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Abstract Objective The eukaryotic tree of life has been subject of numerous studies ever since the nineteenth century, with more supergroups and their sister relations being decoded in the last years. In this study, we reconstructed the phylogeny of eukaryotes using complete 18S rDNA sequences and their individual secondary structures simultaneously. After the sequence-structure data was encoded, it was automatically aligned and analyzed using sequence-only as well as sequence-structure approaches. We present overall neighbor-joining trees of 211 eukaryotes as well as the respective profile neighbor-joining trees, which helped to resolve the basal branching pattern. A manually chosen subset was further inspected using neighbor-joining, maximum parsimony, and maximum likelihood analyses. Additionally, the 75 and 100 percent consensus structures of the subset were predicted. Results All sequence-structure approaches show improvements compared to the respective sequence-only approaches: the average bootstrap support per node of the sequence-structure profile neighbor-joining analyses with 90.3, was higher than the average bootstrap support of the sequence-only profile neighbor-joining analysis with 73.9. Also, the subset analyses using sequence-structure data were better supported. Furthermore, more subgroups of the supergroups were recovered as monophyletic and sister group relations were much more comparable to results as obtained by multi-marker analyses.

The manufacturing industry increasingly needs to shift toward energy-efficient production patterns to address both environmental pollution reduction and higher energy prices. Despite efforts to enhance energy efficiency and to close the energy efficiency gap in small- and medium-sized enterprises (SMEs) limited awareness, lack of know-how, and restricted financial and human resources still hinder large-scale adoption. This is partly because current research on energy flow analysis (EFA) often lacks structured and detailed methodologies and neglects a suitable evaluation of potential greenhouse gas (GHG) emission reductions. In response, we derived a framework from existing literature and applied action research to tailor it to identify energy flows and cost-effective reduction potentials for GHG emissions in SMEs. The framework aims to improve awareness, transparency, and decision-making regarding energy consumption and environmental protection within SMEs by combining GHG accounting with comprehensive EFA to enhance energy efficiency. Furthermore, the framework outlines specific activities to guide SMEs through the implementation process. Two case studies were conducted in partnership with companies operating in the automotive and environmental sectors to develop and validate the framework. Although the case studies focused on non-energy-intensive industries, they revealed untapped potential for reducing energy consumption. Both case studies showed reductions in energy use through the identified measures, achieving reductions of 900,000 kWh and 1,300,000 kWh, respectively. At 2021 energy price levels, these reductions led to a significant drop in energy costs: a 16% drop in case one and 22% in case two. Additionally, insights from the case studies have led to further refinements of the framework, emphasizing the necessity of tailored solutions and resource considerations for SMEs. These findings helped to identify opportunities for future research directions toward optimizing energy management practices in SMEs.

Abstract The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.

Abstract Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV’s counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base preference for the interaction and a unique Vif-ribose contact. This establishes the biological significance of an RNA ligand. Further assessment of interactions between A3G, Vif, and RNA ligands show that the A3G-Vif assembly and subsequent ubiquitination can be controlled by amino acid mutations at the interface or by polynucleotide modification, suggesting that a specific chemical moiety would be a promising pharmacophore to inhibit the A3G-Vif interaction.

Abstract COVID-19 mRNA vaccines induce protective adaptive immunity against SARS-CoV-2 in most individuals, but there is wide variation in levels of vaccine-induced antibody and T-cell responses. However, the mechanisms underlying this inter-individual variation remain unclear. Here, using a systems biology approach based on multi-omics analyses of human blood and stool samples, we identified several factors that are associated with COVID-19 vaccine-induced adaptive immune responses. BNT162b2-induced T cell response is positively associated with late monocyte responses and inversely associated with baseline mRNA expression of activation protein 1 (AP-1) transcription factors. Interestingly, the gut microbial fucose/rhamnose degradation pathway is positively correlated with mRNA expression of AP-1, as well as a gene encoding an enzyme producing prostaglandin E2 (PGE2), which promotes AP-1 expression, and inversely correlated with BNT162b2-induced T-cell responses. These results suggest that baseline AP-1 expression, which is affected by commensal microbial activity, is a negative correlate of BNT162b2-induced T-cell responses.

We demonstrate theoretically and experimentally an electromagnetic lensing concept using the magnetic vector potential—in a region free of classical electromagnetic fields—via the Aharonov–Bohm (AB) effect. This toroid-shaped lens with poloidal current flow allows for electromagnetic lensing which can be tuned to be convex or concave with a spherical aberration coefficient of opposite polarity to its focal length. This field-free lens combines the advantages of traditional electromagnetic and electrostatic field-based lenses and opens up additional possibilities for the optical design of charged-particle systems. More generally, these results demonstrate that the AB effect can shape charged particle wavefronts beyond simple step shifts if topologies beyond simple flux lines are considered and further supports the physical significance of the magnetic vector potential.

