A diverse community of microbes naturally exists on the phylloplane, the surface of leaves. It is one of the most prevalent microbial habitats on earth and bacteria are the most abundant members, living in communities that are highly dynamic. Today, one of the key challenges for microbiologists is to develop strategies to culture the vast diversity of microorganisms that have been detected in metagenomic surveys.Hedera helix (common ivy), a widespread evergreen, using five growth media: Luria–Bertani (LB), LB01, yeast extract–mannitol (YMA), yeast extract–flour (YFlour), and YEx. We also included a comparison with the uncultured phylloplane, which we showed to be dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Inter-sample (beta) diversity shifted from LB and LB01 containing the highest amount of resources to YEx, YMA, and YFlour which are more selective. All growth media equally favoured Actinobacteria and Gammaproteobacteria, whereas Bacteroidetes could only be found on LB01, YEx, and YMA. LB and LB01 favoured Firmicutes and YFlour was most selective for Betaproteobacteria. At the genus level, LB favoured the growth of Bacillus and Stenotrophomonas, while YFlour was most selective for Burkholderia and Curtobacterium. The in vitro plant growth promotion (PGP) profile of 200 isolates obtained in this study indicates that previously uncultured bacteria from the phylloplane may have potential applications in phytoremediation and other plant-based biotechnologies.We isolated bacteria from the phylloplane of H. helix phylloplane, including an evaluation of its culturability using five different growth media. We further provide a collection of 200 bacterial isolates underrepresented in current databases, including the characterization of PGP profiles. Here we highlight the potential of simple strategies to obtain higher microbial diversity from environmental samples and the use of high-throughput sequencing to guide isolate selection from a variety of growth media.This study gives first insights into the total bacterial community of the The online version contains supplementary material available at 10.1186/s12866-021-02119-z. Broadly, leaf microbial communities mainly comprise bacteria belonging to the phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Further, Proteobacteria species have been reported to comprise about half of the phyllosphere community suggesting that, at higher taxonomic levels, phyllosphere bacterial communities are similar across various host plant species [Hedera helix (common ivy) is explored using culture-dependent and -independent techniques. H. helix is an evergreen plant known for its hardiness and climbing ability [An abundant and diverse community of microorganisms naturally exists on the surface of above-ground parts of plants, known as the phyllosphere . The phy species –8. Incre species , and thi ability , and hasTo enhance our understanding about the diversity and function of microbial communities living in the phylloplane, culture-independent approaches are indispensable. Nevertheless, one of the key challenges for microbiologists remains to develop strategies to culture the vast diversity of microorganisms. There has been a recent resurgence in the application of classical culture techniques to interrogate the microbial world, with particular success in environments such as the human gut –14. In gH. helix phylloplane, specifically evaluating the use of Luria–Bertani (LB) [Here, we deployed microbial community metabarcoding to evaluate the culturing efficiency of bacteria from the ani (LB) , LB01 (1ani (LB) , yeast eani (LB) , and YExH. helix host plants, phylloplane samples were analysed in a culture-dependent and -independent way. Metabarcoding was applied to picture the H. helix phylloplane bacterial diversity. Broad characterization of the culturable fraction, using both metabarcoding of Petri dish rinsates and Sanger sequencing of individual isolates, was carried out using five different growth media. The metabarcoding effort of both uncultured and cultured samples yielded 177,872 high-quality 300 bp V3–V4 16S rRNA gene sequences, representing 1482 amplicon sequence variants (ASVs). Bacterial intra-sample  diversity was estimated by rarefaction analysis . Visual examination of the PCoA plot shows that inter-sample bacterial diversity shifts from LB, and to a lesser extent LB01, which contain the highest amount of resources , to YMA and YFlour, which are more selective, and to YEx, which is more limited but also most varied in resources. Differences in carbon-to-nitrogen ratio and carbon sources between the growth media likely contributed to the alpha and beta diversity and prevailing taxonomic groups observed, as it is the case in other bacterial (culture) systems [In an effort to expand the library of phylloplane bacterial isolates from s Figure  and by c systems –25.Fig.Arabidopsis thaliana , Lactuca sativa (lettuce), Glycine max (soy bean), Trifolium repens (white clover), and Oryza sativa (rice) that their phyllosphere communities are mainly comprised of bacteria belonging to phylum Proteobacteria (with classes Alphaproteobacteria and Gammaproteobacteria in particular), Actinobacteria, Bacteroidetes, and Firmicutes [H. helix. This strengthens the observation that the phyllosphere microbiome composition on higher taxonomic level is stabilized by factors such as host plant species and geographical location [In order to take a closer look at the composition of the bacterial communities from uncultured and cultured samples, ASVs were taxonomically assigned using the latest version of the Ribosomal Database Project (RDP) database. From this, differences between the uncultured phylloplane and the cultured samples were evident Fig. . For uncrmicutes , 3–8. Melocation . Host pllocation , 27, whilocation , 28, 29.H. helix phylloplane and the general finding that the vast majority of cultured bacteria are affiliated with these phyla [Bacillus and Stenotrophomonas, with average relative abundances of 19.2 and 16.3%, respectively, while YFlour was most selective for Burkholderia (13.9%) and Curtobacterium (14.7%). Rhizobium was found on all growth media except on LB. In the uncultured phylloplane an average of 18.1% of ASVs could not be classified at the genus level. For growth medium samples this was 4.7% for LB, 5.4% for LB01, 9.0% for YEx, 11.0% for YFlour, and 6.8% for YMA. This suggests that more potentially novel bacterial species were cultured on YFlour, while LB and LB01 yielded the highest abundance of known bacteria.For growth medium samples, all ASVs were taxonomically classified within the phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. This may be expected, given the overall taxonomic structure of the se phyla . All selH. helix, the proportion of shared and unique ASVs for each growth medium was determined. The highest proportion of ASVs (76.3%) was unique for the growth media, 21.9% was shared between at least two of the growth media and only 1.8% was shared between all growth media . Not surprisingly, LB and LB01 contained the highest concentration of (nitrogen) resources, making it easier for r-selected species to grow.Another, more traditional, method to assess culturing efficiency is to count the viable bacterial colonies growing on Petri dishes. A comparison of the viable count of phylloplane colonies growing on the selected growth media, expressed in colony-forming units (CFU) per gram of fresh leaf material, is shown in Fig. A. thaliana [Curtobacterium (41) and Methylobacterium (37). Frigoribacterium (16), Bacillus (13), Rathayibacter (11), Sphingomonas (10), and Pantoea (9) were also common. That one-fifth of cultured bacteria were classified within the genus Curtobacterium may not be surprising, as this genus is ubiquitously reported to be associated within phyllosphere habitats [Curtobacterium species could be isolated from every sample [Methylobacterium [Frigoribacterium [, Sphingomonas [Pantoea [Curtobacterium and Methylobacterium species in this study were able to produce IAA and acetoin, while correlations with ACC deaminase production were not evident. PGP profiles can help to select bacterial isolates with specific PGP traits that can be exploited in microbe-assisted plant biomass production, plant protection, and phytoremediation [Evaluation of PGP potential was determined as indole-3-acetic acid (IAA), 3-hydroxy-2-butanone (acetoin), and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production by 200 bacterial isolates cultured with the selected growth media Fig. a. IAA isthaliana , 33, andthaliana . In our habitats –38. In oy sample . Also, pacterium , Frigoriacterium , Sphingoacterium  and Methngomonas , and Pan[Pantoea . Most isngomonas , and Panediation . For exaediation . Follow-H. helix. Recently, this strategy also proved to be successful in the human gut [Here we highlight the potential of simple strategies to obtain higher microbial diversity from environmental samples. Next to relatively minor tweaks such as the addition of (host) plant extracts  or the uuman gut  and a druman gut , and likCurtobacterium (41), Frigoribacterium (16), Rathayibacter (11), Glaciibacter (5), Cellulomonas (4), Frondihabitans (4), Microbacterium (4), Nocardioides (3), Cellulosimicrobium (2), Leifsonia (2), Nocardia (2), Sediminihabitans (2), Arthrobacter (1), Brevibacterium (1), Flexivirga (1), Gordonia (1), Herbiconiux (1), Micrococcus (1), Patulibacter (1), and Rhodococcus (1). This is interesting given the fact that Actinobacteria members are well-known for their secondary metabolite production [Frigoribacterium and Glaciibacter are typical psychrophilic genera containing a rare group of B-type peptidoglycan [Frondihabitans species are well-adapted to colder and ultraviolet light-exposed environments such as the phylloplane [Our isolation of phylloplane bacteria resulted in a collection of 200 bacterial isolates underrepresented in current databases. Most isolates (104/200) were taxonomically classified within the phylum Actinobacteria, which represented 20 out of a total of 37 genera, including oduction  and abunoduction . For exadoglycan , 50, andlloplane .Additionally, in the context of the coordinated efforts to expand our understanding about plant-associated bacteria and life in general, several isolates from this study were selected for whole-genome sequencing in the framework of the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) project the “Genomic Encyclopedia of Bacteria and Archaea (GEBA)” . A furthH. helix phylloplane and contributes as case study of bacterial culturability of this habitat assessed using high-throughput sequencing technology, including an evaluation of the growth media LB, LB01, YMA, YFlour, and YEx. We provide a collection of 200 bacterial isolates underrepresented in current databases, including the characterization of PGP profiles to facilitate better understanding of the putative ecological roles of phylloplane bacteria that can also guide selection of inoculants for plant–microbe biotechnologies. In the context of international microbial culture collection initiatives aiming to culture at least one member of each functionally diverse group of the microbiota associated with plant hosts, this study highlights the potential of simple strategies to obtain higher microbial diversity from environmental samples and the use of high-throughput sequencing to guide isolate selection from a variety of growth media.This study gives first insights into the total bacterial community of the n = 80, 20 per site) from H. helix plants ranging in age from three to 6 months old were collected at four sites around Hasselt, Belgium ; 50.928680, 5.332674 (B); 50.940104, 5.438675 (C); 50.921694, 5.433951 (D)). The distances in km between the sites are: A–B, 1.4; A–C, 8.5; A–D, 8.4; B–C, 7.6; B–D, 7.2; C–D, 2.1. The soil type at the four sites was characterized to be sandy loam with an average pH of 6.4 ± 0.1  and average soil organic matter content of 958 ± 154 mg kg− 1 . Permission for sampling was obtained and performed in accordance with institutional and international guidelines. Plant leaves were identified as specimens belonging to H. helix