bluuebunny/crossref_metadata_2025 huggingface dataset:
Unilateral Perforant Path Transection Does Not Alter Lateral Entorhinal Cortical or Hippocampal CA3 Arc Expression
Authors: Cooper Tara L. , Thompson John J. , Turner Sean M. , Watson Cory , Lubke Katelyn N. , Logan Carly N. , Maurer Andrew P. , Burke Sara N.
Published: April 2022

It is well established that degradation of perforant path fibers is associated with age-related cognitive dysfunction and CA3 hyperactivity. Whether this fiber loss triggers a cascade of other functional changes within the hippocampus circuit has not been causatively established, however. Thus, the current study evaluated the effect of perforant path fiber loss on neuronal activity in CA3 and layer II of the lateral entorhinal cortex (LEC) in relation to mnemonic similarity task performance. Expression of the immediate early gene Arc was quantified in rats that received a unilateral right hemisphere transection of the perforant path or sham surgery that cut the cortex but left the fibers intact. Behavior-related expression of Arc mRNA was measured to test the hypothesis that fiber loss leads to elevated activation of CA3 and LEC neurons, as previously observed in aged rats that were impaired on the mnemonic similarity task. Transection of perforant path fibers, which has previously been shown to lead to a decline in mnemonic similarity task performance, did not alter Arc expression. Arc expression in CA3, however, was correlated with task performance on the more difficult discrimination trials across both surgical groups. These observations further support a link between CA3 activity and mnemonic similarity task performance but suggest the reduced input from the entorhinal cortex to the hippocampus, as observed in old age, does not causatively elevate CA3 activity.
A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish
Authors: Ye Ding , Tu Yi-Xuan , Wang Houpeng , He Mudan , Wang Yaqing , Chen Zhengfang , Chen Zhen-Xia , Sun Yonghua
Published: April 2022

Zebrafish (Danio rerio) has been used as a promising animal model to study gonadal development and gametogenesis. Although previous studies have identified critical molecules participating in zebrafish gonad differentiation, a landscape view of the biological processes involved in this process is still lacking. Here we isolated intact zebrafish differentiating gonads, at 25 days post-fertilization (dpf) and 30 dpf, and conducted RNA-seq analysis between the juvenile gonads that tended to develop into ovaries or testes. Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the level of biological process. During ovary differentiation, the biological processes related to metabolic activities in production of energy and maternal substances, RNA degradation, and DNA repair were enriched. During testis differentiation, the biological processes related to cell proliferation, differentiation, and morphogenesis were enriched, with a total of 15 signaling pathways. Notably, we reveal that the immune-related processes are extensively involved in the regulation of testis development. Overall, this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.
The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication
Authors: Zhang Hongbo , Domma Anthony J. , Goodrum Felicia D. , Moorman Nathaniel J. , Kamil Jeremy P.
Published: April 2022

The serine/threonine protein kinase AKT is a critical mediator of growth factor signaling that broadly impacts mRNA translation, metabolism, programmed cell death and stress responses. Human cytomegalovirus (HCMV), a large dsDNA virus in the herpesvirus family, dramatically remodels host cell signaling pathways during lytic infection. Here, we show that AKT accumulates in an inactive form during HCMV infection, as indicated by hypo-phosphorylation at Thr308 and Ser473, as well as nuclear localization of FOXO3a. Moreover, we find that expression of constitutively active myristoylated AKT (myr-AKT) causes a 1-log defect in viral replication, accompanied by defects in viral DNA synthesis and viral late gene expression. These findings suggest that HCMV shutoff of AKT is not merely incidental to viral factors such as pUL38 that maintain high levels of mTORC1 activity independently of extracellular growth factor signals. Short interfering RNA knockdown of FOXO3a, but not FOXO1, phenocopied the replication defects seen during expression of myr-AKT, corroborating a role for FOXO3a during HCMV infection. Accordingly, a chimeric FOXO3a-estrogen receptor fusion protein, whose nuclear localization is regulated by the small molecule ligand 4-hydroxytamoxifen instead of AKT activity, rescues virtually all the replication defects induced by myr-AKT expression. Taken together, our results argue that HCMV dampens AKT signaling to promote nuclear localization of FOXO transcription factors, which are required for efficient viral replication.

