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SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenespersists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenesmay “dampen” the expression of genes for methionine import but likely does not turn them “OFF”.

RNAs are often studied in nonnative sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods—a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added postprobing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5′ and 3′ ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced nonnative interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to nonnative sequence elements.

Electrospinning has been widely used as a versatile technique to generate nanofibers of various materials. It is also helpful in teaching topics ranging from macromolecular chemistry to physics, safety, and sustainability at various levels of difficulty and student involvement. Simple and safe hands-on experiments/manual assays can be realized for less than 30 euros to demonstrate polymer viscosity and nanofiber alignment and solubility. Students can further study (super)hydrophobicity and even upcycle packaging waste into useful filter materials but also improve the electrospinning setup from a manual assay to an inexpensive Arduino-based 3D printed research platform. Alternatively, the latter can be used for teacher demonstrations of more challenging experiments that can also be easily done using a commercial syringe-pump.

Reiff and colleagues found that perceived similarity between one’s present and future self is positively associated with life satisfaction 10 years later, using a difference-score approach to operationalize similarity. This study further evaluated the similarity effect by reproducing the original longitudinal association with a difference-score method, by using more sophisticated analyses (i.e., polynomial regression and response surface analyses), and by replicating the association in a newly collected sample. We were able to reproduce and replicate the findings based on a difference-score approach. However, we did not find a similarity effect in either sample using more sophisticated approaches. The current results show that previously reported support for the association between perceived similarity and well-being is driven by the statistical main effects of personality reports.

Biopsy-based transcript diagnostics may identify molecular antibody-mediated rejection (AMR) when microvascular inflammation (MVI) is absent. In this single-center cohort, biopsy-based transcript diagnostics were validated in 326 kidney allograft biopsies. A total of 71 histological AMR and 35 T cell–mediated rejection (TCMR) cases were identified as molecular AMR and TCMR in 55% and 63%, respectively. Among 121 cases without MVI (glomerulitis + peritubular capillaritis = 0), 45 (37%) donor-specific antibody (DSA)-positive and 76 (63%) DSA-negative cases were analyzed. Twenty-one out of the 121 (17%) cases showed borderline changes, or TCMR, while BK nephropathy was excluded. None of the 45 DSA-positive patients showed molecular AMR. Among 76 DSA-negative patients, 2 had mixed molecular AMR/TCMR. All-AMR phenotype scores (sum of R4-R6) exhibited median values of 0.13 and 0.12 for DSA-positive and DSA-negative patients, respectively (P= .84). A total of 13% (6/45) DSA-positive and 11% (8/76) DSA-negative patients showed an all-AMR phenotype score > 0.30 (P= .77). Patients with a higher all-AMR phenotype score showed 33% more histologic TCMR (P= .005). The median all-AMR phenotype scores of glomerular basement membrane double contours = 0 and glomerular basement membrane double contours > 0 biopsies were 0.12 and 0.10, respectively (P= .35). Biopsy-based transcript diagnostics did not identify molecular AMR in cases without MVI. Follow-up biopsies and outcome data should evaluate the clinical relevance of subthreshold molecular alterations.

Nosocomial-associated diarrhea due to Clostridioides difficileinfection (CDI) is diagnosed after sample precultivation by the detection of the toxins in enzyme immunoassays or via toxin gene nucleic acid amplification. Rapid and direct diagnosis is important for targeted treatment to prevent severe cases and recurrence. We developed two singleplex and a one-pot duplex fluorescent 15 min isothermal recombinase polymerase amplification (RPA) assays targeting the toxin genes A and B (tcdAand tcdB). Furthermore, we adapted the singleplex RPA to a 3D-printed microreactor device. Analytical sensitivity was determined using a DNA standard and DNA extracts of 20 C. difficilestrains with different toxinotypes. Nineteen clostridial and gastrointestinal bacteria strains were used to determine analytical specificity. Adaptation of singleplex assays to duplex assays in a 50 μL volume required optimized primer and probe concentrations. A volume reduction by one-fourth (12.4 μL) was established for the 3D-printed microreactor. Mixing of RPA was confirmed as essential for optimal analytical sensitivity. Detection limits (LOD) ranging from 119 to 1411 DNA molecules detected were similar in the duplex tube format and in the singleplex 3D-printed microreactor format. The duplex RPA allows the simultaneous detection of both toxins important for the timely and reliable diagnosis of CDI. The 3D-printed reaction chamber can be developed into a microfluidic lab-on-a-chip system use at the point of care.

