bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of oral baits and distribution methods for Tasmanian devils (Sarcophilus harrisii)
Authors: Dempsey Sean , Pye Ruth J. , Gilbert Amy T. , Fountain-Jones Nicholas M. , Moffat Jennifer M. , Benson-Amram Sarah , Smyser Timothy J. , Flies Andrew S.
Published: April 2022

Context

Diseases are increasingly contributing to wildlife population declines. Tasmanian devil (Sarcophilus harrisii) populations have locally declined by 82% largely due to the morbidity and mortality associated with two independent transmissible devil facial tumours (DFT1 and DFT2). Toxic baits are often used as a management tool for controlling vertebrate pest populations in Australia, but in other areas of the world oral baits are also used to deliver vaccines or pharmaceuticals to wildlife. Oral vaccine bait products have been distributed for more than 50 years at a landscape scale to protect wildlife from rabies virus and contributed to the elimination of fox rabies from more than ten European nations. An oral bait vaccine to protect against devil facial tumours has been proposed as a management tool to improve the population health, resiliency and fitness of wild Tasmanian devils.

Aim

Our goal was to evaluate the potential use of edible baits as vehicles for vaccine delivery to Tasmanian devils.

Method

We tested placebo versions of baits that are already used in Australia.

Key results

Captive devils consumed all types of placebo baits but exhibited a preference for ruminant- and fish-based baits. Captive devils also consumed inert capsules inserted into placebo baits. Bait fate trials in the field revealed that baits were generally removed within 24 hours. Tasmanian pademelons (Thylogale billardierii), brushtail possums (Trichosurus vulpecula), and Eastern quolls (Dasyurus viverrinus) were the most common nontarget bait competitors at six private properties in southern Tasmania; wild devils removed approximately 5% of ground baits at these sites. We also evaluated an automated bait dispenser, which reduced nontarget uptake and resulted in over 50% of the baits being removed by devils.

Conclusions

This study demonstrates that captive and wild devils will accept and consume placebo versions of commercial baits. Bait dispensers or modified baits or baiting strategies are needed to increase bait uptake by devils.

Implications

Bait dispensers can be used at a regional scale to deliver baits to Tasmanian devils. These could act as vehicles for preventive or therapeutic vaccines to mitigate the impacts of disease on devil populations.

Short summary

This study aimed to test oral baits as potential vaccine delivery vehicles for Tasmanian devils. Captive and wild devils consumed placebo versions of commercial baits used on mainland Australia. Abundant non-target species, such as brushtail possums, Tasmanian pademelons, and eastern quolls consumed most baits in the wild. Implementation of automated bait dispensers increased bait uptake by devils to over 50% at the same regional field sites.
The tissue-specific chromatin accessibility landscape of Papaver somniferum
Authors: Xu Yu , Jia Yanyan , Wang Bo , Guo Li , Guo Mengyao , Che Xiaofei , Ye Kai
Published: April 2022

Accessible chromatin regions (ACRs) at promoters, enhancers, and other gene regulatory regions allow transcription factors (TFs) to bind, which regulate gene transcription involved in plant development and metabolism. Papaver somniferum has been widely applied in clinical medicine as one of the most important and oldest medicinal plants due to its unique and effective active ingredients. However, the transcriptional regulatory mechanism of tissue-specific distribution of active ingredients remains unknown. In this study, transcriptome and chromatin accessibility analysis by RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) was performed to investigate these underlying molecular mechanisms. We identified tissue-specific chromatin Tn5 hypersensitive site (THS) and gene expression by examining the variation of THS and transcripts across six tissues (capsule, stem, fine root, tap root, leaf, and petal). Our results provide insight into the epigenetic mechanism of transcriptional plasticity for P. somniferum organ development. Sequence motif analysis within accessible chromatin regions for co-expressed gene modules revealed enriched binding sites of hub transcription factors that regulate tissue-specific functions. Furthermore, we identified regulatory elements for tissue-specific accumulation of morphine and noscapine in P. somniferum. This is the first tissue-specific chromatin accessibility landscape of P. somniferum providing an important resource for functional epigenetic analysis and future molecular breeding in P. somniferum for variety improvement.
Gene Expression Changes in Therapeutic Ultrasound-Treated Human Chronic Wound Tissue
Authors: Boerman Olivia , Abedin Zahidur , DiMaria-Ghalili Rose Ann , Weingarten Michael S. , Neidrauer Michael , Lewin Peter A. , Spiller Kara L.
Published: April 2022

