bluuebunny/crossref_metadata_2025 huggingface dataset:
Dorsal skull meningeal lymphatic vessels drain blood-solutes after intracerebral hemorrhage
Authors: Virenque Anaïs , Balin Raz , Noe Francesco M.
Published: March 2021

Drainage of intraparenchymal hematoma is crucial for the treatment of intracerebral hemorrhage (ICH). We investigated here the possible function of the meningeal lymphatic vessels (mLVs) in ICH resolution. Meningeal lymphatics have been reported to be involved in cerebrospinal fluid drainage, but their role in the drainage and clearance of brain parenchyma has not been characterized in details. Using two preclinical models of ICH, mimicking focal cortical ischemic hemorrhage and subcortical extended hemorrhage, we characterized the dynamics of blood drainage through the dorsal mLVs by two-photon real time imaging in awake mice. After ICH induction, we observe the flow of blood-derived components within the mLVs, suggesting that meningeal lymphatics can play a role in facilitating the drainage of the hemorrhage. We also found that local formation of new mLVs is directly correlated with ICH-related neuroinflammation levels. These findings suggest that meningeal lymphatics could provide a valuable therapeutic target for ICH resolution.

In vivo awake imaging reveals the direct drainage of extravasated blood-solutes from brain parenchyma into dorsal meningeal lymphatic vessels, following focal or diffuse intracranial hemorrhage
Location, location, location– choice of Voxel-Based Morphometry processing pipeline drives variability in the location of neuroanatomical brain markers
Authors: Zhou Xinqi , Wu Renjing , Zeng Yixu , Qi Ziyu , Ferraro Stefania , Yao Shuxia , Kendrick Keith M. , Becker Benjamin
Published: March 2021

Fundamental and clinical neuroscience has benefited from the development of automated computational analyses of Magnetic Resonance Imaging (MRI) data, such as Voxel-based Morphometry (VBM). VBM determines regional gray matter variations with high spatial resolution and results are commonly interpreted in a regional-specific manner, for instance with respect to which specific brain regions differ in volume between women and men. In excess of 600 papers using VBM are now published every year and a number of different automated VBM processing pipelines are frequently used in analyses although it remains to be fully and systematically assessed whether they come up with the same answers. Here we have therefore examined variability between four commonly used VBM pipelines in two large brain structural datasets. Spatial similarity, reproducibility and reliability of the processed gray matter brain maps was generally low between pipelines. Examination of sex-differences and age-related changes in gray matter volumes revealed considerable differences between the pipelines in terms of the specific regions identified as well as meta-analytic characterization of their function. In contrast, applying machine learning-based multivariate analyses allowed an accurate prediction of sex or age based on the gray matter maps across pipelines, although prediction accuracy differed strongly between them. Together the findings suggest that the choice of pipeline alone leads to considerable variability in brain structural analyses which poses a serious challenge for reproducibility as well as interpretation.
Genome structure ofBrachionus asplanchnoidis, a Eukaryote with intrapopulation variation in genome size
Authors: Stelzer C.P. , Blommaert J. , Waldvogel A.M. , Pichler M. , Hecox-Lea B. , Mark Welch D.B.
Published: March 2021

Eukaryotic genomes vary greatly in size due to variation in the proportion of non-coding DNA, a pattern that emerges both in comparisons at a larger taxonomic scale and at the level of individuals within a species. The rotiferBrachionus asplanchnoidisrepresents one of the most extreme cases of intraspecific genome size variation among Eukaryotes, displaying almost 2-fold variation within a geographic population. Here we used a whole-genome sequencing approach to identify the underlying DNA sequence differences by assembling a high-quality reference genome draft for one individual of the population and aligning short-reads of 15 individuals from the same geographic population. We identified large, contiguous copy number variable regions (CNVs), which exhibited significant coverage differences among individuals, and whose coverage overall scaled with genome size. CNVs were mainly composed of tandemly repeated satellite DNA, with only few interspersed genes or other sequences, and were characterized by an elevated GC-content. Judging from their distributions across contigs, some CNVs are fragments of accessory (B-)chromosomes while others resemble large insertions to normal chromosomes. CNV patterns in offspring of two parents with divergent genome size, and CNV patterns in several individuals from an inbred line differing in genome size demonstrated inheritance and accumulation of CNVs across generations. Our study provides unprecedented insights into genome size evolution at microevolutionary time scales and thus paves the way for studying genome size evolution in contemporary populations rather than inferring patterns and processesa posteriorifrom species comparisons.
Inner hair cell dysfunction inKlhl18mutant mice leads to low frequency progressive hearing loss
Authors: Ingham Neil J , Banafshe Navid , Panganiban Clarisse , Crunden Julia L , Chen Jing , Steel Karen P
Published: March 2021

