Vibrio is ubiquitous in marine environments and uses numerous evolutionary characteristics and survival strategies in order to occupy its niche. Here, a newly identified species, Vibrio fujianensis, was deeply explored to reveal a unique environmental adaptability. V. fujianensis type strain FJ201301T shared 817 core genes with the Vibrio species in the population genomic analysis, but possessed unique genes of its own. In addition, V. fujianensis FJ201301T was predicated to carry 106 virulence-related factors, several of which were mostly found in other pathogenic Vibrio species. Moreover, a comparative transcriptome analysis between the low-salt (1% NaCl) and high-salt (8% NaCl) condition was conducted to identify the genes involved in salt tolerance. A total of 913 unigenes were found to be differentially expressed. In a high-salt condition, 577 genes were significantly upregulated, whereas 336 unigenes were significantly downregulated. Notably, differentially expressed genes have a significant association with ribosome structural component and ribosome metabolism, which may play a role in salt tolerance. Transcriptional changes in ribosome genes indicate that V. fujianensis may have gained a predominant advantage in order to adapt to the changing environment. In conclusion, to survive in adversity, V. fujianensis has enhanced its environmental adaptability and developed various strategies to fill its niche.The genus  Vibrio is ubiquitous and abundant in oceanic, estuarine, and freshwater environments [Vibrio species reported to date. Many Vibrio spp. are well-known bacterial pathogens, causing disease in humans or marine animals. Vibrio cholerae [Vibrio parahaemolyticus causes severe gastroenteritis in humans through consumption of contaminated seafood [Vibrio can also cause severe bacteremia, skin, and soft tissue infection [The genus ronments ,2,3. Thecholerae  is the c seafood . Some otnfection .Vibrio adapted to its environment in various strategies. Firstly, Vibrio can obtain a better survival ability in their environment by forming biofilm or growing rapidly to a certain population density. Many Vibrio isolates use population density to control the gene expression via a quorum-sensing system; for instance, the quorum-sensing transcription factor AphA directly regulates natural competence in V. cholerae [V. cholerae also possesses multiple quorum-sensing systems that control virulence and biofilm formation among other traits [Vibrio spp. can develop adaptive strategies to survive in extreme conditions of salinity stress and temperature, such as the mechanism of osmoregulation and osmotic balance, modification of the lipid composition, activity of ion pumps, and increasing of the secondary metabolite production [Vibrio spp. to survive in the changing environment is genetic variation via positional mutations or the horizontal transfer of foreign genes [In the process of evolution, cholerae . V. cholr traits . Secondloduction . In addign genes .Vibrio fujianensis was recently identified as a novel Vibrio species; the type strain FJ201301T was isolated from aquaculture water in Fujian Province, China, in 2013 [V. fujianensis has evolved many characteristics and survival strategies to occupy its niche. For instance, the strain of V. fujianensis is able to grow under a wide range of pH (pH 5–10) and salt concentrations (1%–10% w/v) [V. fujianensis can result in a cross-agglutination reaction with the specific serum of V. cholerae O139 serogroup, which suggests the two species have a similar or the same O-antigen component. It is necessary to understand the evolutionary characteristics and genomic diversity of a new Vibrio species. In the present study, a comparative genomics analysis was performed based on the V. fujianensis draft genome and other reference genomes of the genus Vibrio. Two different salt stress conditions, 1% (w/v) and 8% (w/v), were selected for RNA-Seq-based transcriptome analysis. We investigated the comparative differentially expressed gene (DEG) profiles of V. fujianensis with low-salt or high-salt stress, so as to gain an increased understanding of the molecular mechanisms underlying the species’ environmental response. in 2013 . As a no10% w/v) . In addiV. fujianensis FJ201301T was used throughout this study. The strain was cultured as recommended for Luria-Bertani  agar at 30 °C for 18 h. The total RNA extraction from the two conditionally cultured strains was performed using the TRIZOL extraction reagent , in accordance with the protocol mentioned before [d before .V. fujianensis FJ201301T (accession number: GCA_002749895.1) was sequenced previously. In order to represent low-salt stress and high-salt stress, 1% and 8%  concentrations were chosen, respectively. The RNA samples were extracted separately in each condition to measure them three times in duplicate. Transcriptome sequencing was performed using the Illumina HiSeq™ 2000 platform , with an average yield of 10.52 Mb raw data per sample. SOAPnuke software [The whole genome sequence of software . The whoVibrio bacteria and Aeromonas hydrophila ATCC 7966T are available in the GenBank  server pipeline [https://blast.ncbi.nlm.nih.gov/Blast) [Vibrio O-PS gene cluster was performed by Easyfig 2.2.3 software [The O-PS gene cluster sequence of v/Blast)  was applsoftware .http://www.mgc.ac.cn/VFs/) was used to annotate the virulence genes. The virulence-related genes of the protein sequences were searched against the VFDB using BLAST+ [The Virulence Factors Database (VFDB)    was used library , the DEG−5. The GO term functional analysis and KEGG pathway enrichment analysis were performed in subsequent analyses.The DEGs were annotated against the Swiss-Prot , Gene OnV. fujianensis FJ201301T was aligned and compared to a set of 119 corresponding sequences of other Vibrio species and strains. Aeromonas hydrophila ATCC 7966T served as an outgroup species. A phylogenetic tree suggested that the Vibrio genus should be divided into seven different clades  indicated that interspecies distinction among Vibrio species in the Cincinnatiensis clade was apparent . The first two homologous regions contained four genes, namely, manB, manC, wbfR, and wbfS  lower than the genome average (43.4 mol%), while the homologous fragment  had a GC content of 38.2 mol%, which was far lower than the average GC content of the genome (data are not shown). This atypical GC content provided strong evidence that the homologous fragment had recently gone through a genetic recombination event, by horizontal gene transfer, with V. cholerae O139 serogroup strains, a different bacterial species in the long-term evolution.The O-PS gene cluster analysis A showed ribution B revealeand wbfS C, encodi to wbfP C. Gene gV. fujianensis FJ201301T and other common pathogens were predicted against the VFDB. A putative virulence-associated gene pool included a total of 281 kinds of virulence genes  were not detected in V. fujianensis FJ201301T. The unknown protein related to O-antigen has been described only in V. fujianensis FJ201301T and in V. cholerae MO45  that may participate in changes in the ribosome formation, eight of which were significantly upregulated, while the remaining 24 genes were significantly downregulated . TranscrVibrio, many of which have been identified in recent years. It has been shown that the Vibrio genus is characterized by a remarkable biodiversity, having evolved to develop complex lifestyles. In this study, the phylogenetic tree based on the 16S rRNA gene sequences of 119 Vibrio species defined seven phylogenetic clades  were found, which somehow would cause differences in some aspects, like the GC content, genome structure, proportion of core genes and accessory genes. Each Vibrio strain contained a large number of strain-specific genes, which may be related to different strains inhabiting a limited host and environmental niche, or related to distinct types of diseases with different severity [A gene family is a group of genes derived from the same ancestor and consisting of two or more copies of a gene through gene duplication  and specseverity ,41.M. tuberculosis were implicated in the adaptation of limited nutritional conditions in macrophages or for counteracting the microbicidal host cell responses, and M. tuberculosis EspC and EspA mutants have shown an inhibition of the bacterial growth or the biological metabolism [V. fujianensis, and these genes were universally shared in common pathogenic Vibrio species. By assembling surface antigen structure [V. fujianensis has a strong adaptability to new environments. It carries a number of virulence-associated factors in the virulence-gene pool among pathogenic VibrioV. fujianensis and other pathogenic Vibrio.Virulence-associated genes are believed to allow strains to carry adaptation and pathogenicity . Studiestabolism . In our tructure , secretitructure , and devVibrio [V. cholerae, type VI secretion system genes are coregulated with genes involved in exogenous DNA uptake. On the other hand, cases of serum cross-agglutination between different species are reported from time to time; a well-known example is the cross-reaction between E. coli and Shigella in Enterobacter species [V. fujianensis FJ201301T and V. cholerae O139 serogroup MO45 shared highly homologous gene coding regions. This finding was a reasonable explanation for the cross-agglutination between V. fujianensis and V. cholerae O139 serum. With combined atypical GC content values, we propose that between the V. fujianensis FJ201301T and V. cholerae O139 serogroup MO45, a horizontal gene transfer of a partial O-PS gene cluster may have occurred. Sally et al. [Pseudomonas aeruginosa over the course of chronic respiratory tract infections. The O-antigen of V. cholerae is proven to play a role in critical defense by increasing drag force to impede attackers [V. cholerae specific typing phage [Vibrio species was associated with the specific environmental colonization ability [V. fujianensis FJ201301T and V. cholerae O139 can greatly enhance the environmental adaptability of V. fujianensis FJ201301T to quickly reproduce and tenaciously survive in nature.Horizontal gene transfer (HGT) is considered to be an important mode of acquiring new genes and accelerating evolution. Mobile genetic elements, such as plasmids, phages (prophages), and integrons, are found to be a crucial factor for horizontal gene transfer, even contributing to the virulence and antibiotic resistance of Vibrio . RecentlVibrio  showed tVibrio  have con species ,51,52. Iy et al.  demonstrttackers . O-antigng phage . Another ability , which cV. fujianensis in response to salt stress tolerance are mainly reflected in the molecular function represented by catalytic activity. Transcriptional changes involved in the carbohydrate metabolism, membrane transport, amino acid metabolism, and signal transduction were found to be important for the high salt stress in V. fujianensis. The KEGG pathway enrichment analysis considered the “ribosome pathway” (ko03010) as a significant enrichment; this result is consistent with the high-salt transcription and expression of Shewanella algae in our previous study [+ concentration in the environment changed. Transcriptome research in response to salt stress in Betula halophila [B. halophila. Proteomic analyses in C. albicans revealed a link between ribosomal gene expression and environmental adaptation [Escherichia coli, in which the maturation or function of the ribosome is impaired [70 transcription [+/H+ antiporter makes it effective for the survival of Vibrio species in a saline environment. NhaB has been proven to be a possible mechanism for regulating Na+/H+ antiporter activity [Vibrio biofilm formation. In general, microorganisms rely on a salt rejection strategy called osmoadaptation, which involves compatible solute accumulation, for example, the accumulation of K+ ions or some low molecular mass organic solutes. Usually, the formation of organic solutes makes conditions more favorable for the osmotic balance in cells, like sugar, alcohol, amino acid, and their derivatives [+/H+ antiporter is used to pump extra Na+ ions out of the cell to maintain a proper Na+ concentration in the cell. Fu et al. [Shewanella algae.Transcriptome changes for us study . Eiji etus study  found thalophila  showed taptation . Salt toimpaired . Changesimpaired ,61. Micrcription . The Na+activity . To our activity ,64. Marsactivity  found thivatives . In addiu et al.  also fouV. fujianensis species might help develop its innate adaptability, as well as its potential ability to evolve quickly and thrive in the ecological niche. Several genetic motility genes might be attributed to HGT in the evolutionary process.In this study, changes in the ribosome pathway may be related to salt tolerance via transcriptome sequencing. Strain-specific genes in the


