bluuebunny/crossref_metadata_2025 huggingface dataset:
TheDrosophilaTumour Suppressor Lgl and Vap33 activate the Hippo pathway by a dual mechanism, involving RtGEF/Git/Arf79F and inhibition of the V-ATPase
Authors: Portela Marta , Mukherjee Swastik , Paul Sayantanee , La Marca John E. , Parsons Linda M. , Veraksa Alexey , Richardson Helena E.
Published: July 2023

The tumour suppressor, Lethal (2) giant larvae (Lgl), is an evolutionarily conserved protein that was discovered in the vinegar fly,Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity and tissue architecture. Our previous studies have revealed a new role for Lgl in linking cell polarity and tissue growth through regulation of the Notch (proliferation and differentiation) and the Hippo (negative tissue growth control) signalling pathways. Moreover, Lgl regulates vesicle acidification, via the Vacuolar ATPase (V-ATPase), and we showed that Lgl inhibits V-ATPase activity through Vap33 (a Vamp (v-SNARE)-associated protein, involved in endo-lysosomal trafficking) to regulate the Notch pathway. However, how Lgl acts to regulate the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Using anin vivoaffinity-purification approach we found that Vap33 binds to the actin cytoskeletal regulators RtGEF (Pix, a Rho-type guanine nucleotide exchange factor) and Git (G protein-coupled receptor kinase interacting ArfGAP), which also bind to the Hpo protein kinase, and are involved in the activation of the Hippo pathway. Vap33 genetically interacts with RtGEF and Git in Hippo pathway regulation. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether our data suggests that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism, 1) through interaction with RtGEF/Git/Arf79F, and 2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.
SMU_1361c regulates the oxidative stress response ofStreptococcus mutans
Authors: Yu Shuxing , Huang Jun , Liu Yaqi , Li Jing , Zhang Qiong , Wang Yan , Gong Tao , Ma Qizhao , Zou Jing , Li Yuqing
Published: July 2023

Dental caries is the most common chronic infectious diseases around the world and disproportionately affects the marginalized socioeconomic group.Streptococcus mutans, considered a primary etiological agent of caries, must depend on the coordinated physiological response to tolerate the oxidative stress generated by commensal species within dental plaque, which is a critical aspect of its pathogenicity. Here, we identified and characterized a novel TetR family regulator SMU_1361c, encoded by the TnSmu2 operon, which appears to be acquired by the bacteria via horizontal genes transfer. Surprisingly,smu_1361cfunctions as a transcriptional repressor to regulate gene expression outside its operon, involved in the oxidative stress response ofS. mutans. Thesmu_1361coverexpression strain UA159/pDL278-1361cwas more susceptible to oxidative stress and less competitive against hydrogen peroxide generated by commensal speciesStreptococcus gordoniiandStreptococcus sanguinis. Transcriptomics analysis revealed thatsmu_1361coverexpression resulted in the significant downregulation of 22 genes mainly belonging to three gene clusters responsible for the oxidative stress response. The conversed DNA binding motif of SMU_1361c was determined by electrophoretic mobility shift and DNase I footprinting assay with purified SMU_1361c protein, therefore,smu_1361cis directly involved in gene transcription related to the oxidative stress response. Crucially, our finding provides a new understanding of howS. mutansdeals with the oxidative stress that is required for pathogenesis and will facilitate the development of new and improved therapeutic approaches for dental caries.
Temporal Dynamics of Apoptosis-Induced Proliferation in Pupal Wing Development: Implications for Regenerative Ability
Authors: Ahmed-de-Prado Sara , Estella Carlos , Baonza Antonio
Published: July 2023

The ability of animals to regenerate damaged tissue is a complex process that involves various cellular mechanisms. As animals age, they lose their regenerative abilities, making it essential to understand the underlying mechanisms that limit regenerative ability during aging.Drosophila melanogasterwing imaginal discs are epithelial structures that can regenerate after tissue injury. While significant research has focused on investigating regenerative responses during larval stages, particularly regarding the regulation and function of the JNK pathway, our comprehension of the regenerative potential of pupal wings and the underlying mechanisms contributing to the decline of regenerative responses remains limited. This study explores the temporal dynamics during pupal development of the proliferative response triggered by the induction of cell death, a typical regenerative response. Our results indicate that the apoptosis-induced proliferation response can be initiated as late as 30 hours after pupa formation (APF), when in normal circumstances cell proliferation ceases at around 20 hours APF. Furthermore, our data revealed that after 35 hours APF, cell death alone fails to induce further proliferation. Interestingly, the failure of reinitiating the cell cycle beyond this time point is not attributed to an incapacity to activate the JNK pathway. Instead, one of the constraining factors in the apoptotic-induced proliferation process during pupal development seems to be the activity level of ecdysone-responsive genes.

