bluuebunny/crossref_metadata_2025 huggingface dataset:
Opsin gene expression plasticity and spectral sensitivity in male damselflies could mediate female colour morph detection
Authors: Roberts Natalie S. , Svensson Erik I. , Liénard Marjorie A.
Published: August 2023

The visual systems of Odonata are characterized by many opsin genes, which form the primary light-sensitive photopigments of the eye. Female-limited colour polymorphisms are also common in Odonata, with one morph typically exhibiting male-like (androchrome) colouration and one or two morphs exhibiting female-specific colouration (gynochromes). These colour polymorphisms are thought to be maintained by frequency-dependent sexual conflict, in which males form search images for certain morphs, causing disproportionate mating harassment. Here, we investigate opsin sensitivity and gene expression plasticity in mate-searching males of the damselflyIschnura elegansduring adult maturation and across populations with different female morph frequencies. We find evidence for opsin-specific plasticity in relative and proportional opsin mRNA expression, suggesting changes in opsin regulation and visual sensitivity during sexual maturation. In particular, expression of the long-wavelength sensitive opsin LWF2 changed over development and varied between populations with different female morph frequencies. UV-Vis analyses indicate that short- and long-wavelength opsins absorb wavelength of light between 350 and 650 nm. Assuming opponency between photoreceptors with distinct short- and long-wavelength sensitivities, these sensitivities suggest male spectral visual discrimination ability of andromorph and gynomorph females. Overall, our results suggest that opsin sensitivity and expression changes contribute to visual tuning that could impact conspecific discrimination.
A robust method for measuring aminoacylation through tRNA-Seq
Authors: Davidsen Kristian , Sullivan Lucas B
Published: August 2023

Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge tRNA-Seq method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.
Memory Retrieval Effects as a Function of Differences in Phenomenal Experience
Authors: Schmidt Austin H. , Kirwan C. Brock
Published: August 2023

Conscious experience and perception are restricted to a single perspective. There is evidence to suggest differences in phenomenal experience can produce observable differences in behavior, however it is not well understood how these differences might influence memory. We used fMRI to scan n=49 participants while they encoded and performed a recognition memory test for faces and words. We calculated a cognitive bias score reflecting individual participants’ propensity toward either Visual Imagery or Internal Verbalization based on their responses to the Internal Representations Questionnaire (IRQ). We found weak positive correlations between memory performance for faces and a bias toward visual imagery and between memory performance for words and bias toward internal verbalization. There were typical patterns of activation differences between words and faces during both encoding and retrieval. There was no effect of internal representation bias on fMRI activation during encoding. At retrieval, however, a bias toward visualization was positively correlated with memory-related activation for both words and faces in inferior occipital gyri. Further, there was a crossover interaction in a network of brain regions such that visualization bias was associated with greater activation for words and verbalization bias was associated with greater activation for faces, consistent with increased effort for non-preferred stimulus retrieval. These findings suggest that individual differences in cognitive representations affect neural activation across different types of stimuli, potentially affecting memory retrieval performance.
Where top-down meets bottom-up: Cell-type specific connectivity map of the whisker system
Authors: Rault N. , Bergmans T. , Delfstra N. , Kleijnen BJ , Zeldenrust F. , Celikel T.
Published: August 2023