Higher levels of inflammatory biomarkers are associated with an increased risk of perioperative atrial fibrillation and myocardial injury after non-cardiac surgery (MINS). Colchicine is an anti-inflammatory drug that might reduce the incidence of these complications.

Governmental measures against the spread of the Corona virus have been met with varying levels of opposition in many countries worldwide. Existing research has claimed that some of this opposition is linked to esoteric and anthroposophical beliefs. This research note tests this in an observational study using election results from the 2021 parliamentary election in Germany and new data on the distribution of natural healers, homeopathic doctors and Steiner schools. Results show that counter to common expectations, there is no evidence that esoteric beliefs systematically lead to increased support for the established right-wing AFD. Rather, some indicators for esoteric beliefs – in particular, the presence of homeopathic doctors and Waldorf schools – are related to higher support for the new fringe party dieBasis, a single-issue party campaigning against governmental Corona measures.

RNA molecules convey biological information both in their linear sequence and in their base-paired secondary and tertiary structures. Chemical probing experiments, which involve treating an RNA with a reagent that modifies conformationally dynamic nucleotides, have broadly enabled examination of short- and long-range RNA structure in diverse contexts, including in living cells. For decades, chemical probing experiments have been interpreted in a per-nucleotide way, such that the reactivity measured at each nucleotide reports the average structure at a position over all RNA molecules within a sample. However, there are numerous important cases where per-nucleotide chemical probing falls short, including for RNAs that are bound by proteins, RNAs that form complex higher order structures, and RNAs that sample multiple conformations.

Although not canonically polyadenylated, the long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is stabilized by a highly conserved 76-nt triple helix structure on its 3′ end. The entire MALAT1 transcript is over 8000 nt long in humans. The strongest structural conservation signal in MALAT1 (as measured by covariation of base pairs) is in the triple helix structure. Primary sequence analysis of covariation alone does not reveal the degree of structural conservation of the entire full-length transcript, however. Furthermore, RNA structure is often context dependent; RNA binding proteins that are differentially expressed in different cell types may alter structure. We investigate here the in-cell and cell-free structures of the full-length human and green monkey (Chlorocebus sabaeus) MALAT1 transcripts in multiple tissue-derived cell lines using SHAPE chemical probing. Our data reveal levels of uniform structural conservation in different cell lines, in cells and cell-free, and even between species, despite significant differences in primary sequence. The uniformity of the structural conservation across the entire transcript suggests that, despite seeing covariation signals only in the triple helix junction of the lncRNA, the rest of the transcript's structure is remarkably conserved, at least in primates and across multiple cell types and conditions.

The role of catheter tip shape on the safety and efficacy of radiofrequency (RF) ablation has been overlooked, although differences have been observed in clinical and research fields.

Patient-derived organoids (PDOs) are now used to study many diseases, including prostate cancer. Here, we present a protocol for the transduction of human epithelial prostate cells and PDOs. We describe the steps for producing lentiviruses and transducing PDOs with high efficiency to obtain either overexpression or knockdown of specific genes. More generally, this protocol represents an efficient lentiviral transduction technique to study cell biology using various organoid models.

Direct-acting oral anticoagulant use for stroke prevention in atrial fibrillation is limited by bleeding concerns. Asundexian, a novel, oral small molecule activated coagulation factor XIa (FXIa) inhibitor, might reduce thrombosis with minimal effect on haemostasis. We aimed to determine the optimal dose of asundexian and to compare the incidence of bleeding with that of apixaban in patients with atrial fibrillation.

The parts produced by Laser Metal Deposition (LMD) usually contain deposition defects such as pores, cavities and micro-cracks, which have a negative influence on the quality and mechanical properties. This work examines the use of ultrasonic vibrations to reduce the most common deposition defects. A series of tests are carried out (with and without ultrasonic assistance) to obtain comparative values. These values are analysed for particular purposes and the results show that the ultrasonic-assisted process leads to higher efficiency in powder utilization. Pores and micro-cracks are successfully reduced and crystal grains are visibly refined. However, the improvement of these geometric and microstructural properties does not lead to an increase in tensile strength and hardness. The fundamental investigation presented in this work proves that ultrasonic wave contributes to an efficient and effective LMD process.

In order to keep trace of information and grow up, the infant brain has to resolve the problem about where old information is located and how to index new ones. We propose that the immature prefrontal cortex (PFC) uses its primary functionality of detecting hierarchical patterns in temporal signals as a second feature to organize the spatial ordering of the cortical networks in the developing brain itself. Our hypothesis is that the PFC detects the hierarchical structure in temporal sequences in the shape of ordinal patterns and uses them to index information hierarchically in different parts of the brain. Henceforth, we propose that this mechanism for detecting ordinal patterns participates also in the hierarchical organization of the brain during development; i.e., the bootstrapping of the connectome. By doing so, it gives the tools to the language-ready brain for manipulating abstract knowledge and for planning temporally ordered information; i.e., the emergence of causality and symbolic thinking. In this position paper, we will review several neural models from the literature that support serial ordering and propose an original one. We will confront then our ideas with evidence from developmental, behavioral, and brain results.

AbstractThe literature on autocracies has argued that repression of protest is either a result of the political environment in which protest occurs, or depends on particular characteristics of the protest events themselves. We argue that the interaction of both matters. Authoritarian regimes vary in how they legitimize their rule, and they should be particularly thin-skinned if protesters challenge the basis of their legitimacy. Using event-level data on mass mobilization in autocracies between 2003 and 2015, we use text classification methods to extract protest issues from newspaper reports. Our analysis shows that dictators are more likely to repress protest against incumbents when they claim legitimacy based on the person of the leader. Overall, our study shows that protest issues are not universal in triggering repression; rather, they need to be considered together with the political context in which they are raised.

In the field of Model-Driven Engineering, Triple Graph Grammars (TGGs) play an important role as a rule-based means of implementing consistency management. From a declarative specification of a consistency relation, several operations including forward and backward transformations, (concurrent) synchronisation, and consistency checks can be automatically derived. For TGGs to be applicable in realistic application scenarios, expressiveness in terms of supported language features is very important. A TGG tool is schema compliant if it can take domain constraints, such as multiplicity constraints in a meta-model, into account when performing consistency management tasks. To guarantee schema compliance, most TGG tools allow application conditions to be attached as necessary to relevant rules. This strategy is problematic for at least two reasons: First, ensuring compliance to a sufficiently expressive schema for all previously mentioned derived operations is still an open challenge; to the best of our knowledge, all existing TGG tools only support a very restricted subset of application conditions. Second, it is conceptually demanding for the user to indirectly specify domain constraints as application conditions, especially because this has to be completely revisited every time the TGG or domain constraint is changed. While domain constraints can in theory be automatically transformed to obtain the required set of application conditions, this has only been successfully transferred to TGGs for a very limited subset of domain constraints. To address these limitations, this paper proposes a search-based strategy for achieving schema compliance. We show that all correctness and completeness properties, previously proven in a setting without domain constraints, still hold when schema compliance is to be additionally guaranteed. An implementation and experimental evaluation are provided to support our claim of practical applicability.

Trace gas analysis provides a wide range of insights into environmental processes, particularly with regards to global warming and air quality. With the urgent need to identify sources and accurately measure the harmful emissions negatively impacting our planet, Laser Dispersion Spectroscopy (LDS) offers a unique approach. LDS technology measures optical molecular dispersion via a differential phase measurement of light and, operating in the mid-infrared, provides highly sensitive and robust measurements. This enables highly precise, real-time gas measurements even in adverse environmental conditions such as rain, fog, snow or dust. The technology can be used in both extractive and open-path formats, with real-world applications including emissions monitoring on oil and gas sites, measuring the impact of agricultural activities and monitoring carbon capture storage facilities.

The next generation of railway systems will require more and more accurate information for the planning of rail operation. These are essential for the introduction of automatic processes of an optimized traffic planning, the optimal use of infrastructure capacity and energy, and, overall, the introduction of data-driven approaches into rail operation. Train trajectories collection constitutes a primary source of information for offline procedures such as timetable generation, driving behaviour analysis and models’ calibration. Unfortunately, current train trajectory data are often affected by measurement errors, missing data and, in many cases, incongruence between dependent variables. To overcome this problem, a trajectory reconstruction problem must be solved, before using trajectories for any further purpose. In the present paper, a new hybrid stochastic trajectory reconstruction is proposed. On-board monitoring data on train position and velocity (kinematics) are combined with data on power used for traction and feasible acceleration values (dynamics). A fusion of those two types of information is performed by considering the stochastic characteristics of the data, via smoothing techniques. A promising potential use is seen especially in those cases where information on continuous train positions is not available or unreliable (e.g. tunnels, canyons, etc.).

Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb–siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb–siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb–siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb–siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.