Anopheles darlingi is an important Latin American malaria vector, but we know little about its mating behaviors. Flight tones play important roles in mosquito reproduction. Several mosquito species utilise flight tones for mate localisation and attraction. Typically, the female wingbeat frequency (WBF) is lower than males, and stereotypic acoustic behaviors are instrumental for successful copulation. Mosquito WBFs are usually an important species characteristic, with female flight tones used as male attractants in surveillance traps for species identification. An. darlingi WBFs and examined male acoustic responses to immobilised females. We characterised An. darlingi were recorded individually to determine their WBF distributions. Male-female acoustic interactions were analysed using tethered females and free flying males. Tethered and free flying male and female An. darlingi females are smaller than males. However, the male’s WBF is ~1.5 times higher than the females, a common ratio in species with larger females. When in proximity to a female, males displayed rapid frequency modulations that decreased upon genitalia engagement. Tethered females also modulated their frequency upon male approach, being distinct if the interaction ended in copulation or only contact. Contrary to most mosquito species, An. darlingi flight acoustics, showing that its precopulatory acoustics are similar to other mosquitoes despite the uncommon male:female size ratio, suggesting that WBF ratios are common communication strategies rather than a physical constraint imposed by size. This is the first report of  Anopheles darlingi is a major malaria vector,,An. darlingi is an efficient malaria vector, able to maintain high levels of transmission even when present at low densities.An. darlingi populations is suggested to enable adaptation of this species to a range of habitats.,Malaria is one of the most important vector-borne diseases worldwide, with 219 million cases reported in 2017.,,Aedes aegypti) - males intercept females as they attempt to blood-feed.,An. darlingi males form swarms.Disruption of pre-mating reproductive behaviors has been proposed as a means to control mosquito populations by exploiting important mating-specific cues to prevent male-female interaction.Ae. aegypti, Anopheles gambiae, Anopheles albimanus and Culex quinquefasciatus interact acoustically pre-copula.,,,,,,Ae. aegypti, Cx. quinquefasciatus, An. gambiae, An. coluzzi, and An. albimanus.,,,Ae. aegypti, Cx. quinquefasciatus, and An. gambiae males and females modulate their WBFs to match in a shared harmonic during courtship, a phenomenon known as harmonic convergence.,,,Male and female ,,,,,,,An. darlingi, males and females collected in the field are similarly sized.An. darlingi, an understudied species, will contribute to our knowledge of mosquito mating behaviors.Mosquito WBFs can be an important characteristic of a species,An. darlingi WBF and examined acoustic behaviors upon exposure to the opposite sex. We find that lab reared males are larger than females, unlike other mosquito species. Although larger, males broadcast a significantly higher WBF than females, showing that the WBF ratio between males and females is not determined by body size, contrary of what has been assumed in the past.,,An. darlingi despite the particular size of males and females of this species. These findings support the idea that the acoustic dynamic of mosquito mating behavior is a process of communication rather than a byproduct of motion. Moreover, as flight tones are currently being used in the development of novel surveillance strategies,,,,-An. darlingi reproductive behaviors and WBF characterisation will aid their surveillance and control.To examine precopulatory behavioral interactions in an understudied malaria vector, we characterised Mosquitoes - An. darlingi from Universidad Peruana Cayetano Heredia - ICEMR insectary  were used in our experiments. This colony has been maintained since 2012.,ad libitum. All recordings were conducted at 26ºC and 80% relative humidity (RH). Five to seven-day-old mosquitoes were used in all experiments.Audio recording set up - Mosquitoes were recorded in a 4L plastic cage using a particle velocity microphone . A USB audio interface  was used to amplify and digitise recordings at a sample rate of 11025 Hz/24 bits.Experimental procedures - We first recorded mosquitoes individually to determine WBFs when in free flight and when tethered. As similar aged individuals were difficult to obtain from this colony , the same mosquitoes were utilised to determine WBF under both conditions. Tethered mosquitoes were immobilised as in Pantoja-Sánchez et al.Supplementary data ]. Ten-fifteen males were then introduced into the cage. Females were replaced upon copulation; spermathecae were subsequently dissected to determine insemination status. We recorded all trials with a camera (FLIR-FLEA 3 1.3 MPColor USB 3 Vision with a Fujinon - FF125HA-1B 12.5 mm lens) and identified and timed behaviors in real time. Mosquito wings were measured as in van den Heuvel et al.Signal and statistical analysis - Flight-tone audio recordings were analysed using spectrograms . To evaluate acoustic interactions, audio segments occurring during observed mating attempts were analysed. From the spectrograms, we used the male second harmonic and the female third to examine flight-tones during male-female behavioral interactions due to frequency resolution but present our results in terms of their fundamental WBFs for simplicity, as done previously.max-Fmin)/2) and female measurements by tree (ΔF = (Fmax-Fmin)/3) to express results in terms of fundamental frequency equivalent to the WBF. We further assessed female behavior by examining the rate of increase (ΔF/Δt) after male detection.To compare frequencies between flight conditions (tethered and free flight), and to discriminate differences between sexes, we used t-tests. Normality for the variable WBF was determined with a Shapiro-Wilk test. A linear regression was used to determine the relationship between wing size and WBF. Residuals were tested for normality, homogeneity of variance and independence using Shapiro-Wilks, Bartlett and Durbin-Watson tests, respectively. We compared male-female contact and copulation interactions using the non-parametric Mann-Whitney U test to assess similarities and differences in the distributions of the variables analysed. Results are reported as mean ± standard deviation for variables that followed a normal distribution, and as median (interquartile range - IQR) for variables that did not. Signal processing was performed using Matlab . Statistical analysis was performed using the car packageBody sizes of An. darlingi adults - Using dry weight to examine body size in field collected An. darlingi, Lounibos et al.An. darlingi dry weight is highly correlated with wing length,An. darlingi (2518 ± 88 μm) were significantly smaller than males (2635 ± 14 μm) (t-test: t(29) = 3.82, p < 0.01; ,An. darlingi (size ratio 1.046) is distinct compared to other species in the same genus such as An. albimanus (size ratio: 0.95)Ae. aegypti (size ratio: 0.81)Wing beat frequencies of male and female An. Darling - We recorded individual adults to characterise their flight tones under two conditions: when tethered and in free flight as some reports suggest that tethering impacts WBFs while others report no affect.,,,± 78 Hz (n = 30), and 709 ± 42 Hz (n = 30) in free flight (t-test: t(29) = -9.36, p < 0.01). The mean WBF of tethered females was 362 ± 33 Hz (n = 30), and 456 ± 33 Hz (n = 30) in free flight (t-test: t(29) = -10.97, p < 0.01; (29) = 12.31, p < 0.01) and 1.55 times higher in free flight (t-test: t(29) = 26.10, p < 0.01) similar to what has been observed in other species such as Ae. aegypti and An. albimanus.,2 = 0.071, F28 = 2.14, t = -1.462, p = 0.1549; females: R2 = 0.001, F28 = 0.055, t = 0.23427, p = 0.8165).Male-female acoustic interactions during a mating attempt - To investigate acoustic interactions between sexes, we exposed free flying males to a tethered-flying female. We analysed all interactions where the male’s flight-tone was detected. Two types of interactions were observed: (1) male-female leg contact and (2) copulation, defined as visible genitalia engagement. However, sperm transfer was never detected upon female dissection, possibly due to the inability to achieve a proper angle for insemination.max-Fmin) of 396.51 Hz (IQR 296.84 - 460.69 Hz). RFM was followed by a decrease in the modulation . In all interactions, when nearing the female, RFM of the male flight tone occurred followed by modulation of the female frequency . In copudulation A or by mdulation B, with a,11,17 = 83, Z = 0.49, p = 0.62; 11,17 = 47, Z = 2.18, p = 0.028; Female flight-tones were also analysed. From the 40 interactions described above, 12 were excluded from our analysis as females stopped their wing beat upon male contact. Despite being tethered, female signal modulation was detected in the remaining 28 interactions . Females reacted to the male approach by modulating their flight-tone frequency - we observed an initial WBF increase similar to descriptions in other mosquito species.,An. darlingi. We show how An. darlingi bioacoustics are similar to other species despite their uncommon intraspecific male/female size ratio.,,Mosquito mating has been widely used as a target to develop novel control strategies.Cx. quinquefasciatus, An. gambiae, An. coluzzi and Ae. aegypti, indicate the importance of this behavior.,,Ae. aegypti, RFM cessation coincides with formation of the ventral-ventral copulation position.An. darlingi RFM ends when a pair engage their genitalia; the male decreases the frequency modulation of his tone or ceases flying. This might be a general Anopheline behavior, as mating pairs fall to the ground after couple formation.,An. darlingi females also modulated their flight tone. When males neared or made contact, tethered females increased their WBF at a higher rate than when the interaction resulted in copulation. This might be reflective of active female acceptance or rejection, although more experiments are necessary to test this hypothesis. Tethered Culex females modulate their WBF as a result of physical contact by the male.An. darlingi females initiates during the male approach or only upon physical contact will require higher resolution videos.When exposed to a tethered female, male RFM occurred when approaching and contacting the female. Similar observations in ,,An. darlingi, this study contributes to the overall knowledge of the flight acoustics of this vector and mosquitoes in general. We hope this study will promote future investigations on species of medical relevance for Latin America and aid their surveillance.Contrary to what has long been assumed,


