bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploratory analysis of immunization records highlights decreased SARS-CoV-2 rates in individuals with recent non-COVID-19 vaccinations
Authors: Pawlowski Colin , Puranik Arjun , Bandi Hari , Venkatakrishnan AJ , Agarwal Vineet , Kennedy Richard , O’Horo John C. , Gores Gregory J. , Williams Amy W. , Halamka John , Badley Andrew D. , Soundararajan Venky
Published: July 2020

Multiple clinical studies are ongoing to assess whether existing vaccines may afford protection against SARS-CoV-2 infection through trained immunity. In this exploratory study, we analyze immunization records from 137,037 individuals who received SARS-CoV-2 PCR tests. We find that polio, Hemophilus influenzae type-B (HIB), measles-mumps-rubella (MMR), varicella, pneumococcal conjugate (PCV13), geriatric flu, and hepatitis A / hepatitis B (HepA-HepB) vaccines administered in the past 1, 2, and 5 years are associated with decreased SARS-CoV-2 infection rates, even after adjusting for geographic SARS-CoV-2 incidence and testing rates, demographics, comorbidities, and number of other vaccinations. Furthermore, age, race/ethnicity, and blood group stratified analyses reveal significantly lower SARS-CoV-2 rate among black individuals who have taken the PCV13 vaccine, with relative risk of 0.45 at the 5 year time horizon (n: 653, 95% CI: (0.32, 0.64), p-value: 6.9e-05). These findings suggest that additional pre-clinical and clinical studies are warranted to assess the protective effects of existing non-COVID-19 vaccines and explore underlying immunologic mechanisms. We note that the findings in this study are preliminary and are subject to change as more data becomes available and as further analysis is conducted.
Ocular Dominance Plasticity: Measurement Reliability and Variability
Authors: Min Seung Hyun , Gong Ling , Baldwin Alex S. , Reynaud Alexandre , He Zhifen , Zhou Jiawei , Hess Robert F.
Published: July 2020

Abstract

In the last decade, studies have shown that short-term monocular deprivation strengthens the deprived eye’s contribution to binocular vision. However, the magnitude of the change in eye dominance after monocular deprivation (i.e., the patching effect) has been found to be different between for different methods and within the same method. There are three possible explanations for the discrepancy. First, the mechanisms underlying the patching effect that are probed by different measurement tasks might exist at different neural sites. Second, test-retest variability in the measurement might have led to inconsistencies, even within the same method. Third, the patching effect itself in the same subject might fluctuate across separate days or experimental sessions. To explore these possibilities, we assessed the test-retest reliability of the three most commonly used tasks (binocular rivalry, binocular combination, and dichoptic masking) and the repeatability of the shift in eye dominance after short-term monocular deprivation for each of the task. Two variations for binocular phase combination were used, at one and many contrasts of the stimuli. Also, two variations of the dichoptic masking task was tested, in which the orientation of the mask grating was either horizontal or vertical. This makes five different measurement methods in all. We hope to resolve some of the inconsistencies reported in the literature concerning this form of visual plasticity. In this study, we also aim to recommend a measurement method that will allow us to better understand its physiological basis and the underpinning of visual disorders.
Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures
Authors: Salahudeen Ameen A. , Choi Shannon S. , Rustagi Arjun , Zhu Junjie , de la O Sean M. , Flynn Ryan A. , Margalef-Català Mar , Santos António J. M. , Ju Jihang , Batish Arpit , van Unen Vincent , Usui Tatsuya , Zheng Grace X.Y. , Edwards Caitlin E. , Wagar Lisa E. , Luca Vincent , Anchang Benedict , Nagendran Monica , Nguyen Khanh , Hart Daniel J. , Terry Jessica M. , Belgrader Phillip , Ziraldo Solongo B. , Mikkelsen Tarjei S. , Harbury Pehr B. , Glenn Jeffrey S. , Garcia K. Christopher , Davis Mark M. , Baric Ralph S. , Sabatti Chiara , Amieva Manuel R. , Blish Catherine A. , Desai Tushar J. , Kuo Calvin J.
Published: July 2020

