bluuebunny/crossref_metadata_2025 huggingface dataset:
Portable X-ray Fluorescence for Measuring Lead in the Hair of Wild Mammals
Authors: Jones Evie M. , Bengsen Andrew J. , Specht Aaron J. , Koch Amelia J. , Hamede Rodrigo K. , Jones Menna E. , Hampton Jordan O.
Published: March 2023

Lead exposure threatens scavenging wildlife globally. For inexpensive estimation of lead concentration in bones from avian scavengers, portable X-ray fluorescence (XRF) devices have been trialed. However, portable XRF has not been validated for tissue lead measurement in non-human mammalian scavengers. We evaluated portable XRF for hair lead measurement in the endangered Tasmanian devil (Sarcophilus harrisii). We first analyzed large (∼1.0 g) hair samples from 39 deceased animals from southeastern Tasmania via portable XRF and then inductively-coupled plasma mass spectrometry (ICP-MS) (validation study). We then measured lead concentrations via portable XRF only in 61 small (∼0.1 g) hair samples from live devils from a plantation landscape (plantation study). Portable XRF measures of hair lead levels were positively correlated with ICP-MS values (R2= 0.61). In the validation study, 95% of sampled Tasmanian devils had relatively low lead levels (< 2 mg/kg), but the remaining two showed elevated lead levels (> 15 mg/kg). Mean lead levels in the plantation study and validation study were not significantly different. Our preliminary results suggest that portable XRF can provide a useful measure of differences in lead levels in wildife hair over a coarse scale. We provide recommendations for further research and refinement of this method.

Synopsis

Portable XRF can provide inexpensive and non-destructive analysis of environmental contaminants in wildlife. We present the first evaluation of portable XRF for analysis of lead contamination in mammalian scavenger hair.
Constitutively synergistic multiagent drug formulations targeting MERTK, FLT3, and BCL-2 for treatment of AML
Authors: Kelvin James M , Jain Juhi , Thapa Aashis , Qui Min , Birnbaum Lacey A , Moore Samuel G , Zecca Henry , Summers Ryan J , Costanza Emma , Uricoli Biaggio , Wang Xiaodong , Jui Nathan T , Fu Haian , Du Yuhong , DeRyckere Deborah , Graham Douglas K , Dreaden Erik C
Published: March 2023

Although high-dose, multi-agent chemotherapy has improved leukemia survival rates in recent years, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. Development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. To address this challenge, we developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML andMLL-rearranged precursor B-cell ALL (infant ALL). In a novel, high-throughput combination drug screen, the MERTK/FLT3 inhibitor MRX-2843 synergized with venetoclax and other BCL-2 family protein inhibitors to reduce AML cell densityin vitro. Neural network models based on drug exposure and target gene expression were used to identify a classifier predictive of drug synergy in AML. To maximize the therapeutic potential of these findings, we developed a combination monovalent liposomal drug formulation that maintains ratiometric drug synergy in cell-free assays and following intracellular delivery. The translational potential of these nanoscale drug formulations was confirmed in a genotypically diverse set of primary AML patient samples and both the magnitude and frequency of synergistic responses were not only maintained but were improved following drug formulation. Together, these findings demonstrate a systematic, generalizable approach to combination drug screening, formulation, and development that maximizes therapeutic potential, was effectively applied to develop a novel nanoscale combination therapy for treatment of AML, and could be extended to other drug combinations or diseases in the future.
LiverZap: A chemoptogenetic tool for global and locally restricted hepatocyte ablation to study cellular behaviours in liver regeneration
Authors: Ambrosio Elizabeth M. G. , Bailey Charlotte S. L. , Unterweger Iris A. , Christensen Jens B. , Bruchez Marcel P. , Lundegaard Pia R. , Ober Elke A.
Published: March 2023

The liver restores its mass and architecture after injury. Yet, investigating morphogenetic cell behaviours and signals that repair tissue architecture at high spatiotemporal resolution remains challenging. We developed LiverZap, a tuneable chemoptogenetic liver injury model in zebrafish. LiverZap employs formation of a binary FAP-TAP photosensitiser followed by brief near-infrared illumination inducing hepatocyte-specific death and recapitulating mammalian liver injury types. The tool enables local hepatocyte ablation and extendedliveimaging of regenerative cell behaviours, critical for studying cellular interactions at the interface of healthy and damaged tissue. Applying LiverZap, we show that targeted hepatocyte ablation in a small region-of-interest is unexpectedly sufficient to trigger Liver Progenitor-like Cell (LPC)-mediated regeneration, challenging the current understanding of LPC activation. Associated dynamic bilary network rearrangement and E-cadherin relocalisation suggest modulation of cell adhesion as an integral step of LPC-mediated liver regeneration. This precisely targetable live cell ablation model will enable addressing of key regeneration paradigms.
Rhythmic tapping to a moving beat: motion kinematics overrules motion naturalness
Authors: Pérez Oswaldo , Monache Sergio Delle , Lacquaniti Francesco , Bosco Gianfranco , Merchant Hugo
Published: March 2023

