factor for anthracnose and other fungal diseases. However, studies on the combined stress impact on onions have been ignored. In the present review, we have presented an overview of the anthracnose-twister disease, the waterlogging effect on host physiology, host-pathogen interaction under waterlogging stress, and appropriate management strategies to mitigate the combined stress effects. Crucial soil and crop management strategies can help cope with the negative impact of concurrent stresses. Raised bed planting with drip irrigation, the use of plant bio-regulators along with nutrient management, and need-based fungicide sprays would be the most reliable and feasible management options. The most comprehensive solution to withstand combined stress impacts would be a genetic improvement of commercial onion cultivars.

Anthracnose of Onion (Allium cepa L.): A Twister Disease.

The onion (Allium cepa L.) is a lucrative and high-value vegetable-spice crop in India, but it is sensitive to several of diseases caused by fungi, bacteria, viruses, and nematodes, of which a fungal disease, anthracnose, caused by Colletotrichum spp., is a major issue for both onion producers and researchers since it severely affects the bulb production. Twister disease is currently one of the most common problems in onion production, particularly in humid regions, and it reduces productivity while also lowering the value and profitability. Twister disease is visualised by white or pale-yellow water-soaked oval depressed lesions on leaf blades, which are the first symptoms. Lesions expand as the disease advances, and numerous black-coloured, slightly elevated structures/fruiting bodies appear in the middle area, arranged in concentric rings. Curling, twisting, chlorosis of the leaves, and aberrant extension of the neck or pseudo-stem occurs, followed by rotting of the bulb. In an unmanaged crop, an excess gibberellin production by Colletotrichum gloeosporioides and Gibberella moniliformis is suspected to induce twisting and aberrant neck elongation, which will ruin onion productivity. It is difficult and environmentally unfriendly to control these infections. Since, to the best of our knowledge, this is the first review on onion anthracnose, we tried to consolidate information. This review updates our knowledge of the pathogen, including the disease cycle, infection pathways, and disease management techniques. As a result, growers will be benefit from the application of cultural, biological, and chemical measures and the use of resistant varieties.

The Effects of Combining High-Top Shoes with Twister Wrap Orthoses on Balance Parameters of Children with Spastic Diplegic Cerebral Palsy.

BACKGROUND: Cerebral palsy (CP) is a non-progressive encephalopathy before, during, or after childbirth with almost the most common type, i.e. spastic diplegic, leading to a frequent walking problem, In-toeing. Orthoses can reduce the consequences of CP. OBJECTIVE: This study aimed to evaluate the effect of combining twister wrap orthoses (TWO) with high-top shoes on the balance parameters of children with spastic diplegic CP. MATERIAL AND METHODS: In this quasi-experimental study, twenty children (aged 6.8 ± 0.5 years) with spastic diplegic CP with in-toeing gait participated. The tests were conducted in three conditions: 1) in bare-foot, 2) with high-top shoes, and 3) with high-top shoes plus TWO and the orthoses effects on balance parameters were compared. RESULTS: High-top shoes positively decreased center of pressure (COP) sway in the anterior-posterior (AP) direction. Both high-top shoes and high-top shoes with TWO conditions compared to bare-foot conditions significantly improved standing balance by decreasing the ellipse area. Pairwise, wearing TWO and high-top shoes significantly reduced the COP sway in the medial-lateral (ML) and AP directions with a significant difference between using the combined orthoses and the shoe without TWO in ML of COP displacement. CONCLUSION: High-top shoes alone and the combination of high-top shoes with TWO conditions may enhance the stability of children with spastic diplegia more than barefoot. Although the use of combined orthoses induced significant improvement in the ML direction of COP displacement.

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage.

Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA-binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2'-hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase-mediated general acid-base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small-molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme.

Computer-Aided Design of Active Pseudoknotted Hammerhead Ribozymes.

Pseudoknots are important motifs for stabilizing the structure of functional RNAs. As an example, pseudoknotted hammerhead ribozymes are highly active compared to minimal ribozymes. The design of new RNA sequences that retain the function of a model RNA structure includes taking in account pseudoknots presence in the structure, which is usually a challenge for bioinformatics tools. Our method includes using "Enzymer," a software for designing RNA sequences with desired secondary structures that may include pseudoknots. Enzymer implements an efficient stochastic search and optimization algorithm to sample RNA sequences from low ensemble defect mutational landscape of an initial design template to generate an RNA sequence that is predicted to fold into the desired target structure.

Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme.

Self-cleaving ribozymes engineered to be activated or inhibited by a small molecule binding to an RNA aptamer inserted within a ribozyme (aptazymes) have proven to be useful for controlling gene expression in living cells. In mammalian cells, an aptazyme embedded in the 5' or 3' untranslated region of an mRNA functions as a synthetic riboswitch to chemically regulate gene expression. However, the variety of aptazyme architectures and the ribozyme scaffolds that have been used for mammalian riboswitches has been limited. In particular, fewer synthetic riboswitches that activate gene expression in response to a small molecule (ON-switches) in mammalian cells have been reported compared to OFF-switches. In this work, we developed mammalian riboswitches that function as guanine-activated ON-switches based on a novel aptazyme architecture in which an aptamer and a ribozyme are fused in tandem. The riboswitch performance was optimized by fine-tuning the stability of a critical stem that controls the ribozyme structure and function, yielding switches with ON/OFF ratios greater than 6.0. Our new aptazyme architecture expands the RNA device toolbox for controlling gene expression in mammalian cells.

