bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptomic changes predict metabolic alterations in LC3 associated phagocytosis in aged mice
Authors: Dhingra Anuradha , Tobias John W , Philp Nancy J , Boesze-Battaglia Kathleen
Published: March 2023

LC3b (Map1lc3b) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b, to pro-mote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis and neuroprotection. In a mouse model of retinal lipid steatosis - mice lacking LC3b (LC3b-/-), we observed increased lipid deposition, metabolic dysregulation and enhanced inflammation. Herein we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT andLC3b-/-mice revealed 1533 DEGs, with ~73% upregulated and 27% down-regulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.
Predicting metabolite response to dietary intervention using deep learning
Authors: Wang Tong , Holscher Hannah D. , Maslov Sergei , Hu Frank B. , Weiss Scott T. , Liu Yang-Yu
Published: March 2023

Due to highly personalized biological and lifestyle characteristics, different individuals may have different metabolite responses to specific foods and nutrients. In particular, the gut microbiota, a collection of trillions of microorganisms living in the gastrointestinal tract, is highly personalized and plays a key role in the metabolite responses to foods and nutrients. Accurately predicting metabolite responses to dietary interventions based on individuals' gut microbial compositions holds great promise for precision nutrition. Existing prediction methods are typically limited to traditional machine learning models. Deep learning methods dedicated to such tasks are still lacking. Here we develop a method McMLP (Metabolite response predictor using coupled Multilayer Perceptrons) to fill in this gap. We provide clear evidence that McMLP outperforms existing methods on both synthetic data generated by the microbial consumer-resource model and real data obtained from six dietary intervention studies. Furthermore, we perform sensitivity analysis of McMLP to infer the tripartite food-microbe-metabolite interactions, which are then validated using the ground-truth (or literature evidence) for synthetic (or real) data, respectively. The presented tool has the potential to inform the design of microbiota-based personalized dietary strategies to achieve precision nutrition.
Manuscript: Detecting differences in Size Spectra
Authors: Pomeranz Justin , Junker James R. , Gjoni Vojsava , Wesner Jeff S.
Published: March 2023

The distribution of body size in communities is remarkably consistent across habitats and taxa and can be represented by size spectra, which are described by a power law. The focus of size spectra analysis is to estimate the exponent (λ) of the power law.

Many methods have been proposed for estimatingλmost of which involve binning the data, summing abundance within bins, and then fitting a ordinary least squares (OLS) regression in log-log space. However, recent work has shown that binning procedures may return biased estimates of size spectra exponents compared to procedures that directly estimateλusing maximum likelihood estimation (MLE). Despite this variability in estimates, it is unclear if the relative change across environmental gradients is consistent across methodologies. Here, we used simulation to compare the ability of two binning methods (NAS, ELBn) and MLE to 1) recapture known values ofλ, and 2) recapture parameters in a linear regression measuring the change inλacross a hypothetical environmental gradient. We also compared the methods using two previously published body size datasets across a pollution gradient and a temperature gradient

Maximum likelihood methods always performed better than common binning methods, which demonstrated consistent bias depending on the simulated values ofλ. This bias carried over to the regressions, which were more accurate whenλwas estimated using MLE compared to the binning procedures. Additionally, the variance in estimates using MLE methods is markedly reduced when compared to binning methods.

The uncertainty and variation in estimates when using binning methods is often greater than or equal to the variation previously published in experimental and observational studies, bringing into question the effect size of previously published results. However, while the methods produced different slope estimates from previously published datasets, they were in qualitative agreement on the sign of those slopes (i.e., all negative or all positive). Our results provide further support for the direct estimation ofλusing MLE (or similar procedures) over the more common methods of binning.
Population-level annotation of lncRNA transcription in Arabidopsis reveals extensive variation associated with transposable element-like silencing
Authors: Kornienko Aleksandra E. , Nizhynska Viktoria , Morales Almudena Molla , Pisupati Rahul , Nordborg Magnus
Published: March 2023