Adrenal myelolipoma (MLS) is a rare, benign and non-functional neoplasm, composed of adipose tissue and myeloid. We report a rare case of adrenal myelolipoma of a 20-year-old female revealed with chronic abdominal pain. Computed tomography (CT) scan of the abdomen guided diagnosis and surgical resection was performed given symptomatic and bulky mass. Histological examination confirmed the diagnosis. At 18 months after the surgery, the patient had no evidence of recurrence. The diagnosis of MLS is radiological. Therapeutic abstention is the rule for a small, asymptomatic tumor. The surgical removal is indicated when it is bulky (exceeds 7cm), symptomatic or hormonal activity. Adrenal myelolipoma (MLS) is a rare, benign, non-functional tumor. Composed of mature adipose tissue and hematopoietic elements, the MLS are often a fortuitous discovery and detected during imaging procedures. Usually respected, surgical removal is indicated when tumor is bulky, compressive or at risk of haemorrhage. We report a case of adrenal myelolipoma and review of the literature.2. An ultrasonogram (US) revealed adrenal masses. Computed tomography (CT) scan showed an 8.35 × 5.73cm right adrenal mass, composed of fat tissue, which has a -112 HU value  guideline, Zeiger lolipoma . The clilolipoma . It is vlolipoma . Ultrasololipoma . On CT slolipoma . MRI alllolipoma .Imaging studies are accurate in diagnosing AML in up to 90% of the cases . If CT sAdrenal myelolipomas are uncommon benign tumors, most often asymptomatic and discovered incidentally in imaging diagnostics. Computed tomography can yield the diagnosis. Smaller, asymptomatic myelolipomas can be observed expectantly. The bulky (greater than 7cm), symptomatic, metabolically active tumor scan recommend surgical resection. Mini-invasive techniques performed by experienced laparoscopic surgeons are an effective and feasible way for their removal.The authors declare no competing interests.


Bothrops asper represents the clinically most important snake species in Central America and Northern South America, where it is responsible for an estimated 50–80% of snakebites. Compositional variability among the venom proteomes of B. asper lineages across its wide range mirrors clinical differences in their envenomings. Bothropic antivenoms generated in a number of Latin American countries commonly exhibit a certain degree of paraspecific effectiveness in the neutralization of congeneric venoms. Defining the phylogeographic boundaries of an antivenom's effectivity has implications for optimizing its clinical use. However, the molecular bases and impact of venom compositions on the immune recognition and neutralization of the toxic activities of across geographically disparate populations of B. asper lineages has not been comprehensively studied.B. asper lineages (B. asper (sensu stricto), B. ayerbei and B. rhombeatus) distributed in south-western (SW) Colombia, of six Latin American antivenoms, produced against homologous  and Costa Rica (ICP)), and heterologous (Argentina (BIOL), Perú (INS-PERU) and Venezuela (UCV)) bothropic venoms. In vivo neutralization assays of the lethal, hemorrhagic, coagulant, defibrinogenating, myotoxic, edematogenic, indirect hemolytic, and proteolytic activities of the three SW Colombian B. asper lineage venoms were carried to compare the preclinical efficacy of three  antivenoms frequently used in Colombia. Antivenomics showed that all the six antivenom affinity matrices efficiently immunoretained most of the B. asper lineages venom proteins and exhibited impaired binding towards the venoms' peptidomes. The neutralization profile of the INS-COL, PROBIOL and ICP antivenoms towards the biological activities of the venoms of SW Colombian B. asper (sensu stricto), B. ayerbei and B. rhombeatus lineages was coherent with the antivenomics outcome. In addition, the combination of in vitro (antivenomics) and in vivo neutralization results allowed us to determine their toxin-specific and venom neutralizing antibody content. Noteworthy, heterologous INS-PERU, BIOL, and UCV bothropic antivenoms had equal or higher binding capacity towards the venoms components of SW Colombian B. asper lineages that the homologous Colombian and Costa Rican antivenoms.Third-generation antivenomics was applied to quantify the cross-immunorecognizing capacity against the individual components of venoms of three in vitro and in vivo preclinical outcome showed that antivenoms manufactured in Colombia and Costa Rica effectively neutralize the major toxic activities of SW Colombian B. asper lineage venoms. The antivenomics profiles of the heterologous antivenoms manufactured in Argentina, Venezuela, and Perú strongly suggests their (pre)clinical adequacy for the treatment of B. asper lineage envenomings in SW Colombia. However, their recommendation in the clinical setting is pending on in vivo neutralization testing and clinical testing in humans. Bothrops asper is a highly adaptable snake species complex, which is considered the most dangerous snake throughout much of its distribution range from the Atlantic lowland of eastern México to northwestern Perú. Antivenoms are the only scientifically validated treatment of snakebite envenomings. Venom variation is particularly common in wide ranging species, such as B. asper, and may result in variable clinical presentations of envenomings, as is the case for the B. asper species complex, potentially undermining the efficacy of snakebite treatments depending on the immunization mixture used in the generation of the antivenom. Conversely, phylogenetic conservation of antigenic determinants confers an unpredictable degree of paraspecificity to homologous antivenoms produced for a geographic area, but also to heterologous congeneric antivenoms, towards the venom components of allopatric conspecific populations. This work aimed at comparing the preclinical profile of a panel of Latin American homologous and heterologous antivenoms against the venoms of B. asper lineages distributed in SW Colombia. The outcome of this study strongly suggests the suitability of considering the heterologous antivenoms BIOL (Argentina), UCV (Venezuela) and INS-PERU (Perú) as alternatives to homologous Colombian INS-COL and PROBIOL and Costa Rican ICP antivenoms for the treatment of envenomings by B. asper (sensu stricto) in W Colombia and Ecuador, B. ayerbei in Cauca and Nariño (Colombia), and B. rhombeatus in Cauca river valley, SW Colombia.The combined  B. asper represents the only species of the genus, in SW Colombia three close phylogenetically related B. asper lineages, B. asper (sensu stricto), B. rhombeatus, and B. ayerbei, are responsible for most severe cases of snakebite accidents and exhibit remarkable differences in the physiopathological profile of their envenomings. This work aimed to assess the immunorecognition characteristics of a panel of antivenoms manufactured in Colombia, Costa Rica, Argentina, Perú and Venezuela towards the venoms of the three SW Colombian B. asper lineages. Additionally, combined quantitative in vitro and in vivo data show that the homologous antivenoms produced in Colombia  and Costa Rica (ICP) effectively neutralize the lethality and the major toxic activities tested of the three SW Colombian B. asper lineage venoms. Heterologous Argentinian (BIOL), Venezuelan (UCV) and Peruvian (INS-PERU) antivenoms also showed comparable, even higher, effective immunocapturing ability towards the venom proteomes of SW Colombian B. asper (sensu stricto), B. rhombeatus, and B. ayerbei, than the Colombian and Costa Rican antivenoms. These results are in line with previous studies highlighting the notable conservation of paraspecific antigenic determinants across the phylogeny of genus Bothrops, and advocate for considering the heterologous Argentinian, Venezuelan and Peruvian antivenoms as further therapeutic alternatives for the treatment of B. asper spp. snakebites in Colombia.Snakebite envenoming is an important occupational health problem, particularly in rural areas of developing countries. The timely administration of an effective antivenom remains the mainstay of snakebite management. However, the use of antivenoms is often limited by non-availability due to high cost or by lack of effectiveness. Antivenom shortage can be addressed through the generation of novel polyspecific antivenoms of wide clinical efficacy against the venoms of the medically-relevant snake species within the geographical range where these antivenoms are intended to be deployed, but also by optimizing the paraspecific use of current antivenoms. In Colombia, antivenoms are supplied by two manufacturers, one public, the Instituto Nacional de Salud (INS), and one private, Laboratorios Probiol (PROBIOL). However, the antivenom supply in Colombia has traditionally been insufficient, a circumstance that has led the Colombian Ministerio de Salud y Protección Social to issue several resolutions and decrees to announce this health emergency in the country, and to import antivenoms produced in México and Costa Rica. Contrary to these countries, where  Snakebite envenoming is an occupational hazard and a WHO category A neglected tropical disease (NTD)  that annBothrops, Bothriechis, Bothrocophias and Porthidium. Among them, Bothrops species have the highest epidemiological importance. Particularly B. asper, a species complex widely distributed from México to Perú, where it is commonly known as nauyaca, barba amarilla, terciopelo, equis/talla equis, equis patiana, cacica, pelo de gato, boquidorá, mapaná, damá, tapa, etc. (http://snakedatabase.org/species/Bothrops/asper) .,22.21,22et al.  and Sala , where Ri corresponds to the area of the same protein “i” in the chromatogram of the fraction retained and eluted from the affinity column. This result was corrected for toxins such as SVMP which elute with difficulty from the