bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective neuritogenesis inAbcd1/2deficient rat neurons due to intrinsic and astrocyte-dependent mechanisms
Authors: Golbano Arantxa , Pardo Luis , Menacho Carmen M. , Rierola Marina , Claro Enrique , Wood Levi B. , Masgrau Roser , Galea Elena
Published: October 2022

X-linked adrenoleukodystrophy (X-ALD) is a rare neurometabolic and demyelinating disorder caused by loss of function mutations of the ABCD1 transporter that imports very-long-chain fatty acids (VLCFA) into the peroxisome for beta-oxidation. Impaired ABCD1 function results in VLCFA accumulation, which ultimately causes lethal forms of X-ALD in children (CCALD) and adults (CAMN). Because X-ALD is a genetic disorder, we looked for signs of altered neurodevelopmental pathways in the transcriptomes of brain cortical tissues free of pathology from patients that died of CALD or CAMN. Several categories related to brain development, axonal growth, synaptic signaling and synaptic compartments were significantly dysregulated in both CALD and CAMN, suggesting that congenital circuit abnormalities might be structural in brains of mutated ABCD1 carriers. We partially dissected the cellular origin of dysregulated pathways using rat neuronal and astrocytic cultures in which X-ALD was modeled by silencing of Abcd1 and Abcd2 by RNA interference. Abcd2 was silenced lest it compensated for Abcd1 loss. Abcd1/2 deficient neurons presented higher rates of death, reduced sizes and defective formation of spines, dendrites and axons. The aberrant neuron development was caused by cell-autonomous and astrocyte-dependent mechanisms, and involved Wnt signaling, as suggested by the rescue of the expression of a synaptic gene upon pharmacological activation of the Wnt pathway. As recently proposed for neurogenetic disorders such as Huntington’s disease, our data suggest that X-ALD has a neurodevelopmental component that may cause psychiatric alterations and prime neural circuits for neurodegeneration. If this is the case, therapies aimed at restoring neural-circuit function in neurodevelopmental disorders may be reprofiled for X-ALD therapeutics.
READemption 2: Multi-species RNA-Seq made easy
Authors: Sauerwein Till , Bischler Thorsten , Förstner Konrad U.
Published: October 2022

Dual or Multi RNA-seq simultaneously analyze the transcriptomes of two or more interacting species to gain insights about their interplay. The RNA of the interacting species is collected and sequenced together and only separated in silico by mapping the reads to the corresponding genomes. We developed READemption 2.0, to our knowledge the first tool that performs all necessary steps to handle RNA-seq data from any number of species. These steps comprise basic quality filtering and adapter trimming of raw reads, aligning the reads to reference genomes, generating nucleotide-wise coverage files, creating gene-wise read counts and performing differential gene expression analysis. These results can be visualized by additional subcommands of the software. READemption 2.0 allows users to produce meaningful results with default settings that follow conventional standards. Furthermore, many parameters can be adjusted to meet the users’ specific needs, e.g. keeping or discarding species cross-mapped reads or normalizing the data.
Holographic-(V)AE: an end-to-end SO(3)-Equivariant (Variational) Autoencoder in Fourier Space
Authors: Visani Gian Marco , Pun Michael N. , Angaji Arman , Nourmohammad Armita
Published: October 2022

Group-equivariant neural networks have emerged as a data-efficient approach to solve classification and regression tasks, while respecting the relevant symmetries of the data. However, little work has been done to extend this paradigm to the unsupervised and generative domains. Here, we presentHolographic-(Variational) Auto Encoder (H-(V)AE), a fully end-to-end SO(3)-equivariant (variational) autoencoder in Fourier space, suitable for unsupervised learning and generation of data distributed around a specified origin in 3D. H-(V)AE is trained to reconstruct the spherical Fourier encoding of data, learning in the process a low-dimensional representation of the data (i.e., a latent space) with a maximally informative rotationally invariant embedding alongside an equivariant frame describing the orientation of the data. We extensively test the performance of H-(V)AE on diverse datasets. We show that the learned latent space efficiently encodes the categorical features of spherical images. Moreover, H-(V)AE’s latent space can be used to extract compact embeddings for protein structure microenvironments, and when paired with a Random Forest Regressor, it enables state-of-the-art predictions of protein-ligand binding affinity.
Semi-supervised machine learning for sensitive open modification spectral library searching
Authors: Arab Issar , Fondrie William E. , Laukens Kris , Bittremieux Wout
Published: October 2022

