bluuebunny/crossref_metadata_2025 huggingface dataset:
Light-induced condensates show accumulation-prone and less dynamic properties in the nucleus compared to the cytoplasm
Authors: Hamada Yuta , Kitamura Akira
Published: June 2023

Biomolecular condensates, including membraneless organelles, are ubiquitously observed in subcellular compartments. However, the accumulation and dynamic properties of arbitrarily induced condensates remain elusive. Here, we show the size, amount, and dynamic properties of subcellular condensates using various fluorescence spectroscopic imaging analyses. Spatial image correlation spectroscopy showed that the size of blue-light-induced condensates of cryptochrome 2-derived oligomerization tag (CRY2olig) tagged with a red fluorescent protein in the nucleus was not different from that in the cytoplasm. Fluorescence intensity measurements showed that the condensates in the nucleus were more prone to accumulation than those in the cytoplasm. Single-particle tracking analysis showed that the condensates in the nucleus are predisposed to be stationary dynamics compared to those in the cytoplasm. Therefore, the subcellular compartment may, in part, affect the characteristics of self-recruitment of biomolecules in the condensates and their movement property.
Random effects adjustment in machine learning models for cardiac surgery risk prediction: a benchmarking study
Authors: Dong Tim , Sinha Shubhra , Fudulu Daniel P , Chan Jeremy , Zhai Ben , Narayan Pradeep Narayan , Caputo Massimo , Judge Andy , Dimagli Arnaldo , Benedetto Umberto , Angelini Gianni D.
Published: June 2023

Objectives

There is an ongoing debate over whether a procedural specific (e.g. Society of Thoracic Surgeons (STS)) or universal model (e.g. EuroSCORE II (ES II)) should be used for patient selection in cardiac surgery. Recently, we showed that ES II suffers from severe performance drift across several important metrics and that ML approaches such as Xgboost and Random Forest are substantially more resistant to dataset drift. With the growing interest in big data and its leverage through the use of ML approaches that are not limited by linear statistical assumptions, the number of clinical variables can theoretically increase exponentially. In addition, the variations and residual confounding that historically hindered the usefulness of cardiac risk stratification scores can potentially be taken into account. Here, we assess these possibilities on a large United Kingdom (UK) database.

Methods

A retrospective analysis of prospectively routinely gathered data on adult patients undergoing cardiac surgery in the UK between 2012-2019. We temporally split the data 70:30 into a training and validation subset. Two sets of seven ML mortality prediction models, with and without variable selection were assessed for consensus Clinical Effective Metric (CEM) overall performance and performance within each of CEM’s consistuent metrics. Confounding and potential causal relationships between covariates and outcomes were evaluated using bayesian network analysis.

Results

A total of 227,087 adults underwent cardiac surgery during the study period with a mortality rate of 2.76%. For non-variable selected (NVS) risk scores with 102 variables, Xgboost with adjustment for hospital variation was superior to the Xgboost without adjustment (p < 2e-16). Both NVS and the 18 variables selected (VS) Xgboost with adjustment for hospital variation risk scores were superior to the Xgboost (ES II 18 variables) model (p < 6.3e-15), with NVS Xgboost with adjustment for hospital variation having the best performance, followed by the VS Xgboost with adjustment for hospital variation (CEM Difference: 0.0150 and 0.0023, respectively).

Conclusions

We have identified an ML adjusted risk score comprising 102 variables that increases risk stratification performance on hold out dataset, removing the need to perform variable selection and reduction. This paves the way for further research that utilises this new set of variables with hospital-based adjustments for the safer selection of patients undergoing cardiac surgery.
Medical Devises Regulation in Zimbabwe: An Evaluation of operational readiness
Authors: Chiku Charles , Maruta Talkmore , Mbiba Fredrick , Manasa Justen
Published: June 2023

Background

Regulation of medical devices has seriously lagged, especially in Resource-Limited Settings (RLS). There are disparities in regulating medical devices; in the African region, it is below the global average. This may translate into poor access to quality-assured medical devices and result in undesirable health outcomes. Operational readiness to regulate medical devices in Zimbabwe at the Medicines Control Authority of Zimbabwe (MCAZ), the designated National Regulatory Authority (NRA), is vital for planning and implementation. The study aimed to assess the readiness of the MCAZ to regulate medical devices by identifying the strengths and gaps and proposing an institutional development plan that can be monitored and evaluated to assess progress over time.

Methods

Quantitative study was conducted using the World Health Organization (WHO) Global Benchmarking Tool+ medical devices (GBT+ medical devices) methodology to evaluate the medical devices regulatory oversight at the MCAZ. Data were collected between June and August 2022 using standard checklists to assess the quality of implementation of medical devices’ regulatory functions; National Regulatory System (RS), Registration and Market Authorisation (MA), Vigilance (VL), Market Surveillance and Control (MC), Licensing Establishment (LI), Regulatory Inspection (RI), Laboratory Testing(LT), and Clinical Trials (CTs) Oversight.