column due to their high binding affinity to the immobilized antivenom. In this case, the percentage of non-immunocaptured toxin “i” (% NRtoxin“i”) was calculated as the ratio between the chromatographic areas of the same peak recovered in the non-retained fraction (NRtoxin“i”) and in a reference venom (Vtoxin“i”) containing the same amount of total protein that the parent venom sample and run under identical chromatographic and experimental conditions, using the equation %NRtoxin "i" = (NRtoxin "i"/Vtoxin "i") x 100 .et al.  by the [total amount of antibody (IgG or F(ab’)2) (in mg) per antivenom vial] [50]×LD50, where “n” is the number of LD50s used as challenge dose to determine the ED50 and, "n-1" is used because at the endpoint of the neutralization assay, the remaining activity of one LD50 remains unneutralized causing the death of 50% of mice  ,51,52. T of mice ,54. For ntivenom .2 inhalation, their skin removed, and the area of the hemorrhagic lesion photographed. The images were processed with software Inkscape v. 0.92 as described by Jenkins et al.  for B. rhombeatus, and [INS-COL > (BIOL ~ INS-PERU) > UCV > ICP > PROBIOL] for B. ayerbei. In terms of percentage of toxin-binding antibodies, the order from highest to lowest relative abundance was [INS-PERU > (BIOL ~ INS-COL) > UCV > ICP > PROBIOL] for B. asper (sensu stricto), [INS-COL > (BIOL ~ INS-PERU) > (ICP ~ UCV ~ PROBIOL)] for B. rhombeatus, and [(INS-COL ~ INS-PERU) > BIOL > ( ICP ~ UCV ~ PROBIOL)] for B. ayerbei.The immunoreactivity of the six antivenoms against the venom components of o et al. ,32 was io et al. –45  . Chromatayerbei) . Table 4B. asper lineage venoms, the results displayed in B. asper lineage venoms was comparatively assessed by ELISA: INS-COL showed the highest titer against all the venoms and the lower levels of cross-reactivity of ICP and PROBIOL against the three venoms were similar and indistinguishable between themselves , INS-PERU , UCV , BIOL (B. asper of undisclosed geographic origin) [Although none of the antivenom affinity matrices outshined all the others in its ability to immunocapture each and every one of the components of the three emselves . In addiemselves , calculaRH > BAY . These f brazili ), ICP (C origin) .in vivo lethality neutralization assay [B. asper (sensu stricto), B. rhombeatus, and B. ayerbei. All three antivenoms followed the "antivenomics rule" regarding their potency to neutralize the lethal effect of the three B. asper lineage venoms, as well as the coagulant and proteolytic effects   .50s of INS-COL and ICP are statistically indistinguishable from each other and significantly (p<0.05) higher (more effective) than that of PROBIOL towards each Colombian B. asper lineage venom tested , B. rhombeatus, and B. ayerbei venoms  during the incubation time of the antivenom/venom mixture, affecting vascular permeability and edema formation, has been invoked to explain this phenomenon. The fact that this event was mainly observed in experiments involving the antivenom with the most impurities of plasma proteins, PROBIOL  . Previouumstance . Proteol PROBIOL , would s2+-dependent SVMPs present in B. asper venom [B. asper lineage venoms (ED50 = 42.3 ± 7.7 mg venom/g antivenom), followed in order of potency by ICP and PROBIOL , respectively (Proteolytic activity of the three venoms was similar . This efer venom . INS-COLectively .B. asper (sensu stricto) and B. rhombeatus venoms, but not in B. ayerbei venom (2s [B. asper (sensu stricto) and B. rhombeatus (50 of 24.0 ± 0.5 mg venom/g antivenom (B. asper (sensu stricto) and B. rhombeatus venoms even at a ratio of 2000 μL antivenom/mg venom.Indirect hemolytic activity was recorded in ei venom , as has enom  reported a comparative study of the neutralizing capacity of four polyvalent antivenoms manufactured in Colombia , Venezuela (UCV), and México  against the pharmacological and enzymatic effects of the venoms of B. asper and Porthidium nasutum from Antioquia (northwest Colombia) and Chocó (west Colombia) [B. asper venom pooled from 40–45 specimens from different regions of Antioquia and Chocó. Conversely, antivenom PROBIOL presented the lowest neutralizing capacity towards the same activities, and the Venezuelan UCV antivenom had intermediate neutralization abilities. Understanding the basis of the effectivity of antivenoms against homologous and heterologous venoms demands the quantitative assessment of its toxin-resolved immunorecognition profile. Here we have applied third-generation antivenomics to compare the specific and paraspecific immunoreactivity of six bothropic antivenoms against three Colombian B. asper lineage venoms. The antivenomics outcome showed that all the major toxin families, i.e., SVMP, PLA2, CRISP, SVSP, CTL [B. asper lineage venoms is not biased towards any particular family of toxins but is well balanced among the different venom protein families. Therefore, our study provides a solid experimental ground to rationalize the reported immunological profiles of INS-COL, UCV, and PROBIOL [B. asper venoms from different geographic Colombian ecoregions. These results strongly suggest the feasibility of adding these antivenoms to the list of candidates for the treatment of snakebite accidents caused by the species of the B. asper complex from SW Colombia, B. asper (sensu stricto), B. rhombeatus and B. ayerbei in the Departments of Nariño and Cauca.Our data confirm and expand previous studies. Otero olombia) . In theiVSP, CTL , were im PROBIOL , and othin vitro qualitative (Western blot) and semi-quantitative (ELISA) evidence that Brazilian polyspecific pentabothropic (SAB) or antibothropic-lachesic F(ab')2 antivenoms exhibited variable paraspecific immunoreactivity towards nineteen venoms of bothropic snakes, including B. brazili, B. alternatus, B. atrox, B. bilineatus, B. castelnaudi, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. itapetiningae, B. jararaca, B. jararacussu, B. leucurus, B. marajoensis, B. moojeni, B. neuwiedi, B. pirajai, B. pradoi, and Bothrocophias hyoprorus. The remarkable para-specificity exhibited by antivenoms generated against immunization mixtures that included venoms from phylogenetic distant Bothrops species may be ascribed to large conservation of immunoreactive epitope on venom toxins across much of the natural history of Bothrops, a genus that had its roots in South America during the middle Miocene, 14.07 (CI95: 16.37–11.75) Mya [B. asper as the first species complex to split from the B. atrox group in the Pliocene, around 3.02–2.32 Mya [Bothrops phylogeny emerged also from studies of the paraspecific effectiveness of the pentabothropic polyvalent antivenom SAB  produced by Instituto Butantan  [B. jararaca (50%), B. jararacussu (12.5%), B. moojeni (12.5%), B. alternatus (12.5%) and B. neuwiedi (12.5%) [B. diporus (Argentina), B. jararaca (Brazil), B. matogrossensis (Bolivia), B. atrox (Perú and Colombia) and B. asper (Costa Rica) also showed a pattern of extensive cross-neutralization of all the venoms tested, with quantitative differences in the values of the effective doses of the antivenoms [Bothrops atrox, B. brazili, B. barnetti and B. pictus [Bothrops spp. venoms from Perú and Costa Rica.Queiroz and co-workers  have rep.75) Mya ,89  ,79,91–94 (12.5%) . Furthertivenoms ,97. Simi. pictus . Both anBothrops include taxa that represent the main medically important venomous snakes in their range [Bothrops venoms in Central and South America, and also highlight quantitative differences in their ED50s. The combination of antivenomics and in vivo neutralization assays provides relevant information to delineate the species spectrum and geographic range of clinical applicability of an antivenom. Now, the challenge is to conduct an extensive study to define the matrix of preclinical effectiveness of all the bothropic antivenoms against all the venoms of  species within the genus Bothrops.All the species complex groups within genus ir range ,99. Our S1 FigA displays the fractionation by reverse-phase HPLC of the venom components. Proteins eluting in each peak (1–14) were assigned using the venomics information reported by Mora-Obando et al. [B-G represent RP-HPLC fractionations of the immunoretained fractions recovered in the affinity columns of immobilized antivenoms INS-COL (B), PROBIOL (C), ICP (D), INS-PERU (E), UCV (F), BIOL (G) incubated with increasing amounts of venom (100–1200 μg). Panels H and I display to chromatographic separations of the venom fraction retained in the mock matrix control and the naïve equine immunoglobulins control, respectively.Panel o et al. . Abbrevi(TIF)Click here for additional data file.S2 FigA displays the fractionation by reverse-phase HPLC of the venom components. Proteins eluting in each peak (1–14) were assigned using the venomics information reported by Mora-Obando et al. [B-G represent RP-HPLC fractionations of the immunoretained fractions recovered in the affinity columns of immobilized antivenoms INS-COL (B), PROBIOL (C), ICP (D), INS-PERU (E), UCV (F), BIOL (G) incubated with increasing amounts of venom (100–1200 μg). Panels H and I display to chromatographic separations of the venom fraction retained in the mock matrix control and the naïve equine immunoglobulins control, respectively.Panel o et al. . Abbrevi(TIF)Click here for additional data file.S3 FigA displays the fractionation by reverse-phase HPLC of the venom components. Proteins eluting in each peak (1–14) were assigned using the venomics information reported by Mora-Obando et al. [B-G represent RP-HPLC fractionations of the immunoretained fractions recovered in the affinity columns of immobilized antivenoms INS-COL (B), PROBIOL (C), ICP (D), INS-PERU (E), UCV (F), BIOL (G) incubated with increasing amounts of venom (100–1200 μg). Panels H and I display to chromatographic separations of the venom fraction retained in the mock matrix control and the naïve equine immunoglobulins control, respectively.Panel o et al. . Abbrevi(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S3 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S4 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S5 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S6 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S7 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S8 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S9 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S10 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S11 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S12 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S13 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S14 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S15 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S16 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S17 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S18 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S19 TableMaximal binding for each RP-HPLC fraction is highlighted in yellow background. Green background, amount (in μg) of toxin family proteins immunoretained in the affinity columns.(XLSX)Click here for additional data file.S20 Table(XLSX)Click here for additional data file.


