bluuebunny/crossref_metadata_2025 huggingface dataset:
Bifidobacterium breveUCC2003 induces a distinct global transcriptomic programme in neonatal murine intestinal epithelial cells
Authors: Kiu Raymond , Treveil Agatha , Harnisch Lukas C. , Caim Shabhonam , Leclaire Charlotte , van Sinderen Douwe , Korcsmaros Tamas , Hall Lindsay J.
Published: March 2020

Bifidobacteriumis an important gut microbiota member during early life that is associated with improved gut health. However, the underlying health-driving mechanisms are not well understood, particularly howBifidobacteriummay modulate the intestinal barrier via programming of intestinal epithelial cells (IECs). In this study, we sought to investigate the global impact of model strainBifidobacterium breveUCC2003 on the neonatal IEC transcriptome, including gene regulation and pathway modulation. Small IECs from two-week-old neonatal mice administeredB. breveUCC2003 for three consecutive days or PBS (control group) were subjected to global RNASeq, with various bioinformatic approaches used to determine differentially expressed genes, pathways and affected cell types between control and experimental groups. Whilst colonisation withB. brevehad minimal impacts on the neonatal microbiota, we observed extensive regulation of the IEC transcriptome; ~4,000 genes significantly up-regulated, including key genes associated with epithelial barrier function. Enrichment of cell differentiation and cell proliferation pathways were observed, along with an overrepresentation of stem cell marker genes, indicating an increase in the regenerative potential of the epithelial layer. Expression of distinct immune-associated pathway members (e.g. Toll-like Receptors) were also affected after neonatalB. brevecolonisation. In conclusion,B. breveUCC2003 plays a central role in driving universal transcriptomic changes in neonatal IECs that enhances cell replication, differentiation and growth, predominantly in the stem cell compartment. This study enhances our overall understanding of the benefits ofB. brevein driving intestinal epithelium homeostatic development during early life, with potential avenues to develop novel live biotherapeutic products.
Frisky CALF sometimes outruns LASSO
Authors: Jeffries Clark D. , Ford John R. , Tilson Jeffrey L. , Perkins Diana O. , Bost Darius M. , Filer Dayne L. , Wilhelmsen Kirk C.
Published: March 2020

Regularized regression analysis is a mature analytic approach to identify weighted sums of variables that predict outcomes. Typically, the number of subjects (N) is smaller than the number of predictors (p). Here, we present a novel coarse approximation linear function (CALF) to frugally select important predictors, build linear models, and discover causality.

CALF is a linear regression strategy applied to normalized data that employs only a few (such as 2 to 20) nonzero weights, each +1 or -1. Metrics can be Welch t-test p-value, area under curve (AUC) of receiver operating characteristic, or Pearson correlation, depending upon data type and user preferences. For quantitative approximations, a linear fit (adding an intercept value and rescaling ±1 weights by a common multiplier) can be added to optimize mean squared error (MSE).

Real medical data of five types were used to generate examples with goals of binary classification or real variable approximation. Predictors considered were real, sets of integers, or ternary values of single nucleotide polymorphisms. When applied to real data, CALF approximations outperformed in p-value, AUC, correlation, or MSE a popular regularized linear regression algorithm, namely, basic LASSO. It appears that using LASSO without considering CALF might risk wasting resources.

Availability

R version: Comprehensive R Archive (CRAN):https://cran.r-project.org/web/packages/CALF/index.html

Python 3.x version: GitHub:https://github.com/jorufo/CALF_Python

Contact

clark_jeffries@med.unc.edu
A novel platform of RNA 2′-O-methylation high-throughput and site-specific quantification tools revealed its broad distribution on mRNA
Authors: Tang Yao , Wu Yifan , Wang Sainan , Lu Xiaolan , Gu Xiangwen , Li Yong , Yang Fan , Xu Ruilin , Wang Tao , Jiao Zichen , Wu Yan , Liu Liwei , Chen Jian-Qun , Wang Qiang , Chen Qihan
Published: March 2020

Ribose 2′-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain largely unknown due to the lack of accurate and efficient identification tools. To overcome this gap, we established NJU-seq (an accurate high-throughput single-base method) and Nm-VAQ (a site-specific quantification tool). We identified thousands of new Nm sites on mRNA of human and mouse cell lines, in which 68 of 84 selected sites were further validated to be 2′-O-methylated more than 1%. Unlike rRNA, the methylated ratios of most validated mRNA Nm sites were lower than 30%. In addition, mRNA 2′-O-methylation was dynamic-changing according to the circumstance, which was presented with MHV infection. Furthermore, Nm sites of lung surgery samples revealed commonness of lung cancer pathogenesis, providing potential new diagnostic markers.
Accuracy and efficiency of germline variant calling pipelines for human genome data
Authors: Zhao Sen , Agafonov Oleg , Azab Abdulrahman , Stokowy Tomasz , Hovig Eivind
Published: March 2020

