bluuebunny/crossref_metadata_2025 huggingface dataset:
Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout
Authors: Aufdembrink Lauren M. , Khan Pavana , Gaut Nathaniel J , Adamala Katarzyna P. , Engelhart Aaron E.
Published: February 2020

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which Nucleic Acid Sequence Based Amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.
Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri
Authors: Kenny Nathan J , Francis Warren R. , Rivera-Vicéns Ramón E. , Juravel Ksenia , de Mendoza Alex , Díez-Vives Cristina , Lister Ryan , Bezares-Calderon Luis , Grombacher Lauren , Roller Maša , Barlow Lael D. , Camilli Sara , Ryan Joseph F. , Wörheide Gert , Hill April L , Riesgo Ana , Leys Sally P.
Published: February 2020

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits such as nervous systems arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system, which with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range allows exploration of genomic changes both within sponges and in early animal evolution.
A Src family kinase maintains latent sensitization in rats, a model of inflammatory and neuropathic pain
Authors: Chen Wenling , Marvizón Juan Carlos
Published: February 2020

Latent sensitization is a long-term model of chronic pain in which hyperalgesia is continuously suppressed by opioid receptors. This is demonstrated by the induction of mechanical allodynia by opioid antagonists. Different intracellular signals may mediate the initiation, maintenance and expression of latent sensitization. Our criterion for the involvement of a signal in the maintenance of latent sensitization is that it inhibitors should permanently eliminate the allodynia produced by an opioid antagonist. We hypothesized that Src family kinases (SFKs) maintain latent sensitization and tested this hypothesis in rats with latent sensitization induced by complete Freund’s adjuvant (CFA) or by spared nerve injury. After measures of mechanical allodynia returned to baseline, the SFK inhibitor PP2 or vehicle were injected intrathecally. The opioid antagonist naltrexone injected intrathecally 15 min later produced allodynia in control rats but not in rats injected with PP2. PP2 or vehicle were injected daily for two more days and naltrexone was injected five days later. Again, naltrexone induced allodynia in the control rats but not in the rats injected with PP2. Results were similar when latent sensitization was induced either with CFA or spared nerve injury. We concluded that an SFK, likely Fyn, maintains latent sensitization induced by inflammation or nerve injury.

Perspective

This article presents evidence that a Src family kinase, likely Fyn, maintains latent sensitization induced by inflammation or nerve injury. If latent sensitization is a valid model of chronic pain, inhibiting its maintenance with Src family kinase inhibitors may cure chronic pain.
O-GlcNAcylation, oxidation and CaMKII contribute to atrial fibrillation in type 1 and type 2 diabetes by distinct mechanisms
Authors: Mesubi Olurotimi O. , Rokita Adam G. , Abrol Neha , Wu Yuejin , Chen Biyi , Wang Qinchuan , Granger Jonathan M. , Tucker-Bartley Anthony , Luczak Elizabeth D. , Murphy Kevin R. , Umapathi Priya , Banerjee Partha S. , Boronina Tatiana N. , Cole Robert N. , Maier Lars S. , Wehrens Xander H. , Pomerantz Joel L. , Song Long-Sheng , Ahima Rexford S. , Hart Gerald W. , Zachara Natasha E. , Anderson Mark E.
Published: February 2020

Diabetes mellitus and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF in patients. However, the mechanism(s) underlying this clinical association is unknown. Elevated proteinO-GlcNAcylation (OGN) and reactive oxygen species (ROS) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by OGN (OGN-CaMKII) and ROS (ox-CaMKII). We induced type 1 (T1D) and type 2 diabetes (T2D) in a portfolio of genetic mouse models capable of dissecting the role of OGN and ROS at CaMKII and the type 2 ryanodine receptor (RyR2), an intracellular Ca2+channel implicated as an important downstream mechanism of CaMKII- mediated arrhythmias. Here we show that T1D and T2D significantly increased AF, similar to observations in patients, and this increase required CaMKII. While T1D and T2D both require ox-CaMKII to increase AF, they respond differently to loss of OGN-CaMKII or OGN inhibition. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF, and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by diverse mechanisms and targets, including CaMKII and RyR2. The proarrhythmic consequences of OGN- and ox-CaMKII differ between T1D and T2D. These results provide new and unanticipated insights into the mechanisms for increased AF in diabetes mellitus, and suggest successful future therapies will need to be different for AF in T1D and T2D.
Keratins and Plakin family cytolinker proteins control the length of epithelial microridge protrusions
Authors: Inaba Yasuko , Chauhan Vasudha , van Loon Aaron Paul , Choudhury Lamia Saiyara , Sagasti Alvaro
Published: February 2020

