bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual long-axis reorganization of hippocampus in youth
Authors: Zeng Debin , Li Qiongling , Li Deyu , He Yirong , Dong Xiaoxi , Li Shaoxian , Bi Shenghan , Liao Xuhong , Zhao Tengda , Chen Xiaodan , Xia Yunman , Lei Tianyuan , Sun Lianglong , Men Weiwei , Wang Yanpei , Wang Daoyang , Hu Mingming , Pan Zhiying , Tan Shuping , Gao Jia-Hong , Qin Shaozheng , Tao Sha , Dong Qi , He Yong , Zuo Xi-Nian , Li Shuyu
Published: November 2023

The reorganization of human hippocampus, especially its interaction with cortex, remains largely undefined in youth. The organization of a single hippocampal long-axis has been predominantly characterized as monotonic1–6, despite recent indications of nonmonotonic features in neuron density7and geometric eigenmodes8. While the human cortical hierarchy has been well recognized for significant developmental and evolutionary advantages9–12, hippocampus has been typically considered an evolutionarily conserved brain structure1,13,14, and overlooked regarding its integrative role of cortical hierarchical processing during development. Here, we corroborated the presence and significance of a dual long-axis representation of the hippocampal connectome and geometry including both linear and quadratic gradients along its long-axis in youth. This finding was robust across two independent large-scale developmental cohorts. Charting development of the dual long-axis gradients underscored their specific contributions to the cortical hierarchy maturation from the frontoparietal and salience/ventral attention networks. The observed developmental variability in spontaneous brain activities in youth parallels the gradients of myelin content. During childhood through adolescence to early adulthood, the hippocampus reorganized the dual long-axis by gradually relaxing its geometric constraints on the intrinsic network organization of cortical spontaneous activity for refined executive functions. Molecular processes underlying such reorganization of the dual long-axis in hippocampus are linked to neural growth, stress hormone regulation, and neuroactive signaling. Our findings enrich the understanding of hippocampal-cortical reorganizational principles across structural, functional, and molecular dimensions as well as its maturation, and define the plasticity distribution within the human hippocampus at systems level, holding potentials to enhance and translate neurodevelopment and neuropsychiatric healthcare.
Chikungunya virus infection disrupts MHC-I antigen presentation via nonstructural protein 2
Authors: Ware Brian C. , Parks M. Guston , Morrison Thomas E.
Published: November 2023

Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding a chimeric protein of VENUS fused to a CD8+T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Bothin vivoandex vivoinfected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kband H2-DbMHC proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation could be rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to β2-microglobulin in the CHIKV genome, which bypasses the need for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation.

AUTHOR SUMMARY

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), are re-emerging global public health threats with no approved vaccines or antiviral therapies. Infection with these viruses causes debilitating musculoskeletal disease for months to years that is associated with the persistence of viral RNA and antigen. Prior studies using a mouse model found that CD8+T cells, which recognize viral peptides in the context of major histocompatibility class I (MHC-I) displayed on the surface of infected cells, have a limited role in the control and clearance of CHIKV infection in joint-associated tissues, suggesting that CHIKV infected cells evade these critical effectors of the anti-viral immune response. Here, we show that MHC-I antigen presentation is inefficient in CHIKV-infected joint tissue fibroblasts, and that a protein encoded by CHIKV and produced in infected cells, nonstructural protein 2 (nsP2), disrupts the surface display of MHC-I molecules and antigen recognition of infected cells by CD8+T cells. Our findings support a role for CHIKV nsP2 in the evasion of the CD8+T cell response and establishment of persistent infection.
Human Endogenous Retrovirus Expression is Dynamically Regulated in Parkinson’s Disease
Authors: Gordevičius Juozas , Goralski Thomas , Bergsma Alexis , Parham Andrea , Kuhn Emily , Meyerdirk Lindsay , McDonald Mitch , Milčiūtė Milda , Putten Elizabeth Van , Marshall Lee , Brundin Patrik , Brundin Lena , Labrie Viviane , Henderson Michael , Pospisilik John Andrew
Published: November 2023

