bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel framework for single-cell Hi-C clustering based on graph-convolution-based imputation and two-phase-based feature extraction
Authors: Zhen Caiwei , Wang Yuxian , Han Lu , Li Jingyi , Peng Jinghao , Wang Tao , Hao Jianye , Shang Xuequn , Wei Zhongyu , Peng Jiajie
Published: May 2021

The three-dimensional genome structure plays a key role in cellular function and gene regulation. Singlecell Hi-C technology can capture genome structure information at the cell level, which provides the opportunity to study how genome structure varies among different cell types. However, few methods are well designed for single-cell Hi-C clustering, because of high sparsity, noise and heterogeneity of single-cell Hi-C data. In this manuscript, we propose a novel framework, named ScHiC-Rep, for singlecell Hi-C data representation and clustering. ScHiC-Rep mainly contains two parts: data imputation and feature extraction. In the imputation part, a novel imputation workflow is proposed, including graph convolution-based, random walk with restart-based and genomic neighbor-based imputation. In the feature extraction part, a two-phase feature extraction method is proposed, including linear phase for chromosome level and non-linear phase for cell level feature extraction. The evaluation results show that the proposed framework outperforms existing state-of-the-art approaches on both human and mouse datasets.
Breaking the Scale: Allometric scaling analysis in Carnivoran families
Authors: Navaratnam Stefanie , Phillips Julie Baker
Published: May 2021

The analysis of scaling relationships, allometric scaling, has a long history of importance for modelling and predicting biological phenomena. Individual organisms are not truly independent, and as a result phylogenetic corrections are necessary to increase the accuracy of scaling relationships. The relationships between body mass and gestation length have not been previously reported at the Family level, as it was previously thought species had insufficient time to diverge evolutionarily leaving phylogenetic corrections unnecessary. Using a Carnivora supertree, we perform a phylogenetically generalised least squares (PGLS) analysis using life history information largely from the Pantheria dataset. Our results suggest that allometric relationships are maintained in four families: Canidae, Felidae, Herpestidae and Otariidae. Conversely, several evolutionary mechanisms may contribute to the lack of a significant scaling parameter in other families, such as diverse reproductive strategies or positive selection for genes affecting adiposity. In addition, low sample sizes or the inclusion of paternal body masses could alter the presence of significant scaling. Our results suggests that PGLS analyses are informative at the family level, and the absence of scaling can provide insight to understanding of the evolutionary mechanisms that work on the family taxonomic level or below.

CCS CONCEPTS

• Applied computing → Molecular evolution; • Computing methodologies → Modeling methodologies.

ACM Reference Format

Stefanie Navaratnam and Julie Baker Phillips. 2021. Breaking the Scale: Allometric scaling analysis in Carnivoran families. InBCB: ACM Conference on Bioinformatics, Computational Biology, and Health Informatics, August 01–04, 2021, Virtual due to COVID-19.ACM, New York, NY, USA, 7 pages.https://doi.org/10.1145/1122445.1122456
Molecular and cell biological analysis of SwrB inBacillus subtilis
Authors: Phillips Andrew M. , Sanchez Sandra , Sysoeva Tatyana A. , Burton Briana M. , Kearns Daniel B.
Published: May 2021

Swarming motility is flagellar-mediated movement over a solid surface and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion but the mechanism by which it might perform this function is unknown. Here we show that SwrB likely acts sub-stoichiometrically as it localizes as puncta at the membrane in numbers fewer than that of flagellar basal bodies. Moreover the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely co-localized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs.

IMPORTANCE

Type III secretion systems (T3SS) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moroever, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here we explore a poorly understood activator the flagellar T3SS activation inBacillus subtiliscalled SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.
Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch
Authors: Guo Jiao , Jia Xiaoying , Liu Yang , Cao Junyuan , Xiao Gengfu , Wang Wei
Published: May 2021

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever, for which there are limited treatment options. Successful LASV entry requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of GP1 mutant proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus residue (L84N, K88E, L10F, and H170S) reduced the binding affinity of GP1LASVfor LAMP1. Moreover, all the mutations caused decreases in GPC-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84Nor GPCK88Edecreased sharply in multiple cell types, whereas L107F and H170S had only mild effects on infectivity. Notably, in LAMP1 knockout cells, all four mutants showed reduced pseudovirus infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order L84N > L107F > K88E > H170S.