Results

The MCAZ attained maturity level 1, with an RS score of 79%, MA-44%, VL-27%, MC-40%, LI-62%, RI-68%, LT-88%, and CT-18%. Condoms and gloves were the only regulated medical devices. Indicators on legal provisions, regulations, and guidelines across the regulatory functions were below the optimum requirement.

Conclusion

The legal provisions, regulations, and guidelines are inadequate for effectively regulating medical devices. The medical devices regulation requires review for it to be robust and fit-for-purpose, responsive, oriented to the outcome, predictable based on a standard and transparent approach, and the level of scrutiny proportionate to the risk classification of the medical device.
Machine learning of dissection photographs and surface scanning for quantitative 3D neuropathology
Authors: Gazula Harshvardhan , Tregidgo Henry F. J. , Billot Benjamin , Balbastre Yael , William-Ramirez Jonathan , Herisse Rogeny , Deden-Binder Lucas J , Casamitjana Adrià , Melief Erica J. , Latimer Caitlin S. , Kilgore Mitchell D. , Montine Mark , Robinson Eleanor , Blackburn Emily , Marshall Michael S. , Connors Theresa R. , Oakley Derek H. , Frosch Matthew P. , Young Sean I. , Van Leemput Koen , Dalca Adrian V. , FIschl Bruce , Mac Donald Christine L. , Keene C. Dirk , Hyman Bradley T. , Iglesias Juan Eugenio
Published: June 2023

We present open-source tools for 3D analysis of photographs of dissected slices of human brains, which are routinely acquired in brain banks but seldom used for quantitative analysis. Our tools can:(i)3D reconstruct a volume from the photographs and, optionally, a surface scan; and(ii)produce a high-resolution 3D segmentation into 11 brain regions per hemisphere (22 in total), independently of the slice thickness. Our tools can be used as a substitute forex vivomagnetic resonance imaging (MRI), which requires access to an MRI scanner,ex vivoscanning expertise, and considerable financial resources. We tested our tools on synthetic and real data from two NIH Alzheimer’s Disease Research Centers. The results show that our methodology yields accurate 3D reconstructions, segmentations, and volumetric measurements that are highly correlated to those from MRI. Our method also detects expected differences betweenpost mortemconfirmed Alzheimer’s disease cases and controls. The tools are available in our widespread neuroimaging suite “FreeSurfer” (https://surfer.nmr.mgh.harvard.edu/fswiki/PhotoTools).
Ketamine rescues anhedonia by cell-type and input specific adaptations in the Nucleus Accumbens
Authors: Lucantonio Federica , Li Shuwen , Lu Jaden , Roeglin Jacob , Bontempi Leonardo , Shields Brenda C. , Zarate Carlos A. , Tadross Michael R. , Pignatelli Marco
Published: June 2023

Ketamine’s role in providing a rapid and sustained antidepressant response, particularly for patients unresponsive to conventional treatments, is increasingly recognized. A core symptom of depression, anhedonia, or the loss of enjoyment or interest in previously pleasurable activities, is known to be significantly alleviated by ketamine. While several hypotheses have been proposed regarding the mechanisms by which ketamine alleviates anhedonia, the specific circuits and synaptic changes responsible for its sustained therapeutic effects are not yet understood. Here, we show that the nucleus accumbens (NAc), a major hub of the reward circuitry, is essential for ketamine’s effect in rescuing anhedonia in mice subjected to chronic stress, a critical risk factor in the genesis of depression in humans. Specifically, a single exposure to ketamine rescues stress-induced decreased strength of excitatory synapses on NAc D1 dopamine receptor-expressing medium spiny neurons (D1-MSNs). By using a novel cell-specific pharmacology method, we demonstrate that this cell-type specific neuroadaptation is necessary for the sustained therapeutic effects of ketamine. To test for causal sufficiency, we artificially mimicked ketamine-induced increase in excitatory strength on D1-MSNs and found that this recapitulates the behavioral amelioration induced by ketamine. Finally, to determine the presynaptic origin of the relevant glutamatergic inputs for ketamine-elicited synaptic and behavioral effects, we used a combination of opto- and chemogenetics. We found that ketamine rescues stress-induced reduction in excitatory strength at medial prefrontal cortex and ventral hippocampus inputs to NAc D1-MSNs. Chemogenetically preventing ketamine-evoked plasticity at those unique inputs to the NAc reveals a ketamine-operated input-specific control of hedonic behavior. These results establish that ketamine rescues stress-induced anhedonia via cell-type-specific adaptations as well as information integration in the NAc via discrete excitatory synapses.
Formation of Müller glia-derived progenitor cells in retinas depleted of microglia
Authors: El-Hodiri Heithem M. , Bentley James , Reske Alana , Palazzo Isabella , Campbell Warren A. , Halloy Nicklaus R. , Fischer Andy J.
Published: June 2023

