bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA methyltransferase 1 (DNMT1) function is implicated in the age-related loss of cortical interneurons
Authors: Hahn Anne , Bayer Cathrin , Pensold Daniel , Tittelmeier Jessica , Marx-Blümel Lisa , González-Bermúdez Lourdes , Linde Jenice , Groß Jonas , Salinas-Riester Gabriela , Lingner Thomas , von Maltzahn Julia , Spehr Marc , Pieler Tomas , Urbach Anja , Zimmer-Bensch Geraldine
Published: March 2020

Increased life expectancy in modern society comes at the cost of age-associated disabilities and diseases. Aged brains not only show reduced excitability and plasticity, but also a decline in inhibition. Age-associated defects in inhibitory circuits likely contribute to cognitive decline and age-related disorders. Molecular mechanisms that exert epigenetic control of gene expression, contribute to age-associated neuronal impairments. Both DNA methylation, mediated by DNA methyltransferases (DNMTs), and histone modifications maintain neuronal function throughout lifespan. Here we provide evidence that DNMT1 function is implicated in the age-related loss of cortical inhibitory interneurons. Deletion ofDnmt1in parvalbumin-positive interneurons attenuates their age-related decline in the cerebral cortex. Moreover, DNMT1-deficient mice show improved somatomotor performance and reduced aging-associated transcriptional changes. A decline in the proteostasis network, responsible for the proper degradation and removal of defective proteins, is suggested to be essentially implicated in age- and disease-related neurodegeneration. Our data suggest that DNMT1 acts indirectly on interneuron survival in aged mice by modulating the proteostasis network during life-time.
Bbs5 functions as the docking compartment of Bbsome binding to Klc3 regulated by Bbs5-Ser246PKC-phosphorylation during mouse spermatogenesis
Authors: Yu Dahai , He Lixia , Zhou Xin , Wang Xiuxia , Yu Bingzhi
Published: March 2020

A mitochondrial and a fibrous sheath form the midpiece of the mammalian sperm flagellum encircling most of the axoneme. It has been documented that Kinesin light chain 3 (KLC3) was involved although the formation procedure remains unclear. Yeast-two-hybrid dataset showed an interaction between Klc3 and Bardet-Biedl Syndrome 5 (BBS5) Protein, another molecular associated with cilia and flagella forming. In this study, we presumed that the most conserved IFT complex BBsome was involved in spermatogenesis via the interaction of one of its subunits, Bbs5 with Klc3. Firstly, the interaction between Klc3 and Bbs5 was confirmed with Co-IP. Secondly, we identified PKC phosphorylation sitesin vitroby LC-MS/MS, Ser19and Ser246of Bbs5, examined the phosphorylation status of Bbs5 Ser19and Ser246in mouse testis. Co-IP was performed to find which PKC isoforms phosphorylate Bbs5. In addition, we tried to discuss the roles of Ser19and Ser246of Bbs5 in the Klc3-bbs5 interaction and in mouse spermatogenesis based on our early findings.
One-Day Construction Of Multiplex Arrays to Harness Natural CRISPR Systems
Authors: Cooper Robert M. , Hasty Jeff
Published: March 2020

CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable – we assembled 9-spacer arrays with one day’s worth of work. We used this method to harness the endogenous CRISPR system of the highly competent bacteriumAcinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense vs. DNA acquisition in naturally competentA. baylyi.
Plasticity and Artificial Selection for Developmental Mode in a Poecilogonous Sea Slug
Authors: Caplins Serena A.
Published: March 2020

Developmental mode describes the means by which larvae are provisioned with the nutrients they need to proceed through development and typically results in a trade-off between offspring size and number. The sacoglossan sea slugAlderia willowiexhibits intraspecific variation for developmental mode (= poecilogony) that is environmentally modulated with populations producing more yolk-feeding (lecithotrophic) larvae during the summer, and more planktonic feeding (planktotrophic) larvae in the winter. I found significant family level variation in the reaction norms between 17 maternal families ofA. willowiwhen reared in low (16 ppt) versus high (32 ppt) salinity. I documented a significant response to selection for lecithotrophic larvae, the proportion of which increased 32% after three generations of selection in high salinity, and 18% after 2 generations in low salinity (realized heritability: 0.365 ± 0.024). The slope of the reaction norm was maintained following one generation of selection for lecithotrophy and one generation of selfing. The rapid response to selection favoring one developmental mode may speak to the rarity of intraspecific variation for developmental mode, which could fix for one mode over another much more readily than has generally been assumed from studies of less plastic organisms.
A gut sensor for sugar preference
Authors: Buchanan Kelly L. , Rupprecht Laura E. , Sahasrabudhe Atharva , Kaelberer M. Maya , Klein Marguerita , Villalobos Jorge , Liu Winston W. , Yang Annabelle , Gelman Justin , Park Seongjun , Anikeeva Polina , Bohórquez Diego V.
Published: March 2020

