Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Impact of endosymbionts on tick physiology and fitness.

Ticks transmit pathogens and harbour non-pathogenic, vertically transmitted intracellular bacteria termed endosymbionts. Almost all ticks studied to date contain 1 or more of Coxiella, Francisella, Rickettsia or Candidatus Midichloria mitochondrii endosymbionts, indicative of their importance to tick physiology. Genomic and experimental data suggest that endosymbionts promote tick development and reproductive success. Here, we review the limited information currently available on the potential roles endosymbionts play in enhancing tick metabolism and fitness. Future studies that expand on these findings are needed to better understand endosymbionts' contributions to tick biology. This knowledge could potentially be applied to design novel strategies that target endosymbiont function to control the spread of ticks and pathogens they vector.

MicroRNAs Contribute to Host Response to Coxiella burnetii.

MicroRNAs (miRNAs), a class of small noncoding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or upregulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is downregulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy-conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, downregulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen.

Ergonomic risk and physiological assessment of plogging activity.

BACKGROUND: Plogging, an environment friendly trash workout is a combination of jogging with litter collection. People who are involved in the plogging carry a baggage for collecting the litter. Walking with a weight on one side causes the opposite side of the body to engage for stability and are also exposed to repetitive bending during the activity. OBJECTIVES: The purpose of this study is to evaluate the postural and physiological aspects of plogging activity. METHODS: Thirty six subjects performed the litter collection in stoop, semi-squat, full squat and lunge postures respectively. Postures were analyzed using Rapid Entire Body Assessment (REBA). Physiological aspects of plogging, as well as a comparison of physical activity assessment during jogging and plogging, were investigated using a Polar M430 optical heart rate monitor. Statistical analysis were performed using SPSS version 23. RESULTS: Mean±SD of full squat (5.13±0.59) and lunge (6.64±1.15) posture was found to have lesser risk score in comparison with the other two postures such as stoop (10.31±0.88) and semi-squat (8.11±1.40). Analysis from the Kruskal-Wallis and post hoc test showed that there is no significant interaction between the postures (p < 0.05). Paired Sample t-test showed that the energy expenditure for plogging and jogging are found to be similar (p > 0.05), but the fat percentages of calories burned is more in plogging (p < 0.05). Howerver plogging can be considered as a strenous activity as the % Cardiovascular strain of the activity had a mean value of (99.261%). CONCLUSIONS: Ergonomic interventions are needed to play a vital role in minimizing the musculoskeletal related injuries and the physical strain of the task.

Quantifying microstructural evolution via time-dependent reduced-dimension metrics based on hierarchical n-point polytope functions.

We devise reduced-dimension metrics for effectively measuring the distance between two points (i.e., microstructures) in the microstructure space and quantifying the pathway associated with microstructural evolution, based on a recently introduced set of hierarchical n-point polytope functions P_{n}. The P_{n} functions provide the probability of finding particular n-point configurations associated with regular n polytopes in the material system, and are a special subset of the standard n-point correlation functions S_{n} that effectively decompose the structural features in the system into regular polyhedral basis with different symmetries. The nth order metric Ω_{n} is defined as the L_{1} norm associated with the P_{n} functions of two distinct microstructures. By choosing a reference initial state (i.e., a microstructure associated with t_{0}=0), the Ω_{n}(t) metrics quantify the evolution of distinct polyhedral symmetries and can in principle capture emerging polyhedral symmetries that are not apparent in the initial state. To demonstrate their utility, we apply the Ω_{n} metrics to a two-dimensional binary system undergoing spinodal decomposition to extract the phase separation dynamics via the temporal scaling behavior of the corresponding Ω_{n}(t), which reveals mechanisms governing the evolution. Moreover, we employ Ω_{n}(t) to analyze pattern evolution during vapor deposition of phase-separating alloy films with different surface contact angles, which exhibit rich evolution dynamics including both unstable and oscillating patterns. The Ω_{n} metrics have potential applications in establishing quantitative processing-structure-property relationships, as well as real-time processing control and optimization of complex heterogeneous material systems.

Genome-Wide Identification of Novel sRNAs in Streptococcus mutans.

Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, noncoding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA SmsR4. Our data demonstrate that SmsR4 regulates the enzyme IIA (EIIA) component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. IMPORTANCE Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.

A Peroxide-Responding sRNA Evolved from a Peroxidase mRNA.