IMPORTANCE

Lassa virus requires pH-dependent receptor switch to infect host cells; however, the underlying molecular mechanisms of this process are not well known. Here, we identify four residues, L84, K88, L107, and H170 that contribute to the interaction with the second receptor lysosome-associated membrane protein 1 (LAMP1). Mutant any of the four residues would impair the binding affinity to LAMP1, decrease the glycoprotein mediated membrane fusion, and reduce the pseudovirus infectivity.
The host factor ANP32A is required for influenza A virus vRNA and cRNA synthesis
Authors: Nilsson-Payant Benjamin E. , tenOever Benjamin R. , te Velthuis Aartjan J.W.
Published: May 2021

Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is bound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the complementary RNA (cRNA), and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases and differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from a cRNA, but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32 is not only needed for the actively replicating polymerase, but also for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation.

IMPORTANCE

Zoonotic avian influenza A viruses pose a constant threat to global health and they have the potential to cause highly pathogenic pandemic outbreaks. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments and in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses.
A multisensory circuit for gating intense aversive experiences
Authors: Asok Arun , Leroy Félix , Parro Cameron , de Solis Christopher A. , Ford Lenzie , Fitzpatrick Michelle N. , Kalmbach Abigail , Neve Rachael , Rayman Joseph B , Kandel Eric R.
Published: May 2021

The ventral hippocampus (vHPC) is critical for both learned and innate fear, but how discrete projections control different types of fear is poorly understood. Here, we report a novel excitatory circuit from a subpopulation of the ventral hippocampus CA1 subfield (vCA1) to the inhibitory peri-paraventricular nucleus of the hypothalamus (pPVN) which then routes to the periaqueductal grey (PAG). We find that vCA1→pPVN projections modulatebothlearned and innate fear. Fiber photometric calcium recordings reveal that activity in vCA1→pPVN projections increases during the first moments of exposure to an unconditioned threat. Chemogenetic or optogenetic silencing of vCA1→pPVN cell bodies or vCA1→pPVN axon terminals in the pPVN enhances the initial magnitude of both active and passive unconditioned defensive responses, irrespective of the sensory modalities engaged by a particular innate threat. Moreover, silencing produces a dramatic impact on learned fear without affecting milder anxiety-like behaviors. We also show that vCA1→pPVN projections monosynaptically route to the PAG, a key brain region that orchestrates the fear response. Surprisingly, optogenetic silencing of vCA1 terminals in the pPVN titrates the level of c-Fos neural activity in the PAG differently for learned versus innate threats. Together, our results show how a novel vCA1→pPVN circuit modulates neuronal activity in the PAG to regulate both learned and innate fear. These findings have implications for how initial trauma processing may influence maladaptive defensive behaviors across fear and trauma-related disorders.

One Sentence Summary

A multisensory gate for high intensity aversive experiences.
INfrastructure for a PHAge REference Database: Identification of large-scale biases in the current collection of phage genomes
Authors: Cook Ryan , Brown Nathan , Redgwell Tamsin , Rihtman Branko , Barnes Megan , Clokie Martha , Stekel Dov J. , Hobman Jon , Jones Michael A. , Millard Andrew
Published: May 2021

Background

With advances in sequencing technology and decreasing costs, the number of bacteriophage genomes that have been sequenced has increased markedly in the last decade.

Materials and Methods

We developed an automated retrieval and analysis system for bacteriophage genomes, INPHARED (https://github.com/RyanCook94/inphared), that provides data in a consistent format.

Results

As of January 2021, 14,244 complete phage genomes have been sequenced. The data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated only on 30 bacterial genera. There is further bias with significantly more lytic phage genomes than temperate within the database, resulting in ~54% of temperate phage genomes originating from just three host genera. Within phage genomes, putative antibiotic resistance genes were found in higher frequencies in temperate phages than lytic phages.