Summary Paragraph/Abstract

Animals innately prefer caloric sugars over non-caloric sweeteners. Such preference depends on the sugar entering the intestine.1–4Although the brain is aware of the stimulus within seconds,5–8how the gut discerns the caloric sugar to guide choice is unknown. Recently, we discovered an intestinal transducer, known as the neuropod cell.9,10This cell synapses with the vagus to inform the brain about glucose in the gut in milliseconds.10Here, we demonstrate that neuropod cells distinguish a caloric sugar from a non-caloric sweetener using the electrogenic sodium glucose co-transporter 1 (SGLT1) or sweet taste receptors. Activation of neuropod cells by non-caloric sucralose leads to ATP release, whereas the entry of caloric sucrose via SGLT1 stimulates glutamate release. To interrogate the contribution of the neuropod cell to sugar preference, we developed a method to record animal preferences in real time while using optogenetics to silence or excite neuropod cells. We discovered that silencing these cells, or blocking their glutamatergic signaling, renders the animals unable to recognize the caloric sugar. And, exciting neuropod cells leads the animal to consume the non-caloric sweetener as if it were caloric. By transducing the precise identity of the stimuli entering the gut, neuropod cells guide an animal’s internal preference toward the caloric sugar.
Oligomeric amyloid beta prevents myelination in a clusterin-dependent manner
Authors: Beiter Rebecca M. , Ennerfelt Hannah E. , Rivet-Noor Courtney , Merchak Andrea , Bai Robin , Johanson David M. , Slogar Erica , Sol-Church Katia , Phillips Jennifer L. , Bathe Tim , Overall Christopher C. , Lukens John R. , Prokop Stefan , Gaultier Alban
Published: March 2020

White matter loss has been described as a common occurrence in Alzheimer’s disease (AD) patients for multiple decades. However, it remains unclear why oligodendrocyte progenitor cells (OPCs) fail to repair myelin deficits in these patients. Here, we show that clusterin, a risk factor for late-onset AD, is produced by OPCs and inhibits their differentiation into oligodendrocytes. Specifically, we demonstrate that a unique subset of OPCs produces clusterin. We show that phagocytosis of debris, including amyloid beta (Aβ) and myelin, drives the upregulation of clusterin in OPCs. We confirm,in vivo, that Aβ oligomers drive clusterin upregulation and that OPCs phagocytose Aβ. Furthermore, we show that clusterin is a potent inhibitor of OPC differentiation and prevents the production of myelin proteins. Finally, we demonstrate that clusterin inhibits OPC differentiation by significantly reducing the production of IL-9 by OPCs. Our data reveals that clusterin may be responsible for the lack of myelin repair observed in AD and is a promising therapeutic target for AD-associated cognitive decline.
Molecular structure of a prevalent amyloid-β fibril polymorph from Alzheimer’s disease brain tissue
Authors: Ghosh Ujjayini , Thurber Kent R. , Yau Wai-Ming , Tycko Robert
Published: March 2020

Amyloid-β (Aβ) fibrils exhibit self-propagating, molecular-level polymorphisms that may underlie variations in clinical and pathological characteristics of Alzheimer’s disease. We report the molecular structure of a specific brain-derived polymorph that has been identified as the most prevalent polymorph of 40-residue Aβ fibrils in cortical tissue of Alzheimer’s disease patients. This structure, developed from cryo-electron microscopy and supported by solid state NMR data, differs qualitatively from all previously described Aβ fibril structures, both in its molecular conformation and its organization of cross-β subunits. Knowledge of this brain-derived fibril structure may contribute to the development of structure-specific amyloid imaging agents and aggregation inhibitors with greater diagnostic and therapeutic utility.
Examining the relationship between social cognition and neural synchrony during movies in children with and without autism
Authors: Lyons KM , Stevenson RA , Owen AM , Stojanoski B
Published: March 2020

Children who have been diagnosed with autism spectrum disorder (ASD) often show a marked deficit in measures of social cognition. In autistic adults, measures of social cognition have been shown to relate to differences in brain synchronization (as measured by fMRI) when individuals are processing naturalistic stimuli, such as movies. However, whether children with social impairments, with or without a diagnosis of ASD, differ in their neural responses to movies has not yet been investigated. In the current study, neural synchrony, measured using fMRI, was examined in three groups of children aged 7 to 12, who differed with respect to scores on a measure of autistic traits associated with social impairment and whether or not they had been diagnosed with ASD. While watching the movie ‘Despicable Me’, those diagnosed with ASD had significantly less neural synchrony in areas that have been previously shown to be associated with social cognition (e.g. areas related to ‘theory of mind’), and plot following (e.g. the lateral prefrontal cortex), than those who did not have an ASD diagnosis. In contrast, two groups who differed in their degree of social impairment, but did not have a diagnosis of ASD, showed no significant differences in neural synchrony across the whole brain. These results shed some light on how autistic traits may contribute to an individual’s conscious experience of the world, and how, for children with ASD, that experience may differ markedly from that of those without ASD.
Single-Cell Atlas of Early Human Brain Development Highlights Heterogeneity of Human Neuroepithelial Cells and Early Radial Glia
Authors: Eze Ugomma C. , Bhaduri Aparna , Haeussler Maximilian , Nowakowski Tomasz J. , Kriegstein Arnold R.
Published: March 2020