IMPORTANCE

Evidence from a diverse herpesvirus infection models suggests that the PI3K / AKT signaling pathway suppresses reactivation from latency, and that inactivation of the pathway stimulates viral lytic replication. Here, we show that AKT accumulates in an inactive state during HCMV infection of lytically permissive cells while the presence of constitutive AKT activity causes substantial viral replication defects. Although AKT phosphorylates a diverse array of cellular substrates, we identify an important role for the Forkhead box class O transcription factors. Our findings show that when FoxO3a nuclear localization is decoupled from its negative regulation by AKT, the viral replication defects observed in the presence of constitutively active AKT are reversed. Collectively, our results reveal that HCMV inactivates AKT to promote the nuclear localization of FOXO transcription factors, which strongly implies that FOXOs play critical roles in transactivating cellular and/or viral genes during infection.
Novel CSF biomarkers of GLUT1 deficiency syndrome: implications beyond the brain’s energy deficit
Authors: Peters Tessa M.A. , Merx Jona , Kooijman Pieter C. , Noga Marek , de Boer Siebolt , van Gemert Loes A. , Salden Guido , Engelke Udo F.H. , Lefeber Dirk J. , van Outersterp Rianne E. , Berden Giel , Boltje Thomas J. , Artuch Rafael , Pías Leticia , García-Cazorla Ángeles , Barić Ivo , Thöny Beat , Oomens Jos , Martens Jonathan , Wevers Ron A. , Verbeek Marcel M. , Coene Karlien L.M. , Willemsen Michèl A.A.P.
Published: April 2022

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic CSF profiles from 11 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from O-glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O-glucosylation. Since many proteins are O-glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake.
mRNA-1273 but not BNT162b2 induces antibodies against polyethylene glycol (PEG) contained in mRNA-based vaccine formulations
Authors: Carreño Juan Manuel , Singh Gagandeep , Tcheou Johnstone , Srivastava Komal , Gleason Charles , Muramatsu Hiromi , Desai Parnavi , Aberg Judith A. , Miller Rachel L. ,   PARIS study group, Pardi Norbert , Simon Viviana , Krammer Florian
Published: April 2022

Two messenger RNA (mRNA)-based vaccines are widely used globally to prevent coronavirus disease 2019 (COVID-19). Both vaccine formulations contain PEGylated lipids in their composition, in the form of polyethylene glycol [PEG] 2000 dimyristoyl glycerol for mRNA-1273, and 2 [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide for BNT162b2. It is known that some PEGylated drugs and products for human use that contain PEG, are capable of eliciting immune responses, leading to detectable PEG-specific antibodies in serum. In this study, we determined if any of the components of mRNA-1273 or BNT162b2 formulations elicited PEG-specific antibody responses in serum by enzyme linked immunosorbent assay (ELISA). We detected an increase in the reactivity to mRNA vaccine formulations in mRNA-1273 but not BNT162b2 vaccinees’ sera in a prime-boost dependent manner. Furthermore, we observed the same pattern of reactivity against irrelevant lipid nanoparticles from an influenza virus mRNA formulation and found that the reactivity of such antibodies correlated well with antibody levels against high and low molecular weight PEG. Using sera from participants selected based on the vaccine-associated side effects experienced after vaccination, including delayed onset, injection site or severe allergic reactions, we found no obvious association between PEG antibodies and adverse reactions. Overall, our data shows a differential induction of anti-PEG antibodies by mRNA-1273 and BNT162b2. The clinical relevance of PEG reactive antibodies induced by administration of the mRNA-1273 vaccine, and the potential interaction of these antibodies with other PEGylated drugs remains to be explored.
Stepwise origin and evolution of a transcriptional activator and repressor system integrating nutrient signaling in plants
Authors: Jamsheer K Muhammed , Gazara Rajesh Kumar , Jindal Sunita , Kumar Manoj
Published: April 2022

