bluuebunny/crossref_metadata_2025 huggingface dataset:
Influenza A viruses serially passaged in different MDCK cell lines exhibit limited sequence variation across their genomes, with the exception of the hemagglutinin gene
Authors: Barnard Karen N. , Wasik Brian R. , Alford-Lawrence Brynn K. , Hayward Jessica J. , Weichert Wendy S. , Voorhees Ian E.H. , Holmes Edward C. , Parrish Colin R.
Published: February 2020

New methods for deep sequence analysis provide an opportunity to follow the emergence and dynamics of virus mutations in real time. Although viruses are commonly grown in cell culture for research and for vaccine development, the cells used to grow the virus are often not derived from the same tissue or even the same host that the virus naturally replicates in. The selective pressures of culturing virusin vitroare still only partially understood. MDCK cells are the standard cell for growing influenza viruses yet are derived from the epithelium of the canine kidney and are also heterogenous. We passaged human H3N2, H1N1 pandemic, and canine H3N2 influenza A viruses (IAV) in different lineages of MDCK cells, as well as lines engineered to express variant Sia receptors, including α2,3- and α2,6-linkages orN-glycolylneuraminic acid (Neu5Gc) orN-acetylneuraminic acid (Neu5Ac) forms. MDCK-Type II cells had lower infection efficiency and virus production, and infection appeared more dependent on protease activation of the virus. When viruses were passaged in the different cells, they exhibited only small numbers of consensus-level mutations, and most were within the HA gene. Both human IAVs showed selection for single nucleotide minority variants in the HA stem across cell types, as well as low frequency variants in the HA receptor binding site of virus passaged in cells expressing Neu5Gc. Canine H3N2 also showed minority variants near the receptor-binding site in cells expressing Neu5Gc and also in those expressingα2,6-linkages.

IMPORTANCE

The genetic variation and adaptability of viruses are fundamental properties that allow their evolutionary success in the face of differing host environments and immune responses. The growth of viruses in cell culture is widely used for their study and for preparing vaccines. However, the selection pressures that cell passaging imposes on viruses are often poorly understood. We used deep sequence analysis to define, in detail, how three different influenza A viruses respond to passaging in different lineages of canine MDCK cells that are commonly used for their growth, as well as in variant cells engineered to express different forms of their cell surface receptor, sialic acid. This analysis revealed that most mutations occur in the HA gene and few sequence changes in the virus population reached high proportions. This is relevant for understanding the selective pressures of virus growth in cell culture and how it shapes evolutionary patterns.
AutoGeneS: Automatic gene selection using multi-objective optimization for RNA-seq deconvolution
Authors: Aliee Hananeh , Theis Fabian
Published: February 2020

Tissues are complex systems of interacting cell types. Knowing cell-type proportions in a tissue is very important to identify which cells or cell types are targeted by a disease or perturbation. When measuring such responses using RNA-seq, bulk RNA-seq masks cellular heterogeneity. Hence, several computational methods have been proposed to infer cell-type proportions from bulk RNA samples. Their performance with noisy reference profiles highly depends on the set of genes undergoing deconvolution. These genes are often selected based on prior knowledge or a single-criterion test that might not be useful to dissect closely correlated cell types. In this work, we introduceAutoGeneS, a tool that automatically extracts informative genes and reveals the cellular heterogeneity of bulk RNA samples. AutoGeneS requires no prior knowledge about marker genes and selects genes by simultaneously optimizing multiple criteria: minimizing the correlation and maximizing the distance between cell types. It can be applied to reference profiles from various sources like single-cell experiments or sorted cell populations. Results from human samples of peripheral blood illustrate that AutoGeneS outperforms other methods. Our results also highlight the impact of our approach on analyzing bulk RNA samples with noisy single-cell reference profiles and closely correlated cell types. Ground truth cell proportions analyzed by flow cytometry confirmed the accuracy of the predictions of AutoGeneS in identifying cell-type proportions. AutoGeneS is available for use via a standalone Python package (https://github.com/theislab/AutoGeneS).
In silicocharacterization ofGmSOS1provides a comprehensive understanding for its role in soybean salt tolerance
Authors: Mei Zhi-Chao , Yang Ling-Yan , Liu Zhi-Min , Tang Qi-Li , Hou Xin-Zhao , Xie Li-Jun , Wei Zhu-Jun
Published: February 2020

