bluuebunny/crossref_metadata_2025 huggingface dataset:
Expansion microscopy resolves the 3D thylakoid structure
Authors: Bos Peter R. , Berentsen Jarne , Wientjes Emilie
Published: May 2023

The light-harvesting reactions of photosynthesis take place on the thylakoid membrane inside chloroplasts. The thylakoid membrane is folded into appressed membranes, the grana, and non-appressed membranes that interconnect the grana, the stroma lamellae. This folding is essential for the correct functioning of photosynthesis. Electron microscopy and atomic force microscopy are commonly used to study the thylakoid membrane, but these techniques have limitations in visualizing a complete chloroplast and its organization. To overcome this limitation, we applied expansion microscopy (ExM) on isolated chloroplasts. ExM is a technique that involves physically expanding a sample in a swellable hydrogel to enhance the spatial resolution of fluorescence microscopy. Using all-protein staining, we have visualized the 3D structure of spinach thylakoids with a high level of detail. We were able to resolve stroma lamellae that were 60 nm apart and observe their helical wrapping around the grana. Furthermore, we accurately measured the dimensions of grana from top-views of chloroplasts, which allow for precise determination of the grana diameter. Ultimately, we constructed a 3D model of a complete chloroplast, which provides a foundation for structure-based modeling of photosynthetic adaptations. Our results demonstrate that ExM is a fast and reliable technique for studying thylakoid organization with a high level of detail.
Particulate matter composition drives differential molecular and morphological responses in lung epithelial cells
Authors: Engels Sean M. , Kamat Pratik , Pafilis G. Stavros , Li Yukang , Agrawal Anshika , Haller Daniel J. , Phillip Jude M. , Contreras Lydia M.
Published: May 2023

Particulate matter (PM) is a ubiquitous component of indoor and outdoor air pollution that is epidemiologically linked to many human pulmonary diseases. PM has many emission sources, making it challenging to understand the biological effects of exposure due to the high variance in chemical composition. However, the effects of compositionally unique particulate matter mixtures on cells have not been analyzed using both biophysical and biomolecular approaches. Here, we show that in a human bronchial epithelial cell model (BEAS-2B), exposure to three chemically distinct PM mixtures drives unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability and DNA damage responses and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization and structure, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Lastly, we observed that particulate matter mixtures with high contents of heavy metals, such as cadmium and lead, induced larger drops in viability, increased DNA damage, and drove a redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of cellular morphology provides a robust approach to gauge the effects of environmental stressors on biological systems and determine cellular susceptibilities to pollution.
Changes in Seasonal Footwear Elicited Alterations in Gait Kinematics but Not Stability
Authors: Garrah Sydney N. , Best Aaron N. , Wu Amy R.
Published: May 2023

During daily walking, humans might contend with various perturbations from slippery surfaces in the winter to uneven sidewalks in the summer. Inertial sensors enable investigations of how humans maintain balance under these natural conditions, but conducting these outdoor studies has practical considerations that might influence study results, such as the selection of footwear under different weather conditions. Our study investigates the effects of winter and summer shoe types on gait patterns, specifically whether different shoe types induce changes in gait stability measures under the same walking environment. Twelve healthy adult participants walked indoors with winter and summer shoes while their gait kinematics were recorded using an inertial sensor-based motion capture system. Spatiotemporal measures, body kinematics, stability measures (minimum margin of stability and local divergence exponent), and stepping regressions were calculated to evaluate differences between walking in summer and winter shoes. Statistical significance was determined by paired t-tests. Varying shoe types altered spatiotemporal and kinematic measures, such as increased stride time and stance time while wearing winter shoes, but increased step width and reliance on stepping were the only stability-related changes found. Our study provides insights into the influence of footwear for inertial sensor-based gait studies in real environments, aiding the analysis and interpretation of those studies to augment our understanding of natural stability behavior.
13C tracer analysis reveals the landscape of metabolic checkpoints in human CD8+T cell differentiation
Authors: Kirchmair Alexander , Nemati Niloofar , Lamberti Giorgia , Trefny Marcel P. , Krogsdam Anne , Siller Anita , Hörtnagl Paul , Schumacher Petra , Sopper Sieghart , Sandbichler Adolf M. , Zippelius Alfred , Ghesquière Bart , Trajanoski Zlatko
Published: May 2023

Naïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start proliferation and generation of effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells. Here we used13C metabolomics and transcriptomics to study the metabolic dynamics of CD8+T cells in their complete course of differentiation from naïve over stem-like memory to effector cells. The quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression ofENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of T cell regulatory genes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which paradoxically was not linked to changes in mitochondrial abundance. We further investigated the metabolic phenotype of exhausted T cells, finding that decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of suppressive and quiescence-associated genes, includingTXNIPandKLF2. Thus, these results identify multiple features critical for the metabolic reprogramming that supports quiescence, proliferation and effector function of CD8+T cells during differentiation. Further, an impairment of the same processes in exhaustion suggests that targeting these control points may be useful for both modulation of differentiation and prevention of exhaustion.
Energy Guided Diffusion for Generating Neurally Exciting Images
Authors: Pierzchlewicz Paweł A. , Willeke Konstantin F. , Nix Arne F. , Elumalai Pavithra , Restivo Kelli , Shinn Tori , Nealley Cate , Rodriguez Gabrielle , Patel Saumil , Franke Katrin , Tolias Andreas S. , Sinz Fabian H.
Published: May 2023

In recent years, most exciting inputs (MEIs) synthesized from encoding models of neuronal activity have become an established method to study tuning properties of biological and artificial visual systems. However, as we move up the visual hierarchy, the complexity of neuronal computations increases. Consequently, it becomes more challenging to model neuronal activity, requiring more complex models. In this study, we introduce a new attention readout for a convolutional data-driven core for neurons in macaque V4 that outperforms the state-of-the-art task-driven ResNet model in predicting neuronal responses. However, as the predictive network becomes deeper and more complex, synthesizing MEIs via straightforward gradient ascent (GA) can struggle to produce qualitatively good results and overfit to idiosyncrasies of a more complex model, potentially decreasing the MEI’s model-to-brain transferability. To solve this problem, we propose a diffusion-based method for generating MEIs via Energy Guidance (EGG). We show that for models of macaque V4, EGG generates single neuron MEIs that generalize better across architectures than the state-of-the-art GA while preserving the within-architectures activation and requiring 4.7x less compute time. Furthermore, EGG diffusion can be used to generate other neurally exciting images, like most exciting natural images that are on par with a selection of highly activating natural images, or image reconstructions that generalize better across architectures. Finally, EGG is simple to implement, requires no retraining of the diffusion model, and can easily be generalized to provide other characterizations of the visual system, such as invariances. Thus EGG provides a general and flexible framework to study coding properties of the visual system in the context of natural images.1
Micelle-like clusters in phase-separated Nanog condensates: A molecular simulation study
Authors: Mizutani Azuki , Tan Cheng , Sugita Yuji , Takada Shoji
Published: May 2023

The phase separation model for transcription suggests that transcription factors (TFs), coactivators, and RNA polymerases form biomolecular condensates around active gene loci and regulate transcription. However, the structural details of condensates remain elusive. In this study, for Nanog, a master TF in mammalian embryonic stem cells known to form protein condensatesin vitro, we examined protein structures in the condensates using residue-level coarse-grained molecular simulations. Human Nanog formed micelle-like clusters in the condensate. In the micelle-like cluster, the C-terminal disordered domains, including the tryptophan repeat (WR) regions, interacted with each other near the cluster center primarily via hydrophobic interaction. In contrast, hydrophilic disordered N-terminal and DNA-binding domains were exposed on the surface of the clusters. Electrostatic attractions of these surface residues were responsible for bridging multiple micelle-like structures in the condensate. The micelle-like structure and condensate were dynamic and liquid-like. Mutation of tryptophan residues in the WR region which was implicated to be important for a Nanog function resulted in dissolution of the Nanog condensate. Finally, to examine the impact of Nanog cluster to DNA, we added DNA fragments to the Nanog condensate. Nanog DNA-binding domains exposed to the surface of the micelle-like cluster could recruit more than one DNA fragments, making DNA-DNA distance shorter.

