bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying the effect of vancomycin on HA-MRSA strains using bacteriological and physiological media
Authors: Rajput Akanksha , Poudel Saugat , Tsunemoto Hannah , Meehan Michael , Szubin Richard , Olson Connor A. , Seif Yara , Lamsa Anne , Dillon Nicholas , Vrbanac Alison , Sugie Joseph , Dahesh Samira , Monk Jonathan M. , Dorrestein Pieter C. , Knight Rob , Pogliano Joe , Nizet Victor , Feist Adam M. , Palsson Bernhard O.
Published: May 2020

Healthcare-associated methicillin-resistantStaphylococcus aureus(HA-MRSA) USA100 strains are of major concern due to their evolving antibiotic resistant. They are resistant to a broad class of antibiotics like macrolides, aminoglycosides, fluoroquinolones, and many more. The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (CA-MHB) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behaviour of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. The study reveals the vancomycin resistance behavior of USA100 strains in dual media conditions using wide-ranging experiments.
Development of a vaccine against the newly emerging COVID-19 virus based on the receptor binding domain displayed on virus-like particles
Authors: Zha Lisha , Zhao Hongxin , Mohsen Mona O. , Hong Liang , Zhou Yuhang , Li Zehua , Chen Hongquan , Liu Xuelan , Chang Xinyue , Zhang Jie , Li Dong , Wu Ke , Vogel Monique , Bachmann Martin F , Wang Junfeng
Published: May 2020

The ongoing coronavirus COVID-19 pandemic is caused by a new coronavirus (SARS-CoV-2) with its origin in the city of Wuhan in China. From there it has been rapidly spreading to many cities inside and outside China. Nowadays more than 33 millions with deaths surpassing 1 million have been recorded worldwide thus representing a major health issue. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrated that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus (CuMVTT) resulted in a vaccine candidate that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized COVID-19 virusin vitro.
Extracting transition rates in single-particle tracking using analytical diffusion distribution analysis
Authors: Vink Jochem N.A. , Brouns Stan J.J. , Hohlbein Johannes
Published: May 2020

Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. Observed diffusion coefficientsin vivo, for example, enable researchers to determine whether biomolecules are moving alone, as part of a larger complex or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models especially for molecules that rapidly interchange between different diffusional states. Here, we present analytic diffusion distribution analysis (anaDDA), a framework that allows extracting transition rates from distributions of observed diffusion coefficients. We show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times, to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalysed experimental data of DNA polymerase I diffusing in liveE. coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behaviour of biomolecules in living cells.
Bayesian combination of mechanistic modeling and machine learning (BaM3): improving personalized tumor growth predictions
Authors: Mascheroni Pietro , Savvopoulos Symeon , Alfonso Juan Carlos López , Meyer-Hermann Michael , Hatzikirou Haralampos
Published: May 2020

In clinical practice, a plethora of medical examinations are conducted to assess the state of a patient’s pathology producing a variety of clinical data. However, exploiting these data faces the following challenges: (C1) we lack the knowledge of the mechanisms involved in regulating these data variables, and (C2) data collection is sparse in time since it relies on patient’s clinical presentation. (C1) implies that only a small subset of the relevant variables can be modeled by virtue of mathematical modeling. This limitation allows models to be effective in analyzing the qualitative dynamics of the system, but limits their predictive accuracy. On the other hand, statistical learning methods are well-suited for quantitative reproduction of data, but they do not provide mechanistic understanding of the investigated problem. Moreover, due to (C2) any algorithm is challenged in learning the corresponding disease dynamics. Herein, we propose a novel method, based on the Bayesian coupling of mathematical modeling and machine learning (BaM3), aiming at improving individualized predictions by addressing the aforementioned challenges. As a proof of concept, we evaluate the proposed method on a synthetic dataset for brain tumor growth and analyze its performance in predicting two major clinical outputs, namely tumor burden and infiltration. The BaM3method results in improved predictions in almost all simulated patients, especially for those with a late clinical presentation. In addition, we test the proposed methodology in two settings dealing with real patient cohorts. In both cases, namely cancer growth in chronic lymphocytic leukemia and ovarian cancer, BaM3predictions show excellent agreement with reported clinical data.
Integrated neural dynamics for behavioral decisions and attentional control in the prefrontal cortex
Authors: Erez Yaara , Kadohisa Mikiko , Petrov Philippe , Sigala Natasha , Buckley Mark J. , Kusunoki Makoto , Duncan John
Published: May 2020

