bluuebunny/crossref_metadata_2025 huggingface dataset:
Return of intracranial beta oscillations and traveling waves with recovery from traumatic brain injury
Authors: Vaz Alex , Wathen Connor , Miranda Stephen , Thomas Rachel , Darlington Timothy , Jabarkheel Rashad , Tomlinson Samuel , Arena John , Bond Kamila , Salwi Sanjana , Ajmera Sonia , Bachschmid-Romano Ludovica , Gugger James , Sandsmark Danielle , Diaz-Arrastia Ramon , Schuster James , Ramayya Ashwin G. , Cajigas Iahn , Pesaran Bijan , Chen H. Isaac , Petrov Dmitriy
Published: July 2024

Traumatic brain injury (TBI) remains a pervasive clinical problem associated with significant morbidity and mortality. However, TBI remains clinically and biophysically ill-defined, and prognosis remains difficult even with the standardization of clinical guidelines and advent of multimodality monitoring. Here we leverage a unique data set from TBI patients implanted with either intracranial strip electrodes during craniotomy or quad-lumen intracranial bolts with depth electrodes as part of routine clinical practice. By extracting spectral profiles of this data, we found that the presence of narrow-band oscillatory activity in the beta band (12-30 Hz) closely corresponds with the neurological exam as quantified with the standard Glasgow Coma Scale (GCS). Further, beta oscillations were distributed over the cortical surface as traveling waves, and the evolution of these waves corresponded to recovery from coma, consistent with the putative role of waves in perception and cognitive activity. We consequently propose that beta oscillations and traveling waves are potential biomarkers of recovery from TBI. In a broader sense, our findings suggest that emergence from coma results from recovery of thalamo-cortical interactions that coordinate cortical beta rhythms.
Using deep learning predictions reveals a large number of register errors in PDB deposits
Authors: Rodríguez Filomeno Sánchez , Simpkin Adam J. , Chojnowski Grzegorz , Keegan Ronan M. , Rigden Daniel J.
Published: July 2024

The accuracy of the information in the Protein Data Bank (PDB) is of great importance for the myriad downstream applications that make use of protein structural information. Despite best efforts, the occasional introduction of errors is inevitable, especially where the experimental data are of limited resolution. We have previously established a novel protein structure validation approach based on spotting inconsistencies between the residue contacts and distances observed in a structural model and those computationally predicted by methods such as AlphaFold 2. It is particularly well-suited to the detection of register errors. Importantly, the new approach is orthogonal to traditional methods based on stereochemistry or map-model agreement, and is resolution-independent. Here we identify thousands of likely register errors by scanning 3-5Å resolution structures in the PDB. Unlike most methods, application of our approach yields suggested corrections to the register of affected regions which we show, even by limited implementation, lead to improved refinement statistics in the vast majority of cases. A few limitations and confounding factors such as fold-switching proteins are characterised, but we expect our approach to have broad application in spotting potential issues in current accessions and, through its implementation and distribution in CCP4, helping ensure the accuracy of future deposits.
A Burst-Dependent Algorithm for Neuromorphic On-Chip Learning of Spiking Neural Networks
Authors: Stuck Michael , Wang Xingyun , Naud Richard
Published: July 2024

The field of neuromorphic engineering addresses the high energy demands of neural networks through brain-inspired hardware for efficient neural network computing. For on-chip learning with spiking neural networks, neuromorphic hardware requires a local learning algorithm able to solve complex tasks. Approaches based on burst-dependent plasticity have been proposed to address this requirement, but their ability to learn complex tasks has remained unproven. Specifically, previous burst-dependent learning was demonstrated on a spiking version of the XOR problem using a network of thousands of neurons. Here, we extend burst-dependent learning, termed ‘Burstprop’, to address more complex tasks with hundreds of neurons. We evaluate Burstprop on a rate-encoded spiking version of the MNIST dataset, achieving low test classification errors, comparable to those obtained using backpropagation through time on the same architecture. Going further, we develop another burst-dependent algorithm based on the communication of two types of error-encoding events for the communication of positive and negative errors. We find that this new algorithm performs better on the image classification benchmark. We also tested our algorithms under various types of feedback connectivity, establishing that the capabilities of fixed random feedback connectivity is preserved in spiking neural networks. Lastly, we tested the robustness of the algorithm to weight discretization. Together, these results suggest that spiking Burstprop can scale to more complex learning tasks and can thus be considered for self-supervised algorithms while maintaining efficiency, potentially providing a viable method for learning with neuromorphic hardware.
Bone mineral density affects tumor growth by shaping microenvironmental heterogeneity
Authors: Whitman Matthew A. , Mantri Madhav , Spanos Emmanuel , Estroff Lara A. , De Vlaminck Iwijn , Fischbach Claudia
Published: July 2024