Sensorimotor computation integrates bottom-up world state information with top-down knowledge and task goals to form action plans. In the rodent whisker system, a prime model of active sensing, evidence shows neuromodulatory neurotransmitters shape whisker control, affecting whisking frequency and amplitude. Since neuromodulatory neurotransmitters are mostly released from subcortical nuclei and have long-range projections that reach the rest of the central nervous system, mapping the circuits of top-down neuromodulatory control of sensorimotor nuclei will help to systematically address the mechanisms of active sensing. Therefore, we developed a neuroinformatic target discovery pipeline to mine the Allen Institute’s Mouse Brain Connectivity Atlas. Using network connectivity analysis, we identified new putative connections along the whisker system and anatomically confirmed the existence of 42 previously unknown monosynaptic connections. Using this data, we updated the sensorimotor connectivity map of the mouse whisker system and developed the first cell-type-specific map of the network. The map includes 157 projections across 18 principal nuclei of the whisker system and neuro-modulatory neurotransmitter-releasing. Performing a graph network analysis of this connectome, we identified cell-type specific hubs, sources, and sinks, provided anatomical evidence for monosynaptic inhibitory projections into all stages of the ascending pathway, and showed that neuromodulatory projections improve network-wide connectivity. These results argue that beyond the modulatory chemical contributions to information processing and transfer in the whisker system, the circuit connectivity features of the neuromodulatory networks position them as nodes of sensory and motor integration.
Octopus arm search strategies over complex surfaces
Authors: Sivitilli Dominic M. , Zulch Ariella , Gire David H
Published: August 2023

Despite the extreme flexibility of the octopus’s arms and their resulting near infinite possible configurations, the octopus effectively controls its arms during a wide variety of behaviors, including locomotion, foraging, excavation, exploration, and manipulation. If appropriately characterized, the octopus’s biomechanical properties and control strategies could be implemented in the development of a soft robotic limb with the same range of capabilities. When operating without visual feedback, the octopus must rely on the complex chemotactile sensory system within its suckers, and in these conditions sucker recruitment plays a prominent role in search behavior, causing the arm to conform to surface features in the environment. However, how this mechanism is used to search over the complex and convoluted surfaces in the octopus’s natural habitat is unknown. Here, we investigate the strategies the octopus uses to search for a reward hidden among a row of multiple small openings of a task space, and how it uses multiple arms to search three parallel versions of this task space. We found that when the arm encounters multiple openings in a surface, it performs a distal to proximal search pattern, starting with the farthest openings within reach then working its way proximally with a preference for searching each opening in succession. This strategy would allow the octopus to use its highly flexible limbs to perform an exhaustive search pattern over complex surfaces.
Epigenetic regulation of Leukocyte associated immunoglobulin-like receptors 1 and 2 by interferon signaling in macrophages and T cells
Authors: Dorando Hannah K. , Mutic Evan C. , Li Joanna Y. , Perrin Ezri P. , Wurtz Mellisa , Quinn Chaz C. , Payton Jacqueline E.
Published: August 2023

Background

Inhibitory immune receptors are important for maintaining immune homeostasis. We recently identified epigenetic alterations in two members of this group, LAIR1 and LAIR2, in patients with inflammatory tissue damage and recurrent skin and soft tissue infections. We therefore hypothesized that the expression of LAIR1 and LAIR2 may be controlled by immune stimuli acting on discrete transcriptional regulatory elements.

Methods

We used flow cytometry, qRT-PCR, and RNAseq to assay LAIR1 and LAIR2 expression in human and murine immune cell subsets at baseline and post-treatment with immune mediators, including type I and II interferons, tumor necrosis factor-alpha (TNF-ɑ), and lipopolysaccharide (LPS). Using chromatin immunoprecipitation sequencing (ChIP-seq), we identified candidate transcriptional regulatory elements of LAIR genes and evaluated their regulatory activity using luciferase reporters.

Results

Both human and murine macrophages significantly upregulate LAIR1 expression as they differentiate from monocytes to macrophages. In response to interferons, LAIR1 protein levels increase, while LPS causes a relative reduction. Regulatory elements flanking LAIR genes exhibit distinct patterns of enhancer activity with variable responses to immune stimuli. These responses are related to discrete sets of transcription factors in inflammatory pathways that correlate with cell-specific LAIR expression patterns. In addition, we identifiedLAIR1andLAIR2regulatory elements that act as foci of 3D genome interactions with other highly active regulatory elements.