Long non-coding RNAs (lncRNAs) are understudied and underannotated in plants. In mammals, lncRNA loci are nearly as ubiquitous as protein-coding genes, and their expression is highly variable between individuals of the same species. UsingArabidopsis thalianaas a model, we aimed to understand the true scope of lncRNA transcription across plants from different regions and study its natural variation. We used transcriptome deep sequencing datasets spanning hundreds of natural accessions and several developmental stages to create a population-wide annotation of lncRNAs, revealing thousands of previously unannotated lncRNA loci. While lncRNA transcription is ubiquitous in the genome, most loci appear to be actively silenced and their expression is extremely variable between natural accessions. This high expression variability is largely caused by the high variability of repressive chromatin levels at lncRNA loci. High variability was particularly common for intergenic lncRNAs (lincRNAs), where pieces of transposable elements (TEs) present in 50% of these lincRNA loci are associated with increased silencing and variation, and such lncRNAs tend to be targeted by the TE silencing machinery. We create a population-wide lncRNA annotation inA. thalianaand improve our understanding of plant lncRNA genome biology, raising fundamental questions about what causes transcription and silencing across the genome.

One-sentence summary

lncRNA loci are plentiful in theA. thalianagenome, but their expression is extremely variable and largely repressed, with TE pieces enriched in intergenic lncRNAs aiding variability and silencing.
Transposon-encoded nucleases use guide RNAs to selfishly bias their inheritance
Authors: Meers Chance , Le Hoang , Pesari Sanjana R. , Hoffmann Florian T. , Walker Matt W.G. , Gezelle Jeanine , Sternberg Samuel H.
Published: March 2023

Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605elements undergo ‘peel-and-paste’ transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements fromGeobacillus stearothermophilusthat encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.
Distinct value computations support rapid sequential decisions
Authors: Mah Andrew , Schiereck Shannon S. , Bossio Veronica , Constantinople Christine M.
Published: March 2023

The value of the environment determines animals’ motivational states and sets expectations for error-based learning1–3. How are values computed? Rein-forcement learning systems can store or “cache” values of states or actions that are learned from experience, or they can compute values using a model of the environment to simulate possible futures3. These value computations have distinct trade-offs, and a central question is how neural systems decide which computations to use or whether/how to combine them4–8. Here we show that rats use distinct value computations for sequential decisions within single tri-als. We used high-throughput training to collect statistically powerful datasets from 291 rats performing a temporal wagering task with hidden reward states. Rats adjusted how quickly they initiated trials and how long they waited for re-wards across states, balancing effort and time costs against expected rewards. Statistical modeling revealed that animals computed the value of the environ-ment differently when initiating trials versus when deciding how long to wait for rewards, even though these decisions were only seconds apart. Moreover, value estimates interacted via a dynamic learning rate. Our results reveal how distinct value computations interact on rapid timescales, and demonstrate the power of using high-throughput training to understand rich, cognitive behav-iors.
Damage-induced basal epithelial cell migration modulates the spatial organization of redox signaling and sensory neuron regeneration
Authors: Fister Alexandra M. , Horn Adam , Lasarev Michael , Huttenlocher Anna
Published: March 2023

Epithelial damage leads to early reactive oxygen species (ROS) signaling, which regulates sensory neuron regeneration and tissue repair. How the initial type of tissue injury influences early damage signaling and regenerative growth of sensory axons remains unclear. Previously we reported that thermal injury triggers distinct early tissue responses in larval zebrafish. Here, we found that thermal but not mechanical injury impairs sensory axon regeneration and function. Real-time imaging revealed an immediate tissue response to thermal injury characterized by the rapid Arp2/3-dependent migration of keratinocytes, which was associated with tissue-scale ROS production and sustained sensory axon damage. Isotonic treatment was sufficient to limit keratinocyte movement, spatially restrict ROS production and rescue sensory neuron function. These results suggest that early keratinocyte dynamics regulate the spatial and temporal pattern of long-term signaling in the wound microenvironment during tissue repair.
N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters
Authors: Bhanja Anshuman , Seeley-Fallen Margaret K. , Lazzaro Michelle , Upadhyaya Arpita , Song Wenxia
Published: March 2023

