bluuebunny/crossref_metadata_2025 huggingface dataset:
Farmed crickets raised with dermestids suffer from reduced and delayed growth, but not enough to explain reports of dramatic yield loss
Authors: Muzzatti MJ , Ritchie MW , Bess EC , Bertram SM , MacMillan HA
Published: June 2024

The mass production of crickets for food and feed is an expanding North American industry. Facilities that mass rear insects are at risk of pest infestations because the optimal environmental conditions for rearing beneficial species may also support the development of pest species. Here, we present the first recorded results detailing the interactions between dermestids and farmed crickets. Cricket farms have reported extremely low harvest yield during heavy dermestid infestations, but the exact reasons for this low yield are unknown. Many dermestid larvae are covered in dense, detachable, barbed setae called hastisetae, which are used by the larvae as an active trapping system against arthropod predators. We designed a series of experiments to test the hypotheses that dermestids (Dermestes aterDeGeer) may be directly impacting cricket (Gryllodes sigillatusWalker) yield through the physical effects of hastisetae ingestion and/or indirectly impacting cricket yield through competition for fishmeal, a primary source of protein in conventional cricket feed. Our predictions that cricket life history and survival would be negatively affected by dermestids were largely refuted. Females fed infested diets grew less mass, but not smaller body size, compared to females fed uninfested diets. We also found that while crickets experienced delayed growth early in life after living with dermestids, they were able to tolerate living with, and consuming, dermestid larvae. We discuss how these findings have led to new hypotheses concerning how dermestid infestations drive reductions in cricket farm yield.
Sublethal insecticide exposure of larvae affects the blood-feeding behaviour of adult mosquitoes
Authors: Zeferino Tiago G. , Acerbi Gwendoline , Koella Jacob C.
Published: June 2024

Because of their widespread use for the control of disease vectors and agricultural pests, insecticides have become ubiquitous in the environment, including in water bodies harbouring mosquito larvae. These are therefore continuously exposed to sublethal doses. Since this has long-lasting effects on the mosquitoes’ physiology and life-history, we expected that it may also affect behaviours that underlie the mosquitoes’ population dynamics and disease epidemiology, such as egg-laying preference, blood-feeding motivation, and host-seeking behaviour. Using an insecticide-sensitive and a resistant strain ofAnopheles gambiae, an important malaria vector, we evaluated the effects of sublethal exposure to permethrin throughout larval development on the resistance to the insecticide in adults, on host-seeking behaviour, on the motivation to blood-feed, and on egg-laying behaviour. Resistance, assessed by rates of knock-down and mortality, were similar between exposed and unexposed mosquitoes. However, exposure to sublethal doses of insecticide caused female mosquitoes to split their egg clutches into two parts and increased the motivation of mosquitoes to seek blood meals through permethrin-treated nets, regardless of their sensitivity to the insecticide. Furthermore, it enhanced the natural preference of resistant strains for permethrin-treated nets and increased their blood-meal size. Our results thus suggest that sublethal insecticide concentrations in larval breeding sites have important epidemiological implications.
Susceptibility Patterns of Multiple Antibiotic-Resistant Bacteria from Wound and Urine Samples to the Extract ofSpondias Mombin(Linn)
Authors: Okiti AF , Oladunmoye MK , Ogundare AO
Published: June 2024

This study evaluates the antibacterial activity ofSpondias mombinL. against multiple antibiotic-resistant bacteria isolated from wound and urine samples of patients attending five (5) selected hospitals in Akure, Ondo State, Nigeria. A total of 313 bacterial isolates were isolated from 353 samples of wound and urine using standard bacteriological procedures, withPseudomonas aeruginosaandStaphylococcus aureusbeing the most predominant in urine and wound samples, respectively. The methanolic extract ofS. mombinwas the most effective against wound isolates, while the aqueous extract was the most effective against urine isolates. The results showed that the methanol extract ofS. mombinhad a zone of inhibition of 24.00±0.00, 30.67±0.33 and 19.33±0.33 mm respectively, againstS. aureus, S. epidermidisandP. aeruginosaat 100 mg/ml. The aqueous extract had a zone of inhibition of 24.67±0.33, 27.33±0.33, 18.67±0.33, 24.67±0.33, 23.67±0.33, 21.33±0.33 and 21.67±0.33 mm againstEscherichia coli, Klebsiella pneumoniae, P. aeruginosa, Proteus mirabilis, S. aureus, S. saprophyticusandTrichomonas vaginalisrespectively, at 100 mg/ml. The phytochemical constituents of the extracts include alkaloids, anthraquinones, cardiac glycosides, flavonoids, phenols, saponins, steroids and tannins. These compounds may be responsible for the antibacterial activity ofS. mombinagainst the multiple antibiotic-resistant bacterial isolates. The findings of this study demonstrates the potential ofS. mombinas an alternative treatment for multiple antibiotic-resistant bacteria from wound and urine.
Candida albicansenhanceStaphylococcus aureusvirulence by progressive generation of new phenotypes
Authors: Arévalo-Jaimes Betsy Verónica , Torrents Eduard
Published: June 2024

