Helicobacter pylori colonizes the gastric epithelial cells of at least half of the world’s population, and it is the strongest risk factor for developing gastric complications like chronic gastritis, ulcer diseases, and gastric cancer. To successfully colonize and establish a persistent infection, the bacteria must overcome harsh gastric conditions. H. pylori has a well-developed mechanism by which it can survive in a very acidic niche. Despite bacterial factors, gastric environmental factors and host genetic constituents together play a co-operative role for gastric pathogenicity. The virulence factors include bacterial colonization factors BabA, SabA, OipA, and HopQ, and the virulence factors necessary for gastric pathogenicity include the effector proteins like CagA, VacA, HtrA, and the outer membrane vesicles. Bacterial factors are considered more important. Here, we summarize the recent information to better understand several bacterial virulence factors and their role in the pathogenic mechanism. Helicobacter pylori, one of the most common bacteria to colonize the gastric epithelium of about half of the world’s population, has been estimated to accompany humans for at least 100,000 years [00 years . Geograp00 years . A recen00 years .H. pylori colonizes specific sites like the antrum and corpus. H. pylori has well-developed adaptation mechanisms to survive in the harsh gastric acid conditions and to establish a permanent infection .H. pylori [H. pylori infection, which is a significant factor causing non-iatrogenic peptic ulcer diseases. The risk of peptic ulcer development increases with previous history of H. pylori infection even after its successful eradication compared with non-infected individuals [H. pylori infection compared to non-cured patients [H. pylori eradication status found that the recurrence rate of gastric ulcers and peptic ulcers were 4% and 6%, respectively, in successfully cured patients compared to 59% and 67%, respectively, in non-cured patients [Once the permanent infection is established in the stomach, several gastro-duodenal complications like chronic gastritis, peptic ulcer diseases, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma may develop . However. pylori . It has ividuals . Howeverpatients . The respatients . Howeverpatients .H. pylori encounters extremely harsh conditions of pH around 2.0. However, H. pylori possesses several factors like urease, bacterial shape and flagella mediating motility to interact with the harsh gastric environment  and carbamate production, which is spontaneously decomposed to yield another ammonia (NH3) and carbonic acid (H2CO3). The carbonic acid is broken down to CO2 and water (H2O) molecules. Ammonia in its protonated form (NH4+) neutralizes stomach acidity and plays an important role in providing a favorable nearly neutral micro-environment around H. pylori [2 is converted to bicarbonate (HCO3−) and H+ in the periplasmic space by periplasmic α-carbonic anhydrase, maintaining the periplasmic pH close to 6.1 via an acid acclimation mechanism. In this way, NH3 and CO2 production provides the necessary environment for H. pylori’s gastric survival [Urease-catalyzed urea hydrolysis (endogenous and exogenous) results in ammonia , which contributes to the development and progression of several cancers. A recent study showed that the H. pylori urease activated the PI3K-AKT-mTOR pathway in gastric cells. The activation of this pathway increases HIF-α expression [H. pylori urease was found to drive the differentiation of endothelial cells by producing reactive oxygen species and activating the lipoxygenase pathway via pro-inflammatory properties, contributing to H. pylori infection progression to gastric carcinogenesis [H. pylori urease was shown to bind to major histocompatibility complex (MHC) class II molecules and induce cell apoptosis [In addition to its role in acid neutralization, several in vitro studies uncovered urease’s role and its catalytic products in pathogenicity establishment. Ammonia production disrupts the tight cell junctions, breaches the cellular integrity, and damages the gastric epithelium ,19. CO2 agocytes . Urease agocytes . Chronicpression . Moreoveogenesis . Furtherpoptosis .H. pylori were outcompeted by wild type helical H. pylori [H. pylori’s efficient colonization.A study of the bacterial shape’s role in movement showed that a mutation in the cell shape determinants causing the bacteria to adopt a straight rod morphology reduced the speed of bacterial movement by 7–21% . Moreove. pylori . These sThe characteristic helical shape of bacteria which provides the bacterial motility also helps to cope with the harsh acidic conditions and adapt to different gastric environments . After tH. pylori normally possesses two to six sheathed flagella about 3 μm long at one pole [The flagellum is the locomotory organ that enables the bacterium to move in the ecological niche. one pole . Despiteone pole .H. pylori, providing a synergistic effect for pathological outcome severity in patients [The number of flagella plays a crucial role in the bacterial cell speed; bacteria with more flagella can move faster compared to cells with fewer flagella. One study demonstrated a 19% increase in bacterial speed in bacteria possessing four flagella in a viscous environment compared to cells with three flagella . The bacpatients .After infection, the toll-like receptor (TLR) 5 recognizes the flagellin composed of D0, D1, D2, and D3 domains. TLR5 recognition is important for the immunological events necessary for the inflammatory process. A recent study by Forstneric et al. demonstrated the role of flagellin in the TLR5 recognition evasion. In the study, the residues within the domain D0 play a crucial role in flagellin’s escape from TLR5 recognition .Therefore, urease, bacterial shape, the number of flagella, and motility allow the bacteria to escape the harsh gastric conditions and help establish persistent infections.H. pylori outer membrane protein (HopQ), and other proteins interact with the receptors found on the host epithelial cells, playing a key role in pathological events of persistent infections , SabA, outer inflammatory protein (OipA), fections . This inst cells ,81,82.BabA, an outer membrane adhesin protein, plays a crucial role in bacterial attachment, and it makes a significant contribution to cellular pathogenicity (reviewed by Ansari and Yamaoka ). BabA iH. pylori adheres to the epithelium with the help of BabA as an adhesin on the bacterial surface that interacts with di-fucosylated glycan found on Leb and mono-fucosylated glycan found on H1-antigen, A-antigen, and B-antigen of blood groups O, A, and B respectively [bab gene sequence analysis revealed that there is variability in the middle sequence region and extensive 5′ and 3′ region conservation, particularly between babA and babB. The sequence variability in the middle region of babA suggests that this is the location of BabA’s receptor-binding function [bab gene sequences undergo RecA-dependent intragenomic recombination between these homologous genes , resulting in chimeric genes that may provide benefits to the organisms. The formation of the chimeric gene babB/A has been shown to convert non-Leb binding strains to Leb binding strains, or it can abolish babA-dependent adhesion [ectively ,86,87. Afunction ,88. The adhesion ,90.cagPAI)-independent double-strand breaks in the host cells [H. pylori strains [The BabA binding with Leb has been shown to enhance CagA translocation  and indust cells . In Westst cells ,92. A re strains .H. pylori strains can be of two types. The “specialists” prefer to bind only blood group O-specific glycans, while the “generalists” bind blood group O and blood group A and B-specific glycans [Based on the binding preferences with ABO blood group antigens, the BabA-positive  glycans . The cap glycans .H. pylori chronic infection in human hosts [The OMP SabA binds to gangliosides with fucose substitutions of the N-acetyllactosamine like the dimeric sialyl-Lex antigen. SabA also binds to N-acetyllactosamine-based antigens with terminal α3-linked Neu5Ac, with preferential binding to gangliosides with long N-acetyllactosamine chains . Recentlan hosts .H. pylori colonization density in Leb-negative individuals was reported based on interactions between SabA adhesin and sialylated gastric glycoconjugates [sabA gene sequence can undergo homologous recombination with the homologous gene called sabB, and it can also happen to some extent with hopQ [An enhanced njugates . The sabith hopQ .sabA expression and an increased risk of chronic gastritis [H. pylori-mediated gastric inflammation was shown to alter the gastric mucosa glycosylation with an upregulation of sialyl-Lex antigens and promoting the SabA mediated bacterial attachment to the gastric mucosa [H. pylori, allowing bacterial persistence and gastric pathogenicity establishment [sabA, the cag-pathogenicity island, and babA. According to some authors, the combination of SabA with other virulence factors like OipA and BabA gives the best predictive model by which gastric cancer patients can be distinguished from duodenal ulcer patients and normal individuals [Several studies reported an association between astritis ,98,99, iastritis ,100. H. c mucosa . Therefoividuals .H. pylori OMP family, and it is encoded by the oipA gene. The number of CT dinucleotide repeats in the 5′ signal peptide regulates the expression of OipA via a slipped strand mispairing system. The status is designated as “on” when a functional protein is expressed, while expression of a non-functional protein is designated as “off” [OipA is another virulence protein belonging to the as “off” .oipA gene’s association with chronic gastritis development. Strains expressing functional OipA were most frequently isolated from patients with gastric cancer in Western countries [The strains expressing a functional OipA are reportedly involved in bacterial adherence to the gastric epithelial cells with mucosal damage  and assoountries .oipA possess a higher risk for peptic ulcers and gastric cancer development rather than developing gastritis or functional dyspepsia [cagPAI is exhibited by the OipA positive strains [oipA together with cagA have been suggested to contribute to proliferation and damage to the normal cellular integrity associated with β-catenin signaling [H. pylori OipA was reported to reduce IL-10 expression and dendritic cell (DC) maturation, contributing to chronic H. pylori infection establishment [Furthermore, the results of another study reported that patients infected with strains with “on” status yspepsia . A recen strains . The exiignaling ,110. Morlishment .H. pylori OMP. Sequence analysis of hopQ from unrelated strains revealed two genotypes classified as type I and type II, which exhibit a high level of genetic diversity [H. pylori HopQ contributes to bacterial adherence to epithelial cells interacting with various members of the carcinoembryonic antigen-related cell adhesion molecules (CEACAM) family