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable class of cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that Keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions from mucosal epithelial cells arranged in elaborate fingerprint-like patterns. Developing microridges on zebrafish skin cells contained both Actin and Keratin filaments. Keratin filaments maintained microridges upon F-actin disruption, and overexpressing Keratins lengthened microridges. Envoplakin and Periplakin, Plakin family cytolinkers that bind to F-actin and Keratins, localized to microridges and were required for their morphogenesis. Strikingly, Plakin protein levels directly determined microridge length. An actin-binding domain of Periplakin was required to initiate microridge morphogenesis, whereas Periplakin-Keratin binding was required to stabilize and elongate microridges. Our results thus separate microridge morphogenesis into two steps with differential requirements for cytoskeletal elements, expand our understanding of intermediate filament functions, and identify microridges as cellular protrusions that integrate actin and intermediate filaments.
μCB-seq: Microfluidic cell barcoding and sequencing for high-resolution imaging and sequencing of single cells
Authors: Chen Tyler N , Gupta Anushka N , Zalavadia Mansi D , Streets Aaron
Published: February 2020

Single-cell RNA sequencing (scRNA-seq) enables the investigation of complex biological processes in multicellular organisms with high resolution. However, many phenotypic features that are critical to understanding the functional role of cells in a heterogeneous tissue or organ are not directly encoded in the genome and therefore cannot be profiled with scRNA-seq. Quantitative optical microscopy has long been a powerful approach for characterizing diverse cellular phenotypes including cell morphology, protein localization, and chemical composition. Combining scRNA-seq with optical imaging has the potential to provide comprehensive single-cell analysis, allowing for functional integration of gene expression profiling and cell-state characterization. However, it is difficult to track single cells through both measurements; therefore, coupling current scRNA-seq protocols with optical measurements remains a challenge. Here, we report Microfluidic Cell Barcoding and Sequencing (μCB-seq), a microfluidic platform that combines high-resolution imaging and sequencing of single cells. μCB-seq is enabled by a novel fabrication method that preloads primers with known barcode sequences inside addressable reaction chambers of a microfluidic device. In addition to enabling multi-modal single-cell analysis, μCB-seq improves gene detection sensitivity, providing a scalable and accurate method for information-rich characterization of single cells.
Similar visual perception in GCaMP6 transgenic mice despite differences in learning and motivation
Authors: Groblewski Peter A. , Ollerenshaw Douglas R. , Kiggins Justin , Garrett Marina , Mochizuki Chris , Casal Linzy , Cross Sissy , Mace Kyla , Swapp Jackie , Manavi Sahar , Williams Derric , Mihalas Stefan , Olsen Shawn R.
Published: February 2020

To study mechanisms of perception and cognition, neural measurements must be made during behavior. A goal of theAllen Brain Observatoryis to map activity in distinct cortical cell classes during visual processing and behavior. Here we characterize learning and performance of five GCaMP6-expressing transgenic lines trained on a visual change detection task. We used automated training procedures to facilitate comparisons across mice. Training times varied, but most transgenic mice learned the task. Motivation levels also varied across mice. To compare mice in similar motivational states we subdivided sessions into over-, under-, and optimally motivated periods. When motivated, the pattern of perceptual decisions were highly correlated across transgenic lines, although overall d-prime was lower in one line labeling somatostatin inhibitory cells. These results provide important context for using these mice to map neural activity underlying perception and behavior.
Spatiotemporal Dynamics of Orthographic and Lexical Processing in the Ventral Visual Pathway
Authors: Woolnough Oscar , Donos Cristian , Rollo Patrick S. , Forseth Kiefer J. , Lakretz Yair , Crone Nathan E. , Fischer-Baum Simon , Dehaene Stanislas , Tandon Nitin
Published: February 2020