Candida albicansandStaphylococcus aureushave been co-isolated from several biofilm- associated diseases, including those related to medical devices. This association confers advantages to both microorganisms, resulting in detrimental effects on the host. To elucidate this phenomenon, the present study investigated colony changes derived from non-physical interactions betweenC. albicansandS. aureus. We performed proximity assays by confronting colonies of the yeast and the bacteria on agar plates at six different distances for 9 days. We found that colony variants ofS. aureusoriginated progressively after prolonged exposure toC. albicansproximity, specifically in response to pH neutralization of the media by the fungi. The new phenotypes ofS. aureuswere more virulent in aGalleria mellonellalarvae model compared to colonies grown withoutC. albicansinfluence. This event was associated with an upregulation ofRNAIIIandAgrAexpression, suggesting a role for α-toxin. Our findings indicate thatC. albicansenhancesS. aureusvirulence by inducing the formation of more aggressive colonies.

Importance

For decades, it has been known thatC. albicansincreaseS. aureusvirulence, resulting in a “lethal synergism”. However, it was only recently identified that this outcome is driven by the sustained activation of the staphylococcalagrsystem in response toC. albicansenvironmental modifications. Our experimental design allowed us to observe individual changes over time caused by the proximity of both microorganisms. As a result, we report for first time thatC. albicansexposure induces the generation and favors the growth ofS. aureuscolony variants with increased expression of virulence factors. Our findings highlight the importance to understanding the intricate connection between environmental responses, virulence and fitness inS. aureuspathogenesis.
Control of innate olfactory valence by segregated cortical amygdala circuits
Authors: Howe James R. , Chan Chung-Lung , Lee Donghyung , Blanquart Marlon , Lee James H. , Romero Haylie K. , Zadina Abigail N. , Lemieux Mackenzie E. , Mills Fergil , Desplats Paula A. , Tye Kay M. , Root Cory M.
Published: June 2024

Animals exhibit innate behaviors that are stereotyped responses to specific evolutionarily relevant stimuli in the absence of prior learning or experience. These behaviors can be reduced to an axis of valence, whereby specific odors evoke approach or avoidance responses. The posterolateral cortical amygdala (plCoA) mediates innate attraction and aversion to odor. However, little is known about how this brain area gives rise to behaviors of opposing motivational valence. Here, we sought to define the circuit features of plCoA that give rise to innate attraction and aversion to odor. We characterized the physiology, gene expression, and projections of this structure, identifying a divergent, topographic organization that selectively controls innate attraction and avoidance to odor. First, we examined odor-evoked responses in these areas and found sparse encoding of odor identity, but not valence. We next considered a topographic organization and found that optogenetic stimulation of the anterior and posterior domains of plCoA elicits attraction and avoidance, respectively, suggesting a functional axis for valence. Using single cell and spatial RNA sequencing, we identified the molecular cell types in plCoA, revealing an anteroposterior gradient in cell types, whereby anterior glutamatergic neurons preferentially expressVGluT2and posterior neurons expressVGluT1. Activation of these respective cell types recapitulates appetitive and aversive behaviors, and chemogenetic inhibition reveals partial necessity for responses to innate appetitive or aversive odors. Finally, we identified topographically organized circuits defined by projections, whereby anterior neurons preferentially project to medial amygdala, and posterior neurons preferentially project to nucleus accumbens, which are respectively sufficient and necessary for innate attraction and aversion. Together, these data advance our understanding of how the olfactory system generates stereotypic, hardwired attraction and avoidance, and supports a model whereby distinct, topographically distributed plCoA populations direct innate olfactory responses by signaling to divergent valence-specific targets, linking upstream olfactory identity to downstream valence behaviors, through a population code. This suggests a novel amygdala circuit motif in which valence encoding is represented not by the firing properties of individual neurons, but by population level identity encoding that is routed through divergent targets to mediate distinct behaviors of opposing appetitive and aversive responses.
Same trait, different genes: pelvic spine loss in three brook stickleback populations in Alberta, Canada
Authors: Mee Jonathan A. , Ly Carolyn , Pigott Grace C.
Published: June 2024

The genetic basis of phenotypic or adaptive parallelism can reveal much about constraints on evolution. This study investigated the genetic basis of a canonically parallel trait: pelvic spine reduction in sticklebacks. Pelvic reduction has a highly parallel genetic basis in threespine stickleback in populations around the world, always involving a deletion of the pel1 enhancer ofPitx1. We conducted a genome-wide association study to investigate the genetic basis of pelvic spine reduction in three populations of brook stickleback in Alberta, Canada. Pelvic reduction did not involvePitx1in any of the three populations. Instead, pelvic reduction in one population involved a mutation in an exon ofTbx4, and it involved a mutation in an intron ofLmbr1in the other two populations. Hence, the parallel phenotypic evolution of pelvic spine reduction across stickleback genera, and among brook stickleback populations, has a non-parallel genetic basis. This suggests that there may be redundancy in the genetic basis of this adaptive polymorphism, but it is not clear whether a lack of parallelism indicates a lack of constraint on the evolution of this adaptive trait. Whether the different pleiotropic effects of different mutations have different fitness consequences, or whether certain pelvic reduction mutations confer specific benefits in certain environments, remains to be determined.