by the first author, and verified by all co-authors; voucher specimens are available from Hasselt University. Leaves were cut from the plants at shoulder height using sterile forceps, put in sterile tubes (five leaves per tube) filled with autoclaved phosphate buffer  and immediately transferred to the laboratory. Leaf weight was determined gravimetrically and microbial cells were detached from the leaf surface by sonication , followed by shaking on an orbital shaker . Next, 16 resulting leaf wash suspensions  were centrifuged  and the resuspended pellets were randomly pooled into four samples. For each sample, an aliquot was immediately stored at − 80 °C until DNA isolation; another aliquot was stored overnight at 4 °C for culturing of phylloplane bacteria.Leaves (n = 4) were centrifuged  and genomic DNA was isolated using a NucleoSpin Soil kit . The V3–V4 hypervariable region of the bacterial 16S rRNA gene was PCR-amplified using 341F (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′) primers with attached GS FLX Titanium adaptors, the sequencing key TCAG, and a sample-specific multiplex identifier. PCR products were purified by gel electrophoresis  and the 514 bp bacterial amplicon was excised and further purified using the UltraClean GelSpin DNA extraction kit . Samples were brought to an equimolar concentration (1010 molecules μL− 1) using the Quant-iT PicoGreen dsDNA assay kit . Correct amplicon size and integrity were checked on an Agilent 2100 Bioanalyzer system , followed by sequencing on a Genome Sequencer FLX system  with GS FLX Titanium series reagents by Macrogen Europe .Leaf wash suspensions (n = 4) were pooled, diluted 1/100 and inoculated on 120 × 120 mm square Petri dishes containing LB [2O2 [4. Followed by centrifugation , resuspended pellets were pooled into four samples per growth medium. Pellets were immediately stored at − 80 °C until DNA isolation. Genomic DNA was isolated as described previously and the V3–V4 hypervariable regions of the bacterial 16S rRNA genes were PCR-amplified, purified and prepared for sequencing.Leaf wash suspension aliquots of 10 μL  were checked for purity by streaking, and grown for 24 h in their respective liquid growth medium at 30 °C on a shaker (150 rpm), washed and resuspended in 2 mL of sterile 10 mM MgSO4 solution to obtain suspensions containing bacteria in mid-exponential phase (OD600 nm = 0.4). Next, 20 μL of this bacterial suspension was used for the detection of IAA production using the Salkowski’s reagent method [Bacterial phylloplane colonies were randomly picked from two replicates per growth medium. Isolated colonies  for uncultured bacterial phylloplane samples  and growth medium samples .


Malania oleifera Chun & S. K. Lee have been highly sought after medically because its seeds have high oil content (>60%), especially the highest known proportion of nervonic acid (>55%). Objective of the Study. The objective was to explore the effects of different doses of Malania oleifera Chun oil (MOC oil) on the learning and memory of mice and to evaluate whether additional DHA algae oil and vitamin E could help MOC oil improve learning and memory and its possible mechanisms.  The fruits of  After 30 days of oral administration of the relevant agents to mice, behavioral tests were conducted as well as detection of oxidative stress parameters  and biochemical indicators  in the hippocampus.  Experimental results demonstrated that MOC oil treatment could markedly improve learning and memory of mouse models in behavioral experiments and increase the activity of GSH-PX in hippocampus and reduce the content of MDA, especially the dose of 46.27 mg/kg. The addition of DHA and VE could better assist MOC oil to improve the learning and memory, and its mechanism may be related to the inhibition of oxidative stress and restrain the activity of AChE and also increase the content of ACh.  Our results demonstrated that MOC oil treatment could improve learning and memory impairments. Therefore, we suggest that MOC oil is a potentially important resource for the development of nervonic acid products. Nervonic acid NA C24:1, Δ15), a very long-chain monounsaturated fatty acid, is a major component of mammalian brain nerve fibers and nerve cells , Δ15, a . NervoniMalania oleifera Chun & S. K. Lee, a member of the family Olacaceae [Malania oleifera Chun oil (MOC oil), extracted from the seeds of M. oleifera, was also notable for its substantial phytochemical and phytopharmaceutical value [M. oleifera seeds also produce glycoprotein malaria, which has high cytotoxic activity against tumor cells and is one of the most effective toxins of plant origin [M. oleifera have always been used to make edible oil for consumption by local people [M. oleifera are not common in the wild owing to habitat disturbances and overexploitation. Moreover, with the development of China's agriculture and planting industry, M. oleifera may be planted on a large scale in the future. Since MOC oil is rich in nervonic acid, it is a promising source of natural nervonic acid to promote neurohealth. However, there is no animal test study on the improvement of learning and memory function by MOC oil.lacaceae , was cural value . It has al value , 15. Theal value , 17. Stul people . UnfortuDocosahexaenoic acid (DHA) has been shown to play a key role in neuronal physiology, including neurotransmitter release regulation  and synaα-tocopherol, α-TOH), a group of fat-soluble vitamins with well-known antioxidant functions, has been studied in patients with moderately severe Alzheimer's disease, and was shown to be effective in slowing clinical progression [Vitamin E  with a 12 h light/dark cycle. All experimental procedures followed the guidance of the Ethical Committee for Research on Laboratory Animals of Sichuan University and were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals approved by the European Community Guidelines. All animals were allowed 5 days to acclimatize themselves to the experimental rooms and had free access to food and water prior to experimental procedures.n = 20 per group) as follows: (1) control group : orally corn oil only; (2) low-dose group : application of corn oil with 23.14 mg/kg MOC oil; (3) medium-dose group : application of corn oil with 46.27 mg/kg MOC oil; and (4) high-dose group : application of corn oil with 92.54 mg/kg MOC oil.The nervonic acid content of the MOC oil used in this experiment was about 46.9%. According to the recommended daily dose of NA (2.17 mg/kg) for human , each adAfter the first phase of the experiment, the most effective dose of MOC oil was obtained. Based on the optimal dose, the second phase of the experiment was set up to compare the effects of different combinations of MOC oil, DHA algae oil, and vitamin E on memory of animal models. There was study on adding DHA algae oil to walnut oil, and the stability of the overall composition was determined by the Schaal oven oxidation test. It was found that the 15% DHA algae oil group had the best overall stability and the longest shelf life. Therefore, in this experiment, 85% MOC oil + 15% DHA algal oil was set.n = 20 per group) as follows: (1) control group : orally corn oil only; (2) nervonic acid group : application of corn oil with 21.67 mg/kg nervonic acid; (3) MOC oil group : application of corn oil with 46.27 mg/kg MOC oil; (4) MOC oil and DHA algae oil group : application of corn oil with 46.27 mg/kg MOC oil and 8.17 mg/kg DHA algae oil; and (5) MOC oil, DHA algae oil, and vitamin E group : application of corn oil with 46.27 mg/kg MOC oil and 8.17 mg/kg DHA algae oil and 1.00 mg/kg vitamin E.For the second stage, to explore whether DHA algae oil and vitamin E can help MOC oil improve learning and memory of mice model. All animals were randomly divided into five groups  was dissolved in 0.9% saline to a concentration of 12 mg/mL. Vitamin E was purchased from Sigma-Aldrich Chemical Company, USA. Nervonic acid powder was purchased from Yuanye Biotechnology Co., Ltd. . MOC oil was provided by Kunming Kuteli Biotechnology Co., Ltd.  and stored at 4°C for further use. Malondialdehyde (MDA), acetylcholine (ACh), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), acetyl cholinesterase (AChE), and choline acetyltransferase (ChAT) levels were detected using a commercially available kit, following the manufacturer's instructions . All other chemicals were of analytical grade and obtained from Solarbio.The nervonic acid content of the MOC oil used in this experiment was about 46.9%, and the fatty acid compositions and contents identified of MOC oil were as shown in 3) with a stainless-steel grid floor connected to an electronic stimulant . An insulation platform (4.5 cm in a diameter) was positioned in the middle of the grid floor. The memory acquisition phase was carried out by placing each mouse on the insulation platform and allowing it to free exploration in the box for 3 minutes to acclimatization. Then, the mice were firstly exposed to electric foot shock  for 5 minutes. When subjected to a slight electric shock, the animal's first reaction was to jump on the insulation platform to avoid electric shock. The training was repeated for 5 consecutive days. On the 6th day, the samples were given to the stomachs, and the platform jumping training was repeated one hour later. After the training, the mice in each group (n = 10 per group) were injected intraperitoneally with 120 mg/kg of cycloheximide immediately to create memory consolidation disorder models [To assess a conditional contextual response related to learning and memory capability of animal models, the step-down passive avoidance test was a general accepted experimental method , 30. Thir models . The retn = 10 per group) [The MWMT was conducted to evaluate the discriminative ability and spatial memory of mice model consistent with previous studies , 33. Ther group) . All micThe general condition and behavior of the mice were observed daily throughout the experiment. The body weights of the animals were weighed once a week, and the amounts of the agents were adjusted according to the change in body weight data. On the same day after the last behavioral assessment, the mice were immediately euthanized for cervical dislocation to obtain spleen, liver, kidney, and hippocampus for organ coefficient, oxidative stress parameters, and biochemical indicators analysis. The CA1 region of the hippocampus plays a central role in the long-term memory consolidation . In the t-test, while multiple comparisons were made using a one-way ANOVA followed by the Student–Newman–Keuls (SNK) post hoc test. All data were presented as the means ± SEM, and statistical differences were accepted at the 5% level (P < 0.05), unless otherwise indicated.All analyses were performed using SPSS 26.0 software  and GraphPad PRISM 7  statistical programs. Two-sample comparisons were performed using a Student P > 0.05).At the first stage, the animals in each group developed well, and no abnormal signs and death were observed during the experiment. The results showed that there was no significant difference of weight gain and relative organ indexes of the liver, kidney, and spleen of animals among four groups as shown in (1) Step-Down Passive Avoidance Test. As exhibited in P < 0.01). However, retention latency in the MOC oil-L and MOC oil-H was longer in comparison with the control group but without significant difference (P > 0.05). As indicated in P < 0.05, P < 0.01, and P < 0.01, resp.). These findings demonstrated that MOC oil treatment could improve the impairment of memory consolidation in passive avoidance.