Author Summary

Animals have the remarkable ability to regenerate damaged tissues, but this regenerative potential diminishes with age. Understanding the mechanisms underlying age-related decline in regenerative abilities is crucial. Drosophila melanogaster wing imaginal discs provide a valuable model for studying tissue regeneration. While significant research has focused on regenerative responses during larval stages, our understanding of the regenerative potential and mechanisms in pupal wings remains limited.

In this study, we investigate the temporal dynamics of the proliferative response triggered by cell death during late during the development, in pupal development. Our findings reveal that the apoptosis-induced proliferation response can occur during pupal development, even after normal cell proliferation has ceased. However, at late stages of pupal development this response does not occur. We have found that, the inability to reinitiate the cell cycle beyond this time point is influenced by the activity of the hormone ecdysone and its-responsive genes.

These findings shed light on the dynamic processes involved in tissue regeneration during pupal development. This study expands our understanding of the complex interplay between cell death, proliferation, and gene activity during tissue regeneration, providing valuable insights for future research in regenerative biology.
Emergence of novel non-aggregative variants under negative frequency-dependent selection inKlebsiella variicola
Authors: Nucci Amandine , Janaszkiewicz Juliette , Rocha Eduardo P.C. , Rendueles Olaya
Published: July 2023

Klebsiella variicolais an emergent human pathogen causing diverse infections, including in the urinary tract. However, little is known about the evolution and maintenance of genetic diversity in this species, the molecular mechanisms and their population dynamics. Here, we characterized the emergence of a novel rdar-like morphotype which is contingent both on the genetic background and the environment. We show that mutations in either the nitrogen assimilation control gene(nac)or the type III fimbriae regulator,mrkH,suffice to generate rdar-like colonies. These morphotypes are primarily selected for the reduced inter-cellular aggregation as a result of loss-of-function yielding reduced fimbriae expression. Additionally, these clones also display increased growth rate and reduced biofilm formation. Direct competitions between rdar and wild type clone show that mutations inmrkHprovide large fitness advantages. In artificial urine, the morphotype is under strong negative frequency-dependent selection and is able to socially exploit wild type strains. An exhaustive search formrkHmutants in public databases revealed thatca8% of natural isolates analysed had truncated MrkH proteins many of which were due to insertions of IS elements, including a reported clinical isolate with rdar morphology. These strains were all isolated from human, mostly from urine. The decreased aggregation of these mutants could have important clinical implications as such clones could better disperse within the host allowing colonisation of other body sites and leading to systemic infections.

One-sentence Summary

Report of the emergence of a novel non-aggregative colony morphology inK. variicolaand the first example of social exploitation in theKlebsiellagenus.
Estimating temporally variable selection intensity from ancient DNA data II
Authors: Lyu Wenyang , Dai Xiaoyang , Beaumont Mark , Yu Feng , He Zhangyi
Published: July 2023

Recent technological innovations, such as next generation sequencing and DNA hybridisation enrichment, have made it possible to recover DNA information from historical and archaeological biological materials, which has motivated the development of various statistical approaches for inferring selection from allele frequency time series data. Recently, He et al. (2023a,b) introduced methods that can utilise ancient DNA (aDNA) data in the form of genotype likelihoods, therefore enabling the modelling of sample uncertainty arising from DNA molecule damage and fragmentation. However, their performance suffers from the underlying dependency on the allele age. Here we introduce a novel particle marginal Metropolis-Hastings within Gibbs framework for Bayesian inference of time-varying selection from aDNA data in the form of genotype like-lihoods. To circumvent the performance issue encountered in He et al. (2023a,b), we devise a novel numerical scheme for backward-in-time simulation of the Wright-Fisher diffusion and mix forward- and backward-in-time simulations in the particle filter for likelihood computation. Our framework also enables us to reconstruct the underlying population allele frequency trajectories, integrate temporal information in genotype likelihood calculations and test hypotheses on the drivers of past selection events. We conduct extensive evaluations through simulations and show its utility with an application to aDNA data from pigmentation loci in ancient horses.
High-throughput 3D imaging flow cytometry of adherent 3D cell cultures
Authors: Yamashita Minato , Tamamitsu Miu , Kirisako Hiromi , Goda Yuki , Chen Xiaoyao , Hattori Kazuki , Ota Sadao
Published: July 2023