Small RNAs (sRNAs) are important gene regulators in bacteria, but it is unclear how new sRNAs originate and become part of regulatory networks that coordinate bacterial response to environmental stimuli. Using a covariance modeling-based approach, we analyzed the presence of hundreds of sRNAs in more than a thousand genomes across Enterobacterales, a bacterial order with a confluence of factors that allows robust genome-scale sRNA analyses: several well-studied organisms with fairly conserved genome structures, an established phylogeny, and substantial nucleotide diversity within a narrow evolutionary space. We discovered that a majority of sRNAs arose recently, and uncovered protein-coding genes as a potential source from which new sRNAs arise. A detailed investigation of the emergence of OxyS, a peroxide-responding sRNA, revealed that it evolved from a fragment of a peroxidase messenger RNA. Importantly, although it replaced the ancestral peroxidase, OxyS continues to be part of the ancestral peroxide-response regulon, indicating that an sRNA that arises from a protein-coding gene would inherently be part of the parental protein's regulatory network. This new insight provides a fresh framework for understanding sRNA origin and regulatory integration in bacteria.

Plakophilin3 loss leads to increased adenoma formation and rectal prolapse in APCmin mice.

Plakophilin3 (PKP3) loss leads to tumor progression and metastasis of colon cancer cells. The goal of this report was to determine if PKP3 loss led to increased disease progression in mice. We generated a colonocyte-specific knockout of PKP3 in APCmin mice, which led to increased adenoma formation, the formation of rectal prolapse, and a significant decrease in survival. The observed increase in rectal prolapse formation and decrease in survival correlated with an increase in the expression of Lipocalin2 (LCN2). Increased disease progression was observed even upon treatment with 5-fluorouracil (5FU). These results suggest that an increase in LCN2 expression might lead to therapy resistance and that LCN2 might serve as a potential therapeutic target in colorectal cancer.

A small RNA is functional in Escherichia fergusonii despite containing a large insertion.

Bacterial small RNAs (sRNAs) are important regulators of gene expression; however, the impact of natural mutations on sRNA functions has not been studied extensively. Here we show that the sRNA MgrR contains a unique 53 bp insertion in Escherichia fergusonii, a close relative of Escherichia coli and Salmonella enterica. The insertion is a repetitive extragenic palindromic (REP) sequence that could block transcription, but full-length MgrR is produced in E. fergusonii, showing that the insertion has not affected sRNA production. Additionally, despite containing the large insertion, the sRNA appears to be functional because deletion of mgrR made E. fergusonii more susceptible to H2O2. The molecular details of MgrR's roles in H2O2defence are yet to be defined, but our results suggest that having an alternative function allowed the sRNA to be retained in E. fergusonii despite it sustaining a large, potentially disruptive mutation.

Complete Mitochondrial Genome Sequence of the Gulf Coast Tick (Amblyomma maculatum).

The complete circularized mitochondrial genome sequence of Amblyomma maculatum is 14,803 bp long. It encodes 13 protein coding genes, 2 rRNA genes, 22 tRNA genes, 2 tick box motifs, and 2 control regions. The gene arrangement and content are consistent with those of previously reported Metastriata tick mitochondrial genomes.

Coxiella burnetii and Related Tick Endosymbionts Evolved from Pathogenic Ancestors.

Both symbiotic and pathogenic bacteria in the family Coxiellaceae cause morbidity and mortality in humans and animals. For instance, Coxiella-like endosymbionts (CLEs) improve the reproductive success of ticks-a major disease vector, while Coxiella burnetii causes human Q fever, and uncharacterized coxiellae infect both animals and humans. To better understand the evolution of pathogenesis and symbiosis in this group of intracellular bacteria, we sequenced the genome of a CLE present in the soft tick Ornithodoros amblus (CLEOA) and compared it to the genomes of other bacteria in the order Legionellales. Our analyses confirmed that CLEOA is more closely related to C. burnetii, the human pathogen, than to CLEs in hard ticks, and showed that most clades of CLEs contain both endosymbionts and pathogens, indicating that several CLE lineages have evolved independently from pathogenic Coxiella. We also determined that the last common ancestorof CLEOA and C. burnetii was equipped to infect macrophages and that even though horizontal gene transfer (HGT) contributed significantly to the evolution of C. burnetii, most acquisition events occurred primarily in ancestors predating the CLEOA-C. burnetii divergence. These discoveries clarify the evolution of C. burnetii, which previously was assumed to have emerged when an avirulent tick endosymbiont recently gained virulence factors via HGT. Finally, we identified several metabolic pathways, including heme biosynthesis, that are likely critical to the intracellular growth of the human pathogen but not the tick symbiont, and show that the use of heme analog is a promising approach to controlling C. burnetii infections.

Male fetal sex affects uteroplacental angiogenesis in growth restriction mouse model†.