Advances in next-generation sequencing technology has enabled whole genome sequencing (WGS) to be widely used for identification of causal variants in a spectrum of genetic-related disorders, and provided new insight into how genetic polymorphisms affect disease phenotypes. The development of different bioinformatics pipelines has continuously improved the variant analysis of WGS data, however there is a necessity for a systematic performance comparison of these pipelines to provide guidance on the application of WGS-based scientific and clinical genomics. In this study, we evaluated the performance of three variant calling pipelines (GATK, DRAGEN™ and DeepVariant) using Genome in a Bottle Consortium, “synthetic-diploid” and simulated WGS datasets. DRAGEN™ and DeepVariant show a better accuracy in SNPs and indels calling, with no significant differences in their F1-score. DRAGEN™ platform offers accuracy, flexibility and a highly-efficient running speed, and therefore superior advantage in the analysis of WGS data on a large scale. The combination of DRAGEN™ and DeepVariant also provides a good balance of accuracy and efficiency as an alternative solution for germline variant detection in further applications. Our results facilitate the standardization of benchmarking analysis of bioinformatics pipelines for reliable variant detection, which is critical in genetics-based medical research and clinical application.
Pyocin efficacy in a murine model ofPseudomonas aeruginosasepsis
Authors: Six Anne , Mosbahi Khedidja , Barge Madhuri , Kleanthous Colin , Evans Thomas , Walker Daniel
Published: March 2020

Synopsis

Background

Bloodstream infections with antibiotic resistantPseudomonas aeruginosaare common and increasingly difficult to treat. Pyocins are naturally occurring protein antibiotics produced byP. aeruginosathat have potential for human use.

Objectives

To determine if pyocin treatment is effective in a murine model of sepsis withP. aeruginosa.

Methods

Recombinant pyocins S5 and AP41 were purified tested for efficacy in aGalleria mellonellainfection model and a murine model ofP. aeruginosasepsis.

Results

Both pyocins produced no adverse effects when injected alone into mice and showed good in vitro antipseudomonal activity. In an invertebrate model of sepsis usingGalleria mellonella, both pyocins significantly prolonged survival. Following injection into mice, both showed extensive distribution into different organs. When administered 5 hours after infection, both pyocins reduced mortality, with pyocin S5 being more effective than AP41.

Conclusions

Pyocins S5 and AP41 show in vivo biological activity and can improve survival in a murine model ofP. aeruginosainfection. They hold promise as novel antimicrobial agents for treatment of multi-drug resistant infections with this microbe.
Proliferation of HIV-infected renal epithelial cells following virus acquisition from infected macrophages
Authors: Hughes Kelly , Akturk Guray , Gnjatic Sacha , Chen Benjamin , Klotman Mary , Blasi Maria
Published: March 2020

Objectives

HIV-1 can infect and persist in different organs and tissues, resulting in the generation of multiple viral compartments and reservoirs. Increasing evidence supports the kidney as such a reservoir. Previous work demonstrated that HIV-1 infected CD4+ T-cells transfer virus to renal tubule epithelial (RTE) cells through cell-to-cell contact. In addition to CD4+ T-cells, macrophages represent the other major target of HIV-1. Renal macrophages induce and regulate inflammatory responses and are critical to homeostatic regulation of the kidney environment. Combined with their ability to harbor virus, macrophages may also play an important role in the spread of HIV-1 infection in the kidney.

Design and Methods

Multiparametric histochemistry analysis was performed on kidney biopsies from individuals with HIV-1 associated nephropathy (HIVAN). Primary monocyte-derived macrophages were infected with a (GFP)-expressing replication competent HIV-1. HIV-1 transfer from macrophages to RTE cells was carried out in a co-culture system and evaluated by fluorescence-microscopy and flow-cytometry. Live imaging was performed to assess the fate of HIV-1 infected RTE cells over time.

Results

We show that macrophages are abundantly present in the renal inflammatory infiltrate of individuals with HIVAN. We observed contact-dependent HIV-1 transfer from infected macrophages to both primary and immortalized renal cells. Live imaging of HIV-1 infected RTE cells revealed four different fates: proliferation, hypertrophy, latency and cell death.

Conclusions

Our study suggests that macrophages may play a role in the dissemination of HIV-1 in the kidney and that proliferation of infected renal cells may contribute to HIV-1 persistence in this compartment.
Mapping the Location of Nucleosomes in Simian Virus 40 Chromatin Using the New England Biolabs FS Library Prep Kit
Authors: Milavetz Barry , Haugen Jacob , Rowbotham Kincaid
Published: March 2020

