bluuebunny/crossref_metadata_2025 huggingface dataset:
Sputnik virophage disrupts the transcriptional regulation of its host giant virus
Authors: Chen Jingjie , Ogata Hiroyuki , Hikida Hiroyuki
Published: August 2024

Sputnik virophages are small double-stranded DNA (dsDNA) viruses that replicate only inside host amoebae infected with giant dsDNA viruses, mimiviruses. Sputnik infection affects mimivirus replication, but their molecular interaction remains poorly understood. Here, we performed a time-course transcriptome analysis ofAcanthamoeba castellaniicells infected with Acanthamoeba polyphaga mimivirus (APMV) (hereafter referred to as Sputnik−cells) and those infected with both APMV and Sputnik 3 virophage (Sputnik+cells). The gene expression patterns of the amoebae were similar between these two conditions, whereas the expression of APMV genes was drastically affected by Sputnik, depending on the timing of their expression. Early expressed APMV genes showed similar expression patterns in both conditions at the early stage of infection. However, at late stages, their expression levels remained higher in Sputnik+cells than in Sputnik−cells, suggesting a prolongation of early gene expression by Sputnik. Late expressed APMV genes showed lower expression at earlier stages in Sputnik+cells, but their expression levels reached or exceeded those in Sputnik−cells at later stages, indicating a delay of gene expression. Overall, our results demonstrated that Sputnik infection drastically alters the transcriptome of APMV rather than amoeba, likely by disturbing the transition from early to late stages of APMV infection.
SARS-COV-2 nucleocapsid protein hijacks multiple components of the host nuclear transport machinery for distinct functions
Authors: Harrison Angela R. , Wang Sui Z. , Wagstaff Kylie M.
Published: August 2024

Many viruses target the host nuclear transport machinery to traffic their own proteins or restrict trafficking of host cargo, including mediators of antiviral immune signalling. Nucleocapsid (N) proteins from several coronaviruses traffic to the nucleus/nucleolus, with roles in cell cycle regulation. While N-protein from severe acute respiratory syndrome virus 2 (SARS-COV-2), the causative agent of the global COVID-19 pandemic, is widely reported to localise to the cytoplasm, we identify multiple, functionally distinct interactions of N-protein with the host’s nuclear transport machinery. Using quantitative cell imaging, including fluorescence recovery after photobleaching, and protein-protein interaction analysis, we describe a sub-population of SARS-COV-2 N-protein that localises more diffusely between the nucleus and cytoplasm, undergoes active nuclear import, and re-localises nuclear import receptors (karyopherins) KPNB1 and KPNA2 to the nucleus and cytoplasm, respectively. Truncation analyses identify at least two distinct KPNA2/KPNB1 binding sites located in the N-terminal and C-terminal regions of N-protein. Interestingly, while mutation of K/R-rich sites within these domains reduces KPNA2/KPNB1 binding and disables re-localisation of KPNA2, re-localisation of KPNB1 and nuclear import of N-protein remain intact, indicating that these are molecularly and functionally distinct mechanisms. siRNA knockdown confirms a role for KPNB1 in N-protein nuclear trafficking, while KPNA2 binding and mislocalisation may be antagonistic. Thus, SARS-COV-2 N-protein binds to karyopherinsviamultiple distinct sites to facilitate import and other functions.
Population shifts in begomoviruses associated with tomato yellow leaf curl disease in western Mediterranean countries
Authors: Granier Martine , Faize Mohamed , Passera Sandie , Urbino Cica , Peterschmitt Michel
Published: August 2024

Tomato yellow leaf curl disease (TYLCD) was reported in western Mediterranean basin since the late 1980s. Based on intensive plant samplings performed in Spain, Italy and Morocco at different periods between the 1990s and 2014, several begomoviruses (family Geminiviridae) were identified as the cause of TYLCD. They comprise the native Tomato yellow leaf curl Sardinia virus (TYLCSV), two strains of Tomato yellow leaf curl virus (TYLCV) introduced from the Middle East, and several types of TYLCV/TYLCSV recombinants including the invasive recombinant TYLCV-IS76 in which the proportion of genome inherited from TYLCSV was unusually short. Although parental and recombinant TYLCD-associated begomoviruses were present in each country, country specificities were detected with respect to their relative prevalence, the infection profiles of individual tomato plants, and the recombination patterns of TYLCV/TYLCSV recombinants. Considering geographic proximities and trade activities between these countries, and the efficient transmission of begomoviruses which are persistently transmitted by the polyphagous and tiny whitefly vectorBemisia tabaci, it was not known if these specificities would be maintained over time. To address this question, tomato samples collected in the three countries between 2015 and 2019 were analysed with PCR tests previously designed to distinguish species, strains and recombinants of TYLCD associated begomoviruses, and with an original PCR test distinguishing emerging resistance-breaking recombinants bearing short TYLCSV-inherited fragments (Srec) from formerly reported recombinants with longer TYLCSV fragments (Lrec). The results show that country specificities are still present, the most striking being the contrasted geographic distribution of Srec recombinants, with TYLCV-IS76 detected in Morocco but not in Italy, and TYLCV-IS141 and a new Srec recombinant (TYLCV-IS60) both detected in Italy and not in Morocco. Nevertheless, besides country specificities, a general population shift was revealed regarding TYLCV/TYLCSV recombinants. Indeed, all the recombinant positive samples, irrespective of their geographic origin, were Srec-positive but Lrec-negative, which suggest that the emergence of Srec recombinants occurred at the expenses of the Lrec recombinants. These results are discussed in relation to the positive selection of Srec recombinants by Ty-1 resistant tomato plants.
CarpeDeam: ADe NovoMetagenome Assembler for Heavily Damaged Ancient Datasets
Authors: Kraft Louis , Söding Johannes , Steinegger Martin , Jochheim Annika , Sackett Peter Wad , Fernandez-Guerra Antonio , Renaud Gabriel
Published: August 2024