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3Din vitrohuman distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoidKRT5+cells contained a distinctITGA6+ITGB4+mitotic population whose proliferation segregated to aTNFRSF12Ahisubfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahisubset of FACS-purified ITGA6+ITGB4+basal cells from human lung or derivative organoids.In vivo, TNFRSF12A+cells comprised ~10% of KRT5+basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facilein vitroorganoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.
Incongruent phylogenies and its implications for the study of diversification, taxonomy and genome size evolution ofRhododendron(Ericaceae)
Authors: Khan Gulzar , Nolzen Jennifer , Schepker Hartwig , Albach Dirk C.
Published: July 2020

PREMISE

RhododendronL. (Ericaceae Juss.), is the most species-rich genus of woody flowering plants with > 1000 species. Despite the interest in the genus and numerous previous phylogenetic analysis, the infrageneric classification for the genus is still debated, partly due to its huge diversity, partly due to homoplasy in key characters and partly due to incongruence between phylogenetic markers. Here, we provide a broad coverage of representative species of allRhododendronsubgenera, sections, and most subsections to resolve its infrageneric phylogeny or highlight areas of incongruence, support previous analyses of diversification patterns and establish a relationship between genome size evolution and its diversification.

METHODS

We generated sequences of two plastid (trnK andtrnL-F) and two nuclear (ITS andrpb2-i) markers for a total of 259Rhododendronspecies, and used likelihood and Bayesian statistics to analyze the data. We analyzed the markers separately to discuss and understand incongruence among the data sets and among previous studies.

RESULTS

We found that the larger a subgenus, the more strongly it is supported as monophyletic. However, the smaller subgenera pose several problems, e.g.,R. subgen.Azaleastrumconsists of two sections inferred to be polyphyletic. The main shift to higher diversification in the genus occurred in the Himalayan/SE Asian clade ofR. subgen.Hymenanthes. We found that polyploidy occurs in almost all subgenera but most polyploid species are withinR. subgen.RhododendronsectionsRhododendronandSchistanthe.

CONCLUSION

Whereas previous reports stated that genome sizes of tropical plants are lower than those of colder and temperate regions in angiosperms in general, our study provides evidence for such a shift to small genome-tropical species within a genus. Taken together, we see the merit in the recognition of the five major clades at the sub generic level but given the amount of incongruence a large amount of species cannot be confidently assigned to one of these five clades. Further, genome-wide data will be necessary to assess whether these currently unassignable taxa are independent taxa, assignable to one of the five major clades or whether they are inter-subgeneric hybrids.
Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks
Authors: Hsia Yang , Mout Rubul , Sheffler William , Edman Natasha I. , Vulovic Ivan , Park Young-Jun , Redler Rachel L. , Bick Matthew J. , Bera Asim K. , Courbet Alexis , Kang Alex , Brunette TJ , Nattermann Una , Tsai Evelyn , Saleem Ayesha , Chow Cameron M. , Ekiert Damian , Bhabha Gira , Veesler David , Baker David
Published: July 2020

A goal ofde novoprotein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric protein assemblies using these structures. We first connect designed helical repeat proteins (DHRs) to designed helical bundle proteins (HBs) to generate a large library of heterodimeric and homooligomeric building blocks; the latter have cyclic symmetries ranging from C2 to C6. All of the building blocks have repeat proteins with accessible termini, which we take advantage of in a second round of architecture guided rigid helical fusion (WORMS) to generate larger symmetric assemblies including C3 and C5 cyclic and D2 dihedral rings, a tetrahedral cage, and a 120 subunit icosahedral cage. Characterization of the structures by small angle x-ray scattering, x-ray crystallography, and cryo-electron microscopy demonstrates that the hierarchical design approach can accurately and robustly generate a wide range of macromolecular assemblies; with a diameter of 43nm, the icosahedral nanocage is the largest structurally validated designed cage to date. The computational methods and building block sets described here provide a very general route to newde novodesigned symmetric protein nanomaterials.
A small, computationally flexible network produces the phenotypic diversity of song recognition in crickets
Authors: Clemens Jan , Schöneich Stefan , Kostarakos Konstantinos , Hennig R. Matthias , Hedwig Berthold
Published: July 2020