The location of nucleosomes in chromatin significantly impacts many biological processes including DNA replication, repair and gene expression. A number of techniques have been developed for mapping nucleosome locations in chromatin including MN-Seq (micrococcal nuclease digestion followed by next generation sequencing), ATAC-Seq (Tagamet chromatin fragmentation followed by next generation sequencing), and ChIP-Seq (chromatin immunoprecipitation and fragmentation followed by next generation sequencing). All of these techniques have been successfully used, but each with its own limitations. Recently, New England Biolabs has marketed a new kit, the NEBNext UltraII FS Library Prep kit, for preparing libraries for next generation sequencing from purified genomic DNA. This kit is based on a novel proprietary DNA fragmentation procedure which appears to cleave DNA that is not bound by proteins. Because DNA is fragmented directly in the FS kit, we tested whether the kit might also be useful for mapping the location of nucleosomes in chromatin. Using Simian Virus 40 (SV40) chromatin isolated at different times in an infection, we have compared nucleosome mapping using the NEB FS kit (FS-Seq) to MN-Seq, ATAC-Seq, and ChIP-Seq. Mapping nucleosomes using FS-Seq generated nucleosome profiles similar to those generated by ATAC-Seq and ChIP-Seq in regulatory regions of the SV40 genome. We conclude that FS-Seq is a simple, robust, cost-effective procedure for mapping nucleosomes in SV40 chromatin that should be useful for other forms of chromatin as well. We also present evidence that the FS kit may be useful for mapping the location of transcription factors in chromatin when sequencing reads between 75 and 99 base pairs in size are analyzed.
Evolutionarily conserved non-protein-coding regions in the chicken genome harbor functionally important variation
Authors: Groß Christian , Bortoluzzi Chiara , de Ridder Dick , Megens Hendrik-Jan , Groenen Martien AM , Reinders Marcel , Bosse Mirte
Published: March 2020

The availability of genomes for many species has advanced our understanding of the non-protein-coding fraction of the genome. Comparative genomics has proven to be an invaluable approach for the systematic, genome-wide identification of conserved non-protein-coding elements (CNEs). However, for many non-mammalian model species, including chicken, our capability to interpret the functional importance of variants overlapping CNEs has been limited by current genomic annotations, which rely on a single information type (e.g. conservation). We here studied CNEs in chicken using a combination of population genomics and comparative genomics. To investigate the functional importance of variants found in CNEs we develop a ch(icken) Combined Annotation-Dependent Depletion (chCADD), a variant effect prediction tool first introduced for humans and later on for mouse and pig. We show that 73 Mb of the chicken genome has been conserved across more than 280 million years of vertebrate evolution. The vast majority of the conserved elements are in non-protein-coding regions, which display SNP densities and allele frequency distributions characteristic of genomic regions constrained by purifying selection. By annotating SNPs with the chCADD score we are able to pinpoint specific subregions of the CNEs to be of higher functional importance, as supported by SNPs found in these subregions are associated with known disease genes in humans, mice, and rats. Taken together, our findings indicate that CNEs harbor variants of functional significance that should be object of further investigation along with protein-coding mutations. We therefore anticipate chCADD to be of great use to the scientific community and breeding companies in future functional studies in chicken.
SARS-CoV-2 and ORF3a: Non-Synonymous Mutations and Polyproline Regions
Authors: Issa Elio , Merhi Georgi , Panossian Balig , Salloum Tamara , Tokajian Sima
Published: March 2020

The effect of the rapid accumulation of non-synonymous mutations on the pathogenesis of SARS-CoV-2 is not yet known. To predict the impact of non-synonymous mutations and polyproline regions identified in ORF3a on the formation of B-cell epitopes and their role in evading the immune response, nucleotide and protein sequences of 537 available SARS-CoV-2 genomes were analyzed for the presence of non-synonymous mutations and polyproline regions. Mutations were correlated with changes in epitope formation. A total of 19 different non-synonymous amino acids substitutions were detected in ORF3a among 537 SARS-CoV-2 strains. G251V was the most common and identified in 9.9% (n=53) of the strains and was predicted to lead to the loss of a B-cell like epitope in ORF3a. Polyproline regions were detected in two strains (EPI_ISL_410486, France and EPI_ISL_407079, Finland) and affected epitopes formation. The accumulation of non-synonymous mutations and detected polyproline regions in ORF3a of SARS-CoV-2 could be driving the evasion of the host immune response thus favoring viral spread. Rapid mutations accumulating in ORF3a should be closely monitored throughout the COVID-19 pandemic.

Importance

At the surge of the COVID-19 pandemic and after three months of the identification of SARS-CoV-2 as the disease-causing pathogen, nucleic acid changes due to host-pathogen interactions are insightful into the evolution of this virus. In this paper, we have identified a set of non-synonymous mutations in ORF3a and predicted their impact on B-cell like epitope formation. The accumulation of non-synonymous mutations in ORF3a could be driving protein changes that mediate immune evasion and favoring viral spread.
Ultrafast and accurate 16S microbial community analysis using Kraken 2
Authors: Lu Jennifer , Salzberg Steven L
Published: March 2020

