bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry
Authors: Weerasinghe Nuwani W. , Habibi Yeganeh , Uggowitzer Kevin A. , Thibodeaux Christopher J. 
Published: September 2020

AbstractLanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding, and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These “biophysical” processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its non-covalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively-folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.
Genome wide functional screen for calcium transients inE. coliidentifies decreased membrane potential adaptation to persistent DNA damage
Authors: Luder Rose , Bruni Giancarlo N. , Kralj Joel M. 
Published: September 2020

1.AbstractCalcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Unlike eukaryotic cells, far less is known about calcium signaling in bacteria, and few genes controlling influx and efflux have been identified. Previous work inEscherichia colishowed calcium influx is induced by voltage depolarization, which were enhanced by mechanical stimulation, suggesting a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live cell screen to monitor calcium dynamics in single cells across a genome wide knockout panel inE. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased calcium transients, and 32 genes knockouts that increased transients. Knockouts involved in energy production and regulation appeared, as expected, as well as knockouts of the voltage sink, the F1Fo-ATPase. Knockouts in exopolysaccharide and outer membrane synthesis showed reduced transients and refined our model of electrophysiology mediated mechanosensation inE. coli. Additionally, knockouts annotated in DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and suggested that only long term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long term DNA damage responses in bacteria, which has implications for mitochondrial DNA damage in eukaryotes.ImportanceAll eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium inEscherichia coli, we created a single cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.
Anatomically distinct OFC-PCC circuits relay choice from value space to action space
Authors: Wang Maya Zhe , Hayden Benjamin Y. , Heilbronner Sarah R. 
Published: September 2020

ABSTRACTEconomic choice necessarily involves the transformation of abstract, object-based representations to concrete, action-based ones. This transformation is both determined and delimited by the neuroanatomical organization of the regions that implement it. In choice, the orbitofrontal cortex (OFC) plays a key role in both abstract valuation and cognitive mapping. However, determining the neural processes underlying this transformation has proven difficult. We hypothesized that difficulty stems from in part from the fact that the OFC consists of multiple functionally distinct zones that are distinguished by their differing contributions to the abstract-concrete transformation, and that these functions reflect their differing long-range projections. Here we identify two such subregions, defined by stronger or weaker bidirectional anatomical connectivity with the posterior cingulate cortex (PCC). We call these regions OFCinand OFCout, respectively. We find that OFCin, relative to OFCout, shows enhanced functional connectivity with PCC, as indicated by both spike-field coherence and mutual information. We find converging evidence that the OFCin-PCC circuit, but not the OFCout-PCC circuit, relays choice signals from an abstract value space to a concrete action space. Moreover, the OFCin-PCC circuit shows a putative bidirectional mutually excitatory pattern. Together, these results support the hypothesis that OFC-PCC subareal organization is critical for understanding the implementation of offer-action transformation in economic choice.
Impact of Rap-Phr system abundance on adaptation ofBacillus subtilis
Authors: Gallegos-Monterrosa Ramses , Christensen Mathilde Nordgaard , Barchewitz Tino , Koppenhöfer Sonja , Priyadarshini B. , Bálint Balázs , Maróti Gergely , Kempen Paul J , Dragoš Anna , Kovács Ákos T. 
Published: September 2020

AbstractMicrobes commonly display great genetic plasticity, which has allowed them to colonize all ecological niches on Earth.Bacillus subtilisis a soil-dwelling organism that can be isolated from a wide variety of environments. An interesting characteristic of this bacterium is its ability to form biofilms that display complex heterogeneity: individual, clonal cells develop diverse phenotypes in response to different environmental conditions within the biofilm. Here, we scrutinized the impact that the number and variety of the Rap-Phr family of regulators and cell-cell communication modules ofB. subtilishas on genetic adaptation and evolution. We examine how the Rap family of phosphatase regulators impacts sporulation in diverse niches using a library of single and doublerap-phrmutants in competition under 4 distinct growth conditions. Using specific DNA barcodes and whole-genome sequencing, population dynamics were followed, revealing the impact of individual Rap phosphatases and arising mutations on the adaptability ofB. subtilis.
Sequencing ultra-rare targets with compound nucleic acid cytometry
Authors: Sun Chen , Chang Kai-Chun , Abate Adam R. 
Published: September 2020

