Internal Sequence Research Articles

Abstract 5335: circAde: a circRNA-based system for prolonged and more effective treatment of cancer

Abstract In recently performed phase I/II clinical studies, we have demonstrated successful delivery of vectorized transgene-encoding mRNA payloads to solids tumors with substantial clinical benefit. In patients, persistent and prolonged transgene expression correlated with positive clinical outcome, and thus temporal expansion of the transgene expression profile is expected to improve the therapeutic benefit even further. Recently, circular RNA (circRNA) has been discovered as a novel class of endogenously expressed RNA. CircRNAs, in contrast to mRNAs, are resistant to exo-nucleolytic decay which results in high intra-cellular stability and persistent expression within cells. Therefore, circRNA-based systems serve as an interesting new technology towards prolonged effects of treatment. Here, we show that our proprietary vector system, circAde, allows high expression of circRNAs through spliceosome-dependent biogenesis. By comparing the protein output from circAde and mRNA-based vectors, we show that the circAde-system out-performs conventional mRNA-based design, both in terms of transgene yield and temporal sustainability of expression. Moreover, using a panel of proof-of-concept-reporters, we show that certain design-features greatly influence the circRNA production and subsequent protein expression from the vector. Without the 5’cap, circRNAs are dependent on IRESs (internal ribosome entry sequences) for effective translation. By conducting two orthogonal IRES screens on 1000+ putative IRES elements, we have identified the most effective IRES sequence in melanoma and lung-cancer cells supporting effective circRNA production and high-yield protein expression, thereby improving our circAde vector system even further. Our results open a new platform towards development of novel, more effective strategies for the treatment of cancer and other settings in which durable transgene expression is desirable. Citation Format: Eoghan T. O'leary, Sara Vikberg, Emilie Foord, Erik D. Wiklund, Lone Ottesen, Victor Levitsky, Thomas B. Hansen. circAde: a circRNA-based system for prolonged and more effective treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5335.

Formation of ER-lumenal intermediates during export of Plasmodium proteins containing transmembrane-like hydrophobic sequences.

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway.

RJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbβ3, a fundamental platelet glycoprotein, and αvβ3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of β-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbβ3 antagonist, capable of preventing arterial thrombosis.

Abstract P3-07-27: In vitro antitumoral activity of endophytic fungi isolated from Lophocereus marginatus against the human breast cancer cell line MCF-7

Abstract Breast cancer is the most frequent malignant tumor among women and is the leading cause of death from cancer worldwide. In 2020, 90,222 deaths from breast cancer were reported in México. However, cancer cells resistant to antineoplastic drugs are frequent, which compromises the patient’s survival. For this reason, in oncology, it is essential to search for new sources of anticancer compounds. Endophytic fungi are currently considered potential reservoirs for compounds with antitumor activity. More than 100 different classes of secondary metabolites have been reported with activity against different types of cancer, including breast cancer. Therefore, the objective of this study was to evaluate the activity of methanol extracts of endophytic fungi isolated from Lophocereus marginatus against the MCF-7 breast cancer cell line, using as controls the MA-104 cell line human peripheral blood mononuclear cells. Fungal strains were isolated from stems of L. marginatus and molecularly identified from ribosomal DNA internal transcript spacer region sequencing. Extraction of the secondary metabolites was performed from the maceration of mycelium fungus in methanol. The solvent was removed with a rotary evaporator and the extract was reconstituted with dimethyl sulfoxide (DMSO). Next, cells were incubated with the extract at concentrations ranging from 31.25 µg/mL to 250 µg/mL for 48 h at 37 °C and 5% CO2. Growth inhibition was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay, using 1% DMSO was used as a negative control. IC50 values and selectivity indexes (SI) were then calculated. It was found that tumor cells growth inhibition by the extracts increased in a concentration-dependent manner. A. versicolor PME-H005 strain extract showed the highest antitumor activity, with up to 58.9% growth inhibition at 250 µg/mL and IC50 value of 95.21 ± 1 µg/mL against MCF-7 cells. The highest SI was obtained with MCF-7 cell s with 2.77, as compared with normal PBMC with an IC50 of 264 ± 1.5 µg/mL. In addition, the M. anisopliae PME-H007 strain presented a high SI value of 2.1 and an IC50 of 245.9 ± 1.9 µg/mL, using the MCF-7 cell line. This study shows the potential of the endophytic fungus A. versicolor PME-H005 isolated from L. marginatus for production of secondary metabolites with antitumor activity against MCF-7 cells. Citation Format: Jesica M. Ramírez Villalobos, Cesar I. Romo Sáenz, Karla S. Morán Santibañez, Patricia Tamez Guerra, Orquídea Pérez González, Reyes Tamez Guerra, Cristina Rodríguez-Padilla, Ricardo A. Gomez Flores. In vitro antitumoral activity of endophytic fungi isolated from Lophocereus marginatus against the human breast cancer cell line MCF-7 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-27.