Reading is a rapid, distributed process that engages multiple components of the ventral visual stream. However, the neural constituents and their interactions that allow us to identify written words are not well understood. Using direct intracranial recordings in a large cohort of humans, we comprehensively isolated the spatiotemporal dynamics of visual word recognition across the entire left ventral occipitotemporal cortex. The mid-fusiform cortex is the first region that is sensitive to word identity and to both sub-lexical and lexical frequencies. Its activation, response latency and amplitude, are highly dependent on the statistics of natural language. Information about lexicality and word frequency propagates posteriorly from this region to traditional visual word form regions and to earlier visual cortex. This unique sensitivity of mid-fusiform cortex to the lexical characteristics of written words points to its central role as an orthographic lexicon, which accesses the long-term memory representations of visual word forms.
A machine-vision approach for automated pain measurement at millisecond timescales
Authors: Jones Jessica , Foster William , Twomey Colin , Burdge Justin , Ahmed Osama , Wojick Jessica A. , Corder Gregory , Plotkin Joshua B. , Abdus-Saboor Ishmail
Published: February 2020

Objective and automatic measurement of pain in mice remains a barrier for discovery in both basic and translational neuroscience. Here we capture rapid paw kinematics during pain behavior in mice with high-speed videography and automated paw tracking with machine and deep learning approaches. Our statistical software platform, PAWS (Pain Assessment at Withdrawal Speeds), uses a univariate projection of paw position over time to automatically quantify fast paw dynamics at the onset of paw withdrawal and also lingering pain-related behaviors such as paw guarding and shaking. Applied to innocuous and noxious stimuli across six inbred mouse strains, a linear discriminant analysis reveals a two-dimensional subspace that separates painful from non-painful stimuli on one axis, and further distinguishes the severity of pain on the second axis. Automated paw tracking combined with PAWS reveals behaviorally-divergent mouse strains that display hypo- and hyper-sensitivity to mechanical stimuli. To demonstrate the efficacy of PAWS for detecting hypersensitivity to noxious stimuli, we chemogenetically activated pain-aversion neurons in the amygdala, which further separated the behavioral representation of pain-related behaviors along a low-dimensional path. Taken together, this automated pain quantification approach should increase the ease and objectivity of collecting rigorous behavioral data, and it is compatible with other neural circuit dissection tools for determining the mouse pain state.
Kaposi’s sarcoma-associated herpesvirus fine-tunes the temporal expression of late genes by manipulating a host RNA quality control pathway
Authors: Ruiz Julio C. , Devlin Anne , Kim Jiwoong , Conrad Nicholas K.
Published: February 2020

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic nuclear DNA virus that expresses its genes using the host cell transcription and RNA processing machinery. As a result, KSHV transcripts are subject to degradation by at least two host-mediated nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) and an ARS2-dependent decay pathway. Here, we present global analyses of viral transcript levels to further understand the roles of these decay pathways in KSHV gene expression. Consistent with our recent report that the KSHV ORF57 protein increases viral transcript stability by impeding ARS2-dependent decay, ARS2 knockdown has little effect on viral gene expression 24 hours after lytic reactivation of wild-type virus. In contrast, inactivation of PPD results in premature accumulation of late transcripts. The up-regulation of late transcripts does not require the primary late gene-specific viral transactivation factor, suggesting that cryptic transcription produces the transcripts that then succumb to PPD. Remarkably, PPD inactivation has no effect on late transcripts at their proper time of expression. We show that this time-dependent PPD evasion by late transcripts requires the host factor NRDE2, which has previously been reported to protect cellular RNAs by sequestering decay factors. From these studies, we conclude that KSHV uses PPD to fine-tune the temporal expression of its genes by preventing their premature accumulation.

Importance

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposi’s sarcoma and other lymphoproliferative disorders. Nuclear expression of KSHV genes results in exposure to at least two host-mediated nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) and an ARS2-mediated decay pathway. Perhaps unsurprisingly, we previously found that KSHV uses specific mechanisms to protect its transcripts from ARS2-mediated decay. In contrast, here we show that PPD is required to dampen the expression of viral late transcripts that are prematurely transcribed, presumably due to cryptic transcription early in infection. At the proper time for their expression, KSHV late transcripts evade PPD through the activity of the host factor NRDE2. We conclude that KSHV fine-tunes the temporal expression of its genes by modulating PPD activity. Thus, the virus both protects from and exploits the host nuclear RNA decay machinery for proper expression of its genes.
Ecology of domestic dogs Canis familiaris as an emerging reservoir of Guinea worm Dracunculus medinensis infection
Authors: McDonald Robbie A. , Wilson-Aggarwal Jared K. , Swan George J.F. , Goodwin Cecily E.D. , Moundai Tchonfienet , Sankara Dieudonne , Biswas Gautam , Zingeser James A.
Published: February 2020