Antigen-induced B-cell receptor (BCR) signaling is critical for initiating and regulating B-cell activation. The actin cytoskeleton plays essential roles in BCR signaling. Upon encountering cell-surface antigens, actin-driven B-cell spreading amplifies signaling, while B-cell contraction following spreading leads to signal attenuation. However, the mechanism by which actin dynamics switch BCR signaling from amplification to attenuation is unknown. Here, we show that Arp2/3-mediated branched actin polymerization is required for B-cell contraction. Contracting B-cells generate centripetally moving actin foci from lamellipodial F-actin networks in the B-cell plasma membrane region contacting antigen-presenting surfaces. Actin polymerization driven by N-WASP, but not WASP, initiates these actin foci and facilitates non-muscle myosin II recruitment to the contact zone, creating actomyosin ring-like structures. Furthermore, B-cell contraction increases BCR molecular density in individual clusters, leading to decreased BCR phosphorylation. Increased BCR molecular density reduced levels of the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. These results suggest that N-WASP-activated Arp2/3, coordinating with myosin, generates centripetally moving foci and contractile actomyosin ring-like structures from lamellipodial networks, enabling contraction. B-cell contraction attenuates BCR signaling by pushing out both stimulatory kinases and inhibitory phosphatases from BCR clusters, providing novel insights into actin-facilitated signal attenuation.
Comparative analysis between RNA-seq and single-molecule RNA FISH indicates that the pyrimidine nucleobase idoxuridine (IdU) globally amplifies transcriptional noise
Authors: Calia Giuliana Pia , Chen Xinyue , Zuckerman Binyamin , Weinberger Leor
Published: March 2023

Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability, but the physiological roles of noise have remained difficult to determine in the absence of generalized noise-modulation approaches. Previous single-cell RNA sequencing (scRNA-seq) suggested that the pyrimidine-base analog (5′-iodo-2′ deoxyuridine, IdU) could generally amplify noise without substantially altering mean expression levels but scRNA-seq technical drawbacks potentially obscured the penetrance of IdU-induced transcriptional noise amplification. Here we quantify global-vs.-partial penetrance of IdU induced noise amplification by assessing scRNAseq data using numerous normalization algorithms and directly quantifying noise using single-molecule RNA FISH (smFISH) for a panel of genes from across the transcriptome. Alternate scRNA-seq analyses indicate IdU-induced noise amplification for ~90% of genes, and smFISH data verified noise amplification for ~90% of tested genes. Collectively, this analysis indicates which scRNA-seq algorithms are appropriate for quantifying noise and argues that IdU is a globally penetrant noise enhancer molecule that could enable investigations of the physiological impacts of transcriptional noise.
Hippocampal sharp wave-ripple dynamics in NREM sleep encode motivation for anticipated physical activity
Authors: Buhler Chelsea M. , Basso Julia C. , English Daniel Fine
Published: March 2023

Physical activity is an integral part of every mammal’s daily life, and as a driver of Darwinian fitness, required coordinated evolution of the body and brain. The decision to engage in physical activity is driven either by survival needs or by motivation for the rewarding qualities of physical activity itself. Rodents exhibit innate and learned motivation for voluntary wheel running, and over time run longer and farther, reflecting increased incentive salience and motivation for this consummatory behavior. Dynamic coordination of neural and somatic physiology are necessary to ensure the ability to perform behaviors that are motivationally variable. Hippocampal sharp wave-ripples (SWRs) have evolved both cognitive and metabolic functions, which in modern mammals may facilitate body-brain coordination. To determine if SWRs encode aspects of exercise motivation we monitored hippocampal CA1 SWRs and running behaviors in adult mice, while manipulating the incentive salience of the running experience. During non-REM (NREM) sleep, the duration of SWRs before (but not after) running positively correlated with future running duration, and larger pyramidal cell assemblies were activated in longer SWRs, suggesting that the CA1 network encodes exercise motivation at the level of neuronal spiking dynamics. Inter-Ripple-intervals (IRI) before but not after running were negatively correlated with running duration, reflecting more SWR bursting, which increases with learning. In contrast, SWR rates before and after running were positively correlated with running duration, potentially reflecting a tuning of metabolic demand for that day’s anticipated and actual energy expenditure rather than motivation. These results suggest a novel role for CA1 in exercise behaviors and specifically that cell assembly activity during SWRs encodes motivation for anticipated physical activity.