Abnormally increased angiotensin II activity related to maternal angiotensinogen (AGT) genetic variants, or aberrant receptor activation, is associated with small-for-gestational-age babies and abnormal uterine spiral artery remodeling in humans. Our group studies a murine AGT gene titration transgenic (TG; 3-copies of the AGT gene) model, which has a 20% increase in AGT expression mimicking a common human AGT genetic variant (A[-6]G) associated with intrauterine growth restriction (IUGR) and spiral artery pathology. We hypothesized that aberrant maternal AGT expression impacts pregnancy-induced uterine spiral artery angiogenesis in this mouse model leading to IUGR. We controlled for fetal sex and fetal genotype (e.g., only 2-copy wild-type [WT] progeny from WT and TG dams were included). Uteroplacental samples from WT and TG dams from early (days 6.5 and 8.5), mid (d12.5), and late (d16.5) gestation were studied to assess uterine natural killer (uNK) cell phenotypes, decidual metrial triangle angiogenic factors, placental growth and capillary density, placental transcriptomics, and placental nutrient transport. Spiral artery architecture was evaluated at day 16.5 by contrast-perfused three-dimensional microcomputed tomography (3D microCT). Our results suggest that uteroplacental angiogenesis is significantly reduced in TG dams at day 16.5. Males from TG dams are associated with significantly reduced uteroplacental angiogenesis from early to late gestation compared with their female littermates and WT controls. Angiogenesis was not different between fetal sexes from WT dams. We conclude that male fetal sex compounds the pathologic impact of maternal genotype in this mouse model of growth restriction.

Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host.

Bartonella bacilliformis, the etiological agent of Carrión's disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión's disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

Modulation of Bacterial Fitness and Virulence Through Antisense RNAs.

Regulatory RNAs contribute to gene expression control in bacteria. Antisense RNAs (asRNA) are a class of regulatory RNAs that are transcribed from opposite strands of their target genes. Typically, these untranslated transcripts bind to cognate mRNAs and rapidly regulate gene expression at the post-transcriptional level. In this article, we review asRNAs that modulate bacterial fitness and increase virulence. We chose examples that underscore the variety observed in nature including, plasmid- and chromosome-encoded asRNAs, a riboswitch-regulated asRNA, and asRNAs that require other RNAs or RNA-binding proteins for stability and activity. We explore how asRNAs improve bacterial fitness and virulence by modulating plasmid acquisition and maintenance, regulating transposon mobility, increasing resistance against bacteriophages, controlling flagellar production, and regulating nutrient acquisition. We conclude with a brief discussion on how this knowledge is helping to inform current efforts to develop new therapeutics.

A CsrA-Binding, trans-Acting sRNA of Coxiella burnetii Is Necessary for Optimal Intracellular Growth and Vacuole Formation during Early Infection of Host Cells.

Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of C. burnetii One of them, CbsR12 (Coxiella burnetiismall RNA 12), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro Moreover, through a combination of in vitro and cell culture assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds and upregulates translation of carA transcripts coding for carbamoyl phosphate synthetase A, an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of metK transcripts coding for S-adenosylmethionine synthetase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of cvpD transcripts, coding for a type IVB effector protein, in mammalian cell culture. Finally, we found that CbsR12 is necessary for expansion of Coxiella-containing vacuoles and affects growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK synthesis. CbsR12 is one of only a few identified trans-acting sRNAs that interacts with CsrA.IMPORTANCE Regulation of metabolism and virulence in C. burnetii is not well understood. Here, we show that C. burnetii small RNA 12 (CbsR12) is highly transcribed in the metabolically active large-cell variant compared to the nonreplicative small-cell variant. We show that CbsR12 directly regulates several genes involved in metabolism, along with a type IV effector gene, in trans In addition, we demonstrate that CbsR12 binds to CsrA-2 in vitro and induces autoaggregation and biofilm formation when transcribed ectopically in Escherichia coli, consistent with other CsrA-sequestering sRNAs. These results implicate CbsR12 in the indirect regulation of a number of genes via CsrA-mediated regulatory activities. The results also support CbsR12 as a crucial regulatory component early on in a mammalian cell infection.

Development of a Vertically Integrated Trainee Program: Linking Future and Young Physicians.

Supplemental digital content is available in the text.

Genome-wide screening of potential RNase Y-processed mRNAs in the M49 serotype Streptococcus pyogenes NZ131.