expressed on the gastric epithelium specifically during gastritis and gastric cancer development [HopQ is another iversity ,113. H. elopment ,115,116.H. pylori HopQ type I genotype is associated with increased peptic ulcer development in the US population [In 2005, Cao et al. reported that the pulation . Consequpulation  and in gpulation ,119. Simpulation .H. pylori HopQ (type I and II) has been recently found in the development of B-cell non-Hodgkin lymphoma and HopQ type II with a higher prevalence [The recent studies highlighted that the interaction between HopQ and CEACAM contributes to gastric colonization  and facievalence .Therefore, OMP adhesins provide bacterial attachment to host cells and provide an additional role in pathogenicity.H. pylori cagPAI is an approximately 40 kb long chromosomal region that contains up to 32 genes encoding a multicomponent bacterial type IV secretion system (T4SS) and an effector protein like CagA [cagPAI are associated with the development of several disorders like chronic gastritis, peptic ulcer diseases, and gastric cancer [cagPAI compared to strains recovered from non-ulcer patients [ike CagA ,124,125.ike CagA ,42,43,44ike CagA ,126. At ike CagA ,128. Thec cancer ,132,133.patients .cagPAI components are essential for T4SS function or they are important for CagA translocation (Some of the location . CagT iscagL gene (HP0539), is expressed on the surface of H. pylori in a T4SS-dependent manner [cagL deletion was shown to phenocopy complete cagPAI knockout in many respects because of its role as an essential T4SS component [H. pylori CagL may also be responsible for H. pylori-induced IL-8 expression via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway, H. pylori-induced hummingbird phenomena (elongation of the cells), and the bridging of the T4SS to its human target cells [Another T4SS component, CagL, a pilin-like component encoded by the t manner ,125 and t manner ,49. The t manner . This RGt manner ,137,138.t manner ,140. Part manner ,142. Theomponent ,143. Thiomponent . Therefoomponent ,147,148.et cells .H. pylori strains possessing cagPAI, and it is absent from less virulent cagPAI negative strains. The synthesized CagA is translocated into the gastric epithelial cells via T4SS. Although H. pylori synthesizes an abundant amount of CagA, a relatively low amount of it is translocated inside the host epithelial cells [H. pylori isolates recovered from Western countries usually possess CagA with EPIYA-A, -B, and -C where the EPIYA-C region can be up to three units long, while the CagA in H. pylori strains recovered from the East Asian countries possess EPIYA-A, -B, and -D sequences. The first two EPIYA sequences, EPIYA-A and EPIYA-B, are carried by almost all CagA, and the third EPIYA sequence (either EPIYA-C in Western strains or EPIYA-D in East Asian strains) is associated with geographic, genotypic, and virulence characteristics [H. pylori infection could enhance YAP expression activation in concert with E-cadherin suppression in chronic gastritis tissues infected with H. pylori compared to H. pylori negative patients [The effector protein CagA is 125–145 kDa. It is expressed by the highly virulent al cells . The traal cells ,151. Theeristics . After ieristics . The phoeristics ,154,155 eristics ,53. A repatients .The CagA with EPIYA-D motif has stronger binding affinity with SHP2 than the EPIYA-C motif . TherefoH. pylori pore-forming cytotoxin that plays a crucial role in pathogenicity by interacting with gastric epithelial cells [VacA is an important al cells ,164. Theal cells .H. pylori isolates indicates the s1a, s1b, s1c, and s2 sub-families of the “s“ region. m1 and m2 are sub-families of the “m“ region. i1, i2, and i3 are sub-families of the “i“ region [The three heterogenic regions of VacA have significant sequence variation. The “s” region represents the sequence variation in the amino-terminal signal sequence. The “m” region represents the amino acid variation located in the middle of the p55 domain. The “i” region was recently identified in a survey conducted in the Iranian population, and it represents the amino acid variation in the intermediate region located between the “s“ and “m“ region in the p33 domain ,167,168.“ region ,170. The“ region .vacA transcription than s2 type strains [The studies have revealed that the combination of different sequences in the three regions can determine the capability of vacuolation. Moreover, the genotype possessing the combination s1/m1 exhibits high vacuolating activity, and the genotype s1/m2 has intermediate activity. On the other hand, no vacuolating activity was shown by the genotype s2/m2 . This fi strains . TherefoH. pylori isolates from ulcer patients, a higher prevalence of VacA with s1a, m1, and i1 genotypes have been detected compared to patients with non-ulcer diseases [H. pylori harboring vacA with s1 or m1 regions [vacA i1 type harboring H. pylori are associated with a high risk for gastric cancer development (OR = 10.9–15.0) [H. pylori VacA is significantly associated with the risk of extra-gastric diseases such as colorectal cancer development, particularly in African Americans [In clinical diseases . A meta- regions . On the .9–15.0) . The ant.9–15.0) . A recen.9–15.0) . Furthermericans .H. pylori inside the gastric epithelial cells [H. pylori VacA can play an important role in a transient receptor potential membrane channel mucolopin 1 (TRPML1) activity that inhibits the lysosomal and autophagic killing of bacterial cells to promote the establishment of an intracellular niche that allows for bacterial survival [VacA plays several roles in cellular pathogenicity , and it al cells ,180,181.al cells . This susurvival .H. pylori [H. pylori HtrA is highly resistant to temperature and pH variations, which indicates its ability for adaptation and causing colonization and persistent infection even in harsh stomach conditions [Organisms are continuously exposed to oxidative and heat stress that can kill them. However, organisms tolerate these stresses and degrade intracellular irreversibly misfolded proteins that may be toxic to the organisms ,63. The . pylori ,189,190.nditions ,192.H. pylori HtrA protein was found to cleave the proteins of tight junctions like occludin, claudin-8, and the E-cadherin molecules on the gastric epithelial cells, which are important adherens junction proteins and tumor suppressors. Moreover, their destruction has been strongly connected with the progression and metastasis of gastric tumors in humans [H. pylori across the polarized gastric epithelial linings [H. pylori is one of the highly conserved genes to be found in isolates from Europe, Asia, North America, and South America. This conservation suggests that there is little or no effect on the htrA gene region over the course of H. pylori and human co-evolution like the H. pylori MLST housekeeping genes [The extracellularly exported HtrA enables the bacteria to deliver the virulence factor like CagA to the basolateral membrane of host cells despite infection initiation at the apical side ,66,67,68n humans ,70,71. T linings ,70,193. H. pylori during their growth in logarithmic phase [H. pylori OMVs have been reported to protect the pathogen from the toxic effect of the reactive oxygen species respiratory burst [Outer membrane vesicles (OMVs) are characterized as blebs of 20–300 nm in diameter which are naturally secreted by several Gram-negative bacteria including ic phase ,195,196.ic phase ,200,201.ic phase . Moreovery burst .H. pylori OMVs have been also found to induce dendritic cells by upregulating the expression of co-stimulatory molecules and expression of more heme oxygenase-1 via the activation of Akt-Nrf2 and mTOR-κB Kinase–NF-κB pathways [H. pylori OMVs in the gastric juice of patients with gastric cancer [H. pylori cells and the H. pylori-derived OMVs compared to healthy controls. Furthermore, these H. pylori-derived OMVs also induced inflammation in the mouse model, inducing gastric cancer development [OMVs have been detected intracellularly and extracellularly in gastric biopsy specimens ,200,203.pathways ,75. Morec cancer ,207. In H. pylori γ-glutamyl transpeptidase (gGT) is an enzyme that catalyzes the transpeptidation and hydrolysis of the γ-glutamyl moiety of glutathione and glutathione-conjugated compounds to amino acids [H. pylori infection in mice, the deletion of gGT has no inhibitory effect on the ability to grow on the culture media [H. pylori infection indicates that in infection with gGT-deficient strains, the colonization is reduced in comparison to the infection with the isogenic wild type strains. The consistent results in these two animal models also suggest that the presence of gGT provides at least some advantage to allow H. pylori to infect epithelial cells [H. pylori gGT provides a growth advantage within the gut by salvaging extracellular glutathione to obtain cysteine for subsequent protein synthesis and the activity is greatest at a neutral pH [no acids . Althougre media . A studyal cells . The stuutral pH ,210. Recutral pH . Moreoveutral pH .H. pylori gGT induces apoptosis, inhibits gastric cell proliferation, arrests the cell cycle, and generates reactive oxygen species [H. pylori with higher activity of gGT exhibits more severe gastro-duodenal diseases. species ,213,214  species . Further species . The enhH. pylori infection and associated gastric cancer [cagPAI encoding the T4SS, another putative gene cluster with low G + C content of around 35% encoding the T4SS in H. pylori is located within the integrating conjugative elements (ICE). Recently, we reported the role of the specific variants of H. pylori ICE T4SS (ICEhptfs) in pathogenicity. In the study, the cagPAI-mediated acute inflammation in the antrum and body was ICEhptfs-dependent, and this dependency was also attributed to the higher atrophy score in antrum [H. pylori CGT utilization for the promotion of gastric carcinogenesis [Despite of the above-mentioned well-characterized virulence factors, several studies have depicted the association of other virulence factors in the severity of gastric complications. A recent study conducted by Capitani et al. described the possible role of the HP1454, a secreted protein and component of OMVs, in the Th1 and Th17-mediated inflammatory response in the chronic c cancer . In addin antrum . The expogenesis .In recent years, the whole genome sequencing (WGS)-based approach from cultured bacterial isolates has emerged as one of the most comprehensive tools for surveillance study, drug resistance determination, and evolutionary analysis of infectious diseases ,223,224.Listeria monocytogenes strains to determine disease epidemiology, the presence of drug resistance genes, and to determine virulence factors [Plesiomonas shigelloides [Recently, the WGS has been used to characterize a variety of pathogens to determine the novel or existing virulence factors. In a study, Hurley et al. performed WGS-based characterization of 100  factors . Similarelloides . Furtherelloides . The resH. pylori [H. pylori . Full cagL and cagI sequences were analyzed for single nucleotide variation and amino acid changes. The WGS results identified several putative novel variants of CagL and CagI sequences, proving its usefulness in virulence determination [H. pylori to search for the novel virulence factors using genomic information from strains isolated from gastric cancer and matched controls like isolates from the duodenal ulcers. Berthenet et al. used GWAS in their recent study to illustrate the association of the presence of the cagPAI genes cag11 (cagV), cag12 (cagU), and cag 20 (cagI) in H. pylori isolated from patients with gastric cancer [Currently, the WGS tool is mostly being used to determine the presence of antibiotic resistance genes in . pylori ,231,232 . pylori . Howevermination . The seamination . Recentlc cancer .H. pylori promotes carcinogenesis by influencing the composition of the microbial population (microbiota) in the stomach [Escherichia coli, Lactobacillus spp., Nitrospirae spp., Clostridium spp., Veillonella spp., Haemophilus spp., and Staphylococcus spp. that convert nitrogen compounds to potentially carcinogenic N-nitroso compounds in gastric fluid enhance cancer development [Streptococcus spp., Prevotella spp., and Neisseria spp. are associated with a lower risk for gastric cancer development [Pasteurella stomatis, Spodoptera exigua, Parvimonas micra, Streptococcus anginosus, and Dialister pneumosintes exhibit a strong co-occurrence in gastric carcinogenesis [Lactobacillus spp., Escherichia coli, Shigella spp., Nitrospirae spp., Burkholderia fungorum, and Lachnospiraceae spp.) in gastric cancer patients, where Nirospirae spp. was observed in all gastric cancer patients but not in chronic gastritis patients [H. pylori-positive gastric precancerous lesions, which suggests the possible association of these phyla with the progression of H. pylori related gastric lesions [H. pylori with Methylobacillus and Arthrobacter, respectively, in severe gastritis has been observed, whereas co-excluding interactions with members of the phylum Firmicutes  and co-occurrence interactions with Prevotella, Moryella, and another helicobacter (H. ganmani) have been reported [ stomach ,238,239; stomach ,243,244.elopment ,246,247,elopment . In cont lesions . The co-H. pylori infections contribute the highest risk for developing severe gastric diseases. Therefore, the virulence factors characterized in the isolated strains will provide a clue for the disease prediction in the populations. Currently, WGS and GWAS-based investigations and their meaningful results for the putative virulence and drug resistance determinants have encouraged researchers to perform more extensive and multidisciplinary efforts for better understanding of virulence factor involvement in the onset and progression of gastric complications.This study depicts the importance of several virulence factors playing a role in the severity of gastric complications.


Disruption of the monoaminergic system, e.g. by sleep deprivation (SD), seems to promote certain diseases. Assessment of monoamine levels over the circadian cycle, during different sleep stages and during SD is instrumental to understand the molecular dynamics during and after SD. To provide a complete overview of all available evidence, we performed a systematic review. A comprehensive search was performed for microdialysis and certain monoamines , certain monoamine metabolites , 5-hydroxyindoleacetic acid (5-HIAA)) and a precursor (5-hydroxytryptophan (5-HTP)) in PubMed and EMBASE. After screening of the search results by two independent reviewers, 94 publications were included. All results were tabulated and described qualitatively. Network-meta analyses (NMAs) were performed to compare noradrenaline and serotonin concentrations between sleep stages. We further present experimental monoamine data from the medial prefrontal cortical (mPFC). Monoamine levels varied with brain region and circadian cycle. During sleep, monoamine levels generally decreased compared to wake. These qualitative observations were supported by the NMAs: noradrenaline and serotonin levels decreased from wakefulness to slow wave sleep and decreased further during Rapid Eye Movement sleep. In contrast, monoamine levels generally increased during SD, and sometimes remained high even during subsequent recovery. Decreases during or after SD were only reported for serotonin. In our experiment, SD did not affect any of the mPFC monoamine levels. Concluding, monoamine levels vary over the light-dark cycle and between sleep stages. SD modifies the patterns, with effects sometimes lasting beyond the SD period. Circadian rhythms (CRs) and sleep are influenced by multiple external (e.g. light) and internal (e.g. accumulation of hypnogenic substances) factors . Our relUnusual working hours can result in low sleep quality, consumption of stimulants and/or hypnotics, and stress. These factors could all contribute to sleep-deprivation associated disorders  4567.456Sleep deprivation (SD) induces severe cognitive impairments such as loss of attention, increased reaction times, impaired multitasking and planning, slurred speech, impaired memory, and poor emotion regulation 910. Main9122While the behavioural impact of SD is well-known, our knowledge of the responsible underlying neurological mechanism is still limited. Sleep is embedded in a complex network of interconnected brain regions using multiple neurotransmitters and neuromodulators, within which the monoaminergic pathways seem responsible for sleep-wake modulation 16. In ad1718in vivo, based on the simple principle of diffusion , p < 0.01 and I2 = 66%. The overall effect for wakefulness versus REM was –1.61 (SMD) with CI 95% , p < 0.01 and I2 = 68% (appendix 5–6).For serotonin, concentrations during SWS and REM both showed significant decreases compared to wake; p < 0.0001  SWS , REM ; I2 = 99.5%). Our sensitivity analyses again confirmed these findings; the overall effect for SWS versus wakefulness was –1.54 (SMD) with CI 95% , p = 0.01 and I2 = 53%. The overall effect for wakefulness versus REM was –2.58 (SMD) with CI 95% , p < 0.01 and I2 = 80% (appendix 7–8).For noradrenaline, the concentrations during SWS and REM also showed significant decreases compared to wake; p < 0.0001 . Similar patterns were observed for serotonin levels in the frontal cortex and in the hippocampus, albeit with a lower amplitude (115). The SD-induced changes in the hippocampus could be specific to parts of this brain region. Serotonin levels were observed to decrease during SD in the ventral hippocampus, while increases were observed in the posterior hippocampus. Findings during recovery were inconsistent; both a decrease and an increase compared to baseline were observed for serotonin .2(3) = 8.486, p = 0.037 , the dorsal and median raphe nuclei (serotonin), the ventral periaqueductal grey, the substantia nigra and the ventral tegmental area (dopamine). More specifically, monoamines were thought to inhibit sleep-promoting regions such as the ventrolateral optic area (VLPO) 16. RecenWhile our systematic review focusses on monoamines, these neuromodulators do not act in isolation. For instance, the SCN provides input to the above-mentioned brainstem nuclei to synchronize sleep-wake regulation with the environmental light-dark cycle. Glutamate and acetylcholine release in the SCN depends on input from the laterodorsal and pedunculopontine tegmental nuclei , and theWe exclusively addressed the release of monoaminergic neurotransmitters. Neurotransmitters exert their actions via binding to receptors. It is important to also analyze patterns in the expression of these receptors. Circadian variations in receptor expression have been shown for e.g. adrenergic, muscarinergic, opioidergic, gabaergic, and dopaminergic receptors . BesidesOur review shows that while monoamine fluctuations differed between brain regions and monoamines, overall the monoamine concentrations seem to be higher during the active dark phase than during the inactive light phase. However, fluctuations also vary with factors such as sex, age, BMI, genetic status, temperature, season, and humidity 146147. S146148150151152153148156The conclusions we can draw are limited by the overall amount of evidence; the number of studies per condition is low. Mainly SD studies and adrenaline studies are currently underrepresented in the literature . New literature has probably appeared since we performed our search in September 2017. As systematic reviews generally take over a year from start to completion , a lag tOverall conclusions on monoamine neurochemistry in relation to sleep and wake are further limited by the variations in experimental designs between the included studies. Heterogeneity was observed for e.g. species, group size, brain region, experimental duration, L/D cycle, type of SD (reviewed by ), flow rThe SD method itself could also affect monoamine concentrations, for example via stress. SD can be stressful for the animals because of e.g. social isolation, humidity, and/or restricted or forced locomotion. In SD studies, it is challenging to implement appropriate controls. While some SD techniques are considered less stressful (e.g. gentle handling) than others (e.g. disk over water), they all are intrusive and probably stressful when chronic.Several studies have analysed SD-induced stress in animals 39160161162160161The overall risk of bias for the studies included in our review is difficult to estimate because of poor reporting of experimental procedures. For example, reporting of power calculations and randomisation was mostly absent. Many details of the microdialysis technique were reported well; sample time, perfusion rate, matrix type, and analysis technique were reported in more than 96% of the included studies, and probe length and membrane type were reported in more than 85%. However, reporting of recovery and verification of probe placement could have been better. The list of questions we developed for risk of bias assessment of microdialysis studies (appendix 4) seems to provide an adequate reflection of the technique-related modulating factors . We recoWe compared the noradrenaline and serotonin concentrations between the 3 sleep-wake stages usually distinguished in rodents  with NMAs. Only noradrenaline and serotonin were analysed, as the total number of studies of other monoamines was low, and the heterogeneity between study designs was considered too high for meta-analyses in general. The results of our NMAs were consistent with pairwise comparisons of sleep stages with classic meta-analysis techniques. NMAs have previously been used for clinical trials; to compare treatment effects for more than two treatments, and even to rank a series of treatments in efficacy without direct comparisons being available . The NMAOur primary data suggest that monoamine levels in the medial prefrontal cortex (mPFC) are stable during and after SD. The mPFC is involved in several sleep-dependent processes