PlantSOS1encodes plasma membrane Na+/H+antiporter, which helps in the exclusion of Na+and improves plant salt tolerance. However, detailed studies ofSOS1in the important oil crop, soybean (Glycine max), are still lacking. In the present study, we carried out a comprehensivein silicoanalysis ofSOS1in soybean. Referring to the analysis of physicochemical properties and structural characteristics, the GmSOS1 is an acidic protein with instability and hydrophobicity. Subcellular localization ofGmSOS1supports the presumption that GmSOS1 is a plasma membrane Na+/H+antiporter. Post-translational modification site prediction indicates 4 amino acids that may be phosphorylated. Further, the protein-protein interaction network and co-functional network signify the potential role ofGmSOS1in salt stress tolerance. Although the interaction betweenGmSOS1andGmHKT1remains elusive, some of the intermediary signaling components of SOS pathway in soybean have been predicted. In addition,in silicoexpression analysis based on transcriptome datasets using publicly available database revealed thatGmSOS1was differentially expressed in tissues and different times. Due to the analysis of its regulation mechanism, we found transcription factors such as WRKY and ERF as well as three miRNAs can regulate the expression ofGmSOS1. Phylogenetic analysis using the homologous amino acid sequence of SOS1s from 26 species was performed to study the conserved motifs among these SOS1 members. Overall, we provide an extensive analysis of theGmSOS1and it promises the primary basis for the study in development and response to salt tolerance.
Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe
Authors: McDevitt Allan D. , Coscia Ilaria , Browett Samuel S. , Ruiz-González Aritz , Statham Mark J. , Ruczyńska Inka , Roberts Liam , Stojak Joanna , Frantz Alain C. , Norén Karin , Ågren Erik O. , Learmount Jane , Basto Mafalda , Fernandes Carlos , Stuart Peter , Tosh David G. , Sindicic Magda , Andreanszky Tibor , Isomursu Marja , Panek Marek , Korolev Andrey , Okhlopkov Innokentiy M. , Saveljev Alexander P. , Pokorny Boštjan , Flajšman Katarina , Harrison Stephen W. R. , Lobkov Vladimir , Ćirović Duško , Mullins Jacinta , Pertoldi Cino , Randi Ettore , Sacks Benjamin N. , Kowalczyk Rafał , Wójcik Jan M.
Published: February 2020

Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using Genotyping-by-Sequencing (GBS)) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g. Iberia). In addition, we tested multiple post-glacial re-colonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to an increased mammalian fauna on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.
Microbial association networks give relevant insights into plant pathobiomes
Authors: Pauvert Charlie , Fort Tania , Calonnec Agnès , Faivre d’Arcier Julie , Chancerel Emilie , Massot Marie , Chiquet Julien , Robin Stéphane , Bohan David A. , Vallance Jessica , Vacher Corinne
Published: February 2020

Interactions between plant pathogens and other plant-associated microorganisms regulate disease. Deciphering the networks formed by these interactions, termed pathobiomes, is crucial to disease management. Our aim was to investigate whether microbial association networks inferred from metabarcoding data give relevant insights into pathobiomes, by testing whether inferred associations contain signals of ecological interactions. We used Poisson Lognormal Models to construct microbial association networks from metabarcoding data and then investigated whether some of these associations corresponded to interactions measurable in co-cultures or known in the literature, by using grapevine (Vitis vinifera) and the fungal pathogen causing powdery mildew (Erysiphe necator) as a model system. Our model suggested that the pathogen species was associated with 23 other fungal species, forming its putative pathobiome. These associations were not known as interactions in the literature, but one of them was confirmed by our co-culture experiments. The yeastBuckleyzyma aurantiacaimpeded pathogen growth and reproduction, in line with the negative association found in the microbial network. Co-cultures also supported another association involving two yeast species. Together, these findings indicate that microbial networks can provide plausible hypotheses of ecological interactions that could be used to develop microbiome-based strategies for crop protection.
Investigation of microbial community interactions between lake Washington methanotrophs using genome-scale metabolic modeling
Authors: Islam Mohammad Mazharul , Le Tony , Daggumati Shardhat Reddy , Saha Rajib
Published: February 2020

Background

The role of methane in global warming has become paramount to the environment and the human society, especially in the past few decades. Methane cycling microbial communities play an important role in the global methane cycle, which is why the characterization of these communities is critical to understand and manipulate their behavior. Methanotrophs are a major player in these communities and are able to oxidize methane as their primary carbon source.

Results

Lake Washington is a freshwater lake characterized by a methane-oxygen countergradient that contains a methane cycling microbial community. The major microbial members include methanotrophs such asMethylobacter Tundripaludum 21/22andMethylomonas sp. LW13. In this work, these methanotrophs are studied via developing highly curated genome-scale metabolic models. Each model was then integrated to form a community model with a multi-level optimization framework. The metabolic interactions for the community were also characterized. While both organisms are competitors for methane,Methylobacterwas found to display altruistic behavior in consuming formaldehyde produced byMethylomonasthat inhibits its growth. The community was next tested under carbon, oxygen, and nitrogen limited conditions to observe the systematic shifts in the internal metabolic pathways and extracellular metabolite exchanges. Each condition showed variable differences within the methane oxidation pathway, serine cycle, pyruvate metabolism, and the TCA cycle as well as the excretion of formaldehyde and carbon di-oxide from the community. Finally, the community model was simulated under fixed ratios of these two members to reflect the opposing behavior of the community in synthetic and natural communities. The simulated community demonstrated a noticeable switch in intracellular carbon metabolism and formaldehyde transfer between community members in natural vs. synthetic condition.