RNase Y is a major endoribonuclease in Group A streptococcus (GAS) and other Gram-positive bacteria. Our previous study showed that RNase Y was involved in mRNA degradation and processing in GAS. We hypothesized that mRNA processing regulated the expression of important GAS virulence factors via altering their mRNA stabilities and that RNase Y mediated at least some of the mRNA-processing events. The aims of this study were to (1) identify mRNAs that were processed by RNase Y and (2) confirm the mRNA-processing events. The transcriptomes of Streptococcus pyogenes NZ131 wild type and its RNase Y mutant (Δrny) were examined with RNA-seq. The data were further analyzed to define GAS operons. The mRNA stabilities of the wild type and Δrny at subgene level were determined with tiling array analysis. Operons displaying segmental stability in the wild type but not in the Δrny were predicted to be RNase Y processed. Overall 865 operons were defined and their boundaries predicted. Further analysis narrowed down 15 mRNAs potentially processed by RNase Y. A selection of four candidates including folC1 (folylpolyglutamate synthetase), prtF (fibronectin-binding protein), speG (streptococcal exotoxin G), ropB (transcriptional regulator of speB), and ypaA (riboflavin transporter) mRNAs was examined with Northern blot analysis. However, only folC1 was confirmed to be processed, but it is unlikely that RNase Y is responsible. We conclude that GAS use RNase Y to selectively process mRNA, but the overall impact is confined to selected virulence factors.

Correction: Accumulation and expression of horizontally acquired genes in Arcobacter cryaerophilus that thrives in sewage.

[This corrects the article DOI: 10.7717/peerj.3269.].

LytTR Regulatory Systems: A potential new class of prokaryotic sensory system.

The most commonly studied prokaryotic sensory signal transduction systems include the one-component systems, phosphosignaling systems, extracytoplasmic function (ECF) sigma factor systems, and the various types of second messenger systems. Recently, we described the regulatory role of two separate sensory systems in Streptococcus mutans that jointly control bacteriocin gene expression, natural competence development, as well as a cell death pathway, yet they do not function via any of the currently recognized signal transduction paradigms. These systems, which we refer to as LytTR Regulatory Systems (LRS), minimally consist of two proteins, a transcription regulator from the LytTR Family and a transmembrane protein inhibitor of this transcription regulator. Here, we provide evidence suggesting that LRS are a unique uncharacterized class of prokaryotic sensory system. LRS exist in a basal inactive state. However, when LRS membrane inhibitor proteins are inactivated, an autoregulatory positive feedback loop is triggered due to LRS regulator protein interactions with direct repeat sequences located just upstream of the -35 sequences of LRS operon promoters. Uncharacterized LRS operons are widely encoded by a vast array of Gram positive and Gram negative bacteria as well as some archaea. These operons also contain unique direct repeat sequences immediately upstream of their operon promoters indicating that positive feedback autoregulation is a globally conserved feature of LRS. Despite the surprisingly widespread occurrence of LRS operons, the only characterized examples are those of S. mutans. Therefore, the current study provides a useful roadmap to investigate LRS function in the numerous other LRS-encoding organisms.

Identification of novel MITEs (miniature inverted-repeat transposable elements) in Coxiella burnetii: implications for protein and small RNA evolution.

BACKGROUND: Coxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever. C. burnetii's genome contains an abundance of pseudogenes and numerous selfish genetic elements. MITEs (miniature inverted-repeat transposable elements) are non-autonomous transposons that occur in all domains of life and are thought to be insertion sequences (ISs) that have lost their transposase function. Like most transposable elements (TEs), MITEs are thought to play an active role in evolution by altering gene function and expression through insertion and deletion activities. However, information regarding bacterial MITEs is limited. RESULTS: We describe two MITE families discovered during research on small non-coding RNAs (sRNAs) of C. burnetii. Two sRNAs, Cbsr3 and Cbsr13, were found to originate from a novel MITE family, termed QMITE1. Another sRNA, CbsR16, was found to originate from a separate and novel MITE family, termed QMITE2. Members of each family occur ~ 50 times within the strains evaluated. QMITE1 is a typical MITE of 300-400 bp with short (2-3 nt) direct repeats (DRs) of variable sequence and is often found overlapping annotated open reading frames (ORFs). Additionally, QMITE1 elements possess sigma-70 promoters and are transcriptionally active at several loci, potentially influencing expression of nearby genes. QMITE2 is smaller (150-190 bps), but has longer (7-11 nt) DRs of variable sequences and is mainly found in the 3' untranslated region of annotated ORFs and intergenic regions. QMITE2 contains a GTAG repetitive extragenic palindrome (REP) that serves as a target for IS1111 TE insertion. Both QMITE1 and QMITE2 display inter-strain linkage and sequence conservation, suggesting that they are adaptive and existed before divergence of C. burnetii strains. CONCLUSIONS: We have discovered two novel MITE families of C. burnetii. Our finding that MITEs serve as a source for sRNAs is novel. QMITE2 has a unique structure and occurs in large or small versions with unique DRs that display linkage and sequence conservation between strains, allowing for tracking of genomic rearrangements. QMITE1 and QMITE2 copies are hypothesized to influence expression of neighboring genes involved in DNA repair and virulence through transcriptional interference and ribonuclease processing.