Conclusion

We provide a mechanism to reproducibly extract complete phage genomes and highlight some of the biases within this data, that underpins our current understanding of phage genomes.
Phylogenomic study ofStaphylococcus aureusandStaphylococcus haemolyticusclinical isolates from Egypt
Authors: Montelongo Cesar , Mores Carine R. , Putonti Catherine , Wolfe Alan J. , Abouelfetouh Alaa
Published: May 2021

Antibiotic resistantStaphylococcusinfections are a global concern, with increasing cases of resistantStaphylococcus aureusandStaphylococcus haemolyticusfound circulating in the Middle East. While extensive surveys have described the prevalence of resistant infections in Europe, Asia, and North America, the population structure of resistant staphylococcal Middle Eastern clinical isolates is poorly characterized. We performed whole genome sequencing of 56S. aureusand 10S. haemolyticusisolates from Alexandria Main University Hospital. Supplemented with additional publicly available genomes from the region (34S. aureusand 6S. haemolyticus), we present the largest genomic study of staphylococcal Middle Eastern isolates. These genomes include 20S. aureusmultilocus sequence typing (MLST) types and 9S. haemolyticusMLSTs, including 3 and 1 new MLSTs, respectively. Phylogenomic analyses of each species core genome largely mirrored MLSTs, irrespective of geographical origin. The hospital-acquiredspat037/SCCmecIII/MLST CC8 clone represented the largest clade, comprising 22% ofS. aureusisolates. Similar to other regional genome surveys ofS. aureus, the Middle Eastern isolates have an open pangenome, a strong indicator of gene exchange of virulence factors and antibiotic resistance genes with other reservoirs. We recommend stricter implementation of antibiotic stewardship and infection control plans in the region.

Impact Statement

Staphylococci are under-studied despite their prevalence within the Middle East. Methicillin-resistantStaphylococcus aureus(MRSA) is endemic to hospitals in this region, as are other antibiotic-resistant strains ofS. aureusandS. haemolyticus. To provide insight into the strains currently in circulation within Egypt, we performed whole genome sequencing of 56S. aureusand 10S. haemolyticusisolates from Alexandria Main University Hospital (AMUH). Through analysis of these genomes, as well as other genomes of isolates from the Middle East, we were able to produce a more complete picture of the current diversity than traditional molecular typing strategies. Furthermore, theS. haemolyticusgenome analyses provide the first insight into strains found in Egypt. Our analysis of resistance and virulence mechanisms carried by these strains provides invaluable insight into future plans of antibiotic stewardship and infection control within the region.

Data Summary

Raw sequencing reads and assembled genomes can be found at BioProject Accession number PRJNA648411 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA648411).
Aplp1 and the Aplp1-Lag3 Complex facilitates transmission of pathologic α-synuclein
Authors: Mao Xiaobo , Gu Hao , Kim Donghoon , Kimura Yasuyoshi , Wang Ning , Xu Enquan , Wang Haibo , Chen Chan , Zhang Shengnan , Jia Chunyu , Liu Yuqing , Bian Hetao , Karuppagounder Senthilkumar S. , Jia Longgang , Ke Xiyu , Chang Michael , Li Amanda , Yang Jun , Rastegar Cyrus , Sriparna Manjari , Ge Preston , Brahmachari Saurav , Kim Sangjune , Zhang Shu , Shimoda Yasushi , Saar Martina , Workman Creg J. , Vignali Dario A. A. , Muller Ulrike C. , Liu Cong , Ko Han Seok , Dawson Valina L. , Dawson Ted M.
Published: May 2021

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid β precursor-like protein 1 (Aplp1) forms a complex with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFFin vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology advances our understanding of the molecular mechanism of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson’s disease and related α-synucleinopathies.

One Sentence Summary

Aplp1 forms a complex with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-synuclein.

Graphical Abstract

Aplp1 and the Aplp1-Lag3 complex facilitates transmission of pathologic α-synuclein.

Aplp1 is a receptor that drives pathologic α-syn transmission, and genetic depletion of Aplp1 can significantly reduce the α-synuclein pathogenesis. Aplp1 and Lag3 forms an Aplp1-Lag3 complex that accounts for substantial binding of pathologic α-syn to cortical neurons. Together Aplp1 and Lag3 play a major role in pathologic α-syn internalization, transmission and toxicity. Double knockout of Aplp1 and Lag3 and or a Lag3 antibody that disrupts the Aplp1 and Lag3 complex almost completely blocks α-syn PFF-induced neurodegeneration.
Activation of extrasynaptic kainate receptors drives hilar mossy cell activity
Authors: Ramos Czarina , Lutzu Stefano , Yamasaki Miwako , Yanagawa Yuchio , Sakimura Kenji , Tomita Susumu , Watanabe Masahiko , Castillo Pablo E.
Published: May 2021

Mossy cells (MCs) of the dentate gyrus (DG) are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place field encoding, pattern separation and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished inGluK2KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs, and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.