Conclusions

Our findings define the complex regulatory landscapes of human and mouse LAIR genes and reveal new insights into the transcriptional regulatory mechanisms that control the expression of these important immune modulatory proteins.
MousiPLIER: A Mouse Pathway-Level Information Extractor Model
Authors: Zhang Shuo , Heil Benjamin J. , Mao Weiguang , Chikina Maria , Greene Casey S. , Heller Elizabeth A.
Published: August 2023

High throughput gene expression profiling is a powerful approach to generate hypotheses on the underlying causes of biological function and disease. Yet this approach is limited by its ability to infer underlying biological pathways and burden of testing tens of thousands of individual genes. Machine learning models that incorporate prior biological knowledge are necessary to extract meaningful pathways and generate rational hypothesis from the vast amount of gene expression data generated to date. We adopted an unsupervised machine learning method, Pathway-level information extractor (PLIER), to train the first mouse PLIER model on 190,111 mouse brain RNA-sequencing samples, the greatest amount of training data ever used by PLIER. mousiPLER converted gene expression data into a latent variables that align to known pathway or cell maker gene sets, substantially reducing data dimensionality and improving interpretability. To determine the utility of mousiPLIER, we applied it to a mouse brain aging study of microglia and astrocyte transcriptomic profiling. We found a specific set of latent variables that are significantly associated with aging, including one latent variable (LV41) corresponding to striatal signal. We next performed k-means clustering on the training data to identify studies that respond strongly to LV41, finding that the variable is relevant to striatum and aging across the scientific literature. Finally, we built a web server (http://mousiplier.greenelab.com/) for users to easily explore the learned latent variables. Taken together this study provides proof of concept that mousiPLIER can uncover meaningful biological processes in mouse transcriptomic studies.

Significance statement

Analysis of RNA-sequencing data commonly generates differential expression of individual genes across conditions. However, genes are regulated in complex networks, not as individual entities. Machine learning models that incorporate prior biological information are a powerful tool to analyze human gene expression. However, such models are lacking for mouse despite the vast number of mouse RNA-seq datasets. We trained a mouse pathway-level information extractor model (mousiPLIER). The model reduced the data dimensionality from over 10,000 genes to 196 latent variables that map to prior pathway and cell marker gene sets. We demonstrated the utility of mousiPLIER by applying it to mouse brain aging data and developed a web server to facilitate the use of the model by the scientific community.
Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts
Authors: Yocca Alan E. , Platts Adrian , Alger Elizabeth , Teresi Scott , Mengist Molla F. , Benevenuto Juliana , Felipe V. Ferrão Luis , Jacobs MacKenzie , Babinski Michal , Magallanes-Lundback Maria , Bayer Philipp , Golicz Agnieszka , Humann Jodi L , Main Dorrie , Espley Richard V. , Chagné David , Albert Nick W. , Montanari Sara , Vorsa Nicholi , Polashock James , Díaz-Garcia Luis , Zalapa Juan , Bassil Nahla V. , Munoz Patricio R. , Iorizzo Massimo , Edger Patrick P.
Published: August 2023

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures) as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium - a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.
Density Physics-Informed Neural Network reveals sources of cell heterogeneity in signal transduction
Authors: Jo Hyeontae , Hong Hyukpyo , Hwang Hyung Ju , Chang Won , Kim Jae Kyoung
Published: August 2023

The transduction time between signal initiation and final response provides valuable information on the underlying signaling pathway, including its speed and precision. Furthermore, multimodality in transduction-time distribution informs that the response is regulated by multiple pathways with different transduction speeds. Here, we developed Density physics-informed neural network (Density-PINN) to infer the transduction-time distribution, challenging to measure, from measurable final stress response time traces. We applied Density-PINN to single-cell gene expression data from 16 promoters regulated by unknown pathways in response to antibiotic stresses. We found that promoters with slower signaling initiation and transduction exhibit larger cell-to-cell heterogeneity in response intensity. However, this heterogeneity was greatly reduced when the response was regulated by slow and fast pathways together. This suggests a strategy for identifying effective signaling pathways for consistent cellular responses to disease treatments. Density-PINN can also be applied to understand various time delay systems, including infectious diseases.
Local climate change velocities explain multidirectional range shifts in a North American butterfly assemblage
Authors: da Silva Carmen R.B. , Diamond Sarah E.
Published: August 2023