Lay Summary

In this study, we looked for the genetic basis of a well-studied trait in stickleback fish: the pelvic spines. This structure (i.e. the pelvic girdle and attached spines) has a shared developmental basis (and is homologous to) the pelvic bones and hind limbs of all tetrapods (including humans). We know from studying mice, fish, humans, and even manatees that there are several genes that could affect the development of pelvic spines and hind limbs. In one species of stickleback, the threespine stickleback, however, a single gene calledPitx1is always involved in the loss of pelvic spines in populations that have adapted to freshwater lakes. This replicated evolution of the same trait in the same environmental conditions is called parallel evolution. It’s remarkable thatPitx1is always the gene underlying this adaptive loss of spines in freshwater threespine stickleback populations. We were interested in whether this “genetic parallelism” extended to other species of stickleback that have also evolved the loss of pelvic spines. We looked at three populations of brook stickleback (which are never found in the ocean), each of which contains individuals with and without pelvic spines. We found that thePitx1genetic parallelism does not extend to brook stickleback, and, in fact, the genetic basis of pelvic spine loss differs between populations. In Muir Lake and Astotoin Lake, pelvic spine loss results from a mutation in theLmbr1gene, and in Shunda Lake, pelvic spine loss results from a mutation in theTbx4gene.
Polymerization of Tspan7 into Helical Transmembrane Skeletons for Membrane Tether Stabilization
Authors: Wang Dongju , Jia Xia , Zhang Qi , Xu Kui , Zheng Yi , Zhang Qiangfeng Cliff , Liu Nan , Wang Hong-Wei , Yu Li
Published: June 2024

Membrane tethers, thin tubular extensions of the plasma membrane, form during cell migration, adhesion, and signaling. Various types exist, though their maintenance mechanisms are not fully understood. This study employs live cell imaging,in vitroreconstitution, andin situcryo-EM analysis to show that Tspan7, typically a freely diffusing transmembrane protein, forms long cylindrical polymers in right-handed helical formations on retraction fibers. These polymers assemble into spiral tubule structures, acting as a “transmembrane skeleton”, enriching Tspan7 on retraction fibers and promoting their formation. Our findings reveal a novel assembly strategy for transmembrane proteins and a mechanism for maintaining membrane tethers.
The Ptch methylase installs an m3U modification on 28S rRNA for efficient protein synthesis in flies and men
Authors: Chen Jie , Bai Yaofu , Huang Yuantai , Cui Min , Wang Yiqing , Gu Zhenqi , Wu Xiaolong , Li Yubin , Rong Yikang S.
Published: June 2024

The ribosomal RNA (rRNA) is one of the most heavily modified RNA species in nature. Although we have advanced knowledge of the sites, functions and the enzymology of many of the rRNA modifications from all kingdoms, we lack basic understanding on many of those that are not universally present. A single N3modified Uridine base (m3U) was identified on the 28S rRNA from human and frog over thirty years ago, which is absent in bacteria or yeast. Here we show that the equivalent m3U is present in Drosophila, and that the Ptch enzyme and its human homolog are both necessary and sufficient for carrying out the modification. The Ptch-modified U is at a functional center of the large ribosome, and consistentlyptch-mutant cells suffer loss of ribosomal functions. Ptch, proposed to be the most druggable RNA methyltransferases in human, represents a unique target where ribosomal functions could be specifically compromised in cancer cells.
MORC2 phosphorylation fine tunes its DNA compaction activity
Authors: Tan Winnie , Park Jeong Veen , Venugopal Hariprasad , Lou Jie Qiong , Dias Prabavi Shayana , Baldoni Pedro L. , Dite Toby , Moon Kyoung-Wook , Keenan Christine R. , Gurzau Alexandra D. , Leis Andrew , Yousef Jumana , Vaibhav Vineet , Dagley Laura F. , Ang Ching-Seng , Corso Laura , Davidovich Chen , Vervoort Stephin J. , Smyth Gordon K. , Blewitt Marnie E. , Allan Rhys S. , Hinde Elizabeth , D’Arcy Sheena , Ryu Je-Kyung , Shakeel Shabih
Published: June 2024

Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.
Non-canonical altPIDD1 protein: unveiling the true major translational output of thePIDD1gene
Authors: Comtois Frédérick , Jacques Jean-François , Métayer Lenna , Ouedraogo Wend Yam DD , Ouangraoua Aïda , Denault Jean-Bernard , Roucou Xavier
Published: June 2024