Author summary

In eukaryotic transcription regulation, enhancer elements far from the promoter are known to modulate transcriptional activity, but the molecular mechanism remains elusive. One of these models, the phase separation model, suggests that transcription factors (TFs), coactivators, and transcription machinery form biomolecular condensates that include enhancer and promoter elements, making enhancer-promoter communication possible via the protein network in the condensate. Nanog, one of the core TFs involved in mammalian embryonic stem cells, has been reported to have the ability to form condensates. In this study, we addressed the structural details of Nanog condensates by performing residue-level coarse-grained molecular simulations. We found that Nanog formed micelle-like clusters via primarily hydrophobic interactions between tryptophan repeat regions in the C-terminal disordered domains within the condensate. On the other hand, highly charged N-terminal and DNA-binding domains were exposed to the surface of the micelle and were responsible for bridging many micelles into a condensate. The micelle-like clusters and condensate were dynamic and liquid-like. In addition, Nanog condensates could induce DNA-DNA attraction mediated by micelle-like structures.
Causes for and consequences of winning intergroup encounters in colobus monkeys (Colobus vellerosus)
Authors: Wikberg Eva C. , Lucci Sara , Glotfelty Emily , Campos Fernando , Sicotte Pascale
Published: May 2023

The outcome of an intergroup encounter depends on the relative competitive abilities of the participating groups and the value of the resource for which they compete. However, few studies have been able to assess the consequences of winning intergroup encounters. We used behavioral and demographic data from 94 adult and subadult individuals in 8 groups ofColobus vellerosusat Boabeng-Fiema, Ghana to investigate the factors that predict winning intergroup encounters, and whether intergroup encounter success determines access to food and female reproductive output. In support of the hypothesis that groups with high-quality males would be more likely to win encounters, winning the encounter was best predicted by rates of displays by the alpha male. Food trees that were contested during intergroup encounters were more likely to have young leaves or other higher quality food items and to be important food species. Feeding was more likely to occur during and after the intergroup encounter if the focal animal’s group had won the encounter. The percentage of encounters won was correlated with the group’s dominance rank but not with home range size or the immature-to-female ratio. In populations such as the Boabeng-Fiema colobus in which male quality seems to be associated with winning intergroup encounters and gaining immediate access to food, one of the drivers for female transfer between groups may be differences in alpha male quality between groups and across time.
On the role of melanistic coloration on thermoregulation in the crepuscular geckoEublepharis macularius
Authors: Hastings Brandon T. , Melnyk Anastasiya , Ghyabi Mehrdad , White Emma , Barroso Frederico M. , Carretero Miguel A , Lattanzi David , Claude Julien , Chiari Ylenia
Published: May 2023