Beat induction is the cognitive ability that allow humans to listen to a regular pulse in music and move in synchrony with it. Although auditory rhythmic cues are known to induce more consistent synchronization than flashing visual metronomes, this asymmetry can be canceled out by visual moving metronomes. Here, we investigated whether the naturalness of the visual motion or its kinematics could provide a synchronization advantage over flashing metronomes. Subjects tap in sync with visual isochronous metronomes defined by vertically or horizontally accelerating and decelerating motion, either congruent or not with natural gravity, and then continue tapping with no metronome. We found that motion kinematics was the predominant factor determining rhythm synchronization, as accelerating moving metronomes in either cardinal direction produced more precise and predictive tapping than decelerating or flashing conditions. Notably, a Bayesian observer model revealed that error correction during tapping synchronization and regression towards the mean in accuracy during tapping continuation in the absence of external cues are optimal control strategies independently of the moving properties of the visual metronomes. Our results support the notion that accelerating visual metronomes convey a strong sense of beat as seen in the cueing movements of an orchestra director.
ChemEM: flexible docking of small molecules in Cryo-EM structures using difference maps
Authors: Sweeney Aaron , Mulvaney Thomas , Topf Maya
Published: March 2023

The rapid advancement of the “resolution revolution” has propelled cryo-electron microscopy (cryo-EM) to the forefront of structure-based drug discovery. However, the majority of cryo-EM structures are solved at medium resolution (3-4Å), an unexplored territory for small-molecule docking, due to difficulty in positioning ligands and the surrounding side-chains. Therefore, the development of software capable of reliably and automatically docking ligands into cryo-EM maps at such resolutions is of utmost importance. ChemEM is a novel method that employs cryo-EM data, difference mapping, and a physico-chemical scoring function to flexibly dock one or multiple ligands in a protein binding site. To validate its effectiveness, ChemEM was assessed using a highly curated benchmark containing 33 experimental cryo-EM structures, spanning a resolution range of 2.2-5.6 Å. In all but one case, the method placed the ligands in the density in an accurate conformation, often better than the PDB deposited solutions. Even without the use of cryo-EM density, the ChemEM scoring function outperformed the well-established docking software AutoDock Vina. Furthermore, the study demonstrates that useful information is present in the map even at low resolutions. ChemEM unlocks the potential of medium-resolution cryo-EM structures for drug discovery.
Global parameter optimisation and sensitivity analysis of antivenom pharmacokinetics and pharmacodynamics
Authors: Morris Natalie M , Blee Johanna A , Hauert Sabine
Published: March 2023

In recent years it has become possible to design snakebite antivenoms with diverse pharmacokinetic properties. Owing to the pharmacokinetic variability of venoms, the choice of antivenom scaffold may influence a treatment’s neutralisation coverage. Computation offers a useful medium through which to assess the pharmacokinetics and pharmacodynamics of envenomation-treatment systems, as antivenoms with identical neutralising capacities can be simulated. In this study, we simulate envenomation and treatment with a variety of antivenoms, to define the properties of effective antivenoms. Systemic envenomation and treatment were described using a two-compartment pharmacokinetic model. Treatment ofNaja sumatranaandCryptelytrops purpureomaculatusenvenomation was simulated with a set of 200,000 theoretical antivenoms across 10 treatment time delays. These two venoms are well-characterised and have differing pharmacokinetic properties. The theoretical antivenom set varied across molecular weight, dose, kon, koff, and valency. The best and worst treatments were identified using an area under the curve metric, and a global sensitivity analysis was performed to quantify the influence of the input parameters on treatment outcome. The simulations show that scaffolds of diverse molecular formats can be effective. Molecular weight and valency have a negligible direct impact on treatment outcome, however low molecular weight scaffolds offer more flexibility across the other design parameters, particularly when treatment is delayed. The simulations show konto primarily mediate treatment efficacy, with rates above 105M-1s-1required for the most effective treatments. koffhas the greatest impact on the performance of less effective scaffolds. While the same scaffold preferences for improved treatment are seen for both model snakes, the parameter bounds forC. purpureomaculatusenvenomation are more constrained. This paper establishes a computational framework for the optimisation of antivenom design.
Leukemia core transcriptional circuitry is a sparsely interconnected hierarchy stabilized by incoherent feed-forward loops
Authors: Harada Taku , Kalfon Jérémie , Perez Monika W. , Eagle Kenneth , Braes Flora Dievenich , Batley Rashad , Heshmati Yaser , Ferrucio Juliana Xavier , Ewers Jazmin , Mehta Stuti , Kossenkov Andrew , Ellegast Jana M. , Bowker Allyson , Wickramasinghe Jayamanna , Nabet Behnam , Paralkar Vikram R. , Dharia Neekesh V. , Stegmaier Kimberly , Orkin Stuart H. , Pimkin Maxim
Published: March 2023

Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators’ direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.
Developing QSAR Models for the Identification of Inhibitors TargetingMycobacterium tuberculosisEnoyl-ACP Reductase Enzyme
Authors: Karolczak Aureo André , Timmers Luis Fernando S. M. , Caceres Rafael Andrade
Published: March 2023

Tuberculosis is a global concern due to its high prevalence in developing countries and the ability of mycobacteria to develop resistance to current treatment regimens. In this project, we propose the use of QSAR (Quantitative Structure-Activity Relationships) modeling as a means to identify and evaluate the inhibitory activity of candidate molecules for molecular improvement stages and/or in vitro assays. This approach allows for in silico estimation, reducing research time and costs. To achieve this, we utilized the SAR (Structure-Activity Relationships) study conducted by He, Alian, and Montellano (2007), which focused on a series of arylamides tested as inhibitors of the enzyme enoyl-ACP-reductase (InhA) inMycobacterium tuberculosis. We developed both the Hansh-Fujita (classical) and CoMFA (Comparative Molecular Field Analysis) QSAR models. The classical QSAR model produced the most favorable statistical results using Multiple Linear Regression (MLR). It achieved an internal validation correlation factor R2of 0.9012 and demonstrated predictive quality with a Stone-Geisser indicator Q2of 0.8612. External validation resulted in a correlation factor R2of 0.9298 and Q2of 0.720, indicating a highly predictive mathematical model. The CoMFA Model obtained a Q2of 0.6520 in internal validation, enabling the estimation of energy fields around the molecules. This information is crucial for molecular improvement efforts. We constructed a library of small molecules, analogous to those used in the SAR study, and subjected them to the classic QSAR function. As a result, we identified ten molecules with high estimated biological activity. Molecular docking analysis suggests that these ten analogs, identified by the classical QSAR model, exhibit favorable estimated free energy of binding. In conclusion, the QSAR methodology proves to be an efficient and effective tool for searching and identifying promising drug-like molecules.
Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease
Authors: Sinclair Matt , Stein Richard A , Sheehan Jonathan H , Hawes Emily M , O’Brien Richard M , Tajkhorshid Emad , Claxton Derek P
Published: March 2023

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 cause glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robustin vitroscreening platform to define the atomic interactions governing G6P binding as well as explore the energetic perturbations imposed by disease-linked variants. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence elicits changes in G6P binding energy, thermostability and structural properties, suggesting multiple pathways of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic and non-catalytic side chains.
RSV infection does not induce EMT
Authors: Talukdar Sattya N. , McGregor Brett , Osan Jaspreet K. , Hur Junguk , Mehedi Masfique
Published: March 2023

Respiratory syncytial virus (RSV) infection does not cause severe disease in most of us despite suffering from multiple RSV infections in our lives. However, infants, young children, older adults, and immunocompromised patients are unfortunately vulnerable to RSV-associated severe diseases. A recent study suggested that RSV infection causes cell expansion, resulting in bronchial wall thickeningin vitro. Whether the virus-induced changes in the lung airway resemble epithelial-mesenchymal transition (EMT) is still unknown. Here, we report that RSV does not induce EMT in three differentin vitrolung models: the epithelial A549 cell line, primary normal human bronchial epithelial cells, and pseudostratified airway epithelium. We found that RSV increases the cell surface area and perimeter in the infected airway epithelium, which is distinct from the effects of a potent EMT inducer, TGF-β1-driven cell elongation—indicative of cell motility. A genome-wide transcriptome analysis revealed that both RSV and TGF-β1 have distinct modulation patterns of the transcriptome, which suggests that RSV-induced changes are distinct from EMT.
Hypertensive disorders of pregnancy and risk of cardiovascular disease: A Mendelian Randomisation study in the UK Biobank
Authors: Tschiderer Lena , van der Schouw Yvonne T , Burgess Stephen , Bloemenkamp Kitty WM , Seekircher Lisa , Willeit Peter , Onland-Moret N Charlotte , Peters Sanne AE
Published: March 2023

Background

Observational studies show that hypertensive disorders of pregnancy (HDPs) are related to unfavourable maternal cardiovascular disease (CVD) risk profiles later in life. We investigated whether genetic liability to pre-eclampsia/eclampsia and gestational hypertension is associated with CVD risk factors and occurrence of CVD events.