Global eradication of human Guinea worm disease (dracunculiasis) has been set back by the emergence of infections in animals, particularly domestic dogs Canis familiaris. The ecology and epidemiology of this reservoir is unknown. We tracked dogs using GPS, inferred diets using stable isotope analysis and analysed correlates of infection in Chad, where numbers of Guinea worm infections are greatest. Dogs had small ranges that varied markedly among villages. Diets consisted largely of human staples and human faeces. A minority of ponds, mostly <200 m from dog-owning households, accounted for most dog exposure to potentially unsafe water. The risk of a dog having had Guinea worm was reduced in dogs living in households providing water for animals but increased with increasing fish consumption by dogs. Provision of safe water might reduce dog exposure to unsafe water, while prioritisation of proactive temephos (Abate) application to the small number of ponds to which dogs have most access is recommended. Fish might have an additional role as transport hosts for Guinea worm, by concentrating copepods infected with worm larvae.
Development of a knowledge translation platform for ataxia: Impact on readers and volunteer contributors
Authors: Suart Celeste , Graham Katherine , Suart Theresa Nowlan , Truant Ray
Published: February 2020

Background

Dissemination of accurate health research information to patients and families has become increasingly important with the rise of the internet as a means of finding health information. However, the public faces several barriers to accessing research information; including paywalls and technical jargon. One method to bridge this gap between patients, families, and research is using lay summaries. SCAsource is an online knowledge translation platform where peer-reviewed research papers on ataxia are translated into lay summaries. This online platform was launched in September 2018, with the goal of making ataxia research more accessible and understandable to patients and families. A secondary goal is to provide opportunities for ataxia researchers to develop and hone their knowledge translation skills, altogether improving the quality of patient communication in the ataxia community.

Aim

The aim of this study was to measure the impact of SCAsource on its readers and volunteer contributors after one year of activity. This is to ensure SCAsource is meeting its goals of (1) improving access and understanding of ataxia research to lay audiences, and (2) improving knowledge translation skills of volunteer contributors.

Methods

Two online surveys were launched, one for readers and one for volunteers. Each survey had a combination of multiple-choice, Likert-scale type, and open-ended short-answer questions. Descriptive quantitative analysis was used for respondent characteristics and Likert-type data. A grounded theory coding approach was used to analyze narrative feedback data.

Results

We found that SCAsource has mutually beneficial outcomes for both lay person readers and volunteer contributors. Readers have an increased understanding of ataxia research and access to up-to-date information on recent publications. Volunteers develop knowledge translation skills and have increased confidence in communicating results to lay audiences. Areas of improvement were identified to be incorporated into the platform.

Conclusion

We demonstrated that SCAsource improves access to information and understanding of research to lay audiences, while providing opportunities for researchers to develop knowledge translation skills. This framework can potentially be used by other rare disease organizations to launch and evaluate their own knowledge translation websites.
Analysis of MRC-supported early career researcher success in obtaining follow-on research funding
Authors: Viney Ian , Samarasinghe Buddhini , Dolby Kevin
Published: February 2020

This study examined the progress of MRC-funded researchers, with a focus on their success in securing subsequent research income. We report here results from compiling details of the research income of researchers that completed an MRC early career award between 2011 and 2015. Depending on the funding scheme 50-59 per cent of researchers spent on average more than 100,000 GBP per year from new grants secured within three years of finishing their MRC award. In each scheme around 30 per cent of researchers secured less than 50,000 GBP per year research funding in the three years following their MRC award. We apply the same approach to an analysis of progression for the MRC cadre of intramurally-supported Programme Leader Track researchers, and a sample of MRC research grant holders. We also supplement our results with a small number of interviews with researchers that held MRC New Investigator Research Grants. We suggest that these data confirm reasonably good progression rates, with MRC early career schemes demonstrably effective in supporting research careers. They confirm there is a bottleneck with some researchers taking several years to establish their research career, and that expansion of the research base will need balanced new investment across schemes and stages and cannot be driven through growth in early career schemes alone.
Multi-omic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients
Authors: Constantinides Bede , Chau Kevin K , Quan T. Phuong , Rodger Gillian , Andersson Monique , Jeffery Katie JM , Lipworth Sam , Gweon Soon , Peniket Andy , Pike Graham , Millo Julian , Byukusenge Mary , Holdaway Matt , Gibbons Cat , Mathers Amy J , Crook Derrick W , Peto Timothy EA , Walker A Sarah , Stoesser Nicole
Published: February 2020

Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonised with diverse populations of E. coli, Klebsiella pneumoniae and Klebsiella oxytoca, including both antimicrobial-resistant and susceptible strains. Using whole genome sequencing (WGS) of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies which may vary as a result of different inputs and selection pressures. WGS of 46 contemporaneous patient isolates identified one (2%; 95% CI 0.05-11%) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10% of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including blaCTX-M, blaSHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention.
Integrated analysis of the aging brain transcriptome and proteome in tauopathy
Authors: Mangleburg Carl Grant , Wu Timothy , Yalamanchili Hari K. , Guo Caiwei , Hsieh Yi-Chen , Duong Duc M. , Dammer Eric B. , De Jager Philip L. , Seyfried Nicholas T. , Liu Zhandong , Shulman Joshua M.
Published: February 2020

Background

Tau neurofibrillary tangle pathology characterizes Alzheimer’s disease and other neurodegenerative tauopathies. Brain gene expression profiles can reveal mechanisms; however, few studies have systematically examined both the transcriptome and proteome or differentiated Tau- versus age-dependent changes.

Methods

Paired, longitudinal RNA-sequencing and mass-spectrometry were performed in aDrosophilamodel of tauopathy, based on pan-neuronal expression of human wildtype Tau (TauWT) or a mutation causing frontotemporal dementia (TauR406W). Tau-induced, differentially expressed transcripts and proteins were examined cross-sectionally or using linear regression and adjusting for age. Hierarchical clustering was performed to highlight network perturbations, and we examined overlaps with human brain gene expression profiles in tauopathy.

Results

TauWTinduced 1,514 and 213 differentially expressed transcripts and proteins, respectively. TauR406Whad a substantially greater impact, causing changes in 5,494 transcripts and 697 proteins. There was a ~70% overlap between age- and Tau-induced changes and our analyses reveal pervasive bi-directional interactions. Strikingly, 42% of Tau-induced transcripts were discordant in the proteome, showing opposite direction of change. Tau-responsive gene expression networks strongly implicate innate immune activation, despite the absence of microglia in flies. Cross-species analyses pinpoint human brain gene perturbations specifically triggered by Tau pathology and/or aging, and further differentiate between disease amplifying and protective changes.

Conclusions

Our results comprise a powerful, cross-species functional genomics resource for tauopathy, revealing Tau-mediated disruption of gene expression, including dynamic, age-dependent interactions between the brain transcriptome and proteome.
Survey of Australian STEMM Early Career Researchers: job insecurity and questionable research practices are major structural concerns
Authors: Christian Katherine , Johnstone Carolyn , Larkins Jo-ann , Wright Wendy , Doran Michael R.
Published: February 2020

We sought to understand the pressures on Early Career Researchers (ECR) in the Science, Technology, Engineering, Mathematics, & Medicine (STEMM) disciplines, collecting data from 658 ECRs working in Australia. Respondents indicated a “love of science”, but most also indicated an intention to leave their position. Decisions were primarily motivated by job insecurity (52%), while grievances included poor supervision (60%), bullying or harassment (34%), inequitable hiring practices (39%) and poor support for families (9.6%). A concerning rate of “questionable research practices” by colleagues (34.1% to 41.1%) was reported to have impacted ECR career advancement. Our study links recent reports that characterise the health of the research industry, providing direct insight from ECRs on job insecurity, workplace culture challenges, and the logical rise of questionable research practices. Internationally, nationally and institutionally the research community needs to improve job security (care for our people) and the quality of research data (our product).
Tracing the origin of a new organ by inferring the genetic basis of rumen evolution
Authors: Pan Xiangyu , Wang Yu , Li Zongjun , Chen Xianqing , Heller Rasmus , Wang Nini , Zhao Chen , Cai Yudong , Xu Han , Li Songhai , Li Ming , Li Cunyuan , Hu Shengwei , Li Hui , Wang Kun , Chen Lei , Wei Bin , Zheng Zhuqing , Fu Weiwei , Yang Yue , Zhang Tingting , Hou Zhuoting , Yan Yueyang , Lv Xiaoyang , Sun Wei , Li Xinyu , Huang Shisheng , Liu Lixiang , Mao Shengyong , Liu Wenqing , Hua Jinlian , Li Zhipeng , Zhang Guojie , Chen Yulin , Wang Xihong , Qiu Qiang , Dalrymple Brian P. , Wang Wen , Jiang Yu
Published: February 2020