Conclusion

In this work, we attempted to reveal the response of a simplified methane recycling microbial community from Lake Washington to varying environments and also provide an insight into the difference of dynamics in natural community and synthetic co-cultures. Overall, this study lays the ground forin silicosystems-level studies of freshwater lake ecosystems, which can drive future efforts of understanding, engineering, and modifying these communities for dealing with global warming issues.
Highly reproducible characterization ofEscherichia colitRNA epitranscriptome with a simple method of library preparation for deep sequencing
Authors: Wang Ji , Toffano-Nioche Claire , Lorieux Florence , Gautheret Daniel , Lehmann Jean
Published: February 2020

In conventional RNA high-throughput sequencing, modified bases prevent a large fraction of tRNA transcripts to be converted into cDNA libraries. Recent proposals aiming at resolving this issue take advantage of the interference of base modifications with RT enzymes to detect and identify them by establishing signals from aborted cDNA transcripts. Because some modifications, such as methyl groups, do almost not allow RT bypassing, demethylation and highly processive RT enzymes have been used to overcome these obstacles. Working withEscherichia colias a model system, we show that with a conventional (albeit still engineered) RT enzyme and key optimizations in library preparation, all RT-impairing modifications can be highlighted along the entire tRNA length without a demethylation procedure. This is achieved by combining deep-sequencing samples, which allows to establish aborted transcription signal of higher accuracy and reproducibility, with the potential for differentiating tiny differences in the state of modification of all cellular tRNAs. In addition, our protocol provides estimates of the relative tRNA abundance.
Prevalence of pathogenic variants in DNA damage response genes in patients undergoing cancer risk assessment and reporting a personal history of early-onset renal cancer
Authors: Hartman Tiffiney R. , Demidova Elena V. , Lesh Randy W. , Hoang Lily , Richardson Marcy , Forman Andrea , Kessler Lisa , Speare Virginia , Golemis Erica A. , Hall Michael J. , Daly Mary B. , Arora Sanjeevani
Published: February 2020

Purpose

Pathogenic variants (PVs) in a number of genes are known to increase the risk of hereditary renal cancer (hRC). However, many early-onset RC (eoRC) patients undergoing genetic testing lack PVs in hRC genes; thus, their genetic risk remains undefined. To determine if PVs in DNA damage response (DDR) genes are enriched in a convenience sample of eoRC patients undergoing genetic testing.

Materials and Methods

Retrospective review of results for 844 unselected eoRC patients, undergoing genetic testing with a multi-gene cancer panel by Ambry Genetics [between July 2012 and December 2016]. The patients were tested with CancerNext and/or CancerNext Expanded panels for a variety of indications. Identified PVs were compared with patient characteristics.

Results

Mean age of RC diagnosis was 48 years [range 24-60]. In addition to eoRC, 57.9% patients tested reported at least one additional cancer; breast cancer being the most common (40.1% of females, 2.5% of males). PVs in cancer risk genes were identified in 12.8% of patients—3.7% in RC-specific genes, and 8.55% in DDR genes. DDR gene PVs were most commonly identified inCHEK2, BRCA1/2, andATM. Among the 2.1% of patients with aBRCA1/2PV, <50% reported a personal history of hereditary breast/ovarian-associated cancer. No association between age of RC diagnosis, and prevalence of PVs in RC-specific or DDR genes was observed.

Conclusions

Multi-gene panel testing including DDR genes may provide a more comprehensive risk assessment in unselected eoRC patients, and their families. Validation in larger datasets is needed to characterize the association with eoRC.
Polyploidization enhancing genetic recombination of the ancestral diploid genome in the evolution of hexaploid wheat
Authors: Wan Hongshen , Li Jun , Ma Shengwei , Wang Qin , Zhu Xinguo , Liu Zehou , Yang Fan , Yang Manyu , Zheng Jianmin , Li Shizhao , Luo Jiangtao , Yang Wuyun
Published: February 2020

Allopolyploidy increases its evolutionary potential by fixing heterosis and the advantage of gene redundancy. Allelic combinations generated from genetic recombination potentially provide many variations to the selection pools for evolution. May there be any relationship between allopolyploidization and genetic recombination? To study the impact of polyploidy on genetic recombination, we selected wheat as a model and simulated its evolution pathway of allopolyploidy by developing synthetic hexaploid wheat. The change of homologous chromosome recombination were investigated on their diploid DD and tetraploid AABB genomes after their allohexaploidization, respectively. The genetic recombination of the ancestral diploid genome ofAegilops tauschiiwas enhanced significantly more than 2 folds after their hexaploidization. Hexaploidization enhancing genetic recombination of the ancestral diploid D genome was firstly reported to be a new way to increase evolutionary potential of wheat, which is beneficial for wheat to conquer their narrow origination of D genome, quickly spread and make it a major crop of the world. Finally, re-synthetizing hexaploid wheat using diverseAe. tauschiispecies with tetraploid wheat can be considered as a pleiotropic strategy to speed adaptive evolution of bread wheat in breeding processes by increasing both gene allele types and genetic recombination variations.
GreenMaps: a Tool for Addressing the Wallacean Shortfall in the Global Distribution of Plants
Authors: Daru Barnabas H.
Published: February 2020