Methods

We obtained genetic associations with HDPs from a genome-wide association study and used individual-participant-data of women and men from the UK Biobank to obtain genetic associations with CVD risk factors and CVD events (defined as myocardial infarction or stroke). The rationale for including men and nulligravidae was to study genetic liability to HDPs and CVD risk without experiencing the underlying phenotype. We applied Mendelian Randomisation analysis using inverse variance weighted regression in our primary analysis.

Results

We included 264,160 women (mean age 56.4 [SD 8.0]) and 223,043 men (mean age 56.7 [8.2]) with available genetic data. Genetically proxied pre-eclampsia/eclampsia was related to higher risk of CVD with odds ratios of 1.20 (1.01, 1.43) and 1.29 (1.08, 1.53) in women and men, respectively. For genetically proxied gestational hypertension, odds ratios for CVD were 1.22 (1.10, 1.36) and 1.28 (1.16, 1.42) in women and men, respectively. Furthermore, genetically proxied HDPs were associated with higher levels of systolic and diastolic blood pressure and younger age at hypertension diagnosis in both women and men.

Conclusions

Genetic liability to HDPs including pre-eclampsia/eclampsia and gestational hypertension is associated with higher CVD risk, implying biological mechanisms relating to these disorders are causally related to CVD risk in both women and men.
Antimicrobial peptides modulate lung injury by altering the intestinal microbiota
Authors: Abdelgawad Ahmed , Nicola Teodora , Martin Isaac , Halloran Brian A. , Tanaka Kosuke , Adegboye Comfort Y. , Jain Pankaj , Ren Changchun , Lal Charitharth V. , Ambalavanan Namasivayam , O’Connell Amy E. , Jilling Tamás , Willis Kent A.
Published: March 2023

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair.

Graphical Abstract

In Brief

Using a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota.

Highlights

Supraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs).

Intestinal AMP expression has an inverse relationship with the severity of lung injury.

AMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury.

AMPs may mediate a gut-lung axis that modulates lung injury.
Structure-Based Neural Network Protein-Carbohydrate Interaction Predictions at the Residue Level
Authors: Canner Samuel W. , Shanker Sudhanshu , Gray Jeffrey J.
Published: March 2023

Carbohydrates dynamically and transiently interact with proteins for cell-cell recognition, cellular differentiation, immune response, and many other cellular processes. Despite the molecular importance of these interactions, there are currently few reliable computational tools to predict potential carbohydrate binding sites on any given protein. Here, we present two deep learning models named CArbohydrate-Protein interaction Site IdentiFier (CAPSIF) that predict carbohydrate binding sites on proteins: (1) a 3D-UNet voxel-based neural network model (CAPSIF:V) and (2) an equivariant graph neural network model (CAPSIF:G). While both models outperform previous surrogate methods used for carbohydrate binding site prediction, CAPSIF:V performs better than CAPSIF:G, achieving test Dice scores of 0.597 and 0.543 and test set Matthews correlation coefficients (MCCs) of 0.599 and 0.538, respectively. We further tested CAPSIF:V on AlphaFold2-predicted protein structures. CAPSIF:V performed equivalently on both experimentally determined structures and AlphaFold2 predicted structures. Finally, we demonstrate how CAPSIF models can be used in conjunction with local glycan-docking protocols, such as GlycanDock, to predict bound protein-carbohydrate structures.
A novel sustained release therapy of combined VEGF and TNF-α inhibitors leads to pan-ocular protection for months after severe ocular trauma
Authors: Zhou Chengxin , Lei Fengyang , Hui Pui-Chuen , Wolkow Natalie , Dohlman Claes H. , Vavvas Demetrios G. , Chodosh James , Paschalis Eleftherios I.
Published: March 2023

Purpose

To develop a clinically feasible and practical therapy for multi-ocular protection following ocular injury by using a thermosensitive drug delivery system (DDS) for sustained delivery of TNF-α and VEGF inhibitors to the eye.