Parkinson’s disease (PD) is a progressive, debilitating neurodegenerative disease that afflicts approximately every 1000th individual. Recently, activation of genomic transposable elements (TE) has been suggested as a potential driver of PD onset. However, it is unclear where, when, and to what extent TEs are dysregulated in PD. Here, we performed a multi-tissue transcriptional analysis of multiple patient cohorts and identified TE transcriptional activation as a hallmark of PD. We find that PD patients exhibit up-regulation primarily of human endogenous retrovirus (HERV) transcripts in prefrontal cortex tissue, prefrontal neurons as well as in blood, and we demonstrate that TE activation in the blood is highest at the time of PD diagnosis. Supporting a potentially causal association between ERV dysregulation and PD heterogeneity, reduced gene dosage of the TE repressor Trim28 triggers transcriptional changes highly correlated to those measured in animal models of synucleinopathy (PFF-injection), and importantly, to those exhibited by patients themselves. These data identify ERV up-regulation as a common feature of central and peripheral PD etiology, and highlight potential roles for Trim28-dependent TEs in stratifying and monitoring PD and treatment compliance.
Is M1-L121E a good mimic on microbial rhodopsin? A viewpoint from excited-state dynamics
Authors: Li Gaoshang , Meng Jiajia , Yu Shuang , Bai Xiaolu , Dai Jin , Song Yin , Peng Xubiao , Zhao Qing
Published: November 2023

Microbial rhodopsin, an important photoreceptor protein, has been widely used in several fields, such as optogenetics, biotechnology, and biodevicesetc. However, current microbial rhodopsins are all transmembrane proteins, which both complicates the investigation on the photoreaction mechanism and limits their further applications. Therefore, a suitable mimic for microbial rhodopsin can not only provide a better model for understanding the mechanism, but also can extend the applications. The human protein CRABPII turns out to be a good template for design mimics on rhodopsin, due to the convenience in synthesis and the stability after mutations. Recently, Geigeret al.designed a new CRABPII-based mimic M1-L121E on microbial rhodopsin with the correct 13-cis (13C) isomerization after irritation. However, it still remains a question how similar it is compared with the natural microbial rhodopsin, in particular in the aspect of the photoreaction dynamics. In this article, we investigated the excited-state dynamics of this mimic by measuring its transient absorption spectra. Our results reveal that there are two components in the solution of mimic M1-L121E at PH=8, known as protonated Schiff base (PSB) and unprotonated Schiff base (USB) states. In both states, the photoreaction process from 13-cis (13C) to all-trans (AT) is faster than that from the inverse direction. In addition, the photoreaction process in PSB state is faster than that in the USB state. In the end, we compared the isomerization time of the PSB state with the properties of the microbial rhodopsin, and confirmed that the mimic M1-L121E indeed captures the main feature of the rhodopsin and is a good model of microbial rhodopsin in the photoreaction dynamics. However, our results also reveal significant differences in the excited-state dynamics of the mimic relative to the natural microbial rhodopsin, including the slower PSB isomerization rates in both 13C-AT and AT-13C directions, as well as the unusual USB photoreaction dynamics at PH=8. Such unique properties have not been observed in the natural rhodopsin, which could further deepen the understanding in photoreaction mechanism of the photosensitive proteins.
Machine learning-based exploration, expansion and definition of the atropopeptide family of ribosomally synthesized and posttranslationally modified peptides
Authors: Biermann Friederike , Tan Bin , Breitenbach Milena , Nanudorn Pakjira , Kakumu Yuya , Dimitrova Yoana , Walker Allison S. , Ueoka Reiko , Helfrich Eric J. N.
Published: November 2023

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) constitute a diverse class of natural products. Atropopeptides are a recent addition to the fast-growing number of RiPP families. Characterized members of the peptide family feature a particular intricate three-dimensional shape. Here we developed AtropoFinder, a machine learning-based algorithm to chart the biosynthetic landscape of the atropopeptides. AtropoFinder identified more than 650 atropopeptide biosynthetic gene clusters (BGCs). Through bioinformatics and modeling analyses, we pinpointed crucial motifs and residues in leader and core peptide sequences, prompting a refined definition of the atropopeptide RiPP family. Our study revealed that a substantial subset of atropopeptide BGCs harbors multiple tailoring genes, thus suggesting a broader structural diversity than previously anticipated. To verify AtropoFinder, we heterologously expressed four atropopeptide BGCs, which resulted in the identification of novel atropopeptides with varying peptide lengths, number and type of modifications. Most notably, our study resulted in the characterization of an atropopeptide that is more extensively modified than previously identified members, resulting in an even more rigid 3-dimensional shape. Moreover, one characterized atropopeptide BGC encoding a single P450 is involved in the biosynthesis of two peptides with the same sequence but distinct and non-overlapping modification patterns. This work expands the atropopeptide chemical space, advances our understanding of atropopeptide biosynthesis and underscores the potential of machine learning in uncovering the uncharted biosynthetic diversity encoded in RiPP biosynthetic blueprints.
Using developmental dynamics for evolutionary prediction and control
Authors: Milocco Lisandro , Uller Tobias
Published: November 2023