Recent studies have demonstrated the complex coordination of pro-inflammatory signaling and reactive microglia/macrophage on the formation Müller glial-derived progenitor cells (MGPCs) in the retinas of fish, birds and mice. We generated scRNA-seq libraries to identify transcriptional changes in Müller glia (MG) that result from the depletion of microglia from the chick retina. We found significant changes in different networks of genes in MG in normal and damaged retinas when the microglia are ablated. We identified a failure of MG to upregulate Wnt-ligands, Heparin binding epidermal growth factor (HBEGF), Fibroblast growth factor (FGF), retinoic acid receptors and genes related to Notch-signaling. Inhibition of GSK3β, to simulate Wnt-signaling, failed to rescue the deficit in formation of proliferating MGPCs in damaged retinas missing microglia. By comparison, application of HBEGF or FGF2 completely rescued the formation of proliferating MGPCs in microglia-depleted retinas. Similarly, injection of a small molecule inhibitor to Smad3 or agonist to retinoic acid receptors partially rescued the formation of proliferating MGPCs in microglia-depleted damaged retinas. According to scRNA-seq libraries, patterns of expression of ligands, receptors, signal transducers and/or processing enzymes to cell-signaling via HBEGF, FGF, retinoic acid and TGFβ are rapidly and transiently upregulated by MG after neuronal damage, consistent with important roles for these cell-signaling pathways in regulating the formation of MGPCs. We conclude that quiescent and activated microglia have a significant impact upon the transcriptomic profile of MG. We conclude that signals produced by reactive microglia in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming on MG into proliferating MGPCs.
A translational rodent model of individual differences in sensitivity to the aversive properties of ethanol
Authors: Przybysz Kathryn R , Ramirez Lindsey A , Pitock Joseph R , Starr E Margaret , Yang Hyerim , Glover Elizabeth J
Published: June 2023

Background

A strong relationship exists between individual sensitivity to the aversive properties of ethanol and risk for alcohol use disorder (AUD). Despite this, our understanding of the neurobiological mechanisms underlying subjective response to ethanol is relatively poor. A major contributor to this is the absence of preclinical models that enable exploration of this individual variability similar to studies performed in humans.

Methods

Adult male and female Long-Evans rats were trained to associate a novel tastant (saccharin) with acute exposure to either saline or ethanol (1.5 g/kg or 2.0 g/kg i.p.) over three conditioning days using a standard conditioned taste aversion (CTA) procedure. Variability in sensitivity to ethanol-induced CTA was phenotypically characterized using a median split across the populations studied.

Results

When examining group averages, both male and female rats that had saccharin paired with either dose of ethanol exhibited reduced saccharin intake relative to saline controls of ethanol-induced CTA. Examination of individual data revealed a bimodal distribution of responses uncovering two distinct phenotypes present in both sexes. CTA-sensitive rats exhibited a rapid and progressive reduction in saccharin intake with each successive ethanol pairing. In contrast, saccharin intake was unchanged or maintained after an initial decrease from baseline levels in CTA-resistant rats. While CTA magnitude was similar between male and female CTA-sensitive rats, CTA-resistant females were more resistant to the development of ethanol-induced CTA than their male counterparts. Phenotypic differences were not driven by differences in baseline saccharin intake. CTA sensitivity correlated with behavioral signs of intoxication in only a subset of rats.

Conclusions

These data parallel work in humans by revealing individual differences in sensitivity to the aversive properties of ethanol that emerge immediately after initial exposure to ethanol in both sexes. This model can be leveraged in future studies to investigate the neurobiological mechanisms that confer risk for AUD.
TDP-43-stratified single-cell proteomic profiling of postmortem human spinal motor neurons reveals protein dynamics in amyotrophic lateral sclerosis
Authors: Guise Amanda J. , Misal Santosh A. , Carson Richard , Boekweg Hannah , Van Der Watt Daisha , Truong Thy , Liang Yiran , Chu Jen-Hwa , Welsh Nora C. , Gagnon Jake , Payne Samuel H. , Plowey Edward D. , Kelly Ryan T.
Published: June 2023