such as attention, memory, incentive processing, decision making, and emotional regulation, which rely, at least partially, on the monoaminergic pathways 1719165166.19165167Our systematic review provides a complete overview of the previously published SD- and CR-related monoamine data in the mPFC. Concerning CR, in rats, 7 CRs studies show that monoamine levels fluctuated with CRs. Dopamine, noradrenaline, 5-HT, and 5-HIAA levels augmented during the dark phase and decreased or remained stable during light phase. DOPAC levels were higher during the dark phase than during the light phase. Concerning SD, only 1 SD study in mice showed that noradrenaline levels increased during SD.In contrast to these preceding studies, our primary data show no variation in SD- and CR-related mPFC monoamines, except for DOPAC levels, which were higher during baseline light than during baseline dark. The variability of our data is high, probably due to occasional blockages of the microdialysis set-up. However, in the same samples, we did find normal circadian curves and minimal effects of SD for corticosterone . We usedSleep is required for several fundamental physiological processes, extending beyond the monoaminergic pathways. This paper shows that monoamines fluctuate with CRs, sleep stages, and SD. The monoamines are affected by several factors including e.g. brain region, species, sex, age, BMI, genetic status, temperature, season and humidity. Monoamines may not be part of the basic mechanism underlying the regulation of the sleep-wake cycle . HoweverThe additional files for this article can be found as follows:10.5334/jcr.174.s1Appendix 1.Study characteristics of included publications on circadian rhythms.Click here for additional data file.10.5334/jcr.174.s1Appendix 2.Study characteristics of included publications on sleep stages.Click here for additional data file.10.5334/jcr.174.s1Appendix 3.Study characteristic of included publications on sleep deprivation.Click here for additional data file.10.5334/jcr.174.s1Appendix 4.Risk of bias – questions adapted to microdialysis studies.Click here for additional data file.10.5334/jcr.174.s1Appendix 5.Forest plot comparing serotonin concentration (nanomole/L) during wake and slow waves sleep.Click here for additional data file.10.5334/jcr.174.s1Appendix 6.Forest plot comparing serotonin concentration (nanomole/L) during wake and rapid-eye movement sleep.Click here for additional data file.10.5334/jcr.174.s1Appendix 7.Forest plot comparing noradrenaline concentration (nanomole/L) during wake and slow waves sleep.Click here for additional data file.10.5334/jcr.174.s1Appendix 8.Forest plot comparing noradrenaline concentration (nanomole/L) during wake and rapid-eye movement sleep.Click here for additional data file.


Parasteatoda tepidariorum, Pholcus phalangioides, Centruroides sculpturatus, and Mesobuthus martensii) compared with nonarachnopulmonate arachnids . To assess divergence in the roles of homeobox ohnologs, we analyzed the expression of P. tepidariorum homeobox genes during embryogenesis and found pervasive changes in the level and timing of their expression. Furthermore, we compared the spatial expression of a subset of P. tepidariorum ohnologs with their single copy orthologs in P. opilio embryos. We found evidence for likely subfunctionlization and neofunctionalization of these genes in the spider. Overall our results show a high level of retention of homeobox genes in spiders and scorpions post-WGD, which is likely to have made a major contribution to their developmental evolution and diversification through pervasive subfunctionlization and neofunctionalization, and paralleling the outcomes of WGD in vertebrates.Homeobox genes are key toolkit genes that regulate the development of metazoans and changes in their regulation and copy number have contributed to the evolution of phenotypic diversity. We recently identified a whole genome duplication (WGD) event that occurred in an ancestor of spiders and scorpions (Arachnopulmonata), and that many homeobox genes, including two Hox clusters, appear to have been retained in arachnopulmonates. To better understand the consequences of this ancient WGD and the evolution of arachnid homeobox genes, we have characterized and compared the homeobox repertoires in a range of arachnids. We found that many families and clusters of these genes are duplicated in all studied arachnopulmonates ( Developmental programs precisely orchestrate proliferation and differentiation to build multicellular organisms. Many of the key regulatory factors and pathways utilized in development are conserved between species like the Wnt and Delta/Notch signaling pathways and transcription factors (TF) such as those encoded by the homeobox genes  in a common ancestor of arachnopulmonates , we identified 78 homeobox families     online wrachnids  online.The assignment of homeobox genes into families was verified using a maximum likelihood tree constructed using the homeodomain sequences  online. Comparisons of the repertoires of homeobox families between these species suggest particular patterns of retention and loss of homeobox families in arthropod lineages . OverallFamilies that were present in vertebrates, arachnids and the myriapod, but absent in insects were the HNF and Dmbx families , T. castaneum (105), and D. melanogaster (104) . However, of these five genes only Msx is duplicated, though the other two Msx paralogs are not located in the NK clusters. This indicates differential retention/loss between these duplicate NK clusters in P. tepidariorum.One homeobox cluster that is present across protostomes and deuterostomes is the NK cluster . In P. tscaffold A. Each oP. tepidariorum. There is evidence for a duplication of the SINE/Six cluster on scaffolds #121 and #1185 , and Six4/5   B. This ising ISH B  paralogs, which have diverged in the level and timing of their expression A. For exd Pt-Zfh A, as welSeq data A, which dariorum . The Pt-n starts A. TherefPt-Pitx, Pt-Phox, and Pt-Vvl  and two dispersed (Pt-Irx3 and Pt-Irx4)  C. The taPt-Irx4) A, while t levels A.Pt-Lim1/5 paralogs on the same scaffold had very similar expression, with low levels at S3 but stronger expression at S10  that appear to have undergone subfunctionalization. In the Emx family, the expression pattern of the single copy of Po-Emx is subdivided between the four paralogs found in P. tepidariorum . In this family, Pt-Irx1, Pt-Irx2, and Pt-Irx4 appear to have subdivided the expression pattern between them compared with Po-Irx . However, Pt-Irx4 expression extends more laterally in the opisthosomal segments, compared with Pt-Irx1. Furthermore, the onset of Pt-Irx4 expression is earlier and continues until later in embryogenesis compared with Pt-Irx1. The other expression domain of Po-Irx around the dorsal boundary edge of the germ band is shared with the Pt-Irx2 paralog. Finally, Pt-Irx3 has possibly gained a completely new domain in the prosomal appendages of later stages and therefore possibly represents another case of neofunctionalization in P. tepidariorum , expression during embryogenesis was observed in the precheliceral region, which closely resembled the expression of Pt-Pitx2 in precheliceral region of this spider , indicating possible subfunctionalization of Pt-Pitx paralogs.For the single copy of Po-Pitx U–W′, exps spider Y and Z. paralogs X–Z″, indPt-Zfh paralogs have also undergone expression divergence , where one paralog has retained the likely ancestral pattern as compared with M. martensii , P. opilio (3%), pseudoscorpions (19% and 6%), and several mandibulates (19%) (P. tepidariorum or C. sculpturatus is greater than found in either the BUSCO (41%) or OMA (20.5%) data sets (P. tepidariorum and C. sculpturatus compared with mandibulates and ticks with respect to the BUSCO-Ar database.We found many more duplicated homeobox families in arachnopulmonate species (51–59%) compared with other arthropods surveyed, including es (19%) . Indeed,P. opilio, I. scapularis, and T. urticae  is consistent with their predicted order in the protostome–deuterostome ancestor , SINE, TALE, and LIM class genes , as well as venom and silk genes , being responsible for patterning the distal boundary of the arachnid-specific podomere, the patella .The expression of pression , suggestpression U–Z″.Zfh in Drosophila and P. opilio are also very similar, again implying the ancestral expression of this gene , the harvestman Phalangium opilio (PRJNA236471), and the pseudoscorpion Hesperochernes sp. (PRJNA254752).To identify homeobox genes in arachnid species, we analyzed both existing resources and also new transcriptomic data generated in this study. Existing protein predictions were collected for the tick Pholcus phalangioides and the pseudoscorpion Neobisium carcinoides. For P. phalangioides RNA isolation, library preparation and sequencing with Illumina HiSeq2000 was previously described  and the pseudoscorpion Hesperochernes sp. (PRJNA254752) using TransDecoder v3.0.0 (−6) (Longest open reading frames (ORFs) were predicted from the transcriptomes of 0.0 (−6) , and HMM0.0 (−6)  using thP. phalangioides, I. scapularis, P. opilio, and the two pseudoscorpions were then searched for the presence of homeodomain sequences using BLASTP v2.2.28+ (P. tepidariorum (C. sculpturatus (M. martensii (Strigamia maritima (Pros gene (MMa30254) in M. martensii not reported previously by The protein sequences from v2.2.28+  combinedP. tepidariorum and P. phalangioides), two scorpions (C. sculpturatus and M. martensii) two pseudoscorpions (Hesperochernes sp. and N. carcinoides), the harvestman P. opilio, the tick I. scapularis, the myriapod (centipede) S. maritima and three insects Apis mellifera, Tribolium castaneum, and Drosophila melanogaster were aligned with ClustalW . Homeodomain proteins were classified based on the homology of their homeodomains to known homeodomain containing proteins and annotated with nomenclature following that of Phylogenetic analyses, using only unique homeodomain sequence alignments, were performed in RAxML, with support levels estimated using the rapid bootstrap algorithm (1000 replicates)  from 10–100 embryos of each successive developmental stage  was maintained at Oxford Brookes University and fed on a diet of Drosophila vestigial mutants and Gryllodes sigillatus, with a 12:12 h light:dark cycle at 25°C. The culture of P. opilio was maintained at the University of Wisconsin, Madison, WI and fed on a diet of fish flakes supplemented with Acheta domesticus nymphs, with a 14: 10 light: dark cycle at 20°C.An inbred culture of P. tepidariorum embryos and from S7 to S17 for P. opilio. Gene fragments were amplified by PCR and cloned into the TOPO-TA vector (ThermoFisher Scientific). Primer sequences are provided in cDNA was generated using QuantiTech (Qiagen) with RNA extracted (Qiazol) from S1 to S14 P. tepidariorum and P. opilio was performed as previously described (Colourmetric in situ hybridization (ISH) for Supplementary DataClick here for additional data file.