Plants possess a unique transcriptional regulatory system in which two related MYB-related transcription factors (TFs) coordinate gene expression according to phosphate (Pi) and nitrogen (N) availability. The Phosphorus Starvation Response (PSR) type TFs are transcriptional activators integrating the cellular Pi sensing machinery and gene regulation majorly under Pi starvation. The Hypersensitivity To Low Pi-Elicited Primary Root Shortening (HRS) type TFs are transcriptional repressors integrating the Pi and N availability signals through different feedback loops. They are highly connected through multiple signaling loops to finetune the transcriptional responses according to nutrient availability. Molecular functions of these TFs are fairly uncovered in model systems; however, how plants evolved this activator-repressor system is currently unknown. In this study, using sensitive evolutionary analysis, we identified a stepwise origin of the PSR-HRS regulatory system in plants. The PSR TFs were originated before the split of Prasinodermophyta and Chlorophyta. The HRS TFs were originated later in the Streptrophycean algae. We also identified the asymmetric expansion of this TF repertoire in land plants majorly shaped by genome duplication and triplication events. The phylogenetic reconstruction coupled with motif analysis revealed that the origin of the specific accessory motifs is a major contributing factor in the functional divergence which led to the evolution of different sub-families preceding the angiosperm radiation. The spatiotemporal gene expression analysis in different developmental stages and nutrient availability conditions in angiosperms identified a critical role of expression divergence in shaping the functions of these TF families which is essential for adaptive plasticity of plants.
Chlamydia trachomatis effector Dre1 interacts with dynactin to reposition host organelles during infection
Authors: Sherry Jessica , Dolat Lee , McMahon Eleanor , Swaney Danielle L. , Bastidas Robert J. , Johnson Jeffrey R. , Valdivia Raphael H. , Krogan Nevan J. , Elwell Cherilyn A. , Engel Joanne N.
Published: April 2022

Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a specialized membrane-bound compartment, called the inclusion. Chlamydia species express a unique class of effectors, Incs, which are translocated from the bacteria by a Type III secretion system and are inserted into the inclusion membrane where they modulate the host-bacterium interface. C. trachomatis repositions specific host organelles during infection to acquire nutrients and evade host cell surveillance, however the bacterial and host proteins controlling these processes are largely unknown. Here, we identify an interaction between the host dynactin complex and the C. trachomatis Inc CT192 (CTL0444), hereafter named Dre1 for Dynactin Recruiting Effector 1. We show that dynactin is recruited to the inclusion in a Dre1-dependent manner and that loss of Dre1 diminishes the recruitment of specific host organelles, including the centrosome, mitotic spindle, and Golgi apparatus to the inclusion. Inactivation of Dre1 results in decreased C. trachomatis fitness in cell-based assays and in a mouse model of infection. By targeting particular functions of the versatile host dynactin complex, Dre1 facilitates re-arrangement of certain organelles around the growing inclusion. Our work highlights how C. trachomatis employs a single effector to evoke specific, large-scale changes in host cell organization that establish an intracellular replicative niche without globally inhibiting host cellular function.
Performance Comparison Of Agilent New SureSelect All Exon v8 Probes With v7 Probes For Exome Sequencing
Authors: Belova Vera , Shmitko Anna , Pavlova Anna , Afasizhev Robert , Cheranev Valery , Tabanakova Anastasia , Ponikarovskaya Natalya , Rebrikov Denis , Korostin Dmitriy
Published: April 2022

Exome sequencing may become routine in health care it increases the chance of pinpointing the genetic cause of an individual patient’s condition and thus making an accurate diagnosis. It is important for facilities providing genetic services to keep track of changes in the technology of exome capture in order to maximize throughput while reducing cost per sample. In this study, we focused on comparing the newly released exome probe set Agilent SureSelect Human All Exon v8 and the previous probe set v7. In preparation for higher throughput of exome sequencing using the DNBSEQ-G400, we evaluated target design, coverage statistics, and variants across these two different exome capture products. Although the target size of the v8 design has not changed much compared to the v7 design (35.24 Mb vs 35.8 Mb), the v8 probe design allows you to call more of SNVs (+3.06%) and indels (+8.49%) with the same number of raw reads per sample on the common target regions (34.84 Mb). Our results suggest that the new Agilent v8 probe set for exome sequencing yields better data quality than the current Agilent v7 set.
Parallel lipid-carried epitope pillars enhance stimulation of chimeric antigen receptor T cells
Authors: Tong Jiao , Lai Hanyu , Wang Jingxia , Zhu Chenxi , Mai Xinjia , Zhou Dapeng
Published: April 2022