How neural networks evolved to generate the diversity of species-specific communication signals is unknown. For receivers of the signals one hypothesis is that novel recognition phenotypes arise from parameter variation in computationally flexible feature detection networks. We test this hypothesis in crickets, where males generate and females recognize the mating songs with a species-specific pulse pattern, by investigating whether the song recognition network in the cricket brain has the computational flexibility to recognize different temporal features. Using electrophysiological recordings from the network that recognizes crucial properties of the pulse pattern on the short timescale in the cricketGryllus bimaculatus, we built a computational model that reproduces the neuronal and behavioral tuning of that species. An analysis of the model’s parameter space reveals that the network can provide all recognition phenotypes for pulse duration and pause known in crickets and even other insects. Phenotypic diversity in the model is consistent with known preference types in crickets and other insects, and arise from computations that likely evolved to increase energy efficiency and robustness of pattern recognition. The model’s parameter to phenotype mapping is degenerate—different network parameters can create similar changes in the phenotype—which likely supports evolutionary plasticity. Our study suggests that computationally flexible networks underlie the diverse pattern recognition phenotypes and we reveal network properties that constrain and support behavioral diversity.
Statistical model integrating interactions into genotype-phenotype association mapping: an application to reveal 3D-genetic basis underlying Autism
Authors: Li Qing , Cao Chen , Perera Deshan , He Jingni , Chen Xingyu , Azeem Feeha , Howe Aaron , Au Billie , Yan Jun , Long Quan
Published: July 2020

Biological interactions are prevalent in the functioning organisms. Correspondingly, statistical geneticists developed various models to identify genetic interactions through genotype-phenotype association mapping. The current standard protocols in practice test single variants or single regions (that contain multiple local variants) sequentially along the genome, followed by functional annotations that involve various aspects including interactions. The testing of genetic interactions upfront is rare in practice due to the burden of testing a huge number of combinations, which lead to the multiple-test problem and the risk of overfitting. In this work, we developed interaction-integrated linear mixed model (ILMM), a novel model that integrates aprioriknowledge into linear mixed models. ILMM enables statistical integration of genetic interactions upfront and overcomes the problems associated with combination searching.

Three dimensional (3D) genomic interactions assessed by Hi-C experiments have led to unprecedented biological discoveries. However, the contribution of 3D genomic interactions to the genetic basis of complex diseases has yet to be quantified. Using 3D interacting regions asa prioriinformation, we conducted both simulations and real data analysis to test ILMM. By applying ILMM to whole genome sequencing data for Autism Spectrum Disorders, or ASD (MSSNG) and transcriptome sequencing data (GTEx), we revealed the 3D-genetic basis of ASD and 3D-eQTLs for a substantial proportion of gene expression in brain tissues. Moreover, we have revealed a potential mechanism involving distal regulation between FOXP2 and DNMT3A conferring the risk of ASD.

Software is freely available in our GitHub:https://github.com/theLongLab/Jawamix5
Tumor-stromal IL-1β/ NFκB / ESE3 Signaling Axis Drives Pancreatic Cancer Fibrosis, Chemoresistance, and Poor Prognosis
Authors: Zhao Tiansuo , Xiao Di , Jin Fanjie , Wang Hongwei , Liu Jing , Cai Wenrun , Huang Chongbiao , Wang Xiuchao , Gao Song , Yang Shengyu , Hao Jihui
Published: July 2020

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis and pancreatic ductal adenocarcinoma (PDAC) progression. The mechanisms controlling PSC activation is not completely understood. Here we investigated the role of ESE3 (Epithelium-Specific ETS factor 3) in PSC activation. We discovered that in PDAC patients ESE3 expression was increased in PSC while decreased in tumor cells. ESE3 overexpression in PSC promoted PSC activation. Condition medium from ESE3-overexprssing PSC promotes PDAC cell migration, chemoresistance, tumor growth and fibrosis. ESE3 directly induced the transcription of α-SMA, Collagen 1 and IL-1β by binding to ESE3 binding sites on their promoters to activate PSC. On the other hand, IL-1β upregulates ESE3 in PSC through NFκB activation and ESE3 is required for PSC activation by tumor cell derived IL-1β. Clinical data showed ESE3 upregulation in PSC was positively correlated with tumor size, pTNM stage, CA19-9, CEA and CA242 level in serum. ESE3 overexpression in PSC was an independent negative prognostic factor for disease-free survival and overall survival among PDAC patients. Inhibition of the IL-1β/ ESE3 (PSC)/ IL-1β positive feedback loop represents a promising therapeutic strategy to reduce tumor fibrosis and increase chemotherapeutic efficacy in PDAC.
Treated wastewater irrigation promotes the spread of antibiotic resistance into subsoil pore-water
Authors: Kampouris Ioannis D. , Klümper Uli , Cacace Damiano , Kunze Steffen , Berendonk Thomas U.
Published: July 2020

