bluuebunny/crossref_metadata_2025 huggingface dataset:
The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo
Authors: Echterhof Arne , Dharmaraj Tejas , McBride Robert , Berry Joel , Hopkins Max , Selvakumar Hemaa , Miesel Lynn , Chia Ju-Hsin , Lin Kun-Yuan , Shen Chien-Chang , Lee Yu-Ling , Yeh Yu-Chuan , Liao Wei Ting , Suh Gina , Blankenberg Francis G. , Frymoyer Adam R. , Bollyky Paul L.
Published: January 2024

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is great interest in using lytic bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokineticsin vivoremain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of intravenously administered bacteriophage in uninfected mice. A single dose of LPS-5, an antipseudomonal bacteriophage recently used in human clinical trials, was administered intravenously to both wild-type BALB/c and neutropenic ICR mice. Phage concentrations were assessed in peripheral blood and spleen at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance is only minimally affected by neutropenia. Indeed, the half-life of phages in blood in BALB/c and ICR mice is 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that substantial functional inactivation of circulating phages occurs over time. These data indicate that circulating factors, but not neutrophils, inactivate intravenously administered phages.
A multi-institutional study to investigate the sparing effect after whole brain electron FLASH in mice: Reproducibility and temporal evolution of functional, electrophysiological, and neurogenic endpoints
Authors: Drayson Olivia GG , Melemenidis Stavros , Katila Nikita , Viswanathan Vignesh , Kramár Enikö A , Zhang Richard , Kim Rachel , Ru Ning , Petit Benoit , Dutt Suparna , Manjappa Rakesh , Ashraf M. Ramish , Lau Brianna , Soto Luis , Skinner Lawrie , Yu Amu S. , Surucu Murat , Maxim Peter , Zebadua-Ballasteros Paola , Wood Marcelo , Baulch Janet E. , Vozenin Marie-Catherine , Loo Billy W , Limoli Charles L.
Published: January 2024

Purpose

Ultra-high dose-rate radiotherapy (FLASH) has been shown to mitigate normal tissue toxicities associated with conventional dose rate radiotherapy (CONV) without compromising tumor killing in preclinical models. A prominent challenge in preclinical radiation research, including FLASH, is validating both the physical dosimetry and the biological effects across multiple institutions.

Methods

We previously demonstrated dosimetric reproducibility of two different electron FLASH devices at separate institutions using standardized phantoms and dosimeters. In this study, we compared the outcome of FLASH and CONV 10 Gy whole brain irradiation on female adult mice at both institutions to evaluate the reproducibility and temporal evolution of multiple endpoints.

Results

FLASH sparing of behavioral performance on novel object recognition (4 months post-irradiation) and electrophysiologic long-term potentiation (LTP, 5-months post-irradiation) was reproduced between institutions. Interestingly, differences between FLASH and CONV on the endpoints of hippocampal neurogenesis (Sox2, doublecortin), neuroinflammation (microglial activation), and electrophysiology (LTP) at late times were not observed at early times.

Conclusions

In summary, we demonstrated reproducible FLASH sparing effects between two beams and two institutions with validated dosimetry. FLASH sparing effects on the endpoints evaluated manifested at late but not early time points.
Assessing the Adequacy of Morphological Models used in Palaeobiology
Authors: Mulvey Laura P. A. , May Michael R. , Brown Jeremy M. , Höhna Sebastian , Wright April M. , Warnock Rachel C. M.
Published: January 2024