Three-dimensional (3D) cell cultures are indispensable in recapitulatingin vivoenvironments. Among many 3D culture methods, the strategy to culture adherent cells on hydrogel beads to form spheroid-like structures is powerful for maintaining high cell viability and functions through an efficient supply of nutrients and oxygen. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high-throughput, scalable 3D imaging flow cytometry (3D-iFCM) platform for analyzing spheroid models on hydrogel beads. This platform is realized by integrating a single objective lens-based fluorescence light-sheet microscopy with a microfluidic device employing a combination of hydrodynamic and acoustofluidic focusing techniques. This integration enabled an unprecedentedly high-throughput, robust optofluidic 3D imaging, processing 513 cells s-1and a total of more than 104cells within a minute. The large dataset obtained allows us to quantify and compare the nuclear morphology of adhering and suspended cells, revealing adhering cells have smaller nuclei with non-round surfaces. This platform’s high throughput, robustness, and precision for analyzing the morphology of subcellular compartments in 3D culture models holds promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.
Mapping the dynamics of epigenetic adaptation during heterochromatin misregulation
Authors: Larkin Ajay , Kunze Colin , Seman Melissa , Levashkevich Alexander , Curran Justin , Morris-Evans Dionysus , Lemieux Sophia , Khalil Ahmad S. , Ragunathan Kaushik
Published: July 2023

A classical and well-established mechanism that enables cells to adapt to new and adverse conditions is the acquisition of beneficial genetic mutations. Much less is known about epigenetic mechanisms that allow cells to develop novel and adaptive phenotypes without altering their genetic blueprint. It has been recently proposed that histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), normally reserved to maintain genome integrity, can be redistributed across the genome to establish new and potentially adaptive phenotypes. To uncover the dynamics of this process, we developed a precision engineered genetic approach to trigger H3K9me redistribution on-demand in fission yeast. This enabled us to trace genome-scale RNA and chromatin changes over time prior to and during adaptation in long-term continuous cultures. Establishing adaptive H3K9me occurs over remarkably slow time-scales relative to the initiating stress. During this time, we captured dynamic H3K9me redistribution events ultimately leading to cells converging on an optimal adaptive solution. Upon removal of stress, cells relax to new transcriptional and chromatin states rather than revert to their initial (ground) state, establishing a tunable memory for a future adaptive epigenetic response. Collectively, our tools uncover the slow kinetics of epigenetic adaptation that allow cells to search for and heritably encode adaptive solutions, with implications for drug resistance and response to infection.
Patient-specific computational models predict prognosis in B cell lymphoma by quantifying pro-proliferative and anti-apoptotic signatures from genetic sequencing data
Authors: Norris Richard , Jones John , Mancini Erika , Chevassut Timothy , Pepper Chris , Pepper Andrea , Mitchell Simon
Published: July 2023

Genetic heterogeneity and co-occurring driver mutations impact clinical outcomes in blood cancers. Grouping tumours into clusters based on genetic alterations is prognostically informative. However, predicting the emergent effect of co-occurring mutations that impact multiple complex and interacting signalling networks remains challenging. Here, we used mathematical models to predict the impact of co-occurring mutations on cellular signalling and cell fates in diffuse large B cell lymphoma (DLBCL) and multiple myeloma (MM). Simulations predicted adverse impact on clinical prognosis when combinations of mutations induced both pro-proliferative and anti-apoptotic signalling. So, we established a pipeline to integrate patient-specific mutational profiles into personalised lymphoma models. Using this approach, we identified a subgroup (19%) of patients characterised by simultaneous upregulation of anti-apoptotic and pro-proliferative (AAPP) signalling. AAPP patients have dismal prognosis and can be identified within all current genomic and cell-of-origin classifications. Combining personalised molecular simulations with mutational clustering enabled stratification of patients into clinically informative prognostic categories: good (80% progression-free survival at 120 months), intermediate (median progression-free survival of 93 months), and poor (AAPP, median progression-free survival of 26 months). This study shows that personalised computational models enable identification of novel high-risk patient subgroups, providing a valuable tool for future risk-stratified clinical trials.
Genome-wide screen identifies new set of genes for improved heterologous laccase expression inSaccharomyces cerevisiae
Authors: Strawn Garrett , Wong Ryan , Young Barry , Davey Michael , Nislow Corey , Conibear Elizabeth , Loewen Christopher , Mayor Thibault
Published: July 2023

The yeastSaccharomyces cerevisiaeis widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of theTrametes trogiifungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions ofOCA6, a tyrosine phosphatase, along withPMT1 orPMT2, two ER membrane protein-O-mannosyltransferases involved in ER quality control, andSKI3, a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperoneSCJ1led to almost no secreted laccase activity. When we expressedSCJ1from a low-copy plasmid, laccase secretion was restored. However, overexpression of Scj1p had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression.