Chimeric antigen receptor T cells are genetically engineered to express a specific T cell receptor of interest, such as to target a cancer-specific antigen. Signaling events in chimeric antigen receptor T cells are essential for their proliferation, survival, and function. To achieve optimal antitumor efficacy, signaling motifs as well as the structure of the transmembrane domain of the chimeric antigen receptor have to be carefully designed. However, it remains unclear whether the arrangement, and therefore the movement and elasticity, of tumor antigens influence their stimulation of chimeric antigen receptor T cells. Here we show that MUC1 molecules tethered to a glycosylphosphatidylinositol anchor drastically increased its stimulation of chimeric antigen receptor T cells. In particular, MUC1 molecules containing one tandem repeat sequence showed significantly greater stimulatory activity than five tandem repeat sequences. Thus glycosylphosphatidylinositol-anchored, tumor antigen epitope pillars in parallel are attractive targets for the design of chimeric antigen receptor T cells. These novel findings, which we propose as the parallel lipid-carried epitope model, emphasize the importance of epitope arrangement in selecting highly effective chimeric antigen receptor T cells, potentially revolutionizing the applicability of this therapy. Furthermore, our data have implications for the necessity of methodologies to measure the elasticity, movement, and density of antigen pillars in parallel, as key tools to guide future chimeric antigen receptor T cell therapy.
Comparative Analysis of rRNA removal methods for RNA-seq Differential Expression in Halophilic Archaea
Authors: Martinez-Pastor Mar , Sakrikar Saaz , Rodriguez Deyra N. , Schmid Amy K.
Published: April 2022

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high ratio rRNA:mRNA (80-90%: ∼10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA and therefore requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here we show that after the discontinuation of the previous version of RiboZero (Illumina) that was useful to partially deplete rRNA from halophilic archaea, archaeal transcriptomics studies have experienced a standstill. To overcome this limitation, here we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper we provide insights to the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.
Attentional rhythmic blink: Theta/Alpha balance in neural oscillations determines the rhythmicity in visual sampling
Authors: Kawashima Tomoya , Hayashi Masamichi J , Amano Kaoru
Published: April 2022

Brain oscillations in the theta (3–7 Hz) and alpha (7–13 Hz) bands are implicated in visual perception and attention. We show that in an attentional blink paradigm, where the task requires detecting two targets presented in rapid succession, perceptual performance varied with the rhythms at these two frequencies, which we name attentional rhythmic blink. In the absence of distractors, second target detection performance fluctuated at the theta rhythm, but the fluctuation frequency shifted toward alpha rhythm when distractors were interspersed with the targets. We further show, in magnetoencephalography experiments, that a change in the dominant frequency of ongoing neural oscillations accompanied those in perceptual performance, with the parietal theta being more pronounced in the no-distractor and the occipital alpha in the distractor conditions, respectively. We propose that perceptual rhythms may depend on the power balance between ongoing neural oscillations, determined by the task-specific demand.
Echtvar: Compressed variant representation for rapid annotation and filtering of SNPs and indels
Authors: Pedersen Brent S. , de Ridder Jeroen
Published: April 2022

Germline and somatic variants within an individual or cohort are interpreted with information from large cohorts. Annotation with this information becomes a computational bottleneck as population sets grow to terabytes of data. Here, we introduce echtvar, which efficiently encodes population variants and annotation fields into a compressed archive that can be used for rapid variant annotation and filtering. Most variants, including position and alleles are encoded into 32-bits–half the size of previous encoding schemes and at least 4 times smaller than a naive encoding. The annotations, stored separately, are also encoded and compressed. We show that echtvar is faster and uses less space than existing tools and that it can effectively reduce the number of candidate variants. We give examples on germ-line and somatic variants to document how echtvar can facilitate exploratory data analysis on genetic variants. Echtvar is available at https://github.com/brentp/echtvar under an MIT license.
1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm–egg fusion
Authors: Lu Yonggang , Shimada Kentaro , Zhang Jingjing , Ogawa Yo , Tang Shaogeng , Noda Taichi , Shibuya Hiroki , Ikawa Masahito
Published: April 2022

Sperm acrosomal membrane proteins, such as IZUMO1 and SPACA6, play an essential role in mammalian sperm–egg fusion. How their biosynthesis is regulated during spermiogenesis has largely remained unknown. Here, we show that the 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik encodes a type-II transmembrane protein that is expressed in early spermatids but not in mature spermatozoa. 1700029I15Rik is associated with proteins involved in N-glycosylation, disulfide isomerisation, and ER– Golgi trafficking, suggesting its involvement in nascent protein processing. 1700029I15Rik knockout testis has a normal level of sperm plasma membrane proteins, but decreased expression of multiple acrosomal membrane proteins. The knockout sperm exhibit elevated ubiquitinated proteins and upregulated ER-associated degradation; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway in spermiogenesis for the assembly of acrosomal membrane proteins.