The exponential growth of species occurrence data can facilitate dynamic biodiversity analyses. However, raw biodiversity data alone should not be used indiscriminately due to inherent sampling biases, impediments that contribute to Wallacean shortfall (i.e. the paucity of species’ geographic information). It has been suggested that Wallacean shortfall is a common phenomenon across taxa, however, there is no global assessment geared toward overcoming this impediment for plants, despite the fundamental role of plants in ecosystem stability, food security and biodiversity conservation. Here, I present GreenMaps, a new tool that will permit a rapid initial assessment of the Wallacean shortfall for plants by building base maps of species’ predicted distributions upon which citizen science participation could contribute to spatial
validation of the actual range occupied by species. The initial stages of GreenMaps have now been accomplished, providing a massive dataset of modeled range maps for over 194,000 vascular plant species. This will make it the largest and only global assessment of geographic distributions for plant species at scales relevant to research and conservation. Ultimately, GreenMaps will interface with a mobile application to enable volunteers from any region of the world to validate predicted species distributions to be used for the generation of new and improved global map of plant distributions.
Genome-wide identification and analysis of enhancer regulated microRNAs across 31 human cancers
Authors: Tang Fei , Lang Mei , Wang Wenzhu , Li Yanjing , Li Changying , Tan Zheng tang , Yue Junjie , Guo Zhiyun
Published: February 2020

Enhancers are cis-regulatory DNA elements that positively regulate the transcription of target genes in a tissue-specific manner and dysregulation in various diseases such as cancer. Recent studies showed that enhancers can regulate miRNAs and participate in the biological synthesis of miRNAs. However, the network of enhancer-regulated miRNAs across multiple cancers is still unclear. Here, a total of 2,418 proximal enhancer-miRNA interactions and 1,280 distal enhancer-miRNA interactions were identified through the integration of genomic distance, co-expression, and 3D genome data in 31 cancers. The results showed that both proximal and distal interactions exhibited significant tissue-specific feature and there was a noteworthy positive correlation between the expression of miRNA and the number of regulated enhancers in most tissues. Furthermore, it was found that there was a high correlation between the formation of enhancer-miRNA pairs and the expression of eRNAs whether in distal or proximal regulation. The characteristics analysis showed that miRes (enhancers that regulated miRNAs) and non-miRes presented significant differences in sequence conservation, GC content and histone modification signatures. Notably, GC content, H3K4me1, H3K36me3 were present differently between distal regulation and proximal regulation, suggesting they might participate in chromosome looping of enhancer-miRNA interactions. Finally, we introduced a case study, enhancer: chr1:1186391-1186507∼miR-200a was highly relevant to the survival of thyroid cancer patients and a cis-eQTL SNP on enhancer affected the expression TNFRSF18 gene as a tumor suppressor.
Genome wide association study reveals plant loci controlling heritability of the rhizosphere microbiome
Authors: Deng Siwen , Caddell Daniel , Yang Jinliang , Dahlen Lindsay , Washington Lorenzo , Coleman-Derr Devin
Published: February 2020

Host genetics has recently been shown to be a driver of plant microbiome composition. However, identifying the underlying genetic loci controlling microbial selection remains challenging. Genome wide association studies (GWAS) represent a potentially powerful, unbiased method to identify microbes sensitive to host genotype, and to connect them with the genetic loci that influence their colonization. Here, we conducted a population-level microbiome analysis of the rhizospheres of 200 sorghum genotypes. Using 16S rRNA amplicon sequencing, we identify rhizosphere-associated bacteria exhibiting heritable associations with plant genotype, and identify significant overlap between these lineages and heritable taxa recently identified in maize. Furthermore, we demonstrate that GWAS can identify host loci that correlate with the abundance of specific subsets of the rhizosphere microbiome. Finally, we demonstrate that these results can be used to predict rhizosphere microbiome structure for an independent panel of sorghum genotypes based solely on knowledge of host genotypic information.
Evolutionary and ontogenetic changes of the anatomical organization and modularity in the skull of archosaurs
Authors: Lee Hiu Wai , Esteve-Altava Borja , Abzhanov Arkhat
Published: February 2020