SIGNIFICANCE STATEMENT

Darwinian fitness is increased by body-brain coordination through internally generated motivation, though neural substrates are poorly understood. Specific hippocampal rhythms (i.e., CA1 SWRs), which have a well-established role in reward learning, action planning and memory consolidation, have also been shown to modulate systemic [glucose]. Using a mouse model of voluntary physical activity that requires body-brain coordination, we monitored SWR dynamics when animals were highly motivated and anticipated rewarding exercise (i.e., when body-brain coordination is of heightened importance). We found that during non-REM sleep before exercise, SWR dynamics (which reflect cognitive and metabolic functions) were correlated with future time spent exercising. This suggests that SWRs support cognitive and metabolic facets that motivate behavior by coordinating the body and brain.
Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues
Authors: Park Kieop , Jayadev Ranjay , Payne Sara G. , Kenny-Ganzert Isabel W. , Chi Qiuyi , Costa Daniel S. , Ramos-Lewis William , Thendral Siddharthan Balachandar , Sherwood David R.
Published: March 2023

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using theC. elegansutse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor 2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.
Growth-dependent gene expression variation influences the strength of codon usage biases
Authors: Johnson Mackenzie M. , Hockenberry Adam J. , McGuffie Matthew J. , Vieira Luiz Carlos , Wilke Claus O.
Published: March 2023

The most highly expressed genes in microbial genomes tend to use a limited set of synonymous codons, often referred to as “preferred codons.” The existence of preferred codons is commonly attributed to selection pressures on various aspects of protein translation including accuracy and/or speed. However, gene expression is condition-dependent and even within single-celled organisms transcript and protein abundances can vary depending on a variety of environmental and other factors. Here, we show that growth rate-dependent expression variation is an important constraint that significantly influences the evolution of gene sequences. Using large-scale transcriptomic and proteomic data sets inEscherichia coliandSaccharomyces cerevisiae, we confirm that codon usage biases are strongly associated with gene expression but highlight that this relationship is most pronounced when gene expression measurements are taken during rapid growth conditions. Specifically, genes whose relative expressionincreasesduring periods of rapid growth have stronger codon usage biases than comparably expressed genes whose expressiondecreasesduring rapid growth conditions. These findings highlight that gene expression measured in any particular condition tells only part of the story regarding the forces shaping the evolution of microbial gene sequences. More generally, our results imply that microbial physiology during rapid growth is critical for explaining long-term translational constraints.
Dopamine receptor D2 confers colonization resistance via gut microbial metabolites
Authors: Scott Samantha A. , Fu Jingjing , Chang Pamela V.
Published: March 2023

Abstract/Summary Paragraph:

The gut microbiome plays major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1–3, including enterohemorrhagicEscherichia coli(EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea, and acute renal failure (hemolytic uremic syndrome)4,5. Although gut microbes can provide colonization resistance by outcompeting some pathogens or modulating host defense provided by the gut barrier and intestinal immune cells, this phenomenon remains poorly understood. Emerging evidence suggests that small-molecule metabolites produced by the gut microbiota may mediate this process6. Here, we show that tryptophan (Trp)-derived metabolites produced by the gut bacteria protect the host againstCitrobacter rodentium, a murine AE pathogen widely used as a model for EHEC infection7,8, by activation of the host neurotransmitter dopamine receptor D2 (DRD2) within the intestinal epithelium. We further find that these Trp metabolites act through DRD2 to decrease expression of a host actin regulatory protein involved inC. rodentiumand EHEC attachment to the gut epithelium via formation of actin pedestals. Previously identified mechanisms of colonization resistance either directly affect the pathogen by competitive exclusion or indirectly by modulation of host defense mechanisms9,10, so our results delineate a noncanonical colonization resistance pathway against AE pathogens featuring an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization within the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches for improving gut health and treating gastrointestinal infections, which afflict millions globally.
Gut microbial community in proboscis monkeys (Nasalis larvatus): implications for effects of geographical and social factors
Authors: Jose Lilian , Lee Wanyi , Hanya Goro , Tuuga Augustine , Goossens Benoit , Tangah Joseph , Matsuda Ikki , Kumar Vijay Subbiah
Published: March 2023