Methods

A thermosensitive, biodegradable hydrogel DDS (PLGA-PEG-PLGA triblock polymer) loaded with 0.7mg of adalimumab and 1.4 mg of aflibercept was injected subconjunctivally in Dutch-belted pigmented rabbits after corneal alkali injury. The polymer was tuned to transition from liquid to gel upon contact with body temperature without need of a catalyst. Control rabbits received 2mg of IgG loaded DDS or 1.4mg aflibercept loaded DDS. Animals were followed for 3 months and assessed for tolerability and prevention of corneal neovascularization (NV), improvement of corneal re-epithelialization, inhibition of retinal ganglion cell (RGC) and optic nerve axon loss, and inhibition of immune cell infiltration into the cornea. Drug release kinetics was assessedin vivousing aqueous humor protein analysis.

Results

A single subconjunctival administration of dual anti-TNFα/anti-VEGF DDS achieved sustained 3-month delivery of antibodies to the anterior chamber, iris, ciliary body, and retina. Administration after corneal alkali burn suppressed CD45+immune cell infiltration into the cornea, completely inhibited cornea NV for 3 months, accelerated corneal re-epithelialization and wound healing, and prevented RGC and optic nerve axon loss at 3 months. In contrast, anti-VEGF alone or IgG DDS treatment led to persistent corneal epithelial defect, increased infiltration of CD45+immune cells into the cornea, and significant loss of RGCs and optic nerve axons at 3 months. Aqueous humor protein analysis showed first-order release kinetics without adverse effects at the injection site.

Conclusion

Sustained concomitant inhibition of TNF-α and VEGF using a biodegradable, slow-release thermosensitive DDS provides significant ocular protection and prevents corneal neovascularization and irreversible damage to retina and optic nerve after corneal alkali injury. This therapeutic approach has the potential to dramatically improve the outcomes of severe ocular injuries in patients.
Mitochondrial genome evolution in the Diprionidae: Major gene rearrangement in the basal Hymenoptera
Authors: Li Min , Niu Gengyun , Xu Min , Dai Mengxuan , Jiang Xinghong , Ma Yong , Meng Guanliang , Wei Meicai
Published: March 2023

In comparison to other non-parasitic basal lineages, Apocrita have consistently demonstrated a greatly accelerated rate of gene rearrangement. A number of mechanisms or correlates have been proposed for this observation, such as oxidative stress tolerated by exposure to the host immune system might lead to a high proportion of rearranged mt-genomes. Our studies reveal that gene rearrangements involving the protein-coding gene are present in the basal Hymenoptera lineage based on enriched sampling. We speculate the processes of diversification of rearrangements in the vicinity ofnad2involved tRNAs and NCRs by producing the chronogram of Diprionids. Furthermore, we investigated the relationship between rearranged genes and their nucleotide sequences. In conclusion, we demonstrate the great potential of gene order and associated sequence features as phylogenetic markers in the study of Hymenoptera evolution, offering a new perspective on studying organisms that undergo frequent gene rearrangements.
Mistakes in the re-analysis of lipidomic data obtained from a human model of resolving inflammation lead to erroneous conclusions
Authors: Dalli Jesmond
Published: March 2023

Recent years have seen an increased interest in the biology of specialized pro-resolving lipid mediators (SPM) with many investigators evaluating both their endogenous production as well as their biological and pharmacological properties. This increased interest has led to a rapid evolution in our understanding of both the biological and pharmacological activities of these mediators with their endogenous formation and biological activities being documented in a wide range of species that spans the evolutionary tree including fish, planaria and humans. Despite this plethora of evidence in a recent article Homer and colleagues claim that the reanalysis of a published dataset - partly originating from our laboratory - demonstrates that these molecules are absent in human blister exudates, contrary to the evidence published earlier. Based on their observations these Authors conclude that SPM are not relevant to human resolution biology. In this manuscript a reanalysed the dataset using unbiased methodologies and criteria that align with those recently proposed by the UK Consortium on Metabolic Phenotyping was performed together with the re-analysis of the dataset using criteria described by Homer and colleagues. Results from this re-analysis confirmed presence of SPM in human blister exudates and that the methodologies employed for quantitation of these molecules are robust. We also discuss how the results obtained in the article published by Homer and colleagues present several points of concern including the use of employ an arbitrary cut-off value to assign the noise for all the transitions used that does not take into consideration the fluctuation of the signal in each transition and therefore is not truly representative of the background signal. The use of different transitions to those employed in the original analyses and misreporting of findings based on the criteria employed. In conclusion the evidence presented herein demonstrates that correct application of rigorous criteria accepted by the community is essential in ensuring accurate identification of mediators and avoid blatant mistakes which can impact on the scientific development of the field.
Exposure to the persistent organic pollutant 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) disrupts development of the zebrafish inner ear
Authors: Cintrón-Rivera Layra G. , Oulette Gabrielle , Prakki Aishwarya , Burns Nicole M. , Patel Ratna , Cyr Rachel , Plavicki Jessica
Published: March 2023