Species are often expected to shift their distributions poleward to evade warming climates. However, from 18 years of fixed transect monitoring data on 88 species of butterfly in the midwestern United States, we show that butterflies are shifting their centroids in all directions, except towards the region that is warming the fastest (southeast). Butterflies shifted their centroids at a mean rate of 4.87 km yr-1. The rate of centroid shift was significantly associated with local climate change velocity (temperature and precipitation), but not with mean climate change velocity throughout the species’ ranges. Surprisingly, the centroid shift was also unrelated to species traits expected to mediate the shift response including thermal niche breadth (range of climates butterflies experience throughout their distribution) and wingspan (often used as metric for dispersal capability). Contrasting with a number of previous studies, we observed relatively high phylogenetic signal in the rate and direction species shifted their centroids, suggesting that evolutionary history helps to explain multidirectional range shift responses and that some groups of species will be better able to shift their ranges than others. This research shows important signatures of multidirectional range shifts (latitudinal and longitudinal) and uniquely shows that local climate change velocities are more important in driving range shifts than the mean climate change velocity throughout a species’ entire range.
“Patchiness” in Mechanical Stiffness across a Tumor as an Early-Stage Marker for Malignancy
Authors: Mirzakhel Zibah , Reddy Gudur Ashrith , Boman Jennifer , Manns Brianna , Van Teer Savannah , Katira Parag
Published: August 2023

Mechanical phenotyping of tumors, either at an individual cell level or tumor cell population level is gaining traction as a diagnostic tool. However, the extent of diagnostic and prognostic information that can be gained through these measurements is still unclear. In this work, we focus on the heterogeneity in mechanical properties of cells obtained from a single source such as a tissue or tumor as a potential novel biomarker. We believe that this heterogeneity is a conventionally overlooked source of information in mechanical phenotyping data. We use mechanics-based in-silico models of cell-cell interactions and cell population dynamics within 3D environments to probe how heterogeneity in cell mechanics drives tissue and tumor dynamics. Our simulations show that the initial heterogeneity in the mechanical properties of individual cells and the arrangement of these heterogenous sub-populations within the environment can dictate overall cell population dynamics and cause a shift towards the growth of malignant cell phenotypes within healthy tissue environments. The overall heterogeneity in the cellular mechanotype and their spatial distributions is quantified by a “patchiness” index, which is the ratio of the global to local heterogeneity in cell populations. We observe that there exists a threshold value of the patchiness index beyond which an overall healthy cell population of cells will show a steady shift towards a more malignant phenotype. Based on these results, we propose that the “patchiness” of a tumor or tissue sample, can be an early indicator for malignant transformation and cancer occurrence in benign tumors or healthy tissues. Additionally, we suggest that tissue patchiness, measured either by biochemical or biophysical markers, can become an important metric in predicting tissue health and disease likelihood just as landscape patchiness is an important metric in ecology.
Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis inC. elegans
Authors: Taylor Shane K. B. , Hartman Jessica H. , Gupta Bhagwati P.
Published: August 2023

The conserved mesencephalic astrocyte-derived neurotrophic factor (MANF) protects dopaminergic neurons but also functions in several other tissues. Previously, we showed thatCaenorhabditis elegans manf-1null mutants have increased ER stress, dopaminergic neurodegeneration, protein aggregation, slower growth, and a reduced lifespan. The multiple requirements of MANF in different systems suggest its essential role in regulating cellular processes. However, how intracellular and extracellular MANF regulates broader cellular function remains unknown. Here, we report a novel mechanism of action formanf-1that involves the autophagy transcription factor HLH-30/TFEB-mediated signaling to regulate lysosomal function and aging. We generated multiple transgenic strains overexpressing MANF-1 and found that animals had extended lifespan, reduced protein aggregation, and improved neuronal health. Using a fluorescently tagged MANF-1, we observed different tissue localization of MANF-1 depending on the ER retention signal. Further subcellular analysis showed that MANF-1 localizes within cells to the lysosomes. These findings were consistent with our transcriptomic studies and, together with analysis of autophagy regulators, demonstrate that MANF-1 regulates protein homeostasis through increased autophagy and lysosomal activity. Collectively, our findings establish MANF as a critical regulator of the stress response, proteostasis, and aging.
A systematic review of the performance of actigraphy in measuring sleep stages
Authors: Yuan Hang , Hill Elizabeth A. , Kyle Simon D. , Doherty Aiden
Published: August 2023