Low-frequency, low-intensity ultrasound has been previously shown to promote healing of chronic wounds in humans, but mechanisms behind these effects are poorly understood. The purpose of this study was to evaluate gene expression differences in debrided human venous ulcer tissue from patients treated with low-frequency (20 kHz), low-intensity (100 mW/cm2) ultrasound compared to a sham treatment in an effort to better understand the potential biological mechanisms. Debrided venous ulcer tissue was collected from 32 subjects one week after sham treatment or low-frequency, low-intensity ultrasound treatment. Of these samples, 7 samples (3 ultrasound treated and 4 sham treated) yielded sufficient quality total RNA for analysis by ultra-high multiplexed PCR (Ampliseq) and expression of more than 24,000 genes was analyzed. 477 genes were found to be significantly different between the ultrasound and sham groups using cut-off values of p<0.05 and fold change of 2. Gene set enrichment analysis identified 20 significantly enriched gene sets from upregulated genes and 4 significantly enriched gene sets from downregulated genes. Most of the enriched gene sets from upregulated genes were related to cellcell signaling pathways. The most significantly enriched gene set from downregulated genes was the inflammatory response gene set. These findings show that therapeutic ultrasound influences cellular behavior in chronic wounds as early as one week after application. Considering the well-known role of chronic inflammation in impairing wound healing in chronic wounds, these results suggest that a downregulation of inflammatory genes is a possible biological mechanism of ultrasound-mediated venous chronic wound healing. Such increased understanding may ultimately lead to the enhancement of ultrasound devices to accelerate chronic wound healing and increase patient quality of life.
The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein
Authors: Garner Kirsten E. L. , Salter Anna , Lau Clinton K. , Gurusaran Manickam , Villemant Cécile , Granger Elizabeth P. , McNee Gavin , Woodman Philip G. , Davies Owen R. , Burke Brian E. , Allan Victoria J.
Published: April 2022

Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein’s interphase functions. KASH5 interacts with either dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein’s recruitment to other cellular membranes. KASH5’s N-terminal EF-hands are essential, as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the trans-membrane protein KASH5 is an activating adaptor for dynein, and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag
Authors: Yim Willa Wen-You , Yamamoto Hayashi , Mizushima Noboru
Published: April 2022

Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the GFP-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g., GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant RFP accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g., Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.
Rescue of Escherichia coli auxotrophy by de novo small proteins
Authors: Babina Arianne M. , Surkov Serhiy , Ye Weihua , Jerlström-Hultqvist Jon , Larsson Mårten , Holmqvist Erik , Jemth Per , Andersson Dan I. , Knopp Michael
Published: April 2022

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here we show that by up-regulating hisB expression, de novo small proteins (≤ 50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5’ end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries, and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.
A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins
Authors: Loreau Vincent , Rees Renate , Chan Eunice HoYee , Taxer Waltraud , Gregor Kathrin , Mußil Bianka , Pitaval Christophe , Luis Nuno Miguel , Mangeol Pierre , Schnorrer Frank , Görlich Dirk
Published: April 2022