Breast cancer bone metastasis is the leading cause of mortality in patients with advanced breast cancer. Although decreased mineral density is a known risk factor for bone metastasis, the underlying mechanisms remain poorly understood because studying the isolated effect of bone mineral density on tumor heterogeneity is challenging with conventional approaches. Here, we investigate how bone mineral content affects tumor growth and microenvironmental complexityin vivoby combining single-cell RNA-sequencing with mineral-containing or mineral-free decellularized bone matrices. We discover that the absence of bone mineral significantly influences fibroblast and immune cell heterogeneity, promoting phenotypes that increase tumor growth and alter the response to injury or disease. Importantly, we observe that the stromal response to matrix mineral content depends on host immunocompetence and the murine tumor model used. Collectively, our findings suggest that bone mineral density affects tumor growth by altering microenvironmental complexity in an organism-dependent manner.
TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers
Authors: Kundu Manjari , Greer Yoshimi E. , Lobanov Alexei , Ridnour Lisa , Donahue Renee N. , Ng Yeap , Ratnayake Shashi , Voeller Donna , Weltz Sarah , Chen Qingrong , Lockett Stephen J. , Cam Maggie , Meerzaman Daoud , Wink David A. , Weigert Roberto , Lipkowitz Stanley
Published: July 2024

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils.In vivo, TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.
TheLYCOPENE EPSILON CYCLASEuntranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation
Authors: Alagoz Yagiz , Nayak Jwalit J. , Aryal Rishi , Watkins Jacinta L. , Holland Sophie , Tissue David T. , Pogson Barry J. , Cazzonelli Christopher I.
Published: July 2024

Metabolic feedback is proposed to modulate nuclear gene expression and carotenoid biosynthesis in plastids, however few mechanisms have been identified so far in plants. Utilising mutants, overexpression lines, and chemical inhibitors, we demonstrate that ArabidopsisLYCOPENE EPSILON CYCLASE(εLCY) mRNA levels correlate with changes in β-carotenoid accumulation. Transgenic seedlings harbouring theεLCY5’ leader sequence fused toFIREFLY LUCIFERASE(FiLUC) showed reporter responsiveness to metabolic feedback triggered by norflurazon or loss-of-function in the CAROTENOID ISOMERASE (CRTISO). TheεLCY5’UTR harboured three alternative transcription start sites (TSS). The most abundant -133bp sequence generated in dark and light grown seedlings harboured a 5’ conserved domain (CD) with otherBrassicaceaespecies and a viral internal ribosome entry site (IRES) proximal to the start codon.In silicomodelling predicted the 5’UTR formed two energetically separated RNA structural probabilities having a minimal free energy consistent with metabolite-binding RNA riboswitches that was distinguished by hairpin structures within the CD. Site-specific mutations were used to stabilize the 5’UTR into a single RNA shape definition having negligible separation between the mountain plot structure prediction curves and a distal terminator-like hairpin structure. Stabilizing the 5’UTR shape triggered the posttranscriptional repression of FiLUC activity enabled by the CaMV35S promoter in tobacco transient assays and stable transgenic Arabidopsis lines. The stabilised shape fragment became responsive to metabolic feedback induced by norflurazon and incrtisomutant etiolated and de-etiolated seedlings. TheεLCY5’UTR resembles a conformational RNA regulatory switch harbouring a posttranscriptional expression platform and aptamer domain responsive to carotenoid-mediated feedback signalling.
CGPA: multi-context insights from the cancer gene prognosis atlas
Authors: Cao Biwei , Yu Xiaoqing , Gonzalez Gullermo , Murthy Amith R , Li Tingyi , Shen Yuanyuan , Yao Sijie , Conejo-Garcia Jose R. , Jiang Peng , Wang Xuefeng
Published: July 2024