A key analysis task in mass spectrometry proteomics is matching the acquired tandem mass spectra to their originating peptides by sequence database searching or spectral library searching. Machine learning is an increasingly popular post-processing approach to maximize the number of confident spectrum identifications that can be obtained at a given false discovery rate threshold. Here, we have integrated semi-supervised machine learning in the ANN-SoLo tool, an efficient spectral library search engine that is optimized for open modification searching to identify peptides with any type of post-translational modification. We show that machine learning rescoring boosts the number of spectra that can be identified for both standard searching and open searching, and we provide insights into relevant spectrum characteristics harnessed by the machine learning model. The semi-supervised machine learning functionality has now been fully integrated into ANN-SoLo, which is available as open source under the permissive Apache 2.0 license on GitHub athttps://github.com/bittremieux/ANN-SoLo.
Two new “Incertae sedis” syllids (Annelida: Syllidae) from Brazilian oceanic islands
Authors: Nascimento Rodolfo Leandro , Fukuda Marcelo Veronesi , De Paiva Paulo Cesar
Published: October 2022

Oceanic islands present very interesting environments, known by possessing relatively distinct fauna and flora. However, taxonomic accounts from Brazilian oceanic islands focused on important groups, such as the family Syllidae, began to be published only in recent years. In this paper we provide descriptions and illustrations of two new species,Brevicirrosyllissp. nov. from Trindade Islands andWestheidesyllissp. nov. from Rocas Atoll, two incertae sedis genera previously included in the Eusyllinae subfamily. We also provide updated identification keys for both genera.
A novel micro-ECoG recording method for recording multisensory neural activity from the parietal to temporal cortices in mice
Authors: Setogawa Susumu , Kanda Ryota , Tada Shuto , Hikima Takuya , Saitoh Yoshito , Ishikawa Mikiko , Nakada Satoshi , Seki Fumiko , Hikishima Keigo , Matsumoto Hideyuki , Mizuseki Kenji , Fukayama Osamu , Osanai Makoto , Sekiguchi Hiroto , Ohkawa Noriaki
Published: October 2022

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro (μ)-electrocorticography (μECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped μECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current μECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel μECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the μECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the μECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our μECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.
Insulin receptor substrate 4 deficiency mediates the insulin effect on the epithelial magnesium channel TRPM6 and causes hypomagnesemia
Authors: Zhang Jing , An Sung Wan , Neelam Sudha , Bhatta Anuja , Nie Mingzhu , Duran Claudia , Bal Manjot , Latta Femke , Hou Jianghui , Otto Joseph J. , Kozlitina Julia , Lemoff Andrew , Hoenderop Joost , Baum Michel , Wolf Matthias T
Published: October 2022

The kidney is the key regulator of magnesium (Mg2+) homeostasis in the human body. In the distal convoluted tubule (DCT), the apical epithelial magnesium (Mg2+) channel TRPM6, determines how much Mg2+is excreted in the urine. To better understand the regulation of human renal Mg2+absorption we identified novel, potential interaction partners of TRPM6 by pursuing a liquid chromatography – tandem mass spectrometry (LC-MS/MS) proteomics approach.

We found insulin receptor substrate 4 (IRS4) enriched with TRPM6 tagged to glutathione S-transferase (TRPM6-GST) but not GST control. Physical interaction between IRS4 and TRPM6 was confirmed by co-immunoprecipitation. Applying microdissection of mouse tubules, we detectedIrs4mRNA expression mostly in the DCT and to a lower degree in the proximal tubule and thick ascending limb of Henle. Given the overall low abundance ofIrs4mRNA along the tubule we investigated the phenotype ofIrs4knockout mice (Irs4-/-). TheseIrs4-/-mice displayed significantly higher urinary and fecal Mg2+losses and lower blood Mg2+levels than wild-type (WT) mice. Claudin-16, claudin-19, and Hnf1b mRNA and Claudin-16 and Trpm6 protein expression was significantly higher in kidneys of 3 month oldIrs4-/-mice consistent with a compensatory mechanism to conserve Mg2+. Applying whole-cell patch-clamp recording we confirmed the stimulatory role of insulin on TRPM6 channel activity and showed that IRS4 targets the two TRPM6 phosphorylation sites T1391 and S1583 to enhance TRPM6 current density. To test the effect of Mg2+deficiency on metabolism, we performed glucose and insulin tolerance studies, which were mildly abnormal inIrs4-/-mice.