In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory mesocosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58,tetM,qnrS,blaCTX-M-32andblaTEM), theintI1gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the mesocosms. Among the tested genes in the field study,sul1andintI1exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled mesocosm experiments verified the observed field study results: the relative abundance of several genes, includingsul1andintI1,increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs andintI1in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab mesocosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.

Graphical abstract

Highlights

TWW irrigation intensity andsul1&intI1abundance correlate in a real-scale field

ARGs &intI1increase in subsoil pore-water during TWW irrigation in mesocosms

No increase of ARGs &intI1in freshwater irrigated mesocosms

TWW irrigation does not affect the bacterial load of subsoil pore-water
The common house spider,Parasteatoda tepidariorum, maintains silk gene expression on sub-optimal diet
Authors: Miller Jeremy , Vienneau-Hathaway Jannelle , Dendev Enkhbileg , Lan Merrina , Ayoub Nadia A.
Published: July 2020

Orb-web weaving spiders and their relatives spin multiple task-specific fiber types. The unique material properties of each silk type result from differences in amino acid sequence and structure of their component proteins, primarily spidroins (spider fibrous proteins). Amino acid content and gene expression measurements of spider silks suggest some spiders change expression patterns of individual protein components in response to environmental cues. We quantified mRNA abundance of three spidroin encoding genes involved in prey capture in the common house spider,Parasteatoda tepidariorum(Theridiidae), fed different diets. After 10 days of acclimation to the lab on a diet of mealworms, spiders were split into three groups: (1) individuals were immediately dissected, (2) spiders were fed high-energy crickets, or (3) spiders were fed low-energy flies, for 1 month. All spiders gained mass during the acclimation period and cricket-fed spiders continued to gain mass, while fly fed spiders either maintained or lost mass. Using quantitative PCR, we found no significant differences in the absolute or relative abundance of dragline gene transcripts, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), among groups. In contrast, prey-wrapping minor ampullate spidroin (MiSp)gene transcripts were significantly less abundant in fly-fed than lab-acclimated spiders. However, when measured relative toactin, cricket-fed spiders showed the lowest expression ofMiSp. Our results suggest that house spiders are able to maintain silk production, even in the face of a low-quality diet.
First evidence for an aposematic function of a very common color pattern in small insects
Authors: Mora-Castro Rebeca , Alfaro-Córdoba Marcela , Hernández-Jiménez Marcela , Otárola Mauricio Fernández , Méndez-Rivera Michael , Ramírez-Morales Didier , Rodríguez-Rodríguez Carlos E. , Durán-Rodríguez Andrés , Hanson Paul E.
Published: July 2020

Many small parasitoid wasps have a black-orange-black (BOB) color pattern, which is usually present in both sexes. A likely function of this widespread pattern is aposematic (warning) coloration, but this has never been investigated. To test this hypothesis, we presented spider predators(Lyssomanes jemineus), both field-captured and lab-reared individuals, to a species with the BOB pattern and a congeneric all-black species in each of four scelionid genera (Baryconus, Chromoteleia, MacroteleiaandScelio). Each spider/wasp trial was filmed for 40 minutes under controlled conditions and three behavioral responses (detect, attack, avoid) were recorded in each of 136 trials, never using the same predator and prey more than once. In order to better understand the results obtained, two additional studies were performed. First, the reflection spectrum of the cuticle of the wasp and a theoretical visual sensibility model of the spider were used to calculate a parameter we called “absorption contrast” that allowed us to compare the perception contrast between black and orange in each wasp genus as viewed by the spider. Second, acute toxicity trials with the water flea,Daphnia magna, were performed to determine toxicity differences between BOB and non-BOB wasps. By combining the results from the three types of experiments, together with a statistical analysis, we confirmed that BOB color pattern plays an aposematic role.
Modulation of proteasome activity by curcumin and didemethylcurcumin
Authors: Khan Tapan K. , You Youngki , Nelson Thomas J. , Kundu Subrata , Pramanik Saroj , Das Joydip
Published: July 2020