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency hearing impairment in two mutant mouse lines, carrying different mutant alleles of theKlhl18gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Auditory brainstem response (ABR) thresholds (a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window ofKlhl18lowfmutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed thatKlhl18lowfmutant mice had abnormally tapering inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.Klhl18mutant mice exhibit an uncommon low frequency hearing impairment with physiological features consistent with Auditory Neuropathy Spectrum Disorder (ANSD).

SIGNIFICANCE STATEMENT

We describe a novel progressive hearing loss inKlhl18mutant mice that affects the lower frequencies of its’ hearing range. Investigation of two mutant alleles of this gene revealed primary inner hair cell defects affecting the neural output of the cochlea while outer hair cell function appeared normal. The tallest stereocilia of inner hair cells showed an abnormal tapering shape, especially notable in the apical half of the cochlear duct corresponding to the low frequency hearing loss. Our finding of a primary inner hair cell defect associated with raised thresholds for auditory brainstem responses combined with normal outer hair cell function suggests that Klhl18 deficiency and inner hair cell pathology may contribute to Auditory Neuropathy Spectrum Disorder in humans.
Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity
Authors: Lycas Matthew D. , Ejdrup Aske L. , Sørensen Andreas T. , Haahr Nicolai O. , Jørgensen Søren H. , Guthrie Daryl A. , Støier Jonatan F. , Werner Christian , Newman Amy Hauck , Sauer Markus , Herborg Freja , Gether Ulrik
Published: March 2021

The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. Summarized, the data reveal a mechanism for rapid alterations in nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.
Endogenous retrovirus rewired the gene regulatory network shared between primordial germ cells and naïve pluripotent cells in hominoids
Authors: Ito Jumpei , Seita Yasunari , Kojima Shohei , Parrish Nicholas F. , Sasaki Kotaro , Sato Kei
Published: March 2021

Although the gene regulatory network controlling germ cell development is critical for gamete integrity, this network has been substantially diversified during mammalian evolution. Here, we show that several hundred loci of LTR5_Hs, a hominoid-specific endogenous retrovirus (ERV), function as enhancers in both human primordial germ cells (PGCs) and naïve pluripotent cells. PGCs and naïve pluripotent cells exhibit a similar transcriptome signature, and the enhancers derived from LTR5_Hs contribute to establishing such similarity. LTR5_Hs appears to be activated by transcription factors critical in both cell types (KLF4, TFAP2C, NANOG, and CBFA2T2). Comparative transcriptome analysis between humans and macaques suggested that the expression of many genes in PGCs and naïve pluripotent cells has been upregulated by LTR5_Hs insertions in the hominoid lineage. Together, this study suggests that LTR5_Hs insertions have rewired and finetuned the gene regulatory network shared between PGCs and naïve pluripotent cells during hominoid evolution.

Teaser

A hominoid-specific ERV has rewired the gene regulatory network shared between PGCs and naïve pluripotent cells.
Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability
Authors: Ameen G. , Solanki S. , Sager-Bittara L. , Richards J. , Tamang P. , Friesen T.L. , Brueggeman R.S.
Published: March 2021

Disease lesion mimic mutants (DLMMs) are characterized by spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. Thenecmutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barleynec3mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Also, barleynec3mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens suggesting that they are not typical DLMMs. The F2progeny of a cross betweennec3-γ1 and variety Quest segregated as a single recessive gene post inoculation withBipolaris sorokiniana, the causal agent of the disease spot blotch.Nec3was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of wild type and five independentnec3mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated asnec3by independent mutations that result in predicted nonfunctional proteins. Histology studies determined thatnec3mutants had an unstable cutin layer that disrupted normalBipolaris sorokinianagerm tube development.