The human cortex is comprised of diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain, and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.
Kinetics of charged polymer collapse: effects of additional salt
Authors: Ghosh Susmita , Vemparala Satyavani
Published: March 2020

Extensive molecular dynamics simulations, using simple charged polymer models, have been employed to probe the kinetics and dynamics of early-stage collapse of charged polymers and the effect of additional monovalent salt on such kinetics. The exponents characterizing the coarsening dynamics during such early-collapse stage via finite size scaling for the case of charged polymers are found to be different from the neutral polymers, suggesting that the collapse kinetics of charged polymers are inherently different from that neutral polymers. The kinetics of coarsening of the clusters along the collapsed trajectory also depends significantly on the counterion valency and for higher valency counterions, multiple regimes are observed and unlike the neutral polymer case, the collapse kinetics are a function of charge density along the charged polymer. Inclusion of additional salt affects the kinetics and conformational landscape along the collapse trajectory. Addition of salt increases the value of critical charge density required to initiate collapse for all the counterion valencies, though the effect is more pronounced for monovalent counterion systems. The addition of salt significantly affects the collapse trajectory in the presence of trivalent counterions via promotion of transient long-distance loop structures inducing a parallel and hierarchical local collapsed conformation leading to faster global collapsed states. This may play a role in understanding the fast folding rates of biopolymers such as proteins and RNA from extended state to a collapsed state in the presence of multivalent counterions before reorganizing into a native fold.
Endothelial Cells stimulate proliferation of CD140a sorted human Glial Progenitor Cells and their specification towards astrocytic lineage
Authors: Dasgupta Asmita
Published: March 2020

Homeostasis in stem cell niche is established by fine-tuning between interacting signaling pathways of all resident cell types that constitute the niche. In this context, the exact contribution of human endothelial cells in biology of PDGFRα positive human glial progenitor cells (hGPCs) in niche environment is not well understood. Towards the same, insert co-culture system with human umbilical vein endothelial cells (HUVECs) has been adopted. In co-culture with HUVECs, under proliferative condition, hGPCs show increased proliferation and sphere formation, while under differentiating condition hGPCs show increased differentiation to astrocytes with concomitant decrease in differentiation to oligodendrocytes with respect to no co-culture controls. Transcript assay for selected humoral factors reveals bone morphogenic proteins (BMPs), endothelin1, growth arrest specific 6 and interleukin 6 to be in higher abundance in HUVECS than hGPCs, of which BMP4 transcripts were most abundant, indicating possibilities of its being the key mediator of endothelial mediated effects. Concurrently, noggin effectively attenuates HUVEC mediated astrocytic differentiation of PDGFRα sorted fetal hGPCs The results have implication towards safety of transplantation therapies with PDGFRα sorted fetal hGPCs. It is postulated that proliferation and differentiation response as seen in this co-culture system could have defining implications towards the genesis of glioma.
Converting lateral scanning into axial focusing to speed up 3D microscopy
Authors: Chakraborty Tonmoy , Chang Bo-Jui , Daetwyler Stephan , Sapoznik Etai , Chen Bingying , Dean Kevin M. , Fiolka Reto
Published: March 2020

In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of 3D volumetric imaging. Recently, by conjugating either a movable-mirror to the image plane or an electrotuneable lens (ETL) to the back-focal plane respectively, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil based actuators), while ETLs introduce spherical and higher order aberrations, thereby preventing high-resolution imaging. Here, we introduce a novel optical design that can transform a lateral-scan motion into a spherical-aberration-free, high-resolution, rapid axial scan. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in image space. We characterize the optical performance of this remote focusing technique and use it to accelerate axially swept light-sheet microscopy (ASLM) by one order of magnitude, allowing the quantification of rapid vesicular dynamics in 3D.
Beyond endemism, expanding conservation efforts: a relictual Pleistocene distribution and first report of the prickly pear cactus,Opuntia bonaerensis,in Brazil and Uruguay
Authors: Köhler Matias , Esser Luíz F. , Font Fabián , Souza-Chies Tatiana T. , Majure Lucas C.
Published: March 2020