Enterococcus faecalis is commonly present in the gastro-intestinal microbiota of warm-blooded animals and insect guts. These bacteria can also cause infection and disease in immunocompromised individuals. In the present study, we performed whole genome analysis in Enterococcus strains finding that the complete citrate pathway is present in all of the E. faecalis strains isolated from such diverse habitats as animals, hospitals, water, milk, plants, insects, cheese, etc. These results indicate the importance of this metabolic preservation for persistence and growth of E. faecalis in different niches. We also analyzed the role of citrate metabolism in the E. faecalis pathogenicity. We found that an E. faecalis citrate fermentation-deficient strain was less pathogenic for Galleria mellonella larvae than the wild type. Furthermore, strains with deletions in the oxaloacetate decarboxylase subunits or in the α-acetolactate synthase resulted also less virulent than the wild type strain. We also observed that citrate promoters are induced in blood, urine and also in the hemolymph of G. mellonella. In addition, we showed that citrate fermentation allows E. faecalis to grow better in blood, urine and G. mellonella. The results presented here clearly indicate that citrate fermentation plays an important role in E. faecalis opportunistic pathogenic behavior.Citrate is an ubiquitous compound in nature. However, citrate fermentation is present only in a few pathogenic or nonpathogenic microorganisms. The citrate fermentation pathway includes a citrate transporter, a citrate lyase complex, an oxaloacetate decarboxylase and a regulatory system.  In addition, citrate is extensively used as preservative in food and beverages. Different bacteria are able to utilize citrate via the citrate lyase pathway. This metabolism consists primarily of a transport system, which incorporates citrate into the cell, a citrate lyase complex (CL), which disrupts the molecule and an oxaloacetate decarboxylase enzyme (OAD), which produces pyruvate as a final product. Expression of the complete route is also subjected to fine regulation through different systems  1, 2]. , 2. 1, 2Lactococcus lactis , wh, whcit gculation . In addiE. faecalis are not common, Maadani et al. reported that an E. faecalis strain unable to metabolize ethanolamine was less virulent in the C. elegans model [Salmonella species and Listeria monocytogenes [Examples of metabolic pathways associated with virulence in ns model . Ethanolytogenes .E. faecalis a commonly found cause of bacteremia and urinary tract infections [cit cluster reducing approximately 50% the activity of PcitHO promoter and 15% the activity of the catabolic operon PcitCL promoter [E. faecalis infection.Its ability to survive in hospital environments and to infect immunocompromised patients has made fections –25, 48. fections . Under tpromoter . Neverthpromoter  a value E. faecalis to cause infection would not only implicate an organized regulation of several virulence factors and expression of genetic determinants, but also an adaptation of the bacterial cell physiology during the infection process [It has been proposed that the ability of  process . This suE. faecalis grown in blood and urine has shown that several known virulence factors, such as the fsrB (EF1821), gelE (EF1818), cpsC (EF2493), ace (EF1099) and efaA (EF2076) were modulated in their expression under both growth conditions [E. faecalis infection in subdermal chambers, where citrate metabolism induction was also observed [alsS, EF1213), α-acetolactate decarboxylase , pyruvate dehydrogenase complex  and lactate dehydrogenase  were induced , alcohol dehydrogenase  were repressed Click here for additional data file.S2 FigE. faecalis JH2-2 (red circle) and JH2-2-Cit- (green down triangle) strains in LB (empty symbols) or LB supplemented with citrate (closed symbols). (B) Growth of E. faecalis JH2-2-OadA- (yellow up triangle) and JH2-2-OadA-/pOadA (cyan diamond) strains in LB (empty symbols) or LB supplemented with citrate (closed symbols). The data points correspond to the mean ± standard error of three replicates.(A) Growth of (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Streptococcus genus is relevant to both public health and food safety because of its ability to cause pathogenic infections. It is well-represented (>100 genomes) in publicly available databases. Streptococci are ubiquitous, with multiple sources of isolation, from human pathogens to dairy products. The Streptococcus genus has traditionally been classified by morphology, serum types, the 16S ribosomal RNA (rRNA) gene, and multi-locus sequence types subject to in-depth comparative genomic analysis.The Streptococcus species. The core genome nucleotide diversity was calculated and compared to phylogenomic distances within the genus Streptococcus. The core genome was also used as a resource to recruit metagenomic fragment reads from streptococci dominated environments. A conventional 16S rRNA gene phylogeny reconstruction was used as a reference to compare the resulting dendrograms of average nucleotide identity (ANI) and genome similarity score (GSS) dendrograms.Core and pan-genomes described the genomic diversity of 108 strains belonging to 16 Streptococcus. The average identity of the pairwise compared core proteins decreases proportionally to GSS lower scores, across species. The GSS dendrogram recovers most of the clades in the 16S rRNA gene phylogeny while distinguishing between 16S polytomies (unresolved nodes). The GSS is a distance metric that can reflect evolutionary history comparing orthologous proteins. Additionally, GSS resulted in the most useful metric for genus and species comparisons, where ANI metrics failed due to false positives when comparing different species.The core genome, in this work, consists of 404 proteins that are shared by all 108 Understanding of genomic variability and species relatedness is the goal of tools like GSS, which makes use of the maximum pairwise shared orthologous sequences for its calculation. It allows for long evolutionary distances (above species) to be included because of the use of amino acid alignment scores, rather than nucleotides, and normalizing by positive matches. Newly sequenced species and strains could be easily placed into GSS dendrograms to infer overall genomic relatedness. The GSS is not restricted to ubiquitous conservancy of gene features; thus, it reflects the mosaic-structure and dynamism of gene acquisition and loss in bacterial genomes. Streptococcus is a bacterial genus with more than 50 species. The health and environmental importance of streptococci include a diverse range of human and animal pathogens like the etiological agents for caries and meningitis as well as commensal species inhabiting the intestinal and respiratory tracts of animals  gene phylogenies  above 95% for estimating an overall genome-related index . TraditiBacillus genus were determined to share 814 core genes which defined specific genus features like the ability to build endospores  allows for pan-genomic phylogenomics . The cordospores .Streptococcus are differential for causing caries (S. mutans) or health (S. dentisani)  . The recntisani) . The paintisani) .Streptococcus are differential for causing caries (S. mutans) or health (S. dentisani)  . We calcStreptococcus, representing 16 different species were downloaded from National Center for Biotechnology Information (NCBI) Genbank, based on a manually curated genome list  as out-groups for the comparisons of Streptococci GSS values. An inverse (1-GSS) distance matrix was built and used to compute a neighbor-joining tree using the APE library v. 3.5 . In the GSS, gene content is considered and nonmatching RBH adds a 0 value in the numerator. Since self-Score might differ in proteome y v. 3.5  for R v.y v. 3.5 . GSS caly v. 3.5 .cd-hit-est  within each genome was performed using -hit-est  clusteri-hit-est . The res-hit-est .−6, mask low complexity regions of the query sequence only during the search phase and perform an alignment with the Smith–Waterman algorithm to compute the bit score. Hits with an alignment length shorter than 60% of the length of the query sequence were discarded. Detailed scripting procedure of RBH is available  comprised 33,039 protein clusters (families) at 70% identity . AccordiS. pyogenes  of the pan-genome against the core were compared to the over-represented protein categories in the core  (S. pneumoniae (S. mutans was depleted (17 metagenomic fragments) in the caries-free individual (NOCA_01) and abundant (127 metagenomic fragments) in the patient with caries. Recruiting metagenomic sequences against each reference core genome and filtering alignments with high identity levels (≥90%) show that is possible to generate species-specific profiles  . Two orato >20%) . In botheumoniae , but theprofiles .S. pyogenes, S. dysgalactiae, S. equi, S. uberis, S. parauberis, S. agalactiae, and S. pneumoniae. A second clade is the salivarius group formed just by S. thermophilus and S. salivarius. The mutans clade groups the following species: S. mutans, S. infantarius, S. lutetiensis, S. macedonicus, and S. gallolyticus. The S. suis has its clade with multiple strains of the same species. A fifth clade known as the mitis group is the basal group: S. pneumoniae, S. pseudopneumoniae, S. mitis, S. pasteurianus, S. parasanguinis, S. sanguinis, S.gordonii, S. oligofermentans, and S. intermedius. The external groups are B. subtilis and B. licheniformis.A 16S rRNA phylogenetic reconstruction was done as a reference and confirms previously proposed clades  Kawamur. There iAverage nucleotide identity was calculated for all the Streptococci genomes and it was able to discriminate between main pyogenic and suis clades . HoweverS. agalactiae is included interior to the pyogenic clade in the 16S phylogeny and GSS shows S. agalactiae as the basal group for the pyogenic clade. Another rearrangement of GSS is the suis group, which is normally a sister clade to the mitis group, but in the 16S rRNA phylogeny, suis is placed as a sister clade to the pyogenic group. It is noticeable that the GSS dendrogram distances are vast enough to distinguish discrete groups among closely related strains like such as the inner clades of suis, pyogenic, mutans, and mitis groups. There are resolved clades in the GSS dendrogram for strains of S. pneumoniae and S. pseudopneumoniae; whereas, 16S rRNA does not distinguish close relationships, but instead allows polytomies. Also, the suis GSS clade shows resolved branching when comparing to the 16S rRNA phylogeny.The GSS dendrogram has the same clades as the 16S rRNA , howeverStreptococcus species have historically been classified by their cell wall antigenic properties  and promiscuous genomic regions resulting from HGT . If therA long-range genomic comparison, like the genus-wide presented here, amino acids are a far superior choice for deep-diverging organisms like bacteria. The amino acid search of homologous proteins reduces the number of false positives, increases the chance of finding remote homologous sequences, and eliminates missed alignments because of differential codon usage in each species. Sequence conservation in amino acids, due to functional constraints in the proteins, reduces the amount of sequence while adding the possibility of similar changes. The GSS takes all the perks of using amino acids for homologous searching when compared to ANI or individual genes used as phylogenetic markers. Additionally, the GSS offers a real-world advantage for most current bacterial draft genome sequences; it can identify most of the pairwise shared orthologs to estimate a global similarity using bit scores that summarize alignment quality. Finally, the alignment quality is a proxy to the evolutionary story of the compared organisms. The previously exposed reasons support the GSS a reliable option to perform phylogenomic analysis and place newly sequenced bacteria in context while including the genomic mosaicism of bacteria which is closer to their genome dynamics that only account for universal shared genes to make phylogenetic inferences.Streptococcus species. The core genome represents the phylogenetic coherence of the group and each protein can be used as a phylogenetic marker. In addition to their functionality as phylogenetic markers, core genes allow making a metabolic inventory of what proteome functions are essential for this genus. In the streptococci core genome, the most common phylogenetic markers such as ribosomal biogenesis genes are expectedly reported, but new therapeutic targets arise with genes such as phosphatases, transporters, and even hypothetical conserved proteins. The utility of GSS to maximize the amount of comparative homologous information is demonstrated, being practical to resolve genomic similarity relationships among genomes using the maximum amount of genomic information shared by pairs of comparison genomes. The GSS has the resolution to distinguish within similar strains while avoiding false positives as those observed with the ANI in genus-level comparisons. Finally, the GSS can be used as a proxy for the genomic dynamics and evolutionary history of microorganisms.In this work, we present a comparative genomics analysis using the streptococci core genome with a proposal of 404 conserved proteins in 108 strains belonging to 16 10.7717/peerj.6233/supp-1Supplemental Information 1Information included: Strains names, CDS number, NCBI accession numbers, isolation sources.Click here for additional data file.10.7717/peerj.6233/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.6233/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.6233/supp-4Supplemental Information 4GSS calculations jupyter notebook.Click here for additional data file.10.7717/peerj.6233/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.6233/supp-6Supplemental Information 6Additionally, each streptococci species core genome and orthologous genes shared between strains.Click here for additional data file.10.7717/peerj.6233/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.6233/supp-8Supplemental Information 8Calculated by core genome fragment recruitment, the lowest common ancestor (LCA), and 16S rRNA gene abundances.Click here for additional data file.10.7717/peerj.6233/supp-9Supplemental Information 9S. mutans is depleted in the healthy individual while abundant metagenomic reads are identified in caries patient.(A) Histogram showing the species gene relative abundance in metagenomic reads from a caries patient. (B) Metagenomic reads from a patient with caries, and C) Metagenomic reads from a healthy individual (caries-free) were aligned against the core genomes of 10 different species of Streptococci. (D) Histogram representing the species gene relative abundance in metagenomic reads from a healthy individual. Species-specific profiles can be generated by this method. Click here for additional data file.10.7717/peerj.6233/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.6233/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.6233/supp-12Supplemental Information 12Click here for additional data file.


Agaricus bisporus. In order to explore the bacterial composition and the resultant odor changes in freeze-dried A. bisporus during storage under three typical conditions , bacterial diversity and communities were analyzed using metagenomics. Moreover, volatile compounds were determined using SPME-GC-MS. The results demonstrated that the bacterial composition in freeze-dried A. bisporus was dominated by Pseudomonas, followed by Rhizobium and Pedobacter. In addition, Mucilaginibacter, Flavobacterium, and Thermus were a few other genera more dominant in HT samples, Chryseobacterium was the other genera more dominant in AT samples, while, Sphingobacterium and Chryseobacterium were a few other genera more dominant in RT samples. Furthermore, the increase of benzaldehyde content in HT samples may have been induced by the growth of Pseudomonads and the esters production in RT and AT samples might have been induced by Chryseobacterium. This study provided comprehensive information on exogenous bacterial composition and the resultant odor in freeze-dried A. bisporus. These results may be a theoretical basis for quality control and quick quality detection based on volatiles of freeze-dried A. bisporus.Moisture absorption and bacterial growth are critical factors for quality deterioration of freeze-dried  Agaricus bisporus are popular and valuable foods, which are low in calories but higher in minerals, protein, and fiber  until moisture content is less than 5%. The freeze-dried A. bisporus slices were randomly packaged in normal polyethylene (PE) packing bag (20 g/bag) and, respectively, stored in three conditions for 25 days to simulate the storage condition of dried food: constant room temperature and humidity , constant high temperature and high humidity , variable ambient temperature and humidity . Water activity, total viable counts, Pseudomonads and volatile compounds of freeze-dried A. bisporus under different conditions were determined every 5 days during storage.Freeze-dried s method . After sw meter  (Water activity of the samples was determined with an azerland) , and theA. bisporus under three storage conditions were monitored. Samples were homogenized (1:10) with phosphate buffer solution  in the stomacher bag, and mixed using a Stomacher 400  at 300 rpm for 2 min. The homogenate was gradiently diluted in phosphate buffer solution  for the following tests. Total viable counts were determined on plate count agar (PCA) and incubated at 36 ± 1°C for 48 h ± 2 h  in freeze-dried  h ± 2 h . Pseudom h ± 2 h . AverageSamples on day 0 (CK samples) and samples stored for 25 days at RT, HT, and AT  were chosen for 16S rDNA sequencing analysis. Total DNA was extracted from bacterium solution by the TIANamp Bacteria DNA kit  following the manufacturer’s instruction . The V4 Forward primer: 515F GTGCCAGCMGCCGCGGTAA;Reverse Primer: 806R GGACTACHVGGGTWTCTAAT.Y-axis) within each sample (X-axis clustering) . High-throughput sequencing was performed on the Illumina Miseq platform with the 2 × 250 bp paired-end method after the library was quantified. To identify the OTUs presented in these samples, average linkage hierarchical clustering was used to cluster the partial 16S rDNA sequences at 97% sequence identity . After qstering) . Metastastering) . The seqA. bisporus powder (0.5 g) were weighed in a 20 ml vial with 1 μL internal standard of 1-decanol methanol solution (80 mg/mL) (Volatiles were extracted by headspace solid phase microextraction (HS-SPME). Freeze-dried 0 mg/mL) . The fib0 mg/mL) . The anaThe experimental data from the HP-SPME-GC-MS was analyzed using the statistical software, PASW statistic 18. The data are expressed as the mean ± standard deviation (SD). The measured data were analyzed by SAS system, Version 9.0 . Least significant differences (LSD) multiple comparison tests were then performed with a 95% confidence level.A. bisporus in the three storage conditions were shown in p < 0.05).Water activity is a vital parameter in dried foods quality and stability. Water activity, total viable counts, and Pseudomonads of freeze-dried A. bisporus was identified as 837 OTUs. Rarefaction curves, Shannon index curves and alpha diversity index were shown in Sequencing of 16S rDNA genes from the exogenous bacteria of freeze-dried Gammaproteobacteria and Pseudomonadaceae were higher in CK samples than in treated samples. In addition, seven families and 15 