Comparative anatomy studies of the skull of archosaurs provide insights on the mechanisms of evolution for the morphologically and functionally diverse species of crocodiles and birds. One of the key attributes of skull evolution is the anatomical changes associated with the physical arrangement of cranial bones. Here, we compare the changes in anatomical organization and modularity of the skull of extinct and extant archosaurs using an Anatomical Network Analysis approach. We show that the number of bones, their topological arrangement, and modular organization can discriminate birds from non-avian dinosaurs, and crurotarsans. We could also discriminate extant taxa from extinct species when adult birds were included. By comparing within the same framework, juveniles and adults for crown birds and alligator(Alligator mississippiensis),we find that adult and juvenile alligator skulls are topologically similar, whereas juvenile bird skulls have a morphological complexity and anisomerism more similar to those of non-avian dinosaurs and crurotarsans than of their own adult forms. Clade-specific ontogenetic differences in skull organization, such as extensive postnatal fusion of cranial bones in crown birds, can explain this pattern. The fact that juvenile and adult skulls in birds do share a similar anatomical integration suggests the presence of a specific constraint to their ontogenetic growth.
Δ-dN/dS: A New Criteria to Distinguish among Different Selection Modes in Gene Evolution
Authors: Gu Xun
Published: February 2020

One of the most widely-used measures for protein evolution is the ratio of nonsynonymous distance (dN) to synonymous distance (dS). Under the assumption that synonymous substitutions in the coding region are selectively neutral, thedN/dSratio can be used to test the adaptive evolution ifdN/dS>1 statistically significantly. However, due to selective constraints imposed on amino acid sites, most encoding genes demonstratedN/dS<1. As a result,dN/dSof a gene is less than 1, even some sites may have experienced positive selections. In this paper, we develop a new criterion, calledΔ-dN/dS, for positive selection testing by introducing an indexH, which is a relative measure of rate variation among sites. Under the context of strong purifying selection at some amino acid sites, our model predictsdN/dS=1-Hfor the neutral evolution,dN/dS<1-Hfor the nearly-neutral selection, anddN/dS>1-Hfor the adaptive evolution. The potential of this new method for resolving the neutral-adaptive debates has been illustrated by case studies. For over 4000 vertebrate genes, virtually all of them showeddN/dS<1-H, indicating the dominant role of the nearly-neutral selection in molecular evolution. Moreover, we calculated thedN/dSratio for cancer somatic mutations of a human gene, specifically denoted byCN/CS. For over 4000 human genes in cancer genomics, about 55% of genes showed 1-H<CN/CS<1, about 45% of genes showedCN/CS<1, whereas less than 1% of genes showedCN/CS<1-H. Together our analysis suggested driver mutations, i.e., those initiate and facilitate carcinogenesis, confer a selective advantage on cancer cells, leading toCN/CS>1 (strong positive selection) or 1-H<CN/CS<1 (weak positive selection, combined with strong purifying selection), whereas nearly neutral selection due to reduced effective clonal size is highly unlikely in cancer evolution.
Mixed Culture of Bacterial Cell for Large Scale DNA Storage
Authors: Hao Min , Qiao Hongyan , Gao Yanmin , Wang Zhaoguan , Qiao Xin , Chen Xin , Qi Hao
Published: February 2020

DNA emerged as novel material for mass data storage, the serious problem human society is facing. Taking advantage of current synthesis capacity, massive oligo pool demonstrated its high-potential in data storage in test tube. Herein, mixed culture of bacterial cells carrying mass oligo pool that was assembled in a high copy plasmid was presented as a stable material for large scale data storage. Living cells data storage was fabricated by a multiple-steps process, assembly, transformation and mixed culture. The underlying principle was explored by deep bioinformatic analysis. Although homology assembly showed sequence context dependent bias but the massive digital information oligos in mixed culture were constant over multiple successive passaging. In pushing the limitation, over ten thousand distinct oligos, totally 2304 Kbps encoding 445 KB digital data including texts and images, were stored in bacterial cell, the largest archival data storage in living cell reported so far. The mixed culture of living cell data storage opens up a new approach to simply bridge the in vitro and in vivo storage system with combined advantage of both storage capability and economical information propagation.
A Sequence-Pair-Classification-Based Method for Detecting and Correcting Under-Clustered Gene Families
Authors: Yadav Akshay , Fernández-Baca David , Cannon Steven B.
Published: February 2020