De novoassembly of ancient metagenomic datasets is a challenging task. Ultra-short fragment size and characteristic postmortem damage patterns of sequenced ancient DNA molecules leave current tools ill-equipped for ideal assembly. We presentCarpeDeam, a novel damage-awarede novoassembler designed specifically for ancient metagenomic samples. Utilizing maximum-likelihood frameworks that integrate sample-specific damage patterns,CarpeDeamrecovers longer continuous sequences and more protein sequences from both simulated and empirical datasets compared to existing assemblers. As a pioneering ancient metagenome assembler,CarpeDeamopens the door for new opportunities in functional and taxonomic analyses of ancient microbial communities.
Characterization of Postsynaptic Glutamate Transporter Functionality in the Zebrafish Retinal First Synapse Across Different Wavelengths
Authors: Garbelli Marco , Niklaus Stephanie , Neuhauss Stephan C.F.
Published: August 2024

In the zebrafish retina, incident light undergoes wavelength-dependent processing encompassing mechanisms such as color opponency, contrast enhancement, and motion detection prior to neural transmission to the brain proper. In darkness, photoreceptors continuously release glutamate into the synaptic cleft, a process that diminishes in response to increased light intensity, thereby conveying visual signals to ON and OFF bipolar cells. Specifically, in zebrafish, the ON pathway signal transduction is mediated by metabotropic glutamate receptor 6b (mGluR6b) and Excitatory Amino Acid Transporters (EAATs). Here we demonstrate that knockout ofeaat5bandeaat7disrupts electroretinogram responses to short and long-wavelength stimuli while preserving middle-wavelength responses, suggesting wavelength-specific roles. We found differential expression of EAAT5b and EAAT7 in the outer plexiform layer, particularly in the strike zone, crucial for prey capture, supporting task specific involvement of these signaling pathways. In order to investigate this, we developed a virtual hunting assay using UV light stimuli. Such a behavioral assay targeting short and long wavelengths indicate that EAAT5b and EAAT7 influence UV-dependent prey detection and motion sensing differently. Our findings highlight the importance of EAAT5b and EAAT7 in modulating light integration dynamics in the zebrafish retina.
Hyoid bone position and upper airway patency: A computational finite element modeling study
Authors: Salman Diane , Amatoury Jason
Published: August 2024

Background and Objectives

The hyoid bone’s inferior baseline position in obstructive sleep apnea (OSA) has led to surgical hyoid repositioning treatment, yet outcomes vary widely. The influence of baseline hyoid position (phenotype) and surgical hyoid repositioning on upper airway function remains unclear. We aimed to investigate their impact on the upper airway using computational modeling.

Methods

A validated finite element model of the rabbit upper airway was advanced and used to simulate changes in baseline hyoid position and surgical hyoid repositioning, alone and in combination. The hyoid was displaced in cranial, caudal, anterior, anterior-cranial and anterior-caudal directions from 1-4mm. Model outcomes included upper airway collapsibility, measured using closing pressure (Pclose), cross-sectional area (CSA) and soft tissue mechanics (stress and strain).

Results

Graded baseline hyoid position increments increased Pclose for all directions, and up to 29-43% at 4mm (relative to the original baseline hyoid position). Anterior-based surgical hyoid repositioning decreased Pclose (∼-115% at 4mm) and increased ΔCSA (∼+35% at 4mm). Cranial surgical hyoid repositioning decreased ΔPclose (−29%), minimally affecting CSA. Caudal surgical hyoid repositioning increased ΔPclose (+27%) and decreased ΔCSA (−7%). Anterior-cranial and anterior-caudal surgical hyoid repositioning produced the highest stresses and strains. Surgical hyoid repositioning effects on upper airway outcomes were dependent on baseline hyoid position, with more caudal baseline hyoid positions leading to less effective surgeries.

