Enterococcus and Lactobacillus and had a relatively high proportion of members of the Proteobacteria phylum, which has been observed in other carnivorous marsupials. The diversity and structure of the microbiota was not influenced by presence of Chlamydiales which are intracellular bacteria and potential pathogens. The microbiota of the other marsupials was quite varied, which may be related to their health status. Characterization of the northern quoll microbiota will help to better understand the biology of this endangered animal.The Australian northern quoll is an important predatory marsupial carnivore that is currently endangered due to inappropriate fire regimes, predation, and the spread of invasive cane toads. The microbiota of Australian marsupials has not been extensively studied, but is thought to play a role in their health. This study provides an initial characterization of the cloacal microbiota of the northern quoll, as well as other marsupials including possums and kangaroos which were opportunistically sampled. The northern quoll cloaca microbiota was dominated by  Dasyurus hallucatus) is the largest surviving predatory marsupial carnivore in Northern Australia [Rhinella marina is considered one of the greatest threats to the northern quoll, with their arrival associated with population crashes and local extinctions in Kakadu National Park [Predators exert strong effects on ecosystem community structure by influencing the behavior and demography of the species on which they prey ,2. The nustralia  and is custralia . Northerustralia . Their dustralia ,6. The snal Park . In an effort to preserve the species, in 2003, wildlife authorities translocated 64 quolls to Astell and Pobassoo Islands. These islands are located north of the Northern Territory, and are free from cane toads and mammalian predators of the northern quoll, such as feral cats and dingoes. These island populations were monitored to assess population growth, genetic diversity, and condition, via bait and capture methods . It was Chlamydia pecorum infection, a major infectious disease threat to this iconic arboreal marsupial [In 2016, researchers captured quolls from Astell Island for a breeding program at the Territory Wildlife Park. Progeny of those quolls were subsequently trained to avoid eating cane toads, and were reintroduced to Kakadu National Park . The quon = 27) and ocular cavity (n = 7) in 31 individual quolls. Samples from a range of Australian marsupials  were collected and sequenced separately and their microbiota used for a qualitative comparison. Chlamydial detection was previously established in this sample set, revealing a range of previously characterized and novel Chlamydiales genotypes in quolls and other non-koala marsupials [Chlamydia pecorum has previously been correlated with changes to microbial community structure [Chlamydiales detection.In order to provide an initial overview of the northern quoll microbiota, we characterised the microbial community from the cloaca  and three Arnhem rock rats (Zyzomys mainii) from the Northern Territory. Animals were trapped at sites located on Astell Island  and Kakadu National Park  in February and May 2016, respectively. At each location, wire mesh cage traps  baited with a mixture of peanut butter, honey, and oats, were placed in suitable locations  in the late afternoon. Traps were checked within 2 h of sunrise the following morning, and the sex, mass, reproductive status, and microchip number of animals captured inside traps was recorded. At Kakadu, cloacal and ocular samples were taken from three quolls and three rock rats using swabs  in the field, and samples were frozen within 3 h. On Astell Island, quolls were transported to the Territory Wildlife Park as part of a captive breeding program. Upon arrival to the wildlife park, each quoll underwent a thorough examination from a veterinarian. Cloacal and ocular samples were taken from 31 quolls during the examination, and samples were frozen within 20 min. All samples were collected with Copan floqswabs (519C) and stored in 1 mL of 1 × TE. Samples were transported to the University of Technology Sydney for further processing. All work was done in accordance with the University of Technology Sydney Animal ethics approval number ACEC 2105000175.Samples were collected from 34 northern quolls  as per the manufacturer’s instructions. DNA concentration for each sample was measured using a Qubit and a high sensitivity double stranded DNA fluorescence assay .Samples were prepared for 16S rRNA gene sequencing using a two stage PCR protocol. The first PCR used primers that target the bacterial 16S rRNA gene (V3-V4 region), flanked by partial Illumina adaptors at the 5′ end . Two ng https://www.hmpdacc.org/HMMC/) which is a defined mixture of DNA from of 21 bacterial and one yeast species, and the Zymobiomics microbial community DNA standard  which contains eight bacterial and two yeast genomes.Sequencing controls included DNA extraction process negatives, PCR negatives, and two mock community standards, the even mock community from the Human Microbiome Project , the median sample volume was used to add that sample to the pool. The pooled sample was assessed for quality and quantity on a Bioanalyser using a high sensitivity DNA chip . The pooled sample was loaded onto a MiSeq flow-cell at 6pM with 5% PhiX control and sequenced for paired ends using a V2 500 cycle kit. Sequencing was carried out at the University of Technology Sydney.Pseudocheirus peregrinus), two brushtail possums (Trichosurus vulpecula), one short eared possum (Trichosurus caninus), two Eastern Grey kangaroos (Macropus giganteus), two spotted tail quolls (Dasyurus maculatus), one long nosed bandicoot (Perameles nasuta), and one swamp wallaby . Samples were collected in accordance with animal ethics number is ANS1539, awarded by the University of the Sunshine Coast Animal Ethics Committee. Swabs were previously processed for DNA using a QIAamp DNA mini kit (Qiagen) according to the manufacturer’s instructions. In the current study, DNA was quantified using a Quant-iT dsDNA High Sensitivity kit . Sample concentrations were normalised to between 5 and 10 ng/μL by diluting in molecular water.Urogenital swab samples were opportunistically collected from marsupials presenting at collaborating veterinarian or wildlife centres in Queensland and New South Wales, as previously reported . SamplesSequencing libraries were generated using the protocol outlined in Fadrosh et al. . In brieThe two data sets  were initially processed separately for quality filtering and demultiplexing, due to the different adapter design. For the northern quolls data, raw sequencing reads were assessed for quality using FastQC . SequencFor sequences data from the other marsupials, paired end sequences were merged using PEAR , then dehttps://www.mothur.org/wiki/Silva_reference_files). Alignments were trimmed to global positions that captured the majority of the reads, and filtered such that only sequences covering the V4 region (between Escherichia coli positions 515 and 806) were retained, using the pcr.seqs command. Gaps were removed from the filtered alignment before OTU picking.Demultiplexed sequences from both data-sets were combined at this point. Sequences were aligned in the Mothur v1.39.5  using alThe trimmed and filtered sequences were clustered into operational taxonomic units (OTUs) at 97% similarity using the open_ref_otu_clustering.py workflow script in Qiime v1.9.1, which includes removing OTUs which do not align to the 16S rRNA gene or represented by a single sequence, and creating a phylogenetic tree from representative sequences of each OTU using FastTree 2 . Chimerahttps://www.arb-silva.de/projects/ssu-ref-nr/). Taxonomy was assigned to representative sequences from each OTU with the SINA 1.3.1 alignment and classification tool  with theDownstream analyses were performed in R  using thAfter multiple sequence alignment and trimming to global positions, sequence lengths ranged from 226 to 240 base pairs, with a median of 227. These sequences were used for open reference OTU clustering which produced 22,239 non-singleton OTUs.Corynebacterium (35.8%) and Roseburia (13.2%), while field collection negatives were dominated by Bacteroides (38.8%) and Pseudomonas (18.0%). DNA extraction negatives were dominated by Streptotoccus (20.2%) and Delftia (16.2%).Negative sequencing controls were included at each stage of sample collection and processing, including field collection, DNA extraction, and PCR. Negatives from the PCR stage had read depths of 47 and 1753 reads, while process and field negatives ranged between 7 and 12,000 reads. The most common contaminants from PCR negatives were assigned to the In preliminary data analysis, we performed ordination and clustering on all samples with at least 5000 sequences including negative controls. Forty-eight of 137 samples had less than 5000 reads. We observed eight samples with read coverage from approximately 9000 to 20,000 that clustered closely with the negative controls. To avoid samples with possibly high levels of contaminating sequences, only samples with at least twice the read coverage of the highest coverage of a negative control were used, i.e., only samples with at least 25,000 reads were retained for analysis. Of the northern quolls samples collected and processed (56 cloaca and 57 ocular), 34 samples had read depth above 25,000 (27 cloaca and 7 ocular) from 31 individual quolls. Sequence coverage for all other marsupial samples (from a separate sequencing run) ranged from 2 to 841,000. Thirty samples had coverage above 25,000, and these were included for downstream analyses, and were rarefied to 25,572 sequences per sample for subsequent analyses. Sequence data from both sequencing runs has been deposited in the NCBI SRA under BioProject accession number PRJNA473283.Enterococcus  and Lactobacillus genus . Within the Proteobacteria, the ocular sites were dominated by the Pseudomonas genus (19.1 ± 12.8%), while the cloaca contained Escherichia (11.5 ± 14.2%) with lower levels of Pseudomonas (2.6 ± 3.5%). The predominant genera for cloaca and ocular samples can be seen in Enterococcus genus in the cloaca .All taxa are reported in terms of their average relative abundance (percent ± standard deviation). Northern quoll microbiotas were dominated by the Firmicutes  and Proteobacteria  phyla , with smEscherichia (50.4 ± 26.6%) and Helicobacter 24.1 ± 8.5%) dominated the brushtails, while two of the ringtails were dominated by Gemella (61.2 and 91.2%). One of the Eastern Grey kangaroos was dominated by Campylobacter (67.3%), the other by Streptococcus (13.9%) and the swamp wallaby by Porphyromonas (36.6%). The other marsupials studied included two spotted tail quolls, three possum species , two Eastern Grey kangaroos, one swamp wallaby, one long nosed bandicoot, and two Arnhem rock rats. All samples were collected from the cloaca. The composition of microbial communities from these marsupials was also dominated by the Proteobacteria (25.9 ± 25.0%) and Firmicutes (40.0 ± 31.9%) phyla, with lower relative abundances of Bacteroidetes (10.7 ± 16.6%), and Actinobacteria (4.5 ± 6.2%) . DiffereChlamydiales was previously detected in 19 of the 49 samples [n = 7) and six of the cloaca (n = 27) samples tested positive (as previously described) [Chlamydiales were detected included two spotted quolls, two rock rats, one wallaby, six possums, one kangaroo, and one bandicoot. We did not detect Chlamydiales 16S rRNA gene sequences in the data generated in this study from the same animals. samples , which rscribed) . The remChlamydiales was not significantly correlated to diversity or microbial community composition. Shannon diversity was not significantly different in northern quolls regardless of presence of Chlamydiales  ocular samples collected were sequenced to a high enough read depth to be included in the analysis. A similar effect has been seen previously in koalas, where only 9.8% of ocular samples collected and processed were able to be