Gene families are groups of genes that have descended from a common ancestral gene present in the species under study. Current, widely used gene family building algorithms can produce family clusters that may be fragmented or missing true family sequences (under-clustering). Here we present a classification method based on sequence pairs that, first, inspects given families for under-clustering and then predicts the missing sequences for the families using family-specific alignment score cutoffs. We have tested this method on a set of curated, gold-standard (“true”) families from the Yeast Gene Order Browser (YGOB) database, including 20 yeast species, as well as a test set of intentionally under-clustered (“deficient”) families derived from the YGOB families. For 83% of the modified yeast families, our pair-classification method was able to reliably detect under-clustering in “deficient” families that were missing 20% of sequences relative to the full/” true” families. We also attempted to predict back the missing sequences using the family-specific alignment score cutoffs obtained during the detection phase. In the case of “pure” under-clustered families (under-clustered families with no “wrong”/unrelated sequences), for 78% of families the prediction precision and recall was ≥0.75, with mean precision = 0.928 and mean recall = 0.859. For “impure” under-clustered families, (under-clustered families containing closest sequences from outside the family, in addition to missing true family sequences), the prediction precision and recall was ≥0.75 for 63% of families with mean precision = 0.790 and mean recall = 0.869. To check if our method can detect and correct incomplete families obtained using existing family building methods, we attempted to correct 374 under-clustered yeast families produced using the OrthoFinder tool. We were able to predict missing sequences for at least 19 yeast families with mean precision of 0.9 and mean recall of 0.65. We also analyzed 14,663 legume families built using the OrthoFinder program, with 14 legume species. We were able to identify 1,665 OrthoFinder families that were missing one or more sequences - sequences which were previously un-clustered or clustered into unusually small families. Further, using a simple merging strategy, we were able to merge 2,216 small families into 933 under-clustered families using the predicted missing sequences. Out of the 933 merged families, we could confirm correct mergings in at least 534 families using the maximum-likelihood phylogenies of the merged families. We also provide recommendations on different types of family-specific alignment score cutoffs that can be used for predicting the missing sequences based on the “purity” of under-clustered families and the chosen precision and recall for prediction. Finally, we provide the containerized version of the pair-classification method that can be applied on any given set of gene families.
ALG-2 and Peflin Stimulate or Inffibit Copii Targeting and Secretion in Response to Calcium Signaling
Authors: Sargeant John , Seiler Danette , Costain Tucker , Madreiter-Sokolowski Corina , Gordon David E. , Peden Andrew A. , Malli Roland , Graier Wolfgang F. , Ray Jesse C.
Published: February 2020

ER-to-Golgi transport is the first step in the constitutive secretory pathway which, unlike regulated secretion, is believed to proceed non-stop regardless of Ca2+flux. Rowever, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+signaling by adjusting the ER export rate of COPII-sorted cargos up or down by ~50%. At steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. During Ca2+signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication, following secretory increases, or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+mobilization by ATP results in ALG-2-dependent enhancement of secretion. Within the NRK cell model, distinct Ca2+signaling patterns can produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased COPTT outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPTT outer shell and decreased peflin at ERES. PEF protein complexes represent a true regulator of transport as they are dispensable for secretion yet adjust the secretion rate to physiological conditions. Their dynamics affects secretion of important physiological cargoes such as collagen T and significantly impacts ER stress.
Global proteomics ofUbqln2-based murine models of ALS
Authors: Whiteley Alexandra M. , Prado Miguel A. , de Poot Stefanie A.H. , Paulo Joao A. , Ashton Marissa , Dominguez Sara , Weber Martin , Ngu Hai , Szpyt John , Jedrychowski Mark P. , Easton Amy , Gygi Steven P. , Kurz Thimo , Monteiro Mervyn J. , Brown Eric J. , Finley Daniel
Published: February 2020

Familial forms of neurodegenerative diseases commonly involve mutation of aggregation-prone proteins or components of the protein degradation machinery that act on aberrant proteins.Ubqln2encodes a member of the UBL/UBA family of proteasome shuttle factors that is thought to facilitate proteasomal degradation of substrates, and mutation of this gene results in a familial form of ALS/FTD in humans. HowUbqln2dysfunction leads to neurodegeneration, however, remains uncertain. We undertook a comprehensive study to identify proteomic changes uponUbqln2perturbation in multiple murine models ofUbqln2-mediated neurodegenerative disease. By performing quantitative multiplexed proteomics on neural tissues of affected animals, we identified a small group of proteins whose abundance is tightly linked to UBQLN2 function: the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Further studies using cultured cells of human origin, including induced neurons, found similar changes in protein abundance uponUbqln2loss, and pulse-chase studies suggested that PEG10 and TRIM32 are direct clients of UBQLN2. In conclusion, our study provides a deep understanding of the proteomic landscape of ALS-relatedUbqln2mutants and identifies candidate client proteins that are alteredin vivoin disease models and whose degradation is promoted by UBQLN2.
Cytoplasmic sharing through apical membrane remodeling
Authors: Peterson Nora G. , Stormo Benjamin M. , Schoenfelder Kevin P. , King Juliet S. , Lee Rayson R. S. , Fox Donald T.
Published: February 2020

Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, we discovered cytoplasm sharing without membrane breaching in highly resorptiveDrosophilarectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 27kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as failed cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins (normally associated with transport of molecules <1kDa), which are positioned by membrane remodeling GTPases. Our work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.