Complex neural dynamics in the prefrontal cortex contribute to context-dependent decisions and attentional competition. To analyze these dynamics, we apply demixed principal component analysis to activity of a primate prefrontal cell sample recorded in a cued target detection task. The results track dynamics of cue and object coding, feeding into movements along a target present-absent decision axis in a low-dimensional subspace of population activity. For a single stimulus, object and cue coding are seen mainly in the contralateral hemisphere. Later, a developing decision code in both hemispheres may reflect interhemispheric communication. With a target in one hemifield and a competing nontarget in the other, each hemisphere initially encodes the contralateral object, but finally, decision coding is dominated by the task-relevant target. These findings further suggest that exchange of information between hemispheres plays a key role when attentional competition resolves. Tracking complex neural events in a low-dimensional activity subspace illuminates integration of neural codes towards task-appropriate behavior, comprising a building block of learned task structure in the prefrontal cortex.

AUTHOR SUMMARY

Flexible adaptive processing of information is integral for everyday goal-directed behavior. To unravel dynamic representation of task-relevant information, we analyzed population activity of a primate prefrontal cell sample in a cued target detection task. In a low-dimensional neural subspace, with separate axes for cue, object identity and decision, trajectories showed initial coding of cue and object in the contralateral hemisphere, followed by coding of the behavioral decision across both hemispheres. With target and nontarget stimuli in opposite hemifields, the data suggest initial coding of the contralateral object in each hemisphere. Object coding is then rapidly shut off for the nontarget, and followed by bilateral coding of the target decision. The results bring detailed insight into task structure and information flow within and between the two frontal lobes as a decision is made and attentional competition is resolved.
Detect Genomic G-Quadruplexes in Living Animal Cells with a Tiny Artificial Protein Probe
Authors: Zheng Ke-wei , Zhang Jia-yu , He Yi-de , Gong Jia-yuan , Wen Cui-jiao , Chen Juan-nan , Hao Yu-hua , Tan Zheng
Published: May 2020

G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the biological role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. We engineered a 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technology, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123,000 G4 peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ~70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.
Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy
Authors: Götz Ralph , Kunz Tobias C. , Fink Julian , Solger Franziska , Schlegel Jan , Seibel Jürgen , Kozjak-Pavlovic Vera , Rudel Thomas , Sauer Markus
Published: May 2020

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enabled imaging of sphingolipids and their interactions with proteins in the membrane of intracellular organelles with a spatial resolution of 10-20 nm. Because sphingolipids accumulated efficiently in pathogens we used sphingolipid ExM to investigate bacterial infections of human HeLa229 cells byNeisseria gonorrhoeae, Chlamydia trachomatisandSimkania negevensiswith a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allowed us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.
Cancer-associated hypersialylated MUC1 drives the differentiation of monocytes into macrophages with a pathogenic phenotype
Authors: Beatson Richard , Graham Rosalind , Freile Fabio Grundland , Cozzetto Domenico , Kannambath Shichina , Pfeifer Ester , Woodman Natalie , Owen Julie , Nuamah Rosamond , Mandel Ulla , Pinder Sarah , Gillett Cheryl , Noll Thomas , Bouybayoune Ihssane , Taylor-Papadimitriou Joyce , Burchell Joy M.
Published: May 2020

The tumour microenvironment plays a crucial role in the growth and progression of cancer and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies show that TAMs show transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype. These TAMs can recruit and maintain neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This novel macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.
Neural Correlates of Value Are Intrinsically History Dependent
Authors: Lee Sangil , Lerman Caryn , Kable Joseph
Published: May 2020

The authors have withdrawn their manuscript since they no longer stand by the results reported in this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
Myosin 10 and a Cytoneme-Localized Ligand Complex Promote Morphogen Transport
Authors: Hall Eric T. , Stewart Daniel P. , Dillard Miriam , Wagner Ben , Sykes April , Temirov Jamshid , Cheney Richard E. , Mori Motomi , Robinson Camenzind G. , Ogden Stacey K.
Published: May 2020

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. Molecular mechanisms by which these signaling gradients are established and reinforced remain enigmatic. The cytoneme transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signal activation thresholds are achieved across developing tissues. How morphogens are transported along and deployed from cytonemes is not known. Herein we show that the actin motor Myosin 10 promotes cytoneme-based transport of Sonic Hedgehog (SHH) morphogen to filopodial tips, and that SHH movement within cytonemes occurs by vesicular transport. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid signal response, and that SHH cytonemes are promoted by a complex containing a ligand-specific deployment protein and associated co-receptor.