The accuracy of actigraphy for sleep staging is assumed to be poor, but examination is limited. This systematic review aimed to assess the performance of actigraphy in sleep stage classification for adults. A systematic search was performed using MEDLINE, Web of Science, Google Scholar, and Embase databases. We identified eight studies that compared sleep architecture estimates between wrist-worn actigraphy and polysomnography. Large heterogeneity was found with respect to how sleep stages were grouped and the choice of metrics used to evaluate performance. Quantitative synthesis was not possible, so we performed a narrative synthesis of the literature. From the limited number of studies, we found that actigraphy-based sleep staging had some ability to classify different sleep stages compared with polysomnography.
Phenotyping the virulence of SARS-CoV-2 variants in hamsters by digital pathology and machine learning
Authors: Meehan Gavin R , Herder Vanessa , Allan Jay , Huang Xinyi , Kerr Karen , Mendonca Diogo Correa , Ilia Georgios , Wright Derek W , Nomikou Kyriaki , Gu Quan , Arias Sergi Molina , De Lorenzo Giuditta , Cowton Vanessa , Upfold Nicole , Palmalux Natasha , Brown Jonathan , Barclay Wendy , Filipe Ana Da Silva , Furnon Wilhelm , Patel Arvind H , Palmarini Massimo
Published: August 2023

SARS-CoV-2 has continued to evolve throughout the COVID-19 pandemic, giving rise to multiple variants of concern (VOCs) with different biological properties. As the pandemic progresses, it will be essential to test in near real time the potential of any new emerging variant to cause severe disease. BA.1 (Omicron) was shown to be attenuated compared to the previous VOCs like Delta, but it is possible that newly emerging variants may regain a virulent phenotype. Hamsters have been proven to be an exceedingly good model for SARS-CoV-2 pathogenesis. Here, we aimed to develop robust quantitative pipelines to assess the virulence of SARS-CoV-2 variants in hamsters. We used various approaches including RNAseq, RNAin situhybridization, immunohistochemistry, and digital pathology, including software assisted whole section imaging and downstream automatic analyses enhanced by machine learning, to develop methods to assess and quantify virus-induced pulmonary lesions in an unbiased manner. Initially, we used Delta and Omicron to develop our experimental pipelines. We then assessed the virulence of recent Omicron sub-lineages including BA.5, XBB, BQ.1.18, BA.2 and BA.2.75. We show that in experimentally infected hamsters, accurate quantification of alveolar epithelial hyperplasia and macrophage infiltrates represent robust markers for assessing the extent of virus-induced pulmonary pathology, and hence virus virulence. In addition, using these pipelines, we could reveal how some Omicron sub-lineages (e.g., BA.2.75) have regained virulence compared to the original BA.1. Finally, to maximise the utility of the digital pathology pipelines reported in our study, we developed an online repository containing representative whole organ histopathology sections that can be visualised at variable magnifications (https://covid-atlas.cvr.gla.ac.uk). Overall, this pipeline can provide unbiased and invaluable data for rapidly assessing newly emerging variants and their potential to cause severe disease.
De novoProtein Sequence Design Based on Deep Learning and Validation on CalB Hydrolase
Authors: Mu Junxi , Li Zhenxin , Zhang Bo , Zhang Qi , Iqbal Jamshed , Wadood Abdul , Wei Ting , Feng Yan , Chen Haifeng
Published: August 2023