Conclusions

Baseline hyoid position (phenotype) and surgical hyoid repositioning both alter upper airway outcomes, with effects dependent on hyoid displacement direction and magnitude. Baseline hyoid position influences the effectiveness of surgical hyoid repositioning in reducing upper airway collapsibility. These findings provide further insights into the hyoid’s role in upper airway patency and suggest that considering the hyoid’s baseline position and surgical repositioning direction/increment may help improve hyoid surgeries for OSA treatment.
Seed-competent α-synuclein pathology in metachromatic leukodystrophy: the expanding spectrum of α-synucleinopathy in sphingolipidoses
Authors: Ghanem Simona , Hawksworth Jade , Thom Searlait , Hartanto Annabelle E. , O’Neill Joseph , Ponraj Janarthanan , Mansour Said , Attems Johannes , Pyle Angela , Johnson Lauren , Baines Jack , Taylor Robert W. , Outeiro Tiago F. , El-Agnaf Omar M.A. , Erskine Daniel
Published: August 2024

Metachromatic leukodystrophy (MLD) is a rare - typically paediatric - sphingolipid storage disorder resulting from bi-allelic pathogenic variants in theARSAgene, encoding the lysosomal arylsulphatase A (ASA). Heterozygous variants inARSAare associated with risk of Lewy body diseases (LBD), a group of age-associated neurodegenerative disorders characterised by the accumulation of the protein α-synuclein; however, no study has yet determined whether α-synuclein with putative pathological features is observed in MLD brain tissue. We examinedpost-mortembrain tissue from MLD cases (N=5, age 2-33) compared to matched control cases using histological approaches and α-synuclein seeding amplification assay (SAA). Juvenile-onset MLD cases exhibited granular α-synuclein deposits in neurons of regions prone to neuronal pathology in MLD, and seed-competent conformers that generated atypical short, twisted fibrils on SAA. In contrast, infantile-onset MLD cases gave only variably positive reactions on SAA. In summary, this study suggests MLD cases manifest α-synuclein pathology reminiscent of that observed in LBD, even in juvenile populations, further expanding the spectrum of sphingolipid storage disorders associated with the aggregation of α-synuclein. These findings have important implications for understanding the disease process of both LBD and MLD, potentially highlighting novel pathways for therapeutic interventions in both conditions.
Holoprosencephaly and Cyclopia inbmp7bandbmpr1baCrispant zebrafish
Authors: Kyrychenko Valentyn , Rensinghoff Philipp , Bulk Johannes , Frey Constanze , Heermann Stephan
Published: August 2024

The visual system is highly specialized and its function is substantially depending on the proper development of the eyes. Early eye development starts with the definition of a single eye field, which is localized within the anterior neural plate (ANP). This single eye field is split consecutively and two optic vesicles emerge at the sides. These vesicles are then transformed into optic cups, out of which the future retinae are differentiating.

Holoprosencephaly (HPE) is a frequent developmental forebrain disorder, in which the splitting of ANP domains is hampered. HPE is mostly genetically linked and we recently showed that BMP antagonism is important for the eye field and the telencephalic anlage to split. Excessive BMP induction led to retinal progenitors stuck inside a dysmorphic forebrain. In this study, using the zebrafish as a model, we show with acute CRISPR/ Cas9 analysis in the F0 generation, the necessity ofbmp7bandbmpr1bafor proper forebrain development. In Crispants for both genes we found HPE phenotypes, e.g. cyclopia. Further analysis ofbmp7bCrispants indicated that predominantly the eye field is affected, rather than the telencephalic precursor domain.
Massive reduction of RyR1 in muscle spindles of mice carrying recessiveRyr1mutations alters proprioception and causes scoliosis
Authors: Ruiz Alexis , Benucci Sofia , Meier Hervé , Schultz Georg , Buczak Katarzyna , Handschin Christoph , Pena Rodrigo C. G. , Treves Susan , Zorzato Francesco
Published: August 2024

