bluuebunny/crossref_metadata_2025 huggingface dataset:
A conserved pressure-driven mechanism for regulating cytosolic osmolarity
Authors: Velle Katrina B. , Garner Rikki M. , Beckford Tatihana K. , Weeda Makaela , Liu Chunzi , Kennard Andrew S. , Edwards Marc , Fritz-Laylin Lillian K.
Published: March 2023

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages—the discobanNaegleria gruberi, and the amoebozoan slime moldDictyostelium discoideum. Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolateParamecium multimicronucleatum. Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Batch Action PoTential Analyser (BAPTA): an open source tool for automated high throughput analysis of cardiac action potentials
Authors: Leonov Vladislav , Torre Eleonora , Ronchi Carlotta , Crotti Lia , Schwartz Peter J , Rocchetti Marcella , Zaza Antonio , Sala Luca
Published: March 2023

The cardiac action potential (AP) is a key species-specific feature of cardiomyocytes that occurs in response to coordinated actions of ion channels. It represents the first step of the cardiac excitation-contraction coupling and it is crucial for cardiomyocyte (CM) physiology. Changes in the cardiac AP may primarily occur as a consequence of diseases or as a direct or unwanted response to drugs. Our ability to quantify these changes defines the reliability of our measurements and its throughput.

Cardiac AP parameters are often quantified through manual time-consuming data analysis protocols or custom-made and proprietary data analysis pipelines; to the best of our knowledge, no tools are currently available for automated cardiac AP analysis and AP parameter quantification.

Here we introduce a free and open source software tool named Batch Action PoTential Analyser (BAPTA), written in the R language, designed to i) overcome the inherent operator-dependent bias on trace selection affecting reproducibility, ii) vastly improve the throughput of the analyses of large datasets and iii) analyse both spontaneous and triggered APs from CMs of multiple species and origin.

We present here four use-cases in which BAPTA can be used at high throughput to investigate the effects of: 1) a disease (cardiomyopathy) on rat CMs, 2) drugs on mouse pacemaker cells, 3) rate-dependency of AP duration in guinea pig CMs and 4) metabolic electrophysiological maturation in human stem-cell-derived CMs. Overall, BAPTA consistently provides faster, more reproducible and scalable readouts which excellently correlate with manual analyses performed by experienced electrophysiologists.
A genetic variation in the adenosine A2A receptor gene contributes to variability in oscillatory alpha power in wake and sleep EEG and A1adenosine receptor availability in the human brain
Authors: Tichelman Naemi L. , Foerges Anna L. , Elmenhorst Eva-Maria , Lange Denise , Hennecke Eva , Baur Diego M. , Beer Simone , Kroll Tina , Neumaier Bernd , Bauer Andreas , Landolt Hans-Peter , Aeschbach Daniel , Elmenhorst David
Published: March 2023

The EEG alpha rhythm (8-13 Hz) is one of the most salient human brain activity rhythms. Spectral power in the alpha range in wakefulness and sleep varies among individuals based on genetical predisposition, yet knowledge about the underlying genes is scarce. The EEG alpha oscillations are related to cerebral energy metabolism and modulated by the level of attention and vigilance. The neuromodulator adenosine is directly linked to energy metabolism as product of adenosine tri-phosphate (ATP) breakdown and acts as a sleep promoting molecule by activitating A1and A2Aadenosine receptors. We quantified EEG oscillatory alpha power in wakefulness and sleep, as well as A1adenosine receptor availability by positron emission tomography with18F-CPFPX, in a large sample of healthy volunteers carrying different alleles of gene variant rs5751876 ofADORA2Aencoding A2Aadenosine receptors. Oscillatory alpha power was higher in homozygous C-allele carriers (n = 27, 11 females) compared to heterozygous and homozygous carriers of the T-allele (n(C/T) = 23, n(T/T) = 5, 13 females) (F(18,37)= 2.35, p = 0.014, Wilk’s Λ = 0.467). Across considered brain regions an effect ofADORA2Agenotype on A1adenosine receptor binding potential was found (F(18,40)= 2.62, p = 0.006, Wilk’s Λ = 0.459) and after correction for multiple testing this effect was shown to be significant for circumscribed occipital region of calcarine fissures.