AbstractTargeted sequencing enables sensitive and cost-effective analysis by focusing resources on molecules of interest. Existing methods, however, are limited in enrichment power and target capture length. Here, we present a novel method that uses compound nucleic acid cytometry to achieve million-fold enrichments of molecules >10 kbp in length using minimal prior target information. We demonstrate the approach by sequencing HIV proviruses in infected individuals. Our method is useful for rare target sequencing in research and clinical applications, including for identifying cancer-associated mutations or sequencing viruses infecting cells.
Application of an Optimized Annotation Pipeline to theCryptococcus DeuterogattiiGenome Reveals Dynamic Primary Metabolic Gene Clusters and Genomic Impact of RNAi Loss
Authors: Ferrareze Patrícia Aline Gröhs , Maufrais Corinne , Streit Rodrigo Silva Araujo , Priest Shelby J. , Cuomo Christina , Heitman Joseph , Staats Charley Christian , Janbon Guilhem 
Published: September 2020

AbstractEvaluating the quality of ade novoannotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeastsCryptococcus neoformansandCryptococcus deneoformansas test cases. Our results demonstrate that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5 to 10 million reads per RNA-seq replicate. We also demonstrated that the number of introns predicted is an excellenta prioriindicator of the quality of the finalde novoannotation. We then used this pipeline to annotate the genome of the RNAi-deficient speciesCryptococcus deuterogattiistrain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent inC. deuterogattiithan in the other RNAi-proficient speciesC. neoformansandC. deneoformans. In contrast, we observed that antisense transcription was not higher inC. deuterogattiithan in the two otherCryptococcusspecies. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification ofCryptococcusmetabolite assimilation potential.
Breathing new life into the critical oxygen partial pressure (Pcrit): a new definition, interpretation and method of determination
Authors: Seibel B. A. , Andres A. , Birk M. A. , Burns A. L. , Shaw C. T. , Timpe A. W. , Welsh C. J. 
Published: September 2020

AbstractThe critical oxygen partial pressure (Pcrit) is most commonly defined as the oxygen partial pressure below which an animal’s standard metabolic rate can no longer be maintained. It is widely interpreted as measure of hypoxia tolerance, which influences a species’ aerobic scope and, thus, constrains biogeography. However, both the physiology underlying that interpretation and the methodology used to determine Pcritremain topics of active debate. The debate remains unresolved in part because Pcrit, as defined above, is a purely descriptive metric that lacks a clear mechanistic basis. Here we redefine Pcritas the PO2at which physiological oxygen supply is maximized and refer to these values, thus determined, as Pcrit-α. The oxygen supply capacity (α) is a species- and temperature-specific coefficient that describes the slope of the relationship between the maximum achievable metabolic rate and PO2. Thisαis easily determined using respirometry and provides a precise and robust estimate of the minimum oxygen pressure required to sustain any metabolic rate. To determineα, it is not necessary for an individual animal to maintain a consistent metabolic rate throughout a trial (i.e. regulation) nor for the metabolic rate to show a clear break-point at low PO2. We show that Pcrit-αcan be determined at any metabolic rate as long as the organisms’ oxygen supply machinery reaches its maximum capacity at some point during the trial. We reanalyze published representative Pcrittrials for 40 species across five phyla, as well as complete datasets from six additional species, five of which have not previously been published. Values determined using the Pcrit-αmethod are strongly correlated with Pcritvalues reported in the literature. Advantages of Pcrit-αinclude: 1) Pcrit-αis directly measured without the need for complex statistics that hinder measurement and interpretation; 2) it makes clear that Pcritis a measure of oxygen supply, which does not necessarily reflect hypoxia tolerance; 3) it alleviates many of the methodological constraints inherent in existing methods; 4) it provides a means of predicting the maximum metabolic rate achievable at any PO2, 5) Pcrit-αsheds light on the temperature- and size-dependence of oxygen supply and metabolic rate and 6) Pcrit-αcan be determined with greater precision than traditional Pcrit.
A Human Multi-Lineage Hepatic Organoid Model for Liver Fibrosis
Authors: Guan Yuan , Enejder Annika , Wang Meiyue , Fang Zhuoqing , Cui Lu , Chen Shih-Yu , Wang Jingxiao , Tan Yalun , Wu Manhong , Chen Xinyu , Johansson Patrik K. , Osman Issra , Kunimoto Koshi , Russo Pierre , Heilshorn Sarah C. , Peltz Gary 
Published: September 2020