SIGNIFICANCE STATEMENT

Magnesium (Mg2+) is the second most abundant intracellular cation but the regulation of Mg2+homeostasis is not well understood. The kidney is the key organ for regulating Mg2+homeostasis. Insulin is a known stimulator of the apical epithelial Mg2+channel TRPM6. We present a novel modifier of Mg2+absorption with insulin receptor substrate 4 (IRS4) which illuminates further, how insulin activates the TRPM6 channel and modifies Mg2+homeostasis. Applying protein biochemistry, tubular microdissection, whole mouse physiology, and patch-clamp recording, we demonstrate that IRS4 mediates the stimulatory effect of insulin by enhancing phosphorylation of two specific TRPM6 residues.Irs4-/-mice develop increased urinary and stool Mg2+losses, lower serum Mg2+concentration, and display mild impairment in glucose and insulin tolerance.
Vimentin supports directional cell migration by controlling focal adhesions
Authors: Venu Arun P. , Modi Mayank , Aryal Ujjwal , Tcarenkova Elena , Jiu Yaming , Jacquemet Guillaume , Minin Alexander , Cheng Fang , Eriksson John E.
Published: October 2022

Fibroblastic migration is of key importance in wound healing. While the intermediate filament (IF) protein vimentin is required for normal wound healing, we examined whether vimentin-mediated regulation of fibroblast migration could be involved. In wound healing assays triggering cell polarization and directed migration, we observed that vimentin-deficient mouse and rat embryonic fibroblasts lost their directional persistence. We show that vimentin maintains directionality by guiding focal adhesions (FAs) in fibroblasts. Detailed analysis showed that vimentin stabilizes FAs and regulates their disassembly rate. The destabilization of Vim-/- FAs was reflected by smaller FAs. Live cell and super-resolution imaging demonstrate that vimentin interacts dynamically with the key molecules of FAs and, importantly, with FAK, which is crucial for the maturation of FAs. These results demonstrate that vimentin IFs control the maturation, stability, dynamics, arrangement, and overall orientation of FAs, with a net effect on FA coordination during migration.
Knock-sideways by inducible ER retrieval enables a novel approach for studyingPlasmodiumsecreted proteins
Authors: Fierro Manuel A , Hussain Tahir , Campin Liam J , Beck Josh R
Published: October 2022

Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissectingPlasmodiumsecreted protein functions are limited. We report knockER, a novel DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiplePlasmodiumspecies to interrogate the trafficking, topology and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a novel tool for dissecting secreted protein function with sub-compartmental resolution that should be widely amenable to genetically tractable eukaryotes.

Significance

Protein trafficking and secretion through the endomembrane system is a defining feature of eukaryotes. The secretory pathway is central to the unique biology and pathology of the obligate intracellular malaria parasite, however tools for studying secreted protein function are limited. Knock-sideways is a powerful mutagenesis strategy that conditionally sequesters a protein away from its site of function but is generally not applicable to secreted proteins. We developed a simple approach to conditionally sequesterPlasmodiumsecreted proteins in the ER by inducible C-terminal fusion with a KDEL ER-retrieval sequence that can be used for trafficking, topology and loss-of-function studies. The knockER strategy is broadly applicable to functional dissection of proteins that traverse the eukaryotic secretory pathway.
Development in context: interferon response networks regulate human fetal thymic epithelial cell differentiation
Authors: Mohammed Abdulvasey , Solomon Benjamin D. , Slepicka Priscila F. , Hubka Kelsea M. , Nguyen Hanh Dan , Wang Wenqing , Arreola Martin , Chavez Michael G. , Yeh Christine Y. , Kim Doo Kyung , Winn Virginia , Gifford Casey A. , Kedlian Veronika , Park Jong-Eun , Hollander Georg A. , Sebastiano Vittorio , Khatri Purvesh , Teichmann Sarah A. , Gentles Andrew J. , Weinacht Katja G.
Published: October 2022

The thymus instructs T cell immunity and central tolerance, yet its therapeutic potential remains untapped. The quest to regenerate thymic function for clinical application is lagging while the signals that drive thymic epithelial cell differentiation remain incompletely understood. Here, we elucidate pathways instructing commitment and specialization of the human thymic epithelial stroma through complementary single cell transcriptomic approaches. First, we identify gene regulatory networks that define fetal thymic epithelium in the thus far unexplored context of other anterior foregut-derived organs; then, we characterize lineage trajectories within the thymic epithelial compartment across embryonic, fetal, and early postnatal stages. Activation of interferon response gene regulatory networks distinguished epithelial cells of the thymus from those of all other anterior foregut-derived organs. Interferon signals were processed differentially within thymic cortical and medullary lineages, reflected in distinctNFκBandIRFsignatures, respectively. Our study reveals novel, translatable insights into the developmental programs underlying thymic epithelial cell differentiation that may advance the field of regenerative cell therapies.