Geographical range is one of the critical features for species conservation assessment. Nevertheless, species distribution is frequently unknown, undervalued or overlooked. During a broad taxonomic and floristic study of the southern South American prickly pear species (Opuntiaspp.), new records of a previously endemic Argentinian taxon have been found in Uruguay and Brazil. Molecular phylogenetic inference was carried out to further evaluate the identity of the new records, and ecological niche models were implemented to test how the new records would fit in the previous known distribution of the species. Through molecular and morphological evidence, we confirmed the new records ofOpuntia bonaerensisfor Brazilian and Uruguayan floras and discussed its phylogenetic relationship and morphologic similarities with closely related species. Our new records uncovered a distributional pattern congruent with the Neotropical Peripampasic Orogenic Arc, which must be further explored to better determine the biogeographic history of the species. Ecological niche models (ENM) revealed thatO. bonaerensishad a putative ancient distribution across the grasslands and shrublands in the Pampean region largely congruent with the populations found in Brazil and Uruguay, suggesting relictual Pleistocene populations of the species and the role of glacial/interglacial cycles on the distribution of the species. In a prospective climate change scenario, ENM suggests that the species would in general be more restricted to the southernmost limits of the Pampa region and previous outlying records from Mendoza (Argentina) are a putative future refuge forO. bonaerensis. The importance of these new records for biodiversity and conservation assessment efforts that are ongoing at different scales in Brazil and neighbor areas is highlighted.
Porcupine: Rapid and robust tagging of physical objects using nanopore-orthogonal DNA strands
Authors: Doroschak Kathryn , Zhang Karen , Queen Melissa , Mandyam Aishwarya , Strauss Karin , Ceze Luis , Nivala Jeff
Published: March 2020

Molecular tagging is an approach to labeling physical objects using DNA or other molecules that can be used in cases where methods like RFID tags and QR codes are not suitable. No molecular tagging method exists that is inexpensive, fast and reliable to decode, and usable outside a lab setting to create or read tags. To address this, we present Porcupine, an end-user molecular tagging system that features DNA-based tags readable within seconds using a portable nanopore device. Porcupine’s digital bits are represented by the presence or absence of distinct, nanopore-orthogonal DNA strands, which we call molecular bits (molbits). We classify molbits directly from the raw nanopore signal, avoiding basecalling. To extend the tag’s shelf life, decrease readout time, and make tags robust to environmental contamination, molbits are prepared for readout during tag assembly and can be stabilized by dehydration. The result is an extensible, real time, high accuracy tagging system that includes a novel approach to developing nanopore-orthogonal barcodes.

One sentence summary

Porcupine lets end-users label physical objects with custom DNA tags, without requiring a lab to create or read tags, and offers rapid readout using nanopore sequencing.
Reassessment and typification ofOpuntia canterae(Opuntioideae, Cactaceae), an endemic prickly-pear cactus of Uruguay
Authors: Köhler Matias , Majure Lucas C.
Published: March 2020

Background and aims

Opuntiais the most widespread genus of Cactaceae, naturally occurring throughout arid and semi-arid areas of the Americas. Many of the species have taxonomic problems owed to incomplete original descriptions, lack of type designations, a paucity of taxonomic revisions and in general, difficult species delimitation resulting from hybridization, morphological plasticity and poor specimen preparation. However, efforts are being undertaken to fill in the gaps in our distributional, morphological and phylogenetic knowledge of the group. Here, we reassess the nameOpuntia canterae, providing an updated description, typification, photographs, distribution map, conservation assessment and additional notes.

Material and methods

Extensive fieldwork was conducted, along with comprehensive herbarium and literature review. Morphological characters were assessed based on the commonly used characters used for prickly pears. Species delimitation is proposed based on our morphological studies, taxonomic and literature revision, as well as preliminary phylogenetic studies. The IUCN guidelines were followed to provide a conservation assessment of the species.

Key results

Opuntia canteraeis reassessed as a distinct species separated from its previous synonym (O. elata) by the elliptic to long-oblanceolate stem segments, acute to conical flower bud apex and long-obconic fruits. An epitype is here designate for purposes of the precise designation of the name to the taxon. The species is considered endemic to Uruguay and is assessed as Vulnerable (VU) using IUCN criteria, but more fieldwork will be necessary to provide a more precise conservation status.
Goal-dependent tuning of muscle spindle receptors during movement preparation
Authors: Papaioannou Stylianos , Dimitriou Michael
Published: March 2020