Muscle spindles are stretch receptors lying deep within the muscle belly involved in detecting changes in muscle length and playing a fundamental role in motor control, posture and synchronized gait. They are made up of an external capsule surrounding 3-5 intrafusal muscle fibers and a nuclear bag complex. Dysfunction of muscle spindles leads to abnormal proprioceptor function, which has been linked to aberrant bone and cartilage development, scoliosis, kyphosis and joint contractures.RYR1, the gene encoding the calcium release channel of the sarcoplasmic reticulum, is the most common target of mutations linked to human congenital myopathies, a condition often accompanied by skeleton alterations and joint contractures. So far, the link betweenRYR1mutations, altered muscle spindles and skeletal defects has not been investigated. To this end, we investigated heterozygous mice carrying recessiveRyr1mutations isogenic to those present in a severely affected child. Here we show that: (i) the RyR1 protein localizes to the polar regions of intrafusal fibers and exhibits a doubled row distribution pattern, typical for junctional sarcoplasmic reticulum proteins; (ii) muscle spindles of compound heterozygous mice show structural defects; (iii) RyR1 content in intrafusal muscle fibers from dHT mice is reduced by 54%. Such a massive reduction of mutant RyR1 in intrafusal muscle fibers leads to altered expression of intrafusal fiber proteins, severe scoliosis, alteration of gait and inter limb coordination. These results support the hypothesis thatRYR1mutations not only affect the function of extrafusal muscles, but might also affect that of intrafusal muscles. The latter may be one of the underlying causes of skeletal abnormalities seen in patients affected by recessiveRYR1mutations.
Beyond neutral loci: Examining immune gene variation in tigers(Panthera tigris)
Authors: Aditi B.V. , Ramakrishnan Uma
Published: August 2024

Genetic vulnerability of wild populations is generally assessed using neutral loci, which may not accurately reflect a population’s adaptive potential. In contrast, diversity at loci putatively involved in fitness and survival, such as immune genes, could be a better proxy for future survival. Research on immune genes in wild species has traditionally focused on the MHC loci. We investigate five families of non-MHC immune genes -Tumor Necrosis Factor, Interleukin, Toll-like Receptor, Leukocyte Immunoglobulin-Like Receptors, and Chemokine - involved in adaptive and innate immunity in the tiger(Panthera tigris), an endangered carnivore. We compare immune gene diversity to neutral diversity using publicly available resequenced whole genomes from 107 tigers representing all extant subspecies and subpopulations (within India) with different demographic histories.

Immune receptor genes show high nucleotide diversity (mean: 0.0019) compared to neutral loci (0.0008) and immune signalling genes (0.0004), suggesting positive selection. However, heterozygosity of immune genes does not differ significantly from neutral loci, suggesting strong genetic drift. Populations with histories of bottleneck and inbreeding show reduced genetic diversity and high mutation load but some immune loci retain polymorphisms even in inbred populations. Genetic differentiation and site frequency spectra of immune loci correspond to patterns from neutral loci but Toll-like receptor genes show signatures associated with balancing selection. Our findings indicate that genetic drift is likely the predominant evolutionary force shaping genetic variation in bottlenecked endangered species such as tigers, even at immune loci that are typically subject to selection. However, remnants of past balancing selection might preserve important variation, potentially influencing population survival.
Different ophthalmologic findings in induced models of Holoprosencephaly in zebrafish
Authors: Bulk Johannes , Kyrychenko Valentyn , Heermann Stephan
Published: August 2024

Early forebrain development is a fascinating process and the fate of higher brain function but also the fate of visual perception largely depends on it. During gastrulation a single domain for the prospective telencephalon and a single eye field are localized in the anterior most region of the early neuroectoderm, the anterior neural plate (ANP). Importantly, these domains must be split as development proceeds, giving rise to two telencephalic lobes as well as to two optic vesicles, which are transformed into optic cups subsequently. Holoprosencephaly (HPE) unfortunately is a rather frequent developmental disorder of the forebrain, during which the separation of the early precursor domains is hampered. Clinical manifestation can vary a lot, including the accompanying ophthalmologic findings. Here we ask, whether anophthalmia is more severe than cyclopia, both being ophthalmologic findings in HPE. In this brief analysis, we make use of a recently established zebrafish model of HPE in which the early function of BMP antagonists is abrogated by the excessive induction of a BMP ligand. An early induction was resulting in retinal progenitors being stuck in the forebrain with no eye being formed. We attenuated the induction protocol to investigate whether the anophthalmia phenotype could be changed into a cyclopic phenotype. We found synophthalmia and ocular hypotelorism, however, not cyclopia. Based on this we propose that anophthalmia and cyclopia are both the strongest ophthalmologic finding, however, depending on the type of HPE underlying.
Sexual Dimorphism in Jump Kinematics and Choreography in Peacock Spiders
Authors: Narendra Ajay , Seibel Anna , Ramirez-Esquivel Fiorella , Joshi Pranav , McLean Donald James , Robledo-Ospina Luis , Rao Dinesh
Published: August 2024

Jumping requires a rapid release of energy to propel an animal. Terrestrial animals achieve this by relying on the power generated by muscles, or by storing and rapidly releasing elastic energy. Jumping spiders rely on hydraulic pressure and muscular action to propel their jump. Though males and females of jumping spiders vary in size, sex-specific differences in jumping have never been studied. We investigated sexual dimorphism in the jump kinematics of an Australian Peacock spider,Maratus splendens. We recorded locomotory jumps in males and females using high-speed videography (5000 frames per second). We determined the centre of mass of the animals using µCT and tracked its displacement during a jump. We found that although females weighed more than twice as much as males, both had similar accelerations and take-off velocities. Males had shorter jump take-off duration, steeper take-off angle and experienced higher g-force compared to the females. We examine the jump choreography of male and female spiders and explore the factors behind the differences in their jump kinematics.
SatXplor – A comprehensive pipeline for satellite DNA analyses in complex genome assemblies
Authors: Volarić Marin , Meštrović Nevenka , Despot-Slade Evelin
Published: August 2024