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways and known to modulate PKC activities. The therapeutic significance of curcumin is often considered to reside in its anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. However, recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. In this study, we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin), CUIII modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in relative proteasome activity increase at nanomolar concentrations. Also, curcumin was more effective than CUIII in relative proteasome activity inhibition at micromolar concentration. The docking study was conducted on the 20S proteasome catalytic subunit. Estimated Kd values for curcumin and didemethylcurcumin are 0.0054μM and 1.3167μM, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compare to CUIII. The small size of CUIII makes its dock to the narrow cavity of the active site, but the binding interaction is not strong compare to curcumin. This study suggests that curcumin and its didemethyl derivative can be used to modulate proteasome activity. This communication implicates the reason why curcumin and its didemethyl derivative can be used to two different disease mechanisms, neurodegeneration, and cancer.
A novel ultrasound-guided mouse model of sudden cardiac arrest
Authors: Rutledge Cody A. , Chiba Takuto , Redding Kevin , Dezfulian Cameron , Sims-Lucas Sunder , Kaufman Brett A.
Published: July 2020

Aim

Mouse models of sudden cardiac arrest are limited by challenges with surgical technique and reliable venous access. To overcome this limitation, we sought to develop a simplified method in the mouse that uses ultrasound-guided injection of potassium chloride directly into the heart.

Methods

Potassium chloride was delivered directly into the left ventricular cavity under ultrasound guidance in intubated mice, resulting in immediate asystole. Mice were resuscitated with injection of epinephrine and manual chest compressions and evaluated for survival, body temperature, cardiac function, kidney damage, and diffuse tissue injury.

Results

The direct injection sudden cardiac arrest model causes rapid asystole with high surgical survival rates and low surgical duration. Sudden cardiac arrest mice with 8-min of asystole have significant cardiac dysfunction at 24 hours and high lethality within the first seven days, where after cardiac function begins to improve. Sudden cardiac arrest mice have secondary organ damage, including significant kidney injury, but no clear evidence of neurologic dysfunction.

Conclusions

Ultrasound-guided direct injection of potassium chloride allows for rapid and reliable cardiac arrest in the mouse that mirrors human pathology. This technique lowers the barriers to entry for adoption of the mouse model of sudden cardiac arrest, which will improve investigators’ ability to study the mechanisms underlying post-arrest changes.
Genome wide identification of QTL associated with yield and yield components in two popular wheat cultivars TAM 111 and TAM 112
Authors: Yang Yan , Dhakal Smit , Chu Chenggen , Wang Shichen , Xue Qingwu , Rudd Jackie C. , Ibrahim Amir M.H. , Jessup Kirk , Baker Jason , Fuentealba Maria Pilar , Devkota Ravindra , Baker Shannon , Johnson Charles D. , Metz Richard , Liu Shuyu
Published: July 2020

Two drought-tolerant wheat cultivars, ‘TAM 111’ and ‘TAM 112’, have been widely grown in the Southern Great Plains of the U.S. and used as parents in many wheat breeding programs worldwide. This study aimed to reveal genetic control of yield and yield components in the two cultivars under both dryland and irrigated conditions. A mapping population containing 124 F5:7recombinant inbred lines (RILs) was developed from the cross of TAM 112/TAM 111. A set of 5,948 SNPs from the wheat 90K iSelect array and double digest restriction-site associated DNA sequencing was used to construct high-density genetic maps. Data for yield and yield components were obtained from 11 environments. QTL analyses were performed based on 11 individual environments, across all environments, within and across mega-environments. Thirty-six unique consistent QTL regions were distributed on 13 chromosomes including 1A, 1B, 1D, 2A, 2D, 3D, 4B, 4D, 6A, 6B, 6D, 7B, and 7D. Ten unique QTL with pleiotropic effects were identified on four chromosomes and eight were in common with the consistent QTL. These QTL increased dry biomass grain yield by 16.3 g m−2, plot yield by 28.1 g m−2, kernels spike−1by 0.7, spikes m−2by 14.8, thousand kernel weight by 0.9 g with favorable alleles from either parent. TAM 112 alleles mainly increased spikes m−2and thousand kernel weight while TMA 111 alleles increased kernels spike−1, harvest index and grain yield. The saturated genetic map and markers linked to significant QTL from this study will be very useful in developing high throughput genotyping markers for tracking the desirable haplotypes of these important yield-related traits in popular parental cultivars.
Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeonHaloferax volcanii
Authors: Turkowyd Bartosz , Schreiber Sandra , Wörtz Julia , Segal Ella Shtifman , Mevarech Moshe , Duggin Iain G. , Marchfelder Anita , Endesfelder Ulrike
Published: July 2020

