Deciphering the Universe of RNA Structures and trans RNA–RNA Interactions of Transcriptomes In Vivo: From Experimental Protocols to Computational Analyses

Previous investigators have demonstrated that the tumor necrosis factor-α (TNF-α) response to endotoxin is inhibited by exogenous corticosterone or catecholamines both in vitro and in vivo, whereas others have reported that surgical and nonsurgical stress increase the endogenous concentrations of these stress-induced hormones. We hypothesized that elevated endogenous stress hormones resultant from experimental protocols attenuated the endotoxin-induced TNF-α response. We used a chronically catheterized rat model to demonstrate that the endotoxin-induced TNF-α response is 10- to 50-fold greater in nonstressed (NS) rats compared with either surgical-stressed (SS, laparotomy) or nonsurgical-stressed (NSS, tail vein injection) models. Compared with the NS group, the SS and NSS groups demonstrated significantly lower mean peak TNF-α responses at 2 mg/kg and 6 μg/kg endotoxin [NS 111.8 ± 6.5 ng/ml and 64.3 ± 5.9 ng/ml, respectively, vs. SS 3.9 ± 1.1 ng/ml ( P < 0.01) and 1.3 ± 0.5 ng/ml ( P < 0.01) or NSS 5.2 ± 3.2 ng/ml ( P < 0.01) at 6 μg/kg]. Similarly, baseline concentrations of corticosterone and catecholamines were significantly lower in the NSS group [84.5 ± 16.5 ng/ml and 199.8 ± 26.2 pg/ml, respectively, vs. SS group 257.2 ± 35.7 ng/ml ( P< 0.01) and 467.5 ± 52.2 pg/ml ( P < 0.01) or NS group 168.6 ± 14.4 ng/ml ( P < 0.01) and 1,109.9 ± 140.7 pg/ml ( P < 0.01)]. These findings suggest that the surgical and nonsurgical stress inherent in experimental protocols increases baseline stress hormones, masking the endotoxin-induced TNF-α response. Subsequent studies of endotoxic shock should control for the effects of protocol-induced stress and should measure and report baseline concentrations of corticosterone and catecholamines.

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Sowing the Seeds of a Pandemic? Mammalian Pathogenicity and Transmissibility of H1 Variant Influenza Viruses from the Swine Reservoir

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed4010041 ◽

2019 ◽

Vol 4 (1) ◽

pp. 41 ◽

Cited By ~ 5

Author(s):

Joanna Pulit-Penaloza ◽

Jessica Belser ◽

Terrence Tumpey ◽

Taronna Maines

Keyword(s):

North American ◽

Influenza Viruses ◽

Antigenic Drift ◽

Risk Assessments ◽

Pandemic Preparedness ◽

The North ◽

Zoonotic Viruses ◽

Experimental Protocols ◽

Pandemic Strains

Emergence of genetically and antigenically diverse strains of influenza to which the human population has no or limited immunity necessitates continuous risk assessments to determine the likelihood of these viruses acquiring adaptations that facilitate sustained human-to-human transmission. As the North American swine H1 virus population has diversified over the last century by means of both antigenic drift and shift, in vivo assessments to study multifactorial traits like mammalian pathogenicity and transmissibility of these emerging influenza viruses are critical. In this review, we examine genetic, molecular, and pathogenicity and transmissibility data from a panel of contemporary North American H1 subtype swine-origin viruses isolated from humans, as compared to H1N1 seasonal and pandemic viruses, including the reconstructed 1918 virus. We present side-by-side analyses of experiments performed in the mouse and ferret models using consistent experimental protocols to facilitate enhanced interpretation of in vivo data. Contextualizing these analyses in a broader context permits a greater appreciation of the role that in vivo risk assessment experiments play in pandemic preparedness. Collectively, we find that despite strain-specific heterogeneity among swine-origin H1 viruses, contemporary swine viruses isolated from humans possess many attributes shared by prior pandemic strains, warranting heightened surveillance and evaluation of these zoonotic viruses.

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In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding

Nucleic Acids Research ◽

10.1093/nar/gkx617 ◽

2017 ◽

Vol 45 (16) ◽

pp. 9716-9725 ◽

Cited By ~ 26

Author(s):

Danny Incarnato ◽

Edoardo Morandi ◽

Francesca Anselmi ◽

Lisa M. Simon ◽

Giulia Basile ◽

...

Keyword(s):

Rna Structures ◽

Nascent Rna

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Functional interplay between DEAD-box RNA helicases Ded1 and Dbp1 in preinitiation complex attachment and scanning on structured mRNAs in vivo

Nucleic Acids Research ◽

10.1093/nar/gkz595 ◽

2019 ◽

Cited By ~ 4

Author(s):

Neelam Dabas Sen ◽

Neha Gupta ◽

Stuart K. Archer ◽

Thomas Preiss ◽

Jon R Lorsch ◽

...