Understanding, predicting, and controlling the phenotypic consequences of genetic and environmental change is essential to many areas of fundamental and applied biology. In evolutionary biology, the generative process of development is a major source of organismal evolvability that constrains or facilitates adaptive change by shaping the distribution of phenotypic variation that selection can act upon. While the complex interactions between genetic and environmental factors during development may appear to make it impossible to infer the consequences of perturbations, the persistent observation that many perturbations result in similar phenotypes indicates that there is a logic to what variation is generated. Here, we show that a general representation of development as a dynamical system can reveal this logic. We build a framework that allows to predict the phenotypic effects of perturbations, and conditions for when the effects of perturbations of different origin are concordant. We find that this concordance is explained by two generic features of development, namely the dynamical dependence of the phenotype on itself and the fact that all perturbations must be funneled by the same developmental process. We apply our theoretical results to classical models of development and show that it can be used to predict the evolutionary response to selection using information of plasticity, and to accelerate evolution in a desired direction. The framework we introduce provides a way to quantitatively interchange perturbations, opening a new avenue of perturbation design to control the generation of variation, and thus evolution.
Predmoter - Cross-species prediction of plant promoter and enhancer regions
Authors: Kindel Felicitas , Triesch Sebastian , Schlüter Urte , Randarevitch Laura Alexandra , Reichel-Deland Vanessa , Weber Andreas P.M. , Denton Alisandra K.
Published: November 2023

Motivation

The identification ofcis-regulatory elements (CREs) is crucial for the analysis of gene regulatory networks in plants. Several next generation sequencing (NGS)-based methods were developed to identify CREs. However, these methods can be time-consuming and costly. They also involve creating sequencing libraries for the entire genome. Since many research efforts only focus on specific genomic loci, this presents a considerable expenditure. Computational prediction of the outputs of specialized NGS methods to analyze CREs, like Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq), would significantly cut costs and time investment. Yet, no such method is available to date.

Results

We present Predmoter, a deep neural network able to predict base-wise ATAC-seq and histone Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) read coverage for plant genomes. Predmoter uses only the DNA sequence as input. We evaluated our model on two plant genomes, the genome of the dicotArabidopsis thalianaand of the monocotOryza sativa. We trained our models on 10 species with publicly available ATAC-seq data and 15 species with ChIP-seq data. Our best models showed accurate predictions in peak positions and the overall pattern of peaks for ATAC- and Histone H3 trimethylated at lysine 4 (H3K4me3) ChIP-seq. Annotating putatively accessible chromatin regions provides valuable input for the identification of CREs. In conjunction with otherin silicodata, such as predicted binding affinities for transcription factors (TFs), this can significantly narrow down the search space to a manageable number of experimentally verifiable DNA-protein interaction pairs.

Availability and Implementation

The source code for Predmoter is available at:https://github.com/weberlab-hhu/Predmoteralong with documentation for installation and usage. Predmoter uses a single-command inference, Predmoter.py, for both training and prediction. Predmoter takes a fasta file as input and outputs an h5 file and optionally bigWig and bedGraph files.

Highlight

Predmoter will help identifying CREs and so gaining further insight into gene regulatory networks in plants.
Robust spatiotemporal organization of mitotic events in mechanically perturbedC. elegansembryos
Authors: Borne Vincent , Weiss Matthias
Published: November 2023

Early embryogenesis of the nematodeCaenorhabditis elegansprogresses in an autonomous fashion within a protective chitin eggshell. Cell division timing and the subsequent, mechanically guided positioning of cells is virtually invariant between individuals, especially before gastrulation. Here, we have challenged this stereotypical developmental program in early stages by mechanically perturbing the embryo, without breaking its eggshell. Compressing embryos to about 2/3 of their unperturbed diameter only resulted in markedly slower cell divisions. In contrast, compressing embryos to half of their native diameter frequently resulted in a loss of cytokinesis, yielding a non-natural syncytium that still allowed for multiple divisions of nuclei. Although the orientation of mitotic axes was strongly altered in the syncytium, key features of division timing and spatial arrangement of nuclei remained surprisingly similar to unperturbed embryos in the first few division cycles. This suggests that few, very robust mechanisms provide a basic and resilient program for safeguarding the early embryogenesis ofC. elegans.