Importance

Our study showcases a newly developed high throughput screening technique to identify yeast mutant strains that exhibit an enhanced capacity for recombinant protein production. Using a genome-wide approach, we show that vesicle trafficking plays a crucial role in protein production, as the genes associated with this process are notably enriched in our screen. Furthermore, we demonstrate that a specific set of gene deletions, which were not previously recognized for their impact on recombinant laccase production, can be effectively manipulated in combination to increase the production of heterologous proteins. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.
Mmp17-deficient mice exhibit heightened goblet cell effector expression in the colon and increased resistance to chronicTrichuris murisinfection
Authors: Vornewald Pia M. , Forman Ruth , Yao Rouan , Pamar Naveen , Lindholm Håvard T. , Martín-Alonso Mara , Else Kathryn J. , Oudhoff Menno J.
Published: July 2023

Intestinal epithelial homeostasis is maintained by intrinsic and extrinsic signals. The extrinsic signals include those provided by mesenchymal cell populations that surround intestinal crypts and is further facilitated by the extracellular matrix (ECM), which is modulated by proteases such as matrix metalloproteinases (MMPs). Extrinsic signals ensure an appropriate balance between intestinal epithelial proliferation and differentiation. This study explores the role of MMP17, which is expressed by mesenchymal cells, in intestinal homeostasis and during immunity to infection. Mice lacking MMP17 expressed high levels of goblet-cell associated genes, such as CLCA1 and RELM-β, which are normally associated with immune responses to infection. Nevertheless,Mmp17KO mice did not have altered resistance during a bacterialCitrobacter rodentiuminfection. However, when challenged with a low dose of the helminthTrichuris muris,Mmp17KO mice had increased resistance, without a clear role for an altered immune response during infection. Mechanistically, we did not find changes in traditional modulators of goblet cell effectors such as the NOTCH pathway or specific cytokines. Instead, we found elevated BMP signaling inMmp17KO mouse large intestinal crypts that we propose to alter the goblet cell maturation state. Together, our data suggests that MMP17 extrinsically alters the goblet cell maturation stateviaa BMP signaling axis, which is sufficient to alter clearance in a helminth infection model.
Mapping Single-molecule Protein Complexes in 3D with DNA Nanoswitch Calipers
Authors: Shrestha Prakash , Yang Darren , Shih William M. , Wong Wesley P.
Published: July 2023

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force-spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices we call DNA Nanoswitch Calipers, which we have previously introduced, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5Åagreement to previously reported structures.
3D bioprinting directly affects proteomic signature and myogenic maturation in muscle pericytes-derived human myo-substitute
Authors: Reggio Alessio , Fuoco Claudia , Paolis Francesca De , Testa Stefano , Celikkin Nehar , Bernardini Sergio , Baldi Jacopo , Biagini Roberto , Seliktar Dror , Cirillo Carmine , Grumati Paolo , Cannata Stefano , Costantini Marco , Gargioli Cesare
Published: July 2023

Skeletal muscle tissue engineering (SMTE) has recently emerged to address major clinical challenges such as volumetric muscle loss. Here, we report a rotary wet-spinning (RoWS) biofabrication technique for producing human myo-substitutes with biomimetic architectures and functions. We show how the proposed technique may be used to establish a well-tailored, anisotropic microenvironment that promotes exceptional myogenic differentiation of human skeletal muscle-derived pericytes (hPeri). Using high-resolution mass spectrometry-based proteomics with the integration of literature-derived signaling networks, we uncovered that i) 3D biomimetic matrix environment (PEG-Fibrinogen) confers a lower mitogenicity microenvironment compared to standard 2D cultures, favoring the formation of contractile-competent bundles of pericytes-derived myotubes in an anchoring-independent 3D state, and ii) the bioprinting method promotes an upregulation of muscle matrix structural protein besides increasing contractile machinery proteins with respect to 3D bulk cultures. Finally,in vivoinvestigations demonstrate that the 3D bioprinted myo-substitute is fully compatible with the host ablated muscular tissue, exhibiting myo-substitute engraftment and muscle regeneration in a mouse VML model. Overall, the results show that 3D bioprinting has a superior capability for controlling the myogenic differentiation process on a macroscale and, with future refining, may have the potential to be translated into clinical practice.
A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria
Authors: Sullivan Geraldine J. , Barquist Lars , Cain Amy K.
Published: July 2023