Unbiased proteomics has been employed to interrogate central nervous system (CNS) tissues (brain, spinal cord) and fluid matrices (CSF, plasma) from amyotrophic lateral sclerosis (ALS) patients; yet, a limitation of conventional bulk tissue studies is that motor neuron (MN) proteome signals may be confounded by admixed non-MN proteins. Recent advances in trace sample proteomics have enabled quantitative protein abundance datasets from single human MNs (Cong et al., 2020b). In this study, we leveraged laser capture microdissection (LCM) and nanoPOTS (Zhu et al., 2018c) single-cell mass spectrometry (MS)-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control donor spinal cord tissues, leading to the identification of 2515 proteins across MNs samples (>900 per single MN) and quantitative comparison of 1870 proteins between disease groups. Furthermore, we studied the impact of enriching/stratifying MN proteome samples based on the presence and extent of immunoreactive, cytoplasmic TDP-43 inclusions, allowing identification of 3368 proteins across MNs samples and profiling of 2238 proteins across TDP-43 strata. We found extensive overlap in differential protein abundance profiles between MNs with or without obvious TDP-43 cytoplasmic inclusions that together point to early and sustained dysregulation of oxidative phosphorylation, mRNA splicing and translation, and retromer-mediated vesicular transport in ALS. Our data are the first unbiased quantification of single MN protein abundance changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein abundance changes in human neurologic diseases.
Condition-dependent fitness effects of large synthetic chromosome amplifications
Authors: Keller Abigail , Gao Lucy L. , Witten Daniela , Dunham Maitreya J.
Published: June 2023

Whole-chromosome aneuploidy and large segmental amplifications can have devastating effects in multicellular organisms, from developmental disorders and miscarriage to cancer. Aneuploidy in single-celled organisms such as yeast also results in proliferative defects and reduced viability. Yet, paradoxically, CNVs are routinely observed in laboratory evolution experiments with microbes grown in stressful conditions. The defects associated with aneuploidy are often attributed to the imbalance of many differentially expressed genes on the affected chromosomes, with many genes each contributing incremental effects. An alternate hypothesis is that a small number of individual genes are large effect ‘drivers’ of these fitness changes when present in an altered copy number. To test these two views, we have employed a collection of strains bearing large chromosomal amplifications that we previously assayed in nutrient-limited chemostat competitions. In this study, we focus on conditions known to be poorly tolerated by aneuploid yeast—high temperature, treatment with the Hsp90 inhibitor radicicol, and growth in extended stationary phase. To identify potential genes with a large impact on fitness, we fit a piecewise constant model to fitness data across chromosome arms, filtering breakpoints in this model by magnitude to focus on regions with a large impact on fitness in each condition. While fitness generally decreased as the length of the amplification increased, we were able to identify 91 candidate regions that disproportionately impacted fitness when amplified. Consistent with our previous work with this strain collection, nearly all candidate regions were condition specific, with only five regions impacting fitness in multiple conditions.
Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration
Authors: Lee Soah , Roest Alison S. Vander , Blair Cheavar A. , Kao Kerry , Bremner Samantha B. , Childers Matthew C , Pathak Divya , Heinrich Paul , Lee Daniel , Chirikian Orlando , Mohran Saffie , Roberts Brock , Smith Jacqueline E. , Jahng James W. , Paik David T. , Wu Joseph C. , Gunawardane Ruwanthi N. , Spudich James A. , Ruppel Kathleen , Mack David , Pruitt Beth L. , Regnier Michael , Wu Sean M. , Bernstein Daniel
Published: June 2023

Rationale

Over 200 mutations in the sarcomeric protein β-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation.

Objective

This study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions.

Methods

We developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered.

Results

At the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7WT/G256E) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM.

Conclusions

MYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.
An exploratory study of behavioral traits and the establishment of social relationships in female laboratory rats
Authors: Hakataya Shiomi , Katsu Noriko , Okanoya Kazuo , Toya Genta
Published: June 2023

There is growing evidence that social relationships influence individual fitness through various effects. Clarifying individual differences in social interaction patterns and determinants for such differences will lead to better understanding of sociality and its fitness consequences for animals. Behavioral traits are considered one of the determining factors of social interaction. The purpose of this study was to explore the effects of individual behavioral traits on social relationship building in rats, a highly social species. Initially, the following behavioral characteristics were measured in individuals: tameness (glove test), activity (open field test), exploration (novel object test), sociability (three-chamber test), and boldness (elevated plus maze test). We then used DeepLabCut to behaviorally track three groups of four individuals (12 total) and analyze social behaviors such as approach and avoidance behaviors. Principal component analysis based on behavioral test results detected behavioral traits interpreted as related to exploration, boldness, activity, and tameness, but not sociability. In addition, behavioral tracking results showed consistent individual differences in social behavior indices such as isolation time and partner preference. Furthermore, we found that different components were correlated with different phases of social behavior; exploration and boldness were associated with the early stages of group formation, whereas activity was associated with later stages of relationship building. From these results, we derived hypothesize that personality traits related to the physical and social environment have a larger influence in the relationship formation phase, and the behavioral trait of activity becomes important in the maintenance phase of relationships. Future studies should examine this hypothesis by testing larger group sizes and ensuring there is no bias introduced into group composition.
Scots pine – panmixia and the elusive signal of genetic adaptation
Authors: Bruxaux Jade , Zhao Wei , Hall David , Curtu Alexandru Lucian , Androsiuk Piotr , Drouzas Andreas D. , Gailing Oliver , Konrad Heino , Sullivan Alexis R. , Semerikov Vladimir , Wang Xiao-Ru
Published: June 2023

Scots pine is the foundation species of diverse forested ecosystems across Eurasia and displays remarkable ecological breadth, occurring in environments ranging from temperate rainforests to arid tundra margins. Such expansive distributions can be favored by various demographic and adaptive processes and the interactions between them.