Proteogenomics has enabled the detection of novel proteins encoded in non-canonical or alternative open reading frames (altORFs) in genes already coding a reference protein. Reanalysis of proteomic and ribo-seq data revealed that the p53-induced death domain-containing protein (orPIDD1) gene encodes a second 171 amino acid protein, altPIDD1, in addition to the known 910 amino acid-long PIDD1 protein. The two ORFs overlap almost completely, and the translation initiation site of altPIDD1 is located upstream of PIDD1. AltPIDD1 has more translational and protein level evidence than PIDD1 across various cell lines and tissues. In HEK293 cells, the altPIDD1 to PIDD1 ratio is 40 to 1, as measured with isotope-labeled (heavy) peptides and targeted proteomics. AltPIDD1 localizes to cytoskeletal structures labeled with phalloidin, including stress fibres, lamellipodia and filopodia, interacts with cytoskeletal proteins and is cleaved during apoptosis. Unlike most non-canonical proteins, altPIDD1 is not evolutionarily young but emerged in placental mammals. Overall, we identifyPIDD1as a dual-coding gene, with altPIDD1, not the annotated protein, being the primary product of translation.

Summary blurb

This research uncoversPIDD1as a dual-coding gene, revealing a previously unknown protein, altPIDD1, as the primary product with higher expression and cytoskeletal interactions.
LasR regulates protease IV expression at suboptimal growth temperatures inPseudomonas aeruginosa
Authors: Done Rachel E. , Robertson Joshua K. , Prezioso Samantha M. , Goldberg Joanna B.
Published: June 2024

The opportunistic pathogenPseudomonas aeruginosacauses debilitating lung infections in people with cystic fibrosis, as well as eye, burn, and wound infections in otherwise immunocompetent individuals. Many ofP. aeruginosa’s virulence factors are regulated by environmental changes associated with human infection, such as a change in temperature from ambient to human body temperature. One such virulence factor is protease IV (PIV). Interestingly,pivexpression is higher at ambient temperatures (22-28°C) compared to human body temperature (37°C). We found thatpivexpression was thermoregulated at stationary phase, but not exponential phase, and thatpivis thermoregulated at the level of transcription. Protein levels of known transcriptional regulators ofpiv, the quorum sensing regulator LasR and the gene-silencing histone nucleoid silencing proteins MvaT/MvaU, were not thermoregulated. Using a transcriptional reporter forpiv, we show that LasR activatespivexpression at stationary phase at 25°C but not 37°C, while MvaT/MvaU are not required forpivthermoregulation. We also identified alasbox in thepivpromoter, which is important forpivthermoregulation. We propose that LasR directly regulatespivat stationary phase at 25°C but has a negligible impact at 37°C. Here, we show thatpivis uniquely regulated by LasR in a temperature-dependent manner. Our findings suggest that the LasRI quorum sensing regulon ofP. aeruginosamay not be fully characterized and that growth at non-standard laboratory conditions such as lower temperatures could reveal previously unrecognized quorum sensing regulated genes.

IMPORTANCE

Pseudomonas aeruginosais a versatile opportunistic pathogen capable of causing many different types of infections that are often difficult to treat, such as lung infections in people with cystic fibrosis. Temperature regulates the expression of many virulence factors that contribute toP. aeruginosa’s ability to cause infection, yet our mechanistic understanding of virulence factor thermoregulation is poor. In this study, we show that the virulence factor protease IV is thermoregulated at the level of transcription via temperature-dependent upregulation by the quorum sensing regulator, LasR. Mechanistic studies of virulence factor thermoregulation will expand our understanding of howP. aeruginosaexperiences different environments, including the mammalian host. Our work also highlights the importance of growth conditions in studying gene regulation, as it elucidates the regulation of protease IV by LasR, which was previously not well understood.
The effect of long-range linkage disequilibrium on allele-frequency dynamics under stabilizing selection
Authors: Negm Sherif , Veller Carl
Published: June 2024

Stabilizing selection on a polygenic trait reduces the trait’s genetic variance by (i) generating correlations (linkage disequilibria) between opposite-effect alleles throughout the genome and (ii) selecting against rare alleles at polymorphic loci that affect the trait, eroding heterozygosity at these loci. Here, we characterize the impact of the linkage disequilibria, which stabilizing selection generates on a rapid timescale, on the subsequent allele-frequency dynamics at individual loci, which proceed on a slower timescale. We obtain expressions for the expected per-generation change in minor-allele frequency at individual loci, as functions of the effect sizes at these loci, the strength of selection on the trait, its variance and heritability, and the linkage relations among loci. Using whole-genome simulations, we show that our expressions predict allele-frequency dynamics under stabilizing selection more accurately than the expressions that have previously been used for this purpose. Our results have implications for understanding the genetic architecture of complex traits.
Antibiotic resistant bacteria survive treatment by doubling while shrinking
Authors: Campey Adrian , Chait Remy , Tsaneva-Atanasova Krasimira , Pagliara Stefano
Published: June 2024