Significance Statement

Hilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitabilityin vivoand may also promote dentate gyrus hyperexcitability and epileptogenesis.
Deep Learning-Inferred Multiplex ImmunoFluorescence for IHC Image Quantification
Authors: Ghahremani Parmida , Li Yanyun , Kaufman Arie , Vanguri Rami , Greenwald Noah , Angelo Michael , Hollmann Travis J. , Nadeem Saad
Published: May 2021

Reporting biomarkers assessed by routine immunohistochemical (IHC) staining of tissue is broadly used in diagnostic pathology laboratories for patient care. To date, clinical reporting is predominantly qualitative or semi-quantitative. By creating a multitask deep learning framework referred to as DeepLIIF, we present a single-step solution to stain deconvolution/separation, cell segmentation, and quantitative single-cell IHC scoring. Leveraging a uniquede novodataset of co-registered IHC and multiplex immunofluorescence (mpIF) staining of the same slides, we segment and translate low-cost and prevalent IHC slides to more expensive-yet-informative mpIF images, while simultaneously providing the essential ground truth for the superimposed brightfield IHC channels. Moreover, a new nuclear-envelop stain, LAP2beta, with high (>95%) cell coverage is introduced to improve cell delineation/segmentation and protein expression quantification on IHC slides. By simultaneously translating input IHC images to clean/separated mpIF channels and performing cell segmentation/classification, we show that our model trained on clean IHC Ki67 data can generalize to more noisy and artifact-ridden images as well as other nuclear and non-nuclear markers such as CD3, CD8, BCL2, BCL6, MYC, MUM1, CD10, and TP53. We thoroughly evaluate our method on publicly available benchmark datasets as well as against pathologists’ semi-quantitative scoring. The code, the pre-trained models, along with easy-to-run containerized docker files as well as Google CoLab project are available athttps://github.com/nadeemlab/deepliif.
Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extended metabarcodes
Authors: Eenjes Tavish , Hu Yiheng , Irinyi Laszlo , Vi Hoang Minh Thuy , Smith Leon M. , Linde Celeste C. , Milgate Andrew W. , Meyer Wieland , Stone Eric A. , Rathjen John P. , Mashford Benjamin , Schwessinger Benjamin
Published: May 2021

Background

The increased usage of error-prone long-read sequencing for metabarcoding of fungi has not been matched with adequate public databases and concomitant analysis approaches. We address this gap and present a proof-of-concept study for classifying fungal taxa using linked machine learning classifiers. We demonstrate the capability of linked machine learning classifiers to accurately classify species and strains using real-world and simulated fungal ribosomal DNA datasets, including plant and human pathogens. We benchmark our new approach in comparison to current alignment and k-mer based methods based on synthetic mock communities. We also assess real world applications of species identification in complex unlabelled datasets.

Results

Our machine learning approach assigned individual nanopore long-read amplicon sequences to fungal species with high recall rates and low false positive rates. Importantly, our approach successfully distinguished between closely-related species and strains when individual read errors were higher than the genetic distance between individual taxa, which the alignment and k-mer methods could not do. The machine learning approach showed an ability to identify key species with high recall rates, even in complex samples of unknown species composition.

Conclusions

A proof of concept machine learning approach using a tree-descent approach on a decision tree of classifiers can identify known taxa with high accuracy, and precisely detect known target species from complex samples with high recall rates. We propose this approach is suitable for detecting the known knowns of pathogens or invasive species in any environment of mostly unknown composition, including agriculture and wild ecosystems.
PAM-interacting domain swapping is extensively utilized in nature to evolve CRISPR-Cas9 nucleases with altered PAM specificities
Authors: Wang Qiuyan , Bian Xiaomeng , Du Jinli , Lv Yawen , Tao Lin , Xie Tian
Published: May 2021

It has been anticipated that protospacer adjacent motif (PAM) specificity of the CRISPR-Cas9 nucleases protospacer appears to be modular. Here we present the finding that naturally occurring domain swapping has been extensively involved in altering its PAM specificities. Sequence analysis of streptococcal Cas9 sequences revealed conservation of three distinct PAM-interacting motifs, with phylogenetic analysis of full-length Cas9 and the PID demonstrating that PAM-interacting domain (PID) domain swapping was extensively utilized to diversify its PAM specificity. An extended analysis of 582 representative Cas9 sequences revealed that this PID swapping was broadly present in most of the investigated genera. Mimicking the natural PID domain swapping, a functional chimeric enzyme, based on the scaffold of compact Staphylococcus aureus Cas9, with novel NNAAAA PAM specificity was developed. In summary, our findings shed new light on a rich source of exchangeable PID domains in Cas9 genes, which can be mined for domain swapping aiming to an effective PAM refinement.
Evaluation of Antibiotic Tolerance inPseudomonas aeruginosafor Aminoglycosides and its Predicted Gene Regulations Through In-silico Transcriptomic Analysis
Authors: Kumar Abishek , Thankappan Bency , Jayaraman Angayarkanni , Gupta Akshita
Published: May 2021