Single cell transcriptomics of anterior foregut-derived organs identifies pathways governing thymic epithelial commitment and specialization.
Pharmacologic Inhibition of DYRK1A Results in MYC Hyperactivation and ERK Hyperphosphorylation rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition
Authors: Hurtz Christian , Ayyadevara V. S. S. Abhinav , Wertheim Gerald , Chukinas John A , Loftus Joseph P , Lee Sung June , Kumar Anil , Bhansali Rahul S , Swaminathan Srividya , Geng Huimin , Milne Thomas , Hua Xianxin , Bernt Kathrin M , Besson Thierry , Shi Junwei , Crispino John D. , Carroll Martin , Tasian Sarah K
Published: October 2022

KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.
EEG-based speaker-listener neural coupling reflects speech-selective attentional mechanisms beyond the speech stimulus
Authors: Li Jiawei , Hong Bo , Nolte Guido , Engel Andreas K. , Zhang Dan
Published: October 2022

When we pay attention to someone, do we focus only on the sound they make, the word they use, or do we form a mental space shared with the speaker we want to pay attention to? Some would argue that the human language is no other than a simple signal, but others claim that human beings understand each other not only by relying on the words that have been said but also formed a shared ground in the specific conversation. This debate was raised early, but the conclusion remains vague. Our study aimed to investigate how attention modulates the neural coupling between the speaker and the listener in a cocktail party paradigm. The temporal response function (TRF) method was employed to reveal how the listener was coupled to the speaker at the neural level. The results showed that the neural coupling between the listener and the attended speaker peaked 5 seconds before speech onset at the delta band over the left frontal region, and was correlated with speech comprehension performance. In contrast, the attentional processing of speech acoustics and semantics occurred primarily at a later stage after speech onset and was not significantly correlated with comprehension performance. These findings suggest that our human brain might have adopted a predictive mechanism to achieve speaker-listener neural coupling for successful speech comprehension.

Three key points

Listener’s EEG signals coupled to the speaker’s 5 s before the speech onset, which revealed a “beyond the stimulus” attentional modulation.

Speaker-listener attentional coupling is correlated to the listener’s comprehension performance, but the speech-listener’s coupling didn’t.

The implementation of temporal response function methods and the neural language methods yielded novel perspectives to the analysis of the inter-brain studies.
Auxin production in the green alga Chlamydomonas involves an extracellular L-amino acid oxidase and supports algal-bacterial mutualism with methylobacteria
Authors: Calatrava Victoria , Hom Erik F. Y. , Llamas Angel , Fernández Emilio , Galván Aurora
Published: October 2022

Interactions between algae and bacteria are widespread in aquatic and terrestrial ecosystems and play fundamental roles in nutrient cycling and biomass production. However, the chemical basis for many of these interactions is poorly characterized and understood. Recent studies have shown that the plant auxin indole acetic acid (IAA) can mediate chemical crosstalk between algae and bacteria, resembling its role in plant-bacterial associations. While algae have been shown to produce IAA, molecular pathways for IAA synthesis in algae have remained elusive. Here, we report a mechanism for IAA production from L-tryptophan mediated by the extracellular enzyme L-amino acid oxidase (LAO1) in the model alga Chlamydomonas reinhardtii. Under inorganic nitrogen limitation but in the presence of L-tryptophan and other amino acids, high levels of IAA are generated in an LAO1-dependent manner. Thus, LAO1 plays a dual role in scavenging nitrogen from L-amino acids and in producing the phytohormone IAA, which subsequently inhibits algal cell multiplication and chlorophyll degradation. We show that these inhibitory effects can be relieved in the presence of Methylobacterium spp., well-known plant growth-promoting bacteria (PGPB), whose growth is mutualistically enhanced by the presence of the alga. These findings reveal a complex interplay of microbial auxin production and degradation by algal-bacterial consortia under nitrogen limitation and draws attention to potential ecophysiological roles of terrestrial microalgae and PGPB in association with land plants.
Volatile metabolites in lavage fluid are correlated to Valley fever disease severity in murine model lung infections
Authors: Keppler Emily A. Higgins , Van Dyke Marley C. Caballero , Mead Heather L. , Lake Douglas F. , Magee D. Mitchell , Barker Bridget M. , Bean Heather D.
Published: October 2022