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology andin vivobiochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology.

Here we have developed a reliable protocol for the study of the archaeonHaloferax volcanii. We have generated an autofluorescence-freeH. volcaniistrain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimalH. volcaniicultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in livingH. volcaniicells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.
A-to-I RNA editing in SARS-COV-2: real or artifact?
Authors: Picardi Ernesto , Mansi Luigi , Pesole Graziano
Published: July 2020

ADAR1-mediated deamination of adenosines in long double stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2 infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions and are, in the majority of cases, nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.
TheE. coliCas1/2 endonuclease complex reduces CRISPR/Cascade guide array stability
Authors: Ye Zhixia , Lebeau Juliana , Moreb Eirik A. , Menacho-Melgar Romel , Lynch Michael D.
Published: July 2020

CRISPR based interference has become common in various applications from genetic circuits to dynamic metabolic control. InE. colithe native CRISPR Cascade system can be utilized for silencing by deletion of thecas3nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of protospacer sequences from guide arrays utilized for dynamic silencing. We report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. Acas1deletion improves guide array stability. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of protospacers from guide arrays. Subsequently, mutant guide arrays can be amplified through selection. Replacing a constitutive promoter driving Cascade complex expression with a tightly controlled inducible promoter improves guide array stability, while minimizing leaky gene silencing.

Highlights

Cas1/2 endonuclease complex mediates CRISPR/Cascade protospacer loss inE. coli

Tightly controlled Cascade operon expression increases guide array stability.
Zn2+influx activates ERK and Akt signaling pathways through a common mechanism
Authors: Anson Kelsie J. , Corbet Giulia A. , Palmer Amy E.
Published: July 2020

Zinc (Zn2+) is an essential metal in biology and its bioavailability is highly regulated. Many cell types exhibit fluctuations in Zn2+that appear to play an important role in cellular function. However, the detailed molecular mechanisms by which Zn2+dynamics influence cell physiology remain enigmatic. Here, we use a combination of fluorescent biosensors and cell perturbations to define how changes in intracellular Zn2+impact kinase signaling pathways. By simultaneously monitoring Zn2+dynamics and kinase activity in individual cells, we quantify changes in labile Zn2+and directly correlate changes in Zn2+with ERK and Akt activity. Under our experimental conditions, Zn2+fluctuations are not toxic and do not activate stress-dependent kinase signaling. We demonstrate that while Zn2+can non-specifically inhibit phosphatases leading to sustained kinase activation, ERK and Akt are predominantly activated via upstream signaling, and through a common node via Ras. We provide a framework for quantification of Zn2+fluctuations and correlate these fluctuations with signaling events in single cells to shed light on the role that Zn2+dynamics play in healthy cell signaling.

Significance Statement

While zinc (Zn2+) is a vital ion for cell function and human health, little is known about the role it plays in regulating cell signaling. Here, we use fluorescent tools to study the interaction between Zn2+and cell signaling pathways that play a role in cell growth and proliferation. Importantly, we use small, non-toxic Zn2+concentrations to ensure that our Zn2+changes are closer to what cells would experience in the body and not stress-inducing. We also demonstrate that these signaling changes are driven by Ras activation, which contradicts one of the major hypotheses in the field. Our sensors shed light on how cells respond to a very important micronutrient in real time.
Widespread non-apoptotic activation ofDrosophilaCaspase-2/9 limits JNK signaling, macrophage proliferation, and growth of wound-like tumors
Authors: Xu Derek Cui , Yamada Kenneth M. , Baena-Lopez Luis Alberto
Published: July 2020