Pseudomonas aeruginosacauses chronic infections like cystic fibrosis, endocarditis, bacteremia and sepsis, which are life-threatening and difficult to treat. The lack of antibiotic response inP. aeruginosais due to adaptive resistance mechanism, which prevents the entry of antibiotics into cytosol of the cell to achieve tolerance. Among different groups of antibiotics, aminoglycosides are used as a parental antibiotic for treatment ofP. aeruginosa. This study aims to determine the kinetics of antibiotic tolerance and gene expression changes inP. aeruginosaexposed to amikacin, gentamicin, and tobramycin. These antibiotics were exposed toP. aeruginosaat their MICs and the experimental setup was monitored till 72 hours, followed by the measurement of optical density in the interval of every 12 hours. The growth ofP. aeruginosain MICs of antibiotics represents the kinetics of antibiotic tolerance in amikacin, gentamicin, and tobramycin. Transcriptomic profile of antibiotic exposedP. aeruginosaPA14 was taken from Gene Expression Omnibus (GEO), NCBI as microarray datasets. The gene expressions of two datasets were compared by test versus control. Tobramycin exposedP. aeruginosafailed to develop tolerance in MICs 0.5µg/mL, 1µg/mL and 1.5µg/mL. Whereas amikacin and gentamicin treatedP. aeruginosadeveloped tolerance in MICs. This depicts the superiorin vitroresponse of tobramycin over the gentamicin and amikacin. Further,in silicotranscriptomic analysis of tobramycin treatedP. aeruginosaresulted in low expression of 16s rRNA Methyltransferase E, B & L, alginate biosynthesis genes and several proteins of Type 2 Secretory System (T2SS) and Type 3 Secretory System (T3SS). The Differentially Expressed Genes (DEGs) of alginate biosynthesis, and RNA Methyltransferases suggests increased antibiotic response and low probability of developing resistance. The use of tobramycin as a parental antibiotic with its synergistic combination might combatP. aeruginosawith increased response.
Fractions of traditionally brewed rice beverage ameliorate anxiety and improve spatial memory in mice
Authors: Bhaskar Bhuwan , Adak Atanu , Khan Mojibur R.
Published: May 2021

Rice beverages are traditionally prepared and consumed popularly by the different ethnic groups of North East India. To investigate its effects on behavior, mice were treated with different fractions of rice beverage that included the beverage as a whole, insoluble and soluble fractions. Intragastric treatments of these fractions were given to the mice (n=6 per group) for 30 days and behavioral studies were performed on elevated plus and Y maze to evaluate anxiety and spatial memory, respectively. Next generation sequencing of metagenomic DNA of the beverage indicated presence of 157 OTUs and 26 bacterial genera were dominant with an abundance of 0.1%. The insoluble fraction treated animals showed lowest anxiety like symptoms. Spatial memory improved in all the treatments compared to the control, of which the rice beverage treatment showed the highest levels (p<0.05). Gas chromatography and mass spectroscopy-based metabolite profiling of the beverage revealed 10 alcohols, 29 sachharides, 43 acids and 13 amino acids. Findings of this study suggest a positive effect of rice beverage components on anxiety and spatial memory of mice.
Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain
Authors: Kowalski-Jahn Maria , Schihada Hannes , Turku Ainoleena , Huber Thomas , Sakmar Thomas P. , Schulte Gunnar
Published: May 2021

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.
Targetron-assisted delivery of exogenous DNA sequences intoPseudomonas putidathrough CRISPR-aided counterselection
Authors: Velázquez Elena , Al-Ramahi Yamal , Tellechea Jonathan , Krasnogor Natalio , de Lorenzo Víctor
Published: May 2021