A correlation between individual differences in oscillatory alpha power and adenosine receptor availability was found for the subgroup of female participants only. In conclusion: a genetic variation in the adenosinergic system affects individual alpha power, although a direct modulatory effect via the A1AR has been demonstrated for females only.
Temporal single-cell transcriptomic analysis of thesox1a:eGFPtransgenic line identified the lateral floor plate progenitor cells as the origin of intraspinal serotonergic neurons
Authors: Chen Fushun , Köhler Melina , Cucun Gokhan , Takamiya Masanari , Kizil Caghan , Cosacak Mehmet Ilyas , Rastegar Sepand
Published: March 2023

The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo,sox1genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function ofsox1aandsox1bleads to a significant decline in a subtype of V2 neurons, called V2s, in zebrafish. Here, a single-cell RNA sequencing approach was used to analyse the transcriptome ofsox1alineage progenitors and neurons in the zebrafish spinal cord at four different time points during the first five days of embryonic development, using the Tg(sox1a:eGFP) line. In addition to the previously describedsox1a-expressing neurons, we found thatsox1ais also expressed in late-developing intraspinal serotonergic neurons (ISNs). Analysis of developmental trajectories from single-cell data and depletion of lateral floor plate (LFP) cells bynkx2.9morpholino knockdown suggest that ISNs arise from LFP precursor cells. Pharmacological inhibition of the Notch signalling pathway indicates that this pathway is required for the negative regulation of the development of LFP progenitor cells into ISN populations. Our results show that the zebrafish LFP is a precursor domain that longitudinally gives rise to ISNs in addition to the previously described KA” and V3 interneurons.
Microbial community changes correlate with impaired host fitness ofAurelia auritaafter environmental challenge
Authors: Pinnow Nicole , Chibani Cynthia M. , Güllert Simon , Weiland-Bräuer Nancy
Published: March 2023

Climate change globally endangers certain marine species, but at the same time, such changes may promote species that can tolerate and adapt to varying environmental conditions. Such acclimatization can be accompanied or possibly even be enabled by a host’s microbiome; however, few studies have so far directly addressed this process. Here we show that acute, individual rises in seawater temperature and salinity to sub-lethal levels diminished host fitness of the benthicAurelia auritapolyp, demonstrated by up to 34 % reduced survival rate, shrinking of the animals, and almost halted asexual reproduction. Changes in the fitness of the polyps to environmental stressors coincided with microbiome changes, mainly within the phyla Proteobacteria and Bacteroidota. The absence of bacteria amplified these effects, pointing to the crucial importance of a balanced microbiota to cope with a changing environment. In a future ocean scenario, mimicked by a combined but milder rise of temperature and salinity, the fitness of polyps was severely less impaired, together with condition-specific changes in the microbiome composition. Our results show that the effects on host fitness correlate with the strength of environmental stress, while salt-conveyed thermotolerance might be involved. Further, a specific, balanced microbiome ofA. auritapolyps is essential for the host’s acclimatization. Microbiomes may provide a means for acclimatization, and microbiome flexibility can be a fundamental strategy for marine animals to adapt to future ocean scenarios and maintain biodiversity and ecosystem functioning.
Transcriptional buffering and 3ʹUTR lengthening are shaped during human neurodevelopment by shifts in mRNA stability and microRNA load
Authors: Mufteev Marat , Rodrigues Deivid C. , Yuki Kyoko E. , Narula Ashrut , Wei Wei , Piekna Alina , Liu Jiajie , Pasceri Peter , Rissland Olivia S. , Wilson Michael D. , Ellis James
Published: March 2023

The contribution of mRNA half-life is commonly overlooked when examining changes in mRNA abundance during development. mRNA levels of some genes are regulated by transcription rate only, but others may be regulated by mRNA half-life only shifts. Furthermore, transcriptional buffering is predicted when changes in transcription rates have compensating shifts in mRNA half-life resulting in no change to steady-state levels. Likewise, transcriptional boosting should result when changes in transcription rate are accompanied by amplifying half-life shifts. During neurodevelopment there is widespread 3ʹUTR lengthening that could be shaped by differential shifts in the stability of existing short or long 3ʹUTR transcript isoforms. We measured transcription rate and mRNA half-life changes during induced human Pluripotent Stem Cell (iPSC)-derived neuronal development using RATE-seq. During transitions to progenitor and neuron stages, transcriptional buffering occurred in up to 50%, and transcriptional boosting in up to 15%, of genes with changed transcription rates. The remaining changes occurred by transcription rate only or mRNA half-life only shifts. Average mRNA half-life decreased two-fold in neurons relative to iPSCs. Short gene isoforms were more destabilized in neurons and thereby increased the average 3ʹUTR length. Small RNA sequencing captured an increase in microRNA copy number per cell during neurodevelopment. We propose that mRNA destabilization and 3ʹUTR lengthening are driven in part by an increase in microRNA load in neurons. Our findings identify mRNA stability mechanisms in human neurodevelopment that regulate gene and isoform level abundance and provide a precedent for similar post-transcriptional regulatory events as other tissues develop.
Subset-specific and temporal control of effector and memory CD4+ T cell survival
Authors: Shanmuganad Sharmila , Ferguson Autumn , Paranjpe Aditi , Cianciolo Eileen Elfers , Katz Jonathan D. , Herold Marco J , Hildeman David A.
Published: March 2023