For decades, 16S ribosomal RNA sequencing has been the primary means for identifying the bacterial species present in a sample with unknown composition. One of the most widely-used tools for this purpose today is the QIIME (Quantitative Insights Into Microbial Ecology) package. Recent results have shown that the newest release, QIIME 2, has higher accuracy than QIIME, MAPseq, and mothur when classifying bacterial genera from simulated human gut, ocean, and soil metagenomes, although QIIME 2 also proved to be the most computationally expensive method. Kraken, first released in 2014, has been shown to provide exceptionally fast and accurate classification for shotgun metagenomics sequencing projects. Bracken, released in 2016, then provided users with the ability to accurately estimate species or genus abundances using Kraken classification results. Kraken 2, which matches the accuracy and speed of Kraken 1, now supports 16S rRNA databases, allowing for direct comparisons to QIIME and similar systems. Here we show that, using the same simulated 16S rRNA metagenomic data as previous studies, Kraken 2 and Bracken are up to 300 times faster and also more accurate at 16S profiling than QIIME 2.
Detection of deleterious on-target effects after HDR-mediated CRISPR editing
Authors: Weisheit Isabel , Kroeger Joseph , Malik Rainer , Klimmt Julien , Crusius Dennis , Dannert Angelika , Dichgans Martin , Paquet Dominik
Published: March 2020

CRISPR genome editing is a promising tool for translational research but can cause undesired editing outcomes, both on-target at the edited locus and off-target at other genomic loci. We investigated the occurrence of deleterious on-target effects in human stem cells after insertion of disease-related mutations by homology-directed repair (HDR). We identified large, mono-allelic genomic deletions and loss-of-heterozygosity that escaped standard quality controls in up to 40% of edited clones. To reliably detect such events, we developed simple, low-cost and universally applicable quantitative genotyping PCR (qgPCR) as well as sequencing-based tools and suggest their usage as additional quality controls after editing. This will help to ensure the integrity of edited loci and increase the reliability of CRISPR editing.
Mechano-bactericidal nanopillars require external forces to effectively kill bacteria
Authors: Valiei Amin , Lin Nicholas , Bryche Jean-Francois , McKay Geoffrey , Canva Michael , Charette Paul G. , Nguyen Dao , Moraes Christopher , Tufenkji Nathalie
Published: March 2020

Nanopillars are known to mechanically damage bacteria, suggesting a promising strategy for highly-effective anti-bacterial surfaces. However, the mechanisms underlying this phenomena remain unclear, which ultimately limits translational potential towards real-world applications. Using real-time and end-point analysis techniques, we demonstrate that in contrast to expectations, bacteria on multiple “mechano-bactericidal” surfaces remain viable, unless exposed to a moving air-liquid interface which caused considerable cell death. Reasoning that normal forces arising from surface tension may underlie mechano-bactericidal activity, we developed computational and experimental models to estimate, manipulate, and recreate the impact of these forces. Our experiments together demonstrate that nanopillar surfaces alone do not cause cell death, but require a critical level of external force to deform and rupture bacteria. These studies hence provide fundamental physical insight into the mechanisms by which nanopillar surfaces can serve as effective antibacterial strategies, and describe the use-conditions under which such nanotechnological approaches may provide practical value.

Graphical Abstract
Recent loss of the Dim2 DNA methyltransferase decreases mutation rate in repeats and changes evolutionary trajectory in a fungal pathogen
Authors: Möller Mareike , Habig Michael , Lorrain Cécile , Feurtey Alice , Haueisen Janine , Fagundes Wagner C. , Alizadeh Alireza , Freitag Michael , Stukenbrock Eva H.
Published: March 2020

DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ between eukaryotes. Strains of the plant pathogenic fungusZymoseptoria triticiappeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of thedim2DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation ofdim2occurred recently as someZ. triticiisolates carry a functionaldim2gene. Moreover, we show thatdim2inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with and without functionaldim2. Presence of functionaldim2correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying inactivedim2alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functionaldim2allele in strains with mutateddim2restored normal 5mC levels, demonstratingde novocytosine methylation activity ofdim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lackingdim2. We found that the presence ofdim2alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait inZ. triticipopulations that can impact genome evolution.

Author Summary

Cytosine DNA methylation (5mC) is known to silence transposable elements in fungi and thereby appears to contribute to genome stability. The genomes of plant pathogenic fungi are highly diverse, differing substantially in transposon content and distribution. Here, we show extensive differences of 5mC levels within a single species of an important wheat pathogen. These differences were caused by inactivation of the DNA methyltransferase Dim2 in the majority of studied isolates. Presence of widespread 5mC increased point mutation rates in regions with active or mutated transposable elements during mitosis. The mutation pattern is dependent on the presence of Dim2 and resembles a mitotic version of Repeat-Induced Point mutation (RIP). Thus, loss of 5mC may represent an evolutionary trade-off offering adaptive potential at the cost of transposon control.
TheHoja loca1Mutant and AUX/IAA Function in Phytomer Development
Authors: Richardson A. E. , Sluis A. , Hake S.
Published: March 2020