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understand protein function. However, to date this is still a challenging task, as accessibility of large antibodies to dense tissues is often limited and fluorescent proteins inserted close to a domain of interest may affect function of the tagged protein. These complications are particularly present in the muscle sarcomere, arguably one of the most protein dense structures in nature, which makes studying morphogenesis at molecular resolution challenging. Here, we have employed an efficient pipeline to generate a nanobody toolbox specifically recognising various domains of two largeDrosophilatitin homologs, Sallimus and Projectin. We demonstrate the superior labelling qualities of our nanobodies compared to conventional antibodies in intact muscle tissue. Applying our nanobody toolbox to larval muscles revealed a gigantic Sallimus isoform stretched more than 2 µm to bridge the sarcomeric I-band. Furthermore, N- and C-terminal nanobodies against Projectin identified an unexpected polar orientation of Projectin covering the myosin filaments in larval muscles. Finally, expression of a Sallimus nanobody in living larval muscles confirmed the high affinity binding of nanobodies to target epitopes in living tissue and hence demonstrated their power to reveal thein vivodynamics of sarcomeric protein domains. Together, our toolbox substantiates the multiple advantages of nanobodies to study sarcomere biology. It may inspire the generation of similar toolboxes for other large protein complexes inDrosophilaor mammals.
Evolutionary Origin of Vertebrate OCT4/POU5 Functions in Supporting Pluripotency
Authors: Sukparangsi Woranop , Morganti Elena , Lowndes Molly , Mayeur Hélène , Weisser Melanie , Hammachi Fella , Peradziryi Hanna , Roske Fabian , Hölzenspies Jurriaan , Livigni Alessandra , Godard Benoit Gilbert , Sugahara Fumiaki , Kuratani Shigeru , Montoya Guillermo , Frankenberg Stephen R. , Mazan Sylvie , Brickman Joshua M
Published: April 2022

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse Embryonic Stem Cells (ESCs), we found that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. This specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, ESC phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.
RadGenNets: Deep Learning-Based Radiogenomics Model For Gene Mutation Prediction In Lung Cancer
Authors: Tripathi Satvik , Moyer Ethan Jacob , Augustin Alisha Isabelle , Zavalny Alex , Dheer Suhani , Sukumaran Rithvik , Schwartz Daniel , Gorski Brandon , Dako Farouk , Kim Edward
Published: April 2022

Abstract

In this paper, we present our methodology that can be used for predicting gene mutation in patients with non-small cell lung cancer (NSCLC). There are three major types of gene mutations that a NSCLC patient’s gene structure can change to: epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), and Anaplastic lymphoma kinase (ALK). We worked with the clinical and genomics data for each patient as well CT scans. We preprocessed all of the data and then built a novel pipeline to integrate both the image and tabular data. We built a novel pipeline that used a fusion of Convolutional Neural Networks and Dense Neural Networks. Also, using a search approach, we pick an ensemble of deep learning models to classify the separate gene mutations. These models include EfficientNets, SENet, and ResNeXt WSL, among others. Our model achieved a high area under curve (AUC) score of 94% in detecting gene mutation.
A joint learning approach for genomic prediction in polyploid grasses
Authors: Aono Alexandre Hild , Ulbricht Ferreira Rebecca Caroline , Moraes Aline da Costa Lima , de Castro Lara Letícia Aparecida , Pimenta Ricardo José Gonzaga , Costa Estela Araujo , Pinto Luciana Rossini , de Andrade Landell Marcos Guimarães , Santos Mateus Figueiredo , Jank Liana , Lima Barrios Sanzio Carvalho , do Valle Cacilda Borges , Chiari Lucimara , Franco Garcia Antonio Augusto , Kuroshu Reginaldo Massanobu , Lorena Ana Carolina , Gorjanc Gregor , de Souza Anete Pereira
Published: April 2022

Poaceae, among the most abundant plant families, includes many economically important polyploid species, such as forage grasses and sugarcane (Saccharumspp.). These species have elevated genomic complexities and limited genetic resources, hindering the application of marker-assisted selection strategies. Currently, the most promising approach for increasing genetic gains in plant breeding is genomic selection. However, due to the polyploidy nature of these polyploid species, more accurate models for incorporating genomic selection into breeding schemes are needed. This study aims to develop a machine learning method by using a joint learning approach to predict complex traits from genotypic data. Biparental populations of sugarcane and two species of forage grasses (Urochloa decumbens, Megathyrsus maximus) were genotyped, and several quantitative traits were measured. High-quality markers were used to predict several traits in different cross-validation scenarios. By combining classification and regression strategies, we developed a predictive system with promising results. Compared with traditional genomic prediction methods, the proposed strategy achieved accuracy improvements exceeding 50%. Our results suggest that the developed methodology could be implemented in breeding programs, helping reduce breeding cycles and increase genetic gains.
A genetic model for in vivo proximity labeling of the mammalian secretome
Authors: Yang Rui , Meyer Amanda S. , Droujinine Ilia A. , Udeshi Namrata D. , Hu Yanhui , Guo Jinjin , McMahon Jill A. , Carey Dominique K. , Xu Charles , Fang Qiao , Sha Jihui , Qin Shishang , Rocco David , Wohlschlegel James , Ting Alice Y. , Carr Steven A. , Perrimon Norbert , McMahon Andrew P.
Published: April 2022