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We used 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen origin and evolution. Comparative analyses reveal that the rumen and the first-chamber stomachs of camels and cetaceans shared a common tissue origin from the esophagus. The rumen recruited genes from other tissues/organs and up-regulated many esophagus genes to aquire functional innovations involving epithelium absorption, improvement of the ketone body metabolism and regulation of microbial community. These innovations involve such genetic changes as ruminant-specific conserved elements, newly evolved genes and positively selected genes. Our in vitro experiements validate the functions of one enhancer, one positively selected gene and two newly evolved antibacterial genes. Our study provides novel insights into the origin and evolution of a complex organ.
Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy
Authors: Tepperman Adam , Zheng David Jiao , Abou Taka Maria , Vrieze Angela , Lam Austin Le , Heit Bryan
Published: February 2020

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires provision of appropriate experimental conditions in a format that meets the microscopy’s optical requirements. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized live-cell imaging chambers that are compatible with a range of imaging modalities including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and which supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting PDMS microfluidic devices. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with modern microscopy techniques.
Impacts of climate change on agro-climatic suitability of major food crops and crop diversification potential in Ghana
Authors: Chemura Abel , Schauberger Bernhard , Gornott Christoph
Published: February 2020

Crop diversification is a promising climate change adaptation strategy for food production stability. However, without quantitative assessments of where, with which crop mixes and to what extent diversification is possible now and under future climatic conditions, efforts to expand crop diversification under Nationally Determined Contributions (NDCs) and National Action Plans (NAP) are unsystematic. In this study, we used extreme gradient boosting, a machine learning approach to model the current climatic suitability for maize, sorghum, cassava and groundnut in Ghana using yield data and agronomically important variables. We then used multi-model future climate projections for the 2050s and two greenhouse gas emissions scenarios (RCP 2.6 and RCP 8.5) to predict changes in the suitability range of these crops. We achieved a good model fit in determining suitability classes for all crops (AUC=0.81-0.87). Precipitation-based factors are suggested as most important in determining crop suitability, though the importance is crop-specific. Under projected climatic conditions, optimal suitability areas will decrease for all crops except for groundnuts under RCP8.5 (no change: 0%), with greatest losses for maize (12% under RCP2.6 and 14% under RCP8.5). Under current climatic conditions, 18% of Ghana has optimal suitability for two crops, 2% for three crops with no area having optimal suitability for all the four crops. Under projected climatic conditions, areas with optimal suitability for producing two and three crops will decrease by 12% as areas having moderate and marginal conditions for multiple crops increase. We also found that although diversification opportunities are spatially distinct, cassava and groundnut will be more simultaneously suitable for the south while groundnut and sorghum will be more suitable for the northern parts of Ghana under projected climatic conditions.
Effects of carbon-based additive and ventilation rate on nitrogen loss and microbial community during chicken manure composting
Authors: Chang Ruixue , Li Yanming , Chen Qing , Gong Xiaoyan , Qi Zicheng
Published: February 2020

Aerobic composting is a sustainable method for recycling of chicken manure, while its unsuitable porosity and carbon to nitrogen ratio limit the oxygen supply, which must result in high nitrogen loss because of anaerobic micro-zones in the materials. Treatments with five carbon-based additives and two ventilation rates (0.18 and 0.36 L·min-1·kg-1 DM) were set in chicken manure composting, to investigate their effects on biodegradation process, ammonia (NH3) emission, nitrogen loss, physiochemical properties and microbial community. The additives and ventilation rates influenced the CO2 production from the 2nd week, meanwhile varied the physiochemical parameters all the process. No inhibitory effect on the maturity were observed in all treatments. With woody peat as additive, the NH3 emission amount and nitrogen loss rate were shown as 15.86 mg and 4.02 %, when compared with 31.08-80.13 mg and 24.26-34.24 % in other treatments. The high aeration rate increased the NH3 emission and nitrogen loss, which were varied with different additives. The T-RFLP results showed that the additives and the ventilation rates changed the microbial community, while the prominent microbial clones belonged to the class of Bacilli and Clostridia (in the phylum of Firmicutes), and Alphaproteobacteria, Deltaproteobacteria and Gammaproteobacteria (in the phylum of Proteobacteria). Bacillus spp. was observed to be the most dominant bacteria in all the composting stages and treatments. We concluded that woody peat could improve chicken manure composting more than other additives, especially on controlling nitrogen loss. 0.18 L·min-1·kg-1 DM was suitable for chicken manure composting with different additives.
Chronic chemogenetic activation of forebrain excitatory neurons in postnatal life evokes long-lasting changes in mood-related behavior
Authors: Pati Sthitapranjya , Saba Kamal , Salvi Sonali S. , Tiwari Praachi , Chaudhari Pratik R. , Verma Vijaya , Mukhopadhyay Sourish , Kapri Darshana , Suryavanshi Shital , Clement James P. , Patel Anant B. , Vaidya Vidita A.
Published: February 2020