included for analysis . Given tn humans . Enterococcus genus (27.3 ± 22.4%). Enterococcus are commonly found in the mammalian gut microbiota, but usually in much lower proportions (for humans ~1%) [Enterococcus. We observed similarities between the northern quoll cloaca microbiota and the gut microbiota in Tasmanian devils as reported by Cheng et al. . Both anans ~1%) . None ofLactobacillus was the second highest genus in average proportion (13.9 ± 19.0%). This genus dominates the human female vaginal microbiota [Lactobacillus genus were only present at low proportions [Lactobacillus were observed (0.00–5.3%). It is difficult to speculate why such high proportions were observed here compared to other marsupials since only limited studies have been carried out on marsupial urogenital microbiotas so far.Cloacal swabs may represent both the gut and the urogenital microbiota, as swabs were collected from the cloacal opening which serves the digestive and reproductive/urinary tracts. crobiota , and is portions . In the Chlamydiales are detected in a range of native animals in Australia, and while infection with Chamydia sp. is typically associated with disease the impact of Chlamydia-related bacteria (CRB) on host health is currently unclear  [Chlamydiales DNA was previously detected in six of the 27 northern quoll cloacal samples studied here [Chlamydiales was associated with significant shifts in either alpha diversity or community composition, as has been previously described for koalas [Chlamydiales 16S rRNA gene sequences in the data generated here from the same animals, which may be due to differences in primer specificity. The significance of their presence in the quoll population is unclear, and it would be interesting to determine if there is a higher prevalence in predator rich mainland compared to the predator free island populations.mmensal) . Chlamydied here , includiied here . We founr koalas , while aHelicobacter sp. in possums [The cloacal microbiotas of the other marsupials collected were quite varied . Given t possums , which wThe results of this study have provided the first insight into the microbiota of northern quoll populations and a foundation for future microbiota studies. For example, future studies may consider the impact of reduced genetic diversity of the populations we have assessed on the diversity of the microbiota . Underst


Pomacea canaliculata is becoming increasingly important, because of this gastropod’s role as intermediate host and vector of Angiostrongylus cantonensis, the etiologic agent of eosinophilic meningitis in humans and domestic animals. Immune defenses of this gastropod comprise both humoral and cellular components, but they may also involve organs that act as immune barriers to prevent the spread of alien molecules and organisms. Both the kidney and lung are here shown to serve this function, because of (1) their positions in blood circulation, (2) the intricate architecture of their blood spaces, and (3) the proliferative and nodulation reactions of hemocytes to an immune challenge. However, these organs differ in that only the kidney shows permanent hemocyte aggregations. Microcirculation in the kidney was found to flow through an intricate vascular bed containing the permanent aggregations, which occurred either as hemocyte islets anchored by cytoplasmic projections of the renal epithelium or as perivascular accretions. Within 96 h of the injection of yeast cells, hemocyte nodules were formed both in the kidney and lung. Moreover, cell proliferation in renal hemocyte islets was measured by bromodeoxyuridine (BrdU) incorporation. The proportion of BrdU positive nuclei increased 48 h after injection. Signs of nodule regression  and a decrease in the proportion of BrdU positive nuclei were found at 96 h. In addition, the area of renal hemocyte islets was significantly increased 96 h after injection. Nevertheless, the high complexity of the small vascular chambers that constitute the lung’s respiratory lamina would also facilitate hemocyte-antigen contacts, required to elicit cellular aggregation, and hence, nodulation. To our knowledge, this paper includes the first quantitative indication of hemocyte proliferation after an immune challenge among Caenogastropoda.Knowledge on the immune system of  Pomacea canaliculata  is becoming increasingly important, because of the role of this species as an intermediate host and vector of the nematode Angiostrongylus cantonensis , the major etiologic agent of eosinophilic meningitis, a parasitic disease that can be disabling in humans, and may be even fatal  an adequate position of the organ in blood circulation, so as to prevent antigen dissemination, and/or (2) an intricate microcirculation, which would increase the probability of hemocyte-antigen contacts, and/or (3) a locally high hemocyte concentration.P. canaliculata is that this species is one of the two ampullariids whose immune system has been studied to some extent  to observe the mere reaction to foreign cells, with no accompanying damage as it occurs when pathogens are employed  from the Rosedal strain were used. The origin of the strain and the culturing conditions have been reported several times elsewhere , desiccated and powdered P. canaliculata’s eggs, and toilet paper . Collection of the original stock of the Rosedal strain did not require specific permits since it is an invasive, not an endangered species . Procedures for snail culture, sacrifice, and tissue sampling were approved by the Institutional Committee for the Care and Use of Laboratory Animals , Facultad de Ciencias Médicas, Universidad Nacional de Cuyo), Approval Protocol No 55/2015.e e.g.,  . BrieflyAnimals were put in an ice/water bath , both for inducing relaxation and minimizing pain, before carefully cracking the shell.For light microscopy, samples of the kidney (‘posterior kidney’ in N = 3) were fixed in Karnovsky’s fluid  for 6 h and post-fixed in 2% osmium tetroxide overnight. Samples were rinsed in phosphate buffer, dehydrated in a graded acetone series and finally embedded in Spurr’s resin. Then, thin sections (∼200 nm) were stained with toluidine blue and mounted on glass slides with DPX medium  and examined and photographed under a Nikon Eclipse 80i microscope using Nikon DS-Fi1-U3 camera and Nikon NIS-ELEMENT Image Software for image acquisition. Ultrathin sections (∼70 nm) were mounted on copper grids, stained with uranyl acetate and lead citrate and examined with a Zeiss EM 900 transmission electron microscope.For electron microscopy, small pieces of the kidney  was prepared in a suitable apyrogenic buffer  were injected into treated or control animals respectively. All injections were made in the visceral hemocoel through a small hole (∼1 mm) in the shell, which was drilled manually at midway between sutures of the second whorl  were: (1) control animals sacrificed 48 h after vehicle injection; (2) control animals sacrificed 96 h after vehicle injection; (3) treated animals sacrificed 48 h after yeast injection; and (4) treated animals sacrificed 96 h after yeast injection. Sampling times were chosen according to preliminary observations  of 5-bromo-2-deoxyuridine (BrdU) in nuclei. Control and treated animals were injected with BrdU  into the visceral hemocoel 42 and/or 90 h after injection of either rvations .The kidney was dissected out and fixed in 4% paraformaldehyde for 16 h. Lung samples were not processed, because this organ does not normally show hemocyte aggregates and so, the buffer-injected animals would not provide proper control tissues. Then the slides were passed through the following steps: (1) xylene-removal of the embedding medium, (2) rehydration of sections through an ethanol/water series to distilled water, (3) incubation in buffer  for 25 min at 95–100 °C, (4) washing in distilled water for 10 min followed by two steps in phosphate buffered saline , (5) incubation with a 1:10 dilution of an antibody against BrdU  for 30 min at 37 °C in humid chamber, (6) three washing steps (10 min each) in PBS, (7) incubation with a 1:10 dilution of anti-mouse-Ig-fluorescein antibody  in PBS for 30 min at 37 °C in humid chamber, (8) three washing steps in PBS, (9) counterstaining with 4′,6-diamidino-2-phenylindole (DAPI), (10) washing in PBS and (11) mounting with FluorSave® Reagent .N = 3 animals per group) were captured at high magnification (60×) using an Olympus FV1000 confocal microscope and processed with Adobe Photoshop CS6 software. Total hemocyte nuclei (DAPI+) were counted using CellProfiler software (http://www.cellprofiler.org) and BrdU+ cells were counted manually by two independent researchers. Hemocyte nuclei were clearly distinguished from those of epithelial cells because of their size and the conspicuous nucleoli of the latter. Only BrdU+ labels that overlapped >50% with DAPI+ labeled nuclei were counted as BrdU+ nuclei.Six images per animal  was assessed with the one-tailed Mann–Whitney U– test, using GraphPad Prism 5.03 .Cell proliferation was assessed as the proportion of BrdUN = 9 per group) were sacrificed 96 h after injection of either PcBS or yeast cells respectively, and kidney samples were obtained and processed as described above. In preliminary observations, distinct nodules with an outer layer of flattened cells were constantly found at this time (2) were obtained for each animal to calculate the ‘hemocyte aggregates/total renal tissue’ ratio. Then the mean ± SEM of control and treated animals was computed. Statistical significance of differences (P ≤ 0.05) between control and treated animals were assessed by one-tailed Student’s t-test, using GraphPad Prism 5.03 software.For renal islet morphometry, control and treated animals  were frequently seen . As the In some cases, the nodules appeared formed out of two or more foci, which may have been caused by successive waves of cellular aggregation, sometimes resulting in a core showing a more advanced stage of regression, and a cortex made by recently aggregated cells . SometimMeasurements were made in two ways: (1) by a BrdU incorporation assay to measure cell proliferation in the islets (including nodules), both at 48 and 96 h after injections (either vehicle or yeast injection), and (2) by measuring the renal area occupied by hemocyte aggregates , only 96 h after injections (because nodulation did not constantly appear at 48 h).