Genome editing methods based on Group II introns (known as Targetron technology) have been long used as a gene knock-out strategy in a wide range of organisms in a fashion independent of homologous recombination. Yet, their utility as delivery systems has been typically suboptimal because of their reduced efficiency of insertion when they carry exogenous sequences. We show that this limitation can be tackled and Targetron adapted as a general tool in Gram-negative bacteria. To this end, a set of broad host range standardized vectors were designed for conditional expression of the Ll.LtrB intron. After testing the correct functionality of these plasmids inEscherichia coliandPseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. By following this pipeline, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-typeP. putidaKT2440 and its ΔrecAderivative. Finally, we were able to apply this technology to successfully tag this strain with an orthogonal short sequence (barcode) that acts as a unique identifier for tracking this microorganism in biotechnological settings. The results withP. putidaexemplified the value of the Targetron approach for unrestricted delivery of small DNA fragments to the genomes of Gram-negative bacteria for a suite of genome editing endeavours.
Protein secondary structure affects glycan clustering in native mass spectrometry
Authors: Yan Hao , Lockhauserbäumer Julia , Szekeres Gergo Peter , Mallagaray Alvaro , Creutznacher Robert , Taube Stefan , Peters Thomas , Pagel Kevin , Uetrecht Charlotte
Published: May 2021

Infection with human noroviruses (hNoV) for the vast majority of strains requires attachment of the viral capsid to histo blood group antigens (HBGA). The HBGA binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for binding of HBGAs to P dimers and, in some cases, disagreed whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of the reference protein is crucial. We analyzed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of β-sheet has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.
Organophosphate intoxication inC. elegansreveals a new route to mitigate poisoning through the modulation of determinants responsible for nicotinic acetylcholine receptor function
Authors: Izquierdo Patricia G. , Charvet Claude L. , Neveu Cedric , Green A. Christopher , Tattersall John E.H. , Holden-Dye Lindy , O’Connor Vincent
Published: May 2021

Plasticity is a reactive mechanism that allows the adaptation of organisms to changing environmental cues. The exploitation of this physiological process has a clear benefit to promote the recovery from a wide range of neurological disorders. Here, we show that plasticity-promoting regimes provide candidate mechanisms to supplement the classically used antidotes for anti-cholinesterase poisoning. These neurotoxins inhibit acetylcholinesterase, causing the overstimulation of cholinergic transmission at synapses and neuromuscular junctions. The model organismC. elegansexhibits organophosphate-induced mitigating plasticity that impacts on the recovery of neuromuscular phenotypes, initially impaired by the drug. This is underpinned by overstimulation of nicotinic receptors at the neuromuscular junction. Intrinsic determinants of receptor’s location and sensitivity modulate the extent of plasticity in the context of persistent cholinergic stimulation. Our results indicate that pharmacological intervention of nicotinic receptors and/or scaffolding proteins that support receptor function might provide a novel treatment route for anti-cholinesterase poisoning.
Dyrk1agene dosage in glutamatergic neurons has key effects in cognitive deficits observed in mouse models of MRD7 and Down syndrome
Authors: Brault Véronique , Nguyen Thu Lan , Flores-Gutiérrez Javier , Iacono Giovanni , Birling Marie-Christine , Lalanne Valérie , Méziane Hamid , Manousopoulou Antigoni , Pavlovic Guillaume , Lindner Loïc , Selloum Mohammed , Sorg Tania , Yu Eugene , Garbis Spiros D. , Hérault Yann
Published: May 2021

Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases including to autism spectrum disorders, intellectual disability, and epilepsy. Mutation in theDYRK1Agene located on human chromosome 21 (Hsa21) leads to an intellectual disability syndrome associated with microcephaly, epilepsy, and autistic troubles (MRD7). Overexpression of DYRK1A, on the other hand, has been linked with learning and memory defects observed in people with Down syndrome (DS).Dyrk1ais expressed in both glutamatergic and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. Here we investigated the impact ofDyrk1agene copy number variation in glutamatergic neurons using a conditional knockout allele ofDyrk1acrossed with the Tg(Camk2-Cre)4Gsc transgenic mouse. We explored this genetic modification in homozygotes, heterozygotes and combined with the Dp(16Lipi-Zbtb21)1Yey trisomic mouse model to unravel the consequence ofDyrk1adosage from 0 to 3, to understand its role in normal physiology, and in MRD7 and DS. Overall,Dyrk1adosage in glutamatergic neurons did not impact locomotor activity, working memory or epileptic susceptibility, but revealed thatDyrk1ais involved in long-term explicit memory. Molecular analyses pointed at a deregulation of transcriptional activity through immediate early genes and a role of DYRK1A at the glutamatergic post-synapse by deregulating and interacting with key post-synaptic proteins implicated in mechanism leading to long-term enhanced synaptic plasticity. Altogether, our work gives important information to understand the action of DYRK1A inhibitors and have a better therapeutic approach.