Voluntary movements are believed to be advantageously prepared before they are executed, but the neural mechanisms at work have been unclear. For example, there are no overt changes in skeletal muscle activity during movement preparation. Here, using a delayed-reach manual task, we demonstrate a decrease in the firing rate of human muscle afferents (primary spindles) when preparing stretch rather than shortening of the spindle-bearing muscle. This goal-dependent modulation of proprioceptors begun early after target onset but was markedly stronger at the latter parts of the preparatory period. In two additional experiments, whole-arm perturbations during reach preparation revealed a congruent modulation of stretch reflex gains of shoulder and upper arm muscles. Our study shows that movement preparation can involve sensory elements of the peripheral nervous system. We suggest that central preparatory activity can also reflect sensory control, and preparatory tuning of muscle spindle mechanoreceptors is a component of planned reaching movements.
Eukaryotic transposable elements as “cargo carriers”: the forging of metal resistance in the fungusPaecilomyces variotii
Authors: Urquhart Andrew S. , Chong Nicholas F. , Yang Yongqing , Idnurm Alexander
Published: March 2020

The horizontal transfer of large gene clusters by mobile elements is a key driver of prokaryotic adaptation in response to environmental stresses. Eukaryotic microbes face similar environmental stresses yet a parallel role for mobile elements has not yet been established. A stress faced by all microorganisms is the prevalence of toxic metals in their environment. In fungi, identified mechanisms for protection against metals generally rely on genes that are dispersed within an organism’s genome. Here we have discovered a large (∼85 kb) region that confers resistance to several metals in the genomes of some, but not all, strains of a fungus,Paecilomyces variotii. We name this regionHEPHAESTUS(Hϕ) and present evidence that this region is mobile within theP. variotiigenome with features highly characteristic of a transposable element. While large gene clusters including those for the synthesis of secondary metabolites have been widely reported in fungi, these are not mobile within fungal genomes.HEPHAESTUScontains the greatest complement of host-beneficial genes carried by a transposable element in eukaryotes. This suggests that eukaryotic transposable elements might play a role analogous to their bacterial counterparts in the horizontal transfer of large regions of host-beneficial DNA. Genes withinHEPHAESTUSresponsible for individual metal resistances include those encoding a P-type ATPase transporter, PcaA, required for cadmium and lead resistance, a transporter, ZrcA, providing resistance to zinc, and a multicopper oxidase, McoA, conferring resistance to copper. Additionally, a subregion ofHϕconferring resistance to arsenate was identified. The presence of a strikingly similar cluster in the genome of another fungus,Penicillium fuscoglaucum, suggests thatHEPHAESTUSarrived inP. variotiivia horizontal gene transfer.
Quantitative differences in neuronal subpopulations between mouse and human dorsal root ganglia demonstrated with RNAscopein situhybridization
Authors: Shiers Stephanie , Klein Rebecca M. , Price Theodore J
Published: March 2020

Next generation transcriptomics in combination with imaging-based approaches have emerged as powerful tools for the characterization of dorsal root ganglion (DRG) neuronal subpopulations. The mouse DRG has been well-characterized by many independently conducted studies with convergent findings, but few studies have directly compared expression of population markers between mouse and human. This is important because of our increasing reliance on the mouse as a preclinical model for translational studies. While calcitonin gene-related peptide (CGRP) and P2X purinergic ion channel type 3 receptor (P2X3R) have been used to define peptidergic and non-peptidergic nociceptor subpopulations, respectively, in mouse DRG, these populations may be different in other species. To directly test this, as well as a host of other markers, we used multiplex RNAscopein-situhybridization to elucidate the distribution of a multitude of unique and classic neuronal mRNAs in peptidergic (CGRP expressing) and non-peptidergic (P2X3R expressing) nociceptor subpopulations in mouse and human DRG. We found a large overlapping CGRP and P2X3R neuronal subpopulation in human, lumbar DRG that was not present in mouse. We also found differential expression in a variety of mRNAs for Trp-channels, cholinergic receptors, potassium channels, sodium channels, other markers/targets. These data offer insights into the spatial and functional organization of neuronal cell subpopulations in the rodent and human DRG and support the idea that sensory system organizational principles are likely different between both species.
The mesolimbic dopamine signatures of relapse to alcohol-seeking
Authors: Liu Yu , Jean-Richard-dit-Bressel Philip , Yau Joanna Oi-Yue , Willing Alexandra , Prasad Asheeta A. , Power John M. , Killcross Simon , Clifford Colin W.G. , McNally Gavan P.
Published: March 2020

The mesolimbic dopamine system comprises distinct compartments supporting different functions in learning and motivation. Less well understood is how complex addiction-related behaviors emerge from activity patterns across these compartments. Here we show how different forms of relapse to alcohol-seeking are assembled from activity across the ventral tegmental area and the nucleus accumbens. Using gCaMP and dLight fibre photometry, we show that self-administration and two forms of relapse (renewal/context-induced reinstatement and reacquisition) are associated with recruitment across the mesolimbic dopamine system. Using a variety of interventions, we show that this activity is causal to both forms of relapse. Finally, we use dissimilarity matrices to identify mesolimbic dopamine signatures of self-administration, extinction, and relapse. We show that signatures of relapse can be identified from heterogeneous activity profiles across the mesolimbic dopamine system and that these signatures differ for different forms of relapse.
CircGLCE alleviates intervertebral disc degeneration by regulating apoptosis and matrix degradation through the targeting of miR-587/STAP1
Authors: Chen Zhonghui , Zhang Weibing , Deng Ming , Zhou Yan , Li Yaming
Published: March 2020