Dioxins are a class of highly toxic and persistent environmental pollutants that have been shown through epidemiological and laboratory-based studies to act as developmental teratogens. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin congener, has a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. TCDD-induced AHR activation during development impairs nervous system, cardiac, and craniofacial development. Despite the robust phenotypes previously reported, the characterization of developmental malformations and our understanding of the molecular targets mediating TCDD-induced developmental toxicity remains limited. In zebrafish, TCDD-induced craniofacial malformations are produced, in part, by the downregulation ofSRY-box transcription factor 9b(sox9b), a member of the SoxE gene family.sox9b, along with fellow SoxE gene family memberssox9aandsox10, have important functions in the development of the otic placode, the otic vesicle, and, ultimately, the inner ear. Given thatsox9bin a known target of TCDD and that transcriptional interactions exist among SoxE genes, we asked whether TCDD exposure impaired the development of the zebrafish auditory system, specifically the otic vesicle, which gives rise to the sensory components of the inner ear. Using immunohistochemistry,in vivoconfocal imaging, and time-lapse microscopy, we assessed the impact of TCDD exposure on zebrafish otic vesicle development. We found exposure resulted in structural deficits, including incomplete pillar fusion and altered pillar topography, leading to defective semicircular canal development. The observed structural deficits were accompanied by reduced collagen type II expression in the ear. Together, our findings reveal the otic vesicle as a novel target of TCDD-induced toxicity, suggest that the function of multiple SoxE genes may be affected by TCDD exposure, and provide insight into how environmental contaminants contribute to congenital malformations.

Highlights

The zebrafish ear is necessary to detect changes in motion, sound, and gravity.

Embryos exposed to TCDD lack structural components of the developing ear.

TCDD exposure impairs formation of the fusion plate and alters pillar topography.

The semicircular canals of the ear are required to detect changes in movement.

Following TCDD exposure embryos fail to establish semicircular canals.
PhAT: A flexible open-source GUI-driven toolkit for photometry analysis
Authors: Murphy Kathleen Z. , Haile Eyobel , McTigue Anna , Pierce Anne F. , Donaldson Zoe R.
Published: March 2023

Photometry approaches detect sensor-mediated changes in fluorescence as a proxy for rapid molecular changes within the brain. As a flexible technique with a relatively low cost to implement, photometry is rapidly being incorporated into neuroscience laboratories. While multiple data acquisition systems for photometry now exist, robust analytical pipelines for the resulting data remain limited. Here we present thePhotometryAnalysisToolkit (PhAT) - a free open source analysis pipeline that provides options for signal normalization, incorporation of multiple data streams to align photometry data with behavior and other events, calculation of event-related changes in fluorescence, and comparison of similarity across fluorescent traces. A graphical user interface (GUI) enables use of this software without prior coding knowledge. In addition to providing foundational analytical tools, PhAT is designed to readily incorporate community-driven development of new modules for more bespoke analyses, and data can be easily exported to enable subsequent statistical testing and/or code-based analyses. In addition, we provide recommendations regarding technical aspects of photometry experiments including sensor selection and validation, reference signal considerations, and best practices for experimental design and data collection. We hope that the distribution of this software and protocol will lower the barrier to entry for new photometry users and improve the quality of collected data, increasing transparency and reproducibility in photometry analyses.

Basic Protocol 1: Software Environment Installation

Basic Protocol 2: GUI-driven Fiber Photometry Analysis

Basic Protocol 3: Adding Modules
Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency
Authors: Chanou Anna , Weiβ Matthias , Holler Karoline , Straub Tobias , Krietsch Jana , Sanchi Andrea , Ummethum Henning , Lee Clare S. K. , Kruse Elisabeth , Trauner Manuel , Werner Marcel , Lalonde Maxime , Lopes Massimo , Scialdone Antonio , Hamperl Stephan
Published: March 2023