Many antibiotics that are used in healthcare, farming and aquaculture end up in environments with different spatial structures that might promote heterogeneity in the emergence of antibiotic resistance. However, experimental evolution of microbes at sub-inhibitory concentrations of antibiotics has been mainly carried out at the population level which does not allow capturing heterogeneity within bacterial populations. Here we investigate and compare the emergence of resistance to ciprofloxacin inEscherichia coliin well mixed and structured environments using experimental evolution, genomics and microfluidics-based time-lapse microscopy. We discover that resistance to ciprofloxacin and cross-resistance to other antibiotics is stronger in the well-mixed environment due to the emergence of target mutations, whereas efflux regulator mutations emerge in the structured environment. The latter mutants also harbour sub-populations of persisters that survive high concentrations of ciprofloxacin that inhibit bacterial growth at the population level. In contrast, genetically resistant bacteria that display target mutations also survive high concentrations of ciprofloxacin that inhibit their growth via population-level antibiotic tolerance. These resistant and tolerant bacteria keep doubling while shrinking in size in the presence of ciprofloxacin and regain their original size after antibiotic removal, which constitutes a newly discovered phenotypic response. This new knowledge sheds light on the diversity of strategies employed by bacteria to survive antibiotics and poses a stepping stone for understanding the link between mutations at the population level and phenotypic single-cell responses.
Tricellular junction recruitment of the Wave regulatory complex by Sidekick and Lar induces protrusive activity resolving cell intercalation
Authors: Calvary Lisa , Alégot Hervé , Pouchin Pierre , Richard Graziella , Vachias Caroline , Mirouse Vincent
Published: June 2024

Cell intercalation, a fundamental morphogenetic process characterized by the exchange of neighboring cells, plays a pivotal role in epithelial tissue development. While the initiation of new junctions remains poorly understood, recent research indicates the involvement of tricellular junction actors. In this study, we explore the contribution of the WAVE regulatory complex (WRC), a critical regulator of branched F-Actin generation, in tissue elongation and cell intercalation within the Drosophila ovarian follicular epithelium. WRC localizes at tricellular junctions, where it orchestrates the generation of highly dynamic protrusions emanating from one cell and extending between the bicellular junctions of neighboring cells. This protrusive activity is essential for the initiation of new junctions in cells located at the extremities of these junctions. Furthermore, our findings indicate that WRC recruitment at tricellular junctions is a redundant process, involving the cooperative action of the two transmembrane proteins Sidekick and Lar. Disruption of this recruitment impairs protrusive activity, cell intercalation resolution, and tissue elongation, thereby mechanistically bridging molecular, cellular and tissular scales. Consequently, this elucidates a critical mechanism underlying epithelial morphogenesis through actin polymerization at tricellular junctions.
Chromosomal fusions and large-scale inversions are key features for adaptation in Arctic codfish species
Authors: Hoff Siv N.K , Maurstad Marius , Tørresen Ole K. , Berg Paul R. , Præbel Kim , Jakobsen Kjetill S. , Jentoft Sissel
Published: June 2024

The evolutionary impact of structural variants, such as chromosomal inversions, is well documented, especially for their role in local adaptation in high gene flow systems. However, the role of other genomic rearrangements like chromosomal fusions, fissions, and translocations is still relatively unexplored. Here we present six chromosome-level Gadid reference genomes for the non-migratory Atlantic cod (Gadus morhua) i.e., Norwegian coastal cod (NCC), Atlantic haddock(Melanogrammus aeglefinus),burbot (Lota lota), European hake (Merluccius merluccius) as well as two keystone Arctic codfishes: the polar cod (Boreogadus saida) and Arctic cod (Arctogadus glacialis). Within a comparative genomics framework, we uncovered several lineage-specific chromosomal fusions, resulting in a reduced number of chromosomes compared to the ancestral state in the two cold-water adapted codfishes. The identified fusions were not homologous, i.e., indicating that they originate from independent evolutionary events. Additionally, a high number of partly overlapping chromosomal inversions between the two species were detected. Using a smaller population dataset, we uncovered a high degree of conservation for some of the overlapping inversions (including some breakpoint regions), suggesting that these regions are under selection, and potentially of evolutionary importance. With the use of chromosome-level genome assemblies, we demonstrate how large genomic reorganizations are likely to play important roles in speciation processes and thus, in particular to adaptation to freezing environmental conditions. Moreover, we observe that such massive rearrangement events can take place across relatively short evolutionary time scales.
Nutrient Signaling-Dependent Quaternary Structure Remodeling Drives the Catalytic Activation of metazoan PASK
Authors: Dhungel Sajina , Xiao Michael , Rajesh Rajaian Pushpabai , Kikani Chintan
Published: June 2024