Cancer transcriptomic data are used extensively to interrogate the prognostic value of targeted genes, yet basic scientists and clinicians have predominantly relied on univariable survival analysis for this purpose. This method often fails to capture the full prognostic potential and contextual relevance of the genes under study, inadvertently omitting a group of genes we term univariable missed-opportunity prognostic (UMOP) genes. Recognizing the complexity of revealing multifaceted prognostic implications, especially when extending the analysis to include various covariates and thresholds, we present the Cancer Gene Prognosis Atlas (CGPA). This platform greatly enhances gene-centric biomarker research across cancer types by offering an interactive and user-friendly interface for highly customized, in-depth prognostic analysis. CGPA notably supports data-driven exploration of gene pairs and gene-hallmark relationships, elucidating key composite biological mechanisms like synthetic lethality and immunosuppression. It further expands its capabilities to assess multi-gene panels using both public and user-provided data, facilitating a seamless mechanism-to-machine analysis. Additionally, CGPA features a designated portal for discovering prognostic gene modules using curated cancer immunotherapy data. Ultimately, CGPA’s comprehensive, accessible tools allow cancer researchers, including those without statistical expertise, to precisely investigate the prognostic landscape of genes, customizing the model to fit specific research hypotheses and enhancing biomarker discovery and validation through a synergy of mechanistic and data-driven strategies.

Significance

CGPA is a streamlined, interactive platform for multi-context gene-centric prognostic analysis, simplifying biomarker discovery and validation in oncology for clinicians and basic scientists, and bridging a critical gap in translational cancer research.
Non-Negative Connectivity Causes Bow-Tie Architecture in Neural Circuits
Authors: Liu Zhaofan , Du CongCong , Wong-Lin KongFatt , Wang Da-Hui
Published: July 2024

Bow-tie or hourglass architecture is commonly found in biological neural networks. Recently, artificial neural networks with bow-tie architecture have been widely used in various machine-learning applications. However, it is unclear how bow-tie architecture in neural circuits can be formed. We address this by training multi-layer neural network models to perform classification tasks. We demonstrate that during network learning and structural changes, non-negative connections amplify error signals and quench neural activity particularly in the hidden layer, resulting in the emergence of the network’s bow-tie architecture. We further show that such architecture has low wiring cost, robust to network size, and generalizable to different discrimination tasks. Overall, our work suggests a possible mechanism for the emergence of bow-tie neural architecture and its functional advantages.
A tethering mechanism underlies Pin1-catalyzed prolinecis-transisomerization at a noncanonical site
Authors: Williams Christopher C. , Chuck Jonathan , Munoz-Tello Paola , Kojetin Douglas J.
Published: July 2024

The prolyl isomerase Pin1 catalyzes thecis-transisomerization of proline peptide bonds, a non-covalent post-translational modification that influences cellular and molecular processes, including protein-protein interactions. Pin1 is a two-domain enzyme containing a WW domain that recognizes phosphorylated serine/threonine-proline (pS/pT-P) canonical motifs and an enzymatic PPIase domain that catalyzes prolinecis-transisomerization of pS/pT-P motifs. Here, we show that Pin1 uses a tethering mechanism to bind and catalyze prolinecis-transisomerization of a noncanonical motif in the disordered N-terminal activation function-1 (AF-1) domain of the human nuclear receptor PPARγ. NMR reveals multiple Pin1 binding regions within the PPARγ AF-1, including a canonical motif that when phosphorylated by the kinase ERK2 (pS112-P113) binds the Pin1 WW domain with high affinity. NMR methods reveal that Pin1 also binds and acceleratescis-transisomerization of a noncanonical motif containing a tryptophan-proline motif (W39-P40) previously shown to be involved in an interdomain interaction with the C-terminal ligand-binding domain (LBD). Cellular transcription studies combined with mutagenesis and Pin1 inhibitor treatment reveal a functional role for Pin1-mediated acceleration ofcis-transisomerization of the W39-P40 motif. Our data inform a refined model of the Pin1 catalytic mechanism where the WW domain binds a canonical pS/T-P motif and tethers Pin1 to the target, which enables the PPIase domain to exert catalyticcis-transisomerization at a distal noncanonical site.

SIGNIFICANCE

Proline peptide bonds naturally occur incisconformations and isomerize totransconformations on exchange regimes on the order of seconds to minutes. Pin1, a prolyl isomerase, catalyzes the isomerization of proline peptide bonds that contain a specific phospho-motif—a phosphorylated serine or threonine followed by a proline (pS/pT-P)—allowing for switch-like effects on target protein structure and function. One protein substrate of Pin1 is the nuclear receptor peroxisome proliferator activated receptor gamma (PPARγ), which is shown here to undergo Pin1-catalyzed isomerization at a noncanonical proline distal to a canonical pS/pT-P binding site. These studies lay the foundation for understanding the role of Pin1 in mediating PPARγ-regulated transcription and expand understanding of Pin1-catalyzed enzymatic activities and functions.
Proteome-wide 4-hydroxy-2-nonenal signature of oxidative stress in the marine invasive tunicateBotryllus schlosseri
Authors: Kültz Dietmar , Gardell Alison M. , DeTomaso Anthony , Stoney Greg , Rinkevich Baruch , Qarri Andy , Hamar Jens
Published: July 2024