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developedMethylationAccessibility ofTArgetedChromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of specific genomic loci after targeted purification in their native chromatin context. Applying MATAC-Seq to selected early-efficient (EE) and late-inefficient (LI) budding yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an optimal 100-115bp nucleosome-free region in combination with surrounding well-positioned nucleosomes and open +2 linker region is a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during DNA replication of eukaryotic cells. Consequently, our single-molecule assay for chromatin accessibility will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repairin vitroandex vivo.
Congruent downy mildew-associated microbiomes reduce plant disease and function as transferable resistobiomes
Authors: Goossens P. , Spooren J. , Baremans K.C.M. , Andel A. , Lapin D. , Echobardo N. , Pieterse C.M.J. , Van den Ackerveken G. , Berendsen R.L.
Published: March 2023

Root-associated microbiota can protect plants against severe disease outbreaks. In the model-plantArabidopsis thaliana, leaf infection with the obligate downy mildew pathogenHyaloperonospora arabidopsidis(Hpa) results in a shift in the root exudation profile, therewith promoting the growth of a selective root microbiome that induces a systemic resistance againstHpain the above-ground plant parts. Here we show that, additionally, a conserved subcommunity of the recruited soil microbiota becomes part of a pathogen-associated microbiome in the phyllosphere that is vertically transmitted with the spores of the pathogen to consecutively infected host plants. This subcommunity ofHpa-associated microbiota (HAM) limits pathogen infection and is therefore coined a “resistobiome”. The HAM resistobiome consists of a small number of bacterial species and was first found in our routinely maintained laboratory cultures of independentHpastrains. When co-inoculated withHpaspores, the HAM rapidly dominates the phyllosphere of infected plants, negatively impactingHpaspore formation. Remarkably, isogenic bacterial isolates of the abundantly-present HAM species were also found in strictly separatedHpacultures across Europe, and even in early published genomes of this obligate biotroph. Our results highlight that pathogen-infected plants can recruit protective microbiota via their roots to the shoots where they become part of a pathogen-associated resistobiome that helps the plant to fight pathogen infection. Understanding the mechanisms by which pathogen-associated resistobiomes are formed will enable the development of microbiome-assisted crop varieties that rely less on chemical crop protection.
Generalising the HCP multimodal cortical parcellation to UK Biobank
Authors: Williams Logan Z. J. , Glasser Matthew F. , Alfaro-Almagro Fidel , Dahan Simon , Fawaz Abdulah , Coalson Timothy S. , Fitzgibbon Sean P. , Suliman Mohamed A. , Van Essen David C. , Smith Stephen M. , Edwards A. David , Robinson Emma C.
Published: March 2023

The Human Connectome Project Multimodal Parcellation (HCP_MMP1.0) provides a robustin vivomap of the cerebral cortex, which demonstrates variability in structure and function that cannot be captured through diffeomorphic image registration alone. The HCP successfully employed a fully-connected neural network architecture to capture this variation, however it is unclear whether this approach generalises to other datasets with less rich imaging protocols. In this paper we propose and validate a novel geometric deep learning framework for generating individualised HCP_MMP1.0 parcellations in UK Biobank (UKB), an extremely rich resource that has led to numerous breakthroughs in neuroscience. To address substantial differences in image acquisition (for example, 6 minutes of resting-state fMRI per subject for UKB vs. 60 minutes per subject for HCP), we introduce a multi-step learning procedure including pretraining with a convolutional autoencoder. Compared to a fully-connected baseline, our proposed framework improved average detection rate across all areas by 10.4% (99.1% vs 88.7%), and detection of the worst performing area by 51.0% (80.9% vs. 29.9%). Importantly, this was not a result of the framework predicting one consistent parcellation across subjects, as visual inspection indicated that our method was sensitive to atypical cortical topographies. Code and trained models will be made available athttps://www.github.com.
What is hidden in the darkness? Deep-learning assisted large-scale protein family curation uncovers novel protein families and folds
Authors: Durairaj Janani , Waterhouse Andrew M. , Mets Toomas , Brodiazhenko Tetiana , Abdullah Minhal , Studer Gabriel , Akdel Mehmet , Andreeva Antonina , Bateman Alex , Tenson Tanel , Hauryliuk Vasili , Schwede Torsten , Pereira Joana
Published: March 2023