Keyword(s):

Untranslated Region ◽

Double Mutant ◽

Direct Evidence ◽

Translation Efficiency ◽

Rna Structures ◽

Preinitiation Complex ◽

Rna Helicases ◽

Ribosome Binding ◽

Dead Box

Abstract RNA structures that impede ribosome binding or subsequent scanning of the 5′-untranslated region (5′-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5′-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5′-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such ‘conditionally hyperdependent’ mRNAs contain unusually long 5′-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5′-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

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Intragenic recombination influences rotavirus diversity and evolution

Virus Evolution ◽

10.1093/ve/vez059 ◽

2020 ◽

Vol 6 (1) ◽

Cited By ~ 3

Author(s):

Irene Hoxie ◽

John J Dennehy

Keyword(s):

Amino Acid ◽

Homologous Recombination ◽

Strong Evidence ◽

Protein Structures ◽

Intragenic Recombination ◽

Rna Structures ◽

Rotavirus A ◽

Adaptive Mutations ◽

Variation Database ◽

Computational Analyses

Abstract Because of their replication mode and segmented dsRNA genome, homologous recombination is assumed to be rare in the rotaviruses. We analyzed 23,627 complete rotavirus genome sequences available in the NCBI Virus Variation database, and found 109 instances of homologous recombination, at least eleven of which prevailed across multiple sequenced isolates. In one case, recombination may have generated a novel rotavirus VP1 lineage. We also found strong evidence for intergenotypic recombination in which more than one sequence strongly supported the same event, particularly between different genotypes of segment 9, which encodes the glycoprotein, VP7. The recombined regions of many putative recombinants showed amino acid substitutions differentiating them from their major and minor parents. This finding suggests that these recombination events were not overly deleterious, since presumably these recombinants proliferated long enough to acquire adaptive mutations in their recombined regions. Protein structural predictions indicated that, despite the sometimes substantial amino acid replacements resulting from recombination, the overall protein structures remained relatively unaffected. Notably, recombination junctions appear to occur nonrandomly with hot spots corresponding to secondary RNA structures, a pattern seen consistently across segments. In total, we found strong evidence for recombination in nine of eleven rotavirus A segments. Only segments 7 (NSP3) and 11 (NSP5) did not show strong evidence of recombination. Collectively, the results of our computational analyses suggest that, contrary to the prevailing sentiment, recombination may be a significant driver of rotavirus evolution and may influence circulating strain diversity.

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Long-term in vivo experimental protocols for development of implantable dialysis system

The Proceedings of Mechanical Engineering Congress Japan ◽

10.1299/jsmemecj.2018.j1520204 ◽

2018 ◽

Vol 2018 (0) ◽

pp. J1520204

Author(s):

Yoshihiko KOYA ◽

Takashi OTA ◽

Norihisa MIKI

Keyword(s):

Experimental Protocols

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Gender-Related Differences in Counterregulatory Responses to Antecedent Hypoglycemia in Normal Humans1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.85.6.6641 ◽

2000 ◽

Vol 85 (6) ◽

pp. 2148-2157

Author(s):

S. N. Davis ◽

C. Shavers ◽

F. Costa

Keyword(s):