Functional genomics techniques, such as transposon insertion sequencing and RNA sequencing, are key to studying relative differences in mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy number along the chromosome. This can lead to artefacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control, with relative differences in gene-wise read counts being the result of alterations in chromosomal replication dynamics rather than selection or direct gene regulation. We term this artefact ‘chromosomal location bias’ and implement a principled statistical approach to correct for it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated in statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analysing a ciprofloxacin-treated transposon insertion sequencing dataset inEscherichia colias a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress.

IMPORTANCE

Altered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.
Unreviewed science in the news: The evolution of preprint media coverage from 2014-2021
Authors: Fleerackers Alice , Shores Kenneth , Chtena Natascha , Alperin Juan Pablo
Published: July 2023

It has been argued that preprint coverage during the COVID-19 pandemic constituted a paradigm shift in journalism norms and practices. This study examines whether, in what ways, and to what extent this is the case using a sample of 11,538 preprints posted on four preprint servers—bioRxiv, medRxiv, arXiv, and SSRN—that received coverage in 94 English-language media outlets between 2014–2021. We compared mentions of these preprints with mentions of a comparison sample of 397,446 peer reviewed research articles indexed in the Web of Science to identify changes in the share of media coverage that mentioned preprints before and during the pandemic. We found that preprint media coverage increased at a slow but steady rate pre-pandemic, then spiked dramatically. This increase applied only to COVID-19-related preprints, with minimal or no change in coverage of preprints on other topics. In addition, the rise in preprint coverage was most pronounced among health and medicine-focused media outlets, which barely covered preprints before the pandemic but mentioned more COVID-19 preprints than outlets focused on any other topic. These results suggest that the growth in coverage of preprints seen during the pandemic period may imply a shift in journalistic norms, including a changing outlook on reporting preliminary, unvetted research.
Spontaneous Alignment of Myotubes through Myogenic Progenitor Cell Migration
Authors: Mehanna Lauren E. , Osborne Adrianna R. , Peterson Charlotte A. , Berron Brad J.
Published: July 2023

In large volume muscle injuries, widespread damage to muscle fibers and the surrounding connective tissue prevents myogenic progenitor cells (MPCs) from initiating repair. There is a clinical need to rapidly fabricate large muscle tissue constructs for integration at the site of large volume muscle injuries. Most strategies for myotube alignment require microfabricated structures or prolonged orientation times. We utilize the MPC’s natural propensity to close gaps across an injury site to guide alignment on collagen I. When MPCs are exposed to an open boundary free of cells, they migrate unidirectionally into the cell-free region and align perpendicular to the original boundary direction. We study the utility of this phenomenon with biotin - streptavidin adhesion to position the cells on the substrate, and then demonstrate the robustness of this strategy with unmodified cells, creating a promising tool for MPC patterning without interrupting their natural function. We pre-position MPCs in straight-line patterns separated with small gaps. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps, similar to how they migrate and align with a single open boundary. There is a directional component to the MPC migration perpendicular (90°) to the original biotin-streptavidin surface patterns. The expression of myosin heavy chain, the motor protein of muscle thick filaments, is confirmed through immunocytochemistry (ICC) in myotubes generated from MPCs in our patterning process, acting as a marker of skeletal muscle differentiation. The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary patterns, with MPC alignment based on natural regenerative behavior rather than complex fabrication techniques.

Impact Statement

Positioning myogenic progenitor cells (MPCs) into straight-line patterns with intentional spacings initiates the migration of these cells to bridge these gaps, mimicking their behavior in response to small-scale injuries. By creating repetitions of patterned cells and spacings, we have demonstrated rapid migration and alignment of MPCs, which differentiate into a long-range 2D layer of aligned myotubes.
Burkholderia pseudomalleiBicA protein promotes pathogenicity in macrophages by regulating Invasion, Intracellular Survival, and Virulence
Authors: Stockton Jacob L , Khakhum Nittaya , Stevenson Heather L. , Torres Alfredo G
Published: July 2023