Recent technological advances have enabled comprehensive analyses of the previously uncharacterized microbial community in the gastrointestinal tracts of numerous animal species; however, the gut microbiota of several species, such as the endangered proboscis monkey (Nasalis larvatus) examined in this study, remains poorly understood. Our study sought to establish the first comprehensive data on the gut microbiota of free-ranging foregut-fermenting proboscis monkeys and to determine how their microbiota are affected locally by environmental factor, i.e. geographical distance, and social factor, i.e. number of adult females within harem groups and number of adults and subadults within non-harem groups, in a riparian forest in Sabah, Malaysian Borneo. Using 16S rRNA gene sequencing of 264 faecal samples collected from free-ranging proboscis monkeys, we demonstrated the trend that their microbial community composition is not particularly distinctive compared to other foregut and hindgut fermenting primates. The microbial alpha diversity was higher in larger groups and individuals inhabiting diverse vegetation (i.e. presumed to have a diverse diet). For microbial beta diversity, some measures were significant, showing higher values with larger geographical distances between samples. These results suggest that social factors such as increased interindividual interactions, which can occur with larger groups, as well as physical distances between individuals or differences in dietary patterns, may affect the gut microbial communities.
Gestational exposure to unmethylated CpG oligonucleotides dysregulates placental molecular clock network and fetoplacental growth dynamics, and disrupts maternal blood pressure circadian rhythms in rats
Authors: Bradshaw Jessica L. , Cushen Spencer C. , Ricci Contessa A. , Tucker Selina M. , Gardner Jennifer J. , Little Joel T. , Osikoya Oluwatobiloba , Goulopoulou Styliani
Published: March 2023

Bacterial infections and impaired mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. Here, we tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and the placental molecular clock machinery, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the 3rdtrimester (gestational day, GD, 14, 16, 18) and euthanized on GD20 (near term) or with a single dose of CpG ODN and euthanized 4 hours after treatment on GD14. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. Ap-value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost (p≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN (p< 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 (p≥ 0.05). CpG ODN increased placental expression ofPer2andPer3andTnfα(p≤ 0.05) and affected fetoplacental growth dynamics, such as reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared to controls. In conclusion, gestational exposure to unmethylated CpG DNA dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.
A laser capture microdissection transcriptome ofM. truncatularoots responding to rhizobia reveals spatiotemporal tissue expression patterns of genes involved in nodule signaling and organogenesis
Authors: Schnabel Elise , Thomas Jacklyn , El-Hawaz Rabia , Gao Yueyao , Poehlman William , Chavan Suchitra , Pasha Asher , Esteban Eddi , Provart Nicholas , Feltus F. Alex , Frugoli Julia
Published: March 2023

We report a public resource for examining the spatiotemporal RNA expression of 54,893M. truncatulagenes during the first 72 hours of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of over 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response data set generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of ACC synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division.
Cas12a/Cpf1 knock-in mice enable efficient multiplexed immune cell engineering
Authors: Dong Matthew B. , Tang Kaiyuan , Zhou Xiaoyu , Shen Johanna , Chen Krista , Kim Hyunu R. , Zhou Jerry , Cao Hanbing , Vandenbulcke Erica , Zhang Yueqi , Chow Ryan D. , Du Andrew , Suzuki Kazushi , Fang Shao-Yu , Majety Medha , Dai Xiaoyun , Chen Sidi
Published: March 2023

Cas9 transgenic animals have drastically accelerated the discovery of novel immune modulators. But due to its inability to process its own CRISPR RNAs (crRNAs), simultaneous multiplexed gene perturbations using Cas9 remains limited, especially by pseudoviral vectors. Cas12a/Cpf1, however, can process concatenated crRNA arrays for this purpose. Here, we created conditional and constitutive LbCas12a knock-in transgenic mice. With these mice, we demonstrated efficient multiplexed gene editing and surface protein knockdown within individual primary immune cells. We showed genome editing across multiple types of primary immune cells including CD4 and CD8 T cells, B cells, and bone-marrow derived dendritic cells. These transgenic animals, along with the accompanying viral vectors, together provide a versatile toolkit for a broad range ofex vivoandin vivogene editing applications, including fundamental immunological discovery and immune gene engineering.
Transgenerational virulence: Maternal pathogen exposure reduces offspring fitness
Authors: McIntirea Kristina M. , Dziuba Marcin K. , Haywood Elizabeth B. , Robertson Miles L. , Vaandrager Megan , Baird Emma , Corcoran Fiona , Nelson Taleah , Cortez Michael H. , Duffy Meghan A.
Published: March 2023