AUTHOR SUMMARY

At the site of pathogen infection, plant defense mechanisms rely on controlled programmed cell death (PCD) to sequester biotrophic pathogens that require living cells to extract nutrient from the host. However, these defense mechanisms are hijacked by necrotrophic plant pathogens that purposefully induce PCD mechanism to feed from the dead cells facilitating further disease development. Thus, understanding PCD responses is important for resistance to both classes of pathogens. We characterized five independent disease lesion mimic mutants of barley designated necrotic 3 (nec3) that show aberrant regulation of PCD responses upon pathogen challenge. A cytochrome P450 gene was identified asNec3encoding a Tryptamine 5-Hydroxylase that functions as a terminal serotonin biosynthetic enzyme in the Tryptophan pathway of plants. Thenec3mutants have disrupted serotonin biosynthesis resulting in expansive PCD, necrotrophic pathogen susceptibility and cutin layer instability. Thenec3mutants lacking serotonin deposition in pathogen induced necrotic lesions show expansive PCD and disease susceptibility suggesting a role of serotonin to sequester PCD and suppress pathogen colonization. The identification ofNec3will facilitate functional analysis to elucidate the role serotonin plays in the elicitation or suppression of PCD immunity responses to diverse pathogens and effects it has on cutin layer biosynthesis.
Effects of invasion level ofProsopis julifloraon native species diversity and regeneration in Afar region, Northeast Ethiopia
Authors: Shiferaw Wakshum
Published: March 2021

The study aimed to assess (i) the effects ofProsopis juliflorainvasion on the diversity of plant species, and floristic composition at Awash Fentale and the Amibara districts of the Afar region and (ii) the effects ofP. juliflorainvasion on the regeneration potential of native woody species. Sample collection was performed in habitats ofP. juliflorathicket,P. julifloramixed with native species stands, non-invaded woodlands, and open grazing lands. A stratified random sampling technique was used for data collection. Among species of plants, the highest proportion of species, 87 (27.4%), was recorded under non-invaded woodlands, but the lowest proportion of species, 70 (22%), was recorded under open grazing lands. The invasion level ofP. julifloracaused significantly reduced Shannon diversity index. The mean values of Shannon diversity index and species richness underP. julifloramixed with native species (H’=2.22, R=14) and non-invaded woodlands (H’=2.23, R=13) were significantly higher thanP. juliflorathicket (H’=1.96, R=12) and open grazing lands (H’=1.84, R=10). In this study, 102 trees ha-1native woody species were recorded underP. juliflorathicket, but 1252 trees ha-1native species were recorded under non-invaded woodlands. If the present effects of the invasion ofP. julifloraon native species diversity were to continue coupled with a drier climate, plant diversity of the Afar flora region will be highly affected. As a result the ecosystem services will be under threat. Thus, the participation of all stakeholders and multidisciplinary research approaches should be designed for the management of invaded rangelands to reverse the situation.
Expression and protein sequence analyses of zebrafishimpg2aandimpg2b, two proteoglycans of the interphotoreceptor matrix
Authors: Castellini M.E. , Spagnolli G. , Biasini E. , Casarosa S. , Messina A.
Published: March 2021

Photoreceptor outer segments projecting from the surface of the neural retina toward the retinal pigment epithelium (RPE) are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix (IPM) [1,2]. This extracellular compartment is necessary for physiological retinal function. However, specific roles for molecules characterizing the IPM have not been clearly defined [3]. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2)gene in patients affected by autosomal recessive Retinitis Pigmentosa (arRP) [4,5] and autosomal dominant and recessive vitelliform macular dystrophy (VMD) [6,7]. The gene encodes for a proteoglycan synthesized by photoreceptors and secreted in the IPM. However, little is known about the function and structure of this protein. We used the teleost zebrafish (D.rerio) as a model to studyIMPG2expression both during development and in adulthood, as its retina is very similar in humans [8]. In zebrafish, there are two IMPG2 proteins, IMPG2a and IMPG2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among different vertebrate species, showing a significant similarity despite the evolutionary distance between humans and teleosts. In fact, human IMPG2 andD.rerioIMPG2a and IMPG2b share conserved SEA and EGF-like domains. Homology models of these domains were obtained by using the iTasser server. Finally, expression analyses ofimpg2aandimpg2bduring development and in the adult fish showed expression of both mRNAs starting from 3 days post fertilization (dpf) in the outer nuclear layer of zebrafish retina that continues throughout adulthood. This data lays the groundwork for the generation of novel and most needed animal models for the study of IMPG2-related inherited retinal dystrophies.
Social learning about rewards – how information from others helps to adapt to changing environment
Authors: Winiarski M. , Borowska J. , Wołyniak R. M. , Jędrzejewska-Szmek J. , Kondrakiewicz L. , Mankiewicz L. , Chaturvedi M. , Turzyński K. , Wójcik D.K. , Puścian A. , Knapska E.
Published: March 2021