Following their proliferative expansion and differentiation into effector cells like Th1, Tfh, and T central memory precursors (Tcmp), most effector CD4+ T cells die, while some survive and become memory cells. Here, we explored how Bcl-2 family members controlled the survival of CD4+ T cells during distinct phases of mouse acute LCMV infection. During expansion, we found that Th1 cells dominated the response, downregulated expression of Bcl-2, and did not require Bcl-2 for survival. Instead, they relied on the anti-apoptotic protein, A1 for survival. Similarly, Th17 cells in an EAE model also depended on A1 for survival. However, after the peak of the response, CD4+ effector T cells required Bcl-2 to counteract Bim to aid their transition into memory. This Bcl-2 dependence persisted in established memory CD4+ T cells. Combined, these data show a temporal switch in Bcl-2 family-mediated survival of CD4+ T cells over the course of an immune response. This knowledge can help improve T cell survival to boost immunity and conversely, target pathogenic T cells.
The emergence of tuning to global shape properties of radial frequency patterns in the ventral visual pathway
Authors: Lawrence Samuel J. D. , Zamboni Elisa , Vernon Richard J. W. , Gouws André D. , Wade Alex R. , Morland Antony B.
Published: March 2023

Radial frequency patterns - created by sinusoidal modulations of a circle’s radius - are processed globally when radial frequency is low. These closed shapes therefore offer a useful way to interrogate the human visual system for global processing of curvature. Radial frequency patterns elicit greater responses than those to radial gratings in V4 and more anterior face selective regions of the ventral visual pathway. This is largely consistent with work on non-human primates showing curvature processing emerges in V4, but is evident also higher up the ventral visual stream. Rather than contrasting radial frequency patterns with other stimuli, we presented them at varied frequencies in a regimen that allowed tunings to radial frequency to be derived from 8 human participants (3 female). We found tuning to low radial frequency in lateral occipital areas and to some extent in V4. In a control experiment we added a high frequency ripple to the stimuli disrupting the local contour. Low frequency tuning to these stimuli remained in the ventral visual stream underscoring its role in global processing of shape curvature. We then used representational similarity analysis to show that in lateral occipital areas the neural representation was related to stimulus similarity, when it was computed with a model that captured how stimuli are perceived. We show therefore that global processing of shape curvature emerges in the ventral visual stream as early as V4, but is found more strongly in lateral occipital regions, which exhibit responses and representations that relate well to perception.
“Conformational dynamics of C1r inhibitor proteins from Lyme disease and relapsing fever spirochetes”
Authors: Roy Sourav , Booth Charles E. , Powell-Pierce Alexandra D. , Schulz Anna M. , Skare Jon T. , Garcia Brandon L.
Published: March 2023

Borrelial pathogens are vector-borne etiological agents of Lyme disease, relapsing fever, andBorrelia miyamotoidisease. These spirochetes each encode several surface-localized lipoproteins that bind to components of the human complement system. BBK32 is an example of a borrelial lipoprotein that protects the Lyme disease spirochete from complement-mediated attack. The complement inhibitory activity of BBK32 arises from an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical pathway, C1r.Borrelia miyamotoispirochetes encode BBK32 orthologs termed FbpA and FbpB, and these proteins also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever spirochetes, remains unknown. Here we report the crystal structure of the C-terminal domain ofB. hermsiiFbpC to a limiting resolution of 1.5 Å. Surface plasmon resonance studies and assays of complement function demonstrate that FbpC retains potent BBK32-like anti-complement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out 1 µs molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. This study advances our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveals a surprising plasticity in the structures of borrelial C1r inhibitors.
Brain-wide neural activity underlying memory-guided movement
Authors: Chen Susu , Liu Yi , Wang Ziyue , Colonell Jennifer , Liu Liu D. , Hou Han , Tien Nai-Wen , Wang Tim , Harris Timothy , Druckmann Shaul , Li Nuo , Svoboda Karel
Published: March 2023