One-Sentence summary

Cytoneme-based delivery of the Sonic Hedgehog activation signal is promoted by Myosin 10 and BOC/CDON co-receptor function.
Lack of astrocytic glycogen alters synaptic plasticity but not seizure susceptibility
Authors: Duran Jordi , Brewer M. Kathryn , Hervera Arnau , Gruart Agnès , del Rio Jose Antonio , Delgado-García José M. , Guinovart Joan J.
Published: May 2020

Brain glycogen is mainly stored in astrocytes. However, recent studies bothin vitroandin vivoindicate that glycogen also plays important roles in neurons. By conditional deletion of glycogen synthase (GYS1), we previously developed a mouse model entirely devoid of glycogen in the central nervous system (GYS1Nestin-KO). These mice displayed altered electrophysiological properties in the hippocampus and increased susceptibility to kainate-induced seizures. To understand which of these functions is related to astrocytic glycogen, in the present study we generated a mouse model in which glycogen synthesis is eliminated specifically in astrocytes (GYS1Gfap-KO). Electrophysiological recordings of awake behaving mice revealed alterations in input/output curves and impaired long-term potentiation, similar, but to a lesser extent, to those obtained with GYS1Nestin-KOmice. Surprisingly, GYS1Gfap-KOmice displayed no change in susceptibility to kainate-induced seizures as determined by fEPSP recordings and video monitoring. These results confirm the importance of astrocytic glycogen in synaptic plasticity.
Pretreatment of aged mice with retinoic acid restores alveolar regeneration via upregulation of reciprocal PDGFRA signaling
Authors: Gokey Jason J. , Snowball John , Green Jenna , Waltamath Marion , Spinney Jillian J. , Black Katharine E. , Hariri Lida P. , Xu Yan , Perl Anne-Karina T.
Published: May 2020

Objectives

Idiopathic Pulmonary Fibrosis (IPF) primarily affects the aged population and is characterized by failure of alveolar regeneration leading to loss of alveolar type 1 cells (AT1). Aged mouse models of lung repair have demonstrated that regeneration fails with increased age. Mouse and rat lung repair models have shown retinoic acid (RA) treatment can restore alveolar regeneration. Herein we seek to determine the signaling mechanisms by which RA treatment prior to injury supports alveolar differentiation.

Design

Partial pneumonectomy (PNX) lung injury model and next generation sequencing of sorted cell populations are used to uncover molecular targets regulating alveolar repair.In-vitroorganoids generated from Mouse or IPF patient epithelial cells co-cultured with young, aged, or RA pretreated murine mesenchyme are used to test potential targets.

Main outcome measurements

Known alveolar epithelial cell differentiation markers, including HOPX and AGER for AT1 cells are used to assess outcome of treatments.

Results

Gene expression analysis of sorted fibroblasts and epithelial cells isolated from lungs of young, aged, and RA treated aged mice predicted increased PDGFA signaling that coincided with regeneration and alveolar epithelial differentiation. Addition of PDGFA induced AT1 and AT2 alveolar differentiation in both mouse and human IPF lung organoids generated with aged fibroblasts and PDGFA monoclonal antibody blocked AT1 cell differentiation in organoids generated with young murine fibroblasts.

Conclusions

Our data support the concept that reciprocal PDGFA signaling activates regenerative fibroblasts that support alveolar epithelial cell differentiation and repair, providing a potential therapeutic strategy to influence the pathogenesis of IPF.

Key Question

Which epithelial-mesenchymal crosstalk pathways are activated by RA pretreatment of aged lungs that support realveolarization after partial pneumonectomy surgery?

Bottom Line

Increased PDGFA/PDGFRA signaling in aged lungs promotes regenerative activation of interstitial matrixfibroblast which is required for AT2 to AT1 differentiation and alveolar regeneration.

Read On

In-vitroand in-vivo analysis demonstrated that PDGFA signaling supports alveolar matrixfibroblast and AT1 epithelial cell differentiation, both necessary for alveolar regeneration in aged lungs.
An estimator of the Opportunity for Selection that is independent of mean fitness
Authors: Waples Robin S.
Published: May 2020