Body coloration in ectotherms serves multiple biological functions, including avoiding predators, communicating with conspecific individuals, and involvement in thermoregulation. As ectotherms rely on environmental sources of heat to regulate their internal body temperature, stable melanistic body coloration or color change can be used to increase or decrease heat absorption and heat exchange with the environment. While the function of melanistic coloration for thermoregulation has been found to increase solar radiation absorption for heating in many diurnal ectotherms, research on crepuscular and nocturnal ectotherms is lacking. Since crepuscular and nocturnal ectotherms generally absorb heat from the substrate, coloration is likely under different selective pressures than in diurnal ectotherms. We tested if the proportion of dorsal melanistic body coloration is related to differences in body temperature heating and cooling rates in the crepuscular geckoEublepharis maculariusand whether changes in environmental temperature trigger color changes in this species. Temperature measurements of the geckos and of the environment were taken using infrared thermography and temperature loggers. Color data were obtained using objective photography and a newly developed custom software package. We found that body temperature reflected substrate temperatures, and that the proportion of melanistic coloration has no influence on heating or cooling rates or on color changes. These findings suggest that, inE. macularius, melanistic coloration may not be used for thermoregulation. Future research should further test the function of melanistic coloration in other crepuscular and nocturnal vertebrates to understand the evolution of melanistic pattern in animals active in low light conditions.
Epstein-Barr virus miR-BARTs 7 and 9 modulate viral cycle, cell proliferation and proteomic profiles in Burkitt lymphoma
Authors: Caetano Brunno Felipe Ramos , Rocha Viviana Loureiro , Rossini Bruno Cesar , Santos Lucilene Delazari dos , de Oliveira Deilson Elgui
Published: May 2023

The Epstein-Barr Virus (EBV) encodes viral microRNAs (miRs) that contribute to the pathogenesis of nasopharyngeal and gastric carcinomas, but their potential roles in lymphomas are still to be elucidated. This study sought to assess the impact of knocking down EBV miRs BART 7 and BART9 in EBV-positive Akata cell lines using CRISPR/Cas9 technology. Compared to cells harboring the wild-type (WT) EBV genomes, Akata cells subjected to CRISPR/Cas9-mediated knockdown of EBV BART 7 and BART9 showed a significant reduction in the expression of viral miRs, confirming the validity of the experimental model. Knocking down both BART7 and BART9 caused a significant reduction in cell viability and proliferation rates while increasing the expression of EBV lytic genes. Global proteomic analysis shows that knocking down EBV BART7 significantly decreased the expression of ubiquitin/proteasome proteins while increasing RNA binding proteins (RBPs). On the other hand, BART9 knockdown caused a decrease in proteins associated with oxidoreductase activity, including the metabolism of fatty acids. Our results unravel previously unknown roles for EBV miRs BART7 and BART9 on cellular pathways relevant to both viral biology and lymphomagenesis.
Regulation of RNA methylation by therapy treatment, promotes tumor survival
Authors: Bukhari Syed IA , Truesdell Samuel S , Datta Chandreyee , Choudhury Pritha , Wu Keith Q , Shrestha Jitendra , Maharjan Ruby , Plotsker Ethan , Elased Ramzi , Laisa Sadia , Bhambhani Vijeta , Lin Yue , Kreuzer Johannes , Morris Robert , Koh Siang-Boon , Ellisen Leif W. , Haas Wilhelm , Ly Amy , Vasudevan Shobha
Published: May 2023

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5′UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.

One sentence summary

m6A enzyme translation upon therapy stress, promotes tumor survival
Disrupted memory T cell expansion in HIV-exposed uninfected infants is preceded by premature skewing of T cell receptor clonality
Authors: Dzanibe Sonwabile , Wilk Aaron J. , Canny Susan , Ranganath Thanmayi , Alinde Berenice , Rubelt Florian , Huang Huang , Davis Mark M. , Holmes Susan , Jaspan Heather B. , Blish Catherine A. , Gray Clive M.
Published: May 2023

While preventing vertical HIV transmission has been very successful, the increasing number of HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Immune developmental differences between iHEU and iHUU remains poorly understood and here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations and differences in the emergence of NK cell populations and T cell memory differentiation between iHEU and iHUU. Specific NK cells observed at birth were also predictive of acellular pertussis and rotavirus vaccine-induced IgG and IgA responses, respectively, at 3 and 9 months of life. T cell receptor Vβ clonotypic diversity was significantly and persistently lower in iHEU preceding the expansion of T cell memory. Our findings show that HIV/ARV exposure disrupts innate and adaptive immunity from birth which may underlie relative vulnerability to infections.
Longitudinal analysis of a dietary mouse model of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)
Authors: Saenz Marissa , McDonough Jillian C. , Bloom-Saldana Elizabeth , Irimia Jose M. , Cauble Emily L. , Castillo Ashly , Fueger Patrick T. , Treviño Lindsey S.
Published: May 2023