Behavior requires neural activity across the brain, but most experiments probe neurons in a single area at a time. Here we used multiple Neuropixels probes to record neural activity simultaneously in brain-wide circuits, in mice performing a memory-guided directional licking task. We targeted brain areas that form multi-regional loops with anterior lateral motor cortex (ALM), a key circuit node mediating the behavior. Neurons encoding sensory stimuli, choice, and actions were distributed across the brain. However, in addition to ALM, coding of choice was concentrated in subcortical areas receiving input from ALM, in an ALM-dependent manner. Choice signals were first detected in ALM and the midbrain, followed by the thalamus, and other brain areas. At the time of movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.
Brain state dynamics reflect emotion transitions induced by music
Authors: Sachs Matthew E. , Ochsner Kevin N. , Baldassano Christopher
Published: March 2023

Our ability to shift from one emotion to the next allows us to adapt our behaviors to a constantly-changing and often uncertain environment. Although previous studies have identified cortical and subcortical regions involved in affective responding, no studies have asked whether and how these regions track and represent transitionsbetweendifferent emotional states and modulate their responses based on the recent emotional context. To this end, we commissioned new musical pieces designed to systematically move participants through different emotional states during fMRI. Using a combination of data-driven (Hidden Markov Modeling) and hypothesis-driven methods, we show that spatiotemporal patterns of activation along the temporoparietal axis reflect transitions between music-evoked emotions. Furthermore, self-reported emotions and the subsequent neural response patterns were sensitive to the emotional context in which the music was heard. The findings highlight the role of temporal and parietal brain regions in not only processing low-level auditory signals, but in linking changes in these signals with our on-going, contextually-dependent emotional responses.
Early striatal hyperexcitability in anin vitrohuman striatal microcircuit model carrying the Parkinson’sGBA-N370Smutation
Authors: Do Quyen B. , Ng Bryan , Gomez Ricardo Marquez , Beccano-Kelly Dayne , Ibarra-Aizpura Naroa , Caiazza Maria-Claudia , Lang Charmaine , Baleriola Jimena , Bengoa-Vergniory Nora , Wade-Martins Richard
Published: March 2023

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson’s disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chamber microfluidic platform and established a cortico-striato-nigral microcircuit recapitulating the striatal presynaptic triadin vitrousing induced pluripotent stem cell (iPSC)-derived neurons. We found that, although cortical glutamatergic projections facilitated MSN synaptic activity, dopaminergic transmission was essential for excitability maturation of MSNsin vitro. Replacement of wild-type iPSC-dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-relatedGBA-N370Smutation induced early hyperexcitability in iPSC-MSNs through reduction of voltage-gated sodium and potassium intrinsic currents. Such deficits were resolved in aged cultures or with antagonism of protein kinase A activity in nigrostriatal iPSC-DaNs. Hence, our results highlight the unique utility of modelling striatal neurons in a modular and highly physiological circuit which is essential to reveal mechanistic insights of the loss of electrical functional integrity in the striata ofGBA1PD patients.
Clinical Antibiotic-Resistance Plasmids Have Small Effects on Biofilm Formation and Population Growth inEscherichia coliin vitro
Authors: Brülisauer Laura , Leon-Sampedro Ricardo , Hall Alex R.
Published: March 2023

Antimicrobial resistance (AR) mechanisms encoded on plasmids can affect other phenotypic traits in bacteria, including biofilm formation. These effects may be important contributors to the spread of AR and the evolutionary success of plasmids, but it is not yet clear how common such effects are for clinical plasmids/bacteria, and how they vary among different plasmids and host strains. Here, we used a combinatorial approach to test the effects of clinical AR plasmids on biofilm formation and population growth in clinical and laboratoryEscherichia colistrains. In most of the 25 plasmid-bacterium combinations tested, we observed no significant change in biofilm formation upon plasmid introduction, contrary to the notion that plasmids frequently alter biofilm formation. In a few cases we detected altered biofilm formation, and these effects were specific to particular plasmid-bacterium combinations. By contrast, we found a relatively strong effect of a chromosomal streptomycin-resistance mutation (inrpsL) on biofilm formation. Further supporting weak and host-strain-dependent effects of clinical plasmids on bacterial phenotypes in the combinations we tested, we found growth costs associated with plasmid carriage (measured in the absence of antibiotics) were moderate and varied among bacterial strains. These findings suggest some key clinical resistance plasmids cause only mild phenotypic disruption to their host bacteria, which may contribute to the persistence of plasmids in the absence of antibiotics.
Probabilistic inference of epigenetic age acceleration from cellular dynamics
Authors: Dabrowski Jan. K. , Yang Emma. J. , Crofts Samuel. J. C. , Hillary Robert. F. , Simpson Daniel. J. , Mccartney Daniel. L. , Marioni Riccardo. E. , Latorre-Crespo Eric , Chandra Tamir
Published: March 2023