The colonial ascidianBoytryllus schlosseriis an invasive marine chordate that thrives under conditions of anthropogenic climate change. We show that the B. schlosseri expressed proteome contains unusually high levels of proteins that are adducted with 4-hydroxy-2-nonenal (HNE). HNE represents a prominent posttranslational modification resulting from oxidative stress. Although numerous studies have assessed oxidative stress in marine organisms HNE protein modification has not previously been determined in any marine species. LC/MS proteomics was used to identify 1052 HNE adducted proteins in B. schlosseri field and laboratory populations. Adducted amino acid residues were ascertained for 1849 modified sites, of which 1195 had a maximum amino acid localization score. Most HNE modifications were at less reactive lysines (rather than more reactive cysteines). HNE prevelance on most sites was high. These observations suggest that B. schlosseri experiences and tolerates high intracellular reactive oxygen species levels, resulting in substantial lipid peroxidation. HNE adducted B. schlosseri proteins show enrichment in mitochondrial, proteostasis, and cytoskeletal functions. Based on these results we propose that redox signaling contributes to regulating energy metabolism, the blastogenic cycle, oxidative burst defenses, and cytoskeleton dynamics during B. schlosseri development and physiology. A DIA assay library was constructed to quantify HNE adduction at 72 sites across 60 proteins that represent a holistic network of functionally discernable oxidative stress bioindicators. We conclude that the vast amount of HNE protein adduction in this circumpolar tunicate is indicative of high oxidative stress tolerance contributing to its range expansion into diverse environments.

NEW & NOTEWORTHY

Oxidative stress results from environmental challenges that increase in frequency and severity during the Anthropocene. Oxygen radical attack causes lipid peroxidation leading to HNE production. Proteome-wide HNE adduction is highly prevalent inBotryllus schlosseri, a widely distributed, highly invasive, and economically important biofouling ascidian and the first marine species to be analyzed for proteome HNE modification. HNE adduction of specific proteins physiologically sequesters reactive oxygen species, which enhances fitness and resilience during environmental change.
AI-driven Discovery of Morphomolecular Signatures in Toxicology
Authors: Jaume Guillaume , Peeters Thomas , Song Andrew H. , Pettit Rowland , Williamson Drew F. K. , Oldenburg Lukas , Vaidya Anurag , de Brot Simone , Chen Richard J. , Thiran Jean-Philippe , Le Long Phi , Gerber Georg , Mahmood Faisal
Published: July 2024

Early identification of drug toxicity is essential yet challenging in drug development. At the preclinical stage, toxicity is assessed with histopathological examination of tissue sections from animal models to detect morphological lesions. To complement this analysis, toxicogenomics is increasingly employed to understand the mechanism of action of the compound and ultimately identify lesion-specific safety biomarkers for whichin vitroassays can be designed. However, existing works that aim to identify morphological correlates of expression changes rely on qualitative or semi-quantitative morphological characterization and remain limited in scale or morphological diversity. Artificial intelligence (AI) offers a promising approach for quantitatively modeling this relationship at an unprecedented scale. Here, we introduce GEESE, an AI model designed to impute morphomolecular signatures in toxicology data. Our model was trained to predict 1,536 gene targets on a cohort of 8,231 hematoxylin and eosin-stained liver sections fromRattus norvegicusacross 127 preclinical toxicity studies. The model, evaluated on 2,002 tissue sections from 29 held-out studies, can yield pseudo-spatially resolved gene expression maps, which we correlate with six key drug-induced liver injuries (DILI). From the resulting 25 million lesion-expression pairs, we established quantitative relations between up and downregulated genes and lesions. Validation of these signatures against toxicogenomic databases, pathway enrichment analyses, and human hepatocyte cell lines asserted their relevance. Overall, our study introduces new methods for characterizing toxicity at an unprecedented scale and granularity, paving the way for AI-driven discovery of toxicity biomarkers.

