bluuebunny/crossref_metadata_2025_split huggingface dataset:
A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles
Authors: Yang Emily , Metzloff Matthew , Langmüller Anna M. , Xu Xuejiao , Clark Andrew G. , Messer Philipp W. , Champer Jackson 
Published: May 2021

ABSTRACTGene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive inDrosophila melanogasterthat utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The selected gRNA target sites were close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.
A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia
Authors: Shih Han-Wei , Alas Germain C.M. , Paredez Alexander R. 
Published: May 2021

AbstractTranscriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. In response to encystation stimuli, Giardia lamblia cells shift from G1+S phase of the cell cycle to G2+M. By 2-4 hours, cyst wall proteins are upregulated, indicating that key regulatory steps occur within the first four hours of encystation. However, all characterized transcription factors (TFs) in Giardia have only been investigated at later time points of encystation. How TFs initiate encystation and link it to the cell cycle remains enigmatic. Here, we systematically screened six putative early upregulated TFs for nuclear localization, established their dynamic expression profiles, and determined their functional role in regulating encystation. We found a critical repressor, GLP4 that increases rapidly after 30 min of encystation stimuli and downregulates encystation specific markers, including Cyst Wall Proteins (CWPs) and enzymes in the cyst N-acetyl galactosamine (GalNAc) pathway. Depletion of GLP4 increases cyst production. Importantly, we observe that G2+M cells exhibit higher levels of CWP1 resulting from the activation of MYB2, a TF previously linked to encystation in Giardia. GLP4 upregulation occurs in G1+S cells, suggesting a role in repressing MYB2 and encystation specific genes in the G1+S phase of the cell cycle. Furthermore, we demonstrate that depletion of GLP4 upregulates MYB2 and promotes encystation while overexpression of GLP4 downregulates MYB2 and represses encystation. Together, these results suggest that Giardia employs a dose-dependent transcriptional response that involves the cell cycle regulated repressor GLP4 to orchestrate MYB2 and entry into the encystation pathway.ImportanceTransition between life cycle stages is a common feature among parasitic pathogens and its regulation must be optimized to balance persistence of infection with transmission. The early transcription factors (TFs) regulating commitment to differentiate are totally unknown in Giardia. In this work, we identified GLP4, a previously uncharacterized GARP-like TF, as an early-acting transcriptional repressor that inhibits G1+S cells from entering the encystation pathway. GLP4 is therefore a key regulator controlling the balance between proliferative growth and terminal differentiation into infective cysts.
Loss of N1-Methylation of G37 in tRNA Induces Ribosome Stalling and Reprograms Gene Expression
Authors: Masuda Isao , Hwang Jae-Yeon , Christian Thomas , Maharjan Sunita , Mohammad Fuad , Gamper Howard , Buskirk Allen R. , Hou Ya-Ming 
Published: May 2021

AbstractN1-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m1G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m1G37 deficiency on protein synthesis. Using E. coli as a model, we show that m1G37 deficiency induces ribosome stalling at codons that are normally translated by m1G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the A site. Enzyme and cell-based assays show that m1G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m1G37 deficiency similar to those in the starvation-induced stringent response, consistent with the notion that loss of tRNA aminoacylation activates the response. This work demonstrates a previously unrecognized function of m1G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.
Widespread occurrence of hybrid internal-terminal exons in human transcriptomes
Authors: Fiszbein Ana , McGurk Michael , Calvo-Roitberg Ezequiel , Kim GyeungYun , Burge Christopher B. , Pai Athma A. 
Published: May 2021

AbstractAlternative RNA processing is a major mechanism for diversifying the human transcriptome. Messenger RNA isoform differences are predominantly driven by alternative first exons, cassette internal exons and alternative last exons. Despite the importance of classifying exons to understand isoform structure, there is a lack of tools to look at isoform-specific exon usage using RNA-sequencing data. We recently observed that alternative transcription start sites often arise near annotated internal exons, creating “hybrid” exons that can be used as both first or internal exons. To investigate the creation of hybrid exons, we built the HIT (Hybrid-Internal-Terminal) exon pipeline that systematically classifies exons depending on their isoform-specific usage. Using a combination of junction reads coverage and probabilistic modeling, the HIT index identified thousands of hybrid first-internal and internal-last exons that were previously misclassified. Hybrid exons are enriched in long genes with at least ten internal exons, have longer flanking introns and strong splice sites. The usage of hybrid exons varies considerably across human tissues, but they are predominantly used in brain, testis and colon cells. Notably, genes involved in RNA splicing have the highest fraction of intra-tissue hybrid exons. Further, we found more than 100,000 inter-tissue hybrid exons that changed from internal to terminal exons across tissues. By developing the first method that can classify exons according to their isoform contexts, our findings demonstrate the existence of hybrid exons, expand the repertoire of tissue-specific terminal exons and uncover unexpected complexities of the human transcriptome.
Tightly constrained genome reduction and relaxation of purifying selection during secondary plastid endosymbiosis
Authors: Uthanumallian Kavitha , Iha Cintia , Repetti Sonja I. , Chan Cheong Xin , Bhattacharya Debashish , Duchene Sebastian , Verbruggen Heroen 
Published: May 2021