Being a part of a social structure is key for survival and reproduction. Living with conspecifics boosts evolutionary fitness, by providing essential information about the environment. Nonetheless, studying neural mechanisms of social learning has not yet been established under laboratory conditions. To examine how socially passed information about the reward affects the behavior of individuals we used Eco-HAB, an automated system for tracing voluntary behavior of group-housed mice living under semi-naturalistic conditions. We show that a scent of a rewarded individual has profound effects on the conspecifics’ ability to find the reward in both familiar and novel environments. Importantly, the animals display clear and stable individual differences in social behavior. As a result, socially conveyed information has different effects on individual mice. Further, we show that disrupting neuronal plasticity in the prelimbic cortex with nanoparticles gradually releasing TIMP metallopeptidase inhibitor 1, disrupts animals’ social behavior and results in decreased ability to adapt to environmental changes. The experimental paradigm we developed can be further used to study neuronal mechanisms of social learning.
Local association ofTrypanosoma cruzichronic infection foci and enteric neuropathic lesions at the tissue micro-domain scale
Authors: Khan Archie A. , Langston Harry C. , Costa Fernanda C. , Olmo Francisco , Taylor Martin C. , McCann Conor J. , Kelly John M. , Lewis Michael D.
Published: March 2021

Gastrointestinal (GI) disease affects a substantial subset of chronic Chagas disease (CD) patients, but the mechanism of pathogenesis is poorly understood. The lack of a robust, predictive animal model of chronicT. cruziinfection that exhibits functional digestive disease has held back research. To address this, we combined GI tracer assays and bioluminescencein vivoinfection imaging systems for diverse parasite strains to discover models exhibiting chronic digestive transit dysfunction. We identified the colon as a specific site of both tissue parasite persistence and delayed transit. Digestive CD mice exhibited significant retention of faeces in both sated and fasted conditions. Histological and immunofluorescence analysis of the enteric nervous system (ENS) revealed a dramatic reduction in the number of neurons and a loss of immunoreactivity of the enteric neural network in the colon. This model therefore recapitulates key clinical manifestations of human digestive CD. We also exploited dual bioluminescent-fluorescent parasites to analyse rare chronic infection foci in the colon at the single cell level, revealing co-localisation with ENS lesions. This indicates that long-termT. cruzi-host interactions in the colon drive pathogenesis and thus chronic disease may be preventable using anti-parasitic chemotherapy.
Identification and characterization of two transmembrane proteins required for virulence of Ustilago maydis
Authors: Weiland Paul , Altegoer Florian
Published: March 2021

Smut fungi comprise a large group of biotrophic phytopathogens infecting important crops such as wheat and corn. Through the secretion of effector proteins, the fungus actively suppresses plant immune reactions and modulates its host’s metabolism. Consequently, how soluble effector proteins contribute to virulence is already characterized in a range of phytopathogens. However, membrane-associated virulence factors have been much less studied to date. Here, we investigated six transmembrane (TM) proteins that show elevated gene expression during biotrophic development of the maize pathogen Ustilago maydis. We show that two of the six proteins, named Vmp1 and Vmp2 (virulence-associated membrane protein), are essential for the full virulence of U. maydis. The deletion of the corresponding genes lead to a substantial attenuation in the virulence of U. maydis. Furthermore, both are conserved in various related smuts and contain no domains of known function. Our biochemical analysis clearly shows that Vmp1 and Vmp2 are membrane-associated proteins, potentially localizing to the U. maydis plasma membrane. Mass photometry and light scattering suggest that Vmp1 mainly occurs as a monomer, while Vmp2 is dimeric. Notably, the large and partially unstructured C-terminal domain of Vmp2 is crucial for virulence while not contributing to dimerization. Taken together, we here provide an initial characterization of two membrane proteins as virulence factors of U. maydis.
Balance between extracellular matrix production and macrophage survival by aSalmonella-specific SPI-2 encoded transcription factor
Authors: Echarren María Laura , Figueroa Nicolás R. , Vitor-Horen Luisina , Pucciarelli M. Graciela , Portillo Francisco García-del , Soncini Fernando C.
Published: March 2021