Sympathetic Nerve Activity ◽

Muscle Sympathetic Nerve Activity ◽

Gender Effects ◽

Central Importance ◽

Marked Contrast ◽

Men 2 ◽

Experimental Protocols ◽

Type 1 Dm

Compared to men, inherent counterregulatory responses are reduced in healthy and type 1 diabetic women. Despite this, the prevalence of hypoglycemia in patients with type 1 diabetes (type 1 DM) is gender neutral. The aim of this study was to determine the in vivo mechanism(s) responsible for this apparent clinical paradox. The central importance of antecedent hypoglycemia in causing subsequent counterregulatory failure is now established. We, therefore, hypothesized that a gender-related difference to the blunting effects of prior hypoglycemia may exist, and this could explain why type 1 DM women do not have an increased prevalence of hypoglycemia despite reduced counterregulatory responses. Fifteen healthy male and female individuals (eight men and seven women) underwent four separate 2-day experimental protocols in a randomized fashion. Day 1 involved identical morning and afternoon 2-h hyperinsulinemic (9 pmol/kg·min) glucose clamp studies with 5.1 ± 0.1, 3.9 ± 0.1, 3.3± 0.1, or 2.9 ± 0.1 mmol/L. Day 2 consisted of a single 2-h hypoglycemic clamp of 2.9 ± 0.1 mmol/L. Insulin levels were similar on both days of each protocol in men and women. After day 1 euglycemia (5.1 ± 0.1 mmol/L), day 2 counterregulatory responses were significantly increased (P < 0.01) in men relative to women. In women, counterregulatory responses were resistant to the effects of day 1 hypoglycemia. Antecedent hypoglycemia of 3.9, 3.3, and 2.9 ± 0.1 mmol/L produced 3 ± 2%, 5 ± 2%, and 25 ± 4% aggregate reductions in day 2 neuroendocrine, muscle sympathetic nerve activity, and metabolic counterregulatory responses. In marked contrast, identical day 1 hypoglycemia of 3.9, 3.3, and 2.9 ± 0.1 mmol/L in men produced significantly greater reductions in day 2 counterregulatory responses of 30 ± 6%, 39 ± 6%, and 52 ± 6%, respectively. The net effect of the differential gender effects of antecedent hypoglycemia was to overcome the usually increased (50%) sympathetic nervous system (SNS) counterregulatory responses to hypoglycemia found in men. We conclude that 1) antecedent hypoglycemia produces less blunting of counterregulatory responses to subsequent hypoglycemia in women relative to men; 2) two episodes of antecedent hypoglycemia can overcome the greater SNS response to hypoglycemia usually found in men; and 3) the reduced susceptibility of women to the blunting effects of antecedent hypoglycemia may be the mechanism explaining why, despite inherently reduced SNS counterregulatory responses, female type 1 DM patients have a similar prevalence of hypoglycemia compared to men.

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Self-assembly of large RNA structures: learning from DNA nanotechnology

DNA and RNA Nanotechnology ◽

10.1515/rnan-2015-0002 ◽

2016 ◽

Vol 2 (1) ◽

Cited By ~ 3

Author(s):

Jaimie Marie Stewart ◽

Elisa Franco

Keyword(s):

Self Assembly ◽

De Novo ◽

Dna Nanotechnology ◽

De Novo Design ◽

Dna Nanostructures ◽

Rna Structures ◽

Functional Nanostructures ◽

Rna And Dna ◽

Unique Chemical

AbstractNucleic acid nanotechnology offers many methods to build self-assembled structures using RNA and DNA. These scaffolds are valuable in multiple applications, such as sensing, drug delivery and nanofabrication. Although RNA and DNA are similar molecules, they also have unique chemical and structural properties. RNA is generally less stable than DNA, but it folds into a variety of tertiary motifs that can be used to produce complex and functional nanostructures. Another advantage of using RNA over DNA is its ability to be encoded into genes and to be expressed in vivo. Here we review existing approaches for the self-assembly of RNA and DNA nanostructures and specifically methods to assemble large RNA structures. We describe de novo design approaches used in DNA nanotechnology that can be ported to RNA. Lastly, we discuss some of the challenges yet to be solved to build micron-scale, multi stranded RNA scaffolds.

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Intragenic Recombination Influences Rotavirus Diversity and Evolution

10.1101/794826 ◽

2019 ◽

Author(s):

Irene Hoxie ◽

John J. Dennehy

Keyword(s):

Amino Acid ◽

Homologous Recombination ◽

Strong Evidence ◽

Protein Structures ◽

Intragenic Recombination ◽

Rna Structures ◽

Rotavirus A ◽

Adaptive Mutations ◽

Variation Database ◽

Computational Analyses

AbstractBecause of their replication mode and segmented dsRNA genome, homologous recombination is assumed to be rare in the rotaviruses. We analyzed 23,627 complete rotavirus genome sequences available in the NCBI Virus Variation database, and found 109 instances of homologous recombination, at least 11 of which prevailed across multiple sequenced isolates. In one case, recombination may have generated a novel rotavirus VP1 lineage. We also found strong evidence for intergenotypic recombination in which more than one sequence strongly supported the same event, particularly between different genotypes of segment 9, which encodes the serotype protein, VP7. The recombined regions of many putative recombinants showed amino acid substitutions differentiating them from their major and minor parents. This finding suggests that these recombination events were not overly deleterious, since presumably these recombinants proliferated long enough to acquire adaptive mutations in their recombined regions. Protein structural predictions indicated that, despite the sometimes substantial amino acid replacements resulting from recombination, the overall protein structures remained relatively unaffected. Notably, recombination junctions appear to occur non-randomly with hot spots corresponding to secondary RNA structures, a pattern seen consistently across segments. In total, we found strong evidence for recombination in nine of eleven rotavirus A segments. Only segment 7 (NSP3) and segment 11 (NSP5) did not show strong evidence of recombination. Collectively, the results of our computational analyses suggest that, contrary to the prevailing sentiment, recombination may be a significant driver of rotavirus evolution and may influence circulating strain diversity.

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