(2) Morris Water-Maze Test (MWMT). The spatial memory reproduction ability of the animals was assessed using MWMT, and the results showed that, during the 5 consecutive days of training blocks, mice who received daily doses of MOC oil-treated found the hidden platform sooner than the control ones but without statistically significant difference . In the probe trial, the time animals staying on the remembered-platform in the MOC oil-L, MOC oil-M, and MOC oil-H groups was significantly more than that of the control group  and 46.27 mg/kg MOC oil (P < 0.01) treatment also resulted in marked improvement of the number crossing the original platform area; moreover, 92.54 mg/kg MOC oil treatment would increase the crossing number compared to control group but the difference was not statistically significant (P > 0.05). These findings demonstrated that MOC oil treatment could attenuate learning and memory impairments and improve the retention of spatial memory, especially in the MOC oil-L and MOC oil-M groups.P < 0.05), MOC oil-M (P < 0.01), and MOC oil-H groups (P < 0.01) were significantly reduced . ACh content in the hippocampus of MOC oil treatment groups showed a slight increase but without significant difference  would be used as the basis for investigation to explore the effects of the formula related to MOC oil on the memory of the animal models.P > 0.05).At the second stage, body weights at sacrifice and relative liver/kidney/spleen-to-body weights of mice model in each group are summarized in (1) Step-Down Passive Avoidance Test. P > 0.05, P < 0.01, P < 0.01, P < 0.05, and P < 0.01, resp., P > 0.05).(2) Morris Water-Maze Test (MWMT). In the acquisition phase, the escape latency to reach the platform gradually decreased during the training process in all groups, whereas no significant difference was observed (P < 0.05), which means that 21.67 mg/kg NA and 83.5% MOC oil + 14.7% DHA algae oil + 1.8% VE treatment could improve the acquisition of spatial memory. In the probe phase, compared to the control group, the time that the animals stayed in the circular area was statistically prolonged in NA and all other groups compared with that of the control group , suggesting that DHA and VE might have an auxiliary effect on enhancing memory.observed . In addiP < 0.01, P < 0.05, P < 0.05, and P < 0.01, resp., P < 0.05, P < 0.05, P < 0.05, P < 0.05, P < 0.05, P < 0.05, and P < 0.05, resp.); the AChE activity of the MOC oil group decreased, but the difference was not statistically significant (P > 0.05). Compared to those in control group, the ChAT activity in NA group and MOC oil + DHA + VE group was markedly increased . In addition, there was a slight increase in ACh content, SOD, GSH-PX, and ChAT activity in the hippocampus of the mouse models with additional DHA and VE compared with the pure MOC oil group, but the difference was not statistically significant. MDA content and AChE activity in the MOC oil + DHA and MOC oil + DHA + VE groups were lower than those in the MOC oil group but without significant difference (P > 0.05, Figures MDA levels in all groups exhibited significantly decreased (The principal findings of this study are that MOC oil containing high amounts of nervonic acid could ameliorate memory impairment of mice. The formula of 83.5% MOC oil + 14.7% DHA algae oil + 1.8% vitamin E can prevent lipid oxidation in the hippocampal tissue of mouse models, thereby greatly improving the learning and memory function of mice, which has the potential for further function products' development.Malania oleifera Chun oil [Malania oleifera Chun oil for 30 consecutive days had no adverse effects on the growth and life activities of mice, suggesting that the long-term moderate intake of MOC oil is safe.Records have shown that Zhuang residents in Guangnan County, Yunnan Province have historically taken Chun oil , 36, mosIn the first stage, the step-down passive avoidance test has been employed to assess short-term memory and discrimination capability of animal models on the basis of previous studies. Cycloheximide is a commonly used protein biosynthesis inhibitor. Previous studies have shown that intraperitoneal injection of cycloheximide will impair the memory consolidation function of animals. Therefore, all animals were injected intraperitoneally with 120 mg/kg cycloheximide 10 minutes before the test to establish memory consolidation impairment mice model . The incThe brain consumes an appreciable lot of oxygen and limits its antioxidant capacity; in addition, it has a large amount of polyunsaturated fatty acids and redox active transition metal ions, so it is highly susceptible to oxidative damage. Oxidative damage to synapses in the hippocampus has previously been reported to cause cognitive deficits in mice . StudiesACh is one of the important neurotransmitters of the central cholinergic system . Its metWe proposed an explanation of the mechanisms underlying the interactions of MOC oil: MOC oil could increase the activity of GSH-PX and reduce the content of MDA in the hippocampus of mice, enhance antioxidant capacity, and inhibit lipid peroxidation and oxidative stress damage effectively, thereby improving the learning and memory function.Malania oleifera Chun oil is a potentially important resource for the development of neuronic acid products.In summary, we confirmed that MOC oil-treated mice showed better learning and memory function in behavioral tests. MOC oil could increase the activity GSH-PX in hippocampus and reduce the content of MDA and enhance the learning and memory function of mice. DHA algae oil and vitamin E could clearly enhance the effect of MOC oil on enhancing GSH-PX activity, reducing MDA content and reducing oxidative stress in the hippocampus of mice. At the same time, this formula could effectively reduce the activity of AChE and increase the activity of ChAT, thereby increasing the content of ACh and improving learning and memory functions, which was more efficient together than MOC oil-treated alone. Therefore, we suggest that