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalyzed proximity labeling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labeling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labeling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and healthy and diseased adult states.
Whole-body metabolic modelling predicts isoleucine dependency of SARS-CoV-2 replication
Authors: Thiele Ines , Fleming Ronan M.T.
Published: April 2022

We aimed at investigating host-virus co-metabolism during SARS-CoV-2 infection. Therefore, we extended comprehensive sex-specific, whole-body organ resolved models of human metabolism with the necessary reactions to replicate SARS-CoV-2 in the lung as well as selected peripheral organs. Using this comprehensive host-virus model, we obtained the following key results: 1. The predicted maximal possible virus shedding rate was limited by isoleucine availability. 2. The supported initial viral load depended on the increase in CD4+ T-cells, consistent with the literature. 3. During viral infection, the whole-body metabolism changed including the blood metabolome, which agreed well with metabolomic studies from COVID-19 patients and healthy controls. 4. The virus shedding rate could be reduced by either inhibition of the guanylate kinase 1 or availability of amino acids, e.g., in the diet. 5. The virus variants achieved differed in their maximal possible virus shedding rates, which could be inversely linked to isoleucine occurrences in the sequences. Taken together, this study presents the metabolic crosstalk between host and virus and emphasis the role of amino acid metabolism during SARS-CoV-2 infection, in particular of isoleucine. As such, it provides an example of how computational modelling can complement more canonical approaches to gain insight into host-virus crosstalk and to identify potential therapeutic strategies.
Multisite Test-Retest Reliability and Compatibility of Brain Metrics derived from FreeSurfer Versions 7.1, 6.0, and 5.3
Authors: Haddad Elizabeth , Pizzagalli Fabrizio , Zhu Alyssa H. , Bhatt Ravi R. , Islam Tasfiya , Gari Iyad Ba , Dixon Daniel , Thomopoulos Sophia I. , Thompson Paul M. , Jahanshad Neda
Published: April 2022

Automatic neuroimaging segmentation and parcellation tools provide convenient and systematic methods for extracting numerous features from brain MRI scans, and are becoming standard practice for large-scale coordinated studies. One such tool, FreeSurfer, provides an easy-to-use pipeline to extract metrics describing cortical and subcortical morphometry. Over the past two decades, there have been over 25 stable releases of FreeSurfer, and different versions are used across published works. Despite this, the reliability and compatibility of metrics derived from the most recent major version releases have yet to be assessed empirically. Here, we use test-retest data from three public brain MRI datasets to assess within-version reliability and between-version compatibility across 42 regional outputs from three versions of FreeSurfer: the latest, v7.1, and two previous stable releases - v5.3, and v6.0. We find v7.1 was less compatible with older versions for measuring cortical thickness. In particular, the thickness of the cingulate gyrus had low compatibility (intraclass correlation coefficient (ICC) between 0.37 and 0.61) between versions. Temporal and frontal poles, and the medial orbitofrontal surface area metrics, also showed low to moderate compatibility with v7.1. While our work compares all three versions, our sub-comparisons between the older versions (v5.3 and v6.0) replicates earlier findings of low compatibility of pallidum and putamen volumes. Low between-version compatibility was not always indicative of low within-version reliability – all versions showed good to excellent reliability across most regional measures (ICC>0.8). Age associations, quality control metrics, and Dice coefficients in an independent sample of 106 individual scans, processed with all three versions of FreeSurfer, revealed differences in results of downstream statistical analysis. As neuroimaging studies adopt more recently released software, we provide researchers with a reference to highlight the regions and metrics that may yield findings inconsistent with published works using older FreeSurfer software. An interactive viewer for the results is provided at http://data.brainescience.org/Freesurfer_Reliability/
Local recruitment of DNA repair proteins enhances CRISPR-ssODN-HDR editing
Authors: Jillette Nathaniel , Zhu Jacqueline J. , Cheng Albert W.
Published: April 2022