Salmonella from patients in China, Salmonella isolates were collected from fecal and blood samples of patients. In this study, 42.8% (136/318) clinical isolated Salmonella were resistant to colistin. MIC distribution for colistin at serotype level among the two most prevalent serotypes originating from humans in China indicated that Salmonella Enteritidis  were significantly less susceptible than Salmonella Typhimurium . mcr genes and mutations in PmrAB confer little for rate of colistin resistant Salmonella isolated from human patients. Phylogenetic tree based on core-genome single nucleotide polymorphisms (SNPs) was separately by the serotypes and implied a diffused distribution of MICs in the same serotype isolates. Relatvie expression levels of colistin resistant related pmr genes were significantly higher in non-mcr colistin resistant S. Typhimurium than in colistin sensitive S. Typhimurium, but no discernable differences between colistin resistant and sensitive S. Enteritidis, indicating a different mechanism between colistin resistant S. Typhimurium and S. Enteritidis. In conclusion, colistin susceptibility and colistin resistant mechanism of clinical isolated Salmonella were closely associated with specific serotypes, at least in the two most prevalent serotype Enteritidis and Typhimurium. We suggest clinical microbiology laboratory interpreting Salmonella colistin MIC results in the serotype level.To investigate the prevalence, probable mechanisms and serotype correlation of colistin resistance in clinical isolates of  Paenibacillus genus  have since discovery in 2015 been frequently observed, and their continued widespread dissemination has became a challenge to public health worldwide  by performing a slide agglutination test (State Serum Institute (SSI), Copenhagen, Denmark).A total of 318 isolates of patients . SalmoneE. coli ATCC 25922 was used as a control. The experiment was conducted in duplicate on at least two separate occasions. The higher MIC was accepted for analysis in the duplicative test within one double dilution difference, or a third replicate would be measured  dilutions were used for colistin MIC determination according to the CLSI-EUCAST joint recommendations. measured . The resmcr-1 to mcr-9 from previous studies (pmrA and pmrB were amplified by primers pmrAB-F and pmrAB-R (pmrA and pmrB were compared to that of S. Typhimurium LT2 (GenBank: GCA_000006945.2), a colistin-susceptible Salmonella reference strain.The isolates were screened by using primers targeting  studies . For the pmrAB-R . The posSalmonella spp. isolates (including 64 colistin resistant and 72 susceptible isolates) were extracted using Gentra Puregene Yeast/Bact. Kit . Genomes were sequenced using the Illumina HiSeq 2500-PE150 platform . Quality-trimmed raw sequence data was assembled by using SPAdes 3.13.0. Annotation was performed by uploading the data to the RAST server (rast.nmpdr.org). All the assembled genomes were deposited in NCBI, the genome accession numbers were listed in Genomic DNA of 136 randomly selected Salmonella genomes were analyzed by using the SeqSero 1.2 software . Cells were collected at 4°C by centrifuging at 10,000 rpm for 1 min, and RNA was extracted using TRIzol Reagent (Invitrogen). DNase I-treated RNA was obtained using an RNeasy Mini Kit , and mRNA expression levels of the target genes were examined using real-time PCR primers listed in S. enterica using the standard curve method.Overnight cultures of the target Salmonella Enteritidis and Salmonella Typhimurium. Due to the low prevalence of isolates with other serotypes, these were excluded from the analyses. Fisher’s exact tests were used to determine if resistant and susceptible phenotypes were equally distributed in S. Enteritidis and S. Typhimurium, and among isolates from blood and intestinal samples. A Mann-Whitney test was performed to determine if MICs were equally distributed among isolates of S. Enteritidis and S. Typhimurium. For the statistical analysis of relative expression levels of pmr genes, values are presented as means ± standard deviation (SD). Rank-sum tests were performed for pair-wise comparisons of groups, and P < 0.05 (two-tailed) was considered significant.Hypothesis testing was performed by stratifying the analyses for isolates of Salmonella spp. isolates ranged from 0.25 mg/L to 16 mg/L  of the bloodstream infection isolates were identified as colistin-resistant, whereas 30% (30/100) of the isolates from intestinal samples were determined as colistin-resistant.The colistin MICs of the tested  16 mg/L . The twomcr genes and sequencing of pmrA and pmrB. Out of 136 colistin-resistant isolates, two were as mcr-1 positive (S039-44712 and S040-44862), none had other mcr genes, and two had pmrAB mutations which affected the amino acid sequence (S132-54197 and S133-54550). The isolates with pmrAB mutations carried a missense point mutation in pmrA (T89S), and five missense mutations in pmrB . However, none of the mutations were predicted to affect the protein function.Resistance mechanisms of all the colistin-resistant isolates were investigated by screening for S. Enteritidis (n = 149) and S. Typhimurium (n = 59) were found to be the overall most prevalent. S. Enteritidis (122/218) was the most common among isolates from bloodstream infection samples, followed by S. Typhimurium (12/218), whereas the inverse was true among isolates from patients with intestinal infections, with 27.0% (27/100) S. Enteritidis and 47.0% (47/100) S. Typhimurium (P < 0.01).18 serotypes were identified among the 318 isolates. S. Enteritidis and S. Typhimurium were 83.9% (125/149) and 15.3% (9/59), respectively. Isolates of S. Enteritidis were significantly less susceptible than isolates of S. Typhimurium (P < 0.01) and had significantly higher MICs (P < 0.01). S. Enteritidis was most prevalent among the colistin-resistant isolates, representing 91.9% (125/136) of all with the resistance phenotype, followed by S. Typhimurium . All colistin-resistant isolates were S. Enteritidis and S. Typhimurium, except for the two isolates (S132-54197 and S133-54550) which were identified as Salmonella Gallinarum (1/2) and Salmonella Goldcoast (1/1), respectively.The rate of colistin-resistant Salmonella serotypes, phylogenetic evolution and MICs, Salmonella serotypes were confirmed and phylogenetic trees based on core-genome SNPs were generated. The whole-genome sequenced isolates were predicted serotypes using SeqSero 1.2 and were compared to the traditional Kauffman-White serotyping. Phylogenetic analysis was employed to confirm the serotyping, by using core-genome SNPs as described above. 63 S. Enteritidis, 45 S. Typhimurium and 28 isolates of other serotypes were identified from the 136 whole-genome sequenced isolates, and were 98.4% (63/64), 97.8% (45/46), and 92.9% (28/30) identical to the traditional Kauffman-White serotyping, respectively.In order to discover the relation between S. Enteritidis isolates  in colistin sensitive S. Typhimurium or S. Enteritidis were compared with non-mcr colistin resistant S. Typhimurium or S. Enteritidis, respectively. Seven isolates of each serotype were colistin resistance (pmr genes were enhanced in colistin resistant S. Typhimurium than in colistin sensitive S. Typhimurium (P < 0.05), but no discernable differences between colistin resistant and sensitive S. Enteritidis , however, there was a sizeable and significant difference in colistin susceptibility and MICs between isolates of the two serotypes. Interestingly, the colistin resistance rate of bloodstream infection isolates was significantly higher compared to that of isolates from intestinal samples (P < 0.01). This difference was attributable to that S. Enteritidis were significantly more common among bloodstream isolates compared to the S. Typhimurium (P < 0.01). This indicates that not only is S. Enteritidis more likely to be colistin-resistant compared to S. Typhimurium, but also more frequently cause severe infections in China.mcr genes and missense mutations causing amino acid substitutions in PmrAB are the most commonly reported colistin resistance mechanisms in Salmonella spp. Surprisingly, in this study, only four isolates were observed with either of these resistance mechanisms, including two S. Typhimurium isolates carried mcr-1 and two isolates had missense mutations in pmrAB. According to previous studies in China, S. Typhimurium appear to more easily acquire mcr genes compared to S. Enteritidis  might play a role in colistin susceptibility. Whether and how different O-antigen confer a different colistin susceptibility phenotype of Salmonella needs to be further investigation.Aside from that unknown colistin resistance mechanisms could account for the difference in colistin resistance rates of Salmonella spp. is lacking in the data presented by EUCAST; currently, only the MIC distribution of S. Dublin is presented. The results in this study indicate a very high rate of colistin resistance among S. Enteritidis in clinical isolates in China, and this could be associated to elevated wild type MICs compared to other serotypes. Further studies including isolates of S. Enteritidis from other countries and other sources are warranted to explore this possibility.An epidemiological cut-off value for colistin regarding WPIS00000000-WPNX00000000.The original contributions presented in the study are publicly available. This data can be found in NCBI under accession Nos. The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affilated Hospital of Zhejiang University. The patients/participants provided their written informed consent to participate in this study.QL and YX conceived and designed the experiments. YW, HF, XY, and YC performed the experiments. QL, BB, and BZ analyzed the data. QL wrote the manuscript. All authors contributed to the article 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.


Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs).Collectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea  and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history.We retrieved CYPs from RNA-seq data of Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.This study discovered 378 full-length CYPs in  Jacobaea species in the Asteraceae family. Thirty-seven structurally related PAs have been detected in Jacobaea vulgaris Gaertn., Jacobaea aquatica (Hill) G.Gaertn., B.Mey. & Scherb and their hybrids [Plants produce a great variety of secondary metabolites (SMs) which are involved in the adaptation of plants to both biotic and abiotic stresses –3. At pr hybrids . As yet,To understand the origin of SM diversity, molecular investigations of SM biosynthetic pathways are promising as it is believed that SM diversity of plants is under genetic control –11. GeneTaxus chinensis (Rehder & E.H.Wilson) Rehder transcriptomes with the aim to discover candidate genes involved in the biosynthesis of diterpenoids including taxol. Chen et al. [Salvia miltiorrhiza Bunge transcriptomes with candidates for terpenoid biosynthesis.A well-curated set of CYP genes from a particular species is essential for functional identification of the encoded enzymes. In recent years, genome/transcriptome-wide identification of CYPs from plants has been performed to explore their involvement in metabolic pathways –24. For N-oxide. Senecionine N-oxide undergoes structural transformations in a position-specific and stereoselective manner resulting in the rearrangement of the skeletal structure and oxidative modifications thereof [Jacobaea species occurs in the shoots while the primary PA senecionine N-oxide is synthesized in the roots [N-oxide to other PAs is brought about via specific one- or two-step reactions including epoxidation, hydroxylation, dehydrogenation and/or O-acetylation [Jacobaea species will be beneficial to identify potential CYP candidates involved in PA biosynthesis.PAs were selected to launch the discovery of structural genes causing SM diversity in our study. So far, the only pathway-specific enzyme of PA biosynthesis that has been identified is homospermidine synthase, which converts spermidine and putrescine into homospermidine, the first specific intermediate in the PA biosynthesis pathway . It is n thereof . It was he roots , 27. Wittylation , 28. TheJ. vulgaris and J. aquatica and established comprehensive information on CYP families. These two closely related species have been well studied for their PA contrasts [Jacobaea species and two other species from the Asteraceae using the CYPs from Arabidopsis thaliana (L.) Heynh. as an outgroup to explore relatedness and evolution of CYPs across five species.We have established de novo transcriptome assemblies for ontrasts , 29, butJacobaea species, which facilitates further exploration of possible functions in PA metabolism. In total, two sets of samples were obtained for both J. vulgaris (Jv1 and Jv2) and J. aquatica (Ja1 and Ja2). After removal of adaptor sequences, ambiguous reads and low-quality reads (Q < 30), paired-end clean reads were further processed. The trimmed reads obtained in this study have been deposited in the NCBI SRA database (accession numbers: SRR10013580-SRR10013584 under the BioProject PRJNA561604).The purpose of this study was to obtain systematic information of CYPs in For each of the four sets, more than 20 million cleaned up paired-end reads were used for the de novo assembly with Trinity Table . The resJacobaea sets were similarly distributed among different functional categories. The MeJA treated set (Ja2) showed a slightly lower percentage in the category “cellular component” but higher percentages in the categories “molecular function” and “biological process” than the other three Jacobaea sets  and multi-nucleotide variants (MNVs) were detected based on mutual read mapping for the four J. vulgaris, and a total of 157 full-length , while the 157 full-length CYPs of J. aquatica were divided into eight clans including 35 families  (Table J. vulgaris (53.8%) and J. aquatica (46.4%) were assigned to CYP71, CYP706, CYP76, CYP72, CYP82 and CYP93 families, of which only CYP72 is non-A-type. Compared with J. vulgaris, for J. aquatica less full-length CYPs were detected, which might be caused by the lower number of genotypes and the lower amount of reads in the J. aquatica samples. However, the proportional distributions of full-length CYPs were similar not only in each CYP clan , but also within each CYP family  of the two Jacobaea species , J. aquatica 404 , H. annuus 462, L. sativa 374) contained more CYP genes than A. thaliana (244). It indicates an expansion and functional diversification of CYP genes encoding metabolic pathways in the Asteraceae during evolution and genome duplications.We compared the numbers of the detected full-length CYPs of a. Table . RoughlyP < 0.001). Numbers of CYPs in single-family CYP clans  were fairly consistent . The significant difference was caused by multiple-family clans  which parallel land plant evolution [P < 0.001). In accordance with the statement of Nelson and Werck-Reichhart [Jacobaea species compared with H. annuus and L. sativa, including CYP703, CYP736, CYP715, CYP734, CYP87, CYP718, CYP724, CYP728, CYP729 and CYP733. Without further information, it is difficult to infer whether the absence/presence is an evolutionary consequence or just due to the unavailability of full-length transcripts in the transcriptomes of Jacobaea.Overall, the distributions of CYPs among different CYP clans over the five species Table  were comvolution  and whiceichhart , the youJ. vulgaris  hierarchies  full-length CYPs were appointed to 33 KO catalogs  full-length CYPs of .1% full-Jacobeae species, namely, CYP71, CYP76, CYP706, CYP93, CYP82 and CYP72, based on their amino acid sequences  G. Don are from different families: CYP71D2, CYP72A1, CYP76B6 [KEGG pathway-based annotation was performed for all full-length CYPs, and no CYPs were designated to alkaloid biosynthetic pathways. Empirically, CYPs from the same family/subfamily often catalyze similar/related reactions . For exa CYP76B6 –37. Alka CYP76B6 , especiaJacobaea, a fast evolution of CYPs was observed resulting in lineage-specific expansion. Notably, CYPs do not always follow the pattern in which H. annuus showed a closer phylogenetic relatedness to Jacobaea species than L. sativa as indicated by Compositae metatrees [H. annuus and L. sativa switched phylogenetic closeness to those of Jacobaea species on the phylogenetic tree  were missing in J. aquatica,  were less than those for J. vulgaris (69), leading to less comprehensive CYP expression. Based on our study, it is not possible to appoint CYP candidates involved in PA biosynthesis. Nonetheless, the collection of CYPs in Jacobaea species can speed up the exploration of function in following studies. As long as whole genome information of Jacobaea species is lacking, 5′ Race and 3′ Race techniques can be employed to obtain a more complete collection of full-length CYPs. The prediction of CYP candidates can be further facilitated by correlating gene expression patterns with PA abundances in plants grown under conditions that generate PA contrasts or in F2 offspring segregating for PA profiles.During plant evolution, individual genes and gene families have been confronted with selection for copy number via duplications, transpositions, and/or deletions . J. vulgntration . The queJ. vulgaris and J. aquatica, respectively. Comparison of CYPs over five species showed strong lineage specific diversification of CYPs, indicating fast evolutionary speed of CYPs within the Asteraceae. Only in the closely related J. vulgaris and J. aquatica, CYPs were found quite often in pairs, confirming a close relationship in the evolutionary history. No genes were found to be involved in alkaloid biosynthesis against KEGG database. Finally, our study presents the first comprehensive overview of CYPs in Jacobaea species, which is beneficial for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.Here we detected 221 and 157 full-length CYPs for J. vulgaris and J. aquatica originating from different parts of the distribution ranges  consisted of the pooled shoots and roots of 59 individuals from nine different populations across Europe including two individuals derived from tissue culture and one population from Canada  was composed from multiple individuals, clones, of one genotype that was kept in tissue culture. For the set Jv2, five individuals from tissue culture derived plants of J. vulgaris treated with methyl jasmonate (MeJA) and five mock treated individuals were used as control. From both MeJA treated and control plants cDNA libraries were obtained that were sequenced separately. The resulting reads were pooled in silico in the later assembly step. Both J. aquatica sets (Ja1 and Ja2) were derived from the same seven individuals pooled from two populations with two individuals originating from tissue culture, of which roots were included in Ja1 but not in Ja2  medium with 0.44 mM benzylaminopurine. To induce roots plants were transferred to MS medium without hormones for 2 weeks. After rooting plants were transferred to pots filled with the soil mixture as indicated above. All plants were kept in a climate room for 6 weeks . Then the plants