Significance Statement

In sexually reproducing species, life begins with the fusion between a sperm and an egg. Multiple sperm acrosomal membrane proteins have been reported indispensable for sperm–egg fusion in mammals, yet the mechanism underlying their biosynthesis remains unknown. The present study demonstrates the existence of a 1700029I15Rik-mediated pathway specifically coordinating the processing and assembly of acrosomal membrane proteins. It represents an intriguing paradigm where the biosynthesis of proteins destined for various subcellular compartments might be orchestrated in a spatiotemporal manner. Given 1700029I15Rik is highly conserved in human, our findings provide potential insights into the aetiology of idiopathic male infertility and the development of a novel contraceptive approach involving molecular interventions in the maturation of gamete fusion-required acrosomal proteins.
Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells
Authors: Jaeger Morgane , Anastasio Amandine , Brustlein Sophie , Vincentelli Renaud , Durbesson Fabien , Char Rémy , Boussand Maud , Lechelon Mathias , Argüello Rafael J. , Marguet Didier , He Hai-Tao , Lasserre Rémi
Published: April 2022

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTe) system, a recombinant optogenetics-based molecular tool targeting the T Cell Receptor (TCR). The LiTe system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation patterns. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTe system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTe system provides new opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with a high spatiotemporal control.
Structural Characterization of L-Galactose Dehydrogenase: A Key Enzyme for Vitamin C Biosynthesis
Authors: Vargas Jhon A. , Leonardo Diego A. , Pereira Humberto D’Muniz , Lopes Adriana R. , Rodriguez Hicler N. , Cobos Marianela , Marapara Jorge L. , Castro Juan C. , Garratt Richard C.
Published: April 2022

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.
A high-efficiency scar-free genome editing toolkit for Acinetobacter baumannii
Authors: de Dios Rubén , Gadar Kavita , McCarthy Ronan R
Published: April 2022

Structured synopsis

Background

The current mutagenesis tools for Acinetobacter baumannii leave selection markers or residual sequences behind, or involve tedious counterselection and screening steps. Furthermore, they are usually adapted for model strains, rather than to multidrug resistant (MDR) clinical isolates.

Objectives

To develop a scar-free genome editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075.

Methods

We prove the efficiency of our adapted genome editing system by deleting the multidrug efflux pumps craA and cmlA5, as well as curing plasmid p1AB5075. We then characterised the antibiotic sensitivity phenotype of the mutants compared to the wild type for chloramphenicol, tobramycin and amikacin by disc diffusion assays and determined their minimum inhibitory concentration for each strain.

Results

We successfully adapted the genome editing protocol to A. baumannii AB5075, achieving a double recombination frequency close to 100% and securing the construction of a mutant within 10 work days. Furthermore, we show that the ΔcraA has a strong sensitivity to chloramphenicol, tobramycin and amikacin, whereas the ΔcmlA5 mutant does not show a significant decrease in viability for the antibiotics tested. On the other hand, the removal of p1AB5075 produced an increased sensitivity to tobramycin and amikacin.

Conclusion

We have adapted a highly efficient genome editing tool for A. baumannii and proved that craA has a broader substrate range than previously thought. On the other hand, whereas cmlA5 is annotated as a chloramphenicol efflux pump and is encoded within an aminoglycoside resistance island, it does not provide resistance to any of those compounds.
Characterization of enterovirus D68 infection in four nonhuman primate species
Authors: Dearing Justin , Conte Abigail , Brooks Catherine , Zimina Anna , Rivas Rhiannon , Birch Sharla M. , Adney Danielle R. , Li Maggie , Rascon Adriana , Belov George A. , Pekosz Andrew , Vermillion Meghan S.
Published: April 2022