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections, to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host-pathogen interactions, we infected C57BL/6J mice with C. immitis RS and C. posadasii Silveira via intranasal inoculation. We measured fungal dissemination and collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid phase microextraction (SPME) and two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated to cytokine abundances and clustered mice by disease severity. These 36 VOCs were also able to separate mice with a moderate to high disease severity by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect Coccidioidal infection and provide clinically relevant information on disease severity.

IMPORTANCE

Coccidioidomycosis, or Valley fever, is a fungal disease endemic to the North and South American arid regions. Forty percent of individuals infected with Valley fever will exhibit symptoms consistent with community-acquired pneumonia. However, misdiagnosis frequently occurs in these cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in receiving an accurate diagnosis. Herein, we used a murine lung infection model as a step towards developing a breath-based diagnostic for Valley fever. We infected C57BL/6J mice with C. immitis RS and C. posadasii Silveira and collected bronchoalveolar lavage fluid for untargeted volatile metabolomics. We observed that volatile metabolites in the bronchoalveolar lavage fluid of Cocci-inoculated mice were significantly correlated to disease severity, as measured by immune response. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test.
Effect of changing the direction of the force vector of the mandible in relation to the skull base: study in rabbits
Authors: Cougo Milton Cristian Rodrigues , Puricelli Edela , de Quevedo Alexandre Silva , Catarina Luciana Santa , Artuzi Felipe Ernesto , Ponzoni Deise
Published: October 2022

The temporomandibular joint has a great capacity for functional adaptation. The aim of this study was to evaluate, bilaterally, the influence of unilateral modification of the direction of the mandibular force vector in relation to the skull base in rabbits. Thirty New Zealand rabbits (Oryctolagus cuniculus L.) were randomly divided into two groups (n = 15/group): test (modification of the mandibular force vector) and control (no modification of the mandibular force vector). The animals were killed at 20, 40, and 60 days postoperatively. Histomorphometric evaluation of the temporal and condylar joint structures bilaterally always showed significant differences (P < 0.05) between the test and control groups on both sides of the TMJ. The results demonstrate that the rabbit temporal bone and mandibular condyle showed significant adaptive capacity as a biological response to mechanical forces on both the operated and opposite sides.
Proteomic analysis of flagella fromChlamydomonasmutants lacking the central pair apparatus reveals loss of radial spoke head proteins
Authors: Wemmer Kimberly A. , Azimzadeh Juliette , Marshall Wallace F.
Published: October 2022

The flagellar axoneme is a complex protein-based machine capable of generating motile forces by coordinating the action of thousands of dynein motors. A key element of the axoneme is the central pair apparatus, consisting of a pair of microtubules surrounded by additional structures. In an effort to better understand the organization of the central pair, we used 2D DIGE to identify proteins that are depleted from flagella isolated from two differentChlamydomonas reinhardtiimutants,pf15andpf18, that lack the central pair. The set of proteins contained almost no components of the central apparatus. We find that three proteins of the radial spoke head RSP1, RSP9, and RSP10, as well as a number of other protein components associated with the outer doublets, are depleted from flagella of mutants lacking the central apparatus. Two of the other proteins depleted frompf15andpf18flagella, the microtubule inner proteins (MIPs) FAP21 and FAP161, are missing from the genome ofThalassiosira, an organism that lacks a central pair and radial spokes, and RNAi of FAP21 in planaria shows that it has a role in ciliary motility. Based on the depletion of radial spoke head proteins, as well as MIPs and other axonemal components, from flagella lacking the central pair apparatus, we hypothesize that the central apparatus may play a role in scaffolding the assembly or retention of radial spokes and other axonemal structures.
Estrogen receptor beta in the central amygdala regulates the deleterious behavioral and neuronal consequences of repeated social stress in female rats
Authors: Smiley Cora E. , Pate Brittany S. , Bouknight Samantha J. , Francis Megan J. , Nowicki Alexandria V. , Harrington Evelynn N. , Wood Susan K.
Published: October 2022