Non-alcoholic fatty liver disease (NAFLD), and resultant non-alcoholic steatohepatitis (NASH), incidence and prevalence are rising globally due to increasing rates of obesity and diabetes. Currently, there are no approved pharmacological treatments for NAFLD, highlighting a need for additional mechanistic studies to develop prevention and/or therapeutic strategies. Diet-induced preclinical models of NAFLD can be used to examine the dynamic changes that occur during NAFLD development and progression throughout the lifespan. To date, most studies utilizing such models have focused exclusively on terminal time points and have likely missed critical early and late changes that are important for NAFLD progression (i.e, worsening). We performed a longitudinal analysis of histopathological, biochemical, transcriptomic, and microbiome changes that occurred in adult male mice fed either a control diet or a NASH-promoting diet (high in fat, fructose, and cholesterol) for up to 30 weeks. We observed progressive development of NAFLD in mice fed the NASH diet compared to the control diet. Differential expression of immune-related genes was observed at an early stage of diet-induced NAFLD development (10 weeks) and persisted into the later stages of the disease (20 and 30 weeks). Differential expression of xenobiotic metabolism related genes was observed at the late stage of diet-induced NAFLD development (30 weeks). Microbiome analysis revealed an increased abundance ofBacteroidesat an early stage (10 weeks) that persisted into the later stages of the disease (20 and 30 weeks). These data provide insight into the progressive changes that occur during NAFLD/NASH development and progression in the context of a typical Western diet. Furthermore, these data are consistent with what has been reported in patients with NAFLD/NASH, supporting the preclinical use of this diet-induced model for development of strategies to prevent or treat the disease.
Predicting gene level sensitivity to JAK-STAT signaling perturbation using a mechanistic-to-machine learning framework
Authors: Cheemalavagu Neha , Shoger Karsen E. , Cao Yuqi M. , Michalides Brandon A. , Botta Samuel A. , Faeder James R. , Gottschalk Rachel A.
Published: May 2023

The JAK-STAT pathway integrates complex cytokine signals via a limited number of molecular components, inspiring numerous efforts to clarify the diversity and specificity of STAT transcription factor function. We developed a computational workflow to make global cytokine-induced gene predictions from STAT phosphorylation dynamics, modeling macrophage responses to IL-6 and IL-10, which signal through common STATs, but with distinct temporal dynamics and contrasting functions. Our mechanistic-to-machine learning model identified select cytokine-induced gene sets associated with late pSTAT3 timeframes and a preferential pSTAT1 reduction upon JAK2 inhibition. We predicted and validated the impact of JAK2 inhibition on gene expression, identifying dynamically regulated genes that were sensitive or insensitive to JAK2 variation. Thus, we successfully linked STAT signaling dynamics to gene expression to support future efforts targeting pathology-associated STAT-driven gene sets. This serves as a first step in developing multi-level prediction models to understand and perturb gene expression outputs from signaling systems.
CRISPR-based engineering of RNA viruses
Authors: Nemudryi Artem , Nemudraia Anna , Nichols Joseph E , Scherffius Andrew M , Zahl Trevor , Wiedenheft Blake
Published: May 2023

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a new recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.