Cellulose is a major component of theSalmonellabiofilm extracellular matrix and it is considered an antivirulence factor because it interferes withSalmonellasurvival inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm extracellular matrix formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work we identified a SPI-2 encodedSalmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, STM1389, also known asorf319, and ofcsgDinside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage survival. An increased expression ofcsgDis observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase coding-gene,bcsA, restored intramacrophage survival to rates comparable to wild type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and in consequence activation of the cellulose synthase. Our findings provide a novel link between biofilm formation andSalmonellavirulence.
Cost-effective long-read assembly of a hybrid Formica aquilonia × Formica polyctena wood ant genome from a single haploid individual
Authors: Nouhaud Pierre , Beresford Jack , Kulmuni Jonna
Published: March 2021

Formica red wood ants are a keystone species of boreal forest ecosystems and an emerging model system in the study of speciation and hybridization. Here we performed a standard DNA extraction from a single, field-collected Formica aquilonia × Formica polyctena haploid male and assembled its genome using ∼60× of PacBio long reads. After polishing and contaminant removal, the final assembly was 272 Mb (4,687 contigs, N50 = 1.16 Mb). Our reference genome contains 98.5% of the core Hymenoptera BUSCOs and was scaffolded using the pseudo-chromosomal assembly of a related species, F. selysi (28 scaffolds, N50 = 8.49 Mb). Around one third of the genome consists of repeats, and 17,426 gene models were annotated using both protein and RNAseq data (97.4% BUSCO completeness). This resource is of comparable quality to the few other single individual insect genomes assembled to date and paves the way to genomic studies of admixture in natural populations and comparative genomic approaches in Formica wood ants.
A linear response theory based method for prediction of large scale protein conformational changes upon ligand binding
Authors: Punia Rajat , Goel Gaurav
Published: March 2021

Prediction of ligand-induced protein conformational transitions is a challenging task due to a large and rugged conformational space, and limited knowledge of probable direction(s) of structure change. These transitions can involve a large scale, global (at the level of entire protein molecule) structural change and occur on a timescale of milliseconds to seconds, rendering application of conventional molecular dynamics simulations prohibitive even for small proteins. We have developed a computational protocol to efficiently and accurately predict these ligand-induced structure transitions solely from the knowledge of protein apo structure and ligand binding site. Our method involves a series of small scale conformational change steps, where at each step linear response theory is used to predict the direction of small scale global response to ligand binding in the protein conformational space (dLRT) followed by construction of a linear combination of slow (low frequency) normal modes (calculated for the structure from the previous step) that best overlaps withdLRT. Protein structure is evolved along this direction using molecular dynamics with excited normal modes (MDeNM) wherein excitation energy along each normal mode is determined by excitation temperature, mode frequency, and its overlap withdLRT. We show that excitation temperature (ΔT) is a very important parameter that allows limiting the extent of structural change in any one step and develop a protocol for automated determination of its optimal value at each step. We have tested our protocol for three protein–ligand systems, namely, adenylate Kinase – di(adenosine-5’)pentaphosphate, ribose binding protein –β-D-ribopyranose, and DNAβ-glucosyltransferase – uridine-5’-diphosphate, that incorporate important differences in type and range of structural changes upon ligand binding. We obtain very accurate prediction for not only the structure of final protein–ligand complex (holo-structure) having a large scale conformational change, but also for biologically relevant intermediates between the apo and the holo structures. Moreover, most relevant set of normal modes for conformational change at each step are an output from our method, which can be used as collective variables for determination of free energy barriers and transition timescales along the identified pathway.
Flexible reprogramming ofPristionchus pacificusmotivation for attackingCaenorhabditis elegansin predator-prey competition
Authors: Quach Kathleen T. , Chalasani Sreekanth H.
Published: March 2021

Animals with diverse diets must adapt their food priorities to a wide variety of environmental conditions. This diet optimization problem is especially complex for predators that compete with prey for food. Although predator-prey competition is widespread and ecologically critical, it remains difficult to disentangle predatory and competitive motivations for attacking competing prey. Here, we dissect the foraging decisions of the omnivorous nematodePristionchus pacificusto reveal that its seemingly failed predatory attempts againstCaenorhabditis elegansare actually motivated acts of efficacious territorial aggression. WhileP. pacificuseasily kills and eats larvalC. eleganswith a single bite, adultC. eleganstypically survives and escapes from bites. However, nonfatal biting can provide competitive benefits by reducing access of adultC. elegansand its progeny to bacterial food thatP. pacificusalso eats. We show thatP. pacificusconsiders costs and benefits of both predatory and territorial outcomes to decide which food goal, prey or bacteria, should guide its motivation for biting. These predatory and territorial motivations impose different sets of rules for adjusting willingness to bite in response to changes in bacterial abundance. In addition to biting, predatory and territorial motivations also influence which search tacticP. pacificususes to increase encounters withC. elegans. When treated with an octopamine receptor antagonist,P. pacificusswitches from territorial to predatory motivation for both biting and search. Overall, we demonstrate thatP. pacificusassesses alternate outcomes of attackingC. elegansand flexibly reprograms its foraging strategy to prioritize either prey or bacterial food.
An optogenetic tool to raise intracellular pH in single cells and drive localized membrane dynamics
Authors: Donahue Caitlin E. T. , Siroky Michael D. , White Katharine A.
Published: March 2021

Intracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use non-specific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes timescale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for approximately 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to unstimulated ArchT cells as well as control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and cell behaviors such as membrane ruffling. This tool will be transformative in facilitating experiments required to determine if increased pHi drives these cell behaviors at the single-cell level.
WNK1 Enhances Migration and Invasion in Breast Cancer Models
Authors: Jaykumar Ankita B. , Jung Jiung , Parida Pravat , Dang Tuyen T. , Grzemska Magdalena , Earnest Svetlana , Wichaidit Chonlarat , Goldsmith Elizabeth J. , Pearson Gray W. , Malladi Srinivas , Cobb Melanie H.
Published: March 2021

Metastasis is the major cause of mortality in breast cancer patients. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase WNK1 (with no lysine (K) 1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.
Prioritization of regulatory variants with tissue-specific function in the non-coding regions of human genome
Authors: Dong Shengcheng , Boyle Alan P.
Published: March 2021

Understanding the functional consequences of genetic variation in the non-coding regions of the human genome remains a challenge. We introduce here a computational tool, TURF, to prioritize regulatory variants with tissue-specific function by leveraging evidence from functional genomics experiments, including over three thousand functional genomics datasets from the ENCODE project provided in the RegulomeDB database. TURF is able to generate prediction scores at both organism and tissue/organ-specific levels for any non-coding variant on the genome. We present that TURF has an overall top performance in prediction by using validated variants from MPRA experiments. We also demonstrate how TURF can pick out the regulatory variants with tissue-specific function over a candidate list from associate studies. Furthermore, we found that various GWAS traits showed the enrichment of regulatory variants predicted by TURF scores in the trait-relevant organs, which indicates that these variants can be a valuable source for future studies.
Hippocampal replay reflects specific past experiences rather than a plan for subsequent choice
Authors: Gillespie Anna K. , Astudillo Maya Daniela A. , Denovellis Eric L. , Liu Daniel F. , Kastner David B. , Coulter Michael E. , Roumis Demetris K. , Eden Uri T. , Frank Loren M.
Published: March 2021

Executing memory-guided behavior requires both the storage of information about experience and the later recall of that information to inform choices. Awake hippocampal replay, when hippocampal neural ensembles briefly reactivate a representation related to prior experience, has been proposed to critically contribute to these memory-related processes. However, it remains unclear whether awake replay contributes to memory function by promoting the storage of past experiences, by facilitating planning based on an evaluation of those experiences, or both. We designed a dynamic spatial task which promotes replay before a memory-based choice and assessed how the content of replay related to past and future behavior. We found that replay content was decoupled from subsequent choice and instead was enriched for representations of previously rewarded locations and places that had not been recently visited, indicating a role in memory storage rather than in directly guiding subsequent behavior.
SSR1 is a vital regulator in plant mitochondrial iron-sulfur biosynthesis
Authors: Feng Xuanjun , Han Huiling , Bonea Diana , Liu Jie , Ying Wenhan , Cai Yuanyuan , Zhang Min , Lu Yanli , Zhao Rongmin , Hua Xuejun
Published: March 2021

The ArabidopsisSHORT AND SWOLLEN ROOT1(SSR1) gene encodes a mitochondrial TPR domain-containing protein and was previously reported to function in maintaining mitochondria function. In a screen for suppressors of the short-root phenotype of the loss-of-function mutantssr1-2, two mutations,sus1andsus2(suppressor ofssr1-2), were isolated.sus1andsus2result from G87D and T55M single amino acid substitution in HSCA2 (At5g09590) and ISU1 (At4g22220), both of which are core components in iron-sulfur cluster biosynthesis pathway in mitochondria (ISC). We here demonstrated that SSR1 displayed a strong chaperone-like activity and was able to enhance the binding of HSCA2 to ISU1, an essential step for the normal operation of ISC machinery. Accordingly, the enzymatic activities of several iron-sulfur proteins, the mitochondrial membrane potential and ATP content are reduced inssr1-2. Interestingly,SSR1appears to exist only in plant lineages, possibly conferring adaptive advantages on plant ISC machinery to environment.
High resolution structure of the membrane embedded skeletal muscle ryanodine receptor
Authors: Melville Zephan , Kim Kookjoo , Clarke Oliver B. , Marks Andrew R.
Published: March 2021

The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel, though a recent study resolved the structure with limited resolution in nanodiscs1. In the present study we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, a method that improved the incorporation rate by more than 20-fold compared to a dialysis-based approach. In conjunction with new direct-detection cameras, this allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 Å, respectively. This method offers validation for detergent-based structures of the RyR and lays the groundwork for studies utilizing an electrochemical gradient mimicking the native environment, such as that of the SR, where Ca2+concentrations are millimolar in the lumen and nanomolar in the cytosol of the cell at rest.
KDM6A knockout in human iPSCs alters the genome-wide histone methylation profile at active and poised enhancers, activating expression of ectoderm gene expression pathways
Authors: Maurya Shailendra S. , Yang Wei , Zhang Qiang , Erdmann-Gilmore Petra , Bystry Amelia , Townsend Reid , Druley Todd E.
Published: March 2021

KDM6A is a histone demethylase, known to remove methyl moieties at the lysine residues of histone 3-labeled (H3K27me3) poised enhancers and bivalent promoters, which regulates gene expression during the differentiation of embryonic stem cells and tissue-specific development. However, while tissue- and disease-specific analyses have been performed, little is known about the location and consequences on gene expression of these regulatory regions in human pluripotent cells. Poised enhancers and bivalent promoters function in a coordinated fashion during development, which requires timely and efficient histone modifications. Identification of KDM6A-specific gene-regulatory domains is important for understanding the developmental mechanisms controlled by these histone modifications in pluripotency. In this study, we compared genome-wide histone modification and gene expression differences in isogenic wild type and cas9-mediatedKDM6Aknockout human induced pluripotent stem cells (hiPSC) lines. Here, we report that the absence of KDM6A does not alter the pluripotent phenotype but does substantially alter the histone modification profile at poised and active enhancers, resulting in decreased expression of associated COMPASS complex genes KMT2C and KMT2D and subsequently increasing the expression of gene pathways involved in ectoderm differentiation.
Movement patterns and activity levels are shaped by the neonatal environment in Antarctic fur seal pups
Authors: Nagel Rebecca , Mews Sina , Adam Timo , Stainfield Claire , Fox-Clarke Cameron , Toscani Camille , Langrock Roland , Forcada Jaume , Hoffman Joseph I.
Published: March 2021

Tracking studies of juveniles are rare compared to those of adults, and consequently little is known about the influence of intrinsic and extrinsic factors on activity during this critical life stage. We used hourly GPS data, collected from 66 Antarctic fur seal pups from birth until moulting, to investigate the explanatory power of multiple individual-based and environmental variables on activity levels. Pups were sampled from two nearby breeding colonies of contrasting density during two subsequent years, and a two-state hidden Markov model was used to identify modalities in their movement behaviour, specifically ‘active’ and ‘inactive’ states. We found that movement was typified by central place exploration, with active movement away from and subsequent return to a location of inactivity. The probability of such directed exploration was unaffected by several factors known to influence marine mammal movement including sex, body condition, and temperature. Compared to pups born at the high-density colony, pups at low-density were more active, increased their activity with age, and transitioned earlier into the tussock grass, which offers protection from predators and extreme weather. Our study illustrates the importance of extrinsic factors, such as colony of birth, to early-life activity patterns and highlights the adaptive potential of movement.
A recombinant ‘ACE2 Triple Decoy’ that traps and neutralizes SARS-CoV-2 shows enhanced affinity for highly transmissible SARS-CoV-2 variants
Authors: Tanaka Shiho , Nelson Gard , Olson Anders , Buzko Oleksandr , Higashide Wendy , Shin Annie , Gonzales Marcos , Taft Justin , Patel Roosheel , Buta Sofija , Martin-Fernandez Marta , Bogunovic Dusan , Spilman Patricia , Niazi Kayvan , Rabizadeh Shahrooz , Soon-Shiong Patrick
Published: March 2021

The highly-transmissible SARS-CoV-2 variants now replacing the first wave strain pose an increased threat to human health by their ability, in some instances, to escape existing humoral protection conferred by previous infection, neutralizing antibodies, and possibly vaccination. Thus, other therapeutic options are necessary. One such therapeutic option that leverages SARS-CoV-2 initiation of infection by binding of its spike receptor binding domain (S RBD) to surface-expressed host cell angiotensin-converting enzyme 2 (ACE2) is an ACE2 ‘decoy’ that would trap the virus by competitive binding and thus inhibit propagation of infection. Here, we used Molecular Dynamic (MD) simulations to predict ACE2 mutations that might increase its affinity for S RBD and screened these candidates for binding affinityin vitro. A double mutant ACE2(T27Y/H34A)-IgG1FCfusion protein was found to have very high affinity for S RBD and to show greater neutralization of SARS-CoV-2 in a live virus assay as compared to wild type ACE2. We further modified the double mutant ACE2 decoy by addition of an H374N mutation to inhibit ACE2 enzymatic activity while maintaining high S RBD affinity. We then confirmed the potential efficacy of our ACE2(T27Y/H34A/H374N)-IgG1FCTriple Decoy against S RBD expressing variant-associated E484K, K417N, N501Y, and L452R mutations and found that our ACE2 Triple Decoy not only maintains its high affinity for S RBD expressing these mutations, but shows enhanced affinity for S RBD expressing the N501Y or L452R mutations and the highest affinity for S RBD expressing both the E484K and N501Y mutations. The ACE2 Triple Decoy also demonstrates the ability to compete with wild type ACE2 in the cPass™ surrogate virus neutralization in the presence of S RBD with these mutations. Additional MD simulation of ACE2 WT and decoy interactions with S RBD WT or B.1.351 variant sequence S RBD provides insight into the enhanced affinity of the ACE2 decoy for S RBD and reveals its potential as a tool to predict affinity and inform therapeutic design. The ACE2 Triple Decoy is now undergoing continued assessment, including expression by a human adenovirus serotype 5 (hAd5) construct to facilitate deliveryin vivo.

Summary sentence

An ACE2(N27Y/H34A/H374N)-IgG1FCfusion protein decoy sustains high affinity to all SARS-CoV-2 spike receptor binding domain (RBD) protein variants tested, shows enhanced affinity for the N501Y and L452R variants, and the highest affinity for combined N501Y and E484K variants.
Fresh extension ofVibrio choleraecompetence type IV pili predisposes them for motor-independent retraction
Authors: Chlebek Jennifer L. , Dalia Triana N. , Biais Nicolas , Dalia Ankur B.
Published: March 2021

Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated thatVibrio choleraecompetence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization.

SIGNIFICANCE

Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus ofVibrio choleraeto study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the external pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.