Eriocaulon nepalense was sequenced and assembled in this study. The cp genome of E. nepalense is 150947 bp in length, composed of a pair of 26451 bp inverted repeat regions (IRs), separated by a large single-copy region (LSC) of 81064 bp, and a small single-copy region (SSC) of 16981 bp. The cp genome contained 114 unique genes, including 80 protein-coding genes, 30 tRNA genes, and 4 ribosomal RNA genes. The phylogenetic position of E. nepalense based on the cp genome data is closer to E. decemflorum than E. buergerianum.The complete chloroplast (cp) genome sequence of  Eriocaulon Linn., under the family Eriocaulaceae, consists of 470 species in the world  was applied to perform 2 × 150 bp pair-end sequencing. Clean reads were mapped to published chloroplast genome of Eriocaulon as references . Total genomic DNA was extracted from the fresh leaves of E. nepalense is 150947 bp in length. It comprises a pair of IRs (26451 bp) separated by the LSC (81064 bp) and SSC (16981 bp) regions. The cp genome harbors 114 functional genes, including 80 protein-coding (PCGs), 30 transfer RNA (tRNA) genes, and 4 rRNA genes. Among them, 10 protein-coding and 6 tRNA genes have introns. The average GC content of cp genomes is 35.8%.The complete cp genome sequence of Eriocaulon and closed genera were downloaded from GenBank. Bayesian inference analyses (Ronquist and Huelsenbeck E. nepalense is sister to E. decemflorum.Some available complete cp genome sequences of Eriocaulon, as well as its related genera were consistent with all the previous studies (Darshetkar et al. The sequence alignment and all the settings of Bayesian analyses were the same with previous study by Darshetkar et al. . The phy


Allium spp. extracts had the potential to be used in feeding laying hens to improve productivity, without affecting egg quality, and to modulate the gut microbiota.The growing interest in phytogenic products for use in feed, especially in the poultry sector, is mainly due to the improvement in the productivity parameters and gut microbiota modulation properties. For this reason, phytogenic products are becoming excellent candidates as alternatives to the use of antibiotics in animal production to mitigate the negative effects derived from their use. The aim of this study is to explore the ability of allium extract (containing garlic and onion), used as an ingredient in laying hen feed, to improve performance. The promising results obtained in the present study suggest that Lactobacillus spp. and Bifidobacterium spp., as well as by the reduction in Enterobacteriaceae populations. Finally, the preservation of egg composition was checked by monitoring the content of PTSO, using a new analytical method consisting of the use of solid phase extraction and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Consequently, based on current results, Allium spp. extract rich in organosulfur compounds such as PTSO added to the diet had a beneficial effect on the microbiota and would seem to be a possible alternative to increase productivity, while not affecting the biochemical composition of egg. However, further studies on the effects of allium extract as feed supplement are necessary.The use of allium extract containing propyl propane thiosulfonate (PTSO) as hen feed supplement was evaluated to demonstrate its positive effect on egg production and intestinal microbiota modulation. The study was carried out on 90 laying hens whose feed was supplemented with allium extract for 28 days. Nutritional properties of eggs were not affected, whereas an improvement in productivity was observed based on the increase weight of eggs. In addition, a modulator effect on intestinal microbiota was confirmed by the increase in  Until their ban in 2006 , antibioSome of the major challenges associated with antibiotic-free poultry production include poor growth performance, lower productivity, and increased morbidity as well as mortality in fowl ,5,6. EssAllium spp. as feed supplement in poultry has been extensively studied [Allium spp. [In recent years, the use of extracts from  studied . Organosium spp. ,15,16,17ium spp. . Thiosulium spp. .Escherichia coli and Salmonella spp., as well as against Campylobacter jejuni [−1 [The profile of bioactive compounds in allium extract is conditioned by the type of allium variety and specific extraction process . This par jejuni ,22. In ar jejuni . In addir jejuni . The punjuni [−1 .Lactobacillus spp. and Bifidobacterium spp.), total Enterobacteriaceae count, total anaerobic bacteria, Clostridium spp. and Enterococcus spp.The aim of this study is therefore to evaluate the influence of allium extract intake on laying hens, directly related with PTSO intake, by monitoring egg quality and productivity as well as laying hen health status. For this purpose, a field experiment with 180 laying hens was carried out for 28 days. One half of laying hens (treated group) was supplemented with allium extract, whereas the other half had an allium-free diet (control group). Egg productivity from both groups was compared by studying the number of laid eggs and their weight. In addition, egg quality was determined by the amino acid content and the proximate analysis results. Hen health status was evaluated by feces analyses from each group of microbiota probiotic population  in real conditions. This research is an observational nutritional trial, in which the slaughtering or direct handling of animals did not occur, reducing their stress during the experiment. This test was carried out following the standard commercial procedure in accordance with Royal Decree 348/2000  and comp4OH), potassium sulfate (K2SO4), sodium chloride (NaCl), potassium chloride (KCl), glutathione reduced 98% (GSH) and cysteine hydrochloride 97% (CYS) were purchased from Sigma Aldrich . MeOH, acetone (ACO), acetonitrile (MeCN), ethyl acetate (EtAc) and trichloroacetic acid (TCA) were supplied by Panreac . Lastly, sodium phosphate dibasic (Na2HPO4), monopotassium phosphate (KH2PO4) and peptone solution 1% were purchased from Scharlab . Ultrapure water  was used throughout all of the work.All reagents were of analytical reagent grade. The solvents used as the mobile phase were LC-MS grade. Methanol (MeOH) used as the mobile phase was supplied by VWR BDH Prolabo . Formic acid (FA) , ammonium hydroxide 30%  were used for sample filtration prior to the injection into the chromatographic system. Extraction cartridges containing a mixed-mode cation-exchange polymeric sorbent  were used for SPE. In addition, silica cartridges  and reversed-phase hydrophilic-lipophilic balance polymer cartridges  were tested in the optimization of the SPE procedure.Clarinert®) rich in PTSO (active ingredient) was supplied by DOMCA Co., Granada, Spain. GARLICON