There is intense interest in understanding pathogen virulence – that is, the degree of damage caused to a host by a pathogen. However, despite abundant evidence that effects of stressors can carry across generations, studies of pathogen virulence have focused almost exclusively on a single generation. Here, we describe transgenerational virulence, where parental exposure to a pathogen reduces offspring fitness.Daphniahost exposure to a microsporidian pathogen significantly reduced lifespan and fecundity of offspring. The trans-generational virulent effects of maternal exposure to the microsporidian pathogen matched or even exceeded the within generation effects of a fungal pathogen that is considered highly virulent. Moreover, they did not result simply from immune activation in the host, as the transgenerational effect was seen for infections by the microsporidian but not for infec-tions by two other pathogens. The transgenerational effects of the microsporidian carried to the granddaughters, with grandmaternal exposure influencing neonate size. A parame-terized mathematical model translated these impacts to the population level, finding that transgenerational virulence often reduces host density, infected host density, and infection prevalence, which could have implications for host conservation and spillover risk. Given widespread prior evidence for transgenerational effects of predators, resources, and other factors, we propose that transgenerational virulence may be common.
Single-shot 3D photoacoustic tomography using a single-element detector for ultrafast imaging of hemodynamics
Authors: Zhang Yide , Hu Peng , Li Lei , Cao Rui , Khadria Anjul , Maslov Konstantin , Tong Xin , Zeng Yushun , Jiang Laiming , Zhou Qifa , Wang Lihong V.
Published: March 2023

Imaging hemodynamics is crucial for the diagnosis, treatment, and prevention of vascular diseases. However, current imaging techniques are limited due to the use of ionizing radiation or contrast agents, short penetration depth, or complex and expensive data acquisition systems. Photoacoustic tomography shows promise as a solution to these issues. However, existing photoacoustic tomography methods collect signals either sequentially or through numerous detector elements, leading to either low imaging speed or high system complexity and cost. To address these issues, here we introduce a method to capture a 3D photoacoustic image of vasculature using a single laser pulse and a single-element detector that functions as 6,400 virtual ones. Our method enables ultrafast volumetric imaging of hemodynamics in the human body at up to 1 kHz and requires only a single calibration for different objects and for long-term operations. We demonstrate 3D imaging of hemodynamics at depth in humans and small animals, capturing the variability in blood flow speeds. This concept can inspire other imaging technologies and find applications such as home-care monitoring, biometrics, point-of-care testing, and wearable monitoring.
Human-exoskeleton interaction force estimation in Indego exoskeleton
Authors: Shushtari Mohammad , Arami Arash
Published: March 2023

Accurate interaction force estimation can play an important role in optimization human-robot interaction in exoskeleton. In this work, we propose a novel approach for system identification of exoskeleton dynamics in presence of interaction forces as a whole multi-body system regardless of gait phase or any assumption on human-exoskeleton interaction. We hanged the exoskeleton through a linear spring and excited the exoskeleton joints with chirp commands while measuring the exoskeleton-environment interaction force. Several structures of neural networks have been trained to model the exoskeleton passive dynamics and estimate the interaction force. Our testing results indicated that a deep neural network with 250 neurons and 10 time delays can obtain sufficiently accurate estimation of the interaction force, resulting in 1.23 of RMSE on Z-normalized applied torques and 0.89 of adjustedR2.
Immunogenetic metabolomics revealed key enzymes that modulate CAR-T metabolism and function
Authors: Renauer Paul , Park Jonathan J. , Bai Meizhu , Acosta Arianny , Lee Won-Ho , Lin Guang Han , Zhang Yueqi , Dai Xiaoyun , Wang Guangchuan , Errami Youssef , Wu Terence , Clark Paul , Ye Lupeng , Yang Quanjun , Chen Sidi
Published: March 2023

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we seek to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism, whereby cancer cells suppress T cell function by generating a metabolically unfavorable tumor microenvironment (TME). Specifically, we use anin silicoscreen to identifyADAandPDK1as metabolic regulators, in which gene overexpression (OE) enhances the cytolysis of CD19-specific CD8 CAR-T cells against cognate leukemia cells, and conversely,ADAorPDK1deficiency dampens such effect.ADA-OE in CAR-T cells improves cancer cytolysis under high concentrations of adenosine, the ADA substrate and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics in these CAR-Ts reveal alterations of global gene expression and metabolic signatures in bothADA-andPDK1-engineered CAR-T cells. Functional and immunological analyses demonstrate thatADA-OE increases proliferation and decreases exhaustion in α-CD19 and α-HER2 CAR-T cells. ADA-OE improves tumor infiltration and clearance by α-HER2 CAR-T cells in anin vivocolorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR-T cells, and reveal potential targets for improving CAR-T based cell therapy.