+ hemocyte nuclei / total hemocyte nuclei in the islet section showed a statistically significant 2-fold increase in yeast-injected animals, as compared to controls  no nodules were observed 48 h after injection. Therefore, comparative observations were not made at this time point.The ratio ‘hemocyte aggregates area/total renal tissue area’ was higher in treated than in control animals, 0.26 ± 0.03 The lung is a flattened sac extending over most of the mantle cavity. It is delimited by the gill on its right and posterior borders, and the mantle skirt on the anterior and left borders .Both the roof and the floor of the lung are crossed by main vessels that convey blood from the visceral mass and the gill . We alsoThe lung did not show permanent hemocyte aggregations as those of the kidney , though Hemocyte recruitment towards the blood spaces of the respiratory lamina was also observed (though this was not quantified). Lung nodules, like those in the kidney, were mainly composed of hyalinocytes. They also contained extracellular lacunae, apoptotic bodies, and brown lipofuscin-like concretions, and were sometimes delimited by a layer of flattened cells. Nodules grew out to large sizes, could be multicentered, and were many times visible to the naked eye . No noduP. canaliculata, whose anatomical peculiarities need first to be disclosed to define the way this gastropod can react to immune challenges.Recently This is also certainly the case of the circulatory system of this gastropod and other ampullariids, whose general organization has been studied by 3 kDa; The kidney showed an extensive vascular bed supplied by the ‘perpendicular vessels’ originated from branches of the greater afferent renal vessel. The hemocyte accretions around the afferent perpendicular vessels were also in contact with the system of blood sinuses between the epithelial crypts  which coIn turn, the lung vasculature is composed of the main vessels conveying blood either directly from the viscera or indirectly through the gill vasculature . The curTherefore, both the kidney and the lung may serve a role analogous to that of lymph nodes in vertebrates, where foreign particles and organisms circulating in the lymph can be trapped and inactivated . CuriousA notable feature of the kidney and lung was to host spheroidal hemocyte aggregates or ‘nodules’ which were formed in response to an immune challenge  and 9. SP. canaliculata, nodules were formed on the surface of hemocyte islets and as outgrowths of the perivascular hemocyte accretions .The seemingly universal distribution of nodulation in the Metazoa almost parallels that of phagocytosis, which is certainly the most basal and evolutionary conserved defense mechanism in animals .However, there is a paucity of data in other gastropod clades, even in the Caenogastropoda, which encompasses 60% of species in the widely diverse class Gastropoda. Studies in caenogastropods are restricted to two species of Ampullariidae, M. cornuarietis . In that study, we serially sectioned the lung of this species but did not find any similar tissue band, which might be peculiar to M. cornuarietis.Because of the proposed hematopoietic site in the lung of uarietis , we have3 kDa; In the current study, we have confirmed that the renal hemocyte islets are permanent and rather compact aggregations . BesidesFurthermore, changes in hemocyte proliferation in renal islets after yeast injection were measured by determining the increase in BrdU incorporation in hemocyte nuclei and the increase in the area occupied by hemocytes at the end of the experiment. Statistically significant increases in BrdU incorporation in hemocytes were shown 48 h after challenge  and in tP. canaliculata, either by resident or migrating cells.However, future research should further refine these findings by considering the possible inward and outward hemocyte movements, to and from the islets, as well as the accumulation of newly formed hemocytes. These variables have been studied for the hematopoietic organ of crayfish . HoweverP. canaliculata after challenge, an organ in which preexisting hemocyte aggregates seldom occur and they are likely responses to some occasional infection. Nodules are likely formed by the locally proliferating hemocytes, but also by hemocytes attracted to particular places . Therefore, it is possible that both widespread and localized hematopoiesis occur in this species.Besides this evidence of localized hematopoiesis in the Ampullariidae, it is worth mentioning there is evidence of hematopoietic progenitor cells in the circulation of P. canaliculata was unraveled, i.e., where hemocytes may become in close contact with intruders. Moreover, striking ultrastructural features of the renal hemocyte islets’ architecture have been disclosed, altogether with the unexpected finding of abundant hemocyanin particles into renal blood sinuses. Also, nodular aggregations of hemocytes were shown in both organs as a conspicuous reaction to the immune challenge. And finally, hemocyte proliferation in the renal islets has been quantified following the immune challenge. According to this evidence, the renal hemocyte islets emerge as possibly important, and perhaps the only permanent hematopoietic sites in P. canaliculata.Here the intricate blood circulation in the kidney and lung of 10.7717/peerj.5789/supp-1Figure S1(A) Injections of yeast suspension or buffer in the visceral hemocoel were made through a small hole (∼1 mm) in the shell (red dot), drilled manually at midway between sutures of the second whorl after inducing body withdrawal by gently handling the animal. No exsanguination was induced  . The tip of the injecting needle was introduced ∼4 mm below the shell surface, in the digestive gland close to the testis. Neither spillage of the injected material nor further retraction of the columellar muscle was observed; thus, the whole procedure did not appear to be painful for the animals. (B) Dorsal view of the animal showing shaded areas corresponding to the samples obtained from the kidney and lung. Abbreviations: ctn, ctenidium; kid, kidney; lng, lung; opc, operculum; pod, foot; prc, pericardium; shl, shell; urt, ureter; whl, second whorl of the shell.Click here for additional data file.10.7717/peerj.5789/supp-2Data S1Click here for additional data file.


By forming hydrogen bond between the oxygen-containing functional groups of PEG and citrate ions on the surface of AuNPs, this probe shows good stability. PEG coordinated with Hg2+ synergistically and specifically on the surface of dispersed AuNPs, and the existence of creatinine could induce the aggregation of AuNPs with a corresponding color change and an obvious absorption peak shift within 5 min. This PEG/Hg2+–AuNPs probe towards creatinine shows high sensitivity, and a good linear relationship (R2 = 0.9948) was obtained between A620–522 nm and creatinine concentration, which can achieve the quantitative calculations of creatinine. The limit of detection (LOD) of this PEG/Hg2+–AuNPs probe was estimated to be 9.68 nM, lower than that of many other reported methods. Importantly, the sensitive probe can be successfully applied in a urine simulating fluid sample and a bovine serum sample. The unique synergistic coordination sensing mechanism applied in the designation of this probe further improves its high selectivity and specificity for the detection of creatinine. Thus, the proposed probe may give new inspirations for colorimetric detection of creatinine and other biomolecules.A colorimetric sensor, based on the synergistic coordination effect on a gold nanoparticle (AuNP) platform has been developed for the determination of creatinine. The sensor selects citrate stabilized AuNPs as a platform, polyethylene glycol (PEG) as a decorator, and Hg Creatinine (2-imino-1-methyl-2-imidazoline-4-ketone) is an important clinical analyte that is produced through muscle metabolism and removed by glomerular filtration. The physiological creatinine level of a healthy person is about 3.5 to 34.6 mM in urine and 50 to 140 μM in blood [Nowadays, numerous creatinine detection methods have been reported, such as high performance liquid chromatography (HPLC) , liquid 2+ to form a sensor based on the synergistic coordination effect for creatinine detection. We deduced that, due to the formation of hydrogen bonds between the hydroxyl groups of PEG and citrate ions on the surface of AuNPs, this novel AuNPs based colorimetric sensor would shows high selectivity and greater stability before and after testing. What is more, we added Hg2+, which can coordinate with PEG on the platform of AuNPs, to form a coordination detection system characteristically. Hg2+ links PEG and creatinine synergistically via the electrostatic effect on the surface of AuNPs, which causes a decrease in the inter-particle distance of AuNPs, and then finally leads to the evident aggregation of AuNPs via a synergistic coordination effect together with the obvious change of solution color observed simultaneously by the naked eye. Therefore, this probe can detect whether there is creatinine or not simply by observing distinct color change of the solution or an absorbance shift due to the aggregation of AuNPs. More importantly, after the formation of the coordination detection system, the sites of AuNPs become inactive and hard to aggregate because of the steric effects while it was functionalized. By making use of the synergistic coordination and steric effects on the AuNP surface, this colorimetric probe shows excellent selectivity and specificity towards creatinine. Therefore, we developed a colorimetric method based on AuNPs without using specialized devices in the real application and general testing process. Furthermore, this colorimetric probe may not only possess accurate response ability and excellent stability, but also can achieve the goal of quantitative calculations of creatinine with high specificity. Therefore, this proposed portable AuNPs probe can be expected to be widely applied in early clinical analysis of creatinine content.In recent years, colorimetric sensors that are free of specialized and expensive instruments have attracted increasing attention in the bio-molecule determination field . As repo4·4H2O) was purchased from Tianjin Yingda chemical company and used to synthesize AuNPs. PEG (MW 200) was configured to be 4.5 mM, which was purchased from Shanghai Sinopharm Chemical Reagent Corporation. Different concentrations of creatinine solution (22.5 mM) were prepared by dissolving the desired amount of creatinine in distilled water. Polyvinylpyrrolidone , mercuric nitrate solution was configured to be 0.45 mM. Sodium citrate and other chemicals were of analytical grade and were purchased from Aldrich. The bovine serum was purchased from Shandong Xiaoye Chemical Co., Ltd and was prepared using the reported method, which was the same as the urine simulating fluid sample preparation [Hydrogen tetrachloroaurate gold (III) tetrahydrate .The electronic spectra of gold nanoparticles were recorded by a T6 UV–Vis spectrophotometer . The corresponding color of the solution was recorded by a digital camera. The morphology and size of aunps were recorded by a Philips TEC-NAI G2 F20 transmission electron microscope  operating at 200 kv and dynamic laser scattering (DLS) on a Zetasizer Nano-ZS90  at 25 4 solution (5.2 mM) was added to 79.4 mL of doubly distilled boiling water in a three-necked flask, which was configured to be the solution of HAuCI4 of 1.0 mM. Then, 10 mL of sodium citrate (38.8 mM) was added to 100 mL HAuCl4 solution (1.0 mM) quickly while the HAuCl4 solution was boiling along with vigorous stirring. The solution was allowed to cool to ambient temperature after continuous boiling for 20 min. The absorption spectrum of citrate capped AuNPs before and after detection was recorded by a UV–Vis spectrometer. The concentration of the AuNPs was estimated via Lambert–Beer’s law (the extinction coefficient of ca. 13 nm AuNPs was 2.7 × 108 M−1·cm−1 at 520 nm) [The preparation of citrate capped AuNPs was similar to previous experiments ,34. A 20 520 nm) ,35.2+ (0.1 mM), were added into the citrate capped AuNPs (1.12 nM) solution, respectively , PEG (1.0 mM), and Hg2+ in this creatinine detection system, many other ions, including Zn2+, Mg2+, Br–, SO32–, Ag+, SO42–, CO32–, HCO3–, CH3COO–, Cl–, and Hg2+ were tested under the same experimental conditions, respectively. Moreover, in order to estimate the optimum concentration of Hg2+ in this stable AuNP probe, a series of experiments was monitored to further investigate the influence of Hg2+ concentration under the same experimental condition.In order to investigate the role of HgIn order to examine the selectivity of this AuNP based probe, many other chemicals with similar structures as creatinine, including ascorbic acid, creatine, thymine, nicotinic acid, glucose, urea, PVP, and N-N-hydroxysuccinimide (NHS), were detected under the same optimum experimental conditions. Moreover, a series of samples having different amounts of creatinine were further experimented under the same conditions for the sake of evaluating the sensitivity of this probe. This creatinine colorimetric probe was applied in the urine simulating fluid and real bovine serum samples to investigate its practicality. A series of mimic urine and bovine serum samples were tested by optimum experimental procedures described above. The absorption spectrum and color change of solution were recorded via UV–vis spectrophotometer and a digital camera before and after detection.2+, and creatinine into citrate capped AuNPs solution, the solution immediately changed from red to blue  were added into the AuNPs solution, respectively. After the addition of PEG and Hg2+ or PEG and