Abstract We introduce the Solar Farm Cable Layout Problem (SoFaCLaP), a novel graph-theoretic optimization problem. SoFaCLaP formalizes the task of finding a cost-optimal cable layout in a solar farm where PV string positions are already determined but the positions of other components such as transformers can be picked from a set of candidate positions. The problem statement incorporates a network flow model in which the flow value of a connection represents the number of strings that are (indirectly) connected to a transformer via this connection. A mixed-integer linear program (MILP) formulation is proposed that uses binary variables to indicate which of several available cable types is chosen for each connection. We propose a framework to randomly generate benchmark instances to evaluate any algorithmic approach to SoFaCLaP. In particular, we generate a set of instances based on real-world solar farm characteristics. With an extensive evaluation of the MILP formulation on those instances we establish mixed-integer linear programming as a baseline for future algorithmic approaches to finding solar farm cable layouts.

Summary: Thalamoreticular circuitry plays a key role in arousal, attention, cognition, and sleep spindles, and is linked to several brain disorders. A detailed computational model of mouse somatosensory thalamus and thalamic reticular nucleus has been developed to capture the properties of over 14,000 neurons connected by 6 million synapses. The model recreates the biological connectivity of these neurons, and simulations of the model reproduce multiple experimental findings in different brain states. The model shows that inhibitory rebound produces frequency-selective enhancement of thalamic responses during wakefulness. We find that thalamic interactions are responsible for the characteristic waxing and waning of spindle oscillations. In addition, we find that changes in thalamic excitability control spindle frequency and their incidence. The model is made openly available to provide a new tool for studying the function and dysfunction of the thalamoreticular circuitry in various brain states.

Musculoskeletal disorders remain the most common work-related health problem in the European Union. The most common work-related musculoskeletal disorder reported by workers is backache, especially in the logistics sector. Thus, this article aims to evaluate the effects of a commercial passive low-back exoskeleton during simulated logistics tasks. Thirty participants were recruited for this study. Typical logistics tasks were simulated in a laboratory environment. Cross-over research design was utilized to assess the effects of the exoskeleton on heart rate, trunk inclination, trunk acceleration, throughput, and perceived exertion. Also, usability and acceptance were obtained using a custom questionnaire. We found mostly non-significant differences. Effects on throughput varied widely between workplaces. Usability ratings were poor and acceptance moderate. The study suggests that a holistic evaluation and implementation approach for industrial exoskeletons is necessary. Further, prior to exoskeleton implementation, workplace adaptation might be required.

It is well known that insulating samples can accumulate electric charges from exposure to an electron beam. How the accumulation of charge affects imaging parameters and sample stability in transmission electron microscopy is poorly understood. To quantify these effects, it is important to know how the charge is distributed within the sample and how it builds up over time. In the present study, we determine the spatial distribution and temporal dynamics of charge accumulation on vitreous ice samples with embedded proteins through a combination of modeling and Fresnel diffraction experiments. Our data reveal a rapid evolution of the charge state on ice upon initial exposure to the electron beam accompanied by charge gradients at the interfaces between ice and carbon films. We demonstrate that ice film movement and charge state variations occur upon electron beam exposure and are dose-rate dependent. Both affect the image defocus through a combination of sample height changes and lensing effects. Our results may be used as a guide to improve sample preparation, data collection, and data processing for imaging of dose-sensitive samples.

Immature poxviruses are characterized by nonicosahedral semiordered protein scaffolds critical for morphogenesis. Here, the authors use cryo-EM structures of Vaccinia virus D13 scaffold intermediates to explain their assembly mechanism.

Nakano et al. use high-speed AFM and cryo-EM to study influenza virus RNA synthesis in vitro. They observe the synthesis of two types of RNA: folded single stranded RNA associated to an intact viral ribonucleoprotein (vRNP) and looped, double stranded RNA associated with a partially rearranged vRNP structure.

Abstract During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

The variable regions (V1–V9) of the 18S rDNA are routinely used in barcoding and phylogenetics. In handling these data for trypanosomes, we have noticed a misunderstanding that has apparently taken a life of its own in the literature over the years. In particular, in recent years, when studying the phylogenetic relationship of trypanosomes, the use of V7/V8 was systematically established. However, considering the current numbering system for all other organisms (including other Euglenozoa), V7/V8 was never used. In Maia da Silva et al. [Parasitology 2004, 129, 549–561], V7/V8 was promoted for the first time for trypanosome phylogenetics, and since then, more than 70 publications have replicated this nomenclature and even discussed the benefits of the use of this region in comparison to V4. However, the primers used to amplify the variable region of trypanosomes have actually amplified V4 (concerning the current 18S rDNA numbering system).