CRISPR-Cas technologies enable precise editing of genomic sequences. One major way to introduce precise editing is through homology directed repair (HDR) of DNA double strand breaks (DSB) templated by exogenously supplied single-stranded oligodeoxyribonucleotides (ssODN). Competing pathways determine the outcome of edits. Non-homologous end-joining pathways produce destructive insertions/deletions (indels) at target sites and are dominant over the precise homology directed repair pathways. In this study, we aim to favor HDR and use two strategies to recruit DNA repair proteins (DRPs) to Cas9 cut site, the Casilio-DRP approach that recruits RNA-binding protein-tethered DRPs to target site via aptamers appended to guide RNA; and the 53BP1-DRP approach that recruits DRPs to DSBs via DSB-sensing activity of 53BP1. We conducted two screens using these approaches and identified DRPs such as FANCF and BRCA1 that when recruited to Cas9 cut site, enhance ssODN-templated HDR and increase the proportion of precise edits. This study provides not only new constructs for enhanced CRISPR-ssODN-HDR but also a collection of DRP fusions for studying DNA repair processes.
Genomic surveillance unfolds the dynamics of SARS-CoV-2 transmission and divergence in Bangladesh over the past two years
Authors: Shishir Tushar Ahmed , Jannat Taslimun , Naser Iftekhar Bin
Published: April 2022

The highly pathogenic virus SARS-CoV-2 has shattered the healthcare system of the world causing the COVID-19 pandemic since first detected in Wuhan, China. Therefore, scrutinizing the genome structure and tracing the transmission of the virus has gained enormous interest in designing appropriate intervention strategies to control the pandemic. In this report, we examined 4622 sequences from Bangladesh and found that they belonged to thirty-five major PANGO lineages, while Delta alone accounted for 39%, and 78% were from just four primary lineages. Our research has also shown Dhaka to be the hub of viral transmission and observed the virus spreading back and forth across the country at different times by building a transmission network. The analysis resulted in 7659 unique mutations, with an average of 24.61 missense mutations per sequence. Moreover, our analysis of genetic diversity and mutation patterns revealed that eight genes were under negative selection pressure to purify deleterious mutations, while three genes were under positive selection pressure.

Importance

With 29,122 deaths, 1.95 million infections and a shattered healthcare system from SARS-CoV-2 in Bangladesh, the only way to avoid further complications is to break the transmission network of the virus. Therefore, it is vital to shedding light on the transmission, divergence, mutations, and emergence of new variants using genomic data analyses and surveillance. Here, we present the geographic and temporal distribution of different SARS-CoV-2 variants throughout Bangladesh over the past two years, and their current prevalence. Further, we have developed a transmission network of viral spreads, which in turn will help take intervention measures. Then we analyzed all the mutations that occurred and their effect on evolution as well as the currently present mutations that could trigger a new variant of concern. In short, together with an ongoing genomic surveillance program, these data will help to better understand SARS-CoV-2, its evolution, and pandemic characteristics in Bangladesh.
TNFα-induced metabolic reprogramming drives an intrinsic anti-viral state
Authors: Ciesla Jessica , Moreno Isreal , Munger Joshua
Published: April 2022

Cytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNFα induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNFα activates glycolysis through the induction of muscle-specific hexokinase (HK2). Further, we show that glycolysis is broadly important for TNFα-mediated anti-viral defense, as its inhibition attenuates TNFα’s ability to limit the replication of evolutionarily divergent viruses. Stable-isotope tracing revealed that TNFα-mediated glycolytic activation promotes the biosynthesis of UDP-sugars (essential precursors of protein glycosylation) and that inhibition of glycolysis prevents the accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition also attenuated TNFα’s ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response.
Human genomic DNA is widely interspersed with i-motif structures
Authors: Peña Martinez Cristian David , Zeraati Mahdi , Rouet Romain , Mazigi Ohan , Gloss Brian , Chan Chia-Ling , Bryan Tracy M. , Smith Nicole M. , Dinger Marcel E. , Kummerfeld Sarah , Christ Daniel
Published: April 2022