Burkholderia pseudomallei (Bpm)is the causative agent of melioidosis disease.Bpmis a facultative intracellular pathogen with a complex lifecycle inside host cells. Pathogenic success depends on a variety of virulence factors with one of the most critical being the type 6 secretion system (T6SS).Bpmuses the T6SS to move into neighboring cells, resulting in multinucleated giant cells (MNGCs) formation, a strategy used to disseminate from cell-to-cell. Our prior study using a dual RNA-seq analysis to dissect T6SS-mediated virulence on intestinal epithelial cells identified BicA as a factor upregulated in a T6SS mutant (1). BicA regulates both type 3 secretion system (T3SS) and T6SSs; however, the extent of its involvement during disease progression is unclear. To fully dissect the role of BicA during systemic infection, we used two macrophage cell lines paired with a pulmonaryin vivochallenge murine model. We found that ΔbicAhas a distinct intracellular replication defect in both immortalized and primary macrophages that begins as early as 1 h post-infection. This intracellular defect is linked with the lack of cell-to-cell dissemination and MNGC formation, as well as a defect on T3SS expression. Thein vitrophenotype translatedin vivoas ΔbicAwas attenuated in a pulmonary model of infection; demonstrating a distinct macrophage activation profile and lack of pathological features present in the wild type. Overall, these results highlight the role of BicA in regulating intracellular virulence and demonstrate that specific regulation of secretion systems has a significant effect on host response andBpmpathogenesis.

Importance

Melioidosis is an understudied tropical disease that still results in ∼50% fatalities from those infected patients. It is caused by the Gram-negative bacillusBurkholderia pseudomallei(Bpm). Bpmis an intracellular pathogen that disseminates from the infected cell to target organs, causing disseminated disease. Regulation of secretion systems involved in entry and cell-to-cell spread is poorly understood. In this work, we characterize the role of BicA as a regulator of secretion systems during infection of macrophagesin vitroandin vivo. Understanding how these virulence factors are controlled will help us determine their influence on the host cells and define the macrophage responses associated with bacterial clearance.
ZIF-1-mediated degradation of endogenous and heterologous zinc finger proteins in theC. elegansgerm line
Authors: Schwartz Aaron Z.A. , Abdu Yusuff , Nance Jeremy
Published: July 2023

Rapid and conditional protein depletion is the gold standard genetic tool for deciphering the molecular basis of developmental processes. Previously, we showed that by conditionally expressing the E3 ligase substrate adaptor ZIF-1 inCaenorhabditis eleganssomatic cells, proteins tagged with the first CCCH Zn finger (ZF1) domain from the germline regulator PIE-1 degrade rapidly, resulting in loss-of-function phenotypes. The described role of ZIF-1 is to clear PIE-1 and several other CCCH Zn finger proteins from early somatic cells, helping to enrich them in germline precursor cells. Here, we show that proteins tagged with the PIE-1 ZF1 domain are subsequently cleared from primordial germ cells in embryos and from undifferentiated germ cells in larvae and adults by ZIF-1. We harness germline ZIF-1 activity to degrade a ZF1-tagged heterologous protein from PGCs and show that its depletion produces phenotypes equivalent to those of a null mutation. Our findings reveal that ZIF-1 switches roles from degrading CCCH Zn finger proteins in somatic cells to clearing them from undifferentiated germ cells, and that ZIF-1 activity can be harnessed as a new genetic tool to study the early germ line.
DUX4 is a common driver of immune evasion and immunotherapy failure in metastatic cancers
Authors: Pineda Jose Mario Bello , Bradley Robert K.
Published: July 2023

Cancer immune evasion contributes to checkpoint immunotherapy failure in many patients with metastatic cancers. The embryonic transcription factor DUX4 was recently characterized as a suppressor of interferon-γ signaling and antigen presentation that is aberrantly expressed in a small subset of primary tumors. Here, we report thatDUX4expression is a common feature of metastatic tumors, with ∼10-50% of advanced bladder, breast, kidney, prostate, and skin cancers expressingDUX4.DUX4expression is significantly associated with immune cell exclusion and decreased objective response to PD-L1 blockade in a large cohort of urothelial carcinoma patients.DUX4expression is a significant predictor of survival even after accounting for tumor mutational burden and other molecular and clinical features in this cohort, withDUX4expression associated with a median reduction in survival of over one year. Our data motivate future attempts to develop DUX4 as a biomarker and therapeutic target for checkpoint immunotherapy resistance.
A temperature-sensitive metabolic valve and a transcriptional feedback loop drive rapid homeoviscous adaptation inEscherichia coli
Authors: Hoogerland Loles , van den Berg Stefan , Zoumaro-Djayoon Adja , Geurken Esther , Yang Flora , Bruggeman Frank , Bokinsky Gregory
Published: July 2023