P30® (Feed register number αESP18000180), a powder mix consisting of PTSO (3%) as the active ingredient and sepiolite as the technological additive (97%), was added to hens’ feed at a dose of 1000 mg kg−1 .A commercial allium extract , was kindly provided by the company DOMCA Co . A 2 g LEnterococcus, MacConkey agar for Enterobacteriaceae total count, Wilkins Chalgren agar for the general growth of anaerobes and sulfite-polymyxin-sulfadiazine agar for enumeration of sulfite-reducing clostridia. All media were provided by Biokar Diagnostics S.L.  and subjected to a sterilization process in autoclave at 121 °C for 20 min. Furthermore, a modified Columbia agar medium was used for enumeration of Bifidobacterium spp. [Lactobacillus spp. count [The culture media used for bacterial growth analysis were as follows: Slanetz Bartley agar used for the enumeration of ium spp.  and a LAp. count .TM Algorithm (AB Sciex).PTSO analyses were performed using an Agilent 1290 Infinity LC  equipped with a binary pump, online degasser, autosampler (5 μL loop) and a column thermostat. Mass-spectrometry measurements were performed on a triple quadrupole mass spectrometer API3200  with electrospray ionization (ESI). A Zorbax Eclipse Plus RRHD  chromatographic column was used for the separation. Instrumental data were collected using the Analysts Software version 1.5 with Schedule Multiple Reaction Monitoring (MRM) Amino acid analyses were performed using an Agilent 1260 Infinity LC  system consisting of a quaternary pump, online degasser, autosampler (injection range 0.1–900 μL), a column thermostat and photodiode array detector (PAD). Amino acid separation was performed using an AccQ-Tag chromatographic column . A Universal 320R centrifuge , a vortex-2 Genie , a polytron , an oven with natural air convection , a muffle furnace  and an evaporator system  were also used for sample treatment.The field experiment was carried out in a farm located in Alhendín . Hens were separated into battery cages with a rate of six animals per cage . Housing temperature was maintained at 20 ± 2 °C with an average relative humidity of 78 ± 3% and a photoperiod  of 16 h. A total of 180 Lohmann Brown hens (36-week-old) were randomly allocated in two groups defined as control group (G1) and treated group (G2). Each group was constituted by 90 hens and divided in 15 cages.In a period of 90 days prior to the beginning of the experiment, all hens were given ad libitum access to water and antibiotic-free feed.® was added to the middle of the feed (double mixing procedure). The feed was supplied by Piensos Pisur  and homogenized into a helical solid mixer in order to ensure that both G1 and G2 groups consumed a similar feed. Once homogenized, the additive GARLICON P30The field trial was extended for 28 days (this extension was determined by availability of the farm and the financing of the project). In this period, the access of G1 and G2 to feed was controlled, whereas the access to water was ad libitum. Hens from G1 maintained the standard basal diet, whereas the G2 diet was supplemented with 1000 mg of free n-sulfide compounds allium extract purified (whose main ingredient was PTSO) per kg of feed, which is equivalent to 30 mg of PTSO per kg of feed.Level values of crude protein, methionine, and lysine are not affected by the addition of garlicon. This was taken into account due the important role they play in laying hen feed . Moreove−1 of PTSO, with its antibacterial activity being demonstrated [−1 [Several studies in broilers tested dosages of 45 and 90 mg kgnstrated ,23. In aated [−1 . Based oIndividual weekly feed intake, egg production and egg weight were recorded. Eggs from both groups were collected weekly, counted and weighed. Egg mass per hen per day was calculated as laying percentage (100 percent = 1 egg per hen per day), multiplied by average daily egg weight. Values per week were calculate by multiplying per 7 (days per week). The feed conversion ratio was calculated from weekly egg mass production and weekly feed intake over 28 days to verify the feed efficiency of each group .Proximate analysis consisted of determining total fat, moisture, ash, total protein, total carbohydrates and energy content. Total fat in egg samples was determined using the official method of analysis AOAC 925.32–1925 . MoisturMicrobiological analysis was performed by fecal sampling. This procedure was selected with the objective of minimizing animal manipulation. Other procedures could be a stressful activity (required to collect feces from the cloaca) and could affect egg laying. Analysis of intestinal content was also discharged since this study was purely observational and animal sacrifice was not considered.−1 NaCl, 0.2 g L−1 KCl, 1.15 g L−1 Na2HPO4 and 0.2 g L−1 KH2PO4, and containing CYS in a concentration of 0.5 g L−1 to favor anaerobic bacteria survival. Viable counts of bacteria in the feces samples were then estimated by plating serial 10-fold dilutions in the peptone solution. Media and culture conditions to determine each bacterial group are shown in Fecal samples (25 ± 0.1 g per sample) were collected weekly directly from a conveyor belt placed under each cage in sterile containers classified and labeled for analysis. They were diluted with 9 mL of phosphate buffered saline, pH 7.3, prepared as a mixture of different salts at the following concentrations: 8 g LAlthough several analytical methods for PTSO determination have been established for feed ,29 or miv/v) solution. Samples were finally analyzed by UHPLC-MS/MS using a mobile phase consisting of 0.05% aqueous FA solution (solvent A), and MeOH (solvent B) at a flow rate of 0.4 mL min−1 (gradient mode). Column temperature was set at 25 °C and the injection volume was 5 μL (full loop). The eluent gradient profile was as follows: 10% B (2.5 min) and 100% B (3 min). It was then reduced back to 10% B in 0.5 min, where it was maintained for 2 min for column equilibration. The MS was working with ESI in positive mode (ESI+) under MRM conditions  30 psi; ion spray voltage 5500 V; and GAS 1 and GAS 2 (both of them