1

Reconstructing the evolutionary history of different groups of organisms provides insight into how life originated and diversified on Earth. Phylogenetic trees are commonly used to estimate this evolutionary history, providing a hypothesis of the events. Within Bayesian phylogenetics a major step in estimating a tree is in choosing an appropriate model of character evolution. In the case of most extinct species, our only source of information to decipher their phylogenetic relationships is through the morphology of fossils. We therefore use a model of morphological character evolution, the most common of which being the Mk Lewis model. While it is frequently used in palaeobiology, it is not known whether the simple Mk substitution model, or any extensions to it, provide a sufficiently good description of the process of morphological evolution. To determine whether or not the Mk model is appropriate for fossil data we used posterior predictive simulations, a model adequacy approach, to estimate absolute fit of the model to morphological data sets. We first investigate the impact that different versions of the Mk model have on key parameter estimates using tetrapod data sets. We show that choice of substitution model has an impact on both topology and branch lengths, highlighting the importance of model choice. Next, we use simulations to investigate the power of posterior predictive simulations for morphology. Having validated this approach we show that current variations of the Mk model are in fact performing adequately in capturing the evolutionary dynamics that generated our data. We do not find any preference for a particular model extension across multiple data sets, indicating that there is no ‘one size fits all’ when it comes to morphological data and that careful consideration should be given to choosing models of discrete character evolution. By using suitable models of character evolution, we can increase our confidence in our phylogenetic estimates, which should in turn allow us to gain more accurate insights into the evolutionary history of both extinct and extant taxa.
DNA metabarcoding-based detection of non-indigenous invertebrates in recreational marinas: influence of sample type and seasonal variation
Authors: Lavrador Ana S. , Amaral Fábio G. , Moutinho Jorge , Vieira Pedro E. , Costa Filipe O. , Duarte Sofia
Published: January 2024

Monitoring of marine invertebrate non-indigenous species (NIS) using DNA metabarcoding can be strongly affected by selected sample type due to life history traits, such as habitat preferences and life cycles. Two marinas in the north of Portugal were sampled to assess the impact of sample type (hard and artificial substrates, water eDNA, and zooplankton) and season (spring, autumn, winter) on species and NIS recovery. Using two molecular markers - the mitochondrial cytochrome c oxidase subunit I (COI) and the small subunit ribosomal RNA (18S) - a total of 636 species and 31 NIS were detected. Species numbers were slightly higher in the marina more exposed to maritime traffic, and the highest percentage of exclusive species was detected in zooplankton (up to 24%), as well as the highest numbers of NIS. Regarding season, the highest numbers of species and NIS were detected in the spring and autumn (varying within each marina). Taxonomic composition analysis revealed differences in species richness and community structure among seasons and sample types, particularly between hard and artificial substratesversuseDNA and zooplankton. Of the 31 NIS detected, six are potential first records for Portugal, which await morphology-based validation. No NIS were detected in all sample types nor in all sampled seasons. This highlights the need to employ different sampling approaches and markers, as well as consider seasonal variation and level of exposure to maritime-driven introductions to guarantee a comprehensive metabarcoding-based surveillance of NIS in recreational marinas.
A fungi hotspot deep down the ocean: explaining the presence ofGjaerumia minorin Equatorial Pacific bathypelagic waters
Authors: Pernice Massimo C. , Forn Irene , Logares Ramiro , Massana Ramon
Published: January 2024

A plant parasite associated with the white haze disease in apples, the BasidiomycotaGjaerumia minor,has been found in most samples of the global bathypelagic ocean. An analysis of environmental 18S rDNA sequences on 12 vertical profiles of the Malaspina 2010 expedition shows that the relative abundance of this cultured species actually increases with depth while its distribution is remarkably different between the deep waters of the Pacific and Atlantic oceans, being present in higher concentrations in the former. This is evident from sequence analysis and a microscopic survey with a species-specific newly designed TSA-FISH probe. Several hints point to the hypothesis thatG. minoris transported to the deep ocean attached to particles, and the absence ofG. minorin bathypelagic Atlantic waters could then be explained by the absence of this organism in surface waters of the equatorial Atlantic. The good correlation ofG. minorbiomass with recalcitrant carbon and free-living prokaryotic biomass in South Pacific waters, together with the identification of the observed cells as yeast and not as a resting spore (teliospore), point to the possibility that once arrived at deep layer this species keeps on growing and thriving.
Genome-wide analysis of the class III peroxidase gene family inPhyscomitrium patensand a search for clues to ancient class III peroxidase functions
Authors: Aparato Vincent P. M. , Rabbi Fazle , Madarash Taylor , Brisbourne Wyllie A. , Barker Elizabeth I. , Suh Dae-Yeon
Published: January 2024