Live demo:https://mahmoodlab.github.io/tox-discovery-ui/
A transposable element insertion inIAA16disrupts splicing and causes dicamba resistance inBassia scoparia
Authors: Montgomery Jacob S. , Soni Neeta , Marques Hill Sofia , Morran Sarah , Patterson Eric L. , Edwards Seth A. , Ratnayake Sandaruwan , Hung Yu-Hung , Pandesha Pratheek H. , Slotkin R. Keith , Napier Richard , Dayan Franck , Gaines Todd A.
Published: July 2024

A dicamba-resistant population of kochia (Bassia scoparia) identified in Colorado, USA in 2012 was used to generate a synthetic mapping population that segregated for dicamba resistance. Linkage mapping associating dicamba injury with genotype derived from restriction-site-associated DNA sequencing identified a single locus in the kochia genome associated with resistance on chromosome 4. A mutant version ofAuxin/Indole-3-Acetic Acid 16(AUX/IAA16; a gene previously implicated in dicamba resistance in kochia) was found near the middle of this locus in resistant plants. Long read sequencing of dicamba-resistant plants identified a recently inserted Ty1/Copia retrotransposon near the beginning of the second exon ofAUX/IAA16, leading to disruption of normal splicing. A molecular marker for this insertion allows for rapid detection of resistance. Stable transgenic lines ofArabidopsis thalianaectopically expressing the mutant and wildtype alleles ofAUX/IAA16were developed.Arabidopsis thalianaplants expressing the mutantAUX/IAA16allele grew shorter roots on control media. However, transgenic root growth was less inhibited on media containing either dicamba (5 μM) or IAA (0.5 μM) when compared to non-transgenic plants or those expressing the wildtype allele ofAUX/IAA16.In vitro assays indicate reduced binding affinity and more rapid dissociation of the mutant AUX/IAA with TIR1 in the presence of several auxins, and protein modeling suggests the substitution of the glycine residue in the degron domain of AUX/IAA16 is especially important for resistance. A fitness cost associated with the mutant allele ofAUX/IAA16has implications for resistance evolution and management of kochia populations with this resistance mechanism.

Significance

Auxin mimics are amongst the most important herbicides in modern agriculture. Evolution of weeds that are resistant to these herbicides threatens sustainable crop production. Understanding the basis of auxin herbicide resistance informs the development of improved weed control technologies. Additionally, auxin-resistant mutations and their pleotropic effects help us understand auxin perception and signalling. We describe a transposable element insertion within an herbicide target site gene that alters splicing and reduces synthetic and natural auxin perception.
Non-Invasive Quality Control of Organoid Cultures Using Mesofluidic CSTR Bioreactors and High-Content Imaging
Authors: Charles Seleipiri , Jackson-Holmes Emily , Sun Gongchen , Zhou Ying , Siciliano Benjamin , Niu Weibo , Han Haejun , Nikitina Arina , Kemp Melissa L. , Wen Zhexing , Lu Hang
Published: July 2024

Human brain organoids produce anatomically relevant cellular structures and recapitulate key aspects ofin vivobrain function, which holds great potential to model neurological diseases and screen therapeutics. However, the long growth time of 3D systems complicates the culturing of brain organoids and results in heterogeneity across samples hampering their applications. We developed an integrated platform to enable robust and long-term culturing of 3D brain organoids. We designed a mesofluidic bioreactor device based on a reaction-diffusion scaling theory, which achieves robust media exchange for sufficient nutrient delivery in long-term culture. We integrated this device with longitudinal tracking and machine learning-based classification tools to enable non-invasive quality control of live organoids. This integrated platform allows for sample pre-selection for downstream molecular analysis. Transcriptome analyses of organoids revealed that our mesofluidic bioreactor promoted organoid development while reducing cell death. Our platform thus offers a generalizable tool to establish reproducible culture standards for 3D cellular systems for a variety of applications beyond brain organoids.
Modelling alcohol consumption in rodents using two-bottle choice home cage drinking and optional lickometry-based microstructural analysis
Authors: Hou Shikun , Arce Soto Nathaly M , Glover Elizabeth J
Published: July 2024

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, for 24 hr/day with daily intake measured via change in bottle weight over the 24 hr period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lickometer systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD, has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs.