ONE SENTENCE SUMMARY

Apical membrane remodeling in a resorptiveDrosophilaepithelium generates a shared multinuclear cytoplasm.
Dissimilatory Nitrate Reduction to Ammonium in the Cable BacteriumCa. Electronema Sp. GS
Authors: Marzocchi Ugo , Thorup Casper , Dam Ann-Sofie , Schramm Andreas , Risgaard-Petersen Nils
Published: February 2020

Cable bacteria are filamentous Desulfobulbaceae that split the energy-conserving reaction of sulphide oxidation into two half reactions occurring in distinct cells. Cable bacteria can use nitrate, but the reduction pathway is unknown, making it difficult to assess their direct impact on the N-cycle. Here we show that the freshwater cable bacteriumCa.Electronema sp. GS performs dissimilatory nitrate reduction to ammonium (DNRA).15NO3−-amended sediment withCa.Electronema sp. GS showed higher rates of DNRA and nitrite production than sediment withoutCa.Electronema sp. GS. Electron flux from sulphide oxidation, inferred from electric potential measurements, matched the electron flux needed to drive cable bacteria-mediated nitrate reduction.Ca.Electronema sp. GS expressed a completenapoperon for periplasmic nitrate reduction to nitrite, and genes encoding a periplasmic multiheme cytochrome (pMHC), homolog to a pMHC that can catalyse nitrite reduction to ammonium inCa.Maribeggiatoa. Phylogenetic analysis suggests that the capacity for DNRA was acquired in multiple events through horizontal gene transfer from different organisms, before cable bacteria split into different salinity niches. The architecture of the nitrate reduction system suggests absence of energy conservation through oxidative phosphorylation, indicating that cable bacteria primarily conserve energy through the half reaction of sulfide oxidation.
Unifying the Ambivalent Mechanisms of Protein-Stabilising Osmolytes
Authors: Mukherjee Mrinmoy , Mondal Jagannath
Published: February 2020

The mechanism of protein stabilization by zwiterionic osmolytes has remained a long-standing puzzle. While the prevalent mechanistic hypothesis suggests an ‘osmo-phobic’ model in which osmolytes are assumed to stabilize proteins by preferentially excluding themselves from the protein surface, emerging evidences of preferential binding of popular osmolyte trimethyl amine N-oxide (TMAO) with hydrophobic macromolecules contradict this view. Here we address these contrasting perspectives by investigating the folding mechanism of a set of mini proteins in aqueous solutions of two different osmolytes glycine and TMAO, via free energy simulations. Our results demonstrate that, while both osmolytes are found to stabilize the folded conformation of the mini proteins, their mechanism of actions are mutually diverse: Specifically, glycine always depletes from the surface of all mini proteins, thereby conforming to the osmophobic model; but TMAO is found to display ambivalent signatures of protein-specific preferential binding and exclusion to/from the protein surface. At molecular level, the presence of an extended hydrophobic patch in protein topology is found to be recurrent motif in proteins leading to favorable binding with TMAO. Finally, an analysis based upon the preferential interaction theory and folding free energetics reveals that irrespective of preferential binding vs exclusion of osmolytes, it is therelativepreferential depletion of osmolytes on transition from folded to unfolded conformation of proteins, which drives the overall conformational equilibrium towards the folded state in presence of osmolytes. Taken together, moving beyond the model system and hypothesis, this work brings out ambivalent mechanism of osmolytes on proteins and provides an unifying justification.
Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance inTrypanosoma brucei
Authors: Quintana Juan F. , Bueren-Calabuig Juan , Zuccotto Fabio , de Koning Harry P. , Horn David , Field Mark C.
Published: February 2020

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. African trypanosome pentamidine and melarsoprol sensitivity is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation or via binding to TbAQP2, with subsequent endocytosis, but trafficking and regulation of TbAQPs is uncharacterised. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation also leads to impaired oligomerisation, mislocalisation, and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and contributes towards emergence of drug resistance.
The HPA stress axis shapes aging rates in long-lived, social mole-rats
Authors: Sahm Arne , Hoffmann Steve , Koch Philipp , Henning Yoshiyuki , Bens Martin , Groth Marco , Burda Hynek , Begall Sabine , Ting Saskia , Goetz Moritz , Daele Paul Van , Staniszewska Magdalena , Klose Jasmin , Costa Pedro Fragoso , Platzer Matthias , Szafranski Karol , Dammann Philip
Published: February 2020

Sexual activity and/or reproduction doubles life expectancy in the long-lived rodent genusFukomys. To investigate the molecular mechanisms underlying this phenomenon, we analyzed a total of 636 RNA-seq samples across 15 tissues. This analysis suggests that the differences in life expectancy between reproductive and non-reproductive mole-rats are mainly caused by critical changes in the regulation of the hypothalamic-pituitary-adrenal stress axis, which we further substantiate with a series of independent evidence. In accordance with previous studies, the up-regulation of the proteasome and several so-called “anti-aging molecules”, such as DHEA, is also linked with enhanced life expectancy. On the other hand, several our results oppose crucial findings in short-lived model organisms. For example, we found the up-regulation of the IGF1/GH axis and several other anabolic processes to be compatible with a considerable lifespan prolongation. These contradictions question the extent to which findings from short-lived species can be transferred to longer-lived ones.
Loss of ricePARAQUAT TOLERANCE 3confers enhanced resistance to abiotic stresses and increases grain yield in field
Authors: Alfatih Alamin , Wu Jiu , Jan Sami Ullah , Zhang Zi-Sheng , Xia Jing-Qiu , Xiang Cheng-Bin
Published: February 2020