Satellite DNAs (satDNAs) are tandemly repeated sequences that make up a significant portion of almost all eukaryotic genomes. Although satDNAs have been shown to play a very important role in genome organization and evolution, they are relatively poorly analysed even in model. One of the main reasons for the current lack of in-depth studies on satDNAs is their underrepresentation in genome assemblies. The complexity and highly repetitive nature of satDNAs make their analysis challenging, and there is a need for efficient tools that can ensure accurate annotation and analysis of satDNAs.

We present a novel pipeline, named Satellite DNA Exploration (SatXplor), designed to robustly characterize satDNA elements and analyse their arrays and flanking regions. SatXplor is benchmarked against curated satDNA datasets from diverse species, showcasing its versatility across genomes with varying complexities and different satDNA profile. Component algorithms excel in the identification of tandemly repeated sequences and for the first time enable evaluation of satDNA variation and array annotation with the addition of information about surrounding genomic landscape.

SatXplor is an innovative pipeline for satDNA analysis that can be paired with any tool used for satDNA detection, offering insights into the structural characteristics, array determination and genomic context of satDNA elements. By integrating various computational techniques, from sequence analysis and homology investigation to advanced clustering and graph-based methods, it provides a versatile and comprehensive approach to explore the complexity of satDNA organization and to understand the underlying mechanisms and evolutionary aspects. It is open-source and freely accessible athttps://github.com/mvolar/SatXplor.
Assessing Autophagy Flux in Glioblastoma Temozolomide Resistant Cells
Authors: Clark Courtney , Barzegar-Behrooz Amir , Cordani Marco , Shojaei Shahla , Ghavami Saeid
Published: August 2024

Autophagy is a critical cellular process involved in the degradation and recycling of cytoplasmic components, playing a dual role in cancer by either promoting cell survival or facilitating cell death. In glioblastoma (GB), autophagy has been implicated in resistance to the chemotherapeutic agent Temozolomide (TMZ). This study presents a novel method to accurately measure autophagy flux in TMZ-resistant glioblastoma cells, combining advanced imaging techniques with biochemical assays. By quantifying key autophagy markers such as LC3-II and SQSTM1, our approach provides detailed insights into the dynamic processes of autophagosome formation and clearance under therapeutic stress. This method not only advances our understanding of autophagy in GB chemoresistance but also has significant implications for the development of autophagy-targeted therapies. The ability to monitor and manipulate autophagy flux in real-time offers a promising avenue for monitoring and understnading TMZ resistance and improving patient outcomes in glioblastoma treatment.
The genetic basis of predation resistance inPseudomonasspecies associated with the bactivorous soil amoebaDictyostelium discoideum
Authors: Steele Margaret I. , Peiser Jessica M. , Dawson Simon P. M. , Queller David C. , Strassmann Joan E.
Published: August 2024

Predation is likely to influence the function of bacterial communities and the evolution of bacterial pathogens, because characteristics that permit escape from predators often overlap with traits used for biocontrol of plant pathogens, virulence, or even bioremediation. Soil bacteria are preyed upon by a variety of microorganisms, including the amoebaDictyostelium discoideum,which has led some strains to evolve resistance. We identified genes required for threePseudomonasspecies associated withD. discoideumto evade predation by screening more than 6,000 transposon mutants for loss of resistance. One species required a variety of genes including toxins and secondary metabolism genes, but the other two appear to have functionally redundant mechanisms of resistance, since disruption of genes with pleiotropic effects was required to render them susceptible. We determined that GacA, which positively regulates secondary metabolism, is required for resistance in all three species. Predation resistance also appears to be a social trait based on enrichment of cooperative genes in one species and rescue of mutants by wild type in another. Many genes required for resistance are conserved among both resistant and susceptible species, but several are found in few genomes and some of these have homologs in distantly related species. Gain and loss of resistance appears to be a dynamic process in which regulatory and structural genes are well conserved across species, the specific toxins they regulate may be lost in the absence of predators, and new toxins may be acquired through horizontal gene transfer.
Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB
Authors: DeAngelo Joseph D. , Maron Maxim I. , Roth Jacob S. , Silverstein Aliza M. , Gupta Varun , Stransky Stephanie , Basken Joel , Azofeifa Joey , Sidoli Simone , Gamble Matthew J. , Shechter David
Published: August 2024

Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A) —but not type I PRMT inhibition—leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.
Oxidative stress response mediated by the yeast Rho5 GTPase depends on the proper spatiotemporal distribution of its dimeric GEF
Authors: Bischof Linnet , Heinisch Jürgen J.
Published: August 2024

The small GTPase Rho5 acts as a central hub to mediate the yeast’s response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane,viafusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interactionin vivo. We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.
Human rod photoreceptor outer segments are supported by accessory inner segment structures
Authors: Lewis Tylor R. , Klementieva Natalia V. , Phan Sebastien , Castillo Carson M. , Kim Keun-Young , Cao Lauren Y. , Ellisman Mark H. , Arshavsky Vadim Y. , Alekseev Oleg
Published: August 2024

The first steps in vision take place in photoreceptor cells, which are highly compartmentalized neurons exhibiting significant structural variation across species. The light-sensitive ciliary compartment, called the outer segment, is located atop of the cell soma, called the inner segment. In this study, we present an ultrastructural analysis of human photoreceptors, which reveals that, in contrast to this classic arrangement, the inner segment of human rods extends alongside the outer segment to form a structure hereby termed the “accessory inner segment”. While reminiscent of the actin-based microvilli known as “calyceal processes” observed in other species, the accessory inner segment is a unique structure: (1) it contains an extensive microtubule-based cytoskeleton, (2) it extends far alongside the outer segment, (3) its diameter is comparable to that of the outer segment, (4) it contains numerous mitochondria, and (5) it forms electron-dense structures that likely mediate adhesion to the outer segment. Given that the spacing of extrafoveal human photoreceptors is more sparse than in non-primate species, with vast amounts of interphotoreceptor matrix present between cells, the closely apposed accessory inner segment likely provides structural support to the outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.
TUG protein acts through a disordered region to organize the early secretory pathway
Authors: Parchure Anup , Tejada Helen , Xi Zhiqun , Su Maohan , Julca-Zevallos Omar , Alcazar-Roman Abel R. , Yan You , Liu Xinran , Toomre Derek , Bogan Jonathan S
Published: August 2024

The endoplasmic reticulum (ER)-Golgi Intermediate Compartment (ERGIC) is a distinct compartment in mammalian cells, which forms in part by homotypic fusion of ER-derived vesicles and gives rise to the cis cisterna of the Golgi ribbon. How the ERGIC is regulated is not well understood. Here we show that the TUG protein is essential to maintain this compartment as a distinct organelle. TUG (UBXN9, Aspscr1) is known to regulate the cell type -specific trafficking of GLUT4 glucose transporters, but its role in more ubiquitous trafficking pathways has not been well characterized. TUG localized to the ERGIC and in fibroblasts its deletion enhanced anterograde flux and increased resorption of ERGIC markers into the cis-Golgi, perturbing membrane homeostasis in the early secretory pathway. TUG knockout cells had a compacted Golgi morphology, and ultrastructural studies revealed dilated cisterna with surrounding small vesicles. A central disordered region in TUG mediated its recruitment to ERGIC membranes, and an amino terminal domain was sufficient to induce oligomerization in cells. In TUG knockout cells, ERGIC-dependent processes such as autophagy are disrupted and model cargoes such as CFTR are missorted. Together, these results reveal a novel, TUG-dependent regulatory mechanism in the early secretory pathway, which modulates ERGIC organization and anterograde trafficking. This function is co-opted by GLUT4 and other proteins that employ an unconventional secretion pathway to the plasma membrane.
Auditory Cellular Cooperativity Probed Via Spontaneous Otoacoustic Emissions
Authors: Bergevin Christopher , Whiley Rebecca , Wit Hero , Manley Geoffrey , van Dijk Pim
Published: August 2024

As a sound pressure detector that uses energy to boost both its sensitivity and selectivity, the inner ear is an active non-equilibrium system. The collective processes of the inner ear giving rise to this exquisite functionality remain poorly understood. One manifestation of the active ear across the animal kingdom is the presence of spontaneous otoacoustic emission (SOAE), idiosyncratic arrays of spectral peaks that can be measured using a sensitive microphone in the ear canal.1 Current SOAE models attempt to explain how multiple peaks arise, and generally assume a spatially-distributed tonotopic system. However, the nature of the generators, their coupling, and the role of noise (e.g., Brownian motion) are hotly debated, especially given the inner ear morphological diversity across vertebrates. One means of probing these facets of emission generation is studying fluctuations in SOAE peak properties, which produce amplitude (AM) and frequency modulations (FM). These properties are likely related to the presence of noise affecting active cellular generation elements, and the coupling between generators. To better biophysically constrain models, this study characterizes the fluctuations in filtered SOAE peak waveforms, focusing on interrelations within and across peaks. A systematic approach is taken, examining three species that exhibit disparate inner ear morphologies: humans, barn owls, and green anole lizards. To varying degrees across all three groups, SOAE peaks have intra-(IrP) and interpeak (IPP) correlations indicative of interactions between generative elements. Activity from anole lizards, whose auditory sensory organ is relatively much smaller than that of humans or barn owls, showed a much higher incidence of IPP correlations. Taken together, we propose that these data are indicative of SOAE cellular generators acting cooperatively, allowing the ear to function as an optimized detector.