All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. A quantitative description of how organisms maintain constant fluidity at all growth temperatures has not been achieved. By quantifying both enzymes and metabolic intermediates of theEscherichia colifatty acid and phospholipid synthesis pathways, we discover howE. colimeasures steady-state temperature and restores optimal membrane fluidity within a single generation after temperature shocks. The first element of the system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways. The second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This overshoot strategy accelerates membrane adaptation, and is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a quantitative model, which we further use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across a wide range of temperatures. We hypothesize that core design features of theE. colisystem will prove to be ubiquitous features of homeoviscous adaptation systems.
Proteome-wide copy-number estimation from transcriptomics
Authors: Sweatt Andrew J. , Griffiths Cameron D. , Paudel B. Bishal , Janes Kevin A.
Published: July 2023

Protein copy numbers constrain systems-level properties of regulatory networks, but absolute proteomic data remain scarce compared to transcriptomics obtained by RNA sequencing. We addressed this persistent gap by relating mRNA to protein statistically using best-available data from quantitative proteomics–transcriptomics for 4366 genes in 369 cell lines. The approach starts with a central estimate of protein copy number and hierarchically appends mRNA-protein and mRNA-mRNA dependencies to define an optimal gene-specific model that links mRNAs to protein. For dozens of independent cell lines and primary prostate samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, and empirical protein-to-mRNA ratios. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein interaction complexes, suggesting mechanistic relationships are embedded. We use the method to estimate viral-receptor abundances of CD55–CXADR from human heart transcriptomes and build 1489 systems-biology models of coxsackievirus B3 infection susceptibility. When applied to 796 RNA sequencing profiles of breast cancer from The Cancer Genome Atlas, inferred copy-number estimates collectively reclassify 26% of Luminal A and 29% of Luminal B tumors. Protein-based reassignments strongly involve a pharmacologic target for luminal breast cancer (CDK4) and an α-catenin that is often undetectable at the mRNA level (CTTNA2). Thus, by adopting a gene-centered perspective of mRNA-protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility limits of contemporary proteomics. The collection of gene-specific models is assembled as a web tool for users seeking mRNA-guided predictions of absolute protein abundance (http://janeslab.shinyapps.io/Pinferna).

Significance statement

It is easier to quantify mRNA in cells than it is to quantify protein, but proteins are what execute most cellular functions. Even though protein is synthesized from mRNA in cells, relating a cellular quantity of mRNA to a quantity of protein is challenging. Here, we bring together quantitative measures of mRNA and protein for 4366 genes in 369 different cultured cell types to build equations that predict protein abundance from the abundance of mRNAs expressed. These equations capture facets of biological regulation and work better than existing alternatives that rely on consensus values or ratios. Since mRNA measurements are more widespread than protein, this study makes new analyses possible by protein estimation from mRNA.
Calcineurin is required forCandida glabrataPdr1 transcriptional activation
Authors: Vu Bao Gia , Simonicova Lucia , Moye-Rowley W. Scott
Published: July 2023

Fluconazole is the most commonly used antifungal today. A result of this has been the inevitable selection of fluconazole resistant organisms. This is an especially acute problem in the pathogenic yeastCandida glabrata. Elevated minimal inhibitory concentrations (MICs) for fluconazole inC. glabrataare frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding genePDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes likeCDR1that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure ofC. glabratato fluconazole induced expression of both Pdr1 andCDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation and a hyperactive form of this phosphatase can increase resistance to the echinocandin caspofungin but does not show a similar elevation for fluconazole resistance. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease inCDR1expression and fluconazole resistance, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin resistance together via the control of transcription factor activity.