STATEMENT OF SIGNIFICANCE

Early embryogenesis of the nematodeCaenorhabditis elegansprogresses in an autonomous fashion within a protective chitin eggshell. Cell division timing and cell positioning seemingly runs on autopilot, yielding a stereotypical development. Compressive forces, a potential hazard in the nematode’s native habitat, may jeopardize this. We show that compressing embryos to 2/3 of their native diameter results in markedly slower cell divisions but leaves the early embryonic program otherwise intact. Further compression of embryos impairs the formation of new cells while nuclei still divide in a common cytoplasm (’syncytium’) with basic features of division timing and spatial arrangement being surprisingly similar to unperturbed embryos. This suggests that few robust mechanisms provide a basic program for the early embryonic autopilot.
Intermittent subdiffusion of short nuclear actin rods due to interactions with chromatin
Authors: Speckner Konstantin , Rehfeldt Florian , Weiss Matthias
Published: November 2023

The interior of cellular nuclei, the nucleoplasm, is a crowded fluid that is pervaded by protein-decorated DNA polymers, the chromatin. Due to the complex architecture of chromatin and a multitude of associated non-equilbrium processes, e.g. DNA repair, the nucleoplasm can be expected to feature non-trivial material properties and hence anomalous transport phenomena. Here, we have used single-particle tracking on nuclear actin rods, which are important players in DNA repair, to probe such transport phenomena. Our analysis reveals that short actin rods in the nucleus show an intermittent, anti-persistent subdiffusion with clear signatures of fractional Brownian motion. Moreover, the diffusive motion is heterogeneous with clear signatures of an intermittent switching of trajectories between at least two different mobilities, most likely due to transient associations with chromatin. In line with this interpretation, hyperosmotic stress is seen to stall the motion of nuclear actin rods, whereas hypoosmotic conditions yield a reptation-like motion. Our data highlight the local heterogeneity of the nucleoplasm, e.g. distinct biochemical microenvironments and chromosome territories, that need to be taken into account for an understanding of nucleoplasmic transport and the mechanobiology of nuclei.
Localization of Spatially Extended Brain Sources by Flexible Alternating Projection (Flex-AP)
Authors: Hecker Lukas , Giri Amita , Pantazis Dimitrios , Adler Amir
Published: November 2023

Magnetoencephalography (MEG) and electroencephalography (EEG) are widely employed techniques for thein-vivomeasurement of neural activity with exceptional temporal resolution. Modeling the neural sources underlying these signals is of high interest for both neuroscience research and pathology. The method of Alternating Projection (AP) was recently shown to outperform the well-established recursively applied and projected multiple signal classification (RAP-MUSIC) algorithm. In this work, we further enhanced AP to allow for source extent estimation, a novel approach termed flexible extent AP (FLEX-AP). We found that FLEX-AP achieves significantly lower errors for spatially coherent sources compared to AP, RAP-MUSIC, and the corresponding extension, FLEX-RAP-MUSIC. We also found an advantage for discrete dipoles under forward modeling errors encountered in real-world scenarios. Together, our results indicate that the FLEX-AP method can unify dipole fitting and distributed source imaging into a single algorithm with promising accuracy.
Integrating single-cell RNA-seq datasets with substantial batch effects
Authors: Hrovatin Karin , Moinfar Amir Ali , Zappia Luke , Lapuerta Alejandro Tejada , Lengerich Ben , Kellis Manolis , Theis Fabian J.
Published: November 2023

Integration of single-cell RNA-sequencing (scRNA-seq) datasets has become a standard part of the analysis, with conditional variational autoencoders (cVAE) being among the most popular approaches. Increasingly, researchers are asking to map cells across challenging cases such as cross-organs, species, or organoids and primary tissue, as well as different scRNA-seq protocols, including single-cell and single-nuclei. Current computational methods struggle to harmonize datasets with such substantial differences, driven by technical or biological variation. Here, we propose to address these challenges for the popular cVAE-based approaches by introducing and comparing a series of regularization constraints.