Synopsis

The authors identify the adenosine deaminase gene (ADA) as a regulatory gene that reprograms T cell metabolism. ADA-overexpression (OE) in α-CD19 and α-HER2 CAR-T cells increases proliferation, cytotoxicity, memory, and decreases exhaustion, and ADA-OE α-HER2 CAR-T cells have enhanced clearance of HT29 human colorectal cancer tumorsin vivo.
Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies
Authors: Edman Natasha I , Redler Rachel L , Phal Ashish , Schlichthaerle Thomas , Srivatsan Sanjay R , Etemadi Ali , An Seong , Favor Andrew , Ehnes Devon , Li Zhe , Praetorius Florian , Gordon Max , Yang Wei , Coventry Brian , Hicks Derrick R , Cao Longxing , Bethel Neville , Heine Piper , Murray Analisa N , Gerben Stacey , Carter Lauren , Miranda Marcos , Negahdari Babak , Lee Sangwon , Trapnell Cole , Stewart Lance , Ekiert Damian C , Schlessinger Joseph , Shendure Jay , Bhabha Gira , Ruohola-Baker Hannele , Baker David
Published: March 2023

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways.
NEURD offers automated proofreading and feature extraction for connectomics
Authors: Celii Brendan , Papadopoulos Stelios , Ding Zhuokun , Fahey Paul G. , Wang Eric , Papadopoulos Christos , Kunin Alexander B. , Patel Saumil , Bae J. Alexander , Bodor Agnes L. , Brittain Derrick , Buchanan JoAnn , Bumbarger Daniel J. , Castro Manuel A. , Cobos Erick , Dorkenwald Sven , Elabbady Leila , Halageri Akhilesh , Jia Zhen , Jordan Chris , Kapner Dan , Kemnitz Nico , Kinn Sam , Lee Kisuk , Li Kai , Lu Ran , Macrina Thomas , Mahalingam Gayathri , Mitchell Eric , Mondal Shanka Subhra , Mu Shang , Nehoran Barak , Popovych Sergiy , Schneider-Mizell Casey M. , Silversmith William , Takeno Marc , Torres Russel , Turner Nicholas L. , Wong William , Wu Jingpeng , Yu Szi-chieh , Yin Wenjing , Xenes Daniel , Kitchell Lindsey M. , Rivlin Patricia K. , Rose Victoria A. , Bishop Caitlyn A. , Wester Brock , Froudarakis Emmanouil , Walker Edgar Y. , Sinz Fabian , Seung H. Sebastian , Collman Forrest , da Costa Nuno Maçarico , Reid R. Clay , Pitkow Xaq , Tolias Andreas S. , Reimer Jacob
Published: March 2023

We are now in the era of millimeter-scale electron microscopy (EM) volumes collected at nanometer resolution (Shapson-Coe et al., 2021; Consortium et al., 2021). Dense reconstruction of cellular compartments in these EM volumes has been enabled by recent advances in Machine Learning (ML) (Lee et al., 2017; Wu et al., 2021; Lu et al., 2021; Macrina et al., 2021). Automated segmentation methods produce exceptionally accurate reconstructions of cells, but post-hoc proofreading is still required to generate large connectomes free of merge and split errors. The elaborate 3-D meshes of neurons in these volumes contain detailed morphological information at multiple scales, from the diameter, shape, and branching patterns of axons and dendrites, down to the fine-scale structure of dendritic spines. However, extracting these features can require substantial effort to piece together existing tools into custom workflows. Building on existing open-source software for mesh manipulation, here we present “NEURD”, a software package that decomposes meshed neurons into compact and extensively-annotated graph representations. With these feature-rich graphs, we automate a variety of tasks such as state of the art automated proofreading of merge errors, cell classification, spine detection, axon-dendritic proximities, and other annotations. These features enable many downstream analyses of neural morphology and connectivity, making these massive and complex datasets more accessible to neuroscience researchers focused on a variety of scientific questions.
SMAD4: A Critical Regulator of Cardiac Neural Crest Cell Fate and Vascular Smooth Muscle Differentiation
Authors: Alexander Brianna E. , Zhao Huaning , Astrof Sophie
Published: March 2023

Background

The pharyngeal arch arteries (PAAs) are precursor vessels which remodel into the aortic arch arteries (AAAs) during embryonic cardiovascular development. Cardiac neural crest cells (NCs) populate the PAAs and differentiate into vascular smooth muscle cells (vSMCs), which is critical for successful PAA-to-AAA remodeling. SMAD4, the central mediator of canonical TGFβ signaling, has been implicated in NC-to-vSMC differentiation; however, its distinct roles in vSMC differentiation and NC survival are unclear.