Plant architecture is determined by the iterative building of phytomer units, each containing a portion of stem, an organ and an axillary meristem. Each phytomer can follow different developmental paths underpinning the complexity, and plasticity, of plant form. Auxin plays a central role in the coordination of phytomer development, regulating organ initiation and patterning across all axes. This diversity in auxin function results from changes in the activities and expression of auxin signaling components, including the AUX/IAA repressors. Higher land plants have multigene AUX/IAA families, which leads to functional redundancy. Dominant mutations, which prevent AUX/IAA degradation in response to auxin, have highlighted the importance of these proteins in fine-tuning organ development. Here we report a new dominant AUX/IAA mutant in maize,Hoja loca1(Oja).Ojahas a mutation in the degron motif of ZmIAA28 and specifically affects aerial organ initiation and leaf medio-lateral patterning, but phytomer initiation remains unchanged. Mutant phenotypes are variable and transcriptional profiling of individual meristems identified clusters of genes that may underpin the phenotypic differences. The unique phenotype ofOjaprovides evidence of species-specific sub-functionalization of the AUX/IAAs, and illustrates the crucial role of auxin signaling in the tight coordination of phytomer unit development.
Extended depth of focus multiphoton microscopy via incoherent pulse splitting
Authors: Chen Bingying , Chakraborty Tonmoy , Daetwyler Stephan , Manton James D. , Dean Kevin , Fiolka Reto
Published: March 2020

We present a phase mask that can be easily added to any multi-photon raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point-spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.
Evolution of an interaction between disordered proteins resulted in increased heterogeneity of the binding transition state
Authors: Karlsson Elin , Paissoni Cristina , Erkelens Amanda M. , Tehranizadeh Zeinab Amiri , Sorgenfrei Frieda A. , Andersson Eva , Ye Weihua , Camilloni Carlo , Jemth Per
Published: March 2020

Intrinsically disordered protein (IDP) domains often have multiple binding partners. Little is known regarding molecular changes in the binding mechanism when a new interaction evolves from low to high affinity. Here we compared the degree of native contacts in the transition state of the interaction of two IDP domains, low-affinity ancestral and high-affinity human NCBD and CID. We found that the coupled binding and folding mechanism of the domains is overall similar, but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex while more heterogenous transient interactions, including electrostatic, and an increased disorder characterize the human complex. From an evolutionary perspective, adaptation to new binding partners for IDPs may benefit from this ability to exploit multiple alternative transient interactions while retaining the overall pathway.
Divergent evolution of a protein-protein interaction revealed through ancestral sequence reconstruction and resurrection
Authors: Laursen Louise , Čalyševa Jelena , Gibson Toby J. , Jemth Per
Published: March 2020

The postsynaptic density extends across the postsynaptic dendritic spine with Discs large (DLG) as the most abundant scaffolding protein. DLG dynamically alters the structure of the postsynaptic density, thus controlling the function and distribution of specific receptors at the synapse. PDZ domains make up one of the most abundant protein interaction domain families in animals. One important interaction governing postsynaptic architecture is that between the PDZ3 domain from DLG and cysteine-rich interactor of PDZ3 (CRIPT). However, little is know regarding functional evolution of the PDZ3:CRIPT interaction. Here, we subjected PDZ3 and CRIPT to ancestral sequence reconstruction, resurrection and biophysical experiments. We show that the PDZ3:CRIPT interaction is an ancient interaction, which was present in the last common ancestor of Eukaryotes, and that high affinity is maintained in most extant animal phyla. However, affinity is low in nematodes and insects, raising questions about the physiological function of the interaction in species from these animal groups. Our findings demonstrate how an apparently established protein-protein interaction involved in cellular scaffolding in bilaterians can suddenly be subject to dynamic evolution including possible loss of function.
Physical Interactions Driving the Activation/Inhibition of Calcium/Calmodulin Dependent Protein Kinase II
Authors: Asciutto Eliana K. , Pantano Sergio , General Ignacio J.
Published: March 2020

CaMKII is a protein kinase whose function is regulated by the binding of the Calcium/Calmodulin complex (Ca2+/CaM). It is a major player in the Long Term Potentiation process where it acts as a molecular switch, oscillating between inhibited and active conformations. The mechanism for the switching is thought to be initiated by Ca2+/CaM binding, which allows the trans-phosphorylation of a subunit of CaMKII by a neighboring kinase, leading to the active state of the system. A combination of all-atom and coarse-grained MD simulations with free energy calculations, led us to reveal an interplay of electrostatic forces exerted by Ca2+/CaM on CaMKII, which initiate the activation process. The highly electrically charged Ca2+/CaM neutralizes basic regions in the linker domain of CaMKII, facilitating its opening and consequent activation. The emerging picture of CaMKII’s behavior highlights the preponderance of electrostatic interactions, which are modulated by the presence of Ca2+/CaM and the phosphorylation of key sites.
Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase
Authors: Anselmi M. , Calligari P. , Hub J.S. , Tartaglia M. , Bocchinfuso G. , Stella L.
Published: March 2020