Variation among individuals in number of offspring (fitness,k) sets an upper limit to the evolutionary response to selection. This constraint is quantified by Crow’s Opportunity for Selection (I), which is the variance in relative fitness. Crow’sIhas been widely used but remains controversial because it depends on mean offspring number in a sample. Here I used a generalized Wright-Fisher model that allows for unequal probabilities of producing offspring to evaluate behavior of Crow’sIand related indices under a wide range of sampling scenarios. Analytical and numerical results are congruent and show that rescaling the sample varianceto its expected value at a fixedremoves dependence ofIon mean offspring number, but the result still depends on choice of. A new index is introduced,, which makesÎindependent of samplewithout the need for variance rescaling. ΔIhas a straightforward interpretation as the component of variance in relative fitness that exceeds that expected under a null model of random reproductive success. ΔIcan be used to directly compare estimates of the Opportunity for Selection for samples from different studies, different sexes, and different life stages.
Selective Plane Illumination Microscopy and Computing Reveal Differential Obliteration of Retinal Vascular Plexuses
Authors: Chang Chih-Chiang , Chu Alison , Meyer Scott , Sun Michel M. , Abiri Parinaz , In Baek Kyung , Gudapati Varun , Ding Xili , Guihard Pierre , Ding Yichen , Bostrom Kristina I. , Li Song , Gordon Lynn K. , Zheng Jie J. , Hsiai Tzung K.
Published: May 2020

Murine models of visual impairment provide micro-vascular insights into the 3-D network disarray in retinopathy. Current imaging and analysis tend to be confined to the 2-D retinal vasculature. We hereby integrated selective plane illumination imaging or known as light-sheet fluorescence microscopy (LSFM) with dual-illumination, followed by computational analyses, to reveal the topological network of vertical sprouts bridging the primary and secondary plexuses in a postnatal mouse model of oxygen-induced retinopathy (OIR). We revealed a preferential obliteration of the secondary plexus and bridging vessels despite a relatively unscathed primary plexus. We compared the local versus global vascular connectivity using clustering coefficients and Euler numbers, respectively. The global vascular connectivity in hyperoxia-exposed retinas was significantly reduced (p< 0.05, n = 5 vs. normoxia), whereas the local connectivity was preserved (p> 0.05, n = 5 vs. normoxia). We further applied principal component analysis (PCA) to automatically segment the vertical sprouts, corroborating the preferential obliteration of the interconnection between vertical sprouts and secondary plexuses that were accompanied with impaired vascular branching and connectivity, and reduced vessel volumes and lengths (p< 0.05, n=5 vs. normoxia). Thus, integration of 3-D selective plane illumination with computational analyses allows for early detection of global and spatially-specific vaso-obliteration, but preserved local reticular structure in response to hyperoxia-induced retinopathy.
pegFinder: A pegRNA designer for CRISPR prime editing
Authors: Chow Ryan D. , Chen Jennifer S. , Shen Johanna , Chen Sidi
Published: May 2020

To the Editor

CRISPR technologies have been widely adopted as powerful tools for targeted genomic manipulation1. Recently, a new CRISPR-based strategy for precision genome editing was developed that enables diverse genomic alterations to be directly written into target sites without requiring double-strand breaks (DSBs) or donor templates2. Termed prime editing, this approach involves two key components: 1) a catalytically impaired Cas9 nickase fused to a reverse transcriptase (PE2), and 2) a multifunctional prime editing guide RNA (pegRNA) that specifies the target site and further acts as a template for reverse transcription (RT). pegRNAs are similar to standard single-guide RNAs (sgRNAs), but additionally have a customizable extension on the 3’ end. The 3’ extension is composed of a RT template that encodes the desired edit and a primer binding site (PBS) that anneals to the target genomic site to prime the RT reaction2. These additional components considerably increase the complexity of pegRNA design compared to standard sgRNAs. While many tools have been developed for identifying candidate sgRNAs in a target DNA sequence3–8, no user-friendly web application currently exists for designing pegRNAs. We therefore developed pegFinder, a streamlined web tool that rapidly designs candidate pegRNAs (Figure 1). The pegFinder web portal is freely available athttp://pegfinder.sidichenlab.org/(Supplementary Figure 1).
Characterization Of Ancestral Origin Of Cystic Fibrosis Of Patients With New Reported Mutations In CFTR
Authors: Paz-y-Miño César , Zambrano Ana Karina , Ruiz-Cabezas Juan Carlos , Armendáriz-Castillo Isaac , García-Cárdenas Jennyfer M. , Guerrero Santiago , López-Cortés Andrés , Pérez-Villa Andy , Guevara-Ramírez Patricia , Yumiceba Verónica , Leone Paola E.
Published: May 2020