Genome, host genome integration, and gene expression in Diadegma fenestrale ichnovirus from the perspective of coevolutionary hosts.

Polydnaviruses (PDVs) exhibit species-specific mutualistic relationships with endoparasitoid wasps. PDVs can be categorized into bracoviruses and ichnoviruses, which have independent evolutionary origins. In our previous study, we identified an ichnovirus of the endoparasitoid Diadegma fenestrale and named it DfIV. Here, DfIV virions from the ovarian calyx of gravid female wasps were characterized. DfIV virion particles were ellipsoidal (246.5 nm × 109.0 nm) with a double-layered envelope. Next-generation sequencing of the DfIV genome revealed 62 non-overlapping circular DNA segments (A1-A5, B1-B9, C1-C15, D1-D23, E1-E7, and F1-F3); the aggregate genome size was approximately 240 kb, and the GC content (43%) was similar to that of other IVs (41%-43%). A total of 123 open reading frames were predicted and included typical IV gene families such as repeat element protein (41 members), cysteine motif (10 members), vankyrin (9 members), polar residue-rich protein (7 members), vinnexin (6 members), and N gene (3 members). Neuromodulin N (2 members) was found to be unique to DfIV, along with 45 hypothetical genes. Among the 62 segments, 54 showed high (76%-98%) sequence similarities to the genome of Diadegma semiclausum ichnovirus (DsIV). Three segments, namely, D22, E3, and F2, contained lepidopteran host genome integration motifs with homologous regions of about 36-46 bp between them (Diadegma fenestrale ichnovirus, DfIV and lepidopteran host, Plutella xylostella). Most of the DfIV genes were expressed in the hymenopteran host and some in the lepidopteran host (P. xylostella), parasitized by D. fenestrale. Five segments (A4, C3, C15, D5, and E4) were differentially expressed at different developmental stages of the parasitized P. xylostella, and two segments (C15 and D14) were highly expressed in the ovaries of D. fenestrale. Comparative analysis between DfIV and DsIV revealed that the genomes differed in the number of segments, composition of sequences, and internal sequence homologies.

Mobility of mPing and its associated elements is regulated by both internal and terminal sequences

BackgroundDNA transposable elements are mobilized by a “cut and paste” mechanism catalyzed by the binding of one or more transposase proteins to terminal inverted repeats (TIRs) to form a transpositional complex. Study of the rice genome indicates that the mPing element has experienced a recent burst in transposition compared to the closely related Ping and Pong elements. A previously developed yeast transposition assay allowed us to probe the role of both internal and terminal sequences in the mobilization of these elements.ResultsWe observed that mPing and a synthetic mPong element have significantly higher transposition efficiency than the related autonomous Ping and Pong elements. Systematic mutation of the internal sequences of both mPing and mPong identified multiple regions that promote or inhibit transposition. Simultaneous alteration of single bases on both mPing TIRs resulted in a significant reduction in transposition frequency, indicating that each base plays a role in efficient transposase binding. Testing chimeric mPing and mPong elements verified the important role of both the TIRs and internal regulatory regions. Previous experiments showed that the G at position 16, adjacent to the 5′ TIR, allows mPing to have higher mobility. Alteration of the 16th and 17th base from mPing’s 3′ end or replacement of the 3′ end with Pong 3′ sequences significantly increased transposition frequency.ConclusionsAs the transposase proteins were consistent throughout this study, we conclude that the observed transposition differences are due to the element sequences. The presence of sub-optimal internal regions and TIR bases supports a model in which transposable elements self-limit their activity to prevent host damage and detection by host regulatory mechanisms. Knowing the role of the TIRs, adjacent sub-TIRs, and internal regulatory sequences allows for the creation of hyperactive elements.