creatinine into AuNPs, the color and absorption wavelength of mixture remained unchanged as shown in 2+, the addition of creatinine cause a change in solution color and absorption spectrum. As shown in 2+, and creatinine on the platform of citrate capped AuNPs. Detection experiments were monitored as mentioned above. As shown in 2+–AuNP probe for creatinine colorimetric detection is shown in 2+ can coordinate with PEG synergistically. The linkage of mercury ion between PEG and creatinine on AuNPs results in the gradual decreasing of distance between AuNPs, which finally leads to the transformation of the AuNP state from dispersion to aggregation. Along with the aggregation of AuNPs, the size of AuNPs increases gradually and simultaneously, which finally turns out to be the visible corresponding color change of solution  are shown in 2 = 0.9948 (620–522 nm) and creatinine concentration. The content of creatinine could be calculated quantificationally by the linear relationship obtained  and creatinine concentration could be obtained. The LOD in the urine simulating fluid sample was estimated to be 0.451 μM (R2 = 0.9552), which is lower than normal concentration (50 to 140 μM) in blood, and in bovine serum was estimated to be 0.729 μM (R2 = 0.9892). For comparison, the Jaffe’s reaction [2+–AuNP colorimetric probe shows great potential practicability and availability for actual qualitative and quantitative analysis of creatinine in real samples. Thus, this PEG/Hg2+–AuNP probe is a promising method for wide application of early clinical creatinine testing.As investigated above, this PEG/Hgg fluids  and bovig fluids  were tesreaction  was also2+–AuNP probe exhibited excellent specificity, sensitivity, and practicability in detecting creatinine. PEG coordinated with creatinine synergistically by mercury ions acting as a link on the platform of AuNPs, which finally accounted for AuNP aggregation along with apparent solution color change. The LOD was estimated to be 9.68 nM (R2 = 0.9948) lower than that of many other reported methods. What is more, this probe based on the synergistic coordination effect was applied successfully in urine simulating fluid and bovine serum samples. The same good linear relationship between A620–522 nm and creatinine concentration in serum and urine simulating fluid can be obtained by data analysis, and this PEG/Hg2+–AuNP probe showed high sensitivity and practicability with a LOD of 0.729 μM in bovine serum samples. In conclusion, the PEG/Hg2+–AuNP colorimetric sensor supplies an efficient and simple way for quick creatinine detection and shows great potential for the highly sensitive and selective determination of creatinine.The novel PEG/Hg


Prognostic stratification of elderly patients with chronic obstructive pulmonary disease (COPD) is difficult due to the wide inter-individual variability in the course of the disease. No marker can exactly stratify the evolution and natural history of COPD patients. Studies have shown that leukocyte count is associated with increased risk of mortality in COPD patients. The aim of this study was to evaluate the possible role of relative lymphocyte count as a risk marker for mortality in elderly patients with COPD.p = 0.0012). Survival curve analysis showed higher mortality in patients with relative lymphocyte count ≤20% (p = 0.0005). After adjustment for age and sex, the hazard ratio for mortality risk according to lymphocyte count was 1.79 , even in the analysis limited to the 171 patients without congestive heart failure .This is a3-year prospective study. A total of 218patients, mean age 75.2±7 years, with moderate to severe COPD and free from conditions affecting lymphocyte count were enrolled. The population was divided into two groups according to the relative lymphocyte count, with a cut-off of 20%. Eighty-five patients (39%) had a relative lymphocyte count ≤20%. Three-year mortality rates from any cause in patients with relative lymphocyte count ≤ or > 20% were 68 and 51%, respectively  contains supplementary material, which is available to authorized users. Chronic obstructive pulmonary disease (COPD) is a frequent cause of death in elderly patients.Older age, reduced gas exchange and airflow obstruction, right atrial overload and ventricular overload and hypertrophy and selected comorbidities are the main negative prognostic indicators as they could account for early death in these patients –3.The prognostic stratification of elderly patients with COPD is difficult due to wide inter-individual variability the course of the disease. No marker can exactly stratify the evolution and natural history of COPD patients. Therefore, efforts are made in order to detect new prognostic markers and to verify whether they substantially add further prognostic value to well-recognized indicators , 4, 5. IThe common plasmatic measures and biomarkers such as , C-reactive protein (CRP), white blood cell count (WBC)) are not able to reproducibly predict mortality in COPD patients .Variables reflecting either inflammation or immune depression emerged as possible prognostic markers in patients with COPD. The increase in leukocyte count in peripheral blood shows a statistical trend towards the prediction of long-term all-cause mortality risk in COPD patients but it did not reach a statistical significance in previous studies , 6.More insights come from the analysis of reduced WBCs and, in particular, lymphocyte count. A low relative lymphocyte count is already known to exert a detrimental prognostic role in the setting of acute myocardial infarction, stable coronary heart disease and congestive heart failure –11. It aDespite the established role of low relative lymphocyte count in many diseases, its prognostic value remains to be specifically evaluated in elderly patients with COPD.The aim of the present study was to investigate the prognostic value of relative lymphocyte count in elderly patients with COPD.The study was conducted on elderly patients with COPD who were consecutively admitted to our Institute for Research and Care related to Cardiac and Pulmonary Rehabilitation. The diagnosis of COPD was made according to American Thoracic Society (ATS) criteria . A totalInclusion criteria were: age ≥65 years and presence of severe and very severe COPD, as identified by ATS criteria . Severe . Spirometry was performed with a VMAX 2200 spirometer , meeting the American College of Physicians, American College of Chest Physicians, American Thoracic Society, and European Respiratory Society 2011 recommendations for diagnostic spirometry , arterial blood gases , presence of ischemic heart disease, history of myocardial infarction, congestive heart failure, diabetes, systemic hypertension, cerebro-vascular disease, peripheral vascular disease, use of bronchodilators, mucolytics and aminophylline, heart rate, systolic and diastolic blood pressure. None of the enrolled patients were in long-term oxygen therapy. Furthermore, their PaO2 (mmHg) at rest was 70.4 ± 8 mmHg (range 60–91 mmHg).Patients’ characteristics were reported: age, gender, body mass index, history of smoking (defined as subjects who had regularly smoked at least 5 cigarettes/day during the previous 3 months or who had stopped smoking less than 1 year before admittance into our study), alcohol use, lung function , acute myocardial infarction [31 (25%)], stroke [21 (17%)], cor pulmonare [12 (10%)], respiratory infections [10 (8%)], cancer [9 (7%)], sudden death [4 (3%)], or other causes [5 (4%)].p = 0.0012] (data not shown).Low relative lymphocyte count at baseline was associated with an increased incidence of death from any causes [58 (68%) vs. 68 (51%); In the Additional file Table Kaplan-Meier estimates of the probability of death are shown in Fig. The relationship between tertiles of relative lymphocyte count at study entry and 3-year mortality is shown in Fig. Table Table Relative lymphocyte count was significantly related to survival in elderly patients with moderate to severe COPD. The present findings are in agreement with those of Lehtonen et al. who found that a reduction in both B and T cells could predict mortality in very old people with severe chronic illnesses . The difAnalysis of the causes of death shows that worsening of cardiopulmonary failure was responsible for most of the early deaths, whereas late mortality was related mainly to cardiovascular or cerebrovascular disease and cancer Table . As a siThere are contradictory results regarding lymphocyte number and function in senescence , 27.Rea et al. demonstrated a 10% decrease in both B and T cell number and percentage of absolute lymphocyte count in elderly subjects . AccordiThe mechanisms leading to the reduction in lymphocyte count and immunological impairment are still far from clear. Aging and COPD are associated with psychological stress .It has been long known that psychological and physiological stresses result in a significant increase in systemic cortisol production . The phyThe hypothalamus-hypophysis-adrenal axis is a sensitive feedback system for various pathophysiological conditions leading to neurohumoral activation. Several lines of evidence demonstrate that severe COPD is associated with a generalized increase in circulating catecholamines and cytokines . By incrFurthermore, activation of the inflammatory systems could be responsible for the cachexia and hypermetabolic state found in some patients with severe COPD , 40. A lThis study has some limitations, as follows: 1) lymphocyte subpopulations were not measured, and thus the respective prognostic role of deficits of the cellular and humoral immunity could not be assessed; 2) some degree of uncertainty exists in determining the cause of death in this kind of study, and, theoretically, this could affect the interpretation of the relationship between lymphopenia and mortality; 3) orthopnea and fluid retention are not uncommon in severe COPD complicated by hypoxemia and hypercapnia, and could simulate congestive heart failure. However, the stringent criteria used to diagnose congestive heart failure and the results of survival analysis for patients without congestive heart failure support the reliability of the current conclusions. Nevertheless, the proposed prognostic model should be tested in a population with only moderate COPD in order to limit further the potential confounding effect of congestive heart failure.Relative lymphopenia has a homogeneous and strong effect on mortality across the whole follow-up period, but the inherent mechanisms remain to be clarified. To our knowledge, this study is the first to demonstrate that relative lymphopenia is associated with a poor prognosis in elderly patients with severe COPD. This finding seems worthy of attention because lymphocyte count is a simple, reproducible, widely available and inexpensive prognostic tool. Further research is needed to verify to which extent lymphopenia improves the prognostic definition based on well-established markers, as well as to assess the relationship linking lymphopenia with indices of neurohormonal activation and inflammation. Clarifying these issues would enable quantification of the weight of this new prognostic marker and, possibly, the design of interventions, e.g. immunological therapy, aimed at its correction.Additional file 1:Table S1. Evaluation of the reproducibility of measurements during the follow-up period. (DOCX 17 kb)Additional file 2:Figure S1. Correlation between relative lymphocyte count and 6-min walking test. (TIF 100 kb)Additional file 3:Figure S2. Correlation between relative lymphocyte count and heart rate. (TIF 126 kb)Additional file 4:Figure S3. Hazard ratio as a function of relative lymphocyte count threshold. (TIF 151 kb)


Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew severely affects yield security wheat production in China. Understanding the virulence structure and genetic variations of this pathogen is important for breeding wheat lines resistant to wheat powdery mildew. However, information related to genes controlling resistance remains elusive. This study analyzes the virulence structure and the genetic diversity of pathogenic Bgt populations isolated from northeastern  and northwestern (Gansu) China, two representative wheat producing areas, on 37 wheat cultivars each carrying a known powdery mildew resistance (Pm) gene.Bgt isolates from northeastern China show higher frequencies of virulence genes than populations from Gansu Province. Many of the known Pm genes failed to provide resistance in this study. However, Pm21 provided 100% resistance to all isolates from all three provinces, obtained during two consecutive years, while Pm13 provided 100% resistance in Gansu. Pm13, Pm16, Pm18, and Pm22 also showed partial resistance in northeastern China, while Pm16, Pm18, Pm22, Pm5 + 6 and Pm2 + 6 +? maintained some resistance in Gansu. Genetic diversity among populations in different regions was detected by cluster analyses using expressed sequence tag-simple sequence repeat (EST-SSR). When the genetic similarity coefficient is relatively high, populations from the same regional origin are mostly clustered into one group while populations from different regions exhibit large genetic differences.Pm21 remains the best choice for breeding programs to maintain resistance to Bgt. Only 58% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and frequency of virulence gene data. Blumeria graminis f. sp. tritici (Bgt), is one of the several fungal diseases seriously affecting wheat production [Wheat is a staple food for the majority of the world’s population. China accounted for 11.1% of the global wheat production in 2017, harvesting 134.33 million metric tons [oduction . Virulenoduction , 7, and oduction .Bgt has been carried out continuously [. Numerous studies have been reported using molecular marker technologies in dissecting the genetic diversity of plant pathogens [Bgt [Bgt and found no close correlation between these two factors [Bgt from the perspective of gene expression sequences have been documented while no comparative analysis for the genetic diversity of expression sequences and virulence diversity have been carried out in China. In this study, in order to understand the effective protection provided by powdery mildew resistance (Pm) genes in northeastern China and explore the relationship between genetic polymorphism based on expression sequences and virulence diversity, the virulence of Bgt from northeastern China and from Gansu Province was monitored. The expressed sequence tag (EST)-SSR marker developed by Xu were applied to the analysis of the genetic diversity of Bgt and its regional association [During the last 20 years, virulence monitoring of inuously –12. Numeathogens –15. Seveathogens , simple athogens , inter-sathogens , amplifiathogens , and othens [Bgt . Moreove factors . CurrentBgt were obtained from Liaoning, Heilongjiang and Gansu provinces during 2013–2014 , V21, and V22 (V1e) was less than 30.8% , Pm21, and Pm22 (Pm1e) exhibited good resistance and are good resources for further breeding programs, especially Pm21 which yielded 100% resistance to all the isolates. Therefore, Pm21 was the most promising resistance gene against isolates from northeastern China which were collected in the period 2013–2014.A total of 120 purified pathogenic isolates of Pm13, Pm16, Pm18 (Pm1c), Pm21, Pm22 (Pm1e), Pm5 + 6 and Pm2 + 6 +? showed effective resistance against isolates from Gansu Province with infection scores from 0 to 2. Pm16 and Pm21 were the most effective and resistant genes and were immune to all tested isolates  that were specific to Using the PAGE results of EST-SSR, polymorphism Figs.  and 2, tBgt from different provinces of the northeast and northwest regions. However, at a higher genetic similarity coefficient, the tested isolates were mostly clustered into groups by region, indicating that genetic differences were greatest between Bgt populations isolated from different geographical areas and smallest in Bgt populations isolated from the same area.When the genetic similarity coefficient was 0.82, T4.1 and T2 from Gansu and H1–5-4 from Heilongjiang were clustered individually. With the same similarity coefficient, the remaining 57 isolates were clustered into five groups. Group 1 contained 32 isolates, including 20 isolates from Heilongjiang, eight from Liaoning and four from Gansu. Group 2 consisted of six isolates, including five from Heilongjiang and one from Gansu. Group 3 and 4 were composed of nine and six isolates from Liaoning, respectively. Group 5 contained four isolates from Gansu. Moreover, based on a similarity coefficient of 0.90 the first group could be divided into five sub-groups as follows: the first sub-group consisted of 11 isolates from Heilongjiang; the second sub-group consisted of two isolates from Liaoning, two from Gansu and one from Heilongjiang; the third, fourth and fifth sub-group consisted of eight Heilongjiang isolates, two Gansu isolates and six Liaoning isolates, respectively. There may be some degree of genetic exchange through sexual reproduction among the According to the EST-SSR polymorphisms highlighted by PAGE of 50 isolates, a phylogenetic tree was constructed using genetic NTSYSpc 2.10 Fig. . As can When the similarity coefficient of polymorphism is 0.86, the first large group can be subdivided into three subgroups. Subgroup I includes eight isolates  collected from Heilongjiang, one (W2) from Liaoning and two  from Gansu in 2014. Subgroup II includes two isolates  collected from Gansu. Subgroup III includes eight isolates from Liaoning of which two  were collected in 2014 and six  were collected in 2013. When the similarity coefficient was increased to 0.90 (the third large category) in addition to W8 and H10, the rest of the isolates were clustered into three subgroups. Subgroup I includes 3 isolates  from Liaoning in 2014. Subgroup II includes three isolates  from Heilongjiang in 2013. Subgroup III includes four  from Liaoning and six  from Heilongjiang in 2013.Bgt isolates on 37 differential hosts. Then, a phylogenetic tree was constructed according to the similarity of the infection effect on the hosts  analyzed the virulence of 434 Bgt strains collected from 16 provinces in China from 2015 to 2016. The results showed that the virulence frequencies of Bgt to Pm12, Pm13, Pm16, Pm21 and Pm35 were less than 30%, of which Pm12 were less than 5%, and no isolates were virulent to Pm21 [Bgt strains collected annually from northeast China from 2015 to 2018, and found no isolates were virulent to Pm21 (unpublished). Prolonging the resistance duration of Pm21 gene can be achieved by enriching the diversity of resistance genes by utilizing other effective resistance genes in wheat breeding. Continuous studies including our research in 2008–2014 identified that apart from Pm21, the resistance genes Pm13, Pm16, Pm18, and Pm22 also showed effective resistance to powdery mildew, and can be combined with Pm21 to enhance and extend the resistance of varieties [Bgt can provide a reliable basis for breeding resistance to wheat powdery mildew. Our results also show that although there were many common virulent genes among different geographical groups, the frequency of some virulent genes varied greatly among different regions. For instance, the frequency of V16 was 0% in Gansu vs. 30.8% in Liaoning and 25.0% in Heilongjiang. These differences and characteristics from different regions provide key guidance for reasonable rotation and distribution of wheat varieties, thus effectively reducing the occurrence of powdery mildew.Consistent with previous studies, no isolate was found to be virulent to  to Pm21 . In addiarieties . MoreoveBgt in these three provinces may have a certain level of genetic exchange. However, when the genetic similarity coefficient is relatively high, isolates from different locations are basically clustered into separate groups by region showing that strain populations from different regions exhibit large genetic differences. Years of research have shown that in China pathogens of airborne diseases such as wheat stem rust, and powdery mildew mainly rely on monsoon to spread throughout the country. In spring, pathogens of airborne diseases spreads from south to north through two route, one goes from Yunnan, Guizhou, Guangxi and other places across Sichuan to the spring wheat areas of Gansu and Xinjiang; the other goes from the southern wheat areas such as Fujian and Guangxi across the wheat production areas along the southeast coast to Shandong, and finally spreads to the northeast wheat region. In autumn, the powdery mildew strains spread from the northern wheat region to the southern wheat region according to the reverse direction of the monsoon [Bgt that was revealed by the EST-SSR molecular marker technique and the corresponding virulence diversity are correlated. However, the other 42% of the isolates display great differences in polymorphism and virulence diversity, without any correlation. This result is consistent with previous research results using other molecular marker technologies, such as RAPD [According to the clustering results for gene polymorphisms in isolates from Liaoning, Heilongjiang and Gansu provinces, some of the isolates from different regions were found to be clustered together in a group, indicating that  monsoon . This co as RAPD , 34; ISS as RAPD , 35; AFL as RAPD ; SSR [17 as RAPD . These mBgt, or that multiple genes control one virulence trait together. Second, the internal interaction among genes of Bgt, and the interaction between genes of Bgt and host genes may also change biological traits. In addition, gene expression is influenced by environmental factors, and the virulence of Bgt in wheat is the result of the interaction of genotype and environmental conditions. In view of this, we believe that although EST-SSR and other molecular methods have the advantage of high efficiency and less interference in the dynamic early warning of Bgt and regional correlation analysis, it is not possible to rely solely on molecular methods because the gene sequence of the strain does not correspond well with its pathogenicity. A large number of single-resistance gene cultivar lines are needed to identify the host in the traditional virulence monitoring process relying on inoculation identification, which is time-consuming and laborious, in order to provide effective basic information for the rational prevention and control of wheat powdery mildew disease, disease resistance breeding and variety distribution. However, at present, single-gene line identification is still recommended to be the main method, supplemented by EST-SSR or other molecular markers.The gene expression sequence diversity is not characterized by a simple one-to-one correspondence with its virulence diversity to the host in the same strain. We propose that there may be several reasons: first, it is possible that one gene from the expression sequence controls many virulence traits of Bgt populations from northeastern  and northwestern (Gansu) China, two representative wheat producing areas. The results indicate that Bgt populations from northeastern China showed higher frequencies of virulence than populations from Gansu Province. While many of the known resistance genes failed to provide resistance. Pm21 provided 100% resistance to all the isolates from the three provinces and is therefore provided the best resistance. Pm13, Pm16 (100% resistance in Gansu), Pm18, and Pm22 also maintained acceptable levels of resistance in China. Populations of Bgt from different regions exhibit large genetic differences. The gene expression sequence polymorphisms of Bgt revealed by the EST-SSR molecular marker technique and the corresponding virulence diversity was shown to correlate in only 29 out of 50 isolated tested.This study investigated and analyzed pathogenic Bgt virulence, samples of wheat powdery mildew were collected from main growth areas of wheat in the Liaoning and Heilongjiang provinces in Northeastern China , isolation or purification of single pustules was conducted using standard procedures. The experiments were conducted in triplicates obtain a single pustule, and the single isolate was coded and kept for in vivo propagation [To analyze regional differences in Bgt isolate (as described above) was inoculated on all 37 differential isogenic lines. About 7 days later, when the Chancellor showed a clear level of susceptible symptoms, the infection types (ITs) of all the leaves were observed and recorded using the stringent evaluation standard of Si et al. [Bgt was calculated. To increase the accuracy of the results, each combination of isolate and cultivar was replicated three times.Virulence frequency was analyzed in 37 isogenic lines, each harboring a single known powdery mildew resistance gene Table . The thiTaq PCR Master Mix, 1 μL forward primer, 1 μL reverse primer, 1 μL DNA template (30 ng·μL− 1), 7 μL ddH2O. The PCR procedure was as follows: initial denaturation 5 min at 94 °C; then 35 cycles of: denaturation 30 s at 94 °C; annealing 45 s at the primer pair-specific annealing temperature  following the manufacturer’s protocol. The total PCR reaction volume was 20 μL, consisting of 10 μL 2 × Power http://www.sangon.com/, Shanghai, China).Seven pairs of EST-SSR primers Table  were desThe procedure for polyacrylamide gel electrophoresis (PAGE) was followed as previously published by Chen et al. . The silAdditional file 1: Table S1. Collection dates, sites, and cultivars for all Bgt isolates used in this study (DOCX 29 kb)