The emergence of epigenetic predictors was a pivotal moment in geroscience, propelling the measurement and concept of biological ageing into a quantitative era. However, while current epigenetic clocks have shown strong predictive power, they do not reflect the underlying biological mechanisms driving methylation changes with age. Consequently, biological interpretation of their estimates is limited. Furthermore, our findings suggest that clocks trained on chronological age are confounded by non-age-related phenomena.

To address these limitations, we developed a probabilistic model that describes methylation transitions at the cellular level. Our approach reveals two measurable components, acceleration and bias, that directly relate to perturbations of the underlying cellular dynamics. Acceleration is the proportional increase in the speed of methylation transitions across CpG sites, whereas bias is the degree of global change in methylation affecting all CpG sites uniformly. Using data from 7,028 participants from the Generation Scotland study, we found the age acceleration parameter to be associated with physiological traits known to impact healthy ageing. Furthermore, a genome-wide association study of age acceleration identified four genomic loci previously linked with ageing.
A Systems Serology Approach to the Investigation of Infection-Induced Antibody Responses and Protection in Trachoma
Authors: Barton Amber , Rosenkrands Ida , Pickering Harry , Faal Nkoyo , Harte Anna , Joof Hassan , Makalo Pateh , Ragonnet Manon , Weinreich Olsen Anja , Bailey Robin L. , Mabey David CW , Follmann Frank , Dietrich Jes , Holland Martin J
Published: March 2023

Background

Ocular infections withChlamydia trachomatisserovars A-C cause the neglected tropical disease trachoma. As infection does not confer complete immunity, repeated infections are common, leading to long term sequelae such as scarring and blindness. Here we apply a systems serology approach to investigate whether systemic antibody features are associated with susceptibility to infection.

Methods

Sera from children in five trachoma endemic villages in The Gambia were assayed for 23 antibody features: IgG responses towards twoChlamydia trachomatisantigens and three serovars (elementary bodies and major outer membrane protein MOMP, serovars A-C), IgG responses towards five MOMP peptides (serovars A-C), neutralization and antibody-dependent phagocytosis. Participants were considered resistant if they subsequently developed infection only when over 70% of other children in the same compound were infected.

Results

The antibody features assayed were not associated with resistance to infection (false discovery rate < 0.05). Anti-MOMP SvA IgG and neutralization titer were higher in susceptible individuals (p < 0.05 before multiple testing adjustment). Classification using partial least squares performed only slightly better than chance in distinguishing between susceptible and resistant participants based on systemic antibody profile (specificity 71%, sensitivity 36%).

Conclusions

Systemic infection-induced IgG and functional antibody responses do not appear to be protective against subsequent infection. This may be due to confounding factors increasing both past and future exposure toC. trachomatis, or antibody-dependent enhancement. Ocular responses, IgA, avidity or cell-mediated responses may play a greater role in protective immunity than systemic IgG.
Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizingCluhandSmyd1mRNA
Authors: Singh Sandhya , Gaur Aakash , Kumari Renu , Prakash Shakti , Kumari Sunaina , Chaudhary Ayushi Devendrasingh , Sharma Rakesh Kumar , Prasun Pankaj , Pant Priyanka , Thum Thomas , Jagavelu Kumaravelu , Bharati Pragya , Hanif Kashif , Chitkara Pragya , Kumar Shailesh , Mitra Kalyan , Gupta Shashi Kumar
Published: March 2023