Cratonal tidal deposits of the Paleoproterozoic Mesnard Quartzite, Lake Superior region: interpretation challenges

The Mesnard Quartzite of Paleoproterozoic age is believed to represent among the first preserved cratonal continental rocks in the Lake Superior region, was deposited above Archean basement in the Marquette structural trough and is estimated to contain more than 99% sandstone (now quartzite) beds. The Mesnard Quartzite is interpreted to have been deposited largely by tidal currents. Evidence of tidal current deposition includes lack of wave-influenced structures such as hummocky cross-beds, and presence of strongly unidirectional paleocurrents from cross-beds, mud-drapes on cross-bed foresets, evidence of current reversal including herringbone cross-bedding and sigmoidal cross-bedding. Sandstone bedding types include homogeneous or structureless beds, parallel-laminated beds, cross-beds, and symmetric ripple marks. Unidirectional paleocurrents from cross-beds are largely subparallel to the axis of the Marquette structural trough, leading to the interpretation that the trough was subsiding during deposition. Cross-bed set thickness and symmetric ripple marks both support the interpretation of shallow water marine deposition, likely only a few meters. Although the Mesnard Quartzite is on the order of 110 m thick, there is no evidence of internal sequence boundaries or flooding surfaces, and low accommodation during deposition is inferred. The climate during deposition is interpreted to be humid, and tropical to subtropical based on the quartz arenite composition. Deposition likely occurred in a failed rift basin or in an intracratonic extensional basin.

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions.

Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA (≥50 bp) referred to as copy number variation (CNV). In humans, CNV may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive CNV, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

An on-line seru scheduling algorithm with proactive waiting considering resource conflicts

Seru production systems (SPSs) with quick responsiveness and high flexibility have been applied in many Asian electronic enterprises. An SPS can quickly respond to market demands with changing varieties and fluctuated volumes. Resource conflicts are used to indicate the requirements for a same worker by more than one seru. This paper studies the on-line seru scheduling problem with resource conflicts in limited work space, which decides serus’ built-up sequence over time and aims at minimizing the makespan. To keep a balance between quick responsiveness and production efficiency in an SPS, a proactive waiting strategy is proposed and a nested algorithm is structured with an external framework and an internal sequencing policy. The external framework handles resource conflicts among serus and divides serus into groups in which resource conflicts do not exist. The internal sequencing policy acts on each group and decides the built-up sequence for serus. The nested algorithm is proved to be 2ρ-competitive with two seru families. Performance of the nested algorithm can be enhanced by improving the internal sequencing policy without changing the external framework, which is conformed by computational experiments. With more seru families, experimental results show that the nested-LPT algorithm can adapt to frequent arrivals of orders, complicated resource conflicts, and fluctuating market demands.

Time-Varying Sequence Model

Traditional machine learning sequence models, such as RNN and LSTM, can solve sequential data problems with the use of internal memory states. However, the neuron units and weights are shared at each time step to reduce computational costs, limiting their ability to learn time-varying relationships between model inputs and outputs. In this context, this paper proposes two methods to characterize the dynamic relationships in real-world sequential data, namely, the internal time-varying sequence model (ITV model) and the external time-varying sequence model (ETV model). Our methods were designed with an automated basis expansion module to adapt internal or external parameters at each time step without requiring high computational complexity. Extensive experiments performed on synthetic and real-world data demonstrated superior prediction and classification results to conventional sequence models. Our proposed ETV model is particularly effective at handling long sequence data.