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host specific factors that predict disease severity and progression to the neurologic syndrome. Published animal models of EV-D68 infection to date have been limited to mice, cotton rat and ferrets, and investigation of the susceptibility of nonhuman primate (NHP) species to contemporary EV-D68 isolates has not yet been reported. In this study, we challenged juvenile NHPs – cynomolgus macaques, rhesus macaques, pigtailed macaques, and African green monkeys – with one of five different 2014 or 2018 EV-D68 isolates by the respiratory route. Animals were monitored for clinical respiratory and neurologic signs, and serially collected nasal swabs, bronchoalveolar lavage fluid (BALF) and cerebrospinal fluid (CSF) were evaluated for EV-D68 RNA and infectious virus. Infection with 2014 and 2018 EV-D68 isolates resulted in mild respiratory and gastrointestinal disease in some animals, but no evidence of neurological disease. Neither EV-D68 RNA nor infectious virus could be detected from any sample collected from animals challenged with 2014 EV-D68 isolates. Limited viral shedding – based on viral RNA quantified from nasal swabs and BALF – was detected from some animals infected with 2018 EV-D68 isolates. No virus was detectable in CSF. The rate of seroconversion was 100% for cynomolgus macaques infected with the 2018 EV-D68 isolates, but averaged between 0-50% for the 2014 isolates. Based on the results of this study, there is some evidence that infection with 2018 EV-D68 isolates may be more reliable at establishing limited infection than 2014 EV-D68 isolates. Regardless of virus isolate, however, EV-D68 infection of juvenile NHP species resulted in mild and nonspecific clinical disease and limited viral shedding. These data suggest that further refinements to the NHP model system (e.g., immunosuppression and/or direct viral inoculation) may be required to reproduce EV-D68 infection of the central nervous system and the associated AFM phenotype.
A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers
Authors: Llauger Gabriela , Melero Roberto , Monti Demián , Sycz Gabriela , Huck-Iriart Cristián , Cerutti María L. , Klinke Sebastián , Mikkelsen Evelyn , Tijman Ariel , Arranz Rocío , Alfonso Victoria , Arellano Sofía M. , Goldbaum Fernando A. , Sterckx Yann G. J. , Carazo José-María , Kaufman Sergio B. , Dans Pablo D. , Vas Mariana del , Otero Lisandro H.
Published: April 2022

The fijivirus Mal de Río Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric ΔC-arm version. In addition, although both P9-1 and P9-1ΔC-arm catalyze the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between the ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Río Cuarto crop disease.
Improved metabolic syndrome and timing of weight loss is crucial for influenza vaccine-induced immunity in obese mice
Authors: Honce Rebekah , Vazquez-Pagan Ana , Livingston Brandi , Cherry Sean , Hargest Virginia , Sharp Bridgett , Van de Velde Lee-Ann , Skinner R. Chris , Thomas Paul G. , Schultz-Cherry Stacey
Published: April 2022

Persons with obesity are at higher risk for developing severe complications upon influenza virus infection making vaccination a priority. Yet, studies demonstrate vaccine responses are less effective in obese hosts. In these studies, we examined how the timing of weight loss influenced influenza vaccine efficacy in male and female diet- induced obese mice. Here, we show weight loss post-vaccination is insufficient to rescue poor vaccine efficacy; however, weight loss occurring pre-vaccination successfully improves outcomes at viral challenge. Pre-vaccination weight loss improved vaccine immunogenicity and restored a functional recall response at challenge. Through tracking sera metabolic biomarkers, we propose the metabolic state at the time of vaccination is predictive of vaccine immunogenicity. Altogether, these findings highlight how timing of host-directed interventions is vital when seeking to improve influenza vaccine immunogenicity in obese hosts.
Wide-Field Multicontrast Nonlinear Microscopy for Histopathology
Authors: Castaño Leonardo Uribe , Mirsanaye Kamdin , Golaraei Ahmad , Kontenis Lukas , Done Susan J. , Stambolic Vuk , Akens Margarete K. , Wilson Brian C. , Barzda Virginijus
Published: April 2022

A multicontrast polarimetric wide-field second harmonic generation (SHG) and multiphoton excitation fluorescence (MPF) microscope is optimized for large area imaging of hematoxylin and eosin (H&E) stained and unstained histology slides. The bleaching kinetics of MPF and SHG are examined with various laser intensities at different pulse repetition rates to determine the optimal wide-filed imaging conditions for H&E stained histology slides. Several polarimetric parameters are used to investigate the organization of extracellular matrix collagen in the histology samples.
Calculation of ATP production rates using the Seahorse XF Analyzer
Authors: Desousa Brandon R. , Kim Kristen K.O. , Jones Anthony E. , Ball Andréa B. , Hsieh Wei Y. , Swain Pamela , Morrow Danielle H. , Brownstein Alexandra J. , Ferrick David A. , Shirihai Orian S. , Neilson Andrew , Nathanson David A. , Rogers George W. , Dranka Brian P. , Murphy Anne N. , Affourtit Charles , Bensinger Steven J. , Stiles Linsey , Romero Natalia , Divakaruni Ajit S.
Published: April 2022