Protein design is central to nearly all protein engineering problems, as it can enable the creation of proteins with new biological function, such as improving the catalytic efficiency of enzymes. As one of the key tasks of protein design, fixed-backbone protein sequence design aims to design novel sequence that would fold into a given protein backbone structure. However, current sequence design methods have limitations in terms of low sequence diversity and experimental validation of designed protein function, which cannot meet the needs of functional protein design. We firstly constructed Graphormer-based Protein Design (GPD) model that directly applies Transformer to graph-based representation of 3D protein structure, and added Gaussian noise and sequence random mask to node features to improve the sequence recovery and diversity. Additionally, functional filtering based on the structure folding, solubility, and function were performed to improve the success rate in experiments. The process of “sequence design-functional filtering -functional experiment” was carried out for CalB hydrolase. The experimental results showed that the specify activity of designed protein improved 1.7 times than CalB wild type. This design and filtering platform will be a valuable tool for generating industrial enzymes and protein drugs with specific functions.
The potential of young vegetative quinoa (Chenopodium quinoa) as a new sustainable protein-rich winter leafy crop under Mediterranean climate
Authors: Rubinovich Lior , Dagan Reut , Lugasi Yaron , Galili Shmuel , Asher Aviv
Published: August 2023

The demand for protein products has significantly risen in the last few years. In western countries, animals are the primary source of protein; however, plants could take a share of this market due to lower production costs, among other advantages. Quinoa (Chenopodium quinoaWilld.) is a well- known but under-utilized protein-rich crop, commonly cultivated for grain production. These plants were recently evaluated for their use as a non-traditional, green leafy crop. Here we assessed the potential of young vegetative quinoa as a new sustainable winter leafy crop in Israel—serving as a model for Mediterranean semi-arid regions, by evaluating yield, protein content and quality. Five quinoa accessions were sown on three winter sowing dates over two consecutive years. Plants were harvested when they reached 10–13% dry matter (DM). DM yield ranged between 574 and 1,982 kg ha-1and was generally higher in the second year. Protein content ranged from 14.4–34% and was generally higher in the first year. Protein yield ranged from 111–471 kg ha-1and was greatest on the December sowing date. DM and protein yields were positively correlated with plant density. Protein content was negatively correlated with plant density and DM yield. Our findings show that 200 g DM of young vegetative quinoa can meet the protein and most essential amino acid requirements for a 70 kg human adult. Prospects for cultivating young vegetative quinoa in Mediterranean countries as a new sustainable, protein-rich winter leafy crop are therefore high, as supported by its high protein yields and quality, and its requirement for only scant irrigation. Further studies should examine economic and other agrotechnical parameters toward the geographical distribution and expansion of young vegetative quinoa cultivation.
Improvement ofNeisseria gonorrhoeaeculture media to enable growth without CO2
Authors: Menkiti Chukwuma Jude , Snyder Lori A. S.
Published: August 2023

Bacterial culture is the crucial step in diagnosingNeisseria gonorrhoeaeinfections and is the gold standard for determining their antimicrobial resistance profile. However, culture ofNeisseriaspp. can be challenging in resource poor areas, relying on specialist incubators supplying 5% CO2for growth of the bacteria. Even when such incubators are available, the CO2to run them may be scarce; there were CO2shortages during the COVID-19 pandemic, for example. Although culture jars with gas packs or candles can be used, these are inefficient in terms of use of incubator space and researcher time. To achieve simplicity in culturing ofN. gonorrhoeae, the standard Oxoid GC base medium, made with the Kellogg’s glucose and iron supplements was improved with the addition of 0.75 g/L sodium bicarbonate (NaHCO3), which is inexpensive and really available. This improved media was able to sustain gonococcal growth as well as standard GC media in 5% CO2. Chocolate agar and Thayer-Martin agar with sodium bicarbonate was also developed, with all showing good growth ofN. gonorrhoeaewithout the need for atmospheric CO2.
Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria
Authors: Liao Jane , Yeong Vivian , Obermeyer Allie C.
Published: August 2023

Subcellular phase separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates inEscherichia coliby utilizing electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates withinE. colicells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria.