Significance Statement

The inner ear is a complex biomechanical system whose function is not well understood. To further elucidate the role of coupling in emission generation, this study systematically compares fluctuations in sound emitted spontaneously from the ear (spontaneous otoacoustic emission, SOAE) across three vertebrates. Ultimately these data serve to illustrate that the inner ear is a non-equilibrium, active system whose cellular elements work cooperatively. A clearer understanding of SOAE generation and how it manifests across the animal kingdom will significantly advance our understanding of both normal and impaired auditory function.
Arousal threshold reveals novel neural markers of sleep depth independently from the conventional sleep stages
Authors: Picchioni Dante , Yang Fan Nils , de Zwart Jacco A. , Wang Yicun , Mandelkow Hendrik , Özbay Pinar S. , Chen Gang , Taylor Paul A. , Lam Niki , Chappel-Farley Miranda G. , Chang Catie , Liu Jiaen , van Gelderen Peter , Duyn Jeff H.
Published: August 2024

Reports of sleep-specific brain activity patterns have been constrained by assessing brain function as it related to the conventional polysomnographic sleep stages. This limits the variety of sleep states and underlying activity patterns that one can discover. The current study used all-night functional MRI sleep data and defined sleep behaviorally with auditory arousal threshold (AAT) to characterize sleep depth better by searching for novel neural markers of sleep depth that are neuroanatomically localized and temporally unrelated to the conventional stages.

Functional correlation values calculated in a four-min time window immediately before the determination of AAT were entered into a linear mixed effects model, allowing multiple arousals across the night per subject into the analysis, and compared to models with sleep stage to determine the unique relationships with AAT. These unique relationships were for thalamocerebellar correlations, the relationship between the right language network and the right “default-mode network dorsal medial prefrontal cortex subsystem,” and the relationship between thalamus and ventral attention network. These novel neural markers of sleep depth would have remained undiscovered if the data were merely analyzed with the conventional sleep stages.

Significance Statement

The original classification of sleep into distinct stages used behavioral characteristics.

With the proliferation of new techniques, the first experiments might have been to perform correlations with arousal threshold. These experiments have never been performed, either with functional MRI or with any other modern technique. The amount of communication between brain regions as measured by all-night functional magnetic resonance imaging was correlated with arousal threshold. This revealed novel neural markers of sleep depth that would have remained undiscovered if the data were merely analyzed with the conventional stages. This expands our understanding of sleep and its functions beyond the constraints imposed by the conventional stages.
Drosophila melanogasterpigmentation demonstrates adaptive phenotypic parallelism but genomic unpredictability over multiple timescales
Authors: Berardi Skyler , Rhodes Jessica A. , Berner Mary Catherine , Greenblum Sharon I. , Bitter Mark C. , Behrman Emily L. , Betancourt Nicolas J. , Bergland Alan O. , Petrov Dmitri A. , Rajpurohit Subhash , Schmidt Paul
Published: August 2024

Populations are capable of responding to environmental change over ecological timescales via adaptive tracking. However, the translation from patterns of allele frequency change to rapid adaptation of complex traits remains unresolved. We used abdominal pigmentation inDrosophila melanogasteras a model phenotype to address the nature, genetic architecture, and repeatability of rapid adaptation in the field. We show thatD. melanogasterpigmentation evolves as a highly parallel and deterministic response to shared environmental gradients across latitude and season in natural North American populations. We then experimentally evolved replicate, genetically diverse fly populations in field mesocosms to remove any confounding effects of demography and/or cryptic structure that may drive patterns in wild populations; we show that pigmentation rapidly responds, in parallel, in fewer than ten generations. Thus, pigmentation evolves concordantly in response to spatial and temporal climatic gradients. We next examined whether phenotypic differentiation was associated with allele frequency change at loci with established links to genetic variance in pigmentation in natural populations. We found that across all spatial and temporal scales, phenotypic patterns were associated with variation at pigmentation-related loci, and the sets of genes we identified in each context were largely nonoverlapping. Therefore, our findings suggest that parallel phenotypic evolution is associated with an unpredictable genomic response, with distinct components of the polygenic architecture shifting across each environmental gradient to produce redundant adaptive patterns.