To understand the impact of neutral and selective forces on genetic structure in Scots pine, we conducted range-wide population genetic analyses on 2,321 trees from 202 populations using genotyping-by-sequencing, reconstructed the recent demography of the species, and examined signals of genetic adaptation.

We found a high and uniform genetic diversity across the entire range (globalFST0.048), no increased genetic load in expending populations and minor impact of the last glacial maximum on historical population sizes. Genetic-environmental associations identified only a handful of SNPs significantly linked to environmental gradients.

The results suggest that extensive gene flow is predominantly responsible for the observed genetic patterns in Scots pine. The apparent missing signal of genetic adaptation is likely attributed to the intricate genetic architecture controlling adaptation to multi-dimensional environments. The panmixia metapopulation of Scots pine offers a good study system for further exploration into how genetic adaptation and plasticity evolve under gene flow and changing environment.
Spatiotemporal organization of prefrontal norepinephrine influences neuronal activity
Authors: Glaeser-Khan Samira , Savalia Neil K. , Cressy Jianna , Feng Jiesi , Li Yulong , Kwan Alex C. , Kaye Alfred P.
Published: June 2023

Norepinephrine (NE), a neuromodulator released by locus coeruleus neurons throughout cortex, influences arousal and learning through extra-synaptic vesicle exocytosis. While NE within cortical regions has been viewed as a homogenous field, recent studies have demonstrated heterogeneous axonal dynamics and advances in GPCR-based fluorescent sensors permit direct observation of the local dynamics of NE at cellular scale. To investigate how the spatiotemporal dynamics of NE release in the PFC affect neuronal firing, we employed in-vivo two-photon imaging of layer 2/3 of PFC in order to observe fine-scale neuronal calcium and NE dynamics concurrently. We found that local and global NE fields can decouple from one another, providing a substrate for local NE spatiotemporal activity patterns. Optic flow analysis revealed putative release and reuptake events which can occur at the same location, albeit at different times, indicating the potential to create a heterogeneous NE field. Utilizing generalized linear models, we demonstrated that cellular Ca2+ fluctuations are influenced by both the local and global NE field. However, during periods of local/global NE field decoupling, the local field drives cell firing dynamics rather than the global field. These findings underscore the significance of localized, phasic NE fluctuations for structuring cell firing, which may provide local neuromodulatory control of cortical activity.
On the Mathematics of RNA Velocity II: Algorithmic Aspects
Authors: Li Tiejun , Wang Yizhuo , Yang Guoguo , Zhou Peijie
Published: June 2023

In a previous paper [CSIAM Trans. Appl. Math. 2 (2021), 1-55], the authors proposed a theoretical framework for the analysis of RNA velocity, which is a promising concept in scRNA-seq data analysis to reveal the cell state-transition dynamical processes underlying snapshot data. The current paper is devoted to the algorithmic study of some key components in RNA velocity workflow. Four important points are addressed in this paper: (1) We construct a rational time-scale fixation method which can determine the global gene-shared latent time for cells. (2) We present an uncertainty quantification strategy for the inferred parameters obtained through the EM algorithm. (3) We establish the optimal criterion for the choice of velocity kernel bandwidth with respect to the sample size in the downstream analysis and discuss its implications. (4) We propose a temporal distance estimation approach between two cell clusters along the cellular development path. Some illustrative numerical tests are also carried out to verify our analysis. These results are intended to provide tools and insights in further development of RNA velocity type methods in the future.
Development of cell-free transcription-translation systems in three soil Pseudomonads
Authors: Meyerowitz Joseph T. , Larsson Elin M. , Murray Richard M.
Published: June 2023

In vitrotranscription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX-TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth:Pseudomonas synxantha, Pseudomonas chlororaphis, andPseudomonas aureofaciens. One,P. synxantha, was further characterized. A lysate test ofP. synxanthashowed a maximum protein yield of 2.5μM at 125 proteins per DNA template and a maximum protein synthesis rate of 20 nM/min. A set of different constitutive promoters driving mNeonGreen expression were tested in TX-TL and integrated into the genome, showing similar normalized strengths forin vivoandin vitrofluorescence. This correspondence between the TX-TL derived promoter strength and thein vivopromoter strength indicates these lysate-based cell-free systems can be used to characterize and engineer biological parts without genome integration, enabling a faster designbuild-test cycle.
Weak local adaptation to drought in seedlings of a widespread conifer
Authors: Candido-Ribeiro Rafael , Aitken Sally N.
Published: June 2023