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded byPTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations inPTPN11are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics simulations of the N-SH2 domain complexed to 12 different peptides, to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position −2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3 and +5 in an apolar groove of the domain, and interaction of residue −2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues −1, +1, +2 and +4. Finally, negatively charged residues at position +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific of the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core β-sheet and the loop that closes the pY binding pocket.
Evaluating the transcriptional fidelity of cancer models
Authors: Peng Da , Gleyzer Rachel , Tai Wen-Hsin , Kumar Pavithra , Bian Qin , Issacs Bradley , da Rocha Edroaldo Lummertz , Cai Stephanie , DiNapoli Kathleen , Huang Franklin W , Cahan Patrick
Published: March 2020

Background

Cancer researchers use cell lines, patient derived xenografts, engineered mice, and tumoroids as models to investigate tumor biology and to identify therapies. The generalizability and power of a model derives from the fidelity with which it represents the tumor type under investigation, however, the extent to which this is true is often unclear. The preponderance of models and the ability to readily generate new ones has created a demand for tools that can measure the extent and ways in which cancer models resemble or diverge from native tumors.

Methods

We developed a machine learning based computational tool, CancerCellNet, that measures the similarity of cancer models to 22 naturally occurring tumor types and 36 subtypes, in a platform and species agnostic manner. We applied this tool to 657 cancer cell lines, 415 patient derived xenografts, 26 distinct genetically engineered mouse models, and 131 tumoroids. We validated CancerCellNet by application to independent data, and we tested several predictions with immunofluorescence.

Results

We have documented the cancer models with the greatest transcriptional fidelity to natural tumors, we have identified cancers underserved by adequate models, and we have found models with annotations that do not match their classification. By comparing models across modalities, we report that, on average, genetically engineered mice and tumoroids have higher transcriptional fidelity than patient derived xenografts and cell lines in four out of five tumor types. However, several patient derived xenografts and tumoroids have classification scores that are on par with native tumors, highlighting both their potential as faithful model classes and their heterogeneity.

Conclusions

CancerCellNet enables the rapid assessment of transcriptional fidelity of tumor models. We have made CancerCellNet available as freely downloadable software and as a web application that can be applied to new cancer models that allows for direct comparison to the cancer models evaluated here.
Computational Investigation Of Structural Interfaces Of Protein Complexes With Short Linear Motifs
Authors: Upadhyayula Raghavender Surya
Published: March 2020

Protein complexes with short linear motifs (SLiMs) are known to play important regulatory functions in eukaryotes. In this investigation, I have studied the structures deposited in PDB with SLiMs. The structures Were grouped into three broad categories of protein-protein, protein-peptide and the rest as others. Protein-peptide complexes Were found to be most highly represented. The interfaces Were evaluated for geometric features and conformational variables. It was observed that protein-protein and protein-peptide complexes show characteristic differences in residue pairings, which Were quantified by evaluating normalized contact residue pairing frequencies. Interface residues adopt characteristic canonical residue conformations in the Ramachandran space, with a pronounced preference for positive ϕ conformations. It was observed that phosphorylated residues have an unusual propensity to adopt the unusual positive ϕ conformations at the interface.
Production of Hyaluronan by the Trophectoderm is a Prerequisite for Mouse Blastocyst Attachment
Authors: Hadas Ron , Gershon Eran , Cohen Aviad , Elbaz Michal , Ben-Dor Shifra , Kohen Fortune , Dekel Nava , Neeman Michal
Published: March 2020

Embryo implantation requires execution of highly synchronized processes at the feto-maternal interface, initiated by blastocyst attachment to the endometrial epithelium. Hyaluronan is a major ECM component known to regulate adhesion-associated biological processes in various physiological settings. We hypothesized that hyaluronan may facilitate blastocyst attachment. In order to test our hypothesis, we characterized the blastocyst expression of hyaluronan synthesizing and degrading enzymes, as well as the expression of hyaluronan receptors during attachment. The functional impact of hyaluronan was challenged by the use of mouse transgenic blastocysts, in which genes encoding for hyaluronan synthesizing enzymes were deleted using lentiviral incorporation of Cas-9 endonuclease alongside specific short-guide RNAs into the embryonic trophectoderm. Embryos with transgenic trophectoderm were tested for their attachmentin vitro, or assessed for implantationin vivo, upon transfer to foster dams. Deletion of the trophectoderm hyaluronan biosynthesis significantly reduced the number of blastocysts attached to human uterine epithelium cellsin vitro. Reduced attachment was also observedin vivo, in pregnant mice carrying blastocysts with hyaluronan-depleted trophectoderm. In agreement, trophectoderm expression of osteopontin, was downregulated upon depletion of hyaluronan. MRI measurements revealed a decrease in uterine blood vessels permeability. Uterine expression of VEGF-A, PTGS-2 and uterine osteopontin, which constitute the immediate response to blastocyst attachment was also reduced. Furthermore, impaired implantation, associated with a decrease in hyaluronan synthesis in the mural trophectoderm, obtained upon tamoxifen treatment, has been recovered by LIF administration. These results demonstrate that estrogen-regulated hyaluronan-synthesis in the trophectoderm is indispensable for mouse blastocysts attachment to the uterine epithelium.
Antimicrobial Resistance Profile andmcr-1Gene Detection inSalmonellaIsolates from Poultry in Bangladesh: Molecular and Bioinformatics Characterization
Authors: Uddin Md Bashir , Hossain S M Bayejed , Hasan Mahmudul , Alam Mohammad Nurul , Debnath Mita , Begum Ruhena , Roy Sawrab , Harun-Al-Rashid Ahmed , Chowdhury Md. Shahidur Rahman , Rahman Md. Mahfujur , Hossain Md. Mukter , Chowdhury Mohammed Yousuf Elahi , Ahmed Syed Sayeem Uddin
Published: March 2020