AbstractEndosymbiosis, the establishment of a former free-living prokaryotic or eukaryotic cell as an organelle inside a host cell, can dramatically alter the genomic architecture of the endosymbiont. Plastids, the light harvesting organelles of photosynthetic eukaryotes, are excellent models to study this phenomenon because plastid origin has occurred multiple times in evolution. Here, we investigate the genomic signature of molecular processes acting through secondary plastid endosymbiosis – the origination of a new plastid from a free-living eukaryotic alga. We used phylogenetic comparative methods to study gene loss and changes in selective regimes on plastid genomes, focusing on the green lineage that has given rise to three independent lineages with secondary plastids (euglenophytes, chlorarachniophytes,Lepidodinium). Our results show an overall increase in gene loss associated with secondary endosymbiosis, but this loss is tightly constrained by retention of genes essential for plastid function. The data show that secondary plastids have experienced temporary relaxation of purifying selection during secondary endosymbiosis. However, this process is tightly constrained as well, with selection relaxed only relative to the background in primary plastids, but purifying selection remaining strong in absolute terms even during the endosymbiosis events. Selection intensity rebounds to pre-endosymbiosis levels following endosymbiosis events, demonstrating the changes in selection efficiency during different phases of secondary plastid origin. Independent endosymbiosis events in the euglenophytes, chlorarachniophytes, andLepidodiniumdiffer in their degree of relaxation of selection, highlighting the different evolutionary contexts of these events. This study reveals the selection-drift interplay during secondary endosymbiosis, and evolutionary parallels during the process of organelle origination.
ErpY-like lipoprotein of Leptospira outsmart host complement regulation by acquiring complement regulators, activating alternate pathway, and intervening membrane attack complex
Authors: Hota Saswat , Hussain Md Saddam , Kumar Manish 
Published: May 2021

AbstractThe survival of pathogenic Leptospira in the host pivots on its proficiency to circumvent the immune response. These pathogens evade the complement system in serum by enticing and amassing the serum complement regulators onto their surface. ErpY-like lipoprotein, a surface-exposed protein of Leptospira spp., is conserved and exclusively present in the pathogenic spirochete. The recombinant form of this protein is comprehended to interact with multiple extracellular matrix (ECM) components and serum proteins like soluble complement regulators factor H (FH) and factor I (FI). Here, we document that the supplementation of recombinant ErpY-like protein (40 µg/mL) in the host (humans) serum augments the viability of E. coli and saprophytic L. biflexa by more than 2-fold. Pure complement regulators FH and FI, when bound to rErpY-like protein, preserve their respective cofactor and protease activity mandated to cleave the complement component C3b. The supplementation of rErpY-like protein (40 µg/mL) in serum ensued in ∼90 % reduction of membrane attack complex (C5b-9/MAC) deposition through alternate complement pathway (AP) activation. However, rErpY-like protein could moderately reduce (∼16%) MAC deposition in serum through the classical pathway (CP). In addition, the rErpY-like protein solely activated the AP, suggesting its role in the rapid consumption and depletion of the complement components. Blocking the pathogenic L. interrogans surface with anti-rErpY resulted in an increase in MAC formation on the bacterial surface, indicating a specific role of the ErpY-like lipoprotein in complement-mediated immune evasion. This study underscores the role of the ErpY-like lipoprotein of Leptospira in complement evasion.
Vaccine hesitancy and anti-vaccination attitudes during the start of COVID-19 vaccination program: A content analysis on Twitter data
Authors: Küçükali Hüseyin , Ataç Ömer , Palteki Ayşe Seval , Tokaç Ayşe Zülal , Hayran Osman Erol 
Published: May 2021