Highlights

Minimal disordered cationic peptides of varying charge densities can promote protein phase separation in bacterial cells.

Protein net charge and charge-patterning are distinct determinants of phase behavior.

Protein charge distribution can be used to tune the size, number, position, and reversibility of condensates.

Multiple proteins can be selectively recruited to synthetic condensates with disordered cationic peptides.
A patchwork pathway of apparently recent origin enables degradation of the synthetic buffer compound TRIS in bacteria
Authors: Holert Johannes , Borker Aron , Nübel Laura , Daniel Rolf , Poehlein Anja , Philipp Bodo
Published: August 2023

The widely used synthetic buffer compound TRIS was long considered to be biologically inert. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, aPseudomonasstrain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Conjugational transfer of this plasmid intoP. putidaKT2440 enabled this strain to grow with TRIS, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase and a D-serine dehydratase which plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from thePseudomonasandShinellagenera carrying highly similar TRIS degradation gene clusters.

Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways and database searches suggest that the TRIS degradation pathway is now globally distributed. Our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales. This knowledge is valuable for developing new water purification processes in times of increasing water scarcity.
A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall
Authors: Marmont Lindsey S. , Orta Anna K. , Corey Robin A. , Sychantha David , Galliano Ana Fernández , Li Yancheng E. , Baileeves Becca W.A. , Greene Neil G. , Stansfeld Phillip J. , Clemons William M. , Bernhardt Thomas G.
Published: August 2023

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants ofPseudomonas aeruginosaMraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.
Major urinary protein (Mup) gene family deletion drives sex-specific alterations on the house mouse gut microbiota
Authors: Real Madalena V. F. , Colvin Melanie S. , Sheehan Michael J. , Moeller Andrew H.
Published: August 2023

The gut microbiota is shaped by host metabolism. In house mice (Mus musculus), major urinary protein (MUP) pheromone production represents a considerable energy investment, particularly in sexually mature males. Deletion of theMupgene family shifts mouse metabolism towards an anabolic state, marked by lipogenesis, lipid accumulation, and body mass increases. Given the metabolic implications of MUPs, they may also influence the gut microbiota. Here, we investigated the effect of deletion of theMupgene family on the gut microbiota of sexually mature mice. Shotgun metagenomics revealed distinct taxonomic and functional profiles between wildtype and knockout males, but not females. Deletion of theMupgene cluster significantly reduced diversity in microbial families and functions in male mice. Additionally, specific taxa of the Ruminococcaceae family, which is associated with gut health and reduced risk of developing metabolic syndrome, and several microbial functions, such as transporters involved in vitamin B5 acquisition, were significantly depleted in the microbiota ofMup-knockout males. Altogether these results show that major urinary proteins significantly affect the gut microbiota of house mouse in a sex-specific manner.

Importance

The community of microorganisms that inhabit the gastrointestinal track of animals, known as the gut microbiota, can have profound effects on host phenotypes. The gut microbiota is in turn shaped by host genes, including those involved with host metabolism. In adult male house mice, expression of the major urinary protein (Mup) gene cluster represents a substantial energy investment, and deletion ofMupgene family leads to fat accumulation and weight gain in males. We show for the first time that deletingMupgenes also alters the gut microbiota of male, but not female, mice in terms of both taxonomic and functional composition. Male mice withoutMupgenes harbored fewer gut bacterial families and reduced abundances of several species, including bacteria previously shown to reduce obesity risk. Studying the impact of theMupgenes on the gut microbiota will help us understand how these genes influence host phenotype more broadly.
In vivocharacterization of a secologanin transporter fromCatharanthus roseus
Authors: Li Fanfan , Shahsavarani Mohammadamin , Handy-Hart Cody-Jordan , Montgomery Victoria , Beech Robin N. , Liu Lan , Bayen Stéphane , Qu Yang , De Luca Vincenzo , Dastmalchi Mehran
Published: August 2023

Monoterpenoid indole alkaloid (MIA) biosynthesis inCatharanthus roseusis a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, andin plantasilencing in two varieties ofC. roseusresulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel ofin vivobiochemical characterization using theXenopus laevisoocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, bothin plantaand in theX. laevissystem. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasisin planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs.

