bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACTDespite 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

AbstractBrain 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

AbstractGermline 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

AbstractSperm 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 StatementIn 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.
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

AbstractIn 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 synopsisBackgroundThe 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.ObjectivesTo develop a scar-free genome editing tool suitable for chromosomal and plasmid modifications in MDR A. baumannii AB5075.MethodsWe 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.ResultsWe 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.ConclusionWe 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

AbstractHuman 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

ABSTRACTThe 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

AbstractPersons 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

AbstractA 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

ABSTRACTOxidative 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

AbstractMost 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

AbstractObjectivePost-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.DesignFemale 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.ResultsEpiphyseal 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.ConclusionThese 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.