Abstract In the Wind Farm Cabling Problem (WCP) the task is to design the internal cabling of a wind farm such that all power from the turbines can be transmitted to the substations and the costs for the cabling are minimized. Cables can be chosen from several available cable types, each of which has a thermal capacity and cost. Until now, solution approaches mainly use Mixed-integer Linear Programs (MILP) or metaheuristics. We present our current state of research on a fast heuristic specifically designed for WCP. We introduce an algorithm that iteratively improves a cable layout by finding and canceling negative cycles in a suitably defined network. Our simulations on publicly available benchmark sets show that the heuristic is not only fast but it tends to produce good results. Currently our algorithm gives better solutions on large wind farms compared to an MILP solver. However, on small to medium instances the solver performs better in terms of solution quality, which represents a starting point for future work.

Pressure to increase effectiveness and efficiency drives the product development process. The software industry has been using agile development approaches due to their advantages when coping with uncertainties and simultaneously increasing value. Therefore, the following work deals with the considerations of agile powertrain development. In order to identify important conditions for the incorporation of agile principles into powertrain development, semistructured interviews were conducted with experts from an engineering company. Having identified agile development challenges, advantages, and principles, the next step is comparing agile principles with the current principles of conventional powertrain development. Furthermore, a basic understanding of the powertrain development process regarding the incorporation of agile principles is established, the main attributes of powertrain development are investigated, and the key disciplines involved are identified. Finally, a model is created to assess whether the application of an agile or conventional development approach is more suitable.

To ask students what a species is always has something rhetorical about it. Too quickly comes the rote answer, often learned by heart without ever thinking about it: “A species is a reproductive community of populations (reproductively isolated from others), which occupies a specific niche in nature” (Mayr 1982). However, do two people look alike because they are twins or are they twins because they look alike? “Two organisms do not belong to the same species because they mate and reproduce, but they only are able to do so because they belong to the same species” (Mahner and Bunge 1997). Unfortunately, most biology (pre-university) teachers have no opinion on whether species are real or conceptual, simply because they have never been taught the question themselves, but rather one answer they still pass on to their students today, learned by heart without ever thinking about it. Species are either real or conceptual and, in my opinion, it is this “or” that we should teach about. Only then can we discuss those fundamental questions such as who or what is selected, who or what evolves and, finally, what is biodiversity and phylogenetics all about? Individuals related to each other by the tree of life.

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

The bacterial flagellar hook is made up of many copies of the protein FlgE. Here, the authors report the full structure of the hook from Campylobacter jejuni and show that its overall structure is different from that of the previously published filament.

Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA.

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca2+ ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca2+]i clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca2+ clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.

In the original publication of this article (Gritzbach et al., 2018), an incorrect version of Algorithm 1 was used. In this correction article the corrected version of Algorithm 1 is shown. The original publication of this article has been corrected.

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

Compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) rDNA secondary structures correlate with Ernst Mayr's biological species concept. This hypothesis also referred to as the CBC species concept recently was subjected to large-scale testing, indicating two distinct probabilities. (1) If there is a CBC then there are two different species with a probability of ∼0.93. (2) If there is no CBC then there is the same species with a probability of ∼0.76. In ITS2 research, however, the main problem is the multicopy nature of ITS2 sequences. Most recently, 454 pyrosequencing data have been used to characterize more than 5000 intragenomic variations of ITS2 regions from 178 plant species, demonstrating that mutation of ITS2 is frequent, with a mean of 35 variants per species, respectively per individual organism. In this study, using those 454 data, the CBC criterion is reconsidered in the light of intragenomic variability, a proof of concept, a necessary criterion, expecting no intragenomic CBCs in variant ITS2 copies. In accordance with the CBC species concept, we could demonstrate that the probability that there is no intragenomic CBC is ∼0.99.

BACKGROUND:Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta. METHODOLOGY/PRINCIPAL FINDINGS:Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses. CONCLUSIONS/SIGNIFICANCE:Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.

Phylogenetic tree reconstruction is traditionally based on multiple sequence alignments (MSAs) and heavily depends on the validity of this information bottleneck. With increasing sequence divergence, the quality of MSAs decays quickly. Alignment-free methods, on the other hand, are based on abstract string comparisons and avoid potential alignment problems. However, in general they are not biologically motivated and ignore our knowledge about the evolution of sequences. Thus, it is still a major open question how to define an evolutionary distance metric between divergent sequences that makes use of indel information and known substitution models without the need for a multiple alignment. Here we propose a new evolutionary distance metric to close this gap. It uses finite-state transducers to create a biologically motivated similarity score which models substitutions and indels, and does not depend on a multiple sequence alignment. The sequence similarity score is defined in analogy to pairwise alignments and additionally has the positive semi-definite property. We describe its derivation and show in simulation studies and real-world examples that it is more accurate in reconstructing phylogenies than competing methods. The result is a new and accurate way of determining evolutionary distances in and beyond the twilight zone of sequence alignments that is suitable for large datasets.