AbstractBackgroundTo characterize fibrogenic mechanisms, genome engineering and a human hepatic organoid system was used to produce anin vitromodel for human liver fibrosis.Methods and resultsHuman hepatic organoids that were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD) developed the key features of ARPKD liver pathology (abnormal bile ducts and hepatic fibrosis) in only 21 days. Second harmonic generation microscopy confirmed that the ARPKD mutation increased collagen abundance and thick collagen fiber production in hepatic organoids; and we demonstrated that these changes mirrored that occurring in ARPKD liver tissue. Transcriptomic and other analyses indicated that the ARPKD mutation generates cholangiocytes with increased TGFβ-associated pathway activation, which are actively involved in collagen fiber generation. The abnormal cholangiocytes promote the expansion of collagen-producing myofibroblasts with markedly increased PDGFRβ protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resembled that of myofibroblasts in liver tissue obtained from patients with commonly occurring acquired forms of liver fibrosis. The involvement of the PDGFRB pathway was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors.ConclusionsBesides providing mechanistic insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.
Sex-specific calibration of memory recall by glucocorticoid receptors on cortical astrocytes
Authors: Taylor WW , Imhoff BR , Sathi ZS , Garza KM , Dias BG 
Published: September 2020

ABSTRACTDysfunctions in memory recall lead to pathological fear; a hallmark of trauma-related disorders, like Post-Traumatic Stress Disorder (PTSD). Heightened recall of an association between a cue and trauma, as well as impoverished recall that a previously trauma-related cue is no longer a threat both result in a debilitating fear toward the cue. Glucocorticoid-mediated action via the glucocorticoid receptor (GR) influences memory recall. This literature has primarily focused on GRs expressed in neurons or ignored cell-type specific contributions. To ask how GR action in non-neuronal cells influences memory recall, we combined auditory fear conditioning in mice and the knockout of GRs in astrocytes in the prefrontal cortex (PFC), a brain region implicated in memory recall. We found that GRs in astrocytes in the PFC calibrate recall in female but not male mice. Specifically, we found that knocking out GRs in astrocytes in the PFC of female mice (AstroGRKO) after fear conditioning resulted in higher recall of fear to the CS+ tone when compared to controls (AstroGRintact). While we did not find any differences in extinction of fear toward the CS+ between these groups, AstroGRKO female mice showed impaired recall of extinction training. We did not observe any significant results in male mice. These results suggest a sex-specific calibration of memory recall by GRs in astrocytes in the PFC. These data demonstrate the need to examine GR action in cortical astrocytes to elucidate the basic neurobiology underlying memory recall and potential mechanisms that underlie female-specific biases in the incidence of PTSD.
Induced Disassembly of a Virus-Like Particle under Physiological Conditions for Venom Peptide Delivery
Authors: Kelly M. Patrick , Napolitano Tanya , Anand Prachi , Ho Justin S. K. , Jabeen Shakeela , Kuppan Jessica , Manir Sujoy , Holford Mandë 
Published: September 2020