This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lickometer system that enables microstructural analysis of drinking behavior.
Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology
Authors: Lee Byungjin , Sunden Fanny , Miller Michael , Pak Bumshik , Krebber Anker , Lutz Stefan , Fordyce Polly Morrell
Published: July 2024

Engineered enzymes with enhanced or novel functions are specific catalysts with wide-ranging applications in industry and medicine. Here, we introduce Droplet Array Microfluidic Enzyme Kinetics (DA-MEK), a high-throughput enzyme screening platform that combines water-in-air droplet microarrays formed on patterned superhydrophilic/omniphobic surfaces with cell-free protein synthesis to enable cost-effective expression and quantitative kinetic characterization of enzyme variants. By printing DNA templates encoding enzyme variants onto hydrophilic spots, stamping slides to add cell-free expression mix, and imaging the resulting arrays, we demonstrate reproducible expression of hundreds of enzyme variants per slide. Subsequent stamping of fluorogenic substrates and time-lapse imaging allows determination of Michaelis-Menten parameters for each variant, with measured catalytic efficiencies spanning 5 orders of magnitude and agreeing well with values obtained via traditional microtiter plate assays. DA-MEK consumes orders of magnitude less reagents than plate-based assays while providing accurate and detailed kinetic information for both beneficial and deleterious mutations. In future work, we anticipate DA-MEK will provide a powerful and versatile platform to accelerate enzyme engineering and enable screening of large variant libraries under diverse conditions.
High microsatellite but no mitochondrial DNA variation in an invasive Japanese mainland population of the parasitoid waspMelittobia sosui
Authors: Abe Jun , Takahashi Jun-ichi , Tsuchida Koji
Published: July 2024

Theory predicts that bottleneck events reduce genetic diversity in invasive populations. The parasitoid waspMelittobia sosuiwas only identified in the subtropical area of Japanese south islands and Taiwan, but recently found also in the temperate area of the Japanese mainland. The species may expand their distribution northward due to factors such as recent global warming. We investigated population genetics in both the native and invasive areas using mitochondrial and nuclear microsatellite DNA. As expected, there was mitochondrial variation in the native area, but not in the invasive area, which only had one haplotype. However, the two areas had a similar level of microsatellite variation, with averagely 43% and 38% alleles uniquely found in the native and invasive populations, respectively. The difference of genetic variation between mitochondrial and microsatellite DNA in the invasive populations may be explained by the faster mutation rate of microsatellites, as well as the population structure ofMelittobia, in which subdivided small inbreeding lineages may facilitate the accumulation of mutations. The high proportion of private alleles suggests that the mainland population diverged from the native populations at least 100 years ago, ruling out the possibility that the mainland population was established recently. Instead, the present study suggests thatM. sosuimight have already existed in the mainland but with a low frequency, or the mainland population was derived from a third population which diverged from the native populations over 100 years ago.
Biology of healthy aging: Biological hallmarks of stress resistance-related and unrelated to longevity in humans
Authors: Badial Komalpreet , Lacayo Patricia , Murakami Shin
Published: July 2024

Stress resistance is tightly associated with longer and healthier lifespans in various model organisms, including nematodes, fruit flies, and mice. However, we lack a complete understanding of stress resistance in humans, and therefore, we investigated how stress resistance and longevity are interlinked in humans. Using more than 180 databases, we identified 541 human genes associated with stress resistance. The curated gene set is highly enriched with genes involved in cellular response to stress. The Reactome analysis identified 398 biological pathways, narrowed down to 172 pathways, using a medium threshold (p-value < 1 × 10−04). We further summarized into 14 pathway categories, e.g., cellular response to stimuli/stress, DNA repair, gene expression, and immune system. There were overlapping categories between stress resistance and longevity, including gene expression, signal transduction, immune system, and cellular responses to stimuli/stress. The categories include the PIP3-AKT-FOXO and mTOR pathways, known to specify lifespans in the model systems. They also include the accelerated aging syndrome genes (WRN and HGPS/LMNA), while the genes were also involved in non-overlapped categories. Notably, nuclear pore proteins are enriched among the stress resistance pathways and overlap with diverse metabolic pathways. This study suggests that stress resistance is closely linked with longevity pathways, but not entirely identical. While most longevity categories intersect with stress-resistance categories, some do not, particularly those related to cell proliferation and beta-cell development. We also note inconsistencies in pathway terminologies with aging hallmarks reported previously and propose them to be more unified and integral.
Temporal Transcriptomic Profiling of the DevelopingXenopus laevisEye
Authors: Hack Samantha J. , Petereit Juli , Tseng Kelly Ai-Sun
Published: July 2024