The Per-Arnt-Sim (PAS) domains are characterized by diverse sequences and feature tandemly arranged PAS and PAS-associated C-terminal (PAC) motifs that fold seamlessly to generate the metabolite-sensing PAS domain. Here, using evolutionary scale sequence, domain mapping, and deep learning-based protein structure analysis, we deconstructed the sequence-structure relationship to unearth a novel example of signal-regulated assembly of PAS and PAC subdomains in metazoan PAS domain-regulated kinase (PASK). By comparing protein sequence, domain architecture, and computational protein models between fish, bird, and mammalian PASK orthologs, we propose the existence of previously unrecognized third PAS domain of PASK (PAS-C) formed through long-range intramolecular interactions between the N-terminal PAS fold and the C-terminal PAC fold. We experimentally validated this novel structural design using residue-level cross-linking assays and showed that the PAS-C domain assembly is nutrient-responsive. Furthermore, by combining structural phylogeny approaches with residue-level cross-linking, we revealed that the PAS-C domain assembly links nutrient sensing with quaternary structure reorganization in PASK, stabilizing the kinase catalytic core of PASK. Thus, PAS-C domain assembly likely integrates environmental signals, thereby relaying sensory information for catalytic control of the PASK kinase domain. In conclusion, we theorize that during their horizontal transfer from bacteria to multicellular organisms, PAS domains gained the capacity to integrate environmental signals through dynamic modulation of PAS and PAC motif interaction, adding a new regulatory layer suited for multicellular systems. We propose that metazoan PAS domains are likely to be more dynamic in integrating sensory information than previously considered, and their structural assembly could be targeted by regulatory signals and exploited to develop therapeutic strategies.
Bacterial motility depends on a critical flagellum length and energy-optimised assembly
Authors: Halte Manuel , Popp Philipp F. , Hathcock David , Severn John , Fischer Svenja , Goosmann Christian , Ducret Adrien , Charpentier Emmanuelle , Tu Yuhai , Lauga Eric , Erhardt Marc , Renault Thibaud T.
Published: June 2024

The flagellum is the most complex macromolecular structure known in bacteria and comprised of around two dozen distinct proteins. The main building block of the long, external flagellar filament, flagellin, is secreted through the flagellar type-III secretion system at a remarkable rate of several tens of thousands amino acids per second, significantly surpassing the rates achieved by other pore-based protein secretion systems. The evolutionary implications and potential benefits of this high secretion rate for flagellum assembly and function, however, have remained elusive. In this study, we provide both experimental and theoretical evidence that the flagellar secretion rate has been evolutionarily optimized to facilitate rapid and efficient construction of a functional flagellum. By synchronizing flagellar assembly, we found that a minimal filament length of 2.5 µm was required for swimming motility. Biophysical modelling revealed that this minimal filament length threshold resulted from an elasto-hydrodynamic instability of the whole swimming cell, dependent on the filament length. Furthermore, we developed a stepwise filament labeling method combined with electron microscopy visualization to validate predicted flagellin secretion rates of up to 10,000 amino acids per second. A biophysical model of flagellum growth demonstrates that the observed high flagellin secretion rate efficiently balances filament elongation and energy consumption, thereby enabling motility in the shortest amount of time. Taken together, these insights underscore the evolutionary pressures that have shaped the development and optimization of the flagellum and type-III secretion system, illuminating the intricate interplay between functionality and efficiency in assembly of large macromolecular structures.

Significance statement

Our study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.
Assessment of Antimicrobial Therapy in EradicatingChlamydia muridarumin Research Mice: Immune Status and its Impact on Outcomes
Authors: Palillo Michael B , Carrasco Sebastian E , Mishkin Noah , Palillo Jack A , Lynch Denise B , Lawton Samira , Aydin Mert , Mourino Anthony , Lipman Neil S , Ricart Arbona Rodolfo J
Published: June 2024

Chlamydia muridarum (Cm) is a moderately prevalent, gram-negative, intracellular bacterium that affects laboratory mice, causing subclinical to severe disease, depending on the host’s immune status. The effectiveness of various antibiotic regimens aimed at eradicating Cm in both immunodeficient and immunocompetent laboratory mice was evaluated. NSG mice were cohoused with Cm-shedding BALB/cJ mice for 14 days to simulate natural exposure. Four groups of 8 infected NSG mice were treated for 7 days with either 0.08% sulfamethoxazole and 0.016% trimethoprim (TMS) in water, 0.0625% doxycycline in feed, 0.124%/0.025% TMS in feed, or 0.12% amoxicillin in feed. A control group was provided standard water and feed. The impact of treatment on gastrointestinal microbiota (GM) was performed using next-generation shotgun sequencing on the last day of treatment. TMS and Amoxicillin had negligible effects on GM, while doxycycline had the largest effect. All antibiotic treated NSG mice exhibited clinical disease, including dehydration, hunched posture, >20% weight loss, and dyspnea, leading to euthanasia 21-40 days post-treatment (32.6 ± 4.2 days; mean ± SD). Untreated controls were euthanized 14-33 days post-exposure (23.75 ± 5.9 days). All mice were fecal PCR positive for Cm at euthanasia. Histological evaluation revealed multifocal histiocytic and neutrophilic bronchointerstitial pneumonia and/or bronchiolitis featuring prominent intralesional chlamydial inclusion bodies in all mice. Subsequently, groups of 8 C57BL/6J, BALB/cJ, NOD.SCID, and NSG mice infected with Cm were treated with 0.124%/0.025% TMS in feed for 7 (BALB/cJ and C57BL/6J) or 21 days (NSG and NOD.SCID). All immunocompetent and NOD.SCID mice were negative for Cm by PCR 14 days post-treatment, remained clinically normal and had no evidence of Cm infection at necropsy, all NSG mice remained Cm positive and were euthanized. While these findings highlight the difficulties in eradicating Cm from highly immunodeficient mice, eradication of Cm from immunocompetent or moderately immunocompromised mice with antibiotics is feasible.
Globoside is an essential intracellular factor required for parvovirus B19 endosomal escape in erythroid progenitor cells
Authors: Bieri Jan , Suter Corinne , Caliaro Oliver , Bartetzko Seraina , Bircher Cornelia , Ros Carlos
Published: June 2024

Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2(PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.
Rhythmic variation in proteomics: challenges and opportunities for statistical power and biomarker identification
Authors: Spick Matt , Isherwood Cheryl M , Gethings Lee , Hassanin Hana , van der Veen Daan R , Skene Debra J. , Johnston Jonathan D
Published: June 2024

Time-of-day variation in the molecular profile of biofluids and tissues is a well-described phenomenon, but – especially for proteomics – is rarely considered in terms of the challenges this presents to reproducible biomarker identification. In this work we demonstrate these confounding issues using a small-scale proteomics analysis of male participants in a constant routine protocol following an 8-day laboratory study, in which sleep-wake, light-dark and meal timings were controlled. We provide a case study analysis of circadian and ultradian rhythmicity in proteins in the complement and coagulation cascades, as well as apolipoproteins, and demonstrate that rhythmicity increases the risk of Type II errors due to the reduction in statistical power from increased variance. For the proteins analysed herein we show that to maintain statistical power if chronobiological variation is not controlled for,nshould be increased (by between 9% and 20%); failure to do so would increase β, the chance of Type II error, from a baseline value of 20% to between 22% and 28%. Conversely, controlling for rhythmic time-of-day variation in study design offers the opportunity to improve statistical power and reduce the chances of Type II errors. Indeed, control of time-of-day sampling is a more cost-effective strategy than increasing sample sizes. We recommend that best practice in proteomics study design should account for temporal variation as part of sampling strategy where possible. Where this is impractical, we recommend that additional variance from chronobiological effects be considered in power calculations, that time of sampling be reported as part of study metadata, and that researchers reference any previously identified rhythmicity in biomarkers and pathways of interest. These measures would mitigate against both false and missed discoveries, and improve reproducibility, especially in studies looking at biomarkers, pathways or conditions with a known chronobiological component.
Comprehensive analysis of end-modified long dsDNA donors in CRISPR-mediated endogenous tagging
Authors: Takagi Rioka , Hata Shoji , Tei Chiharu , Mabuchi Akira , Anzai Ryosuke , Fukuyama Masamitsu , Yamamoto Shohei , Chinen Takumi , Toyoda Atsushi , Kitagawa Daiju
Published: June 2024

CRISPR-mediated endogenous tagging is a powerful gene editing technique for studying protein dynamics and function in their native cellular environment. While the use of 5’ modified DNA donors has emerged as a promising strategy to improve the typically low efficiency of knock-in gene editing, the underlying mechanisms remain poorly understood. In this study, we conducted a comprehensive analysis of end-modified long linear dsDNA donors in CRISPR-mediated endogenous tagging in human non-transformed cells. In-depth analysis of repair patterns reveals that 5’ biotinylation of dsDNA donors significantly reduces imprecise insertions, thereby enhancing homology-directed repair (HDR)-mediated precise insertion efficiency. Notably, the impact of biotinylation on repair patterns resembles that of non-homologous end joining (NHEJ) pathway inhibition, suggesting its role in preventing NHEJ-mediated mis-integration. Moreover, combining biotin modification with NHEJ inhibitor treatment further improves bi-allelic knock-in efficiency. Overall, this study provides novel insights into the mechanisms by which 5’ modifications enhance precise knock-ins and demonstrates their potential for achieving high-efficient, prercise endogenous tagging in human cells.
Role of Phage–Antibiotic Combinations in Reducing ESBL-Producing and Carbapenem-ResistantEscherichia coli
Authors: Shamsuzzaman Md , Kim Shukho , Kim Jungmin
Published: June 2024