SIGNIFICANCE

We have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter inCatharanthus roseus,as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.
ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis
Authors: Cowling Craig L. , Homayouni Arielle L. , Callwood Jodi B. , McReynolds Maxwell R. , Khor Jasper , Ke Haiyan , Draves Melissa A. , Dehesh Katayoon , Walley Justin W. , Strader Lucia C. , Kelley Dior R.
Published: August 2023

Plant root systems play a pivotal role in plant physiology and exhibit diverse phenotypic traits. Understanding the genetic mechanisms governing root growth and development in model plants like maize is crucial for enhancing crop resilience to drought and nutrient limitations. This study focused on identifying and characterizing ZmPILS6, an annotated auxin efflux carrier, as a key regulator of various crown root traits in maize. ZmPILS6-modified roots displayed reduced network area and suppressed lateral root formation, desirable traits during drought and low phosphate conditions. The research revealed that ZmPILS6 localizes to the endoplasmic reticulum and plays a vital role in controlling the spatial distribution of indole-3-acetic acid (IAA or “auxin”) in primary roots. The study also demonstrated that ZmPILS6 can actively efflux IAA when expressed in yeast. Furthermore, the loss of ZmPILS6 resulted in significant proteome remodeling in maize roots, particularly affecting hormone signaling pathways. To identify potential interacting partners of ZmPILS6, a weighted gene co-expression analysis (WGNA) was performed. Altogether, this research contributes to the growing knowledge of essential genetic determinants governing maize root morphogenesis, which is crucial for guiding agricultural improvement strategies.

Significance Statement

Crop yield and stress resilience are significantly influenced by crown root architecture. A reverse genetic screen aimed at identifying novel regulators of maize root morphogenesis led to the discovery of ZmPILS6, an auxin efflux carrier. The loss of ZmPILS6 negatively impacts numerous root traits that are linked to plant physiology and function. Proteomic characterization ofpils6-1roots revealed that this evolutionarily conserved transporter is required for the proper expression of numerous phytohormone pathways, including abscisic acid, gibberellins, and jasmonic acid. Notably, ZmPILS6 appears to have a contrasting role in regulating root morphogenesis compared to its Arabidopsis ortholog, PILS6. This finding emphasizes the need for functional characterization of candidate genes directly within key crops of interest, which cannot always be correctly inferred from other model plants.
Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase
Authors: Cordoba John J. , Mullins Elwood A. , Salay Lauren E. , Eichman Brandt F. , Chazin Walter J.
Published: August 2023

DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ∼20 nucleotides of DNA. Here we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
Protein Ensemble Generation through Variational Autoencoder Latent Space Sampling
Authors: Mansoor Sanaa , Baek Minkyung , Park Hahnbeom , Lee Gyu Rie , Baker David
Published: August 2023

Mapping the ensemble of protein conformations that contribute to function and can be targeted by small molecule drugs remains an outstanding challenge. Here we explore the use of soft-introspective variational autoencoders for reducing the challenge of dimensionality in the protein structure ensemble generation problem. We convert high-dimensional protein structural data into a continuous, low-dimensional representation, carry out search in this space guided by a structure quality metric, then use RoseTTAFold to generate 3D structures. We use this approach to generate ensembles for the cancer relevant protein K-Ras, training the VAE on a subset of the available K-Ras crystal structures and MD simulation snapshots, and assessing the extent of sampling close to crystal structures withheld from training. We find that our latent space sampling procedure rapidly generates ensembles with high structural quality and is able to sample within 1 angstrom of held out crystal structures, with a consistency higher than MD simulation or AlphaFold2 prediction. The sampled structures sufficiently recapitulate the cryptic pockets in the held-out K-Ras structures to allow for small molecule docking.