DNA i-motif structures are formed in the nucleus of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by characterisation of select model genes, the abundance and distribution of such structures in the human genome has remained unclear. Here we utilize high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in human genomic DNA. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified over 650,000 i-motif structures in human genomic DNA. The i-motif structures are widely distributed throughout the human genome and are common among highly expressed genes and in genes upregulated in G0/G1 cell cycle phase. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA.
Architecture of the biofilm-associated archaic CupE pilus fromPseudomonas aeruginosa
Authors: Böhning Jan , Dobbelstein Adrian , Sulkowski Nina , Eilers Kira , von Kügelgen Andriko , Tarafder Abul K. , Alva Vikram , Filloux Alain , Bharat Tanmay A. M.
Published: April 2022

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogenPseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor β-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface ofP. aeruginosacells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance ofcupEgenes in isolates ofP. aeruginosaand the co-occurrence ofcupEwith othercupclusters, suggesting interdependence ofcuppili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation inP. aeruginosa.
Sustained plumage divergence despite weak genomic differentiation and broad sympatry in sister species of Australian woodswallows (Artamus spp.)
Authors: Peñalba Joshua V. , Peters Jeffrey L. , Joseph Leo
Published: April 2022

Plumage divergence can function as a strong premating barrier when species come into secondary contact. When it fails to do so, the results are often genome homogenization and phenotypic hybrids at the zone of contact. This is not the case in the largely sympatric masked woodswallow and white-browed woodswallow species (Passeriformes: Artamidae: Artamus spp) complex in Australia where phenotypic integrity is sustained despite no discernible mitochondrial structure in earlier work. This lack of structure may suggest recent divergence, ongoing gene flow or both, and phenotypic hybrids are reported albeit rarely. Here, we further assessed the population structure and differentiation across the species’ nuclear genomes using ddRAD-seq. As found in the mitochondrial genome, no structure or divergence within or between the two species was detected in the nuclear genome. This coarse sampling of the genome nonetheless revealed peaks of differentiation around the genes SOX5 and Axin1. Both are involved in the Wnt//μ-catenin signaling pathway, which regulates feather development. Reconstruction of demographic history and estimation of parameters supports a scenario of secondary contact. Our study informs how divergent plumage morphs may arise and be sustained despite whole-genome homogenization and reveals new candidate genes potentially involved in plumage divergence.
Orexin cells efficiently decode blood glucose dynamics to drive adaptive behavior
Authors: Viskaitis Paulius , Tesmer Alexander L. , Karnani Mahesh M. , Arnold Myrtha , Donegan Dane , Bracey Ed F. , Grujic Nikola , Peleg-Raibstein Daria , Burdakov Denis
Published: April 2022

Blood glucose variability shapes human brain performance and diverse clinical outcomes. However, it remains poorly understood how blood glucose fluctuations are decoded by genetically-defined neurons to change brain activity and behavior. Recent breakthroughs in genetics and clinical diagnostics identified hypothalamic hypocretin/orexin neurons (HONs) as core determinants of brain activity and adaptive behavior across mammals. Here we show that low-frequency HON population waves are tuned for transmitting information about minute-to-minute temporal features of blood glucose, thus rapidly converting its variability into brain state of behaving mice. Contrary to current theories envisioning glucose-proportional neural responses, the HONs’ response tracked blood glucose gradients, thus generating efficient neural adaptations in anticipation of maximal glucose deviations. Resolving this population response at the single cell level with volumetric multiphoton imaging furthermore revealed glucose-excited and glucose-inhibited HONs, distinctly coupled to body movements in the high-frequency domain. Finally, HON-selective opotogenetics and cell ablation demonstrated that HONs are critical for linking glucose to adaptive behavior. These results provide an insight into how behaviorally influential hypothalamic networks interpret blood glucose variability. This may inform future metrics for efficient prediction of glycemic states in health and disease.
Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana
Authors: Xu Fan , Gonneau Martine , Faucher Elvina , Habrylo Olivier , Lefebvre Valérie , Domon Jean-Marc , Martin Marjolaine , Sénéchal Fabien , Peaucelle Alexis , Pelloux Jérôme , Höfte Herman
Published: April 2022