While over 95% of the population has reported experiencing extreme stress or trauma, females of reproductive age develop stress-induced neuropsychiatric disorders at twice the rate of males. This suggests that estrogen may facilitate neural processes that increase stress susceptibility and underlie the heightened rates of these disorders, like depression and anxiety, that result from stress exposure in females. However, there is contradicting evidence in the literature regarding estrogen’s role in stress-related behavioral outcomes. Estrogen signaling through estrogen receptor beta (ERβ) has been traditionally thought of as anxiolytic, but recent studies suggest estrogen exhibits distinct effects in the context of stress. Furthermore, ERβ is found abundantly in many stress-sensitive brain loci, including the central amygdala (CeA), in which transcription of the vital stress hormone, corticotropin releasing factor (CRF), can be regulated by an estrogen response element. Therefore, these experiments sought to identify the role of CeA ERβ activity during stress on behavioral outcomes in naturally cycling, adult, female Sprague-Dawley rats. Rats were exposed to an ethological model of vicarious social stress, witness stress (WS), in which they experienced the sensory and psychological aspects of an aggressive social defeat encounter between two males. Following stress, rats exhibited stress-induced anxiety-like behaviors in the marble burying task, and, finally, brain analysis revealed increased ERβ and CRF specifically within the CeA following exposure to stress cues. Subsequent experiments were designed to target this receptor in the CeA using microinjections of the ERβ antagonist, PHTPP, prior to each stress session. Sucrose preference, acoustic startle, and marble burying tasks determined that blocking ERβ in the CeA during WS prevented the development of depressive-,anxiety-like, and hypervigilant behaviors. Additionally, brain analysis revealed a long-term decrease of intra-CeA CRF expression in PHTPP-treated WS rats compared to vehicle. These experiments indicate that ERβ signaling in the CeA, through its effects on CRF, contribute to the development of negative valence behaviors that result from exposure to repeated social stress in female rats.
AKIR-1 Regulates Proteasome Localization and Function inCaenorhabditis elegans
Authors: Pispa Johanna , Mikkonen Elisa , Arpalahti Leena , Jin Congyu , Martínez-Fernández Carmen , Cerón Julián , Holmberg Carina I.
Published: October 2022

Regulated protein clearance is vital for cells to maintain protein homeostasis and the conditions essential for survival. The primary machinery for intracellular protein degradation is the ubiquitin– proteasome system (UPS), by which ubiquitin-tagged proteins are degraded by the proteasome. Proteasomes are present both in the cytoplasm and the nucleus, but the mechanisms coordinating proteasome activity and its subcellular localization in a multicellular organism are still unclear. Here, we identified the nuclear protein-encoding geneakir-1as a proteasome regulator in a genome-wideCaenorhabditis elegans(C. elegans) RNAi screen. We show that the depletion ofakir-1causes accumulation of endogenous polyubiquitinated proteins in the nuclei of intestinal cells, concomitant with slowerin vivoproteasomal degradation in this subcellular compartment. Remarkably, the loss ofakir-1does not induce an accumulation of polyubiquitinated proteins in oocyte nuclei, thoughakir-1is essential for the nuclear localization of proteasomes in both cell types. We further show that the importin family memberima-3genetically interacts withakir-1, and affects subcellular distribution of polyubiquitinated proteins in intestinal cells. We show for the first time that conserved AKIR-1 is important for the nuclear transport of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in the maintenance of proteostasis.
Intranasal delivery of NS1-deleted influenza virus vectored COVID-19 vaccine restrains the SARS-CoV-2 inflammatory response
Authors: Zhang Liang , Jiang Yao , He Jinhang , Chen Junyu , Qi Ruoyao , Yuan Lunzhi , Shao Tiange , Chen Congjie , Chen Yaode , Wang Xijing , Lei Xing , Gao Qingxiang , Zhuang Chunlan , Zhou Ming , Ma Jian , Liu Wei , Yang Man , Fu Rao , Wu Yangtao , Chen Feng , Xiong Hualong , Nie Meifeng , Chen Yiyi , Wu Kun , Fang Mujing , Wang Yingbin , Zheng Zizheng , Huang Shoujie , Ge Shengxiang , Cheng Shih Chin , Zhu Huachen , Cheng Tong , Yuan Quan , Wu Ting , Zhang Jun , Chen Yixin , Zhang Tianying , Qi Hai , Guan Yi , Xia Ningshao
Published: October 2022

The emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) variants and “anatomical escape” characteristics threaten the effectiveness of current coronavirus disease (COVID-19) vaccines. There is an urgent need to understand the immunological mechanism of broad-spectrum respiratory tract protection to guide broader vaccines development. In this study, we investigated immune responses induced by an NS1-deleted influenza virus vectored intranasal COVID-19 vaccine (dNS1-RBD) which provides broad-spectrum protection against SARS-CoV-2 variants. Intranasal delivery of dNS1-RBD induced innate immunity, trained immunity and tissue-resident memory T cells covering the upper and lower respiratory tract. It restrained the inflammatory response by suppressing early phase viral load post SARS-CoV-2 challenge and attenuating pro-inflammatory cytokine (IL-6, IL-1B, and IFN-γ) levels, thereby reducing excess immune-induced tissue injury compared with the control group. By inducing local cellular immunity and trained immunity, intranasal delivery of NS1-deleted influenza virus vectored vaccine represents a broad-spectrum COVID-19 vaccine strategy to reduce disease burden.
Large-scale phylogenomics of aquatic bacteria reveal molecular mechanisms for adaptation to salinity
Authors: Jurdzinski Krzysztof T , Mehrshad Maliheh , Delgado Luis Fernando , Deng Ziling , Bertilsson Stefan , Andersson Anders F
Published: October 2022

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transitions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as are molecular adaptations facilitating cross-biome transitions. Here, we conduct large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,276 MAGs). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. Distinct brackish basins co-hosted numerous species despite differences in salinity and geographic distance, the latter having stronger intra-species population structuring effects. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed towards the brackish biome. Transitions were accompanied by changes in isoelectric point distribution and amino acid composition of inferred proteomes, as well as convergent gains or losses of specific gene functions. Therefore, adaptive challenges entailing proteome reorganization and specific changes in gene content result in species-level separation between aquatic biomes.
Systems-level investigation of mucopolysaccharidosis IIIA identifies deficient synaptic activity as a key driver of disease progression
Authors: Douek Alon M , Salavaty Abbas , Kreuder Florian , Stamatis Sebastian-Alexander , Steele Joel R , Hanchapola Iresha , Shah Anup D , Schittenhelm Ralf B , Ramialison Mirana , Currie Peter D , Kaslin Jan
Published: October 2022

Mucopolysaccharidoses are lysosomal storage diseases that collectively represent a major cause of lethal, treatment-refractory childhood dementias 1–7 Clinically-useful interventions are hampered due to an incomplete understanding of their neuropathological origins. Using the zebrafish sgsh model of mucopolysaccharidosis IIIA 8 (MPS IIIA, Sanfilippo syndrome A), we conducted several ‘omics-based analyses, and developed and benchmarked a novel bioinformatic feature classification and ranking model for high-throughput datasets – ExIR – to prioritise important features in the progression of neurological manifestations of the disease. We find that the massive endolysosomal burden resulting from increased lysosomal storage of heparan sulfate and other secondarily accumulating substrates, such as sphingolipids, induces abnormal microtubule organisation and vesicle trafficking in neurons. This results in a gradual impairment of synaptic vesicle localisation at the presynaptic terminal and consequently impaired neuronal activity. Importantly, the endolysosomal phenotype in MPS IIIA zebrafish well-precedes the onset of neural pathology, though the larval MPS IIIA brain was found to be more susceptible to perturbation than wild type siblings. Collectively, these analyses demonstrate the presence of a progressive ‘functional neurodegenerative’ phenotype underpinning neurological disease in MPS IIIA. Our findings provide direct mechanistic evidence linking the well-described lysosomal storage basis for MPS IIIA to its disproportionately severe neural clinical involvement, enabling development and refinement of future therapeutic interventions for this currently untreatable disorder.

Highlights

MPS IIIA represents one of the most common causes of broadly fatal childhood dementia, but the mechanisms underlying disease progression are poorly understood.

The first systems-level analyses of disease state and progression in the CNS of an MPS IIIA animal model were performed.

Experimental data-based Integrative Ranking (ExIR) was developed to provide unbiased prioritisation and classification of biological data as drivers, biomarkers and mediators of biological processes from high-throughput data at a systems level.

Application of ExIR to a transcriptomic and proteomic analyses of a zebrafish model of MPS IIIA implies progressive deficiencies in synaptic activity as a key driver of disease progression correlating with progressive neuronal endolysosomal burden and secondary storage diseases.