ABSTRACTVirus-like nanoparticles (VLPs) show considerable promise for thein vivodelivery of therapeutic compounds such as bioactive venom peptides. While loading and targeting protocols have been developed for numerous VLP prototypes, induced disassembly under physiological conditions of neutral pH, moderate temperature, and aqueous medium, remain a challenge. Here, we implement and evaluate a ring-opening metathesis polymerization (ROMP) general mechanism for controllable VLP disassembly that is independent of cell-specific factors or the manipulation environmental conditions such as pH and temperature that cannot be readily controlledin vivo. The ROMP substrate norbornene is covalently conjugated to surface-exposed lysine residues of a P22 bacteriophage-derived VLP, and ROMP is induced by treatment of water-soluble ruthenium catalyst AquaMet. Disruption of the P22 shell and release of a GFP reporter is confirmed via native agarose electrophoresis and quantitative microscopy and light scattering analyses. Our ROMP disassembly strategy does not depend on the particular structure or morphology of the P22 nanocontainer and is adaptable to other VLP prototypes for the potential delivery of venom peptides for pharmacological applications.Abstract Figure
Computationally assisted design and selection of maneuverable biological walking machines
Authors: Wang Jiaojiao , Park Junehu , Zhang Xiaotian , Park Insu , Kilicarslan Evin , Kim Yongdeok , Bashir Rashid , Gazzola Mattia 
Published: September 2020

ABSTRACTThe intriguing opportunities enabled by the use of living components in biological machines have spurred the development of a variety of muscle-powered bio-hybrid robots in recent years. Among them, several generations of bio-hybrid walkers have been established as reliable platforms to study untethered locomotion. However, despite these advances, such technology is not mature yet, and major challenges remain. This study takes steps to address two of them: the lack of systematic design approaches, common to bio-hybrid robotics in general, and in the case of bio-hybrid walkers specifically, the lack of maneuverability. We then present here a dual-ring biobot, computationally designed and selected to exhibit robust forward motion and rotational steering. This dual-ring biobot consists of two independent muscle actuators and a 4-legged scaffold asymmetric in the fore/aft direction. The integration of multiple muscles within its body architecture, combined with differential electrical stimulation, allows the robot to maneuver. The dual-ring robot design is then fabricated and experimentally tested, confirming computational predictions and turning abilities. Overall, our design approach based on modeling, simulation, and fabrication exemplified in this robot represents a route to efficiently engineer biological machines with adaptive functionalities.
eEF2 kinase enhances the expression of PD-L1 by promoting the translation of its mRNA
Authors: Wu Yu , Xie Jianling , Jin Xin , Lenchine Roman V. , Wang Xuemin , Fang Danielle M. , Nassar Zeyad D. , Butler Lisa M. , Li Jing , Proud Christopher G. 
Published: September 2020

AbstractEmerging advances in cancer therapy have transformed the landscape from conventional therapies towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are ‘game-changers’ for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that inhibits the elongation stage of protein synthesis, actually promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association ofPD-L1mRNAs with translationally active polyribosomes and that translation of thePD-L1mRNA is regulated by a uORF (upstream open reading-frame) within its 5’-UTR (5’-untranslated region) which starts with a non-canonical CUG codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5’-region. This, and other roles of eEF2K in cancer cell biology (e.g., in cell survival and migration), may be exploited for the design of future therapeutic strategies.
Species-Dependent Heterophilic and Homophilic Cadherin Interactions in Intestinal Intermicrovillar Links
Authors: Gray Michelle E. , Johnson Zachary R. , Modak Debadrita , Tyska Matthew J. , Sotomayor Marcos 
Published: September 2020

ABSTRACTEnterocytes are specialized epithelial cells lining the luminal surface of the small intestine that build densely packed arrays of microvilli known as brush borders. These microvilli drive nutrient absorption and are arranged in a hexagonal pattern maintained by intermicrovillar links formed by two non-classical members of the cadherin superfamily of calcium-dependent cell adhesion proteins: protocadherin-24 (PCDH24, also known as CDHR2) and the mucin-like protocadherin (CDHR5). The extracellular domains of these proteins are involved in heterophilic and homophilic interactions important for intermicrovillar function, yet the structural determinants of these interactions remain unresolved. Here we present X-ray crystal structures of the PCDH24 and CDHR5 extracellular tips and analyze their species-specific features relevant for adhesive interactions. In parallel, we use binding assays to identify the PCDH24 and CDHR5 domains involved in both heterophilic and homophilic adhesion for human and mouse proteins. Our results suggest that homophilic and heterophilic interactions involving PCDH24 and CDHR5 are species dependent with unique and distinct minimal adhesive units.