genera were significantly higher in the HT group. Meanwhile, ten families and 12 genera were significantly higher in the RT group, while five families and 13 genera were significantly higher in the AT group. At the family levels, the relative abundance of Cytophagaceae and Rhodobacteraceae were enriched in RT samples, while Sphingobacteriaceae and Rhodospirillaceae were enriched in AT samples. Moreover, HT samples showed higher relative abundance in Comamonadaceae, Dermabacteraceae, and Deinococcaceae. Cluster analysis, PCA, principal coordinate analyses (PCoA) based on the unweighted UniFrac distances, and non-metric multidimensional scaling (NMDS) were shown in The taxa difference with LDA scores greater than 2.0 were shown in A. bisporus was visualized using a heatmap by employing the relative abundance of each family. The findings were compared by using a hierarchical dendrogram . Therefore, the growth of Pseudomonads in the storage at high temperature and humidity may induce the release of benzaldehyde which resulted in the odor deterioration in freeze-dried A. bisporus. Ketones in the freeze-dried A. bisporus increased during storage, which are the important volatile metabolite of Pseudomonas (Chryseobacterium. Thus, the growth of Chryseobacterium abundance in RT and AT samples may induce the esters production which imparts the fruity aroma. Flavobacteria could produce the volatile compounds included alcohols, ketones, aldehydes, esters, and the resultant odors have been described as fishy, foul (Flavobacterium in HT samples significantly increased. This may lead to odor deterioration of freeze-dried A. bisporus and even low market acceptance. However, volatile compounds changes induced by the specific strain remained unclear. Thus, further studies are needed in order to monitor the growth of strains during storage as a comparison of volatile compounds.Freeze-dried  storage . Moreovealdehyde . It was udomonas . Kumar ehy, foul . The relA. bisporus during storage were investigated. Lower water activity in RT and AT samples extended the lag phase of bacterial growth and slowed their propagation, while the total bacterial colonies significantly increased in HT samples owing to the high humidity. This study showed that bacterial diversity in freeze-dried A. bisporus increased during storage, and HT samples had the highest diversity index among these groups. Moreover, beta diversity analysis of the bacterial communities suggested that the exogenous bacteria in freeze-dried A. bisporus exhibit unique composition during storage. Nevertheless, the dominant bacteria population in freeze-dried A. bisporus was Proteobacteria, Bacteroidetes, and Firmicutes. However, at the genus level, the composition was dominated by Pseudomonas, followed by Rhizobium and Pedobacter. The analysis showed that Mucilaginibacter, Flavobacterium, and Thermus were a few other genera more dominant in HT samples, whereas, Sphingobacterium and Chryseobacterium were a few other genera more dominant in RT samples, and Chryseobacterium were the other more dominant in AT samples. Furthermore, the increasing benzaldehyde content in HT samples may have been induced by the growth of Pseudomonads. Moreover, the increasing relative abundance of Chryseobacterium in RT and AT samples may have induced the esters production. The results provided comprehensive information about the biodiversity and the structure of bacterial community in freeze-dried A. bisporus during storage. Furthermore, the study provides significant information for the safety and quality monitor for freeze-dried A. bisporus.In the present study, the exogenous bacterial composition and volatile compounds of freeze-dried QH and FP contributed to the conception and design of the study. LW organized the database and performed the statistical analysis. WY and MM wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas aeruginosa is the major cause of bacterial keratitis, a sight-threatening ocular infection that can occur in contact lens wearers, as well as in others. Here, we report the draft genomes of 8 different P. aeruginosa corneal isolates, adding to the list of publicly available corneal infection-associated P. aeruginosa genomes. Pseudomonas aeruginosa is the major cause of bacterial keratitis, a sight-threatening ocular infection that can occur in contact lens wearers, as well as in others. Here, we report the draft genomes of 8 different P. aeruginosa corneal isolates, adding to the list of publicly available corneal infection-associated P. aeruginosa genomes. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes infections in immunocompromised people. While well known for its ability to cause infections of the respiratory tract, P. aeruginosa is relatively understudied in the context of bacterial keratitis (infection of the cornea). P. aeruginosa is the major cause of bacterial keratitis, occurring in 71% of all infection-positive contact lens wearers   , 10, conAll isolates were incubated at 37°C overnight in 3 ml lysogeny broth (LB) with shaking. Genomic DNA was isolated using a Qiagen DNeasy blood and tissue kit. Samples were then sequenced using an Illumina NextSeq 500 instrument , producing 151-bp paired-end reads. Sequencing libraries were prepared by the sequencing center as described by Baym et al. . Quality12–The final assemblies have an average coverage of 80×, with 110 contigs and 66.2% GC content. All strains were positive for the exo-enzymes PrpL, PASP, and ExoT. All strains except 6487 were positive for ExoU, and only strain 6487 was positive for ExoS.PRJNA558357.The accession numbers and assembly statistics of these isolates are listed in


Rickettsia spp. and Anaplasma/Ehrlichia spp. using molecular techniques, while adult fed ticks were tested individually. Additionally, larval and nymphal ticks were collected in the wild from small mammals, pooled and tested for Rickettsia spp. and Anaplasma/Ehrlichia spp. There were 38 fed adult and 618 larvae/nymphs  Dermacentor spp. ticks collected from livestock and rodents. All individual adult ticks and tick pools were positive for Rickettsia spp. While none of the larvae/nymphs were positive for Anaplasma/Ehrlichia spp., two adult fed ticks were positive. Rickettsia spp. DNA was detected in 91% (30/33) of the pooled eggs tested, and one pool of eggs tested positive for Anaplasma/Ehrlichia spp. Sequencing data revealed Rickettsia spp. shared ≥99% identity with R. raoultii ompA. Anaplasma/Ehrlichia spp. shared ≥89% identity with A. ovis 16S ribosomal RNA. This study identified potential transovarial transmission of Rickettsia spp. and Anaplasma spp. among D. nuttalli ticks. Additional studies are needed to further assess the proportion of transovarial transmission occurring in nature to better understand the burden and disease ecology of tick-borne rickettsiae in Mongolia.Transstadial transmission of tick-borne rickettsiae has been well documented. Few studies, however, have evaluated the role of transovarial transmission of tick-borne rickettsiae, particularly in nature within the host-vector ecosystem. This cross-sectional study aimed to understand the role of transovarial transmission of tick-borne rickettsiae among feeding ticks at different life stages. Tick eggs laid by engorged wild-caught adult female ticks were pooled and tested for  Rickettsia spp., Anaplasma spp., and Ehrlichia spp. Using molecular techniques, we detected rickettsiae among all Dermacentor spp. tick life stages and demonstrated potential vertical transmission of Rickettsia spp., and Anaplasma spp. among wild engorged adult female Dermacentor nuttalli ticks. We believe our findings provide important information regarding the ecology of key rickettsiae associated with tick-borne disease in Mongolia.In this study, we evaluate the probability or likelihood that tick-borne rickettsiae might be transmitted vertically from wild engorged adult female ticks collected throughout the Northern region of Mongolia during the summer of 2016. While significant effort has been directed to study tick-borne rickettsiae, this public health challenge is complicated by the limited knowledge and understanding of tick and tick-borne rickettsiae ecology within Mongolia. Tick-borne rickettsiae of concern to humans and animals in this region of the world are  While significant effort has been directed to study tick-borne rickettsiae, they continue to be a global public health threat. Mongolia is a country known for its rich nomadic and pastoral culture, with populations of people who work very closely with their livestock in environments that are often densely populated with ticks. Additionally, ecotourism is a rapidly growing industry in Mongolia, placing international visitors at risk of exposure to tick-borne rickettsiae , 2. ThisThe mobility of ticks is restricted to questing and travelling via feeding on animals and humans . Tick-boSeveral tick-borne rickettsiae surveillance and case studies have been conducted throughout China, Russia and Mongolia, which have tested humans –10, liveRickettsia spp. [Anaplasma spp. [Ehrlichia spp. [Tick-borne rickettsiae of most concern in the Asian and Eurasian regions of the world are sia spp. , 24, Anasma spp. , 27, 32,hia spp. , 25. Thehia spp. , 17, 19.th to 22nd, 2016. The second component of this study, examined larvae and nymphs removed from trapped small mammals across seven soums in three aimags situated in the Northern region of Mongolia from June 20th to July 23rd, 2016. Using a handheld global positioning system (GPS) unit , latitudinal and longitudinal coordinates were collected at each small mammal collection site , both adult ticks and eggs were stored separately at -80°C until DNA extraction was performed. The whole egg clutch was pooled and tested from each adult female