The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate PME activity in vivo, in studies assessing the role of pectin de-methylesterification in the control of cell expansion. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity in acidic pH conditions for a variety of cell wall extracts and for purified PME preparations, but doesn’t affect PME activity at neutral pH. This suggests that the previously observed in vivo effects reflect the inhibition of PME activity at low pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, application of purified PMEI3 on Arabidopsis thaliana seedlings showed a dose-dependent inhibition of homogalacturonan de-methylesterification and root growth. Purified recombinant PMEI3 is therefore a powerful tool to study the connection between pectin methylesterification and cell expansion.
Quantitative Cell Proteomic Atlas: Pathway-scale targeted mass spectrometry for high-resolution functional profiling of cell signaling
Authors: Cifani Paolo , Kentsis Alex
Published: April 2022

In spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk and feedback remain poorly understood. To enable integrated and quantitative measurements of cellular biochemical activities, we have developed the Quantitative Cell Proteomics Atlas (QCPA). QCPA consists of panels of targeted mass spectrometry assays to determine the abundance and stoichiometry of regulatory post-translational modifications of sentinel proteins from most known physiologic and pathogenic signaling pathways in human cells. QCPA currently profiles 1,913 peptides from 469 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes triplets of isotopically labeled peptides covering known post-translational regulatory sites to determine their stoichiometries and unmodified protein regions to measure total protein abundance. The QCPA framework incorporates analytes to control for technical variability of sample preparation and mass spectrometric analysis, including TrypQuant, a synthetic substrate for accurate quantification of proteolysis efficiency for proteins containing chemically modified residues. The ability to precisely and accurately quantify most known signaling pathways should enable improved chemoproteomic approaches for the comprehensive analysis of cell signaling and clinical proteomics of diagnostic specimens. QCPA is openly available athttps://qcpa.mskcc.org.
Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs
Authors: Bernard Florian , Dargère Delphine , Rechavi Oded , Dupuy Denis
Published: April 2022

Nematode mRNA processing involves a trans-splicing step through which a 21nt sequence from a snRNP replaces the original 5’ end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods.

In this study, we used Oxford Nanopore’s long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms.

We demonstrated that spliced leader (SL) sequences presence at the 5’ end of the messengers affected library preparation and generated sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we found evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These messengers all share the capacity to generate a 5’ terminal hairpin structure mimicking the SL structure providing a mechanistic explanation for their non conformity. Altogether, our data provides the most comprehensive quantitative analysis of SL usage to date in C. elegans.
A butterfly pan-genome reveals a large amount of structural variation underlies the evolution of chromatin accessibility
Authors: Ruggieri Angelo A. , Livraghi Luca , Lewis James J. , Evans Elizabeth , Cicconardi Francesco , Hebberecht Laura , Montgomery Stephen H. , Ghezzi Alfredo , Rodriguez-Martinez José Arcadio , Jiggins Chris D. , McMillan W. Owen , Counterman Brian A. , Papa Riccardo , Van Belleghem Steven M.
Published: April 2022

Despite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual H. charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis-regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for several transcription factor binding site candidates with known function in neuron development in Drosophila. We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution.
Parallel recruitment pathways contribute to synaptonemal complex assembly during mammalian meiosis
Authors: Crichton James H. , Dunce James M. , Baarends Willy M. , Davies Owen R. , Adams Ian R.
Published: April 2022

During meiosis, the synaptonemal complex (SC) assembles between paired chromosomes, binding them together in close apposition, and facilitating recombination. SC assembly is thought to occur through the hierarchical zipper-like recruitment of axial elements, followed by transverse filaments and then central elements. However, the rapidity of SC formation in mammals has hitherto hindered investigation of its assembly mechanisms and their relationship with recombination. Using super-resolution imaging of separation-of-function mouse mutants, we show that, contrary to the hierarchical assembly model, central element protein SYCE2 is recruited to recombination sites early in SC assembly, and independently of SYCP1-containing transverse filaments. Further, SYCE2-TEX12 binds DNA in vitro, and SYCE2-containing bridges physically link paired chromosomes at recombination sites prior to transverse filament recruitment and chromosome synapsis. These data suggest that mammals integrate parallel recruitment pathways to assemble a mature SC: one recruiting central element proteins to recombination sites, and another recruiting transverse filaments to chromosomes.
Acute administration of a dopamine D2/D3 receptor agonist alters behavioral and neural parameters in adult zebrafish
Authors: Nabinger Débora Dreher , Altenhofen Stefani , Buatois Alexis , Facciol Amanda , Peixoto Julia Vasconcellos , da Silva Julia Maria Kuhl , Rübensam Gabriel , Gerlai Robert , Bonan Carla Denise
Published: April 2022

The dopaminergic neurotransmitter system is involved in numerous brain functions and behavioral processes. Alterations in this neurotransmitter system are associated with the pathogenesis of several human neurological disorders. Pharmacological agents that interact with the dopaminergic system allow the investigation of dopamine-mediated cellular and molecular responses and may elucidate the biological bases of such disorders. The zebrafish, a translationally relevant biomedical research organism, has been successfully employed in prior psychopharmacology studies. Here, we evaluate the effects of quinpirole (a dopamine D2/D3 receptor agonist) in adult zebrafish on behavioral parameters and neurotransmitter levels. Adult zebrafish received intraperitoneal injections of 0.5, 1.0, or 2.0 mg/kg of quinpirole or saline (control group) twice with an inter-injection interval of 48h. All tests were performed 24h after the second injection. After acute quinpirole administration, zebrafish exhibited decreased locomotor activity, increased anxiety-like behaviors and memory impairment compared to control. However, the quinpirole administration did not affect social and aggressive behavior. Quinpirole-treated fish exhibited altered swimming patterns: fish showed stereotypic swimming characterized by repetitive behavior, swimming from corner to corner at the bottom of the tank preceded and followed by episodes of immobility. Moreover, analysis of neurotransmitter levels in the brain demonstrated a significant increase in glutamate and a decrease in serotonin, while no alterations were observed in dopamine. These findings demonstrate that dopaminergic signaling altered by quinpirole administration results in significant changes in behavior and neurotransmitter levels in the central nervous system of zebrafish. Thus, we conclude that the use of quinpirole administration in adult zebrafish may be an appropriate tool for the analysis of mechanisms underlying neurological disorders related to the dopaminergic system.
A silk-based platform to stabilize phenylalanine ammonia-lyase (PAL) for orally administered enzyme replacement therapy
Authors: d’Amone Luciana , Trivedi Vikas D. , Nair Nikhil U. , Omenetto Fiorenzo G.
Published: April 2022

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ∼1 in 15,000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and is currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 °C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, enabling the oral administration of pegvaliase for the treatment of PKU.
Generative Moment Matching Networks for Genotype Simulation
Authors: Perera Maria , Montserrat Daniel Mas , Barrabés Míriam , Geleta Margarita , Giró-i-Nieto Xavier , Ioannidis Alexander G.
Published: April 2022

The generation of synthetic genomic sequences using neural networks has potential to ameliorate privacy and data sharing concerns and to mitigate potential bias within datasets due to under-representation of some population groups. However, there is not a consensus on which architectures, training procedures, and evaluation metrics should be used when simulating single nucleotide polymorphism (SNP) sequences with neural networks. In this paper, we explore the use of Generative Moment Matching Networks (GMMNs) for SNP simulation, we present some architectural and procedural changes to properly train the networks, and we introduce an evaluation scheme to qualitatively and quantitatively assess the quality of the simulated sequences.