There is an urgent need for better understanding how populations of trees will respond to predictable changes in climate and the intensification of extreme weather events such as droughts. The distribution of adaptive traits in seedlings is a crucial component of population adaptive potential and its characterization is important for development of management approaches mitigating the effects of climate change on forests. In this study, we used a large-scale common garden drought experiment to characterize the variation in drought tolerance, growth, and plastic responses to extreme summer drought in seedlings of 73 natural provenances of the two main varieties of Douglas-fir (Pseudotsuga menziesiivar.menziesiiand var.glauca), sampled across most of their extensive natural ranges. We detected large differences between the two Douglas-fir varieties for all traits assessed, with var.glaucashowing higher tolerance to drought but slower height growth and less plasticity than var.menziesii. Surprisingly, signals of local adaptation to drought within varieties were weak within var.glaucaand nearly absent within var.menziesii. Temperature-related variables were identified as the main climatic drivers of clinal variation in drought tolerance and height growth species-wide, and in height growth within var.menziesii. Furthermore, our data indicate that higher plasticity under extreme droughts could be maladaptive in var.menziesii. Overall, our study suggests that genetic variation for drought tolerance in seedlings is maintained primarily within rather than among provenances within varieties and does not compromise growth at early stages of plant development. Given these results, assisted gene flow is unlikely to help facilitate adaptation to drought within Douglas-fir varieties, but selective breeding within provenances could accelerate adaptation.
Acyl-AcpB, a FabT co-repressor inStreptococcus pyogenes
Authors: Lambert Clara , d’Orfani Alice , Gaillard Marine , Zhang Qiufen , Gloux Karine , Poyart Claire , Fouet Agnès
Published: June 2023

Membranes are a universal barrier to all cells. Phospholipids, essential bacterial membrane components, are composed of a polar head and apolar fatty acid (FA) chains. Most bacterial FA are synthesized by the FA synthesis pathway (FASII). In streptococcaceae, enterococci andLactococcus lactis, a unique feedback mechanism controls the FASII gene expression. FabT, encoded in the FASII main locus, is the repressor and it is activated by acyl-ACP. Many Streptococci,Enterococcus faecalis, but notL. lactis, possess two ACPS. AcpA encoding gene is within the FASII locus and is, to a certain extent, coregulated with the FASII genes. Acyl-AcpA is the end product of FASII. AcpB encoding gene is in operon withplsX. The role of AcpB as FabT corepressor is controversial.Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possesses AcpB. In this study, we show by comparing gene repression of FASII genes in wild-type,fabTmutant andacpBmutant strains grown in the presence and the absence of exogenous FAs, that AcpB isS. pyogenesFabT main co-repressor. Also,acpBdeletion impacts the membrane FA composition and adhesion to eucaryotic cells, highlighting the role of AcpB.
Robust Optogenetic Inhibition with Red-light-sensitive Anion-conducting Channelrhodopsins
Authors: Oppermann Johannes , Rozenberg Andrey , Fabrin Thomaz , González-Cabrera Cristian , Parker Rafael E. S. , Béjà Oded , Prigge Matthias , Hegemann Peter
Published: June 2023

Channelrhodopsins (ChRs) are light-gated ion channels widely used to optically activate or silence selected electrogenic cells, such as individual brain neurons. Here, we describe identifying and characterizing a set of anion-conducting ChRs (ACRs) from diverse taxa and representing various branches of the ChR phylogenetic tree. TheMantoniella squamataACR (MsACR1) showed high sensitivity to yellow-green light (λmaxat 555 nm) and was further engineered for optogenetic applications. A single amino-acid substitution that mimicked red-light sensitive rhodopsins like Chrimson shifted the photosensitivity 20 nm towards red light and accelerated photocurrent kinetics. Hence, it was named red and accelerated ACR, raACR. Both wild-type and mutant are capable optical silencers at low light intensities in mouse neuronsin vitroandin vivo,while raACR offers a higher temporal resolution.
Neuropathy target esterase activity predicts retinopathy amongPNPLA6disorders
Authors: Liu James , He Yi , Lwin Cara , Han Marina , Guan Bin , Naik Amelia , Bender Chelsea , Moore Nia , Huryn Laryssa A. , Sergeev Yuri , Qian Haohua , Zeng Yong , Dong Lijin , Liu Pinghu , Lei Jingqi , Haugen Carl J. , Prasov Lev , Shi Ruifang , Dollfus Hélène , Aristodemou Petros , Laich Yannik , Németh Andrea H. , Taylor John , Downes Susan , Krawczynski Maciej , Meunier Isabelle , Strassberg Melissa , Tenney Jessica , Gao Josephine , Shear Matthew A. , Moore Anthony T. , Duncan Jacque L. , Menendez Beatriz , Hull Sarah , Vincent Andrea , Siskind Carly E. , Traboulsi Elias I. , Blackstone Craig , Sisk Robert , Utz Virginia , Webster Andrew R. , Michaelides Michel , Arno Gavin , Synofzik Matthis , Hufnagel Robert B
Published: June 2023