A novel unifying explanation of pathobiology and progression of MPS IIIA facilitates identification of clinically targetable features and may be generalised to other neuronopathic storage disorders.
Optimised whole-genome CRISPR interference screens identify ARID1A-dependent growth regulators in human induced pluripotent stem cells
Authors: Usluer Sunay , Hallast Pille , Crepaldi Luca , Zhou Yan , Urgo Katie , Dincer Cansu , Su Jing , Noell Guillaume , Alasoo Kaur , Garwany Omar El , Gerety Sebastian , Newman Ben , Dovey Oliver M. , Parts Leopold
Published: October 2022

Perturbation of gene function is a powerful way to understand the role of individual genes in cellular systems. Whole-genome CRISPR/Cas-based screens have parallelized this approach and identified genes that modulate growth in many contexts. However, the DNA break-induced stress upon Cas9 action limits the efficacy of these screens in important models, such as human induced pluripotent stem cells (iPSCs). Silencing with a catalytically inactive Cas9 is a less stressful alternative, but has been considered less effective so far. Here, we first tested the efficiency of several dCas9 fusion proteins for target repression in human iPSCs, and identified dCas9-KRAB-MeCP2 as the most potent. We then produced monoclonal and polyclonal cell lines carrying this construct from multiple iPSC donors, and optimized genome-wide screens with them. We found silencing in a 200bp window around the transcription start site to be as effective as using wild-type Cas9 for identifying essential genes in iPSCs, but with a reduced cost due to better cell survival. Monoclonal lines performed better, but data from polyclonal lines were of sufficient quality for screening for larger effects. Finally, we performed whole-genome screens to identify dosage sensitivities that depend on the functionality of ARID1A, a commonly mutated cancer and developmental disorder gene. We observed differential growth upon depletion of NF2, TAF6L, EZH2, and PSMB2 genes in ARID1A+/-lines compared to wild type, and an enrichment of proteasome genes. Further, we confirmed that the context-specific growth decrease was phenocopied by treating the cells with a proteasome inhibitor, suggesting a pharmacologically targetable synthetic lethal interaction between the proteasome and ARID1A. We propose that many more plausible targets in challenging cell models can be efficiently identified with our approach.
Inhibition of CHK2 dependent DNA damage response suppresses Zika virus infection through a STING dependent mechanism
Authors: Ma Huailiang , Li Chunfeng , Wang Yanli , Valore Erika V. , Trisal Meera , Hui Harold Sai-yin , Hakimpour Paul , Bowen James R. , Mak Tak W , Suthar Mehul S. , Pulendran Bali
Published: October 2022

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neurological disorders and microcephaly. Recent research has shown that ZIKV causes cell cycle arrest and DNA damage response in neuronal progenitor cells that potentially leads to congenital neurodevelopmental defects. However, the specific role of regulators that control DNA damage response to ZIKV infection and the related mechanisms remain largely unknown. Here through bothin vitroandin vivostudies, we observe that ZIKV induces DNA damage response in both human cell line and mouse embryo. When CHK2 dependent DNA damage response pathway is inhibited by a small molecule inhibitor or genetic deletion, ZIKV production is reduced and the embryonic developmental defects are rescued. Furthermore, we demonstrate that in ZIKV infectedChk2-/-mice, the reduced viral load correlates with elevated antiviral innate immune response which is found to be dependent on the STING signaling pathway. Collectively, our study reveals a specific role of CHK2 for ZIKV infection and pathogenesis, demonstrating a mechanism that inhibition of the CHK2 axis suppresses ZIKV infection through the STING-dependent antiviral pathway, thus providing a new therapeutic strategy against ZIKV. These findings also suggest the intriguing possibility that ZIKV evolved to orchestrate DNA damage response factors to create a beneficial environment for its infection in the host cells.
Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates anin vitromodel of bone marrow adipocyte subtypes
Authors: Tratwal Josefine , Falgayrac Guillaume , During Alexandrine , Bertheaume Nicolas , Bataclan Charles , Tavakol Daniel N. , Campos Vasco , Duponchel Ludovic , Daley George Q. , Penel Guillaume , Chauveau Christophe , Naveiras Olaia
Published: October 2022

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier and are more resilient to environmental and metabolic disruptions.In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thusin vitromodels remain the current standard to study nuances of adipocyte differentiation. Noin vitromodel has yet been described for the study of rBMAds/cBMAds.

Here, we present anin vitromodel of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression ofScd1andFads2differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesisin vitroin a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 modelin vitrosystem and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.