Resistance to apoptosis due to caspase deregulation is considered one of the main hallmarks of cancer. However, the discovery of novel non-apoptotic caspase functions has revealed unknown intricacies about the interplay between these enzymes and tumor progression. To investigate this biological problem, we capitalized on aDrosophilatumor model highly relevant for humans that relies on the concomitant upregulation of EGFR and the JAK/STAT signaling pathway. Our results indicate that widespread non-apoptotic activation of initiator caspases limits JNK signaling and facilitates cell fate commitment in these tumors, thus preventing the overgrowth and exacerbation of malignant features. Intriguingly, these caspase functions are strongly linked to the ability of these enzymes to control the recruitment and subsequent proliferationin situof macrophage-like cells on the tumor. These findings assign novel tumor-suppressor activities to caspases independent of apoptosis, while providing highly relevant molecular details to understanding their diverse contribution during tumor progression.
Polycomb-mediated Genome Architecture Enables Long-range Spreading of H3K27 methylation
Authors: Kraft Katerina , Yost Kathryn E. , Murphy Sedona , Magg Andreas , Long Yicheng , Corces M.Ryan , Granja Jeffrey M. , Mundlos Stefan , Cech Thomas R. , Boettiger Alistair , Chang Howard Y.
Published: July 2020

Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction1-5. This process is essential for embryonic stem cell (ESCs) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spread of H3K27me3 on the linear genome6. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remains unclear7-10. Here, we apply H3K27me3 HiChIP11-13, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture14to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells7-10. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes, such asHmx1, Wnt6andHoxa. Genetic deletion of H3K27me3 loop anchors revealed a coupling of Polycomb-associated genome architecture and H3K27me3 deposition evidenced by disruption of spatial contact between distant loci and altered H3K27me3in cis, both locally and megabases away on the same chromosome. Further, we find that global alterations in PRC2 occupancy resulting from an EZH2 mutant15selectively deficient in RNA binding is accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 acts as a “genomic wormhole”, using RNA binding to enhance long range chromosome folding and H3K27me3 spreading. Additionally, developmental gene loci have novel roles in Polycomb spreading, emerging as important architectural elements of the epigenome.
Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals
Authors: Doddapaneni Harsha , Cregeen Sara Javornik , Sucgang Richard , Meng Qingchang , Qin Xiang , Avadhanula Vasanthi , Chao Hsu , Menon Vipin , Nicholson Erin , Henke David , Piedra Felipe-Andres , Rajan Anubama , Momin Zeineen , Kottapalli Kavya , Hoffman Kristi L. , Sedlazeck Fritz J. , Metcalf Ginger , Piedra Pedro A. , Muzny Donna M. , Petrosino Joseph F. , Gibbs Richard A.
Published: July 2020

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.
Massive-scale biological activity-based modeling identifies novel antiviral leads against SARS-CoV-2
Authors: Huang Ruili , Xu Miao , Zhu Hu , Chen Catherine Z. , Lee Emily M. , He Shihua , Shamim Khalida , Bougie Danielle , Huang Wenwei , Hall Mathew D. , Lo Donald , Simeonov Anton , Austin Christopher P. , Qiu Xiangguo , Tang Hengli , Zheng Wei
Published: July 2020

The recent global pandemic caused by the new coronavirus SARS-CoV-2 presents an urgent need for new therapeutic candidates. While the importance of traditionalin silicoapproaches such as QSAR in such efforts in unquestionable, these models fundamentally rely on structural similarity to infer biological activity and are thus prone to becoming trapped in the very nearby chemical spaces of already known ligands. For novel and unprecedented threats such as COVID-19 much faster and efficient paradigms must be devised to accelerate the identification of new chemical classes for rapid drug development. Here we report the development of a new biological activity-based modeling (BABM) approach that builds on the hypothesis that compounds with similar activity patterns tend to share similar targets or mechanisms of action. In BABM, compound activity profiles established on massive scale across multiple assays are used as signatures to predict compound activity in a new assay or against a new target. We first trained and validated this approach by identifying new antiviral lead candidates for Zika and Ebola based on data from ~0.5 million compounds screened against ~2,000 assays. BABM models were then applied to predict ~300 compounds not previously reported to have activity for SARS-CoV-2, which were then tested in a live virus assay with high (>30%) hit rates. The most potent compounds showed antiviral activities in the nanomolar range. These potent confirmed compounds have the potential to be further developed in novel chemical space into new anti-SARS-CoV-2 therapies. These results demonstrate unprecedented ability using BABM to predict novel structures as chemical leads significantly beyond traditional methods, and its application in rapid drug discovery response in a global public health crisis.
Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mproand cathepsin L
Authors: Sacco Michael Dominic , Ma Chunlong , Lagarias Panagiotis , Gao Ang , Townsend Julia Alma , Meng Xiangzhi , Dube Peter , Zhang Xiujun , Hu Yanmei , Kitamura Naoya , Hurst Brett , Tarbet Bart , Marty Michael Thomas , Kolocouris Antonios , Xiang Yan , Chen Yu , Wang Jun
Published: July 2020