Antimicrobial resistance genemcr-1has been disseminated globally since its first discovery in Southern China in late 2015. However, themcr-1 gene had not been identified previously inSalmonellaisolates from poultry in Bangladesh. Here, we aimed to explore antimicrobial resistance genemcr-1inSalmonellaisolates. Eighty twoSalmonellaisolates were isolated and characterized from suspected poultry specimens received from different zones of the country. A phenotypic disc diffusion assay with 15 antimicrobial agents was performed following CLSI standard. The disk diffusion assay showed that, all of the isolates presented high resistance to colistin (92.68%), oxytetracycline (86.59%), co-trimoxazole (76.83%), ciprofloxacin (73.17%) and enrofloxacin (65.85%). Further, randomly selected 10Salmonellaisolates were analyzed by polymerase chain reaction (PCR) targeting genus-specificinvAand antimicrobial (colistin) resistancemcr-1 genes. Five were confirmed for the presence of themcr-1gene belonging toSalmonellaspp. Further, sequencing followed by phylogenetic analysis revealed divergent evolutionary relation between the LptA and MCR proteins rendering them resistant to colistin. Three-dimensional homology structures of MCR-1 proteins were constructed and verified using different bioinformatics tools. Moreover, molecular docking interactions suggested that, MCR-1 and LptA share a similar substrate binding cavity which could be validated for the functional analysis. The results represent here is the first molecular andin silicoanalysis of colistin resistancemcr-1gene ofSalmonellain poultry in Bangladesh, which may emphasize the importance of the study on antibiotic resistance genes requiring for national monitoring and strategic surveillance in the country.
A novel rare c. -39C>T mutation in thePROS15’UTR causing PS deficiency by creating a new upstream translation initiation codon and inhibiting the production of the natural protein
Authors: Labrouche-Colomer Sylvie , Soukarieh Omar , Proust Carole , Mouton Christine , Huguenin Yoann , Roux Maguelonne , Besse Céline , Boland Anne , Olaso Robert , Constans Joël , Deleuze Jean-François , Morange Pierre-Emmanuel , Jaspard-Vinassa Béatrice , Trégouët David-Alexandre ,   on behalf of the GenMed consortium
Published: March 2020

Inherited Protein S deficiency (PSD) (MIM176880) is a rare automosal dominant disorder caused by rare mutations, mainly located in the coding sequence of the structuralPROS1gene, and associated with an increased risk of venous thromboembolism. To identify the molecular defect underlying PSD observed in an extended French pedigree with 7 PSD affected members in who no candidate deleteriousPROS1mutation was detected by Sanger sequencing ofPROS1exons and their flanking intronic regions or via a MLPA approach, a whole genome sequencing strategy was adopted. This led to the identification of a never reported C to T substitution at c.-39 from the natural ATG codon of thePROS1gene that completely segregates with PSD in the whole family. This substitution ACG->ATG creates a new start codon upstream of the main ATG. We experimentally demonstrated that the variant generates a novel overlapping ORF and inhibits the translation of the wild type protein from the main ORF in HeLa cells. This work describes the first example of 5’UTRPROS1mutation causing PSD through the creation of an upstream ORF, a mutation that is not predicted to be deleterious by standard annotation softwares.
Left-handed DNA-PAINT for improved superresolution imaging in the nucleus
Authors: Geertsema H.J. , Aimola G. , Fabricius V. , Fuerste J.P. , Kaufer B.B. , Ewers H.
Published: March 2020

DNA point accumulation in nanoscale topography (DNA-PAINT) advances super-resolution microscopy with superior resolution and multiplexing capabilities. However, cellular DNA may interfere with this single-molecule localization technique based on DNA-DNA hybridization. Here, we introduce left-handed DNA (L-DNA) oligomers that do not hybridize to naturally present R-DNA and demonstrate that L-DNA PAINT has the same specificity and multiplexing capability as R-DNA PAINT, but greatly improves specific visualization of nuclear target molecules.
Induced Pluripotent Stem Cell-derived CAR-Macrophage Cells with Antigen-dependent Anti-Cancer Cell Functions for Liquid and Solid Tumors
Authors: Zhang Li , Tian Lin , Dai Xiaoyang , Yu Hua , Wang Jiajia , Lei Anhua , Zhao Wei , Zhu Yuqing , Sun Zhen , Zhang Hao , Church George M. , Huang He , Weng Qinjie , Zhang Jin
Published: March 2020