Background

Intervertebral disc degeneration (IDD) can induce profound global socioeconomic burdens. Recent studies have suggested that circular RNAs might have crucial functions in the progression of IDD. The purpose of this study was to identify a specific circular RNA and to investigate its regulatory mechanism in IDD.

Methods

CircGLCE was selected after microarray analyses and was further analysed by RT-qPCR and FISH. After silencing CircGLCE, its function was assessed with RT-qPCR, immunofluorescence analysis and flow cytometry. Based on Sanger sequencing, miR-587 was identified as a direct target of CircGLCE, and it was further examined with RNA pulldown assays, RT-qPCR, dual luciferase assays and FISH. After silencing CircGLCE or miR-587, western blotting, immunofluorescence analysis, and flow cytometry were conducted. STAP1 was assessed by RT-qPCR and luciferase assay, and experiments with silenced and overexpressed miR-587 were performed. A rescue experiment was also included. In an IDD rat model, the in vivo effects of overexpressing CircGLCE on IDD were analysed with imaging techniques, TUNEL staining, FISH, western blotting, H&E staining and immunohistochemistry.

Results

CircGLCE was found to stably exist in the cytoplasm of nucleus pulposus (NP) cells. It was downregulated in IDD. Knockdown of CircGLCE promoted apoptosis and induced the expression of matrix-degrading enzymes in NP cells. CircGLCE served as a miR-587 sponge in NP cells. Inhibiting miR-587 counteracted the IDD-enhancing effect caused by silencing CircGLCE. STAP1 served as the miRNA target that mediated the functions of miR-587. Overexpressing CircGLCE alleviated IDD in vivo.

Conclusions

CircGLCE attenuates IDD by regulating the apoptosis of NP cells and by regulating ECM degradation through the targeting of miR-587/STAP1. CircGLCE may be a potential therapeutic target for IDD treatments.
SPINK1-induced amelioration of impaired autophagy contributes to suppression of trypsinogen activation in a model of acute pancreatitis
Authors: Zhao Yuxiao , Jia Jianlong , Shopit Abdullah , Liu Yang , Wang Jun
Published: March 2020

SPINK1 has been regarded as a reversible trypsinogen inhibitor for the inappropriate activation of trypsin, a key step in the initiation of acute pancreatitis (AP). However, the mechanisms of its action remains largely unclear and controversial. Here, we reported an unexpected effects of SPINK1 on inhibiting trypsinogen activation through the regulation of impaired autophagy in cerulein-stimulated AR42J cells, a well-established in vitro model of acute pancreatitis. Firstly, we found that the impaired autophagic flux was induced and trypsinogen activity enhanced in the above setting. Then, we showed that SPINK1 overexpression could inhibit the level of increased autophagic activity, improving the hindered autophagy flux, and significantly decreased the trypsinogen activity, whereas shRNA-caused downregulation of SPINK1 exacerbated the impairment of autophagic flux and trypsin activity, in the same cerulein-processed cells. More importantly, the trypsinogen activation in this model could be ameliorated by 3-Methyladenine(3-MA), an autophagy inhibitor. Thus, this study revealed, possibly for the first time, that SPINK1 greatly blocked the trypsinogen activation possibly through the modulation of impaired autophagy in cerulein-induced in vitro model of acute pancreatitis.
High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
Authors: Gonzalez Carlos G. , Wastyk Hannah C. , Topf Madeline , Gardner Christopher D. , Sonnenburg Justin L. , Elias Joshua E.
Published: March 2020

Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, in comparison to competing technologies such as metagenomics and 16S rRNA sequencing, it is under-utilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles, and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high throughout proteomic method for processing stool samples in a technically reproducible manner, and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting.

Importance

Widely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insight might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass-spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, roughly 10 times faster than previous methods. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.
Assessment of the Clinical Utility of Plasma Metagenomic Next-Generation Sequencing in a Pediatric Hospital Population
Authors: Lee Rose A. , Al Dhaheri Fatima , Pollock Nira R. , Sharma Tanvi S.
Published: March 2020

Background

Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA (cfDNA) is commercially available, but its role in the workup of infectious diseases is unclear.

Methods

To understand the clinical utility of plasma mNGS, we retrospectively reviewed patients tested at a pediatric institution over 2 years to evaluate the clinical relevance of the organism(s) identified and impact on antimicrobial management. We also investigated the effect of pre-test antimicrobials and interpretation of molecules of microbial cfDNA per microliter (MPM) plasma.

Results

29/59 (49%) mNGS tests detected organism(s), and 28/51 (55%) organisms detected were clinically relevant. Median MPM of clinically relevant organisms was 1533 versus 221 for irrelevant organisms (p=0.01). mNGS test sensitivity and specificity were 53% and 79%, respectively, with a positive predictive value (PPV) of 72% and negative predictive value (NPV) of 50%. 14% of tests impacted clinical management by changing antimicrobial therapy. Immunocompromised status was the only patient characteristic that trended towards a significant clinical impact (p=0.056). No patients with culture-negative endocarditis had organisms identified by mNGS. There were no significant differences in antimicrobial pre-test duration between tests with clinically relevant organism(s) versus those that returned negative, nor was the MPM different between pre-treated and un-treated organisms, suggesting that 10 days of antimicrobial therapy as observed in this cohort did not sterilize testing; however, no pre-treated organisms identified resulted in a new diagnosis impacting clinical management

Conclusions

Plasma mNGS demonstrated higher utility for immunocompromised patients, but given the low PPV and NPV, cautious interpretation and Infectious Diseases consultation are prudent.

We evaluate the test performance characteristics and clinical utility of plasma metagenomic next-generation sequencing in a pediatric hospital cohort and demonstrate sensitivity and specificity of 53% and 79%, with 14% of tests impacting antimicrobial management.
Mechanistic Insights into Crosstalk of Tet(X) and MCR-1, Two Resistance Enzymes Co-produced by A Single Plasmid
Authors: Xu Yongchang , Liu Lizhang , Zhang Huimin , Feng Youjun
Published: March 2020

Tigecycline and colistin are few of last-resort defenses used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-bornetet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically ineffective, posing a risky challenge to global public health. Here, we report that co-carriage oftet(X6) andmcr-1gives co-resistance to both classes of antibiotics by a single plasmid inE. coli. Genomic analysis suggested that transposal transfer ofmcr-1proceeds into the plasmid pMS8345A, in which a new varianttet(X6) is neighbored with Class I integron. The structure-guided mutagenesis finely revealed the genetic determinants of Tet(X6) in the context of phenotypic tigecycline resistance. The combined evidencein vitroandin vivodemonstrated its enzymatic action of Tet(X6) in the destruction of tigecycline. The presence of Tet(X6) (and/or MCR-1) robustly prevents the accumulation of reactive oxygen species (ROS) induced by tigecycline (and/or colistin). Unlike thatmcr-1exerts fitness cost inE. coli, tet(X6) does not. In thetet(X6)-positive strain that co-harborsmcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodeling, and vice versa. Co-production of Tet(X6) and MCR-1 gives no synergistic delayed growth of the recipientE. coli. Similar to that MCR-1 behaves in the infection model ofG. mellonella, Tet(X6) renders the treatment of tigecycline ineffective. Therefore, co-transfer of such two AMR genes is of great concern in the context of “one health” comprising environmental/animal/human sectors, and heightened efforts are required to monitor its dissemination.

Author summary

We report thattet(X6), a new tigecycline resistance gene, is co-carried with the other resistance genemcr-1by a single plasmid. Not only have we finely mapped genetic determinants oftet(X6), but also revealed its biochemical action of tigecycline destruction. Crosstalk of Tet(X6) with MCR-1 is addressed. Tet(X6) tigecycline resistance is independently of MCR-1 colistin resistance, and vice versa. Similar to MCR-1 that renders colistin clinically ineffective, Tet(X6) leads to the failure of tigecycline treatment in the infection model ofG. mellonella. This study extends mechanistic understanding mechanism and interplay of Tet(X6) and MCR-1, coproduced by a single plasmid. It also heightens the need to prevent rapid and large-scaled spread of AMR.
Genomic islands of differentiation in a rapid avian radiation have been driven by recent selective sweeps
Authors: Hejase Hussein A. , Salman-Minkov Ayelet , Campagna Leonardo , Hubisz Melissa J. , Lovette Irby J. , Gronau Ilan , Siepel Adam
Published: March 2020

Numerous studies of emerging species have identified genomic “islands” of elevated differentiation against a background of relative homogeneity. The causes of these islands remain unclear, however, with some signs pointing toward “speciation genes” that locally restrict gene flow and others suggesting selective sweeps that have occurred within nascent species after speciation. Here, we examine this question through the lens of genome sequence data for five species of southern capuchino seedeaters, finch-like birds from South America that have undergone a species radiation during the last ∼50,000 generations. By applying newly developed statistical methods for ancestral recombination graph inference and machine-learning methods for the prediction of selective sweeps, we show that previously identified islands of differentiation in these birds appear to be generally associated with relatively recent, species-specific selective sweeps, most of which are predicted to be “soft” sweeps acting on standing genetic variation. Many of these sweeps coincide with genes associated with melanin-based variation in plumage, suggesting a prominent role for sexual selection. At the same time, a few loci also exhibit indications of possible selection against gene flow. These observations shed new light on the complex manner in which natural selection shapes genome sequences during speciation.