The two commonly used strategies for increasing batch correction in cVAEs, that is Kullback–Leibler divergence (KL) regularization strength tuning and adversarial learning, suffer from substantial loss of biological information. Therefore, we adapt, implement, and assess alternative regularization strategies for cVAEs and investigate how they improve batch effect removal or better preserve biological variation, enabling us to propose an optimal cVAE-based integration strategy for complex systems. We show that using a VampPrior instead of the commonly used Gaussian prior not only improves the preservation of biological variation but also unexpectedly batch correction. Moreover, we show that our implementation of cycle-consistency loss leads to significantly better biological preservation than adversarial learning implemented in the previously proposed GLUE model. Additionally, we do not recommend relying only on the KL regularization strength tuning for increasing batch correction, as it removes both biological and batch information without discriminating between the two. Based on our findings, we propose a new model that combines VampPrior and cycle-consistency loss. We show that using it for datasets with substantial batch effects improves downstream interpretation of cell states and biological conditions. To ease the use of the newly proposed model, we make it available in the scvi-tools package as an external model named sysVI. Moreover, in the future, these regularization techniques could be added to other established cVAE-based models to improve the integration of datasets with substantial batch effects.
LncPlankton V1.0: a comprehensive collection of plankton long non-coding RNAs
Authors: Debit Ahmed , Vincens Pierre , Bowler Chris , de Carvalho Helena Cruz
Published: November 2023

Long considered as transcriptional noise, long non-coding RNAs (lncRNAs) are emerging as central, regulatory molecules in a multitude of eukaryotic species, from plants to animals to fungi. Yet, our knowledge about the occurrence of these molecules in the marine environment, namely in planktonic protists, is still elusive. To fill this gap of knowledge we developed LncPlankton v1.0, which is the first comprehensive database of marine plankton lncRNAs. By integrating the predictions derived from ten distinctive coding potential prediction tools in a majority voting setting, we identified 2,210,359 lncRNAs distributed across 414 marine plankton species from over nine different phyla. A user-friendly, open-access web interface for the exploration of the database was implemented (https://www.lncplankton.bio.ens.psl.eu/). We believe LncPlankton v1.0 will serve as a rich resource for studies of lncRNAs that will contribute to small- and large-scale analyses in a wide range of marine plankton species and allow comparative analysis well beyond the marine environment.
Connectome-Based Attractor Dynamics Underlie Brain Activity in Rest, Task, and Disease
Authors: Englert Robert , Kincses Balint , Kotikalapudi Raviteja , Gallitto Giuseppe , Li Jialin , Hoffschlag Kevin , Woo Choong-Wan , Wager Tor D. , Timmann Dagmar , Bingel Ulrike , Spisak Tamas
Published: November 2023

Abstract

Understanding large-scale brain dynamics is a grand challenge in neuroscience. We propose functional connectome-based Hopfield Neural Networks (fcHNNs) as a model of macro-scale brain dynamics, arising from recurrent activity flow among brain regions. An fcHNN is neither optimized to mimic certain brain characteristics, nor trained to solve specific tasks; its weights are simply initialized with empirical functional connectivity values. In the fcHNN framework, brain dynamics are understood in relation to so-called attractor states, i.e. neurobiologically meaningful low-energy activity configurations. Analyses of 7 distinct datasets demonstrate that fcHNNs can accurately reconstruct and predict brain dynamics under a wide range of conditions, including resting and task states and brain disorders. By establishing a mechanistic link between connectivity and activity, fcHNNs offer a simple and interpretable computational alternative to conventional descriptive analyses of brain function. Being a generative framework, fcHNNs can yield mechanistic insights and hold potential to uncover novel treatment targets.

Key Points

We present a simple yet powerful phenomenological model for large-scale brain dynamics

The model uses a functional connectome-based Hopfield artificial neural network (fcHNN) architecture to compute recurrent “activity flow” through the network of brain regions

fcHNN attractor dynamics accurately reconstruct several characteristics of resting state brain dynamics

fcHNNs conceptualize both task-induced and pathological changes in brain activity as a non-linear alteration of these dynamics

Our approach is validated using large-scale neuroimaging data from seven studies

fcHNNs offers a simple and interpretable computational alternative to conventional descriptive analyses of brain function
Calibrated Identification of Feature Dependencies in Single-cell Multiomics
Authors: Boyeau Pierre , Bates Stephen , Ergen Can , Jordan Michael I. , Yosef Nir
Published: November 2023

Data-driven identification of functional relationships between cellular properties is an exciting promise of single-cell genomics, especially given the increasing prevalence of assays for multiomic and spatial transcriptomic analysis. Major challenges include dealing with technical factors that might introduce or obscure dependencies between measurements, handling complex generative processes that require nonlinear modeling, and correctly assessing the statistical significance of discoveries.