AbstractBackgroundVaccine hesitation, which is defined as one of the most important global health threats by World Health Organization, maintains its universal importance during the COVID-19 period. Due to the increasing appearance of anti-vaccine arguments on social media, Twitter is a useful resource in detecting these contents. In this study, we aimed to identify the prominent themes about vaccine hesitancy and refusal on social media during the COVID-19 pandemic.MethodsIn this qualitative study we collected Twitter contents which contain a vaccine-related keywords and published publicly between 9/12/2020 and 8/1/2021 (n=551,245). A stratified random sample (n=1041) is selected and analyzed by four researchers with content analysis method.ResultsAll tweets included in the study were shared from 1,000 unique accounts of which 2.7% were verified and 11.3% organizational users. 90.5% of the tweets were about vaccines, 22.6% (n=213) of the tweets mentioned at least one COVID-19 vaccine name and the most frequently mentioned COVID-19 vaccine was CorronaVac (51.2%). Yet, it was mostly as “Chinese vaccine” (42.3%). 22.0% (n=207) of the tweets included at least one anti-vaccination theme. Among tweets that included an anti-vaccination theme; poor scientific processes (21.7%), conspiracy theories (16.4%), and suspicions towards manufacturers (15.5%) were the most frequently mentioned themes. The most co-occurred themes were “Poor scientific process” theme come along with “suspicion towards manufacturers” (n=9) and “suspicion towards health authorities” (n=5).ConclusionsThis study may be helpful for health managers to identify the major concerns of the population and organize the preventive measures, through the significant role of social media on early information about vaccine hesitancy and anti-vaccination attitudes.
MATRIX METALLOPROTEINASE 26 (MMP-26) OVEREXPRESSION IN PROSTATIC ADENOCARCINOMA
Authors: da Silva Romildo Luciano , de Lima Ingrid Tavares , Luís Almeida Paes Francisco , Souza da Silva Sandra Maria , Almeida Diniz Gurgel Ana Pavla , Santos Fabiano , da Costa Silva Neto Jacinto 
Published: May 2021

ABSTRACTIntroductionMatrix metalloproteinases (MMP) have been identified as biomarkers for several diseases, including cancer. MMP-26 is constitutively expressed in some cancer cells of epithelial origin. Despite this, there is a lack of studies regarding the expression of MMP-26 on prostatic carcinoma.AimHere, we investigate the expression of the MMP-26 peptide in benign and malign prostatic tissues.Patients and MethodsFor this, 150 specimens, including atrophy (N = 25), prostatic intraepithelial neoplasia (PIN) (N = 25), benign prostatic hyperplasia (BPH) (N = 50), and prostatic adenocarcinoma (PA) (N = 50), were immunohistochemically (IHC) examined for the expression of MMP-26.ResultsMMP-26 expression was positive in 70 (46.7%) out of the 150 samples, being more prevalent in the PA group (46/50 cases,92%), followed by PIN (22/25 cases, 88%). The BPH group showed only 2/50 (4%) positive cases, and the atrophy group showed no reactivity. ROC curve analysis showed that MMP-26 immunoexpression had a higher area under the curve between PA vs atrophy+PIN+BPH (AUC=0.94; 95% CI 0.9-0.98), PA+PIN vs atrophy+BPH (AUC=0.97; 95% CI 0.94-0.99) and PA vs atrophy+BPH (AUC=0.97; 95% CI 0.95-1.00) groups. In addition, the expression and intensity of the MMP-26 reaction showed a significant association with total PSA values (P=0.001).ConclusionsOur results showed that MMP-26 immunoexpression was useful to differentiate a group of benign and malignant samples in prostate tumors. This characteristic could assist in the predictive assessment and, consequently, in the development of new strategies for the diagnosis, prognosis, and treatment of prostate cancer.
A genome-wide CRISPR screen identifies regulators of beta cell function involved in type 2 diabetes risk
Authors: Grotz Antje K , Navarro-Guerrero Elena , Bevacqua Romina J , Baronio Roberta , Thomsen Soren K , Nawaz Sameena , Rajesh Varsha , Wesolowska-Andersen Agata , Kim Seung K , Ebner Daniel , Gloyn Anna L 
Published: May 2021

AbstractIdentification of the genes and processes mediating genetic association signals for complex disease represents a major challenge. Since many of the genetic signals for type 2 diabetes exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of- function screen in human pancreatic beta cells. We focused on the regulation of insulin content as a disease-relevant readout of beta cell function. We identified 580 genes influencing this phenotype: integration with genetic and genomic data provided experimental support for 20 candidate type 2 diabetes effector transcripts including the autophagy receptorCALCOCO2. Our study highlights how cellular screens can augment existing multi-omic efforts to accelerate biological and translational inference at GWAS loci.
Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat
Authors: Stout Andrew J. , Mirliani Addison B. , White Eugene C. , Yuen John S.K. , Kaplan David L. 
Published: May 2021