The Chimera antigen receptor (CAR)-T cell therapy has gained great success in the clinic. However, there are still major challenges for its wider applications in a variety of cancer types including lack of effectiveness due to the highly complex tumor microenvironment, and the forbiddingly high cost due to personalized manufacturing procedures. In order to overcome these hurdles, numerous efforts have been spent focusing on optimizing Chimera Antigen Receptors, engineering and improving T cell capacity, exploiting features of subsets of T cell or NK cells, or making off-the-shelf universal T cells. Here, we developed induced pluripotent stem cells (iPSCs)-derived, CAR-expressing macrophage cells (CAR-iMac). These cells showed antigen-dependent macrophage functions such as expression and secretion of cytokines, polarization toward the pro-inflammatory/anti-tumor state, and phagocytosis of tumor cells, as well as somein vivoanti-cancer cell activity for both liquid and solid tumors. This technology platform for the first time provides an unlimited source of iPSC-derived engineered CAR-macrophage cells which could be utilized to eliminate cancer cells or modulate the tumor microenvironment in liquid and solid tumor immunotherapy.

One sentence summary

We developed CAR-expressing iPSC-induced macrophage cells that have antigen-dependent phagocytosis and pro-inflammatory functions and anti-cancer cell activity for both liquid and solid tumor cells.
Differential small RNA responses against co-infecting insect-specific viruses inAedes albopictusmosquitoes
Authors: Frangeul Lionel , Blanc Hervé , Saleh Maria-Carla , Suzuki Yasutsugu
Published: March 2020

The mosquito antiviral response has been mainly studied in the case of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are abundantly infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are maintained primarily by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs,Aedesflavivirus (AEFV), Menghai Rhabdovirus (MERV) and Shinobi tetra virus (SHTV), we infected adultAedes albopictusfemales and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. Piwi1-4 were abundantly expressed in ovaries and testes in contrast to Piwi5-9, suggesting that Piwi 5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infectedAedes albopictusproduce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.
Individualized System for Augmenting Ventilator Efficacy (iSAVE): A Rapidly deployable system to expand ventilator capacity
Authors: Srinivasan Shriya , Ramadi Khalil B , Vicario Francesco , Gwynne Declan , Hayward Alison , Langer Robert , Frassica Joseph J. , Baron Rebecca M. , Traverso Giovanni
Published: March 2020

The COVID-19 pandemic is overwhelming healthcare systems worldwide. A significant portion of COVID-19 patients develop pneumonia and acute respiratory distress syndrome (ARDS), necessitating ventilator support. Some health systems do not have the capacity to accommodate this surge in ventilator demand, leading to shortages and inevitable mortality. Some clinicians have, of necessity, jerry-rigged ventilators to support multiple patients, but these devices lack protected air streams or individualized controls for each patient. Moreover, some have not been tested under conditions of ARDS. We have developed the Individualized System for Augmenting Ventilator Efficacy (iSAVE), a rapidly deployable platform to more safely use a single ventilator to simultaneously support multiple critically-ill patients. The iSAVE enables patient-specific volume and pressure control and incorporates safety features to mitigate cross-contamination between patients and flow changes due to patient interdependencies within the respiratory circuit. Here we demonstrate through simulated andin vivopig evaluation the capacity of the iSAVE to support a range of respiratory clinical states. By leveraging off-the-shelf components that are readily available to intensive care unit (ICU) caregivers, the iSAVE could potentially be translated for human application to expand the ventilation capacity of hospitals using existing ventilators, minimizing the need to procure additional ventilators.
Internally quenched fluorogenic probe provides selective and rapid detection of cathepsin L activity
Authors: Schleyer Kelton A , Fetrow Ben , Fatland Peter Zannes , Liu Jun , Chaaban Maya , Ma Biwu , Cui Lina
Published: March 2020

Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and/or altered trafficking during disease states such as cancer. The overlapping substrate specificity of cathepsin family members makes selective detection of activity from a single cathepsin difficult, and CTL activity is particularly difficult to parse from its close homologue CTV and the ubiquitous CTB. Despite this, screening campaigns have explored the extended chemical space in the cathepsin binding sites and identified unique substrate structures that offer selectivity for one enzyme over others. In this vein, we present CTLAP, a fluorogenic probe that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL activity probes. CTLAP exhibits intrinsically low background fluorescence, which we attribute to possible self-quenching mechanisms. CTLAP demonstrates markedly higher turn-on ratios (24-fold) and moderately improved enzyme selectivity compared to Z-FR-AMC (10-fold turn-on ratio), a commercially available CTL-selective probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation with the enzyme, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins.