Being Correct Is Not Enough: Efficient Verification Using Robust Linear Temporal Logic

While most approaches in formal methods address system correctness, ensuring robustness has remained a challenge. In this article, we present and study the logic rLTL, which provides a means to formally reason about both correctness and robustness in system design. Furthermore, we identify a large fragment of rLTL for which the verification problem can be efficiently solved, i.e., verification can be done by using an automaton, recognizing the behaviors described by the rLTL formula φ, of size at most O(3 |φ |), where |φ | is the length of φ. This result improves upon the previously known bound of O(5|φ |) for rLTL verification and is closer to the LTL bound of O(2|φ |). The usefulness of this fragment is demonstrated by a number of case studies showing its practical significance in terms of expressiveness, the ability to describe robustness, and the fine-grained information that rLTL brings to the process of system verification. Moreover, these advantages come at a low computational overhead with respect to LTL verification.

Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.

Glossary of Russian-German Literature

The article presents a large fragment of the authors glossary (dictionary) on the literature of Russian Germans (the authors definitions of literary concepts related to the literature of Russian Germans, based on its study in a doctoral dissertation and a number of articles). Writers - Russian Germans - Russian-German bilinguals, therefore the dictionary on the literature of Russian Germans should be presented in German-Russian format (articles in German and Russian). In the original literature of Russian Germans, there are phenomena and facts specific to this subculture. The Dictionary of the Literature of Russian Germans, explaining the meanings of words that may be unfamiliar, incomprehensible, or partially incomprehensible to the reader, makes it possible to significantly expand the circle of connoisseurs of the literature of Russian Germans, facilitates the perception of literary and scientific text.

GREPore-seq: A Robust Workflow to Detect Changes after Gene Editing through Long-range PCR and Nanopore Sequencing

To achieve the enormous potential of gene-editing technology in clinical therapies, both the on-target and unintended editing consequences need to be thoroughly evaluated. However, there is a lack of a comprehensive, pipelined, large-scale and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR-Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. To overcome the high error rates and indels of nanopore sequencing, we developed a pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can detect NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also identifies HDR-mediated large gene knock-in, which excellently correlates with FACS analysis data. Low-level plasmid backbone insertion after HDR editing was also detected. We have established a practical workflow to identify genetic changes, including quantifying dsODN insertions, knock-ins, plasmid backbone insertions, and large fragment deletions after CRISPR editing. This toolkit for nanopore sequencing of pooled long amplicons should have broad applications in assessing on-target HDR editing and inadvertent large indels of over 1 kb. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq).

Qui capite ipse sua in statuit uestigia sese. Lucrezio e lo scetticismo nel libro IV del De rerum natura

In his refutation of skepticism in book IV of De rerum natura (469–521), Lucretius uses argumentative methods typical of Epicurus: the περιτροπή is in many ways similar to that used by the philosopher in book XXV of Περὶ φύσεως, the same book where, in a passage dedicated to the criticism against determinists, can be found a reference to the criterion of the πρόληψις, that Lucretius exploits in his refutation. Moreover, Lucretius develops a strong demonstration concerning the irrefutability of αἴσθησις as a criterion of truth, which finds significant points of contact with a large fragment, transmitted by Diogenes Laertius (X 31–32) and generally traced back to the Canon of Epicurus. The last argument used by the poet is a pragmatic one: for the skeptic it would be impossible to live. The argument is similar to the praxis-based argument used by Epicurus in the Περὶ φύσεως against the partisans of determinism. But the pragmatic argument goes back to a very ancient layer of anti-skeptical polemics, even prior to Epicurus and already present in book Γ of Aristotle’s Metaphysics. Although later influences cannot be excluded, Lucretius appears to be a faithful witness of Epicurus. Probably in a lost section of the Περὶ φύσεως, the philosopher of Samos showed positions going against skeptical or proto-skeptical attitude, contemporary or earlier to the time of the philosopher, probably developed in a Democritean or a Socratic context. Epicurus’ ad hominem strategy is very close to that of the philosopher in the Principal Doctrines XXIII–XXV, and certainly follows Aristotle’ strategy in book Γ of Metaphysics against those who deny the principle of non-contradiction. In a similar way, Epicurus does not have one figure as the objective of his refutation but constructs a hypothetical dialectical opponent capable of embodying a series of philosophical tendencies judged by the founder of Kepos to be extremely dangerous not only for the correct exercise of thought but for the human being’s life itself.

The effects of locking inserts and overtorque on the mechanical properties of a large fragment locking compression plate

Purpose The study was to determine the effect of locking hole inserts and their insertion torque on the fatigue life of a large fragment Locking Compression Plate (LCP) under bending forces. Methods Fatigue strength of the LCP was examined using cyclic three-point bend testing at 80% yield strength of the construct. Locking hole inserts were used in 2, 4, and 6-hole of a 12-hole plate to simulate three different working lengths. Within each working length, plates were tested without locking inserts serving as the control group. In the experimental groups, inserts were tightened to manufacturer recommendations (4 Nm) and using overtorque (8 Nm). Results Significantly fewer cycles to failure were observed in control groups versus the locking hole insert groups for all working lengths (2-hole: 4 Nm p = 0.003, 8 Nm p = 0.003; 4-hole: 4 Nm p = 0.02, 8 Nm p < 0.001; 6-hole: 4 Nm p = 0.004, 8 Nm p < 0.001). There was a statistically significant increase in fatigue strength when using overtorque in the 4-hole (p = 0.04) and 6-hole (p = 0.01) defect groups. This was not shown in the 2-hole defect group (p = 0.99). Conclusions By placing locking inserts in the empty locking regions of Combi holes along the working length, the number of cycles to failure was increased. Tightening inserts to twice the recommended insertion torque further increased cycles to failure in longer working length models. A longer fatigue life has the potential to decease the incidence of plate failure especially in the setting of delayed union due to poor intrinsic healing capacity, fractures in the geriatric population, osteoporosis and periprosthetic fractures.

Birds of Parque Nacional do Caparaó, Atlantic Forest, southeastern Brazil

Parque Nacional do Capara&oacute; is located in the Serra do Capara&oacute; in the southeastern region of Brazil. It lies on the border between the states of Esp&iacute;rito Santo and Minas Gerais. It is a large fragment of Atlantic Forest vegetation, with altitudinal variation from 630 to 2,892 m. We present an annotated list of bird species found in the park based on data collected from 2012 to 2018. We recorded 216 species, including 66 endemic, 22 threatened, and 17 newly recorded species. To complete the list of species, we added data available in several bibliographic sources and digital databases, bringing the total number of bird species in the park to 348 and including 98 endemic and 33 threatened species. This work is the first to present an updated list of birds for the park, and the data presented show the importance of this protected area for conservation of birds in the region.

Endogenous CRISPR-assisted microhomology-mediated end joining enables rapid genome editing in Zymomonas mobilis

Background Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required. Results We developed a novel gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat–Spacer–Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500 bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a σ28 deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem. Conclusions Here, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.

Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice

ABSTRACTCRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.

Finitely Materialisable Datalog Programs with Metric Temporal Operators

DatalogMTL is an extension of Datalog with metric temporal operators that has recently received significant attention. In contrast to plain Datalog, where scalable implementations are often based on materialisation (a.k.a. forward chaining), reasoning algorithms for recursive fragments of DatalogMTL are automata-based and not well suited for practice. In this paper we propose the class of finitely materialisable DatalogMTL programs, for which forward chaining reasoning terminates after finitely many rounds of rule application. We show that, for bounded programs (a large fragment of DatalogMTL where temporal intervals are restricted to not mention infinity), checking whether a program is finitely materialisable is feasible in exponential time, and propose sufficient conditions for finite materialisability that can be checked more efficiently. We finally show that fact entailment over finitely materialisable bounded programs is ExpTime-complete, and hence no harder than Datalog reasoning.