The incidence of Cystic fibrosis (CF) and the frequency of the variants for CFTR depend on the population; furthermore, CF symptomatology is characterized by obstructive lung disease, pancreatic insufficiency among others, reliant on the individual genotype. Ecuadorian population is a mixture of Native Americans, Europeans, and Africans. That population admixture could be the reason for the new mutations reported in a previous study by Ruiz et al. (2019). A panel of 46 Ancestry Informative Markers was used to estimate the ancestral proportions of each available sample (12 samples in total). As a result, the Native American ancestry proportion was the most prevalent in almost all individuals, except for three patients from Guayaquil with the mutation[c.757G>A:p.Gly253Arg; c.1352G>T:p.Gly451Val]who had the highest European composition.
DNA extraction and sequencing of the Mawangdui ancient cadaver protected by formalin
Authors: Huang Hua-Lin , Yin Shikui , Zhao Huifang , Tian Chao , Huang Jufang , Deng Sihao , Li Zhiyuan
Published: May 2020

Mawangdui ancient Cadaver is the first wet corpse found in the world, which is famous for being immortal for over two thousands of years. After being unearthed, the female corpse was immersed in the formalin protective solution for more than 40 years. We used magnetic bead method and formalin fixed paraffing (FFPE) method to extract the DNA of the female corpse, respectively. PCR amplification, sanger sequencing, library building, high throughput sequencing (testing) and data processing were carried out on the DNA samples, and about 0.5% of the whole genome coverage sequencing data was obtained. Comparing the results of DNA trough two extraction and sequencing methods. We found that the FFPE and high throughput sequencing methods is better than others for DNA extraction of the ancient samples which were preserved in formalin, providing a guidance for dealing with formalin preserved ancient samples in the future.
Reclassification of seven honey bee symbiont strains asBombella apis
Authors: Smith Eric A. , Anderson Kirk E. , Corby-Harris Vanessa , McFrederick Quinn S. , Parish Audrey J. , Rice Danny W. , Newton Irene L. G.
Published: May 2020

Honey bees are important pollinators of many major crops and add billions of dollars annually to the US economy through their services. Recent declines in the health of the honey bee have startled researchers and lay people alike as honey bees are agriculture’s most important pollinator. One factor that may influence colony health is the microbial community. Although honey bee worker guts have a characteristic community of bee-specific microbes, the honey bee queen digestive tracts are colonized predominantly by a single acetic acid bacterium tentatively named “Parasaccharibacter apium”. This bacterium is related to flower-associated microbes such asSaccharibacter floricola, and initial phylogenetic analyses placed it as sister to these environmental bacteria. We used a combination of phylogenetic and sequence identity methods to better resolve evolutionary relationships among “P. apium”, strains in the genusSaccharibacter, and strains in the closely related genusBombella. Interestingly, measures of genome-wide average nucleotide identity and aligned fraction, coupled with phylogenetic placement, indicate that many strains labeled as “P. apium” andSaccharibactersp. are all the same species asBombella apis. We propose reclassifying these strains asBombella apisand outline the data supporting that classification below.
In situ detection of protein interactions for recombinant therapeutic enzymes
Authors: Samoudi Mojtaba , Kuo Chih-Chung , Robinson Caressa M. , Shams-Ud-Doha Km , Schinn Song-Min , Kol Stefan , Weiss Linus , Bjorn Sara Petersen , Voldborg Bjorn G. , Campos Alexandre Rosa , Lewis Nathan E.
Published: May 2020

Despite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and post-translational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1 and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method. We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model, and found proteins involved in protein folding, disulfide bond formation and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability. Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.
A Pipeline for Precise and Efficient Genome Editing by sgRNA-Cas9 RNPs inDrosophila
Authors: Nyberg Kevin G. , Nguyen Joseph Q. , Kwon Yong-Jae , Blythe Shelby , Beitel Greg J. , Carthew Richard W.
Published: May 2020

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background ofDrosophila melanogasteras well as to otherDrosophilaspecies. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi againsteyes absent. This allows for screening based on eye morphology rather than color. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.
Plasmids shape the diverse accessory resistomes ofEscherichia coliST131
Authors: Decano Arun Gonzales , Tran Nghia , Al-Foori Hawriya , Al-Awadi Buthaina , Campbell Leigh , Ellison Kevin , Mirabueno Louisse Paola , Nelson Maddy , Power Shane , Smith Genevieve , Smyth Cian , Vance Zoe , Woods Caitriona , Rahm Alexander , Downing Tim
Published: May 2020