VI-VS(Variational Inference for Variable Selection) is a comprehensive framework designed to strike a balance between robustness and interpretability.VI-VSemploys nonlinear generative models to identify conditionally dependent features, all while maintaining control over false discovery rates. These conditional dependencies are more stringent and more likely to represent genuine causal relationships.VI-VSis openly available athttps://github.com/YosefLab/VIVS, offering a no-compromise solution for identifying relevant feature relationships in multiomic data, advancing our understanding of molecular biology.
PANAMA enabled high sensitivity dual nanoflow LC/MS metabolomics and proteomics analysis
Authors: Lin Weiwei , Mousavi Fatemeh , Blum Benjamin C. , Heckendorf Christian F. , Lampl Noah , Heckman Ryan , Guo Hongbo , McComb Mark , Emili Andrew
Published: November 2023

High sensitivity nanoflow liquid chromatography (nLC) is seldom employed in untargeted metabolomics because current sample preparation techniques are inefficient to prevent nanocapillary column performance degradation. Here, we describe an nLC-based tandem mass spectrometry workflow that enables seamless joint analysis and integration of metabolomics (including lipidomics) and proteomics from the same samples without instrument duplication. This workflow is based on robust solid phase micro-extraction step for routine sample clean-up and bioactive molecule enrichment. Our method, termed PANAMA, improves compound resolution and detection sensitivity without compromising depth of coverage as compared with existing widely used analytical procedures. Notably, PANAMA can be applied to a broad array of specimens including biofluids, cell line and tissue samples. It generates high quality, information rich metabolite-protein datasets while bypassing the need for specialized instrumentation.

Motivation

The ability to routinely, sensitively and reproducibly analyze both cellular proteins and metabolite mixtures from the same biospecimens can enhance the discovery of biomolecules associated with basic biochemical processes and pathobiological states. Yet existing mass spectrometry-based profiling methods rely on specialized protocols and duplicated instrumentation platforms, resulting in increased time, sample consumption and costs. We sought to generate an effective platform for both metabolomic and proteomic studies on the same samples by enabling nanoflow liquid chromatography for small molecules. The resulting approach was extensively optimized and benchmarked to provide in depth molecular coverage, along with improved chromatographic separations, sensitivity and reliability as compared to existing methods. The cost benefit ratio of PANAMA is substantial because the platform bypasses the need for specialized instrumentation stemming from incompatible procedures.
Purinergic Ca2+signaling as a novel mechanism of drug tolerance in BRAF mutant melanoma
Authors: Stauffer Philip E. , Brinkley Jordon , Jacobson David , Quaranta Vito , Tyson Darren R.
Published: November 2023

Drug tolerance is a major cause of relapse after cancer treatment. In spite of intensive efforts1–9, its molecular basis remains poorly understood, hampering actionable intervention. We report a previously unrecognized signaling mechanism supporting drug tolerance in BRAF-mutant melanoma treated with BRAF inhibitors that could be of general relevance to other cancers. Its key features are cell-intrinsic intracellular Ca2+signaling initiated by P2X7 receptors (purinergic ligand-gated cation channels), and an enhanced ability for these Ca2+signals to reactivate ERK1/2 in the drug-tolerant state. Extracellular ATP, virtually ubiquitous in living systems, is the ligand that can initiate Ca2+spikes via P2X7 channels. ATP is abundant in the tumor microenvironment and is released by dying cells, ironically implicating treatment-initiated cancer cell death as a source of trophic stimuli that leads to ERK reactivation and drug tolerance. Such a mechanism immediately offers an explanation of the inevitable relapse after BRAFi treatment in BRAF-mutant melanoma, and points to actionable strategies to overcome it.
Mutation of NOTCH1 is selected within normal esophageal tissues, yet leads to selective epistasis suppressive of further evolution into cancer
Authors: Glasmacher Kira A. , Cannataro Vincent L. , Mandell Jeffrey D. , Jackson Mia , Fisk J. Nic , Townsend Jeffrey P.
Published: November 2023