One-Sentence Summary

Programmable CRISPR RNA-guided ribonucleases enable recombinant RNA technology.
The structure of NAD+consuming proteinAcinetobacter baumanniiTIR domain shows unique kinetics and conformations
Authors: Klontz Erik , Obi Juliet O. , Wang Yajing , Glendening Gabrielle , Carr Jahid , Tsibouris Constantine , Buddula Sahthi , Nallar Shreeram , Soares Alexei S. , Beckett Dorothy , Redzic Jasmina S. , Eisenmesser Elan , Palm Cheyenne , Schmidt Katrina , Scudder Alexis H. , Obiorah Trinity , Essuman Kow , Milbrandt Jeffrey , Diantonio Aaron , Ray Krishanu , Deredge Daniel , Snyder M LD. , Snyder Greg A.
Published: May 2023

Toll-like and Interleukin-1/18 receptor resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of theAcinetobacter baumanniiTIR domain protein (AbTir-TIR) with confirmed NAD+hydrolysis and map the conformational effects of its interaction with NAD+using HDX-MS. NAD+results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using 2pFLIM which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIREAprotein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.
Centralin vivomechanisms by whichC. difficile’sproline reductase drives efficient metabolism, growth, and toxin production
Authors: Cersosimo Laura M. , Graham Madeline , Monestier Auriane , Pavao Aidan , Worley Jay N. , Peltier Johann , Dupuy Bruno , Bry Lynn
Published: May 2023

Clostridioides difficile(CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate thein vivoeffects of reductive proline metabolism onC.difficile’svirulence in an enriched gut nutrient environment, we evaluated wild-type and isogenicΔprdBstrains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with theΔprdBmutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease.In vivotranscriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted the pathogen’s metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages ofC. difficilecolonization and subsequent impact on the pathogen’s ability to rapidly expand and cause disease.
How do fluctuating temperatures alter the cost of development?
Authors: Pettersen Amanda K , Nord Andreas , While Geoffrey , Uller Tobias
Published: May 2023

Quantifying how variable temperature regimes affect energy expenditure during development is crucial for understanding how future thermal regimes may impact early life survival and population persistence.

Developmental Cost Theory (DCT) suggests that there is an optimal temperature (Topt) that minimises energy expenditure during development (the “cost of development”). Exposure to fluctuating temperatures around an average of Toptare anticipated to increase either development time or metabolic rate. As a result, embryos will rapidly deplete yolk reserves, and consequently hatch at a smaller size or with less residual yolk to support postnatal survival and growth.

Here, we studied total embryonic energy expenditure (development time and rate of CO2production) and conversion of yolk into tissue in common wall lizards (Podarcis muralis) under three incubation treatments anticipated, based on DCT, to increase the cost of development: no variance (Toptconstant, 24 °C), low variance (22 – 26 °C), and high variance (18 – 30 °C).

As predicted, we found that increasing variance around Toptincreased the cost of development, despite reducing time to hatching. As a consequence, embryos on average hatched with 59% lower residual yolk reserves under high variance versus constant incubation temperatures.

Our results highlight how the relative temperature sensitivities of development time and metabolic rate determine the cost of development, which in turn may predict the ability of egg-laying ectotherms to persist in variable environments. We show that DCT can provide a mechanistic framework for understanding the widespread, but often seemingly idiosyncratic, effects of fluctuating incubation temperatures on hatchling tissue and residual yolk mass.
Cancer cells co-evolve with retrotransposons to mitigate viral mimicry
Authors: Sun Siyu , Hong Jungeui , You Eunae , Tsanov Kaloyan M. , Chacon-Barahona Jonathan , Gioacchino Andrea Di , Hoyos David , Li Hao , Jiang Hua , Ly Han , Marhon Sajid , Murali Rajmohan , Chanda Pharto , Karacay Ali , Vabret Nicolas , De Carvalho Daniel D. , LaCava John , Lowe Scott W. , Ting David T. , Iacobuzio-Donahue Christine A. , Solovyov Alexander , Greenbaum Benjamin D.
Published: May 2023

Overexpression of repetitive elements is an emerging hallmark of human cancers1. Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system2–5. Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolvedShortInterspersedNuclearElements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by eitherLongInterspersedNuclearElements 1 (LINE1/L1) mobility or ADAR1 activity in aTP53mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated withTP53mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.
A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor
Authors: Raicu Ana-Maria , Suresh Megha , Arnosti David N.
Published: May 2023