Plant class III peroxidases (PRX) catalyze generation of reactive oxygen species and oxidation of various compounds including lignin precursors. PRXs function in cell wall metabolism, defense and stress responses. However, gene redundancy and catalytic versatility have impeded detailed functional characterization ofPRXgenes. The genome of the model mossPhyscomitrium patensharbors a relatively small number (49) ofPRXgenes. Conserved architecture of four exons and three ‘001’ introns, found in some algalPRXgenes and in thePpPRXfamily, suggests that this architecture predated divergence of the green algal and land plant lineages. ThePpPRXfamily expanded mainly through whole-genome duplications. All duplicated pairs but one were under purifying selection and generally exhibited similar expression profiles. An expanded phylogenetic tree revealed a conserved land plant-wide clade that contained PRXs implicated in stress responses in non-lignifying cells, providing a clue to ancient functions of land plant PRXs. Functional clustering was not observed, suggesting convergent evolution of specific PRX functions (e.g., lignification) in different plant lineages. With its small complement of PRXs, P. patensmay be useful for functional characterization of land plant PRXs. Several PpPRXs were proposed for further study, includingPpPRX34andPpPRX39in the ancient land plant-wide clade.
k-nonical space: sketching with reverse complements
Authors: Marçais Guillaume , Elder C.S. , Kingsford Carl
Published: January 2024

Sequences equivalent to their reverse complements (i.e., double-stranded DNA) have no analogue in text analysis and non-biological string algorithms. Despite this striking difference, algorithms designed for computational biology (e.g., sketching algorithms) are designed and tested in the same way as classical string algorithms. Then, as a post-processing step, these algorithms are adapted to work with genomic sequences by folding ak-mer and its reverse complement into a single sequence: the canonical representation (k-nonical space). The effect of using the canonical representation with sketching methods is understudied and not understood. As a first step, we use context-free sketching methods to illustrate the potentially detrimental effects of using canonicalk-mers with string algorithms not designed to accommodate for them. In particular, we show that large stretches of the genome (“sketching deserts”) are undersampled or entirely skipped by context-free sketching methods, effectively making these genomic regions invisible to subsequent algorithms using these sketches. We provide empirical data showing these effects and develop a theoretical framework explaining the appearance of sketching deserts. Finally, we propose two schemes to accommodate for these effects: (1) a new procedure that adapts existing sketching methods tok-nonical space and (2) an optimization procedure to directly design new sketching methods fork-nonical space.

The code used in this analysis is freely available athttps://github.com/Kingsford-Group/mdsscope.
Predicting microbial genome-scale metabolic networks directly from 16S rRNA gene sequences
Authors: Øyås Ove , Kobel Carl M. , Vik Jon Olav , Pope Phillip B.
Published: January 2024

Genome-scale metabolic models are key biotechnology tools that can predict metabolic capabilities and growth for an organism. In particular, these models have become indispensable for metabolic analysis of microbial species and communities such as the gut microbiomes of humans and other animals. Accurate microbial models can be built automatically from genomes, but many microbes have only been observed through sequencing of marker genes such as 16S rRNA and thus remain inaccessible to genome-scale modeling. To extend the scope of genome-scale metabolic models to microbes that lack genomic information, we trained an artificial neural network to build microbial models from numeric representations of 16S rRNA gene sequences. Specifically, we built models and extracted 16S rRNA gene sequences from more than 15,000 reference and representative microbial genomes, computed multiple sequence alignments and large language model embeddings for the 16S rRNA gene sequences, and trained the neural network to predict metabolic reaction probabilities from sequences, alignments, or embeddings. Training was fast on a single graphics processing unit and trained networks predicted reaction probabilities accurately for unseen archaeal and bacterial sequences and species. This makes it possible to reconstruct microbial genome-scale metabolic networks from any 16S rRNA gene sequence and enables simulation of metabolism and growth for all observed microbial life.
Paraoxonase-like APMAP maintains endoplasmic reticulum-associated lipid and lipoprotein homeostasis
Authors: Paul Blessy , Merta Holly , Ugrankar-Banerjee Rupali , Hensley Monica , Tran Son , Dias do Vale Goncalo , McDonald Jeffrey G , Farber Steven A. , Henne W Mike
Published: January 2024