Studies on penetrating injuries in Europe are scarce and often represent data from single institutions. The aim of this study was to describe the incidence and demographic features of patients hospitalized for stab injury in a whole nation.This was a retrospective nationwide population-based study on all consecutive adult patients who were hospitalized in Iceland following knife and machete-related injuries, 2000–2015. Age-standardized incidence was calculated and Injury Severity Score (ISS) was used to assess severity of injury.P = 0.826). Most cases were assaults (95.9%) occurring at home or in public streets, and involved the chest (n = 32), abdomen (n = 26), upper limbs (n = 26), head/neck/face (n = 21), lower limbs (n = 10), and the back (n = 6). Median ISS was 9, with 14 patients (19.2%) having severe injuries (defined as ISS > 15). The median length of hospital stay was 2 days (range 0–53). Forty-seven patients (64.4%) underwent surgery and 26 of them (35.6%) required admission to an intensive care unit (ICU), all with ISS scores above 15. Three patients did not survive for 30 days (4.1%); all of them had severe injuries .Altogether, 73 patients  were admitted during the 16-year study period, giving an age-standardized incidence of 1.54/100,000 inhabitants. The incidence did not vary significantly during the study period (Stab injuries that require hospital admission are rare in Iceland, and their incidence has remained relatively stable. One in every five patients sustained severe injuries, two-thirds of whom were treated with surgical interventions, and roughly one-third required ICU care. Although some patients were severely injured with high injury scores, their 30-day mortality was still low in comparison to other studies. Trauma is one of the leading causes of death in all age groups in most developed countries , 2, and Penetrating stab injuries, especially deep injuries involving internal organs and large vessels in the chest or abdomen, can lead to infections, shock, exsanguination, and death . Early dThere have been very few European studies on penetrating trauma published, especially from urban areas. Furthermore, there have been no studies representing whole populations, with most reports representing single trauma centres in larger cities , 18, 19.The aim of this study was to determine the incidence, demographic features, and outcome of penetrating stab injuries in Iceland.This was a retrospective cohort study involving all the patients who were hospitalized in Iceland following a penetrating stab injury some time between 1 January 2000 and 31 December 2015. The mean size of the population over the 16-year study period was 309,360 and at the end of December 2015, Iceland had a population of 332,529 [Iceland is a sparsely populated country with primary trauma care provided in small hospitals in each quarter of the country. The only tertiary trauma centre, Landspitali University Hospital, is located in the capital city (Reykjavik) where about 213,000 people live within 6.3 min of ambulance transport time to the hospital . There in = 15) were included only for calculation of total mortality. Data on these patients were obtained from the National Cause of Death Registry in Iceland and compared to the autopsy databases of the Medical Examiner Office of Iceland and the Department of Pathology at Landspitali Hospital, where all autopsies in Iceland are performed.Included in the study were all patients ≥ 18 years of age who sustained a penetrating stabbing injury (with a knife or machete) and were admitted to hospital. Patients who were treated in the emergency department but discharged without admission were excluded from the study. Patients who died on site or in the emergency department before being admitted to hospital , anatomical site of injury, whether intoxicated with alcohol when injured, surgical procedures, ICU admission, blood transfusions, length of stay ), and 30-day and hospital mortality.Injury classification was done by calculation of anatomical and physiological scores using the Abbreviated Injury Scale (AIS), the Injury Severity Score (ISS), the New Injury Severity Score (NISS) and the Revised Trauma Score (RTS). Of these, AIS, ISS, and NISS are all anatomical scoring systems used to rate the severity of each injury to the body whereas the RTS takes into account the physiological parameters such as Glasgow coma scale, respiratory rate, and systolic blood pressure on first medical contact.Data were entered into a Microsoft Excel spreadsheet. All statistical analyses were performed using R Statistics version 3.4.4. Descriptive analyses were performed for both continuous and categorical variables. Normally distributed variables were summarized as mean with 95% confidence intervals (CI) and compared using Student’s t-test. Variables with skewed distributions were summarized with their median and interquartile range (IQR) and compared using Wilcoxon’s rank sum test. Rates were calculated using age and gender-specific population estimates from Statistics Iceland and they were standardized using the World Health Organization’s World Standard Population. Age standardization was accomplished using the direct method and confidence intervals were calculated assuming the gamma distribution. The incidence of stab injuries by study periods was estimated using Poisson regression.The study was approved by the Icelandic National Bioethics Committee and the Data Protection Authority. As individual patients were not identified, individual consent for participation in the study was not obtained from them.P = 0.826). The incidence is shown in Fig. During the 16-year study period, a total of 88 patients had penetrating stab wounds but 15 of them died on site or in the emergency department before being admitted to hospital, eight due to homicide, six due to suicide, and one for unknown reasons. Out of the 73 patients a total of 23 cases were admitted 2000–2004, 19 cases 2005–2009 and 31 cases 2010–2015. For the whole 16-year study period the age-standardized incidence of penetrating stab injuries in individuals 18 years or more was 1.54 per 100,000 inhabitants (95% CI 1.21–1.94). The incidence of penetrating stabbing injuries did not vary significantly during the study period, incidence rate ratio 1.00 , followed by three cases of self-inflicted injuries (4.1%). In two out of these three cases, the patients were female. The most common cause of injury caused by assault was domestic violence (54.8%), followed by violence in public streets (31.5%), in bars and nightclubs (8.2%), and in the workplace (4.1%). In one case, the cause was unknown.n = 23) and chest tube insertion (n = 19) were the most common interventions, followed by 12 laparotomies or laparoscopies and six emergency thoracotomies and sternotomies. Of the 73 patients, 26 (35.6%) required admission to the ICU  underwent a total of 60 surgical procedures. Table tion n = 9 were thMedian ISS and NISS of all patients admitted were 9 (IQR 2–11) and 9 (IQR 3–11), respectively. Patients admitted to the ICU had significantly higher ISS and NISS scores (median 14.5 (IQR 6–18.5) and 14.5 (IQR 6–23.8), respectively), but 14 patients (19.2%) had severe injuries, with an ISS or NISS over 15. The median length of hospital stay was 2 days  and the median length of ICU stay was one day .Three of the 73 patients died within 30 days of admission, giving a 30-day mortality rate of 4.1%, and the in-hospital mortality rate was the same. Two of the patients who died had sustained injuries to more than one anatomical region, with an ISS of 17 and 75, and the third patient had a severe injury to the thorax only (ISS 25). These three patients all underwent emergency surgery, with one of them succumbing intraoperatively due to haemorrhagic shock. The other two died 11 and 15 days postoperatively from complications related to the initial injury.This population-based study covered all knife and machete-related penetrating stab injuries that required hospitalization in Iceland over a recent 16-year period. The incidence of stab injuries requiring hospitalization in Iceland was very low , which is in line with other Nordic countries such as Finland  . FurtherThirty-day mortality in the present study was very low (4.1%), but our cohort represents a small population compared to previously reported series in the literature , 27, 28.Almost all cases in this study were assaults on men in the early decades of adult life. This is in line with previous studies, where penetrating stab injuries have mainly been observed in young men in the second and third decades of life , 27–29. In the present study, the body regions most commonly affected were the chest and abdomen, which is in line with other studies , 27, 30.It has been reported that a non-operative approach can delay the diagnosis of organ injuries. Thus, extreme caution is recommended when choosing this approach at small/low-volume trauma centres where expertise regarding penetrating trauma may be less than at larger centres . Still, The main strength of this study was its nationwide design, representing a well-defined study population with a modern nationalized healthcare system. Furthermore, it covered a relatively long period of time (16 years) and was based on reliable, detailed data extracted from medical records from all primary and tertiary care centres, from autopsy databases, and from the Cause of Death Registry at Statistics Iceland. The main limitation of this study stems from its relatively small number of cases and its retrospective design. It only covered stab injuries in adult patients, and the age-standardized incidence does not therefore represent all ages. Furthermore, only hospitalized patients were analyzed, and not those who died on the scene or before reaching hospital. Finally, the Trauma and Injury Severity Score, which determines the probability of survival using numbers from the ISS/NISS and RTS, could not be reported as documentation of vital signs — in particular, the respiratory rate and the Glasgow coma scale score — was often missing on first medical contact during the first few years of the study period. The documentation has, however, improved significantly in recent years in Iceland and will be more reliable in future studies.This population-based study, covering a whole nation study and using all adult cases registered, has shown that penetrating stab injuries are rare in Iceland and that the incidence is not increasing. A large proportion of the patients needed surgical interventions, and their 30-day mortality was low despite having moderate-to-severe injuries.