The C-terminal Binding Protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C-terminus. In the mammalian system, CtBP proteins lacking the C-terminal Domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of ∼100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurring Drosophila isoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impactsin vivo. Interestingly, CtBP(S) was able to significantly repress transcription of theMpp6promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBP’s repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfectedMpp6reporter gene. The context-specific differences in activity of these two developmentally-regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.
Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide
Authors: Marquez Melissa D. , Greth Carina , Buzuk Anastasiya , Liu Yaxi , Blinn Catharina M. , Beller Simone , Leiskau Laura , Hushka Anthony , Wu Kassandra , Nur Kübra , Netz Daili J. , Perlstein Deborah L. , Pierik Antonio J.
Published: May 2023

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO-tripeptide present at the C-terminus of clients is necessary and sufficient for binding to the CTCin vitroand directing Fe-S cluster deliveryin vivo. Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications.

One-Sentence Summary

A C-terminal tripeptide guides eukaryotic iron-sulfur cluster insertion into cytosolic and nuclear proteins.
Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs
Authors: Garibyan Mher , Hoffman Tyler , Makaske Thijs , Do Stephanie , March Alexander R , Cho Nathan , Pedroncelli Nico , Lima Ricardo Espinosa , Soto Jennifer , Jackson Brooke , Khademhosseini Ali , Li Song , McCain Megan , Morsut Leonardo
Published: May 2023

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands.

We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.
Benzylisoquinoline alkaloid production in yeast via norlaudanosoline improves selectivity and yield
Authors: Narcross Lauren , Pyne Michael E. , Kevvai Kaspar , Siu Ka-Hei , Dueber John E. , Martin Vincent J. J.
Published: May 2023

The benzylisoquinoline alkaloid (BIA) family of tetrahydroisoquinolines (THIQs) comprises over 2,500 members, including the pharmaceuticals morphine, codeine, and papaverine as well as the antibiotics sanguinarine and chelerythrine used in animal husbandry. Agricultural cultivation can currently supply the demand for the BIAs that accumulate in plants, but broader access to the entire BIA family would open new avenues of research and commercialization. Microbial synthesis presents an attractive option due to cheap feedstock, genetic tractability, and ease of scale-up. Previously we reported titers of the key branch-point BIA (S)-reticuline of 4.6 g/L in yeast, which was achieved through leveraging the Ehrlich pathway 2-oxoacid decarboxylase Aro10 to generate the intermediate 4-hydroxyphenylacetaldehyde (4-HPAA). Here, we establish a superior route to (S)-reticuline by switching the pathway intermediate from 4-HPAA to 3,4-dihydroxyphenylacetaldehyde (3,4-dHPAA) using human monoamine oxidase A (MAO). The resulting (S)-norlaudanosoline route to (S)-reticuline synthesis is more selective, resolving prior issues with off-pathway THIQs synthesized due to concerted enzyme promiscuity. The new pathway is also more efficient, enabling titers of 4.8 g/L (S)-reticuline while improving yields over 40%, from 17 mg/g sucrose to 24 mg/g sucrose in fed-batch fermentations. Finally, we extendde novo(S)-reticuline synthesis to dihydrosanguinarine, achieving 635 mg/L dihydrosanguinarine and sanguinarine in fed-batch fermentation the highest reported titer of these BIAs by a factor of 40.

Highlights

Monoamine oxidase A (MAO) supports high-titer (S)-reticuline synthesis in yeast

MAO route to (S)-reticuline improves specificity compared to Aro10 route

This work represents a 40% increase in highest reported (S)-reticuline yield

653 mg/L (dihydro-) sanguinarine was produced by extending the pathway
Plasmid-free cheater cells commonly evolve during laboratory growth
Authors: Bedore Amber M. , Waters Christopher M.
Published: May 2023

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, secretion of beta-lactamase from resistant cells, and subsequent degradation of nearby penicillin and related antibiotics, allows neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss still occurred. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.