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA-sequencing of whole eyes fromXenopusat three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development includingsox2,pax6,nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and included regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.
Harnessing Dye-induced Photothermal Confinement in Lipid Membranes: A Path to NIR-modulated Artificial Synaptic Vesicles
Authors: Sarker Satya Ranjan , Yamazaki Takeru , Sou Keitaro , Takemura Ichiro , Kurita Yusuke , Nomura Kayoko , Ichimura Mari , Suzuki Takahito , Kai Ayumi , Araki Takumi , Hattori Shinnosuke , Furuyama Taniyuki , Chang Young-Tae , Kiya Taketoshi , Arai Satoshi
Published: July 2024

Optical heating coupled with near-infrared (NIR) light and photothermal materials enables thermal confinement within biospecimens, minimizing undesirable thermal damage. Here, we demonstrated that photothermally heating lipid bilayers embedded with a unique phthalocyanine dye (VPc) efficiently perturbs the bilayers, resulting in increased permeability. Notably, microscopic studies revealed that the mechanism causing changes in membrane permeability may not follow the conventional temperature-sensitive liposome model. Furthermore, the heat generated by NIR laser illumination rarely diffused into the surrounding environment, and the dye was located within the bilayers at the molecular level, where it effectively transferred heat to the lipid bilayer. We prepared VPc-embedded liposomes encapsulating acetylcholine (ACh) and demonstrated the NIR laser-triggered release of ACh, creating a concentration jump across a few cells or within a limited single cell region. This method induced Ca2+flux through ACh receptor stimulation in thermally delicate biospecimens such as C2C12 myotubes and theDrosophilabrain.
European Robin Cryptochrome-4a Associates with Lipid Bilayers in an Ordered Manner, Fulfilling a Molecular-Level Condition for Magnetoreception
Authors: Majewska Marta , Hanić Maja , Bartölke Rabea , Schmidt Jessica , Mouritsen Henrik , Koch Karl-Wilhelm , Solov’yov Ilia A. , Brand Izabella
Published: July 2024

Since the middle of the 20th century, long-distance avian migration has been known to rely partly on the geomagnetic field. However, the underlying sensory mechanism is still not fully understood. Cryptochrome 4a (ErCry4a), found in European Robin (Erithacus rubecula), a night-migratory songbird has been suggested to be a magnetic sensory molecule. It is sensitive to external magnetic fields via the so-called radical-pair mechanism. ErCry4a is primarily located in the outer segments of the double cone photoreceptor cells in the eye, which contain stacked and highly ordered membranes that could facilitate the anisotropic attachment of ErCry4a needed for magnetic compass sensing. Here, we investigate possible interactions of ErCry4a with a model membrane that mimics the lipid composition of outer segments of vertebrate photoreceptor cells by using experimental and computational approaches. Experimental results show that the attachment of ErCry4a to the membrane could be controlled by the physical state of lipid molecules (average area per lipid) in the outer leaflet of the lipid bilayer. Furthermore, polarization modulation infrared reflection absorption spectroscopy allowed us to determine the conformation, motional freedom, and average orientation of the α- helices in ErCry4a in a membrane-associated state. Atomistic molecular dynamics studies supported the experimental results. A ∼1000 kcal mol−1decrease in the interaction energy as a result of ErCry4a membrane binding was determined compared to cases where no protein binding to the membrane occurred. At the molecular level, the binding seems to involve negatively charged carboxylate groups of the phosphoserine lipids and the C-terminal residues of ErCry4a. Our study reveals a potential direct interaction of ErCry4a with the lipid membrane and discusses how this binding could be an essential step for ErCry4a to propagate a magnetic signal further and thus fulfill a role as a magnetoreceptor.
MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks
Authors: Torres Esteban Monica , Stewart Matthew J. , Bragginton Eilis , Jeanrenaud Alain , Smerdon Stephen J. , Stucki Manuel
Published: July 2024

Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.
Inseparable/IER3IP1are essential for cytokinesis inDrosophilaneuroblast and human cells
Authors: Kakade Aishwarya Arun , Gupta Sachin , Varghese Reshmi , Adicherla Harikrishna , Nagarkar-Jaiswal Sonal
Published: July 2024