were separated into shoots and roots, and roots were rinsed with water. Two to three fully grown leaves and ¼ of roots from each plant were wrapped in aluminum foil and flash frozen in liquid nitrogen, respectively. Afterwards all samples were separately ground into powder with liquid nitrogen. Shoot powder was mixed with root powder in a ratio of 3:1 for each plant, and then identical amounts of powder from each individual were pooled for Jv1 and Ja1, respectively, whereas only powdered shoots were pooled for Ja2. All powdered materials were stored at − 80 °C until RNA extraction.For sets Jv1, Ja1 and Ja2, seeds were germinated on the surface of wet potting soil covered by plastic bags and the seedlings were transferred into 9 × 9 × 10 cm pots filled with 50% sandy soil (collected from Meijendel), 50% potting soil  and 1.5 g/L Osmocote slow release fertilizer . Tissue cultured plants of J. vulgaris tissue culture plants were kept on MS medium with agar for 2 weeks after propagation in a climate room . One hundred microliters of MeJA (Sigma-Aldrich) dissolved in 10% ethanol solution (4.5 mmol/L) was added to the surface of medium, reaching a final concentration of 90 μmol/L after diffusion in each tube, while the same volume of 10% ethanol was added to the control group under axenic condition. Shoots of five biological replicates collected at 8 days after the treatment were pooled and ground into fine powder for both induced and control groups, respectively. All powder was stored at − 80 °C until RNA extraction.For set Jv2, replicate Total RNA was extracted with the NucleoSpin® RNA Plant-Macherey-Nagel kit for five samples, namely Jv1, MeJA induced group of Jv2, control group of Jv2, Ja1 and Ja2. The RNA integrity Number (RIN) and RNA concentration were assessed using the Agilent 2100 Bioanalyzer. Strand specific RNAseq libraries were generated using the method described by  with minAfter removal of adapter sequences, the qualities of raw reads were checked using FastQC and the bases with low quality (threshold < 30) were cut off by Trimmomatic via the Galaxy platform . The paiJacobaea sets at the Trinity ‘gene’ level containing a cluster of transcript isoforms.The likely coding regions and open reading frames (ORFs) of transcriptomes were predicted with TransDecoder . The traJ. aquatica were mapped to the transcriptomes of Jv1 and Jv2 separately, whereas the combined reads of J. vulgaris were mapped to the transcriptomes of Ja1 and Ja2 separately. The detection of InDels and structural variants was performed based on the read mapping in CLC Genomics Workbench (version 8.5.1) using default parameters except that the P-Value threshold was set to 0.00001. In addition, SNVs and MNVs were detected also based on the abovementioned read mapping using the Basic Variant Detection tool of the Variant Detectors module in CLC Genomics Workbench with default parameters. SSRs were identified using the MISA MicroSatellite identification tool [The combined reads of ion tool , settingJ. vulgaris were combined and 100% identical transcripts were removed by using the CD-HIT-EST algorithm (version 4.6.8) [J. aquatica, the sample approach was applied to combine CYP-like contigs from sets Ja1 and Ja2.To identify CYP-like contigs from the four transcriptomes, the HMMER program  was usedn 4.6.8) , 58. ForJ. vulgaris were mapped to all CYP-like contigs of J. aquatica in CLC Genomics Workbench using the following parameters: mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.8, similarity fraction 0.97. The consensus sequences of the mapped reads were retained and assembled with the original CYP-like contigs of J. vulgaris in Sequencher (version 5.0), using a minimum match percentage of 97% while minimum overlap was set to 15%. Thereupon, the Sequencher assembly of CYP-like contigs were checked for redundancies using the CD-HIT-EST algorithm with sequence identity of 97% as cutoff. Similarly, to get additional CYP-like contigs for J. aquatica, CYP-like contigs of J. vulgaris were used as references for read mapping, followed by the same steps afterwards.To obtain additional CYP-like contigs, the reads of The likely coding regions of the resultant CYP-like contigs of both species were predicted by TransDecoder . In ordeThe final classification and nomenclature of all full-length CYP proteins were carried out by Prof. Dr. David R. Nelson through comparison with references from a well-annotated plant CYP database which includes both published and confidential sequences, following the CYP nomenclature principle . Cutoff The CYP assemblies were divided into A-type which only comprises the CYP71 clan, and non-A-type which includes all other plant CYP clans. The sequences of A-type and non-A-type were separately submitted to Multiple Expectation Maximization for Motif Elicitation (MEME) to predict motifs and to Motif Alignment and Search Tool (MAST) to discover homologs . The logH. annuus [L. sativa [Jacobaea species based on homologs by the HMM model. Only CYPs longer than 400 amino acids were chosen in this study as the length of the most reliably annotated CYPs of A. thaliana ranges from 457 to 594 amino acids without taking pseudogenes into account. All chosen CYP genes were classified based on the best blast hits by Prof. Dr. David R. Nelson. The CYP protein sequences of A. thaliana were downloaded from the Arabidopsis Cytochrome P450 database [The CYP protein sequences of . annuus  and L. s. sativa  were retdatabase . Multipldatabase , 71 on Xdatabase . BootstrAdditional file 1. Functional annotation and structural analysis of Jv1 transcriptome. The file contains the information of gene functional annotation, InDels, structural variants and simple sequence repeats (SSRs).Additional file 2. Functional annotation and structural analysis of Jv2 transcriptome. The file contains the information of gene functional annotation, InDels, structural variants and simple sequence repeats (SSRs).Additional file 3. Functional annotation and structural analysis of Ja1 transcriptome. The file contains the information of gene functional annotation, InDels, structural variants and simple sequence repeats (SSRs).Additional file 4. Functional annotation and structural analysis of Ja2 transcriptome. The file contains the information of gene functional annotation, InDels, structural variants and simple sequence repeats (SSRs).Additional file 5: Figure S1. WEGO histogram representation of GO classification for transcriptomes of Jv1, Jv2, Ja1 and Ja2. X-axis shows user selected GO terms; left y-axis shows the percentages of genes .Additional file 6: Table S1. List of full-length CYPs of J. vulgaris identified in this study. aCellular location of the protein predicted by TargetP. “C” chloroplast; “S” secreted; “_” any other location; “*” unknown. bnot available. cKEGG Orthology.Additional file 7: Table S2. List of full-length CYPs of J. aquatica identified in this study. aCellular location of the protein predicted by TargetP. “C” chloroplast; “S” secreted; “_” any other location; “*” unknown. bnot available. cKEGG Orthology.Additional file 8: Figure S2. Phylogenetic tree of the CYP76 family from 5 species inferred with the maximum likelihood method. CYP450s are color coded for different species: J. vulgaris (orange), J. aquatica (light blue), H. annuus (dark blue), L. sativa (green), A. thaliana (black). The names of CYP450s of H. annuus and L. sativa were tentatively coded without nomenclature. A. thaliana was used as the outgroup.Additional file 9: Figure S3. Phylogenetic tree of the CYP706 family from 5 species inferred with the maximum likelihood method. CYP450s are color coded for different species: J. vulgaris (orange), J. aquatica (light blue), H. annuus (dark blue), L. sativa (green), A. thaliana (black). The names of CYP450s of H. annuus and L. sativa were tentatively coded without nomenclature. A. thaliana was used as the outgroup.Additional file 10: Figure S4. Phylogenetic tree of the CYP82 family from 5 species inferred with the maximum likelihood method. CYP450s are color coded for different species: J. vulgaris (orange), J. aquatica (light blue), H. annuus (dark blue), L. sativa (green), A. thaliana (black). The names of CYP450s of H. annuus and L. sativa were tentatively coded without nomenclature. A. thaliana was used as the outgroup.Additional file 11: Figure S5. Phylogenetic tree of the CYP93 family from 5 species inferred with the maximum likelihood method. CYP450s are color coded for different species: J. vulgaris (orange), J. aquatica (light blue), H. annuus (dark blue), L. sativa (green), A. thaliana (black). The names of CYP450s of H. annuus and L. sativa were tentatively coded without nomenclature. A. thaliana was used as the outgroup.Additional file 12: Figure S6. Phylogenetic tree of the CYP72 family from 5 species inferred with the maximum likelihood method. CYP450s are color coded for different species: J. vulgaris (orange), J. aquatica (light blue), H. annuus (dark blue), L. sativa (green), A. thaliana (black). The names of CYP450s of H. annuus and L. sativa were tentatively coded without nomenclature. A. thaliana was used as the outgroup.Additional file 13: Table S3. Details of sample sets of J. vulgaris and J. aquatica for deep sequencing analysis. *cDNA library was normalized with duplex-specific thermostable nuclease to remove abundant library molecules.