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprisingMsi1andMsi2are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore,Msi2was significantly enriched in the heart’s cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified,Msi2isoforms 1, 4, and 5 and two novel putative isoforms labeled asMsi2isoforms 6 and 7 to be expressed in the heart. Overexpression ofMsi2isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally,Msi2was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression ofMsi2isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function forMsi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identifiedCluhandSmyd1as direct downstream targets ofMsi2. Overexpression ofCluhorSmyd1inhibitedMsi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show thatMsi2induces hypertrophy, mitochondrial dysfunction, and heart failure.
Spatiotemporal regulation ofde novoand salvage purine synthesis during brain development
Authors: Mizukoshi Tomoya , Yamada Seiya , Sakakibara Shin-ichi
Published: March 2023

The levels of purines, essential molecules to sustain eukaryotic cell homeostasis, are regulated by the coordination of thede novoand salvage synthesis pathways. In the embryonic central nervous system (CNS), thede novopathway is considered crucial to meet the requirements for the active proliferation of neural stem/progenitor cells (NSPCs). However, how these two pathways are balanced or separately utilized during CNS development remains poorly understood. In this study, we showed a dynamic shift in pathway utilization, with greater reliance on thede novopathway during embryonic stages and on the salvage pathway at postnatal–adult stages. The pharmacological effects of various purine synthesis inhibitorsin vitroand the expression profile of purine synthesis enzymes indicated that NSPCs in the embryonic cerebrum mainly utilize thede novopathway. Simultaneously, NSPCs in the cerebellum require both thede novoand the salvage pathways.In vivoadministration ofde novoinhibitors resulted in severe hypoplasia of the forebrain cortical region, indicating a gradient of purine demand along the anteroposterior axis of the embryonic brain, with cortical areas of the dorsal forebrain having higher purine requirements than ventral or posterior areas such as the striatum and thalamus. This histological defect of the neocortex was accompanied by strong downregulation of the mechanistic target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase (S6K)/S6 signaling cascade, a crucial pathway for cell metabolism, growth, and survival. These findings indicate the importance of the spatiotemporal regulation of both purine pathways for mTORC1 signaling and proper brain development.

Significance Statement

Brain development requires a balance ofde novoand salvage purine synthetic pathways. However, the utilization of these pathways during brain development remains poorly understood. This study provides evidence that the spatiotemporal regulation of these two purine synthesis pathways is essential for normal brain development. We revealed that inhibition ofde novopurine synthesis results in the downregulation of mammalian/mechanistic target of rapamycin (mTOR) signaling, leading to malformations in specific embryonic brain regions such as the cerebral neocortex. These results suggest a temporal and spatial gradient of purine demand during embryonic brain development. These findings could improve our understanding of neurological diseases caused by defects in purine metabolism.
Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen
Authors: Wong Chun Hao , Wingett Steven W. , Qian Chen , Taliaferro J. Matthew , Ross-Thriepland Douglas , Bullock Simon L.
Published: March 2023

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.
Potent GST ketosteroid isomerase activity relevant to ecdysteroidogenesis in the malaria vectorAnopheles gambiae
Authors: Musdal Yaman , Ismail Aram , Sjödin Birgitta , Mannervik Bengt
Published: March 2023

Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the ordersDipteraandLepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquitoAnopheles gambiaeis a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity with these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Nobo Asp111 is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.
Logic-based mechanistic machine learning on high-content images reveals how drugs differentially regulate cardiac fibroblasts
Authors: Nelson Anders R. , Christiansen Steven L. , Naegle Kristen M. , Saucerman Jeffrey J.
Published: March 2023

Fibroblasts are essential regulators of extracellular matrix deposition following cardiac injury. These cells exhibit highly plastic responses in phenotype during fibrosis in response to environmental stimuli. Here, we test whether and how candidate anti-fibrotic drugs differentially regulate measures of cardiac fibroblast phenotype, which may help identify treatments for cardiac fibrosis. We conducted a high content microscopy screen of human cardiac fibroblasts treated with 13 clinically relevant drugs in the context of TGFβ and/or IL-1β, measuring phenotype across 137 single-cell features. We used the phenotypic data from our high content imaging to train a logic-based mechanistic machine learning model (LogiMML) for fibroblast signaling. The model predicted how pirfenidone and Src inhibitor WH-4-023 reduce actin filament assembly and actin-myosin stress fiber formation, respectively. Validating the LogiMML model prediction that PI3K partially mediates the effects of Src inhibition, we found that PI3K inhibition reduces actin-myosin stress fiber formation and procollagen I production in human cardiac fibroblasts. In this study, we establish a modeling approach combining the strengths of logic-based network models and regularized regression models, apply this approach to predict mechanisms that mediate the differential effects of drugs on fibroblasts, revealing Src inhibition acting via PI3K as a potential therapy for cardiac fibrosis.