Oxidative stress perturbs lipid homeostasis and contributes to metabolic diseases. Though ignored compared to mitochondrial oxidation, the endoplasmic reticulum (ER) generates reactive oxygen species requiring antioxidant quality control. Using multi-organismal profiling featuringDrosophila, zebrafish, and mammalian cells, here we characterize the paraoxonase-like APMAP as an ER-localized protein that promotes redox and lipid homeostasis and lipoprotein maturation. APMAP-depleted mammalian cells exhibit defective ER morphology, elevated ER and oxidative stress, lipid droplet accumulation, and perturbed ApoB-lipoprotein homeostasis. Critically, APMAP loss is rescued with chemical antioxidant NAC. Organismal APMAP depletion inDrosophilaperturbs fat and lipoprotein homeostasis, and zebrafish display increased vascular ApoB-containing lipoproteins, particles that are atherogenic in mammals. Lipidomics reveals altered polyunsaturated phospholipids and increased ceramides upon APMAP loss, which perturbs ApoB-lipoprotein maturation. These ApoB-associated defects are rescued by inhibiting ceramide synthesis. Collectively, we propose APMAP is an ER-localized antioxidant that promotes lipid and lipoprotein homeostasis.

Key findings summary

APMAP localizes primarily to the ER network in human cells andDrosophilafat body tissue, and is a type II integral membrane protein

Loss of APMAP orDrosophilaAPMAP (dAPMAP) causes ER membrane expansion, elevates CHOP-associated ER stress, promotes LD accumulation, and alters ApoB-lipoprotein secretion

APMAP-depleted cells and dAPMAP-depletedDrosophilafat tissue exhibit defective redox homeostasis; phenotypes associated with APMAP loss are rescued by antioxidant NAC

Zebrafish-based LipoGlo reporter reveals that loss ofapmapin zebrafish causes increased vascular ApoB-containing lipoproteins

Lipidomic profiling indicates that APMAP loss reduces PUFA-phospholipids and elevates intracellular ceramides, which perturbs ApoB maturation
Emergent collective alignment gives competitive advantage to longer cells during range expansion
Authors: van den Berg Nathan , Thijssen Kristian , Nguyen Thu Trang , Sarlet Adrien , Cordero Mireia , Vázquez Alba García , Mitarai Namiko , Doostmohammadi Amin , Jauffred Liselotte
Published: January 2024

Bacteria’s competition for nutrients and territory drives biofilm evolution (1–4). The factors determining the outcome of competition among diverse bacterial species have a broad impact on a wide range of pathological (5), environmental (6), and microbiome interactions (7). While motility-related traits (8–11) and specific molecular mechanisms (12, 13) have been identified as potential winning attributes in bacteria, a shared and universally conserved feature determining competition remains elusive. Here, we demonstrate that a simple morphological feature of individual bacteria, cell aspect ratio, provides a winning trait for the population. Using growth-based range expansion experiments (14), we show that relatively longer bacteria robustly conquer the expanding front, even when initially in minority. Using an agent-based model of dividing bacteria, we reveal that the takeover mechanism is their emergent collective alignment: groups of locally aligned bacteria form “nematic arms” bridging the central region of the colony to the expanding front. Once at the front, bacteria align parallel to it and block the access of shorter bacteria to nutrients and space. We confirm this observation with single-cell experiments and further generalise our findings by introducing a generic continuum model of alignment-dominated competition, explaining both experimental and cell-based model observations. Moreover, we extend our predictions to spherical range expansions (15) and confirm the competitive advantage of being longer, even though the effect is less pronounced than in surface-attached colonies. Our results uncover a simple, yet hitherto overlooked, mechanical mechanism determining the outcome of bacterial competition, which is potentially ubiquitous among various bacteria. With the current advances in genetic engineering, varying aspect ratios can work as a simple tunable mechanism for the on-demand setting of the outcome of bacterial competitions with widespread implications for biofilm control.
An accessible digital imaging workflow for multiplexed quantitative analysis of adult eye phenotypes inDrosophila melanogaster
Authors: Pipkin Heidi J. J. , Lindsay Hunter L. , Smiley Adam T. , Jurmu Jack D. , Arsham Andrew M
Published: January 2024