N-terminal pro-B-type natriuretic peptide (NT-proBNP) serum concentrations with cognitive functions of glioma and meningioma patients.  This study aimed to investigate the association of high-sensitivity C-reactive protein (hsCRP) and  177 brain tumor patients awaiting for brain tumor surgery participated in the study. Patients were assessed preoperatively, using neuropsychological tests for verbal memory, psychomotor speed, mental flexibility, and verbal fluency. The functional status of patients was evaluated using the Karnofsky Performance Index. Blood samples were drawn for evaluation of serum hsCRP and NT-proBNP concentrations upon hospital admission. rho = 0.30, p < 0.05). In meningioma sample, NT-proBNP correlated with decreased psychomotor speed , mental flexibility , worse cumulative learning , and delayed recall . However, the relationship between the NT-proBNP and cognitive functions became nonsignificant when demographic and clinical covariates were included into analysis. Higher hsCRP concentration remained significantly related to slower psychomotor speed (p = 0.02) and worse mental flexibility (p = 0.05) in glioma patients, independently from demographic and clinical covariates. Preoperative cognitive functioning was also predicted by older age, gender, side and location of the tumor, and tumor malignancy, and general functional status of a patient.  The highest NT-proBNP concentration was observed in meningioma patients. Glioma and meningioma patients did not differ in hsCRB concentration. Patients in the highest hsCRP tertile were older and more frequently reported cardiovascular comorbidity. Patients in the highest NT-proBNP tertile were older, more frequently with cardiovascular comorbidity, females, and diagnosed with a meningioma. hsCRP was significantly related to slower psychomotor speed in high-grade glioma patients ( NT-proBNP was not associated with memory, language, and attention/executive cognitive domains of glioma and meningioma patients. Increased hsCRP was related to slower psychomotor speed and worse mental flexibility in glioma patients, indicating that inflammation processes are important for cognitive functioning in glial tumors. Cognitive decline is a common and devastating sequalae of brain tumors (BTs) . CognitiNumerous tumor and treatment-related factors were implicated in the progressive cognitive decline of BT patients. BTs can infiltrate and/or displace normal brain tissue, increase intracranial pressure, cause seizures, and disrupt structural and functional brain connectivity, subsequently leading to cognitive decline , 6. HoweInflammatory response is important for development, progression, and prognosis of malignant , 16 and NT-proBNP is produced by cardiac myocytes in response to volume overload and is used for diagnosis and monitoring of heart failure patients . GreaterIn this study, we aimed to investigate the association of hsCRP and NT-proBNT serum concentrations with memory, language, and attention/executive cognitive domains of glioma and meningioma patients.The study protocol and consent procedure were approved by the Ethics Committee for Biomedical Research of the Lithuanian University of Health Sciences, Kaunas, Lithuania, and were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from each study patient before inclusion in the study.Consecutive patients admitted at the Department of Neurosurgery of Hospital of Lithuanian University of Health Sciences, Kaunas, Lithuania, for glioma or meningioma surgery in a period from October 2015 to May 2017 were invited to participate in this cross-sectional observational cohort study. The study exclusion criteria included severe preoperative cognitive deficits and/or neurological impairment leading to inability to complete the study tasks, established diagnosis of acute infections  or documented chronic inflammatory disorders .Blood samples were drawn for evaluation of serum hsCRP and NT-proBNP concentrations upon hospital admission. Neuropsychological assessment was performed from two to three days before BT surgery by a certified medical psychologist. Medical history, clinical characteristics, and functional status of the study patients were recorded by the study neurosurgeon. Histological BT diagnoses were verified from postoperative pathology reports. Information about tumor laterality and location was obtained by reviewing preoperative imaging (MRI or CT) studies.During the study period, a total of 277 patients with histologically confirmed diagnoses of glioma or meningioma operated at our department were approached and invited to participate in the study. Three (1%) patients refused to participate and 14 (5%) patients were unable to complete the study tasks due to severe neurological/auditory impairment or poor health status. Blood samples were missing for 16 (6%) of patients. Sixty-seven (24.19%) patients were not evaluated for cognitive functioning due to logistic reasons that included shorter than three days of preoperative stay and intensive preparation for surgery schedule. Consequentially, the final study sample was comprised of 177 patients that included 90 (51%) glioma and 87 (49%) meningioma patients.Karnofsky performance index (KPI) was used for the assessment of functional status . The KPIThe Hopkins Verbal Learning Test-Revised (HVLT-R) is a list of learning verbal memory task . The tesTrail Making Test  is a test for assessment of visual attention, psychomotor speed, mental flexibility, and executive functioning . The tasVerbal Fluency Test consists of phonemic and semantic fluency tasks . During Venous blood samples were centrifuged, and serum samples were stored frozen in -80°C. Serum concentrations of hsCRP and NT-proBNP were evaluated at the certified laboratory after the study completion. hsCRP concentration was evaluated using the Beckman Coulter Unicel DXC 600 kit. Serum concentrations of NT-proBNP were assessed using a radioimmunoassay method .Data was analysed using the IBM SPSS Statistics 20 package. Pearson chi-square criteria and nonparametric Kruskal-Wallis test were used for comparison of sociodemographic and clinical characteristics, and hsCRP and NT-proBNP concentrations between meningioma and glioma patients. Before proceeding with further analysis, the dataset was evaluated for outliers: univariate outliers of hsCRP and NT-proBNT and multivariate outliers of cognitive variables based on Mahalanobis distance were removed. Patients were divided into tertiles according to their hsCRP and NT-proBNP levels: for hsCRP: <0.59/0.59-1.40/>1.40 mg/l, for NT-proBNP: <54.10/54.10-163.77/>163.77 ng/l.Pearson chi-square test and one-way ANOVA were used to compare sociodemographic and clinical variables stratified by hcCRP tertiles and NT-proBNP tertiles. Linear regression was used to analyse the relationship of hsCRP and NT-proBNT concentrations with scores on cognitive tests controlling for demographic and clinical variables. Regression analysis was performed for glioma and meningioma patients separately. Covariates used in the regression analysis were patient age (in years), gender , education status , preoperative functional status (KPI score), histological BT diagnosis , BT laterality , and BT location .p = 0.03). When compared to glioma patients, meningioma patients were older (p < 0.01), were more likely to live alone (p = 0.03), have histories of cardiovascular diseases (p = 0.03), had bifrontal tumors, and had greater NT-proBNP concentrations. High-grade glioma patients had lower initial functional status when compared to low-grade glioma and meningioma patients (p < 0.01). Glioma and meningioma patients did not differ in education status, tumor location involving frontal/temporal lobes and hsCRP concentrations.Demographic and clinical characteristics as a function of BT histological diagnosis are presented in p = 0.02) and more frequently reported cardiovascular diseases (p = 0.03) relative to patients in lower tertiles (p = 0.04), more often reported cardiovascular diseases (p < 0.01), were females (p = 0.04) and were diagnosed with meningioma (p < 0.01) when compared to lower tertiles.Patients in the highest hsCRP tertile were significantly older . In meningioma sample, NT-proBNP correlated with decreased psychomotor speed , mental flexibility , worse cumulative learning , and delayed recall . A similar but nonsignificant trend was observed in low-grade glioma patients: NT-proBNP correlated with slower psychomotor speed  and mental flexibility . However, the cognitive functioning of brain tumor patients depends on many factors, that is why linear regression analysis was used to compare cognitive performance in relation to hsCRP and NT-proBNP controlling for clinical and demographic covariates.Initial correlation analysis revealed some weak but significant relationships between hsCRP and NT-proBNP concentrations and cognitive measures in brain tumor patients. hsCRP was significantly related to slower psychomotor speed in high-grade glioma patients (Spearman p = 0.02) and worse mental flexibility (p = 0.05), independently from demographic and clinical covariates. Older age was related to slower psychomotor speed (p < 0.001), decreased mental flexibility (p < 0.001), slower cumulative learning (p = 0.02), worse delayed recall (p < 0.001), and decreased verbal fluency (p = 0.01). The effect of gender was observed on cumulative learning (p = 0.03) and delayed recall (p < 0.001) with females demonstrating better results. The left or bifrontal location of the tumor was related to worse cumulative learning (p < 0.001), delayed recall (p < 0.001), and decreased verbal fluency (p < 0.001). Tumor grade was related to mental flexibility (p = 0.05) and cumulative learning (p = 0.01). Frontal and/or temporal tumor location was not related to cognitive functioning in glioma patients. Decreased functional status of the glioma patient was significantly related to decreased psychomotor speed (p < 0.001), mental flexibility (p -0.03), worse verbal memory performance . NT-proBNP was not related to the cognitive functioning of glioma patients, when demographic and clinical variables were included into analysis.In glioma patients , higher p = 0.06) (p < 0.001), worse verbal learning (p = 0.01), decreased delayed recall (p < 0.001), and verbal fluency (p = 0.02). Females when compared with males, demonstrated better verbal fluency (p = 0.01). Verbal fluency was also related to education (p = 0.05) and left/bifrontal location of the tumor (p = 0.02). Similarly to glioma patients, frontal and/or temporal tumor location of the tumor was not related to cognitive functioning of meningioma patients. Decreased functional status was significantly related to decreased psychomotor speed (p = 0.05), mental flexibility (p < 0.001), worse cumulative learning (p = 0.01), and decreased verbal fluency (p = 0.05).In meningioma patients, the only tendency was observed hsCRP to be related with slower psychomotor speed ( = 0.06) . NT-proBIn this study, we aimed to investigate the possible association of hsCRP (proinflammatory biomarker) and NT-proBNP (cardiac impairment biomarker) with preoperative cognitive functioning of glioma and meningioma patients. Greater NT-proBNP serum concentration was associated with lower psychomotor speed, worse mental flexibility, less effective learning, and decreased delayed recall of verbal information of meningioma patients in univariate correlation analyses. A similar trend was observed in low-grade glioma patients. However, the association became nonsignificant after adjusting for demographic, clinical, and tumor-related variables, indicating that observed relationships were caused by other variables, most likely, presence of comorbid cardiovascular disease, but not NT-proBNP directly. Contrary, hsCRP correlated with slower psychomotor speed in high-grade glioma patients in univariate correlation analysis and this relationship remained significant in linear regression analysis when clinical and demographic factors were considered, indicating the unique impact of inflammation processes on cognitive functioning of glioma patients.To our knowledge, only one previous study investigated the relationship between levels of NT-proBNP and cognitive functioning of BT patients. Bunevicius et al. found that greater NT-proBNP serum concentrations correlated with general cognitive decline in a mixed sample of BT patients ; howeverDaniels et al.  hypothesIn our sample, hsCRP serum concentrations were statistically significantly associated with decreased psychomotor speed and mental flexibility in glioma patients. Numerous studies documented that greater proinflammation cytokine concentrations are associated with worse cognitive functioning , 42, 43.Worse performance on cognitive tests was associated with higher tumor grade, left-sided or bifrontal tumors, and worse general functional status. Campanella et al.  also repAge was consistently related to worse cognitive functioning both in glioma and meningioma patients. These results are expected as it is known that cognitive functioning decreases during the life span, and this change significantly impacts neuropsychological tests performance , 53. HowSeveral limitations of the current study should be acknowledged. The sample size of low-grade glioma patients was small, and the distribution of bilateral tumors was different in glioma and meningioma patients. Histories of cardiovascular disorders and inflammatory disorders were based on medical histories and patient self-report and were not prospectively evaluated. Prospective assessment for concurrent health conditions that can directly impair the studied biomarkers should be considered in the future studies in order to discern possible confounding effect. On the other hand, reasonable sample size, prospective evaluation of cognitive functioning, and adjustment for potential confounders are strengths of this study.NT-proBNP was not associated with memory, language, and attention/executive cognitive domains of glioma and meningioma patients. Increased hsCRP was related to slower psychomotor speed and worse mental flexibility only in glioma patients. Cognitive performance was also related to the functional status of patients, age, gender, side and location of the tumor, and tumor malignancy in case of glioma.