Significance

Cardiac fibrosis is a dysregulation of the normal wound healing response, resulting in excessive scarring and cardiac dysfunction. As cardiac fibroblasts primarily regulate this process, we explored how candidate anti-fibrotic drugs alter the fibroblast phenotype. We identify a set of 137 phenotypic features that change in response to drug treatments. Using a new computational modeling approach termed logic-based mechanistic machine learning, we predict how pirfenidone and Src inhibition affect the regulation of the phenotypic features actin filament assembly and actin-myosin stress fiber formation. We also show that inhibition of PI3K reduces actin-myosin stress fiber formation and procollagen I production in human cardiac fibroblasts, supporting a role for PI3K as a mechanism by which Src inhibition may suppress fibrosis.
CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment
Authors: Muthana Musleh M. , Du Xuexiang , Liu Mingyue , Wang Xu , Wu Wei , Ai Chunxia , Su Lishan , Zheng Pan , Liu Yang
Published: March 2023

Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here we probe the decline of B cells in human CTLA-4 knock-in mice by using anti-human CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.
Combining high-resolution imaging, deep learning, and dynamic modelling to separate disease and senescence in wheat canopies
Authors: Anderegg Jonas , Zenkl Radek , Walter Achim , Hund Andreas , McDonald Bruce A.
Published: March 2023

Maintenance of sufficient healthy green leaf area after anthesis is key to ensuring an adequate assimilate supply for grain filling. Tightly regulated age-related physiological senescence and various biotic and abiotic stressors drive overall greenness decay dynamics under field conditions. Besides direct effects on green leaf area in terms of leaf damage, stressors often anticipate or accelerate physiological senescence, which may multiply their negative impact on grain filling. Here, we present an image processing methodology that enables the monitoring of chlorosis and necrosis separately for ears and shoots (stems + leaves) based on deep learning models for semantic segmentation and color properties of vegetation. A vegetation segmentation model was trained using semi-synthetic training data generated using image composition and generative adversarial neural networks, which greatly reduced the risk of annotation uncertainties and annotation effort. Application of the models to image time-series revealed temporal patterns of greenness decay as well as the relative contributions of chlorosis and necrosis. Image-based estimation of greenness decay dynamics was highly correlated with scoring-based estimations (r ≈ 0.9). Contrasting patterns were observed for plots with different levels of foliar diseases, particularly septoria tritici blotch. Our results suggest that tracking the chlorotic and necrotic fractions separately may enable (i) a separate quantification of the contribution of biotic stress and physiological senescence on overall green leaf area dynamics and (ii) investigation of the elusive interaction between biotic stress and physiological senescence. The potentially high-throughput nature of our methodology paves the way to conducting genetic studies of disease resistance and tolerance.
Actin-microtubule crosstalk imparts stiffness to the contractile ring in fission yeast
Authors: Bellingham-Johnstun Kimberly , Tyree Zoe L. , Martinez-Baird Jessica , Thorn Annelise , Laplante Caroline
Published: March 2023

Actin-microtubule interactions are critical for cell division yet how these networks of polymers mutually influence their mechanical properties and functions in live cells remains unknown. In fission yeast, the post-anaphase array (PAA) of microtubules assembles in the plane of the contractile ring and its assembly relies on the Myp2p-dependent recruitment of Mto1p, a component of equatorial microtubule organizing centers (eMTOCs). The general organization of this array of microtubule and the impact on their physical attachment to the contractile ring remain unclear. We found that Myp2p facilitates the recruitment of Mto1p to the inner face of the contractile ring where the eMTOCs polymerize microtubules without their direct interaction. The PAA microtubules form a dynamic polygon of Ase1p crosslinked microtubules inside the contractile ring. The specific loss of PAA microtubules affects the mechanical properties of the contractile ring of actin by lowering its stiffness. This change in the mechanical properties of the ring has no measurable impact on cytokinesis or on the anchoring of the ring. Our work proposes that the PAA microtubules exploit the contractile ring for their assembly and function during cell division while the contractile ring may receive no benefit from these interactions.
Medium depth influences O2availability and metabolism in cultured RPE cells
Authors: Hass Daniel T. , Zhang Qitao , Autterson Gillian A. , Bryan Richard A. , Hurley James B. , Miller Jason ML.
Published: March 2023

Structured Abstract

Purpose

RPE oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O2availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism.