Significance Statement

Shifts in global climate conditions have heightened our need to understand the dynamics and pace of adaptation in natural populations. In order to anticipate the population-level response to rapidly changing environmental conditions, we need to understand whether trait evolution is predictable over short timescales, and whether the genetic basis of adaptation is shared or distinct across multiple timescales. Here, we explored parallelism in the adaptive response of a complex phenotype,D. melanogasterpigmentation, to shared conditions that varied over multiple spatiotemporal scales. Our results demonstrate that while phenotypic adaptation proceeds as a predictable response to environmental gradients, even over short timescales, the genetic basis of the adaptive response is variable and nuanced across spatial and temporal contexts.
Synthetic mucus biomaterials synergize with antibiofilm agents to combatPseudomonas aeruginosabiofilms
Authors: Yang Sydney , Stern Alexa , Duncan Gregg
Published: August 2024

Bacterial biofilms are often highly resistant to antimicrobials causing persistent infections which when not effectively managed can significantly worsen clinical outcomes. As such, alternatives to standard antibiotic therapies have been highly sought after to address difficult-to-treat biofilm-associated infections. We hypothesized a biomaterial-based approach using the innate functions of mucins to modulate bacterial surface attachment and virulence could provide a new therapeutic strategy against biofilms. Based on our testing inPseudomonas aeruginosabiofilms, we found synthetic mucus biomaterials can inhibit biofilm formation and significantly reduce the thickness of mature biofilms. In addition, we evaluated if synthetic mucus biomaterials could work synergistically with DNase and/or α-amylase for enhanced biofilm dispersal. Combination treatment with these antibiofilm agents and synthetic mucus biomaterials resulted in up to 3 log reductions in viability of matureP. aeruginosabiofilms. Overall, this work provides a new bio-inspired, combinatorial approach to address biofilms and antibiotic-resistant bacterial infections.
Duplexed CeTEAM drug biosensors reveal determinants of PARP inhibitor selectivity
Authors: Pires Maria J. , Lovric Alen , Alam Seher , Fabbrizi Emanuele , Rotili Dante , Altun Mikael , Valerie Nicholas C.K.
Published: August 2024

PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry CeTEAM drug biosensors to systematically characterize binding and cell cycle alterations of 27 PARPi at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs, including the next-generation drug, senaparib. However, benzimidazole carboxamide (niraparib) derivatives demonstrated PARP1-selective tendencies, while pthalazinones (olaparib) favored PARP2. AZD5305, a reported PARP1-selective inhibitor with characteristics of both series, was the exception and appears ∼1600-fold more potent towards PARP1. In agreement with current understanding, we see that PARP trapping phenotypes positively correlate with PARP1/2 binding potency, while some potent binders, such as veliparib, did not – likely reflecting their allosteric influence on DNA retention. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping phenotypes. The PARP1/2 CeTEAM platform thus provides a structural roadmap for the development of selective PARPi and should facilitate the discovery of better targeted therapies. Furthermore, our results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity.
Role of U11/U12 minor spliceosome geneZCRB1in Ciliogenesis and WNT Signaling
Authors: Powell-Rodgers Geralle , Pirzada Mujeeb Ur Rehman , Richee Jahmiera , Jungers Courtney F. , Colijn Sarah , Stratman Amber N. , Djuranovic Sergej
Published: August 2024

Despite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally,zcrb1CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.
G protein-Coupled Receptor Associated Sorting Protein GASP1 mediated trafficking of the Glucagon Like Peptide-1 Receptor contributes to the development of tolerance to incretin drugs
Authors: Gaur Anirudh , Keeshen Caroline M , King Madeline , Dirks Noah , Flisher Marcus , Cummings Bethany P , Huising Mark O , Whistler Jennifer L
Published: August 2024

Incretin mimetic drugs are in widespread use for the treatment of type 2 diabetes and obesity and more recently have been prescribed for weight loss in otherwise healthy individuals. These drugs are all agonists of the glucagon-like peptide 1 receptor (GLP-1R) and function by supplementing effects produced by the endogenous hormone agonist glucagon-like peptide 1 (GLP-1). The therapeutic benefits of these medications, including improved glucose control and weight loss, require continued usage and wane with time. The molecular mechanisms underlying this loss of effect to incretin drugs remain unknown. Following activation by agonist and signaling to G protein, the GLP-1R engages arrestins and is endocytosed. Here we investigated the role of G protein-coupled receptor associated sorting protein 1 (GASP1), a critical regulator of the post-endocytic trafficking of GLP-1R, on tolerance to GLP-1R agonist drug. We found that tolerance to incretin drug was prevented at the cellular, tissue and whole animal level in mice with a selective disruption of the GASP1 protein in beta cells of the pancreatic islet. These studies implicate post-endocytic sorting of the GLP-1R in the loss of effectiveness of incretin therapeutics with prolonged use. These findings also suggest a novel strategy to prevent tolerance by biasing incretin drugs for G protein and away from arrestin engagement.