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore developed a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data. We quantified the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation toin vitroculture conditions. Additionally, we detected substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.
Protein kinase D promotes prostate cancer cell bone metastasis by positively regulating Runx2 in a MEK/ERK1/2-dependent manner
Authors: Roy Adhiraj , Prasad Sahdeo , Chao Yapeng , Zhao Jinjun , Wang Qiming Jane
Published: April 2022

Most cancer patients die because of tumor metastasis not due to tumors at the primary site. Prostate tumors in advance stages frequently metastasize to the bone, which is the main cause of death for the disease. The family of protein kinase D (PKD) has been implicated in prostate cancer development, however, its role in prostate cancer metastasis has not been investigated. In this study, we examined the contribution of PKD to the metastatic potential of prostate cancer cells and the impact of PKD inhibition on prostate cancer bone metastasisin vivo. Our data showed that depletion of PKDs by siRNA or inhibition of PKD by an inhibitor CRT0066101 in a highly invasive prostate cancer PC3-ML cells potently inhibited colony formation and cell migration. Furthermore, depletion or inhibition of PKD significantly blocked invasion of PC3-ML cells and suppressed expression of genes related to bone metastasis. The reduced invasive activity resulted from PKD depletion was in part mediated through the transcription factor Runx2 as its silencing decreased PKD-mediated metastatic gene expression. Mechanistically, our data indicate that PKD modulated Runx2 target gene expression through the MEK/ERK1/2 signaling axis. Additionally, we examined whether PKD inhibitor CRT0066101 could prevent prostate cancer bone metastasis in a mouse model of metastasis, where intracardiac injection of PC3-ML cells led to metastasis of cells to the bone. We found that CRT0066101 potently decreased the frequency of micrometastases in mouse bone. These results indicate that PKDs play an important role in bone metastasis of prostate cancer cells and its inhibition may be beneficial for treatment of advanced stages of cancer.
Subchondral bone and synovial fluid metabolomic profiles are altered in injured and contralateral limbs 7 days after non-invasive joint injury in skeletally-mature C57BL/6 mice
Authors: Hislop Brady , Devine Connor , June Ronald K. , Heveran Chelsea M.
Published: April 2022

Objective

Post-traumatic osteoarthritis (PTOA) is a common long-term outcome following ACL injury. However, early changes to bone and synovial fluid after ACL injury are not sufficiently understood. The objectives of this study were to (1) evaluate whether acute bone loss one week after ACL injury is accompanied by altered subchondral bone plate modulus, (2) determine if bone changes are localized to the injured limb or extend to the contralateral-to-injured limb compared with sham-loaded controls, and (3) identify shifts in synovial fluid metabolism unique to injured limbs.

Design

Female C57Bl\6N mice (19 weeks at injury) were subjected to either a single tibial compression overload to simulate ACL injury (n=8) or a small pre-load (n=8). Mice were euthanized 7 days after injury, and synovial fluid was immediately harvested for metabolomic profiling. Bone microarchitecture, bone formation, and subchondral bone modulus at the proximal tibia were studied using microCT, histomorphometry, and nanoindentation, respectively. Osteoclast number density was assessed at the distal femur. For each bone measure a mixed model ANOVA was generated to determine the effects of injury and loaded side.

Results

Epiphyseal and subchondral bone microarchitecture decreased while subchondral bone tissue modulus was unchanged after ACL injuries. Bone resorption increased but bone formation was not changed. Loss of bone microarchitecture also occurred for the contralateral-to-injured limb, demonstrating that the early response to ACL injury extended beyond the injured joint. While the metabolomic profiles of the injured and contralateral-to-injured limbs had many similarities, there were also distinct metabolic shifts present in only the injured limbs. The most prominent of the pathways was cysteine and methionine metabolism, which is associated with osteoclast activity.

Conclusion

These results add to the understanding of early bone changes following ACL injury. Confirming prior reports, we observe a decline in epiphyseal and subchondral bone microarchitecture. We add the finding that subchondral bone modulus remains unchanged at one week after ACL injury. A potential biomarker of this initial bone catabolic response may be synovial fluid cysteine and methionine metabolism, which was only dysregulated in injured knees. Our results implicate a rapidly changing biological and mechanical environment within both the injured and contralateral joints that has the potential for influencing the progression to PTOA.
HiCHub: A Network-Based Approach to Identify Domains of Differential Interactions from 3D Genome Data
Authors: Li Xiang , Yuan Shuang , Zhu Shaoqi , Xue Hai-Hui , Peng Weiqun
Published: April 2022