Methods

We altered the availability of O2to human primary RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCR), glucose consumption, lactate production,13C6-glucose flux, hypoxia inducible factor (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, trans-epithelial electrical resistance, cell morphology, and pigmentation.

Results

Medium volumes commonly employed during RPE culture limit diffusion of O2to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O2availability decrease acetyl-CoA utilization, increase glycolysis, and alter the size and number of intracellular lipid droplets under lipid-rich conditions.

Conclusions

Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology “under the hood”. As RPE-centric diseases like age-related macular degeneration (AMD) involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O2availability seen with smaller media volumes.
Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins
Authors: Schmidt Anja , Finegan Tara , Häring Matthias , Kong Deqing , Fletcher Alexander G , Alam Zuhayr , Grosshans Jörg , Wolf Fred , Peifer Mark
Published: March 2023

During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. Here we combine genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player,DrosophilaPolychaetoid, homolog of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways but combine to produce robust connections.
NMRQNet: a deep learning approach for automatic identification and quantification of metabolites using Nuclear Magnetic Resonance (NMR) in human plasma samples
Authors: Wang Wanli , Ma Li-Hua , Maletic-Savatic Mirjana , Liu Zhandong
Published: March 2023

Nuclear Magnetic Resonance is a powerful platform that reveals the metabolomics profiles within biofluids or tissues and contributes to personalized treatments in medical practice. However, data volume and complexity hinder the exploration of NMR spectra. Besides, the lack of fast and accurate computational tools that can handle the automatic identification and quantification of essential metabolites from NMR spectra also slows the wide application of these techniques in clinical. We present NMRQNet, a deep-learning-based pipeline for automatic identification and quantification of dominant metabolite candidates within human plasma samples. The estimated relative concentrations could be further applied in statistical analysis to extract the potential biomarkers. We evaluate our method on multiple plasma samples, including species from mice to humans, curated using three anticoagulants, covering healthy and patient conditions in neurological disorder disease, greatly expanding the metabolomics analytical space in plasma. NMRQNet accurately reconstructed the original spectra and obtained significantly better quantification results than the earlier computational methods. Besides, NMRQNet also proposed relevant metabolites biomarkers that could potentially explain the risk factors associated with the condition. NMRQNet, with improved prediction performance, highlights the limitations in the existing approaches and has shown strong application potential for future metabolomics disease studies using plasma samples.
Cat LCA-CRXmodel, homozygous for an antimorphic mutation has a unique phenotype
Authors: Occelli Laurence M. , Tran Nicholas M. , Chen Shiming , Petersen-Jones Simon M.
Published: March 2023

PURPOSE

Human mutations in theCRXtranscription factor are associated with dominant retinopathies often with more severe macular changes. TheCRX-mutant cat (Rdy-A182d2) is the only animal model with the equivalent of the critical retinal region for high acuity vision, the macula. Heterozygous cats (CRXRdy/+) have a severe phenotype modeling Leber congenital amaurosis. This study reports the distinct ocular phenotype of homozygous cats (CRXRdy/Rdy).

METHODS

Gene expression changes were assessed at both mRNA and protein levels. Changes in globe morphology and retinal structure were analyzed.

RESULTS

CRXRdy/Rdycats had high levels of mutantCRXmRNA and protein. The expression of photoreceptor target genes was severely impaired while there were variable effects on the expression of other transcription factors. The photoreceptor cells remained immature and failed to elaborate outer segments consistent with the lack of retinal function. The retinal layers displayed a progressive remodeling with cell loss but maintained overall retinal thickness due to gliosis. Rapid photoreceptor loss largely occurred in the macula-equivalent retinal region. The homozygous cats developed markedly increased ocular globe length.

CONCLUSIONS

The phenotype ofCRXRdy/Rdycats was more severe compared toCRXRdy/+cats by several metrics.

TRANSLATIONAL RELEVANCE

TheCRX-mutant cat is the only model forCRX-retinopathies with a macula-equivalent region. A prominent feature of theCRXRdy/Rdycat phenotype not detectable in homozygous mouse models, was the rapid degeneration of the macula-equivalent retinal region highlighting the value of this large animal model and its future importance in the testing of translational therapies aiming to restore vision.