Author summary

The Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A, DYRK1A, drives cognitive alterations with increased dose in Down syndrome (DS) or with reduced dose in mental retardation disease 7 (MRD7). Here we report that specific and complete loss ofDyrk1ain glutamatergic neurons induced a range of specific cognitive phenotypes and alter the expression of genes involved in neurotransmission in the hippocampus. We further explored the consequences ofDyrk1adosage in glutamatergic neurons on the cognitive phenotypes observed respectively in MRD7 and DS mouse models and we found specific roles in long-term explicit memory with no impact on motor activity, short-term working memory, and susceptibility to epilepsy. Then we demonstrated that DYRK1A is a component of the glutamatergic post-synapse and interacts with several component such as NR2B and PSD95. Altogether our work describes a new role of DYRK1A at the glutamatergic synapse that must be considered to understand the consequence of treatment targeting DYRK1A in disease.
Predicting drug concentrations in PDMS microfluidic organ chips
Authors: Grant Jennifer , Özkan Alican , Oh Crystal , Mahajan Gautam , Prantil-Baun Rachelle , Ingber Donald E.
Published: May 2021

Microfluidic organ-on-a-chip (Organ Chip) cell culture devices are often fabricated using polydimethylsiloxane (PDMS) because it is biocompatible, transparent, elastomeric, and oxygen permeable; however, hydrophobic small molecules can absorb to PDMS, which makes it challenging to predict drug responses. Here, we describe a combined simulation and experimental approach to predict the spatial and temporal concentration profile of a drug under continuous dosing in a PDMS Organ Chip containing two parallel channels separated by a porous membrane that is lined with cultured cells, without prior knowledge of its log P value. First, a three-dimensional finite element simulation of drug loss into the chip was developed that incorporates absorption, adsorption, convection, and diffusion, which predicts changes in drug levels over time and space as a function of potential PDMS diffusion coefficients and log P values. By then experimentally measuring the diffusivity of the compound in PDMS and determining its partition coefficient through mass spectrometric analysis of the drug concentration in the channel outflow, it is possible to estimate the effective log P range of the compound. The diffusion and partition coefficients were experimentally derived for the antimalarial drug and potential SARS-CoV-2 therapeutic, amodiaquine, and incorporated into the model to quantitatively predict the drug-specific concentration profile over time measured in human Lung Airway Chips lined with bronchial epithelium interfaced with pulmonary microvascular endothelium. The same strategy can be applied to any device geometry, surface treatment, orin vitromicrofluidic model to predict the spatial and temporal gradient of a drug in 3D without prior knowledge of the partition coefficient or the rate of diffusion in PDMS. Thus, this approach may expand the use of PDMS Organ Chip devices for various forms of drug testing.
Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
Authors: Barr David A. , Omollo Charles , Mason Mandy , Koch Anastasia , Wilkinson Robert J. , Lalloo David G. , Meintjes Graeme , Mizrahi Valerie , Warner Digby F. , Davies Gerry
Published: May 2021

Detection and accurate quantitation of viableMycobacterium tuberculosisis fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology – including the tendency to clump, and “differential” culturability – and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA+ cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derivedMycobacterium bovis BCGtime-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.
Mesenchymal Lineage Heterogeneity Underlies Non-Redundant Functions of Pancreatic Cancer-Associated Fibroblasts
Authors: Helms Erin J. , Berry Mark W. , Chaw R. Crystal , DuFort Chris C. , Sun Duanchen , Onate M. Kathrina , Oon Chet , Bhattacharyya Sohinee , Sanford-Crane Hannah , Horton Wesley , Finan Jennifer M. , Sattler Ariana , Makar Rosemary , Dawson David W. , Xia Zheng , Hingorani Sunil R. , Sherman Mara H.
Published: May 2021

Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSCs), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progressionin vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed non-redundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific components of the extracellular matrix. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs, and demonstrate unique functions for CAFs of a defined cellular origin.

Statement of significance

By tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity as well as signaling gradients diversify PDAC CAFs.