To unveil the molecular players that maintain neural stem cell homeostasis, we conducted a genetic screen inDrosophilaand isolated an uncharacterized gene that we namedInseparable(Insep).Insepis theDrosophilahomologue of humanIER3IP1, a gene associated with Microcephaly, Epilepsy, and Neonatal Diabetes Syndrome (MEDS-1). We show thatInseploss leads to early larval lethality with small brains and these phenotypes can be rescued by expressing IER3IP1 indicating that their biological function is conserved through evolution. TheInsepdeficient neuroblasts fail to complete cytokinesis and show excessive accumulation of Rab11 vesicles in the cytoplasm. Similarly,IER3IP1depletion in human cells leads to cytokinesis failure and accumulation of Rab11 vesicles. Insep and IER3IP1 localize to Rab11 vesicles and interact with Rab11. The pathogenic mutations in IER3IP1 perturb its localization to Rab11 vesicles and interaction with Rab11. These results suggest that Insep and IER3IP1 work along with Rab11 and may regulate fusion of Rab11 vesicles to the advancing furrow during cytokinesis.
CTCF-DEPENDENT INSULATION OFHoxb13AND THE HETEROCHRONIC CONTROL OF TAIL LENGTH
Authors: Lopez-Delisle Lucille , Zakany Jozsef , Bochaton Célia , Osteil Pierre , Mayran Alexandre , Darbellay Fabrice , Mascrez Bénédicte , Rekaik Hocine , Duboule Denis
Published: July 2024

In mammals, tail length is controlled by several genetic determinants, amongst which Hox13 genes located at the posterior extremities of Hox clusters, whose main function are to terminate the extension of the body axis. In this view, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks all posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing an unusually high number of CTCF sites, suggesting it isolates Hoxb13 from the rest of the cluster, thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes, which were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant ES cells grown as pseudo-embryos in vitro, which allowed us to examine in details the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations are already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.
IGLoo: Profiling the Immunoglobulin Heavy chain locus in Lymphoblastoid Cell Lines with PacBio High-Fidelity Sequencing reads
Authors: Lin Mao-Jan , Langmead Ben , Safonova Yana
Published: July 2024

New high-quality human genome assemblies derived from lymphoblastoid cell lines (LCLs) provide reference genomes and pangenomes for genomics studies. However, the characteristics of LCLs pose technical challenges to profiling immunoglobulin (IG) genes. IG loci in LCLs contain a mixture of germline and somatically recombined haplotypes, making them difficult to genotype or assemble accurately. To address these challenges, we introduceIGLoo, a software tool that implements novel methods for analyzing sequence data and genome assemblies derived from LCLs.IGLoocharacterizes somatic V(D)J recombination events in the sequence data and identifies the breakpoints and missing IG genes in the LCL-based assemblies. Furthermore,IGLooimplements a novel reassembly framework to improve germline assembly quality by integrating information about somatic events and population structural variantions in the IG loci. We appliedIGLooto study the assemblies from the Human Pangenome Reference Consortium, providing new insights into the mechanisms, gene usage, and patterns of V(D)J recombination, causes of assembly fragmentation in the IG heavy chain (IGH) locus, and improved representation of the IGH assemblies.
Deep Learning-based Modeling for Preclinical Drug Safety Assessment
Authors: Jaume Guillaume , de Brot Simone , Song Andrew H. , Williamson Drew F. K. , Oldenburg Lukas , Zhang Andrew , Chen Richard J. , Asin Javier , Blatter Sohvi , Dettwiler Martina , Goepfert Christine , Grau-Roma Llorenç , Soto Sara , Keller Stefan M. , Rottenberg Sven , del-Pozo Jorge , Pettit Rowland , Le Long Phi , Mahmood Faisal
Published: July 2024

In drug development, assessing the toxicity of candidate compounds is crucial for successfully transitioning from preclinical research to early-stage clinical trials. Drug safety is typically assessed using animal models with a manual histopathological examination of tissue sections to characterize the dose-response relationship of the compound – a timeintensive process prone to inter-observer variability and predominantly involving tedious review of cases without abnormalities. Artificial intelligence (AI) methods in pathology hold promise to accelerate this assessment and enhance reproducibility and objectivity. Here, we introduce TRACE, a model designed for toxicologic liver histopathology assessment capable of tackling a range of diagnostic tasks across multiple scales, including situations where labeled data is limited. TRACE was trained on 15 million histopathology images extracted from 46,734 digitized tissue sections from 157 preclinical studies conducted onRattus norvegicus. We show that TRACE can perform various downstream toxicology tasks spanning histopathological response assessment, lesion severity scoring, morphological retrieval, and automatic dose-response characterization. In an independent reader study, TRACE was evaluated alongside ten board-certified veterinary pathologists and achieved higher concordance with the consensus opinion than the average of the pathologists. Our study represents a substantial leap over existing computational models in toxicology by offering the first framework for accelerating and automating toxicological pathology assessment, promoting significant progress with faster, more consistent, and reliable diagnostic processes.

Live Demo:https://mahmoodlab.github.io/tox-foundation-ui/