Biallelic pathogenic variants in thePNPLA6gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism, and hair anomalies.PNPLA6encodes Neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a clinical meta-analysis of a novel cohort of 23 new patients along with 95 reported individuals withPNPLA6variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed acrossPNPLA6-associated clinical diagnoses unambiguously reclassified 10 variants as likely pathogenic and 36 variants as pathogenic, establishing a robust functional assay for classifyingPNPLA6variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus,PNPLA6disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship and the generation of a preclinical animal model pave the way for therapeutic trials, using NTE as a biomarker.
Structural basis of the interaction between ESV1 and LESV fromArabidopsis thalianawith starch glucans
Authors: Osman Rayan , Bossu Mélanie , Dauvillée David , Spriet Corentin , Liu Chun , Zeeman Samuel , D’Hulst Christophe , Bompard Coralie
Published: June 2023

Starch is the major energy storage compound in plants. Whether it is transient or stored, it is accumulated in the form of insoluble, semi-crystalline granules. The structure of these granules is related to the structure of the main component: amylopectin. Amylopectin consists of linear polymers of glucose units linked by α-1,4 bonds, forming double helices that combine to form the semi-crystalline lamellae of the granules, and α-1,6 branching points that form the amorphous lamellae. This particular structure of amylopectin is linked to the action of isoamylases, which cut the excess of branching points and allow the granules to be structured. For a long time, it was thought that the action of these enzymes was responsible for the structuring of starch granules. Recently, two new proteins, LESV and ESV1, have been characterized and are involved in the phase transition of amylopectin (LESV) or in the maintenance of the granule structure (ESV1). These proteins share a tryptophan-rich domain folded into an antiparallel β-sheet that is particularly well suited to bind amylopectin double helices. In this paper we present the structural study of these interactions using integrative structural biology approaches and show that LESV, in contrast to ESV1 can intervenes during amylopectin biosynthesis.
From gonadogenesis to testicular cancer: Unravelling the novel regulators and identification of drug candidates against FGF9 and PTGDS
Authors: Kumar Cash , Singh Vinay Kumar , Roy Jagat Kumar
Published: June 2023

Sex determination is the preliminary step toward gonadogenesis in mammals. Antagonistic interactions of key regulators have been only fragmentarily mentioned so far. Therefore, exploring regulators underlying the phenomena is required to solve questions, especially regarding female gonad development and gonadal disorders in congenital or adults. Inhibiting discrepancies in PPI pathways and combating related disorders are of urgent necessity, for which novel drugs are constantly required. Here, we performedin silicoanalysis using robust bioinformatics methods, which is unprecedented work in sex determination studies, providing large-scale analysis without exorbitant wet lab procedures. Analyzed regulators were overlapped with our RNA-seq data for authentication, to obtain differentially expressed elements. Additionally, CADD approach was used to discover inhibitors for FGF9 and PTGDS to search for potential drugs combating gonadal disorders in adults. Along with druggable properties, only FGF9 and PTGDS had full-length protein structures available, among 25 key genes under investigation. Our large-scale analysis of PPIN, produced highly interacting hub-bottleneck nodes as novel genes. Further, functional enrichment analysis revealed importance of these regulators in gonadogenesis. We identified sex-specific novel genes, miRNAs-target pairs, and lncRNAs-target pairs, which appear to play an important role in regulation of ovary development. CADD with molecular docking, MD simulations, and molecular mechanics confirmed stability of two novel compounds, DB12884 and DB12412 that could potentially inhibit FGF9 and PTGDS respectively. Taken together our study provides valuable information regarding involvement of crucial regulators in antagonistic mechanism of gonadogenesis and their related disorders, which will further assist in refining wet lab experiments.
The effects of data leakage on connectome-based machine learning models
Authors: Rosenblatt Matthew , Tejavibulya Link , Jiang Rongtao , Noble Stephanie , Scheinost Dustin
Published: June 2023

Predictive modeling has now become a central technique in neuroimaging to identify complex brain-behavior relationships and test their generalizability to unseen data. However, data leakage, which unintentionally breaches the separation between data used to train and test the model, undermines the validity of predictive models. Previous literature suggests that leakage is generally pervasive in machine learning, but few studies have empirically evaluated the effects of leakage in neuroimaging data. Although leakage is always an incorrect practice, understanding the effects of leakage on neuroimaging predictive models provides insight into the extent to which leakage may affect the literature. Here, we investigated the effects of leakage on machine learning models in two common neuroimaging modalities, functional and structural connectomes. Using over 400 different pipelines spanning four large datasets and three phenotypes, we evaluated five forms of leakage fitting into three broad categories: feature selection, covariate correction, and lack of independence between subjects. As expected, leakage via feature selection and repeated subjects drastically inflated prediction performance. Notably, other forms of leakage had only minor effects (e.g., leaky site correction) or even decreased prediction performance (e.g., leaky covariate regression). In some cases, leakage affected not only prediction performance, but also model coefficients, and thus neurobiological interpretations. Finally, we found that predictive models using small datasets were more sensitive to leakage. Overall, our results illustrate the variable effects of leakage on prediction pipelines and underscore the importance of avoiding data leakage to improve the validity and reproducibility of predictive modeling.
Similarity metric learning on perturbational datasets improves functional identification of perturbations
Authors: Smith Ian , Smirnov Petr , Haibe-Kains Benjamin
Published: June 2023