Comparison of the Novel Thin Film-Solid Phase Microextraction and Sorptive Extraction Methods for Picual and Hojiblanca Olive Oil Volatile Fraction Analysis in Headspace.

For first time, the new device named thin film solid phase microextraction (TF-SPME) has been used to determine the volatile profile of the Picual and Hojiblanca varieties of extra virgin olive oils. To this end, different traditional sampling methods such as headspace sorptive extraction (HSSE) with polydimethylsiloxane (PDMS) and polyethyleneglycol-modified silicone (EG/Silicone) Twisters® have been compared with the TF-SPME devices coated with different extraction polymeric phases. PARADISe software was used as a non-targeting method to process all data. The best results were obtained by HSSE-PDMS and 2TF-SPME. Moreover, the 2TF-SPME extraction method achieved the most adequate results of linearity for most compounds, according to F-values, while the intermediate precision results were similar for both 2TF-SPME and HSSE-PDMS sampling methods. Different sensitivity was observed between both sampling methods depending on the volatile compound, without being clearly influenced by the polarity of them. Although both sampling methods enabled the main active aroma of olive oil to be determined and for them to be differentiated according to olive variety, the 2TF-SPME method appears to be the most suitable for this goal.

Effects of transcranial direct current stimulation over the Broca's area on tongue twister production.

PURPOSE: The present study aimed to explore the short-term effect of anodal transcranial direct current stimulation (tDCS) on tongue twister production. METHOD: Thirty healthy native Cantonese adult speakers were randomly assigned to the anodal tDCS group or the sham tDCS group. Anodal tDCS of 2 mA was applied over the Broca's area of the brain. The stimulation lasted for 20 min for the anodal tDCS group and 30 s for the sham tDCS group. The participants were instructed to produce a list of tongue twisters before, immediately after and 4 h after tDCS. RESULT: Speech rate and response accuracy measured immediately after stimulation were significantly faster and higher, respectively, than before stimulation. Although there was no change in speech rate measured at 4 h after stimulation, response accuracy at that time point was significantly lower than that measured immediately after stimulation. However, there were no significant differences between the anodal tDCS and sham tDCS groups in either speech rate or response accuracy. CONCLUSION: The findings revealed that a single session of anodal tDCS over the Broca's area did not significantly improve speech production during tongue twister production.

Peripheral Sensory Nerve Tissue but Not Connective Tissue Is Involved in the Action of Acupuncture.

Acupuncture has been used to treat a variety of diseases and symptoms for more than 2,500 years. While a number of studies have shown that nerves are responsible for initiating the effects of acupuncture, several lines of study have emphasized the role of connective tissue in the initiation of acupuncture signals. To determine whether nerves or connective tissue mediate the action of acupuncture, we constructed a robotic acupuncture needle twister that mimicked the twisting of the needle by an acupuncturist, and we examined the role of nerves and connective tissues in the generation of acupuncture effects in rat cocaine-induced locomotion, stress-induced hypertension, and mustard oil-induced visceral pain models. Robotic or manual twisting of acupuncture needles effectively suppressed cocaine-induced hyperactivity, elevated systemic blood pressure or mustard oil-induced visceral pain in rats. These acupuncture effects were completely abolished by injecting bupivacaine, a local anesthetic, into acupoints. However, disruption of connective tissue by injecting type I collagenase into acupoints did not affect these acupuncture effects. Our findings suggest that nerve tissue, but not connective tissue, is responsible for generating the effects of acupuncture.

Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology.

The catalytic properties of RNA have been a subject of fascination and intense research since their discovery over 30 years ago. Very recently, several classes of nucleolytic ribozymes have emerged and been characterized structurally. Among these, the twister ribozyme has been center-stage, and a topic of debate about its architecture and mechanism owing to conflicting interpretations of different crystal structures, and in some cases conflicting interpretations of the same functional data. In the present work, we attempt to clean up the mechanistic "debris" generated by twister ribozymes using a comprehensive computational RNA enzymology approach aimed to provide a unified interpretation of existing structural and functional data. Simulations in the crystalline environment and in solution provide insight into the origins of observed differences in crystal structures, and coalesce on a common active site architecture, and dynamical ensemble in solution. We use GPU-accelerated free energy methods with enhanced sampling to ascertain microscopic nucleobase pK a values of the implicated general acid and base, from which predicted activity-pH profiles can be compared directly with experiments. Next, ab initio quantum mechanical/molecular mechanical (QM/MM) simulations with full dynamic solvation under periodic boundary conditions are used to determine mechanistic pathways through multi-dimensional free energy landscapes for the reaction. We then characterize the rate-controlling transition state, and make predictions about kinetic isotope effects and linear free energy relations. Computational mutagenesis is performed to explain the origin of rate effects caused by chemical modifications and make experimentally testable predictions. Finally, we provide evidence that helps to resolve conflicting issues related to the role of metal ions in catalysis. Throughout each stage, we highlight how a conserved L-platform structural motif, to- gether with a key L-anchor residue, forms the characteristic active site scaffold enabling each of the catalytic strategies to come together not only for the twister ribozyme, but the majority of the known small nucleolytic ribozyme classes.