AbstractCell-cultured meat offers the potential for a more sustainable, ethical, resilient, and healthy food system. However, research and development has been hindered by the lack of suitable serum-free media that enable the robust expansion of relevant cells (e.g., muscle satellite cells) over multiple passages. Recently, a low-cost serum-free media (B8) was described for induced pluripotent stem cells. Here, we adapt this media for bovine satellite cells and show that the addition of a single component, recombinant albumin, renders B8 suitable for the long-term expansion of cells without sacrificing myogenicity. We show that this new media (Beefy-9) maintains robust cell growth over the entire culture period tested (seven passages) with an average growth rate of 39 hours per population doubling. Along with demonstrated efficacy for bovine cells, this work provides a promising starting-point for developing serum-free media for cultures from other meat-relevant species. Ultimately, this work offers a promising foundation for escaping the reliance on serum in cultured meat research, thereby accelerating the field.
TALPID3/KIAA0586 regulates multiple aspects of neuromuscular patterning during gastrointestinal development in animal models and human
Authors: Delalande Jean Marie , Nagy Nandor , McCann Conor J. , Natarajan Dipa , Cooper Julie E. , Carreno Gabriela , Dora David , Campbell Alison , Laurent Nicole , Kemos Polychronis , Thomas Sophie , Alby Caroline , Attié-Bitach Tania , Lyonnet Stanislas , Logan Malcolm P. , Goldstein Allan M. , Davey Megan G. , Hofstra Robert M.W. , Thapar Nikhil , Burns Alan J. 
Published: May 2021

ABSTRACTTALPID3/KIAA0586 is an evolutionary conserved protein, which plays an essential role in protein trafficking. Its role during gastrointestinal (GI) and enteric nervous system (ENS) development has not been studied previously. Here, we analysed chicken, mouse and human embryonic GI tissues with TALPID3 mutations. The GI tract of TALPID3 chicken embryos was shortened and malformed. Histologically, the gut smooth muscle was mispatterned and enteric neural crest cells were scattered throughout the gut wall. Analysis of the Hedgehog pathway and gut extracellular matrix provided causative reasons for these defects. Interestingly, chicken intra-species grafting experiments and a conditional knockout mouse model showed that ENS formation did not require TALPID3, but was dependent on correct environmental cues. Surprisingly, the lack of TALPID3 in enteric neural crest cells (ENCC) affected smooth muscle and epithelial development in a non cell-autonomous manner. Analysis of human gut fetal tissues with aKIAA0586mutation showed strikingly similar findings compared to the animal models demonstrating conservation of TALPID3 and its necessary role in human GI tract development and patterning
Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k
Authors: Middleton A.J. , Teyra J. , Zhu J. , Sidhu S.S. , Day C.L. 
Published: May 2021

SummaryTransfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme, and E3-catalysed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.
Triangulation of microbial fingerprinting in anaerobic digestion reveals consistent fingerprinting profiles
Authors: De Vrieze Jo , Heyer Robert , Props Ruben , Van Meulebroek Lieven , Gille Karen , Vanhaecke Lynn , Benndorf Dirk , Boon Nico 
Published: May 2021

AbstractThe anaerobic digestion microbiome has been puzzling us since the dawn of molecular methods for mixed microbial community analysis. Monitoring of the anaerobic digestion microbiome can either take placeviaa non-targeted holistic evaluation of the microbial community through fingerprinting or by targeted monitoring of selected taxa. Here, we compared four different microbial community fingerprinting methods,i.e., amplicon sequencing, metaproteomics, metabolomics and cytomics, in their ability to characterise the full-scale anaerobic digestion microbiome. Cytometric fingerprinting through cytomics reflects a, for anaerobic digestion, novel, single cell-based approach of direct microbial community fingerprinting by flow cytometry. Three different digester types,i.e., sludge digesters, digesters treating agro-industrial waste and dry anaerobic digesters, each reflected different operational parameters. The α-diversity analysis yielded inconsistent results, especially for richness, across the different methods. In contrast, β-diversity analysis resulted in comparable profiles, even when translated into phyla or functions, with clear separation of the three digester types. In-depth analysis of each method’s featuresi.e., operational taxonomic units, metaproteins, metabolites, and cytometric traits, yielded certain similar features, yet, also some clear differences between the different methods, which was related to the complexity of the anaerobic digestion process. In conclusion, cytometric fingerprinting through flow cytometry is a reliable, fast method for holistic monitoring of the anaerobic digestion microbiome, and the complementary identification of key features through other methods could give rise to a direct interpretation of anaerobic digestion process performance.