Regional mechanism for sustainable development

Technologization and resource consumption of modern regional production complexes actualizes the paradigm of their sustainable development. Ecosystem development of mesoterritoriums requires the use of integrated approaches, the creation of conditions and incentives. The concept of using an integrated mechanism to achieve sustainable development of the region as a technology for managing the territorial economy, focused on improving the efficiency of territorial production complexes, the implementation of priority areas in science, technology and critical technologies is put forward. The components of the management tools for the sustainable development of mesosystems are proposed, representing the internal structure and sequence of stages of interrelated directions of movement of the territorial economy to the desired state. This mechanism can be considered as a set of actions, the implementation of which will lead to socio-economic progress at a new level. A comprehensive regional mechanism of sustainable development is substantiated, which activates the process of economic and social changes in five directions: economic, spatial, social, digital and environmental, ensuring the implementation of the strategy of transformational processes. The proposed integrated regional mechanism of sustainable development can be applied at different levels of territorial management. In the conditions of modern realities of the implementation of the new regionalism, it is advisable to ensure stable trends in territorial development on the basis of platform and ecosystem approaches.

D3GF: A Study on Optimal Defense Performance Evaluation of Drone-Type Moving Target Defense Through Game Theory

Based on the paradigm shift in modern warfare, ground forces conduct pilot operations of wireless unmanned maneuvering systems, such as tactical drones, in the form of manned and unmanned cooperative tactics after deploying the relevant systems in the battlefield. However, security considerations for relevant systems are limited to the scope of using only the existing end-to-end encryption and public key authentication modules, and no defense strategy to actively respond to specialized cyber-electronic warfare threats has been officially established. To drastically reduce both the potential attack surface and security vulnerabilities of drones employed in network-centric-warfare, a proactive defense technology that expires the effectiveness of attacks by avoiding invasive action at the target is expected to be essential. Accordingly, this paper proposes the concept of active moving-target-defense (MTD), an element of cyber deception that minimizes the rate of success of cyber-attacks while conversely maximizing both defense predominance and attack complexity asymmetrically, exclusive according to partially observable Markov decision process (POMDP)-based threat modeling that considers both the internal and external operation sequences of target drones. To optimally design the proposed drone-type MTD based on the Pareto frontier, we additionally advanced and simulated a drone-based defensive deception game framework (D3GF), which represents a general-sum combat framework reflecting decision logics such as the perfect Bayesian Nash equilibrium, stochastic Stackelberg, and partial signal game. This study was conducted to compare and calculate the efficiencies of the drone-type MTD’s deceptive defense, which had not been considered in prior studies, by unique environmental features inside and outside the drone. Furthermore, we conducted a detailed performance evaluation considering game metrics based on sensitivity analysis. Hereafter, the drone-type MTD will be extended into an actual active drone protection technology combining cyber flare-type avoidance strategies and cyber camouflage-type disarrangement strategies by expanding its optimization domain as a hypergame, while integrating it with drone decoy elements.

SR Protein Kinase 1 Inhibition by TAF15.

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases' partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins.

LB1578. Fully Automated Real-Time PCR for Detection of Candida auris

Abstract Background Candida auris is an emerging fungus that presents a serious global health threat. It is often multidrug-resistant and difficult to identify with standard laboratory methods. C. auris outbreaks have been associated with high mortality in healthcare settings. Rapid and accurate screening for C. auris is crucial for preventing transmission and directing treatment. This study evaluated a new fully automated, lab-developed real-time PCR assay for the detection of C. auris. Methods Nasal swabs collected with Aptima Multitest swabs were used as a representative clinical matrix to evaluate C. auris detection using the Open Access™ functionality on the Panther Fusion® instrument. It provided fully automated nucleic acid extraction, amplification, and detection workflow. The primers and probe are published sequences designed by CDC which target the internal transcribed spacer 2 (ITS2) region of the C. auris ribosomal RNA gene. Analytical studies evaluated assay sensitivity, specificity, and inclusivity. An internal control is also included to ensure assay validity. Results The limit of detection (≥ 95% detection rate) of the Candida auris real-time PCR assay is 5 CFU/mL for nasal swabs. Eleven C. auris strains from CDC clinical isolates were reactive for inclusivity study. No cross reactivity or interference was observed from 60 microorganisms of closely related yeast, bacteria and viruses typically present in skin and groin samples. Conclusion A fully automated and high throughput real-time PCR test for C. auris with internal control, utilizing primers and probe sequences published by CDC, demonstrated excellent sensitivity, specificity, and inclusivity. This lab developed test (LDT) provides a rapid, sensitive, and scalable method for detection of C. auris. The results are crucial for timely detection, transmission prevention and targeted treatment of the multidrug-resistant C. auris. Disclosures All Authors: No reported disclosures.