Sequencing of tissues from histologically normal esophagus, among other organs, has revealed that normal tissues harbor somatic variants that are also found in cancers arising from the same tissue types. Our understanding of how somatic mutations commonly found in normal tissue can contribute to tumorigenesis is limited: common somatic mutations may or may not confer phenotypes compatible with oncogenesis. However, the strength of selection for somatic variants that appear in both normal and cancer tissues can be quantified in each context using evolutionary modeling approaches. We studied the evolutionary trajectory from normal esophageal tissue to esophageal squamous-cell carcinoma (ESCC) by analysis of 2171 sequenced samples from previous studies on normal esophageal epithelium and ESCC to reveal the stepwise contributions of somatic mutations to increased cellular division and survival. We also analyzed pairwise selective epistasis between somatically mutated genes that may lead to stepwise substitution patterns. We found thatNOTCH1substitutions are highly selected along the trajectory from embryogenesis to adult normal esophageal tissue, explaining their high prevalence in the tissue. In contrast, there is little to no positive selection forNOTCH1mutations along the trajectory from adult normal tissue to ESCC, suggesting thatNOTCH1substitutions do not drive tumorigenesis. Furthermore, mutations in NOTCH1 exhibit antagonistic epistasis with well-known cancer drivers including TP53, reducing selection for progressive mutations in tumorigenesis. This antagonistic epistasis likely corresponds with a low likelihood of tumor progression in the presence ofNOTCH1mutations in the esophagus.
A novel exopolysaccharide pathway from a freshwaterSphingomonasisolate
Authors: Goetsch Alexandra G. , Ufearo Daniel , Keiser Griffin , Heiss Christian , Azadi Parastoo , Hershey David M.
Published: November 2023

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. To better understand the breadth of polysaccharide production in nature we isolated a bacterium from Lake Michigan calledSphingomonas sp.LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a completewzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming that LM7 assembles a novelwzy/wzx-dependent polysaccharide. We extracted EPS from LM7 cultures and showed that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we found that the EPS pathway we identified diverges from those of adhesive polysaccharides such as the holdfast that are conserved in higherAlphaproteobacteria.Our approach of characterizing complete biosynthetic pathways holds promise for engineering of polysaccharides with valuable properties.

Importance

Bacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced bySphingomonas sp.LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering.
Heterogeneity in Neutrophil Extracellular Traps from Healthy Human Subjects
Authors: Collins Margaret S. , Imbrogno Michelle A. , Kopras Elizabeth J. , Howard James A. , Zhang Nanhua , Kramer Elizabeth L. , Hudock Kristin M.
Published: November 2023

Neutrophil Extracellular Traps (NETs), a key component of early defense against microbial infection, are also associated with tissue injury. NET composition has been reported to vary with some disease states, but the composition and variability of NETs across many healthy subjects provides a critical comparison that has not been well investigated. We evaluated NETs from twelve healthy subjects of varying ages isolated from multiple blood draws over a three and one half-year period to delineate the variability in extracellular DNA, protein, enzymatic activities, and susceptibility to protease inhibitors. We calculated correlations for NET constituents and loss of human bronchial epithelial barrier integrity, measured by transepithelial electrical resistance, after NET exposure. We found that although there was some variability within the same subject over time, the mean numbers of neutrophils, protein, LDH, serine protease activities, and cytokines IL-8, IL-1RA, and G-CSF in isolated NETs were consistent across subjects. Total DNA and double stranded DNA content in NETs were different across donors. NETs had little or no TNFα, IL-17A, or GM-CSF. NET DNA concentration correlated with increased NET neutrophil elastase activity and higher NET IL-1RA concentrations. NET serine protease activity varied considerably within the same donor from day-to-day. Mean response to protease inhibitors was significantly different across donors. NET DNA concentration correlated best with reductions in barrier integrity of human bronchial epithelia. Defining NET concentration by DNA content correlates with other NET components and reductions in NET-driven epithelial barrier dysfunction, suggesting DNA is a reasonable surrogate measurement for these complex structures in healthy subjects.
PARP knockdown promotes synapse reformation after axon injury
Authors: Belew Micah Y. , Huang Wenjia , Florman Jeremy T. , Alkema Mark J. , Byrne Alexandra B.
Published: November 2023

Injured nervous systems are often incapable of self-repairing, resulting in permanent loss of function and disability. To restore function, a severed axon must not only regenerate, but must also reform synapses with target cells. Together, these processes beget functional axon regeneration. Progress has been made towards a mechanistic understanding of axon regeneration. However, the molecular mechanisms that determine whether and how synapses are formed by a regenerated motor axon are not well understood. Using a combination ofin vivolaser axotomy, genetics, and high-resolution imaging, we find that poly (ADP-ribose) polymerases (PARPs) inhibit synapse reformation in regenerating axons. As a result, regeneratedparp(-)axons regain more function than regenerated wild-type axons, even though both have reached their target cells. We find that PARPs regulate both axon regeneration and synapse reformation in coordination with proteolytic calpain CLP-4. These results indicate approaches to functionally repair the injured nervous system must specifically target synapse reformation, in addition to other components of the injury response.
How resource abundance and stochasticity affect organisms’ range sizes
Authors: Mezzini Stefano , Fleming Chris H. , Medici E. Patrícia , Noonan Michael J.
Published: November 2023