Paralithodes camtschaticus) fishery is one of the largest crab fisheries in Alaska. Summer crab distributions have been well documented by decades of bottom trawl surveys. However, crab movement and distribution are poorly understood outside the summer survey period, which creates several management challenges. One important component of fishery management is the existence of no-trawl zones, which are intended to protect crab from bottom trawl fisheries. However, it is difficult to evaluate the placement of no-trawl zones, because most crab bycatch occurs in trawl fisheries during winter when crab distributions are unknown. Daily fishing logs, kept by skippers in the red king crab fleet since 2005, contain detailed information on the spatial distribution of catch and effort of legal sized male crab during the autumn crab fishery. However, data contained in these hand-written logbooks have not been readily accessible. We digitized daily fishing logs from 2005 to 2016 and used spatial information on catch and effort to infer geographic distributions of legal sized male king crab during the crab fishing season. Changes in distribution were tracked across this 12-yr period and comparisons were made between warm and cold temperature regimes. In warm years , crab aggregated in the center of Bristol Bay, Alaska, while in cold years (2007–2013) they were closer to the Alaska Peninsula. The majority of crab were caught in no-trawl areas (63.4% on average), but variations occurred among years and with temperature regime (40.0–86.8% in no-trawl zones). As temperatures continue to shift in the Bering Sea, it will be important to continue monitoring crab distributions outside the summer survey period.Spatial distributions of fished species must be well characterized to avoid local depletions, identify critical habitat, and predict and mitigate interactions with other fisheries. The Bristol Bay red king crab ( Paralithodes camtschaticus, in Bristol Bay, Alaska, has had a long history, beginning with Japanese harvests in the 1920s ) had little effect on catch per pot . The resultant mean string length was reduced to 8.0 ± 3.6 km (±1 SD), and the number of pots per string was reduced to 25.4 ± 12.0 (±1 SD). For each trimmed record, the string midpoint was calculated by averaging the start and end coordinates of the string. The string midpoints were used in all subsequent spatial analyses.i) of the variable X of interest   crab, Tanner crab [Chionoecetes bairdi], snow crab [Chionoecetes opilio]). The Bristol Bay RKC fleet has begun to transition from paper to electronic logbooks, which should make these data more readily available to managers and avoid additional human transcriber errors. RKC are highly mobile species, thus the summer survey cannot fully explain their distribution; DFLs are an important tool that can supplement summer surveys, helping to improve fishery management decisions that concern crab distribution.DFLs can help elucidate the distribution of Bristol Bay RKC during the autumn, when standard survey data area not available. They provide insights into patterns in crab distribution with temperature regime and can help inform the placement of closure areas to achieve crab conservation objectives. DFLs provide accurate spatial data, given the similarity of results derived from observer and DFL data. Given the many uses of DFL data, it is essential that they become more accessible in the future for other Bering Sea and Aleutian Island crab fisheries Click here for additional data file.S1 Figd) of 20, 10, and 5 km and a neighbor number (k) of 20 and 40. Results shown for two sample years, 2005 and 2008. Areas with restrictions on trawling are outlined in red or gray and described in Analyses performed on catch per unit effort data from daily fishing logs in the Bristol Bay red king crab fishery. Analyses were conducted using 5 weighting methods: a distance band ((TIF)Click here for additional data file.S2 Figst 5% is the mean CPUE from the start of the fishery until 5% of the total legal crab in the population were caught each year. Exploitation rate is the percent of legal crab caught each fishing season. Estimates of annual legal crab abundance and exploitation rate were obtained from the 2017 Stock Assessment and Fishery Evaluation Report (scenario 2b) [CPUE Total is the mean annual CPUE for 2005–2016 using daily fishing log data from the Bristol Bay red king crab fishery, while CPUE 1ario 2b) .(PDF)Click here for additional data file.S3 Fig2: x = no catch, small circle = 1–100 crab, medium circle = 101–500 crab, large circle = 501–1,000 crab, and yellow star = >1,000 crab. A Getis-Ord hot spot analysis was performed on daily fishing log data: red = statistically significant (α < 0.01) high catch per unit effort (CPUE), blue = statistically significant low CPUE, yellow = not statistically different than the mean, and white = areas where no fishing occurred. Areas with restrictions on trawling are outlined in red or gray and described in Summer data are from the National Marine Fisheries Service (NMFS) trawl survey and autumn data are from daily fishing logs Click here for additional data file.


The commercial products are known for their safety, but data on the molecular, cellular, and tissue responses are lacking. We aimed to evaluate the bioavailability and the pro-angiogenic features of the products Bioregen® and Bioexpander® in vitro on cultured endothelial cells (ECs) and dermal fibroblasts in vivo when injected into experimental animals. When added to fibroblasts and ECs, Bioexpander® induced cell migration. The two HA preparations were well tolerated, while a transient proangiogenic behavior of Bioexpander®, when implanted subcutaneously in mice, was found. The neovascular response was evident in the first week with higher levels of VEGF and FGF-2 before undergoing regression. In conclusion, our data strengthen the safety of HA synthetic preparations both in vitro and in vivo. Even if a proangiogenic response is documented, it is modest and transient, leading to tissue recovery and absence of an inflammatory infiltrate.Hyaluronic acid (HA) is used in substitutive and aesthetic medicine with various applications. Ultrapure absorbable HA (Bioregen Hyaluronic acid (HA) (a nonsulfated glycosaminoglycan consisting of repeated disaccharide units of D-glucoronic acid and N-acetylglucosamine), glycans (nHA), and oligosaccharides (oHA) exert different effects on the biological functions of cells and tissues, such as in the connective, epithelial, and neural tissues ,2. HA plHA is a naturally occurring glycosaminoglycan which, by virtue of its viscosity, elasticity, and other rheological properties (particularly for its biocompatibility), acts as a lubricating and shock absorbing fluid in skin and joints and as an ocular lubricant. HA is well tolerated and nonimmunogenic and for these reasons its potential use in medicine is wide-ranging. In fact, the medical use of HA covers clinical use in ophthalmology, such as for cataract surgery, retinal reattachment, corneal transplantation, trabeculectomy in glaucoma, joint diseases (particularly osteoarthritis and rheumatoid arthritis), and adjuvant in wound healing .In addition to its use in conventional medicine, in recent years HA has been commonly used in aesthetic medicine as filler and in the treatment of the signs of aging and in resolving skin blemishes ,6. Some It is noteworthy to observe that HA activities strongly depend on polymer size and the degree of cross-linking. High molecular weight HA (HMWHA) shows anti-inflammatory and immune suppressive activities and induces tissue reparative processes as described in wound healing . It has At tissue level, vascularization and organization of the stromal components are perquisites for tissue homeostasis and the biocompatibility of synthetic medical devices. An adequate blood supply guarantees cell survival and correct biochemical functioning without oedema formation, inflammatory cell infiltration, and scar development ,12. ECs ® (resorbable HA) and Regenyal Idea Bioexpander® (a mix of reticulated and free low molecular weight HA) in vitro on both cultured endothelial cells (ECs) and dermal fibroblasts and in vivo when implanted into experimental animals. The expression of inflammatory markers and the unbalance of proangiogenic factors  vs. antiangiogenic proteins  was monitored during this study.The aim of the study was to evaluate the bioavailability and the pro-angiogenic features of two commercial products, BioregenHuman umbilical vein endothelial cells (HUVEC) were purchased form Lonza  and grown in complete endothelial growth medium (EGM-2) . Some experiments were performed using human dermal blood endothelial cells (HDBEC) purchased from Promocell  and grown in complete endothelial cell basal medium . Both cell lines were supplemented with 10% fetal bovine serum (FBS) .2. Both endothelial cells and fibroblasts were split 1:3 twice a week and used until passage 10.Normal human dermal fibroblasts (NHDFs) were from Lonza . The medium used was fibroblast basal medium (FBM) supplemented with 10% FBS. Cells were cultured at 37 °C in 5% CO®—an ultrapure multifractional adsorbable HA—and Regenyal Idea Bioexpander®—a mix of reticulated and intercalated free low molecular weight HA—both provided by Regenyal Laboratories Srl, S. Benedetto del Tronto (AP), Italy. Specifically, Bioregen® is a mix of five different but not crosslinked molecular weights of HA sodium salt of non-animal origin  obtained by bacterial fermentation. Fragmentation was carried out by implementing heat and ultrasounds in a highly alkaline environment, starting from the fraction of 1 MDa. Analytical characterization was performed as described in .Fibroblast and EC proliferation was evaluated according to the same protocol. One thousand cells/well (of a 96-well multiplate) were left to adhere in 10% serum for 3–4 h and then HA preparations (used at increasing concentrations in the range 0.1–10 mg/mL) were added in a medium with 2% serum which represented the basal control condition. All experimental points using cells