Targeted Nanopore Resequencing and Methylation Analysis of LINE-1 Retrotransposons.

Retrotransposition of LINE-1 (L1) elements represents a major source of insertional polymorphisms in mammals, and their mutagenic activity is restricted by silencing mechanisms, such as DNA methylation. Despite a very high level of sequence identity between copies, their internal sequence contains small nucleotide polymorphisms (SNPs) that can alter their activity. Such internal SNPs can also appear in different alleles of a given L1 locus. Given their repetitive nature and relatively long size, short-read sequencing approaches have limited access to L1 internal sequence or DNA methylation state. Here, we describe a targeted method to specifically sequence more than a hundred L1-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. Our protocol, modified from the nanopore Cas9 targeted sequencing (nCATS) strategy, provides a full and haplotype-resolved L1 sequence and DNA methylation levels. It introduces a streamlined and multiplex approach to synthesize guide RNAs and a quantitative PCR (qPCR)-based quality check during library preparation for cost-effective L1 sequencing. More generally, this method can be applied to any type of transposable elements and organisms.

Tracking human skill learning with a hierarchical Bayesian sequence model.

Humans can implicitly learn complex perceptuo-motor skills over the course of large numbers of trials. This likely depends on our becoming better able to take advantage of ever richer and temporally deeper predictive relationships in the environment. Here, we offer a novel characterization of this process, fitting a non-parametric, hierarchical Bayesian sequence model to the reaction times of human participants' responses over ten sessions, each comprising thousands of trials, in a serial reaction time task involving higher-order dependencies. The model, adapted from the domain of language, forgetfully updates trial-by-trial, and seamlessly combines predictive information from shorter and longer windows onto past events, weighing the windows proportionally to their predictive power. As the model implies a posterior over window depths, we were able to determine how, and how many, previous sequence elements influenced individual participants' internal predictions, and how this changed with practice. Already in the first session, the model showed that participants had begun to rely on two previous elements (i.e., trigrams), thereby successfully adapting to the most prominent higher-order structure in the task. The extent to which local statistical fluctuations in trigram frequency influenced participants' responses waned over subsequent sessions, as participants forgot the trigrams less and evidenced skilled performance. By the eighth session, a subset of participants shifted their prior further to consider a context deeper than two previous elements. Finally, participants showed resistance to interference and slow forgetting of the old sequence when it was changed in the final sessions. Model parameters for individual participants covaried appropriately with independent measures of working memory and error characteristics. In sum, the model offers the first principled account of the adaptive complexity and nuanced dynamics of humans' internal sequence representations during long-term implicit skill learning.

Comparative genomics and informational content analysis uncovered internal regions of the core genes rpoD, pepN and gltX for an MLSA with genome-level resolving power within the genus Pseudomonas

In the field of prokaryotic taxonomy, there has been a recent transition towards phylogenomics as the gold standard approach. However, genome-based phylogenetics is still restrictive for its cost when managing large amounts of isolates. Fast, cheap, and taxonomically competent alternatives, like multilocus sequence analysis (MLSA) are thus recommendable. Nevertheless, the criteria for selecting the conserved genes for MLSA have not been explicit for different bacterial taxa, including the broadly diverse Pseudomonas genus. Here, we have carried out an unbiased and rational workflow to select internal sequence regions of Pseudomonas core genes (CG) for a MLSA with the best phylogenetic power, and with a resolution comparable to the genome-based ANI approach. A computational workflow was established to inspect 126 complete genomes of representatives from over 60 Pseudomonas species and subspecies, in order to identify the most informative CG internal regions and determine which combinations in sets of three partial CG sequences have comparable phylogenetic resolution to that of the current ANI standard. We found that the rpoD346-1196-pepN1711-2571-gltX86-909 concatenated sequences were the best performing in terms of phylogenetic robustness and resulted highly sensitive and specific when contrasted with ANI. The rpoD-pepN-gltX MLSA was validated in silico and in vitro. Altogether, the results presented here supports the proposal of the rpoD-pepN-gltX MLSA as a fast, affordable, and robust phylogenetic tool for members of the Pseudomonas genus.