Driven by the development and upscaling of fast genome sequencing and assembly pipelines, the number of protein-coding sequences deposited in public protein sequence databases is increasing exponentially. Recently, the dramatic success of deep learning-based approaches applied to protein structure prediction has done the same for protein structures. We are now entering a new era in protein sequence and structure annotation, with hundreds of millions of predicted protein structures made available through the AlphaFold database. These models cover most of the catalogued natural proteins, including those difficult to annotate for function or putative biological role based on standard, homology-based approaches. In this work, we quantified how much of such “dark matter” of the natural protein universe was structurally illuminated by AlphaFold2 and modelled this diversity as an interactive sequence similarity network that can be navigated athttps://uniprot3d.org/atlas/AFDB90v4. In the process, we discovered multiple novel protein families by searching for novelties from sequence, structure, and semantic perspectives. We added a number of them to Pfam, and experimentally demonstrate that one of these belongs to a novel superfamily of translation-targeting toxin-antitoxin systems, TumE-TumA. This work highlights the role of large-scale, evolution-driven protein comparison efforts in combination with structural similarities, genomic context conservation, and deep-learning based function prediction tools for the identification of novel protein families, aiding not only annotation and classification efforts but also the curation and prioritisation of target proteins for experimental characterisation.
Technical and biological sources of unreliability of Infinium probes on Illumina Methylation microarrays
Authors: Nazarenko Tatiana , Vavourakis Charlotte D. , Jones Allison , Evans Iona , Schreiberhuber Lena , Kastner Christine , Ishaq-Parveen Isma , Redl Elisa , Watson Antony W. , Brandt Kirsten , Carter Clive , Zaikin Alexey , Herzog Chiara , Widschwendter Martin
Published: March 2023

The Illumina Methylation array platform has facilitated countless epigenetic studies on DNA methylation (DNAme) in health and disease, yet relatively few studies have so studied its reliability, i.e., the consistency of repeated measures. Here we focus on the reliability of both type I and type II Infinium probes. We propose a method for excluding unreliable probes based on dynamic thresholds for mean intensity (MI) and ‘unreliability’, estimated by probe-level simulation of the influence of technical noise on methylation β-values using the background intensities of negative control probes. We validate our method in several datasets, including Illumina MethylationEPIC BeadChip v1.0 data from paired whole blood samples taken six weeks apart. Our analysis revealed that specifically probes with low MI exhibit higher β-value variability between repeated samples. MI was associated with the number of C-bases in the respective probe sequence and correlated negatively with unreliability scores. The unreliability scores were substantiated through validation in a new EPIC v1.0 (blood and cervix) and a publicly available 450k (blood) dataset, as they effectively captured the variability observed in β-values between technical replicates. Finally, despite promising higher robustness, the newer version v2.0 of the MethylationEPIC BeadChip retained a substantial number of probes with poor unreliability scores. To enhance current pre-processing pipelines, we developed an R package to calculate MI and unreliability scores and provide guidance on establishing optimal dynamic score thresholds for a given data set.
Behavioral, molecular and neuronal mechanisms involved in recognition memory retrieval under degraded spatial cues in the rat hippocampus
Authors: Miranda Magdalena , Silva Azul , Morici Juan Facundo , Coletti Marcos Antonio , Belluscio Mariano , Bekinschtein Pedro
Published: March 2023

In a constantly changing environment, organisms face the challenge of adapting their behavior by retrieving previous experiences or acquiring new information. Previous research has postulated that this balance between memory generalization and differentiation manifests in a dichotomic manner. When environmental information exceeds a given threshold, activation of a stored representation could initiate retrieval, but below this threshold, a novel event could be encoded with a concomitant remapping of the internal representation in the hippocampus. Here, we examined the hippocampal molecular and neuronal mechanisms underlying retrieval in a cue-degraded environment by combining in vivo electrophysiological recordings and pharmacological manipulations. We developed a memory recognition task that allows a graded decrease in the contextual cues present during retrieval. We found that the manipulation of the number of visual cues was consistent with the activation or not of the contextual memory trace. Retrieval of a specific context memory was reflected by the level of CA3 remapping, demonstrating a clear relationship between remapping and contextual recognition. Also, manipulation of NMDAR activity in the DG-CA3 circuit bidirectionally modulated contextual memory retrieval. The blockade of NMDAR in CA3 impaired recognition in a cue-degraded, but not in a full-cue context, while their activation has the opposite effect. Conversely, blockade of NMDAR in the DG promoted retrieval under an even more cue-degraded environment, while activation had the opposite effect. Our results provide evidence for a flexible interaction between environmental cues and information stored in the hippocampus and give new insights into the biological mechanisms that balance memory encoding and retrieval.
State-dependent GABAergic regulation of striatal spiny projection neuron excitability
Authors: Day Michelle , Belal Marziyeh , Surmeier William C. , Melendez Alexandra , Wokosin David , Tkatch Tatiana , Clarke Vernon R. J. , Surmeier D. James
Published: March 2023

Synaptic transmission mediated by GABAAreceptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouseex vivobrain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.