tick. Mass of egg clutches ranged from 10 to 410 milligrams. Eggs and adult ticks were briefly rinsed with 70% ethanol in sterile 1 mL vials to remove contamination and then air dried on a sterile dish in preparation for processing , 34.Trapping and handling procedures for small mammals were approved by the Duke University Institutional Animal Care and Use Committee (#A086-16-04) in accordance with the Mongolian Institute of Veterinary Medicine, Ulaanbaatar, Mongolia. At each location, live Tomahawk and Sherman traps were placed near holes where there were signs of recent small mammal habitation. All captured small mammals were sedated with ketamine (50 mg/Kg) and inspected for ticks. Ticks were stored in 70% ethanol at room temperature. Specimens were taxonomically identified to genus for larvae and nymphs and species for adults by a trained entomologist. Ticks were air dried and pooled based on life stage , small mammal host, sampling location, and tick genus. Pools (n = 60) were stored at -20°C in new sterile 1 mL vials before genomic DNA was extracted.Rickettsia spp. targeting the citrate synthase gene (gltA) [ompA) [Anaplasma spp. and Ehrlichia spp., the 16S rRNA gene [All ticks and eggs were ground using a sterile pre-chilled mortar and pestle with 500 μL of sterile PBS and 50 mg sterile sand for friction . Contente (gltA)  and the ) [ompA) , as prevRNA gene  was targGel electrophoresis was used to evaluate amplified products using 1% (w/v) agarose gels stained with ethidium bromide at 120 V. Gels were analyzed using the Gel Doc EZ System  with ultra-violet illumination.http://www.ncbi.nlm.nih.gov/BLAST/) for species identification. Rickettsia spp. gltA and ompA sequences were used as confirmation of amplified Rickettsia spp. samples and Anaplasma/Ehrlichia spp. 16S rRNA sequences were used as confirmation of amplified Anaplasma spp. Anaplasma/Ehrlichia spp., and Rickettsia spp. sequences were structured for phylogenetic relatedness using Molecular Evolutionary Genetics Analysis (MEGA) software, version 7.0.A representative subset of positive amplicons were selected and directly sequenced using Sanger sequencing . Sequencing results were then compared against the NCBI nucleotide database using the Standard Nucleotide BLAST application .Dermacentor spp. Due to the size of larval and nymphal ticks collected, and the variety of Dermacentor spp. found in Mongolia, early life stage ticks were only identified to genus. All adult-fed ticks were morphologically identified to be D. nuttalli. Of the early life stage ticks collected from small mammals, 546 (88%) were larvae, 72 (12%) were nymphs. There were 588 (95%) of 618 ticks that were allocated into 42 larval and 18 nymphal pools . A total of 38 adult fed female ticks were collected from sheep and cattle. Of the 38 adult ticks collected, 33 laid eggs.A total of 656 ticks were collected from 15 different locations across nine soums in five aimags. All ticks were morphologically identified as Rickettsia spp. Subsequent PCR testing of paired eggs resulted in 91% (30/33) PCR positive among tick egg pools for Rickettsia spp. Sequencing data revealed Rickettsia spp. shared ≥99% identity with R. raoultii ompA (Accession numbers MH234455 and MH234456) shown in the phylogenetic analysis (gltA sequences shared ≥99% identity with R. raoultii (Accession numbers MH208721 and MH208722) shown in the phylogenetic analysis  and the other engorged adult female tick shared 89% (accession number MG461483) identity with the A. ovis 16S ribosomal RNA gene. Both Anaplasma spp. sequences are shown in the phylogenetic analysis  were PCR-positive for analysis .Rickettsia spp. [Rickettsia spp. and Anaplasma/Ehrlichia spp. plays in the maintenance of tick-borne rickettsiae in Mongolia. Additionally, few studies have assessed tick-borne rickettsiae in tick larvae collected from small mammals [Few studies have evaluated transovarial transmission of tick-borne rickettsiae in Mongolia , 43. As sia spp.  are part mammals , 46.R. raoultii found in this study show similarity with other studies conducted in Mongolia with 100% of Dermacentor spp. tick pools testing positive for R. raoultii [R. raoultii with few other ticks carrying uncharacterized rickettsiae, suggesting that the prevalence of R. raoultii in the areas in which the ticks were collected is quite high.It is often difficult to compare field surveys on tick-borne rickettsiae, because of varying sampling methods and sample sizes. However, the detection of raoultii , 22. TheRickettsia spp. by transovarial transmission. Transovarial transmission of Rickettsia spp. has been demonstrated in controlled laboratory settings and observed in nature. Laboratory demonstration of transovarial transmission has often included controlled concentration of the rickettsiae, artificial climactic conditions , and use of experimental hosts such as rabbits [Rickettsia spp. from mother to progeny has been shown to occur up to 100% across various tick species, including Dermacentor genus [Rickettsia spp. over multiple generations of ticks [Rickettsia spp. to progeny, prevalence has ranged from 30% to 100% [Though this study does not report animal host infection status, it can prove advantageous to test larvae collected from animal hosts as a method of determining if rickettsiae infect ticks by transovarial or transstadial transmission with paired animal health information. A positive infection status in ticks could occur by horizontal transmission of rickettsiae from an infected animal host to the tick. The alternative explanation is that the larvae found on the small mammals may be infected with the  rabbits , mice, g rabbits . Among tor genus , 48. Addof ticks , 49. AmoRickettsia spp. are sustained through both transovarial and transstadial transmission in Dermacentor spp. ticks based on previous epidemiological research of Rickettsia spp. in Inner Mongolia, China, suggesting that there may be similar maintenance of Rickettsia spp. in Mongolia [R. raoultii in D. nuttalli, D. silvarum, D. marginatus, and D. reticulatus ticks. Transovarial transmission ranged from 43% to 100% prevalence depending on generation of tick infection, species of tick, and strain of R. raoultii. In comparison to the previous laboratory-based research reporting 43% to 99.5% of transovarial transmission of R. raoultii in D. nuttalli ticks [D. nuttalli ticks at 91%. Though there has been no conclusive data reporting vertebrate hosts as a reservoir for R. raoultii, it has been suggested that D. marginatus and D. reticulatus serve as both vector and reservoir [D. nuttalli ticks have been implicated as the primary vector for R. raoultii in Mongolia [It is well documented that naturally occurring Mongolia . Previouli ticks , this steservoir . AdditioMongolia .Dermacentor species  ticks may serve not only as the primary vector, but also as the primary reservoir for R. raoultii in the northern region of Mongolia.Our study’s high prevalence of infected egg clutches suggesting transovarial transmission in conjunction with the substantial number of infected larvae found on small mammals, suggests that Anaplasma spp. or Ehrlichia spp. in larvae or nymphs, there was detection of an Anaplasma spp. most similar to A. ovis in two D. nuttalli fed adult ticks and in one tick egg pool. There have been reports of A. phagocytophilum transmitted transovarially with prevalence’s ranging from 10% to 40% [Anaplasma spp. and Ehrlichia spp. are thought to occur at low frequencies or not at all [Anaplasma spp. and Ehrlichia spp. with little success [A. ovis in D. nuttalli ticks [Anaplasma spp. in Dermacentor spp. ticks [A. ovis is not known to cause human disease, the economic burden is great for individuals who rely on raising livestock for their income [A. ovis.Though this study did not identify % to 40% , howevert at all , 54. The success . To our li ticks . There hp. ticks  being asp. ticks , 12, shep. ticks , 14, catp. ticks , and reip. ticks  throughor income . FurtherRickettsia spp. infected within tick pools cannot be calculated if 100% of sample pools are positive [R. raoultii did occur, we were unable to determine how many progeny were infected. Additionally, by only screening infection status of egg mass, we are unable to discuss if infected larvae will hatch. Furthermore, Rickettsia spp. PCR primers have been shown to cross-react with Anaplasma spp. and Ehrlichia spp. However, this study also utilized a general screening assay for Anaplasma/Ehrlichia spp. and confirmation by sequencing, which allowed for greater confidence in the Rickettsia spp. PCR assay.Like many tick pool studies, it is difficult to determine the exact prevalence of disease using this approach. Due to the nature of the maximum likelihood estimation calculation, the proportion of infected ticks with maximum likelihood of being positive . AdditioD. nuttalli ticks can serve as reservoirs for R. raoultii may warrants additional evaluation of transovarial and transstadial transmission of R. raoultii. Studies should focus on assessing tick eggs, either in smaller egg pools or individually, to determine the proportion of transovarial transmission as well as transstadial transmission for R. raoultii in eggs entering larval life stage, and larvae entering nymphal stages in the natural foci of Mongolia. Additionally, the testing of larvae from animal hosts and the environment should be further examined, preferably testing individual ticks instead of tick pools. Also, this report has identified a potentially novel transovarial transmission of A. ovis. Further investigation would be needed to determine the efficiency and prevalence of transovarial transmission of this rickettsiae.The indication that S1 Dataset(PDF)Click here for additional data file.S2 Dataset(PDF)Click here for additional data file.