Chromatin architecture is important for gene regulation. Existing algorithms for the identification of interactions changes focus on loops between focal loci. Here we develop a network-based algorithm HiCHub to detect chromatin interaction changes at larger scales. It identifies clusters of genomic elements in physical proximity in one state that exhibit concurrent decreases in interaction among them in the opposite state. The hubs exhibit concordant changes in chromatin state and expression changes, supporting their biological significance. HiCHub works well with data of limited sequencing coverage and facilitates the integration of the one-dimensional epigenetic landscape onto the chromatin architecture. HiCHub provides an approach for finding extended architectural changes and contributes to the connection with transcriptional output. HiCHub is freely available at https://github.com/WeiqunPengLab/HiCHub.
Structural dynamics of Zika Virus NS1 via a reductionist approach reveal the disordered nature of its beta roll domain in isolation
Authors: Kapuganti Shivani Krishna , Kumar Prateek , Giri Rajanish
Published: April 2022

Flavivirus Non-structural 1 (NS1) protein performs multiple functions such as host immune evasion, interaction with complement system factors, membrane rearrangement, etc. Therefore, it is highly plausible that significant structural and folding dynamics of NS1 might play a role in its multifunctionality. The dimeric structures of NS1 of multiple flaviviruses, including Zika virus (ZIKV), are available. However, its domain-wise dynamics perspective has not been explored so far. Therefore, it is of utmost importance to understand the structural conformations of NS1 and its domains in isolation, possibly highlighting the implications on the overall NS1 protein dynamics. Here, we have employed extensively long molecular dynamic (MD) simulations to understand the role of monomer, dimer, and a reductionist approach in understanding the dynamics of the three structural domains (i.e., β- roll, wing, and β-ladder) in isolation. Further, we experimentally validated our findings using CD spectroscopy and confirmed the intrinsically disordered behavior of NS1 β-roll in isolation and lipid mimetic environments. We also found that the β-ladder domain is highly flexible during long simulations. Therefore, we believe this study may have implications for significant dynamics played by NS1 protein, specifically during oligomerization of NS1.

Graphical Abstract

Schematic representation of the ZIKV NS1 protein and the models that we have used in this study.
Plant-on-Chip: core morphogenesis processes in the tiny plantWolffia australiana
Authors: Li Feng , Yang Jing-Jing , Sun Zong-Yi , Wang Lei , Qi Le-Yao , Sina A , Liu Yi-Qun , Zhang Hong-Mei , Dang Lei-Fan , Wang Shu-Jing , Luo Chun-Xiong , Nian Wei-Feng , O’Conner Seth , Ju Long-Zhen , Quan Wei-Peng , Li Xiao-Kang , Wang Chao , Wang De-Peng , You Han-Li , Cheng Zhu-Kuan , Yan Jia , Tang Fu-Chou , Yang De-Chang , Xia Chu-Wei , Gao Ge , Wang Yan , Zhang Bao-Cai , Zhou Yi-Hua , Guo Xing , Xiang Sun-Huan , Liu Huan , Peng Tian-Bo , Su Xiao-Dong , Chen Yong , Ouyang Qi , Wang Dong-Hui , Zhang Da-Ming , Xu Zhi-Hong , Hou Hong-Wei , Bai Shu-Nong , Li Ling
Published: April 2022

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the tiniest known angiosperm to overcome this challenge and provide an ideal model system for plant morphogenesis. We present a detailed morphological description of the monocotWolffia australiana, as well as high-quality genome information. Further, we developed the Plant-on-Chip culture system and demonstrate the application of advanced technologies such as snRNA-seq, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate howW. australianacan open a new horizon for deciphering the core regulatory mechanisms of plant morphogenesis.

Significance

What is the core morphogenetic process in angiosperms, a plant like a tree indeterminately growing, or a bud sequentially generating limited types of organs?Wolffia australiana, one of the smallest angiosperms in the world may help to make a distinction. Wolffia plantlet constitutes of only three organs that are indispensable to complete life cycle: one leaf, one stamen and one gynoecium. Before the growth tip is induced to flower, it keeps branching from the leaf axil and the branches separate from the main plantlet. Here we present a high-quality genome ofW. australiana, detailed morphological description, a Plant-on-Chip cultural system, and some principle-proof experiments, demonstrating thatW. australianais a promising model system for deciphering core developmental program in angiosperms.