The human gut microbiome includes beneficial, commensal and pathogenic bacteria that possess antimicrobial resistance (AMR) genes that exchange these predominantly through conjugative plasmids.Escherichia coliis a significant component of the gastrointestinal microbiome and is typically non-pathogenic in this niche. In contrast, extra-intestinal pathogenicE. coli(ExPEC) including ST131 may occupy other environments like the urinary tract or bloodstream where they express genes enabling AMR and host cell adhesion like type 1 fimbriae. The extent to which commensalE. coliand uropathogenic ExPEC ST131 share AMR genes remains understudied at a genomic level, and here we examined this using a preterm infant resistome. Here, individual ST131 had small differences in AMR gene content relative to a larger shared resistome. Comparisons with a range of plasmids common in ST131 showed that AMR gene composition was driven by conjugation, recombination and mobile genetic elements. Plasmid pEK499 had extended regions in most ST131 Clade C isolates, and it had evidence of a co-evolutionary signal based on protein-level interactions with chromosomal gene products, as did pEK204 that had a type IV fimbrialpiloperon. ST131 possessed extensive diversity of selective type 1, type IV, P and F17-like fimbriae genes that was highest in subclade C2. The structure and composition of AMR genes, plasmids and fimbriae vary widely in ST131 Clade C and this may mediate pathogenicity and infection outcomes.

Data Summary

The following files are available on the FigShare project “Plasmids_ST131_resistome_2020” :

The set of 794 AMR genes derived from [74] are available (with their protein sequence translation) at FigShare at doi: dx.doi.org/10.6084/m9.figshare.11961402.

The AMR gene profiles per sample determined by their BLAST sequence similarity results against CARD are available at FigShare at doi: dx.doi.org/10.6084/m9.figshare.11961612. This dataset includes the PlasmidFinder results. It also includes other AMR database comparisons (ARG-ANNOT, ResFinder, MegaRes, VFDB and VirulenceFinder).

The BLAST sequence similarity results for thefim, pil, papanducloperons’ genes versus 4,071E. coliST131 assemblies from Decano & Downing (2019) are available at FigShare at doi: dx.doi.org/10.6084/m9.figshare.11961711.

The genome sequences and annotation files for reference genomes NCTC13441, EC958 and SE15, along with the assembled contigs for 83972 and 3_2_53FAA are available at FigShare at doi: dx.doi.org/10.6084/m9.figshare.11961813.

The 4,071E. coliST131 genome assemblies from Decano & Downing (2019) are available at FigShare at doi: 10.6084/m9.figshare.11962278 (the first 1,680 assemblies) and at doi: dx.doi.org/10.6084/m9.figshare.11962557 (the second 2,391 assemblies).
The Multilocus Multispecies Coalescent: A Flexible New Model of Gene Family Evolution
Authors: Li Qiuyi , Scornavacca Celine , Galtier Nicolas , Chan Yao-Ban
Published: May 2020

Abstract

Incomplete lineage sorting (ILS), the interaction between coalescence and speciation, can generate incongruence between gene trees and species trees, as can gene duplication (D), transfer (T) and loss (L). These processes are usually modelled independently, but in reality, ILS can affect gene copy number polymorphism, i.e., interfere with DTL. This has been previously recognised, but not treated in a satisfactory way, mainly because DTL events are naturally modelled forward-in-time, while ILS is naturally modelled backwards-in-time with the coalescent. Here we consider the joint action of ILS and DTL on the gene tree/species tree problem in all its complexity. In particular, we show that the interaction between ILS and duplications/transfers (without losses) can result in patterns usually interpreted as resulting from gene loss, and that the realised rate of D, T and L becomes non-homogeneous in time when ILS is taken into account. We introduce algorithmic solutions to these problems. Our new model, themultilocus multispecies coalescent(MLMSC), which also accounts for any level of linkage between loci, generalises the multispecies coalescent model and offers a versatile, powerful framework for proper simulation and inference of gene family evolution.
3D single cell migration driven by temporal correlation between oscillating force dipoles
Authors: Godeau A.L. , Leoni M. , Comelles J. , Guyomar T. , Lieb M. , Delanoë-Ayari H. , Ott A. , Harlepp S. , Sens P. , Riveline D.
Published: May 2020

Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-driven force dipoles at both sides of the nucleus, that locally and periodically pinch the matrix. The existence of a phase shift between the two dipoles is required for directed cell motion which manifests itself as cycles with finite area in the dipole-quadrupole diagram, a formal equivalence to the Purcell cycle. We confirm this mechanism by triggering local dipolar contractions with a laser. This leads to directed motion. Our study reveals that these cells control their motility by synchronizing dipolar forces distributed at front and back. This result opens new strategies to externally control cell motion as well as for the design of micro-crawlers.
AI-based identification of grain cultivars via non-target mass spectrometry
Authors: Nichani Kapil , Uhlig Steffen , Colson Bertrand , Hettwer Karina , Simon Kirsten , Bönick Josephine , Uhlig Carsten , Rawel Harshadrai M. , Stoyke Manfred , Gowik Petra , Huschek Gerd
Published: May 2020