The compound eye ofDrosophila melanogasterhas long been a model for studying genetics, development, neurodegeneration, and heterochromatin. Imaging and morphometry of adultDrosophilaand other insects is hampered by the low throughput, narrow focal plane, and small image sensors typical of stereomicroscope cameras. When data collection is distributed among many individuals or extended time periods, these limitations are compounded by inter-operator variability in lighting, sample positioning, focus, and post-acquisition processing. To address these limitations we developed a method for multiplexed quantitative analysis of adultDrosophila melanogasterphenotypes. Efficient data collection and analysis of up to 60 adult flies in a single image with standardized conditions eliminates inter-operator variability and enables precise quantitative comparison of morphology. Semi-automated data analysis using ImageJ and R reduces image manipulations, facilitates reproducibility, and supports emerging automated segmentation methods, as well as a wide range of graphical and statistical tools. These methods also serve as a low-cost hands-on introduction to imaging, data visualization, and statistical analysis for students and trainees.
Human vascular organoids with a mosaicAKT1mutation recapitulate Proteus syndrome
Authors: He Siyu , Zhu Yuefei , Chauhan Shradha , Tavakol Daniel Naveed , Lee Jong Ha , Berris Rayna Batya-Leia , Xu Cong , Lee Jounghyun H. , Lee Caleb , Cai Sarah , McElroy Shannon , Vunjak-Novakovic Gordana , Tomer Raju , Azizi Elham , Xu Bin , Lao Yeh-Hsing , Leong Kam W.
Published: January 2024

Vascular malformation, a key clinical phenotype of Proteus syndrome, lacks effective models for pathophysiological study and drug development due to limited patient sample access. To bridge this gap, we built a human vascular organoid model replicating Proteus syndrome’s vasculature. Using CRISPR/Cas9 genome editing and gene overexpression, we created induced pluripotent stem cells (iPSCs) embodying the Proteus syndrome-specific AKTE17Kpoint mutation for organoid generation. Our findings revealed that AKT overactivation in these organoids resulted in smaller sizes yet increased vascular connectivity, although with less stable connections. This could be due to the significant vasculogenesis induced by AKT overactivation. This phenomenon likely stems from boosted vasculogenesis triggered by AKT overactivation, leading to increased vascular sprouting. Additionally, a notable increase in dysfunctional PDGFRβ + mural cells, impaired in matrix secretion, was observed in these AKT-overactivated organoids. The application of AKT inhibitors (ARQ092, AZD5363, or GDC0068) reversed the vascular malformations; the inhibitors’ effectiveness was directly linked to reduced connectivity in the organoids. In summary, our study introduces an innovative in vitro model combining organoid technology and gene editing to explore vascular pathophysiology in Proteus syndrome. This model not only simulates Proteus syndrome vasculature but also holds potential for mimicking vasculatures of other genetically driven diseases. It represents an advance in drug development for rare diseases, historically plagued by slow progress.
Centromere landscapes resolved from hundreds of human genomes
Authors: Gao Shenghan , Zhang Yimeng , Bush Stephen J. , Wang Bo , Yang Xiaofei , Ye Kai
Published: January 2024

High-fidelity (HiFi) sequencing has facilitated the assembly and analysis of the most repetitive region of the genome, the centromere. Nevertheless, our current understanding of human centromeres draws from a relatively small number of telomere-to-telomere assemblies, and so has not yet captured its full diversity. In this study, we investigated the genomic diversity of human centromere higher order repeats (HORs) using both HiFi reads and haplotype-resolved assemblies from hundreds of samples drawn from ongoing pangenome-sequencing projects and reprocessed using a novel HOR annotation pipeline, HiCAT-human. We use this wealth of data to provide a global survey of the centromeric HOR landscape, in particular finding that 23 HORs exhibited significant copy number variability between populations. We detected three centromere genotypes with imbalance population frequencies on each of chromosome 5, 8 and 17. An inter-assembly comparison of HOR loci further revealed that while HOR array structures are diverse, they nevertheless tend to form a number of specific landscapes, each exhibiting different levels of HOR subunit expansion and possibly reflecting a cyclical evolutionary transition from homogeneous to nested structures and back.
GRK2-mediated AKT activation controls cell cycle progression and G2-checkpoint in a p53-dependent manner
Authors: Rivas Verónica , González-Muñoz Teresa , Albitre Ángela , Lafarga Vanesa , Mayor Federico , Penela Petronila
Published: January 2024

Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss, rendering cell viability dependent on the strength of the G2 checkpoint through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53’s scaffolding function, triggering FOXO3a phosphorylation, impaired cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.
Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays
Authors: Van Vranken Jonathan G. , Li Jiaming , Mintseris Julian , Wei Ting-Yu , Sniezek Catherine M. , Gadzuk-Shea Meagan , Gygi Steven P. , Schweppe Devin K.
Published: January 2024

In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to approximate compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified more than one million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell-and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.
Growing a cystic fibrosis-relevant polymicrobial biofilm to probe community phenotypes
Authors: Poirier Sarah , Jean-Pierre Fabrice
Published: January 2024

Mostin vitromodels lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat chronic and polymicrobial biofilm-based infections detected in the airways of persons with cystic fibrosis (pwCF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of pwCF, noin vitromodels thus far had fully integrated critical cystic fibrosis (CF)-relevant lung features. Therefore, a significant knowledge-gap in our capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections remained. To tackle this challenge, we have built a four-species microbial community model includingPseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, andPrevotella melaninogenicagrown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial (Abx) recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of thisin vitromodel resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

In this protocol, we describe a cystic fibrosis (CF)-lung relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.
Characterization of antimalarial activity of artemisinin-based hybrid drugs
Authors: Quadros Helenita Costa , Herrmann Lars , Manaranche Jeanne , Paloque Lucie , Borges-Silva Mariana C. , Dziwornu Godwin Akpeko , D’Alessandro Sarah , Chibale Kelly , Basilico Nicoletta , Benoit-Vical Françoise , Tsogoeva Svetlana B. , Moreira Diogo Rodrigo M.
Published: January 2024

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed and their activity profile was characterized against drug-sensitive and drug-resistantPlasmodium falciparumparasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in the hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide and in inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed some limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

GRAPHICAL ABSTRACT

Hepatic and cell-host-mediated metabolism are responsible for short plasma half-lives of antimalarial artemisinins (ARTs), illustrated here by dihydroartemisinin (DHA). ART-based hybrid drugs that overcome rapid degradation can facilitate activity against ART-resistant parasites, as illustrated by hybrid1.
Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter by Reverse Genetics
Authors: Snyder Anthony J. , Agbemabiese Chantal A. , Patton John T.
Published: January 2024

Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, a need remains for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents aRotavirus Aspecies with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined by Nanopore sequencing, we developed a robust reverse genetics system enabling recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers (∼107plaque-forming units/ml), its growth kinetics were modestly decreased in comparison to laboratory-adapted OSU virus. The reverse genetics system was used to generate rOSU rotavirus that served as an expression vector for foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced genetically stable rOSU that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens of other porcine enteric diseases.
Escherichia coliextra-intestinal population translocation in leukemia patients
Authors: Marin Julie , Walewski Violaine , Dziri Samira , Magnan Mélanie , Denamur Erick , Carbonnelle Etienne , Bridier-Nahmias Antoine
Published: January 2024

Escherichia coli, a commensal species of the human gut, is an opportunistic pathogen which can reach extra-intestinal compartments, including the bloodstream and the bladder, among others. In non-immunosuppressed patients, purifying or neutral evolution ofE. colipopulations has been reported in the gut. Conversely, it has been suggested that when migrating to extra-intestinal compartments,E. coligenomes undergo diversifying selection as supported by strong evidence for adaptation. The level of genomic polymorphism and the size of the populations translocating from the gut to extra-intestinal compartments is largely unknown.

To gain insights in the pathophysiology of these translocations, we investigated the level of polymorphism and the evolutionary forces acting on the genomes of 77E. coliisolated from various compartments in three immunosuppressed patients. We detected a unique strain for each patient across the blood, the urine and the gut. In one case, all isolates recovered were mutators i.e. isolates with a very high mutation rate. In all instances, we observed that translocation encompasses the majority of the genomic diversity present in the gut. The same signature of selection, whether purifying or diversifying, and as anticipated, neutral for mutator isolates, was observed in both the gut and bloodstream. Additionally, we found a limited number of non-specific mutations among compartments for non-mutator isolates. In all cases, urine isolates were dominated by neutral selection. These findings indicate that substantial proportions of populations are undergoing translocation and that they present a complex compartment-specific pattern of selection at the patient level.