The emergence of extended-spectrumβ-lactamase (ESBL)-producingE. coliand carbapenem-resistantE. coli(CREC) poses a significant global health concern. Here, we isolated and characterized two novel phages and studied their effectiveness with antibiotics against ESBL-producingE. coliand CREC. The isolated phages, EC.W1-9 and EC.W15-4, belonged to thePodoviridaeandMyoviridaefamilies, respectively. They are safe for bacterial control as they do not contain integrase or toxin-coding genes. The phage combination considerably enhanced lytic ability, effectively lysing 61.7% of the 60E. coliisolates, compared to lysis in the 41.6% –55% range by individual phages. Phages EC.W1-9 and EC.W15-4 combined demonstrated 100% susceptibility against differentE. colisequence types, including ST73, ST648, ST2311, ST405, ST7962, ST131, ST13003, and ST167. Additionally, studies showed synergy between antibiotics and phage combinations against ESBL-producingE.coli, with susceptibility of 73.3% and 54% for CREC. The combined treatment of isolated phages and antibiotics significantly increased survival rates in BALB/c mice exposed to various ST types of ESBL-producingE. coliand CREC, including ST131, ST648, and ST410. Survival rates against KBN7288 (ST131) increased by approximately 75% and 50% compared to individual phages EC.W1-9 and EC.W15-4, respectively. When phages and antibiotics were combined, survival rates againstE. coliisolates KBN5617 (ST410), KBN6241 (ST410), and KBN4004 (ST648) ranged from 75% – 100%. Finally, this study highlights the importance of phage and phage-antibiotic combinations to prepare phages for killing different ST types of ESBL-producingE. coliand CREC isolates.

IMPORTANCE

When combined with antibiotics, phage therapy shows promise in fighting multidrug-resistant bacteria. However, antagonism between phages and antibiotics has been reported. This research isolates and characterizes two novel phages, EC.W1-9 and EC.W15-4, from thePodoviridaeandMyoviridaefamilies, respectively, and evaluates their effectiveness against ESBL-producingE. coliand CREC. These phages, lacking integrase or toxin-coding genes, showed significant promise in bacterial control. Combined phage treatment lysed 61.7% ofE.coliisolates, outperforming individual phages. The phage combination showed 100% susceptibility against differentE. colisequence types. Additionally, the synergy between phages and antibiotics increased susceptibility rates to 73.3% for ESBL-producingE. coliand 54% for CREC. In BALB/c mice, combined treatments significantly improved survival rates against variousE. coliisolates. Finally. this study emphasizes the potential of phage and phage-antibiotic combinations in targeting various ST types of ESBL-producingE. coliand CREC.
The Molecules Gateway: a homogeneous, searchable database of 150k annotated molecules from Actinomycetes
Authors: Simone Matteo , Iorio Marianna , Monciardini Paolo , Santini Massimo , Cantù Niccolò , Tocchetti Arianna , Serina Stefania , Brunati Cristina , Vernay Thomas , Gentile Andrea , Aracne Mattia , Cozzi Marco , van der Hooft Justin J.J. , Sosio Margherita , Donadio Stefano , Maffioli Sonia I.
Published: June 2024

Natural products are a sustainable resource for drug discovery, but their identification in complex mixtures remains a daunting task. We present an automated pipeline that compares, harmonizes and ranks the annotations of LC-HRMS data by different tools. When applied to 7,400 extracts derived from 6,566 strains belonging to 86 actinomycete genera, it yielded 150,000 molecules after processing over 50 million MS features. The web-based Molecules Gateway provides a highly interactive access to experimental and calculated data for these molecules, along with the metadata related to extracts and producer strains. We show how the Molecules Gateway can be used to rapidly identify known hard to find microbial products, unreported analogs of known families and not yet described metabolites. The Molecules Gateway, which complements available repositories of annotated MS data, is experimentally and computationally homogeneous, and thus amenable to global analyses, which show a large and untapped chemical diversity afforded by actinomycetes.
Bottom-up reconstruction of minimal pyrenoids provides insights into the evolution and mechanisms of carbon concentration by EPYC1 proteins
Authors: Küffner A. M. , Pommerenke B. , Kley L. , Ng J. Z. Y. , Prinz S. , Tinzl-Zechner M. , Claus P. , Paczia N. , Zarzycki J. , Hochberg G. K. A. , Erb T. J.
Published: June 2024

Membraneless organelles play essential roles in cellular processes. In various photosynthetic organisms, they confer carbon concentrating mechanisms (CCMs). One example is the pyrenoid inChlamydomonas reinhardtii, a liquid-phase separated organelle that localizes and improves carbon fixation via the intrinsically-disordered protein (IDP) EPYC1. Modern-day pyrenoids are complex structures, which makes it impossible to study the function of EPYC1, especially whether EPYC1 alone confers carbon concentration, and how EPYC1 could have initiated the evolution of pyrenoids. Here, we developed a bottom-up approach to study the function of EPYC1 and its sequence-function space across evolution. We demonstrate that modern-day EPYC1-sequences, but not other IDPs, induce liquid-phase separation of Rubisco into minimal pyrenoids with functional CCMs. Using ancestral sequence reconstruction, we trace the evolution of pyrenoids and demonstrate that selection acted on carboxylation rate, and in selected cases on specificity, providing unexpected perspectives on the design and function of natural and synthetic pyrenoids.