Importance

Plasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid containing cell is grown in the presence of antibiotic. Here we find that growth of plasmid-containing bacteria during laboratory conditions in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells for viability. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.
A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria
Authors: Gilbert Charlie , Brumwell Stephanie L. , Crits-Christoph Alexander , Kang Shinyoung Clair , Martin-Moldes Zaira , Alsharif Wajd , Esmurria Ariela , Nguyen Mary-Anne , Lee Henry H. , Ostrov Nili
Published: May 2023

Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such asEscherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs forHalomonas alkaliphila, Halomonas neptunia,andShewanella electrodiphila. Furthermore, we expand the list of functional ORIs forDuganella zoogloeoides,Pseudomonas alcaliphila,Shewanella oneidensisandShewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.
Tertiary structure of single-instant RNA molecule reveals folding landscape
Authors: Liu Jianfang , McRae Ewan K.S. , Zhang Meng , Geary Cody , Andersen Ebbe Sloth , Ren Gang
Published: May 2023

The folding of RNA and protein molecules during their synthesis is a crucial self-assembly process that nature employs to convert genetic information into the complex molecular machinery that supports life. Misfolding events are the cause of several diseases, and the folding pathway of central biomolecules, such as the ribosome, is strictly regulated by programmed maturation processes and folding chaperones. However, the dynamic folding processes are challenging to study because current structure determination methods heavily rely on averaging, and existing computational methods do not efficiently simulate non-equilibrium dynamics. Here we utilize individual-particle cryo-electron tomography (IPET) to investigate the folding landscape of a rationally designed RNA origami 6-helix bundle that undergoes slow maturation from a “young” to “mature” conformation. By optimizing the IPET imaging and electron dose conditions, we obtain 3D reconstructions of 120 individual particles at resolutions ranging from 23-35 Å, enabling us first-time to observe individual RNA helices and tertiary structures without averaging. Statistical analysis of 120 tertiary structures confirms the two main conformations and suggests a possible folding pathway driven by helix-helix compaction. Studies of the full conformational landscape reveal both trapped states, misfolded states, intermediate states, and fully compacted states. The study provides novel insight into RNA folding pathways and paves the way for future studies of the energy landscape of molecular machines and self-assembly processes.
Application of polystyrene sulfonate (PSS) for inhibiting toxicity of ALS/FTD-linked dipeptide repeats
Authors: Bratek-Skicki Anna , Ahmed Junaid , Jonckheere Karl , Peeters Eveline , Heeren Kara , Volkov Alex , Hendrix Jelle , Batys Piotr , Bosch Ludo Van Den , Tompa Peter
Published: May 2023

The expansion of GGGGCC (G4C2) repeats in the noncoding region of C9orf72 is the most common genetic cause of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat region is translated into five different dipeptide repeats (DPRs), of which the arginine-rich DPRs (R-DPRs) poly-GR (GRn) and poly-PR (PRn) are highly neurotoxic and are probably primarily responsible for the disease. Here, we characterized the protective effect against R-DPR toxicity of polystyrene sulfonate (PSS), an FDA-approved drug applied in hyperkalemia, in biochemical, cellular, and animal models of ALS/FTD. We found that PSS, in a length-dependent manner, interacts very tightly with R-DPRs, and releases their bound RNA in R-DPR - RNA mixtures. PSS significantly influences the liquid-liquid phase separation (LLPS) of R-DPRs elicited by RNA and reduces their ensuing cell toxicity in Neuro2a cells. PSS is cell penetrable, and it is also effective in countering the toxicity of R-DPRs in zebrafish embryos. Except for the longest (n = 340) variant, PSS is toxic neither to cells nor to mice upon intracerebroventricular injection up to 1 mM concentration. Our results suggest that its polymeric nature endows PSS with an advantageous effect in C9-ALS/FTD and offers a possible remedy against this debilitating neurodegenerative disease.