The amount of space organisms use is thought to be tightly linked to the availability of resources within their habitats, such that organisms living in productive habitats generally require less space than those in resource-poor habitats. This hypothesis has widespread em-pirical support, but existing studies have focused primarily on responses to the mean amount of resources, while responses to the variance around the mean are still largely unknown. This is not a trivial oversight. Organisms adjust to variable environmental conditions, so failing to consider the effects of resource (un)predictability can result in a limited understanding of an organism’s range size, which challenges ecological theory and applied conservation alike. In this study, we leverage the available literature to provide a unifying framework and hypotheses for the effect of mean and variance in resources on range sizes. Next, we use simulated movement data to demonstrate how the combined effects of mean and variance in resource abundance interact to shape predictable patterns in range size. Finally, we use real-world tracking data on a lowland tapir (Tapirus terrestris) from the Brazilian Cerrado to show how this framework can be applied to better understand the movement ecology of free-ranging animals.
The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer
Authors: Khalil Mohamed I. , Yang Canchai , Vu Lexi , Chadha Smriti , Nabors Harrison , James Claire D. , Morgan Iain M. , Pyeon Dohun
Published: November 2023

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the E7 protein by degrading the components of the SKP1-CUL1-F-box (SCF) ubiquitin ligase complex in HPV+ HNC cells. We found thatMARCHF8knockdown in HPV+ HNC cells drastically decreases the E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.

IMPORTANCE

Since HPV oncoprotein E7 is essential for virus replication, HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the components of the E3 ubiquitin ligase complex CUL1 and UBE2L3 play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase MARCHF8 induced by HPV E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.
A blood-brain-barrier penetrant AAV gene therapy rescues neurological deficits in mucolipidosis IV mice
Authors: Sangster Madison , Bishop Martha , Yao Yizheng , Feitor Jessica , Shahriar Sanjid , Miller Maxwell , Chekuri Anil K. , Budnik Bogdan , Bei Fengfeng , Grishchuk Yulia
Published: November 2023

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits and progressive vision loss. Using AAV9 and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the humanMCOLN1gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-orientedMCOLN1gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1in adult symptomaticMcoln1-/-mice at a dose of 1e12 vg per mouse resulted inMCOLN1expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression ofMCOLN1transcripts was also detected in the retina of the mouse that had exhibited significant degeneration at the time of the treatment. However, no increase of retinal thickness was observed after the gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.
IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells
Authors: Wong Victoria A. , Dinh Kristie N. , Chen Guangchun , Wrenshall Lucile E.
Published: November 2023

IL-2Rα KO mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα knock out (KO) mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα knock mice to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα knock out vascular smooth muscle cells had detectable IL-2Rα. Further studies suggested that the source of IL-2Rα protein was likely maternal heterozygous cells present in KO offspring due to maternal microchimerism. Because the KO was generated by using a neomycin resistance gene insert, we treated cells with G418 and were able to eliminate the majority of IL-2Rα expressing cells. This elimination revealed that IL-2Rα KO vascular smooth muscle cells exhibited increased proliferation, decreased size, and hypodiploid DNA content when compared to wildtype cells. Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.
Male-male interactions shape mate selection inDrosophila
Authors: Sten Tom Hindmarsh , Li Rufei , Hollunder Florian , Eleazer Shadé , Ruta Vanessa
Published: November 2023

Males of many species have evolved behavioral traits to both attract females and repel rivals. Here, we explore mate selection inDrosophilafrom both the male and female perspective to shed light on how these key components of sexual selection — female choice and male-male competition — work in concert to guide reproductive strategies. We find that male flies fend off competing suitors by interleaving their courtship of a female with aggressive wing flicks, which both repel competitors and generate a ‘song’ that obscures the female’s auditory perception of other potential mates. Two higher-order circuit nodes – P1a and pC1x neurons – are coordinately recruited to allow males to flexibly interleave these agonistic actions with courtship displays, assuring they persistently pursue females until their rival falters. Together, our results suggest that female mating decisions are shaped by male-male interactions, underscoring how a male’s ability to subvert his rivals is central to his reproductive success.