Early adversity is a key risk factor for the development of adult psychopathology, including anxiety, depression and schizophrenia. Rodent models of early adversity program persistent behavioral, molecular, metabolic, and neurophysiological changes. Perturbed signaling via forebrain Gq-coupled neurotransmitter receptors is a common feature across multiple models of early adversity. We addressed whether enhanced Gq-mediated signaling in forebrain excitatory neurons during postnatal life can evoke long-lasting mood-related behavioral changes. Excitatory hM3Dq DREADD-mediated chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during postnatal life (P2-14) increased anxiety- and despair-like behavior, and evoked sensorimotor gating deficits in adulthood. In contrast, chronic chemogenetic hM3Dq DREADD activation of forebrain excitatory neurons in the juvenile or adult window did not evoke any mood-related behavioral alterations, highlighting the criticality of the postnatal temporal window. The enhanced anxiety-, despair- and schizophrenia-like behavioral changes evoked by chronic chemogenetic activation of forebrain excitatory neurons in postnatal life, was accompanied by an increased cortical and hippocampal metabolic rate of glutamatergic and GABAergic neurons in adulthood. Furthermore, animals with a history of postnatal hM3Dq activation exhibited a decline in the expression of activity-dependent and plasticity-associated markers within the hippocampus, along with perturbed hippocampal excitatory and inhibitory currents in adulthood. These results indicate that Gq signaling mediated activation of forebrain excitatory neurons during the critical postnatal window is sufficient to program altered mood-related behavior, as well as metabolic and neurophysiological changes in forebrain glutamate and GABA systems, recapitulating specific aspects of the consequences of early adversity.
High-throughput laboratory evolution and evolutionary constraints inEscherichia coli
Authors: Maeda Tomoya , Iwasawa Junichiro , Kotani Hazuki , Sakata Natsue , Kawada Masako , Horinouchi Takaaki , Sakai Aki , Tanabe Kumi , Furusawa Chikara
Published: February 2020

Understanding the constraints that shape the evolution of antibiotic resistance is critical for predicting and controlling drug resistance. Despite its importance, however, a systematic investigation for evolutionary constraints is lacking. Here, we performed a high-throughput laboratory evolution ofEscherichia coliunder the addition of 95 antibacterial chemicals and quantified the transcriptome, resistance, and genomic profiles for the evolved strains. Using interpretable machine learning techniques, we analyzed the phenotype-genotype data and identified low dimensional phenotypic states among the evolved strains. Further analysis revealed the underlying biological processes responsible for these distinct states, leading to the identification of novel trade-off relationships associated with drug resistance. We also report a novel constraint that leads to decelerated evolution. These findings bridge the genotypic, gene expression, and drug resistance space and lead to a better understanding of evolutionary constraints for antibiotic resistance.
Cooption of polyalanine tract into a repressor domain in the mammalian transcription factor HoxA11
Authors: Lynch Vincent J. , Wagner Gunter P.
Published: February 2020

An enduring problem in biology is explaining how the functions of genes originated and how those functions diverge between species. Despite detailed studies on the functional evolution of a few proteins, the molecular mechanisms by which protein functions have evolved are almost entirely unknown. Here we show that a polyalanine tract in the homeodomain transcription factor HoxA11 arose in the stem-lineage of mammals and functions as an autonomous repressor module by physically interacting with the PAH domains of SIN3 proteins. These results suggest that long polyalanine tracts, which are common in transcription factors and often associated with disease, may generally function as repressor domains and can contribute to the diversification of transcription factor functions despite the deleterious consequences of polyalanine tract expansion.