Importance

It has been suggested that intra and extra-intestinal compartments differentially constrain the evolution ofE. colistrains. Whether host particular conditions, such as immunosuppression, could affect the strain evolutionary trajectories remain understudied. We found that, in immunosuppressed patients, large fractions ofE. coligut populations are translocating with variable modifications of the signature of selection for commensal and pathogenic isolates according to the compartment and/or the patient. Such multiple site sampling should be performed in large cohorts of patients to get a better understanding ofE. coliextra-intestinal diseases.
Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction
Authors: Dupuis Sunnyjoy , Lingappa Usha F. , Mayali Xavier , Sindermann Eve S. , Chastain Jordan L. , Weber Peter K. , Stuart Rhona , Merchant Sabeeha S.
Published: January 2024

Although the green algaChlamydomonas reinhardtiihas long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigatedC. reinhardtii’scapacity to support a heterotrophic microbe using the established coculture system withMesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga’s fermentative degradation of starch reserves during the night would boostM. japonicumheterotrophy.15NH4assimilation rates and changes in cell size revealed thatM. japonicumcells reduced new biomass synthesis in coculture with the alga but continued to divide – a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12and supported the growth of a B12-dependentC. reinhardtiimutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.
Impaired glycolysis in aged endothelial cells is associated with reduced neovascularisation upon tissue ischemia
Authors: Kiesworo Kevin , Agius Thomas , Lyon Arnaud , Macarthur Michael R , Zhang Jing , Déglise Sébastien , Markmann James F. , Uygun Korkut , Yeh Heidi , Bock Katrien De , Mitchell Sarah J. , Ocampo Alejandro , Allagnat Florent , Longchamp Alban
Published: January 2024

The vascular system experiences an age-associated decline in tissue perfusion and response to ischemic diseases. The factors driving this age-associated neovascularization decline remain unclear. While old endothelial cells (ECs) adopt a pro-angiogenic gene expression profile, we observed a stark reduction in the proliferative capacity of old ECs, while migratory capabilities remain intact. This is paralleled by a drastic decline in glycolytic capacity and ATP production, which likely act as limiting factors to neovascularization. These findings may provide new strategies to restore EC function in aging, thereby improving organ resilience and extending health- and lifespan.
In silico λ-dynamics predicts protein binding specificities to modified RNAs
Authors: Angelo Murphy , Zhang Wen , Vilseck Jonah Z. , Aoki Scott T.
Published: January 2024

RNA modifications shape gene expression through a smorgasbord of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.
Environmental and Population influences on Mummichog (Fundulus heteroclitus) Gut Microbiomes
Authors: Ma Lei , Hahn Mark E. , Karchner Sibel I. , Nacci Diane , Clark Bryan W. , Apprill Amy
Published: January 2024

The mummichog,Fundulus heteroclitus, an abundant estuarine fish broadly distributed along the eastern coast of North America, has repeatedly evolved tolerance to otherwise lethal levels of aromatic hydrocarbon exposure. This tolerance is linked to reduced activation of the aryl hydrocarbon receptor (AHR) signaling pathway. In other animals, the AHR has been shown to influence the gastrointestinal-associated microbial community, or gut microbiome, particularly when activated by the model toxic pollutant 3,3’,4,4’,5-pentachlorobiphenyl (PCB-126) and other dioxin-like compounds. In order to understand host population and PCB-126 exposure effects on mummichog gut microbiota, we sampled two populations of wild fish, one from a PCB-contaminated environment (New Bedford Harbor, MA) and the other from a non-polluted location (Scorton Creek, MA), as well as laboratory-reared F2 generation fish originating from each of these populations. We examined the bacteria and archaea associated with the gut of these fish using amplicon sequencing of small subunit ribosomal RNA genes. Fish living in the PCB-polluted site had high microbial alpha and beta diversity and an altered microbial network structure compared to fish from the non-polluted site. These differences between wild fish were not present in laboratory-reared F2 fish that originated from the same populations. Microbial compositional differences existed between the wild and lab-reared fish, with the wild fish dominated by Vibrionaceae and the lab-reared fish by Enterococceae. These results suggest that mummichog habitat and/or environmental conditions has a stronger influence on the mummichog gut microbiome compared to population or hereditary-based influences. Mummichog are important eco-evolutionary model organisms; this work reveals their importance for exploring host-environmental-microbiome dynamics.
Acid-treatedStaphylococcus aureusinduces acute silkworm hemolymph melanization
Authors: Matsumoto Yasuhiko , Sato Eri , Sugita Takashi
Published: January 2024