Analysis of high-throughput perturbational datasets, including the Next Generation Connectivity Map (L1000) and the Cell Painting projects, uses similarity metrics to identify perturbations or disease states that induce similar changes in the biological feature space. Similarities among perturbations are then used to identify drug mechanisms of action, to nominate therapeutics for a particular disease, and to construct bio-logical networks among perturbations and genes. Standard similarity metrics include correlations, cosine distance and gene set enrichment methods, but these methods operate on the measured features without refinement by transforming the measurement space. We introduce Perturbational Metric Learning (PeML), a weakly supervised similarity metric learning method to learn a data-driven similarity function that maximizes discrimination of replicate signatures by transforming the biological measurements into an intrinsic, dataset-specific basis. The learned similarity functions show substantial improvement for recovering known biological relationships, like mechanism of action identification. In addition to capturing a more meaningful notion of similarity, data in the transformed basis can be used for other analysis tasks, such as classification and clustering. Similarity metric learning is a powerful tool for the analysis of large biological datasets.
A non-oscillatory, millisecond-scale embedding of brain state provides insight into behavior
Authors: Parks David F. , Schneider Aidan M. , Xu Yifan , Brunwasser Samuel J. , Funderburk Samuel , Thurber Danilo , Blanche Tim , Dyer Eva L. , Haussler David , Hengen Keith B.
Published: June 2023

Sleep and wake are understood to be slow, long-lasting processes that span the entire brain. Brain states correlate with many neurophysiological changes, yet the most robust and reliable signature of state is enriched in rhythms between 0.1 and 20 Hz. The possibility that the fundamental unit of brain state could be a reliable structure at the scale of milliseconds and microns has not been addressed due to the physical limits associated with oscillation-based definitions. Here, by analyzing high resolution neural activity recorded in 10 anatomically and functionally diverse regions of the murine brain over 24 h, we reveal a mechanistically distinct embedding of state in the brain. Sleep and wake states can be accurately classified from on the order of 100to 101ms of neuronal activity sampled from 100 μm of brain tissue. In contrast to canonical rhythms, this embedding persists above 1,000 Hz. This high frequency embedding is robust to substates and rapid events such as sharp wave ripples and cortical ON/OFF states. To ascertain whether such fast and local structure is meaningful, we leveraged our observation that individual circuits intermittently switch states independently of the rest of the brain. Brief state discontinuities in subsets of circuits correspond with brief behavioral discontinuities during both sleep and wake. Our results suggest that the fundamental unit of state in the brain is consistent with the spatial and temporal scale of neuronal computation, and that this resolution can contribute to an understanding of cognition and behavior.
3D spheroid culturing ofAstyanax mexicanusliver-derived cell lines recapitulates distinct transcriptomic and metabolic states ofin vivotissue environment
Authors: Biswas Tathagata , Rajendran Naresh , Hassan Huzaifa , Zhao Chongbei , Rohner Nicolas
Published: June 2023

In vitroassays are crucial tools for gaining detailed insights into various biological processes, including metabolism. Cave morphs of the river-dwelling fish species,Astyanax mexicanus, have adapted their metabolism allowing them to thrive in the biodiversity-deprived and nutrient-limited environment of caves. Liver-derived cells from the cave and river morphs ofAstyanax mexicanushave proven to be excellentin vitroresources to better understand the unique metabolism of these fish. However, the current 2D cultures have not fully captured the complex metabolic profile of theAstyanaxliver. It is known that 3D culturing can modulate the transcriptomic state of cells when compared to its 2D monolayer culture. Therefore, in order to broaden the possibilities of thein vitrosystem by modeling a wider gamut of metabolic pathways, we cultured the liver-derivedAstyanaxcells of both surface and cavefish into 3D spheroids. We successfully established 3D cultures at various cell seeding densities for several weeks and characterized the resultant transcriptomic and metabolic variations. We found that the 3D culturedAstyanaxcells represent a wider range of metabolic pathways, including cell cycle changes and antioxidant activities, associated with liver functioning as compared to its monolayer culture. Additionally, the spheroids also exhibited surface and cave-specific metabolic signatures, making it a suitable system for evolutionary studies associated with cave adaptation. Taken together, the liver-derived spheroids prove to be a promisingin vitromodel for widening our understanding of metabolism inAstyanax mexicanusand of vertebrates in general.