Significance Statement

Genome-wide scans can identify differentiated loci between species that may have promoted speciation. So-called “islands of differentiation” have generally been identified and characterized using standard population genetic summary statistics (e.g.,FSTanddxy), which are limited in distinguishing among possible causes of differentiation, such as selection against gene flow and selective sweeps. We introduce a powerful strategy for analyzing such islands, combining new methods for inferring the full Ancestral Recombination Graph and machine learning methods for identifying selective sweeps. We applied our methods to genomic sequences from closely related southern capuchino seedeaters (Neotropical tanagers) and found signatures of recent selective sweeps around pigmentation genes, including many “soft” sweeps that acted on standing variation.
Global Landscape of Native Protein Complexes inSynechocystissp. PCC 6803
Authors: Xu Chen , Wang Bing , Yang Lin , Hu Lucas Zhongming , Yi Lanxing , Wang Yaxuan , Chen Shenglan , Emili Andrew , Wan Cuihong
Published: March 2020

Synechocystissp. PCC 6803 (hereafter:Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Though known as the first fully sequenced phototrophic organism,Synechocystisstill has almost half of its proteome without functional annotations. In this study, we obtained 291 protein complexes, including 24,092 protein-protein interactions (PPIs) among 2062 proteins by using co–fractionation and LC/MS/MS. The additional level of PPIs information not only revealed the roles of photosynthesis in metabolism, cell motility, DNA repair, cell division, and other physiological processes, but also showed how protein functions vary from bacteria to higher plants due to the changed interaction partner. It also allows us to uncover functions of hypothetical proteins, such as Sll0445, Sll0446, S110447 participating in photosynthesis and cell motility, and Sll1334 regulating the expression of fatty acid. Here we presented the most extensive protein interaction data inSynechocystisso far, which might provide critical insights into the fundamental molecular mechanism in Cyanobacterium.
Geological degassing enhances microbial metabolism in the continental subsurface
Authors: Bornemann Till L.V. , Adam Panagiotis S. , Turzynski Victoria , Schreiber Ulrich , Figueroa-Gonzalez Perla Abigail , Rahlff Janina , Köster Daniel , Schmidt Torsten C. , Schunk Ralf , Krauthausen Bernhard , Probst Alexander J.
Published: March 2020

Mantle degassing provides a substantial amount of reduced and oxidized gases shaping microbial metabolism at volcanic sites across the globe, yet little is known about its impact on microbial life under non-thermal conditions. Here, we characterized deep subsurface fluids from a cold-water geyser driven by mantle degassing using genome-resolved metagenomics to investigate how the gases impact the metabolism and activity of indigenous microbes compared to non-impacted sites. While species-specific analyses of Altiarchaeota suggest site-specific adaptations and a particular biogeographic pattern, chemolithoautotrophic core features of the communities appeared to be conserved across 17 groundwater ecosystems between 5 and 3200 m depth. We identified a significant negative correlation between ecosystem depth and bacterial replication, except for samples impacted by high amounts of subsurface gases, which exhibited near-surface activity. Our results suggest that geological degassing leads to higher nutrient flows and microbial activity in the deep subsurface than previously estimated.
Characterization of fluorescent proteins with intramolecular photostabilization
Authors: Henrikus Sarah S. , Tassis Konstantinos , Zhang Lei , van der Velde Jasper H. M. , Gebhardt Christian , Herrmann Andreas , Jung Gregor , Cordes Thorben
Published: March 2020

Genetically encodable fluorescent proteins have revolutionized biological imagingin vivoandin vitro. Since there are no other natural fluorescent tags with comparable features, the impact of fluorescent proteins for biological research cannot be overemphasized. Despite their importance, their photophysical properties, i.e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties. The approach uses direct conjugation of photostablizing compounds such as triplet-state quenchers or redox-active substances to an organic fluorophore, thereby creating high local concentrations of photostabilizer. Here, we introduce an experimental strategy to screen for the effects of covalently-linked photostabilizers on fluorescent proteins. We recombinantly produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel in close proximity to the chromophore. Whereas labelling with photostabilizers such as Trolox, Nitrophenyl, and Cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation of α-GFP to an azobenzene derivative. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the near-UV and blue spectral range. Our study paves the way towards the development and design of a second generation of fluorescent proteins with photostabilizers placed directly in the protein barrel by methods such as unnatural amino acid incorporation.