Research Highlights

We show that a polyalanine track in HoxA11 evolved into a repressor domain in mammals through an increase in alanine repeat number, indicating that transcription factors can evolve novel functions despite the potential deleterious consequences associated with amino acid repeats.
Targeting HIV Env immunogens to B cell follicles in non-human primates through immune complex or protein nanoparticle formulations
Authors: Martin Jacob T. , Cottrell Christopher A. , Antanasijevic Aleksandar , Carnathan Diane G. , Cossette Benjamin J. , Enemuo Chiamaka A. , Gebru Etse H. , Choe Yury , Viviano Federico , Tokatlian Talar , Cirelli Kimberly M. , Ueda George , Copps Jeffrey , Schiffner Torben , Menis Sergey , Schief William R. , Crotty Shane , King Neil P. , Baker David , Silvestri Guido , Ward Andrew B. , Irvine Darrell J.
Published: February 2020

Following immunization, high affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble HIV envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single lymph node within 2 days after immunization. Imaging of LNs collected 7 days post-immunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent germinal centers. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with immune complexes or nanoparticles.
Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase
Authors: Chen J. Z. , Fowler D.M. , Tokuriki N.
Published: February 2020

Metallo-β-lactamases (MBLs) degrade a broad spectrum of β-lactam antibiotics, and are a major disseminating source for multidrug resistant bacteria. Despite many biochemical studies in diverse MBLs, molecular understanding of the roles of residues in the enzyme’s stability and function, and especially substrate specificity, is lacking. Here, we employ deep mutational scanning (DMS) to generate comprehensive single amino acid variant data on a major clinical MBL, VIM-2, by measuring the effect of thousands of VIM-2 mutants on the degradation of three representative classes of β-lactams (ampicillin, cefotaxime, and meropenem) and at two different temperatures (25°C and 37°C). We revealed residues responsible for expression and translocation, and mutations that increase resistance and/or alter substrate specificity. The distribution of specificity-altering mutations unveiled distinct molecular recognition of the three substrates. Moreover, these function-altering mutations are frequently observed among naturally occurring variants, suggesting that the enzymes has continuously evolved to become more potent resistance genes.
Swine Growth Promotion with Antibiotics or Alternatives can Increase Antibiotic Resistance Gene Mobility Potential
Authors: Muurinen Johanna , Richert Jacob , Wickware Carmen , Richert Brian , Johnson Timothy A.
Published: February 2020

Even though the use of antibiotics for food-producing animals may contribute to the emergence of antimicrobial resistance, antibiotics are still used as growth promoters. Due to consumer and regulatory pressures, the use of alternatives to antibiotics as growth promoters is increasing, thus more information is needed on their capability to disseminate antimicrobial resistance compared to antibiotics. We investigated the impacts of carbadox (antibiotic), copper sulfate and zinc oxide (metals) and mushroom powder (natural product) on the pig fecal resistome and microbiome. Antibiotic resistance gene (ARG) and mobile genetic element (MGE) abundances were measured using a high-throughput qPCR array with 382 primer pairs. Bacterial community composition was determined by 16S rRNA gene sequencing. More ARGs co-occurred with MGEs in the growth promoter group samples than in the control group samples. Community composition could not be linked to resistome in the growth promoter group samples, indicating a potential decoupling of ARGs and phylogeny. Additionally, machine-learning methods aided in defining the community and resistome differences in response to treatments. Since increased ARG mobility potential was the primary response to the dietary additives used in this study, we suggest that ARG mobility should be considered when designing antimicrobial use policies and antimicrobial resistance surveillances.
Genetic architecture of oxidative stress tolerance in the fungal wheat pathogenZymoseptoria tritici
Authors: Zhong Ziming , McDonald Bruce A. , Palma-Guerrero Javier
Published: February 2020

Reactive oxygen species are toxic byproducts of aerobic respiration produced during cell growth. They also are an important component of plant defenses to inhibit microbial pathogens. Tolerance to oxidative stress contributes to viability and pathogenicity of plant pathogens. However, the complex molecular network of oxidative stress responses hinders identification of the genes contributing to variation in this trait. Variation in genes affecting responses to oxidative stress is likely to affect the evolutionary potential of pathogen tolerance to host defences. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogenZymoseptoria tritici. By performing quantitative trait locus (QTL) mapping in two crosses, we identified several genomic regions associated with tolerance to oxidative stress, including a QTL having a large effect on growth under oxidative stress. We found evidence for a significant trade-off between growth under non-stressful conditions and growth inhibition under oxidative stress. We identified a large QTL associated with this trade-off and with growth under non-stressful conditions, suggesting that differences in fungal growth could result in different sensitivities to oxidative stress. Our results suggest that genes related to fungal growth could also contribute to variation in oxidative stress tolerance among fungal strains.
Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance
Authors: Bowden Anne Ramsay , Juarez David A. Morales , Sczaniecka-Clift Matylda , Agudo Maria Martin , Lukashchuk Natalia , Thomas John C. , Jackson Stephen P.
Published: February 2020

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens usingTP53wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type andTP53knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.