from the single culture plate were run in triplicate. After 2 days, cells were fixed, stained with H&E and randomly counted at 20× original magnification in 5 fields ,20. Data5 cells/well) and, after confluence, were scratched using a sterile tip. Once gently washed to remove debris cells were treated with HA preparations diluted in a medium with 2% serum for 18 h in the presence of the antimitotic ARA-C (25 ug/mL) . This was to evaluate only cell migration. The results are expressed as the percentage of wound area with respect to time to 0, measured by image analysis software [Cells (monocultures of fibroblasts or ECs) were seeded into 24-well plates   on ice. At different times, compatible with the biomaterial half-life once injected (1–9 days for the absorbable Bioregen® and 1–4 weeks for Bioexander®), mice were sacrificed by CO2 inhalation and implants were harvested. The implants were re-suspended in 1 mL of Drabkin’s reagent  for 18 h on ice, and haemoglobin concentration was determined by absorbance at 540 nm and compared with a standard curve . Some plugs were stored at −80 °C for protein extraction and Western blot analysis.The mice were SC injected in the dorsal midline region with 0.4 mL of Bioregen®, BIO 98 Srl, Milan, Italy) used at the dose of 0.5 mL (i.m.) and followed by tiletamine and zolazepam  given at the dose of 5 mg/kg (i.m.). The deepness of anaesthesia was checked as reflex to pressure. Each eye was protruded by the use of a dental dam and a local anaesthetic (0.4% benoxinate) was instilled just before surgery. The Regenyal Idea Bioexpander® implantation procedure started with a linear intrastromal incision, parallel to the corneoscleral limbus (linear keratotomy), using a surgical blade. The corneal pocket for the plug implant was produced with a 1.5 mm pliable silver spatula with a smooth edge blade in the lower half of the cornea. The HA gel was introduced with a syringe through the keratotomy line, parallel to and under the corneal epithelium in the external third of the stroma, up to 2 mm from the limbus [Angiogenesis was studied in the cornea of male New Zealand white rabbits  as described in . The inve limbus ,22. The Western blot analysis was performed on the depolymerized plugs of HA or in cell culture lysates. The plugs were isolated from the mice and depolymerized. The HA depolymerization was carried out using hyaluronidase (60 U/mL) for 18 h at 37 °C  in phosphate buffer saline (pH 7.4) .® or Regenyal Idea Bioexpander® (at 1 mg/mL concentration) in a medium with 2% serum.Subconfluent fibroblasts and ECs were seeded in 6 cm diameter petri dishes. After adherence, cells were treated for 18 h with BioregenProtein extraction and Western blot were performed as previously described ,12,13. EThe results are either representative or the average of at least three independent experiments done in triplicate starting from different single culture plates. Statistical analysis was performed using ANOVA test followed by Bonferroni test, and Student t test when appropriate (GraphPad). P < 0.05 was considered statistically significant.® and Bioexpander® for 48 h, no impairment of cell survival was found. The number of cells in the presence of the biomaterial was maintained as in the control condition (medium supplemented with 2% FBS), with a modest increase (in the range of 20–30% cell number) at 1 mg/mL of hyaluronic acid for both products  toward migration was assessed with Bioregen®  was accompanied by the physical deposition of the biomaterial on the endothelial cell surface, as evidenced by purple-blue stained material on the cell cultures after cell fixation and H&E staining  exposed to 0.1 and 1 mg/mL of Bioexpander® F, Bioregigration E. On theonitored E.® on both fibroblasts and endothelial cells. These results are substantiated by the fact that the scratch assay is performed in the presence of an antimitotic agent, in order to avoid cell proliferation that can mask cell spreading.In the complex, the results evidence the promigratory effect of Bioexpander® induced an upregulation of FGF-2 and downregulation of the adhesion molecule fibronectin, consistent with a trend to migration  was well tolerated and no adverse effect was observed after ten days . The addition of Bioregen® to Matrigel  did not change the type of response with no evident angiogenesis induction .The process of angiogenesis was further evaluated following the subcutaneous implant in mice through morphological and biochemical analysis of the harvested implants, to mimic the potential use in humans. After sacrifice, the implants (if not resorbed) were exposed and recovered. The subcutaneous injection of Bioregen® . In particular, after one week, in the seven plugs recovered out of eight implants performed, five implants were clearly vascularized .Instead, the formation of a thin and transparent capsule was observed in all the implants of Bioexpanderularized A, one onularized B. After ularized C. The haularized D. The saThe measurement of angiogenesis modulators in the plugs by Western blot indicated the presence of proangiogenic proteins (VEGF and FGF-2) after 1 week and their decrease at 2 weeks , with ef® when implanted subcutaneously, to an extent that was, however, modest and transient.In total, these data indicate the induction of an angiogenic response by Regenyal Idea Bioexpander® when implanted subcutaneously, whereas the biomaterial is inert when implanted in the avascular tissue of the cornea. The neovascular response was evident in the first week before undergoing regression. VEGF and FGF-2 levels were high for the first week, while the global balance of the angiogenesis modulators moved toward inhibition after a longer time (after one week) with decreased VEGF and FGF-2 levels and increased expression of endostatin. Since in the avascular cornea tissue the biomaterial does not affect angiogenesis and the responses observed on cultured endothelial cells are modest and partial, it is suggested that reticulated HA containing low molecular weight molecules  in vivo induces the recruitment and activation of stromal cells, and is able to release factors that finely modulate the angiogenesis process.In this paper, we describe the tolerability of two HA preparations with different chemico-physical features, and the proangiogenic behavior of Regenyal Idea Bioexpander® does not induce proliferation of endothelial cells (HUVEC and HDBEC), but it is deposited on the cell surface in a concentration-dependent manner. Indeed, the biological functions of HA are mediated by cell surface HA receptors or HA-binding proteins [® was demonstrated with no effect on cell proliferation. On the contrary, a slight increase in fibroblast proliferation was observed in skin fibroblasts, as previously demonstrated [It is widely reported in the literature that HA, due its structural role, that is, to keep tissues hydrated and maintain osmotic balance, regulates cellular processes such as adhesion, migration, and proliferation . Differeproteins . HUVEC cnstrated .® (and not Bioregen®) increases fibroblast and HUVEC migration. The most important receptor that triggers hyaluronan-mediated motility (RHAMM), also known as CD168, is present in several cell types including endothelial cells and various tumor cell lines [®. These molecular features elicited by Bioexpander® were shared also by Bioregen®, even if at functional level a promigratory effect is not evident. Probably, other mechanisms are involved in the final outcome of cell functional responses.In this study, we observed that Regenyal Idea Bioexpanderll lines . Intracell lines . The red® implantation, there is a reactive angiogenesis probably mediated through the recruitment of stromal cells that are able to locally increase the levels of proangiogenic factors. This is confirmed by the engagement of the CD40 receptor in endothelial cells that incites the production of adhesion molecules, cytokines, chemokines, and inflammatory mediators which are important for wound healing [®.Literature data document that HA is a strong inducer of angiogenesis, although its biologic activity in tissues has been shown to depend on molecular size, chemical formula, and structure ,31,32. I healing . It has  healing ,34. Our In summary, our study recounts the events of induction and activation of angiogenesis during a foreign body reaction (FBR). In general, the FBR resembles a sterile inflammatory reaction, i.e., inflammation in the absence of accumulating granulocytes, lymphocytes and plasma cells. Besides formation of a capsule and infiltration of cells such as macrophages into the biomaterial and fusion of these cells to giant cells, protrusion of neovascular sprouts into the biomaterial and surrounding tissue is a main characteristic of the FBR . The mac® implants are still visible after 1 month, while Bioregen®, which does not induce any change in the CD-40 in fibroblasts or endothelial cells, is completely resorbable in a short time.The mild inflammatory reaction could also participate in forming a layer of fibrous capsule on the scaffold, thus preventing its fast degradation. This explains why the Bioexpander®) and the other reticulated and not immediately resorbable (Bioexpander®), have an optimal biocompatibility. Skin fibroblast functions and markers are maintained at a physiological level with HA preparations, with no significant modifications. Endothelial cells, being more susceptible to physical stimuli, show a positive response to the biomaterial only in a strict concentration range (0.1–1 mg/mL) with an activated angiogenic phenotype. When implanted subcutaneously, the reticulated biomaterial induces a transient proangiogenic behavior presumably linked to mild stromal/inflammatory cell activation.From our in vitro models, we can confirm that the two HA preparations, one resorbable (BioregenOur data strengthen the beneficial characteristics of HA in aesthetic dermatology. These beneficial characteristics are mainly based on the properties of HA to bind a large number of water molecules, thus improving tissue hydration and resistance to mechanical stress ,6.