The skin microbiome maintains healthy human skin, and disruption of the microbiome balance leads to inflammatory skin diseases such as folliculitis and atopic dermatitis.Staphylococcus aureusandCutibacterium acnesare pathogenic bacteria that simultaneously inhabit the skin and cause inflammatory diseases of the skin through the activation of innate immune responses. Silkworms are useful invertebrate animal models for evaluating innate immune responses. In silkworms, phenoloxidase generates melanin as an indicator of innate immune activation upon the recognition of bacterial or fungal components. We hypothesized thatS. aureusandC. acnesinteract to increase the innate immunity-activating properties ofS. aureus. In the present study, we showed that acidification is involved in the activation of silkworm hemolymph melanization byS. aureus. Autoclaved-killedS. aureus(S. aureus[AC]) alone does not greatly activate silkworm hemolymph melanization. On the other hand, applyingS. aureus[AC] treated withC. acnesculture supernatant increased the silkworm hemolymph melanization. AddingC. acnesculture supernatant to the medium decreased the pH.S. aureus[AC] treated with propionic acid, acetic acid, or lactic acid induced higher silkworm hemolymph melanization activity than untreatedS. aureus[AC].S. aureus[AC] treated with hydrochloric acid also induced silkworm hemolymph melanization. The silkworm hemolymph melanization activity ofS. aureus[AC] treated with hydrochloric acid was inhibited by protease treatment ofS. aureus[AC]. These results suggest that acid treatment ofS. aureusinduces innate immune activation in silkworms and thatS. aureusproteins are involved in the induction of innate immunity in silkworms.
A critical role for Pol II CTD phosphorylation in heterochromatic gene activation
Authors: Kainth Amoldeep S. , Zhang Hesheng , Gross David S.
Published: January 2024

How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that inSIR-regulated heterochromatin ofSaccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription. Here we demonstrate that such activation occurs in a substantial fraction of cells, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene,YFR057w, as a model. Unlike covalent histone modifications, CTD phosphorylation at Ser2, Ser5 and Ser7 is abundant at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P,YFR057wis negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated inctk1Δ bur1cells when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene – an activity that may help supplant the need for histone epigenetic modifications.
Analyzing the implications of protein folding delay caused by translation
Authors: Houben Bert , Duran-Romaña Ramon , Migens Paula Fernández , Rousseau Frederic , Schymkowitz Joost
Published: January 2024

Because of vectorial protein production, residues that interact in the native protein structure but are distantly separated in the primary sequence are unavailable simultaneously. Instead, there is a temporal delay during which the N-terminal interaction partner is vulnerable to off-pathway, non-native interactions. In this analysis, we introduce “FoldDelay” (FD), a metric that integrates the topological pattern of atomic interactions of the native structure with translation kinetics to quantify such time delays. The FD metric reveals that many proteins, particularly at eukaryotic translation rates, exhibit residues with FDs in the range of tens of seconds. These residues, predominantly in well-structured, buried regions, often coincide with predicted aggregation-prone regions. We show a correlation between FD and co-translational engagement by the yeast Hsp70 chaperone Ssb, suggesting that fold-delayed regions have a propensity to misfold. In support of this, we show that proteins with high FDs are more frequently co-translationally ubiquitinated and prone to aggregate upon Ssb deletion. Finally, we find that FD cannot be adequately reduced through codon optimization, highlighting the importance of co-translational chaperones to shield these vulnerable regions. This work offers insights into co-translational proteostasis and the delicate balance between efficient folding and potential misfolding and aggregation during translation.