Importance

While drug resistant microorganisms are a problem in treatment of all infectious disease, this is an especially acute problem with fungi due to the existence of only 3 classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeastCandida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with fluconazole resistance and poor clinical outcomes. Here we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug resistance. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally elevated fluconazole resistance. Previous studies showed that calcineurin is required for resistance to the echinocandin class of antifungal drugs and our data demonstrate this protein phosphatase is also required for azole drug resistance. Calcineurin plays a central role in resistance to two of the three major classes of antifungal drugs inC. glabrata.
Scaling, correlations, and artifacts in biological timeseries
Authors: Faerberg Denis F. , Gurarie Victor , Ruvinsky Ilya
Published: July 2023

Summary

Time is a key variable in biology. Conceptual and methodological advances focused recent studies of timing in biological systems on monitoring trait dynamics in singled individuals. Here we contribute to this effort by analyzing general properties of individual timeseries. We make four broad claims. First, we show that for traits that range from behavior in animals, to growth in plants, to division timing in cell lineages, faster timeseries tend to be comprised of consistently shorter sub-stages, not a few unusually fast sub-stages. We demonstrate that this property constitutes a particular type of scaling that can be readily detected by a straightforward comparison of absolute and relative variability within timeseries data for any trait. We show that correlations within timeseries are necessary and sufficient for the type of scaling we describe and infer that the ubiquitous occurrence of scaling results from natural correlations within the continuous processes that govern trait dynamics. Second, failing to observe scaling indicates a break in correlation and, we argue, a switch in the mechanisms underlying the rate of trait change. Third, we caution that analyses of individual timeseries are highly susceptible to batch effects, but contend that it is possible to detect signal despite the artifactual correlations introduced by batching. Finally, we show that the timing of biological process that scale is overdispersed compared to matched but uncorrelated comparisons; this is an advantage in variable environments. Our results advance understanding of common properties of biological timeseries and offer practical tools for their further study.
Integrative reappraisal of the Amazonian nurse frogAllobates gasconi(Morales 2002) based on topotypical data, with implications for the systematics and taxonomy of a large species complex
Authors: Ferreira Anthony S. , Ferrão Miquéias , Cunha-Machado Antonio S. , Magnusson William E. , Hanken James , Lima Albertina P.
Published: July 2023

Taxonomic uncertainty at the species level compromises our knowledge of biodiversity, conservation and systematics. The impact of such uncertainty is heightened in megadiverse regions such as Amazonia due to high levels of cryptic diversity. We used integrative taxonomy based on newly collected topotypical specimens to redescribe the Amazonian nurse frogAllobates gasconiand infer its phylogenetic relationships. This species was described in 2002 based solely on morphology, but several characters crucial for the reliable diagnosis of species inAllobateswere not considered. Our results show thatA. gasconisensu stricto is not a member of theA. caeruleodactylusclade as previously claimed, but is a member of theA. trilineatusclade.Allobates gasconiis readily distinguished from congeners by a combination of morphological and bioacoustic characters; a revised diagnosis is provided. The type series ofA. gasconicomprises more than one species, and we exclude a paratype from lower Juruá River. The species is restricted geographically to flooded environments in the middle and upper Juruá River in Brazil and in the Ucayali River in Peru. The initial misidentification, subsequent absence of topotypic molecular and acoustic data, and the poor preservation condition of the type series have contributed to taxonomic confusion sinceA. gasconiwas first described. The descriptions of other species ofAllobatespublished more than two decades ago were based mainly on gross morphology and we recommend integrative taxonomic revisions to elucidate their systematics.
Atomic-level architecture ofCaulobacter crescentusflagellar filaments provide evidence for multi-flagellin filament stabilization
Authors: Sanchez Juan C. , Montemayor Eric J. , Ploscariu Nicoleta T. , Parrell Daniel , Baumgardt Joseph K. , Yang Jie E. , Sibert Bryan , Cai Kai , Wright Elizabeth R.
Published: July 2023

Flagella are dynamic, ion-powered machines with assembly pathways that are optimized for efficient flagella production. In bacteria, dozens of genes are coordinated at specific times in the cell lifecycle to generate each component of the flagellum. This is the case forCaulobacter crescentus, but little is known about why this species encodes six different flagellin genes. Furthermore, little is known about the benefits multi-flagellin species possess over single flagellin species, if any, or what molecular properties allow for multi-flagellin filaments to assemble. Here we present an in-depth analysis of several single flagellin filaments fromC. crescentus,including an extremely well-resolved structure of a bacterial flagellar filament. We highlight key molecular interactions that differ between each bacterial strain and speculate how these interactions may alleviate or impose helical strain on the overall architecture of the filament. We detail conserved residues within the flagellin subunit that allow for the synthesis of multi-flagellin filaments. We further comment on how these molecular differences impact bacterial motility and highlight how no single flagellin filament achieves wild-type levels of motility, suggestingC. crescentushas evolved to produce a filament optimized for motility comprised of six flagellins. Finally, we highlight an ordered arrangement of glycosylation sites on the surface of the filaments and speculate how these sites may protect the β-hairpin located on the surface exposed domain of the flagellin subunit.