Detection of food fraud and geographical traceability of ingredients is a continually sought goal for government institutions, producers, and consumers. Herein we explore the use of non-target high-resolution mass spectrometry approaches and demonstrate its utility through a particularly challenging case study – to distinguish wheat and spelt cultivars. By employing a data-independent acquisition (DIA) approach for sample measurement, the spectra are of considerable size and complexity. We utilize artificial intelligence (AI) algorithms (artificial neural networks) to evaluate the extensive proteomic footprint of several wheat and spelt cultivars. The AI model thus obtained is used to classify newer varieties of spelt, processed flour, and bread samples. Additionally, we discuss the validation of such a method coupling DIA and AI approaches. The novel framework for method validation enables calculation of precision parameters for facile comparison of the discriminatory power of the method and in the development of a reliable decision rule.
A Modified ACE2 peptide mimic to block SARS-CoV2 entry
Authors: Saurabh Suman , Purohit Shubh Sanket
Published: May 2020

A 23-residue peptide fragment that forms a part of theα-1 helix of the ACE2 peptidase domain, the recognition domain for SARS-CoV2 on the ACE2 receptor, holds the potential as a drug to block the viral receptor binding domain (RBD) from forming a complex with ACE2. The peptide has recently been shown to bind the viral RBD with good efficiency. Here, we present a detailed analysis of the energetics of binding of the peptide to the SARS-CoV2 RBD. We use equilibrium molecular dynamics simulation to study the dynamics of the complex. We perform end-state binding energy calculations to gain a residue-level insight into the binding process and use the information to incorporate point mutations into the peptide. We demonstrate using binding energy calculations that the peptide with certain point mutations, especially E17L, shows a stronger binding to the RBD as compared to the wild type peptide. We propose that the modified peptide will thus be more efficient in blocking RBD-ACE2 binding.
Haldane’s rule in the placenta: sex-biased misregulation of theKcnq1imprinting cluster in hybrid mice
Authors: Arévalo Lena , Gardner Sarah , Campbell Polly
Published: May 2020

Mammalian hybrids often show striking asymmetries in their phenotypes both between reciprocal crosses, and between sexes in accordance with Haldane’s rule. Hybrid inviability is associated with parent-of-origin placental growth abnormalities for which misregulation of imprinted genes is a strong candidate mechanism. However, direct evidence for the involvement of abnormal imprinting and the mechanisms behind this proposed misregulation is limited. We used transcriptome and reduced representation bisulfite sequencing to evaluate the contribution of imprinted genes to a long-standing example of parent-of-origin placental growth dysplasia in the cross between the house mouse (Mus musculus domesticus) and the Algerian mouse (Mus spretus). We found little evidence for loss of imprinting and imprinted genes with biallelic expression were not misexpressed. Instead, imprinted genes with transgressive expression and methylation were concentrated in theKcnq1cluster, which contains causal genes for prenatal growth abnormalities in both mice and humans. Hypermethylation of the cluster’s imprinting control region, and consequent misexpression of the genesPhlda2andAscl2, is a strong candidate mechanism for hybrid placental undergrowth. Transgressive placental and gene regulatory phenotypes, including expression and methylation in the Kcnq1 cluster, were more extreme in hybrid males. While consistent with Haldane’s rule, male-biased defects are not expected in rodent placenta because the maternal X chromosome is effectively hemizygous in both sexes. In search of an explanation we found evidence of leaky imprinted X-chromosome inactivation in hybrid females. Supplementary expression from the paternal X-chromosome may buffer the females from the effects of X-linked incompatibilities to which males are fully exposed. Sex differences in chromatin structure on the X and sex-biased maternal effects are non-mutually exclusive alternative explanations for adherence to Haldane’s rule in hybrid placenta. The results of this study contribute to understanding of the genetic basis of hybrid inviability in mammals, and the role of imprinted genes in speciation.
ampir: an R package for fast genome-wide prediction of antimicrobial peptides
Authors: Fingerhut Legana C.H.W , Miller David J. , Strugnell Jan M. , Daly Norelle L. , Cooke Ira R.
Published: May 2020

Antimicrobial peptides (AMPs) are key components of the innate immune system that protect against pathogens, regulate the microbiome, and are promising targets for pharmaceutical research. Computational tools based on machine learning have the potential to aid discovery of genes encoding novel AMPs but existing approaches are not designed for genome-wide scans. To facilitate such genome-wide discovery of AMPs we developed a fast and accurate AMP classification framework, ampir. ampir is designed for high throughput, integrates well with existing bioinformatics pipelines, and has much higher classification accuracy than existing methods when applied to whole genome data.