Selective Strategy Differential Evolution for Stochastic Internal Task Scheduling Problem in Cross-Docking Terminals.

This study proposed an algorithm called selective strategy differential evolution (SSDE) to handle the complexity of the stochastic internal task scheduling problem in cross-docking terminals. The aims of this study are to assign workers and transfer equipment to internal operations and sequence those operations under randomness and uncertainty with the purpose to minimise total tardiness. The main feature of SSDE is its ability to adapt itself in order to execute the best search strategy. The proposed algorithm was tested on 16 instances using generated data based on real-case scenarios of a pharmaceutical distribution centre. The results showed the significant performance of SSDE to other existing algorithms in terms of solution quality and computational time. The key success factors of SSDE are the use of various search strategies in a single run and the application of suitable termination conditions.

104. H3K27-altered diffuse midline gliomas with secondary driver molecular alterations

Large-scale sequencing efforts have identified co-occurring driver events, such as activating mutations in FGFR1 and BRAF, in occasional diffuse midline gliomas (DMGs) H3K27-altered, but their significance is incompletely explored. To better understand the clinicopathologic features of these tumors, we identified all H3K27-mutant gliomas from our internal sequencing database (2013-2021) of over 2,000 gliomas and collected detailed clinical information, pathologic features, mutational signatures, and copy number profiles for the resulting cohort. We identified 77 patients (age range 2-68, median 26). The histologic diagnoses were DMG (n=55), anaplastic astrocytoma/glioblastoma (n=19), low-grade glioma (n=1), low-grade glioneuronal tumor (n=1), and ganglioglioma (n=1). Commonly recurring alterations were seen in TP53 (n=42), ATRX (n=17), NF1 (n=15), and PDGFRA (n = 4). Five cases had BRAF V600E (1 ganglioglioma; 4 DMG); 12 had FGFR1 N546K/D or K656E mutations (9 DMG; 3 anaplastic astrocytoma/glioblastoma). BRAF-mutant tumors most commonly arose in the brainstem, while FGFR1-mutant tumors were mostly located within the thalamus. Median overall survival for DMGs lacking BRAF or FGFR1 mutations was 12.9 months, while median survival for FGFR1-mutant gliomas was 14.2 months (p=0.186) and 22.5 months for BRAF-mutant tumors (p=0.285). Two patients with low-grade histology (ganglioglioma and low-grade glioneuronal tumor) were still alive at last follow-up (37 months and 78 months, respectively). Despite the failure to reach statistical significance, improved survival trends were observed for patients with secondary molecular drivers compared with conventional H3K27M DMG, especially in those with low-grade histology. These findings and the possibility of targeted therapy argue for comprehensive sequencing of H3K27-altered gliomas.

Cloning of Maize TED Transposon into Escherichia coli Reveals the Polychromatic Sequence Landscape of Refractorily Propagated Plasmids.

MuDR, the founder member of the Mutator superfamily and its MURA transcripts, has been identified as toxic sequences to Escherichia coli (E. coli), which heavily hindered the elucidation of the biochemical features of MURA transposase and confined the broader application of the Mutator system in other organisms. To harness less constrained systems as alternatives, we attempted to clone TED and Jittery, two recently isolated autonomous Mutator-like elements (MULEs) from maize, respectively. Their full-length transcripts and genomic copies are successfully cloned when the incubation time for bacteria to recover from heat shock is extended appropriately prior to plating. However, during their proliferation in E. coli, TED transformed plasmids are unstable, as evidenced by derivatives from which frameshift, deletion mutations, or IS transposon insertions are readily detected. Our results suggest that neither leaky expression of the transposase nor the presence of terminal inverse repeats (TIRs) are responsible for the cloning barriers, which were once ascribed to the presence of the Shine–Dalgarno-like sequence. Instead, the internal sequence of TED (from 1250 to 2845 bp), especially the exons in this region, was the most likely causer. The findings provide novel insights into the property and function of the Mutator superfamily and shed light on the dissection of toxic effects on cloning from MULEs.