Results

Here, we investigated the role of SMAD4 in cardiac NC differentiation to vSMCs using lineage-specific inducible mouse strains in an attempt to avoid early embryonic lethality and NC cell death. We found that with global SMAD4 loss, its role in smooth muscle differentiation could be uncoupled from its role in the survival of the cardiac NCin vivo. Moreover, we found that SMAD4 may regulate the induction of fibronectin, a known mediator of NC-to-vSMC differentiation. Finally, we found that SMAD4 is required in NCs cell-autonomously for NC-to-vSMC differentiation and for NC contribution to and persistence in the pharyngeal arch mesenchyme.

Conclusions

Overall, this study demonstrates the critical role of SMAD4 in the survival of cardiac NCs, their differentiation to vSMCs, and their contribution to the developing pharyngeal arches.
Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using αvβ3-targeted iron oxide microparticles
Authors: Buck Jessica , Perez-Balderas Francisco , Zarghami Niloufar , Johanssen Vanessa , Khrapitchev Alexandre A , Larkin James R , Sibson Nicola R
Published: March 2023

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly anti-angiogenic therapeutics, is difficult to ascertain using current magnetic resonance imaging (MRI) methods. Imaging of the angiogenic vasculature has been successfully carried out in solid tumours using microparticles of iron oxide (MPIO) conjugated to a peptide (RGD) targeting the integrin αvβ3. The aim of this study was to determine whether RGD-MPIO could be used to identify angiogenic blood vessels in brain metastasesin vivo.A mouse model of intracerebrally implanted brain macrometastasis was established through intracerebral injection of 4T1-GFP cells. T2* weighted imaging was used to visualize MPIO induced hypointense voxelsin vivo,and Prussian blue staining was used to visualize MPIO and endogenous iron histologicallyex vivo.The RGD-MPIO showed target-specific bindingin vivo,but the sensitivity of the agent for visualizing angiogenic vesselsper sewas reduced by the presence of endogenous iron-laden macrophages in larger metastases, resulting in pre-existing hypointense areas within the tumour. Further, our data suggest that peptide-targeted MPIO, but not antibody-targeted MPIO, are taken up by perivascular macrophages within the macrometastatic microenvironment, resulting in additional nonspecific contrast. Whilst pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our findings suggest that the use of antibodies rather than peptides as the targeting ligand may represent a preferable route forward for new angiogenesis-targeted molecular MRI agents.
Identification of a Micropeptide Linked to Cancer Stem Cell Regulation and Chemoresistance
Authors: Fernando Achala , Liyanage Chamikara , Srinivasan Srilakshmi , Janaththani Panchadsaram , Batra Jyotsna
Published: March 2023

Short open reading frames encoding micropeptides (miPEPs) less than 100 amino acids in length have recently emerged as important regulators of diverse biological functions. However, the functional role of cancer-specific miPEPs in cancer progression and therapeutic response remains largely unexplored. Genome-wide association studies have identified an association of Iroquois (IRX) clusters with multiple cancer risk. In this study, we identified 17 miPEPs generated from IRX clusters in prostate, breast, endometrial, and ovarian cancers using SWATH-MS/MS-based proteomic analysis. We found thatIRX4-derived miPEP, IRX4_PEP1, promotes prostate cancer (PCa) cell proliferation, migration, and invasion by interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK). Overexpression of IRX4_PEP1 leads to dysregulation of stem cell pathways by co-interaction with Catenin beta-1 (CTNB1) and upregulation of prominent PCa stem markers, resulting in docetaxel resistance in PCa. IRX4_PEP1 expression is significantly upregulated in prostate tumour tissues compared to normal and is positively correlated with disease aggressiveness.

SIGNIFICANCE

Our findings highlight the critical role of IRX4_PEP1 in regulating PCa stem cells and chemotherapy resistance and suggest it as a novel therapeutic target for the treatment of PCa. Moreover, IRX4_PEP1 expression can serve as a novel potential diagnostic and prognostic biomarker for PCa.