Availability and Implementation

ampir is implemented primarily in R with core feature calculation methods written in C++. Release versions are available via CRAN and work on all major operating systems. The development version is maintained athttps://github.com/legana/ampir

Contact

legana.fingerhut@my.jcu.edu.au;ira.cooke@jcu.edu.au

Supplementary information

Supplementary data are available athttps://github.com/legana/amp_pub
Three-dimensional reconstructions of the internal structures of haustoria in parasitic Orobanchaceae
Authors: Masumoto Natsumi , Suzuki Yuki , Cui Songkui , Wakazaki Mayumi , Sato Mayuko , Kumaishi Kie , Shibata Arisa , Furuta Kaori M. , Ichihashi Yasunori , Shirasu Ken , Toyooka Kiminori , Sato Yoshinobu , Yoshida Satoko
Published: May 2020

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasiteStriga hermonthicainfecting rice and the facultative parasitePhtheirospermum japonicuminfectingArabidopsis. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides structural insights into how organs interact between hosts and parasitic plants.

One-sentence summary

Three-dimensional image reconstruction was used to visualize the spatial organization of cell types in the haustoria of parasitic plants with special reference to their interaction with host roots.
Ultraviolet A1 phototherapy for the treatment of localized scleroderma
Authors: Furuhashi Takuya , Torii Kan , Ikumi Kyoko , Kato Hiroshi , Nishida Emi , Morita Akimichi
Published: May 2020

Ultraviolet (UV)A1 phototherapy is effective for T‐cell‐mediated skin diseases such as atopic dermatitis and mast cell‐mediated skin diseases such as mastocytoma. UVA1 phototherapy is also effective against the sclerotic lesions of systemic sclerosis and morphea. Currently, in Japan, access to UVA1 phototherapy is limited because the UVA1 phototherapy device has not yet been approved. On the basis of our experience, we report three patients with localized scleroderma who responded successfully to UVA1 phototherapy. Efficacy was assessed by histological analysis and elastography. UVA1 successfully ameliorated sclerotic lesions, including morphea, linear scleroderma and morphea lesions in a patient with limited cutaneous systemic sclerosis. No side‐effects were observed during UVA1 phototherapy.
Toll‐like receptor‐2 mediates systemic inflammation in gentamicin‐induced rat nephrotoxicity
Authors: Karimi Zeinab , Pakfetrat Zahra , Roozbeh Jamshid , Janfeshan Sahar
Published: May 2020

Gentamicin is an aminoglycoside antibiotic commonly administrated to patients with Gram‐negative infections. Gentamicin induced nephrotoxicity by functional and structural impairment. Toll‐like receptors (TLRs) as key mediators in the innate and adaptive immune system response involved in gentamicin‐induced nephrotoxicity. The present study aimed to investigate the gene expression of TLR2 and pro‐inflammatory cytokines in the renal tissues and buffy coat of the whole blood in gentamicin‐treated rats. Twenty adult male Sprague Dawley rats weighing 180–200 were randomly divided into gentamicin (100 mg/kg, i.p) and control groups (n = 10). After 10 days, the serum creatinine (Cr) levels and blood urea nitrogen (BUN) were measured. The mRNA levels of TLR2, tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, and monocyte chemoattractant peptide (MCP)‐1 were investigated in the renal tissue and buffy coat by qRT‐PCR. Kidney histological analysis performed by hematoxylin‐eosin (H&E) staining. Functional disturbance is characterized by a significant increase in the serum levels of Cr and BUN in the gentamicin group. Renal tissue slides of the gentamicin group indicated severe glomerular and tubular damage including lobulation of the glomerular tuft, Bowman's space enlargement, acute tubular necrosis, and proximal tubular destruction. The mRNA levels of IL‐1β, TNF‐α, MCP‐1, and TLR2 increased in the buffy coat, but all of them except TLR2 decreased in the renal tissues in the gentamicin group compared with controls. Gentamicin administration induced relative systemic inflammation, which may be related to an increase in the mRNA levels of TLR2 results in gene expression of pro‐inflammatory chemokines and cytokines including IL‐1β, TNF‐α, and MCP‐1 in immune cells.