Plants frequently suffer from environmental stresses in nature and have evolved sophisticated and efficient mechanisms to cope with the stresses. To balance between growth and stress response, plants are equipped with efficient means to switch off the activated stress responses when stresses diminish. We previously revealed such an off-switch mechanism conferred by ArabidopsisPQT3, knockout of which significantly enhances resistance to abiotic stresses. To explore whether the rice homologOsPQT3is functionally conserved, we generated three knockout mutants with CRISPR-Cas9 technology. TheOsPQT3knockout mutants (ospqt3) display enhanced resistance to oxidative and salt stress with elevated expression ofOsGPX1,OsAPX1, andOsSOD1. More importantly, theospqt3mutants show significantly enhanced agronomic performance with higher yield compared with the wild type under salt stress in greenhouse as well as in field conditions. We further showed thatOsPQT3was rapidly down regulated in response to oxidative and other abiotic stresses asAtPQT3. Taken together, these results support our previous findings that AtPQT3 acts as an off-switch in stress response, which is well conserved in rice. Therefore,PQT3locus provides a promising candidate for crop improvement with enhanced stress resistance by gene editing technology.
RiceNIN-LIKE PROTEIN 1Rapidly Responds to Nitrogen Deficiency and Improves Yield and Nitrogen Use Efficiency
Authors: Alfatih Alamin , Wu Jie , Zhang Zi-Sheng , Xia Jing-Qiu , Jan Sami Ullah , Yu Lin-Hui , Xiang Cheng-Bin
Published: February 2020

Nitrogen (N) is indispensable for crop growth and yield, but excessive agricultural application of nitrogenous fertilizers has generated severe environmental problems. A desirable and economical solution to cope with these issues is to improve crop nitrogen use efficiency (NUE). Plant NUE has been a focal point of intensive research worldwide, yet much more has to be learned about its genetic determinants and regulation. Here, we show that riceNIN-LIKE PROTEIN 1(OsNLP1) plays a fundamental role in N utilization. OsNLP1 protein localizes in nucleus and its transcript level is rapidly induced by N starvation. Overexpression ofOsNLP1improves plant growth, grain yield and NUE under different N conditions while knockout ofOsNLP1impairs grain yield and NUE under N limiting conditions. OsNLP1 regulates nitrate and ammonium utilization by cooperatively orchestrating multiple N uptake and assimilation genes. Chromatin immunoprecipitation and yeast-one-hybrid assays show that OsNLP1 can directly bind to the promoter of these genes to activate their expression. Therefore, our results demonstrate that OsNLP1 is a key regulator of N utilization and represents a potential target for improving NUE and yield in rice.

One-sentence summary

OsNLP1 rapidly responds to N availability, enhances N uptake and assimilation, and holds great potential in promoting high yield in rice.
Cultivars of biofortified cowpea and sweet potato: Bioavailability of iron and interaction with vitamin A in vivo and in vitro
Authors: Corrêa Sarah Ramos , Brigide Priscila , Vaz‐Tostes Maria das Graças , Costa Neuza Maria Brunoro
Published: February 2020

The objective of this study was to evaluate the interaction of pro‐vitamin A‐rich sweet potato on iron bioavailability of biofortified cowpeas, using in vitro Caco‐2 cells and in vivo depletion–repletion rat model. Mixtures of conventional rice with cultivars of iron‐biofortified (Aracê, Xiquexique, and Tumucumaque) or conventional (Guariba) cowpeas with or without sweet potato biofortified with pro‐vitamin A carotenoids were evaluated. The ratio of ferritin/total protein in Caco‐2 cells was used as the index of cellular Fe uptake in the in vitro assay. The animal study evaluated the hemoglobin gain, the relative biological value, and the gene expression of transferrin and ferritin proteins by reverse transcription polymerase chain reaction. In the in vitro study, Xiquexique cowpea presented higher bioavailability of iron in the absence of sweet potato, and no difference was observed between the other cultivars of cowpea with and without sweet potato. The in vivo bioavailability (relative biological value of hemoglobin regeneration efficiency) differed statistically only between Guariba groups added to sweet potato and Tumucumaque. Ferritin mRNA expression did not differ between the test and control (ferrous sulfate) groups. Regarding the transferrin mRNA expression, there was a difference between the test and control groups except for the Xiquexique group. The association of rice and beans with sweet potato rich in carotenoids favored the gene expression of proteins involved in the iron metabolism, as well as its bioavailability, corroborating beneficial effects of this mixture. Xiquexique cowpea was shown to be the most promising compared to the other cultivars, exhibiting higher iron content in the digestible fraction, better in vitro bioavailability of iron, and transferrin gene expression.

Practical Application

Data from the study indicated greater in vitro bioavailability of iron for Xiquexique cowpea and sweet potato mixtures, in addition to the greater regeneration efficiency of hemoglobin in vivo as the bioavailability of iron among biofortified beans, highlighting the promising benefits of biofortification.