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mproinhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several Mproinhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mproin complex with calpain inhibitors II and XII, and three analogues ofGC-376, one of the most potent Mproinhibitorsin vitro. The structure of Mprowith calpain inhibitor II confirmed the S1 pocket of Mprocan accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1’ pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1’ pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of threeGC-376analoguesUAWJ246,UAWJ247, andUAWJ248provide insight to the sidechain preference of the S1’, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the α-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mproinhibitors as SARS-CoV-2 antivirals.
Transitions in symbiosis: evidence for environmental acquisition & social transmission within a clade of heritable symbionts
Authors: Drew Georgia C , Budge Giles E , Frost Crystal L , Neumann Peter , Siozios Stefanos , Yañez Orlando , Hurst Gregory DD
Published: July 2020

A dynamic continuum exists from free-living environmental microbes to strict host associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in the modes by which symbioses form is lacking.Arsenophonusis a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genusArsenophonusand the Western honey bee. We then present multiple lines of evidence that this symbiont deviates from a heritable model of transmission. Field sampling uncovered marked spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and individuals in the laboratory. Fluorescent in-situ hybridization showedArsenophonuslocalised in the gut, and detection of the bacterium was rare in screens of early honey bee life stages. We directly show horizontal transmission ofArsenophonusbetween bees under varying social conditions. We conclude that honey bees acquireArsenophonusthrough a combination of environmental exposure and social contacts. Together these findings uncover a key link in theArsenophonusclades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.
Oncogenic Gene Fusions in Non-Neoplastic Precursors as Evidence that Bacterial Infection Initiates Prostate Cancer
Authors: Shrestha Eva , Coulter Jonathan B. , Guzman William , Ozbek Busra , Mummert Luke , Ernst Sarah E. , Maynard Janielle P. , Meeker Alan K. , Heaphy Christopher M. , Haffner Michael C. , De Marzo Angelo M. , Sfanos Karen S.
Published: July 2020

Prostate adenocarcinoma is the second most commonly diagnosed cancer in men worldwide and the initiating factors are unknown. Oncogenic TMPRSS2:ERG (ERG+) gene fusions are facilitated by DNA breaks and occur in up to 50% of prostate cancers1,2. Infection-driven inflammation is implicated in the formation of ERG+ fusions3, and we hypothesized that these fusions initiate in early inflammation-associated prostate cancer precursor lesions, such as proliferative inflammatory atrophy (PIA), prior to cancer development. We investigated whether bacterial prostatitis is associated with ERG+ precancerous lesions in unique cases with active bacterial infections at time of radical prostatectomy. We identified a high frequency of ERG+ non-neoplastic-appearing glands in these cases, including ERG+ PIA transitioning to early invasive cancer. We verified TMPRSS2:ERG genomic rearrangements in precursor lesions using tri-color fluorescencein situhybridization. Identification of rearrangement patterns combined with whole prostate mapping in 3 dimensions confirmed multiple (up to 8) distinct ERG+ precancerous lesions in infected cases. Finally, we identified the pathogen-derived genotoxin colibactin as a potential source of DNA breaks in clinical cases as well as cultured prostate cells. Overall, we provide evidence that bacterial infections initiate driver gene alterations in prostate cancer. Furthermore, infection-induced ERG+ fusions are an early alteration in the carcinogenic process and PIA may serve as a direct precursor to prostate cancer.