nitrogen) were set to 50 psi . Under these conditions, CSSP, GSSP and PTSO were determined in 4.0 min. A scheme of the whole sample procedure is shown in Statistical analysis was performed by comparing the analyzed variables’ behavior throughout the field trial. The study of the influence of allium extract on egg quality and microbiological analysis was determined by two-factor analysis of variance (ANOVA) establishing time and treatment as fixed effects. In addition, when the interaction was significant, results between G1 and G2 at each time were compared by applying Student’s test . All statistical data treatment was performed using STATGRAPHICS Centurion XVI Version 16.2.04 (32 bits).p ≤ 0.05).The experimental unit was the cage. In all statistical analyses, the confidence interval was established at 95% (differences were taken to be significant at Feed intake (g per week), egg mass (g per week) and feed conversion ratio during the experimental period are presented in p > 0.05) throughout different time points of the trial .No relevant differences were observed in feed intake per week and hen for G1 and G2 . Therefore, an increase in the average weight was obtained as result of use PTSO as feed supplement.Laid eggs from G1 and G2 were collected, counted, and weighed on a weekly basis. A total of 845 eggs (411 from G1 and 436 from G2) were evaluated throughout the field trial. A sightly increase in egg mass was observed for G2 from the 14th day , as a result of the use of PTSO as a feed supplement. Finally, Student’s t-test showed no differences related to the number of laid eggs between G1 and G2 throughout the field trial (p > 0.05).The results are shown in p = 0.005), 21st (p = 0.016) and 28th day (p = 0.001).In the same way, there was an impact on feed conversion ratio that was observed by an increase in average weight in G2 by the same feed intake of G1. This trend was observed for the 14th  when used as a feed supplement, and therefore can be traduced into productive capacity improvement. By having a better profile of the intestinal microbiota and possibly of the general immunity, it is possible to obtain an indirect effect derived from the greater absorption of nutrients and intestinal well-being.p > 0.05 in all cases. The results obtained for G1 versus G1 by considering all time results (since there is not time dependence) are shown in The influence of allium extract on egg nutritional parameters was studied by analyzing energetic value, total fat, moisture, ash, total protein and total carbohydrates. No significant differences were seen among time-points of each group, G1 and G2 throughout the trial were observed for egg nutritional parameters, obtaining p > 0.05 in all cases). The results obtained for G1 versus G1 by considering all time results (since there is not time dependence) are shown in p > 0.05 in all cases).Egg amino acid content was also monitored in both experimental groups (G1 and G2) as a quality control parameter. Eggs are characterized by the presence of 17 amino acids: aspartic acid (ASP), serine (SER), glycine (GLY), glutamic acid (GLU), histidine (HIS), arginine (ARG), threonine (THR), alanine (ALA), proline (PRO), CYS, tyrosine (TYR), valine (VAL), methionine (MET), lysine (LYS), isoleucine (ILE), leucine (LEU), phenylalanine (PHE)   from G1 and G2 was analyzed weekly. No significant differences among time-points of each group nor G1 and G2 throughout the trial were observed for anaerobic total bacteria, Clostridium spp. and Enterococcus spp. (p > 0.05). The results obtained for G1 versus G1 by considering all time results (since there is not time dependence) are shown in The effect on intestinal microbiota was studied, since it plays a key role in animal health status and may be influenced by several factors, such as diet, environment, stress or the presence of pathogens ,46. The Lactobacillus spp. and Bifidobaterium spp. confirmed that the use of allium extract containing PTSO as a feed supplement has a significant effect on the intestinal microbiota by reducing the number of Enterobacteriaceae in hen’s feces after 7 days (p = 0.028) of treatment . Enterobacteriaceae includes Salmonella spp., whose prevention is one of the major challenges in poultry production. PTSO supplementation reduces this pathogen, which could translate to a decrease in associated animal pathologies such as salmonellosis. The reduction in the fecal contamination of eggs ensures animal and food safety [The results obtained for total Enterobacteriaceae, reatment . This trd safety .Lactobacillus spp. in G2 compared to G1 at 21st (p = 0.011) and 28th days (p = 0.035). A similar behavior was observed for total Bifidobacteria population, which increased at the 7th (p = 0.041), 14th (p = 0.034), 21st (p = 0.039) and 28th (p = 0.045) days. Lactobacillus spp. and Bifidobacterium spp. population are associated with the inhibition of pathogen growth. They are also closely related to the increase in nutrient absorption efficiency as well as the improvement of the immunostimulatory effect [Lactobacillus spp. and Bifidobacterium spp. populations were increased by diet supplementation with allium extract. Therefore, it could be said that the presence of PTSO in the diet of laying hens triggers a positive intestinal microbiota modulation. The capacity of allium organosulfur compounds, such as PTSO, to modulate the intestinal microbiota is widely known [Lactobacillus spp. and a reduction in Salmonella spp. and Clostridium spp. in feces [Salmonella [Allium spp. derivatives and the evidence that points to the antimicrobial activity against enteropathogens through the organosulfur compounds it contains suggest that this could be a good way to improve gut health in animals [Finally, there was an increase in y effect ,49. As cly known . Recent in feces . Residenlmonella . The cap animals .Twenty egg samples from G1 and G2, respectively, were analyzed weekly. Neither PTSO nor its derivatives (CSSP and GSSP) were detected in any sample. In other words, allium extract residues that could affect egg biochemical composition were not detectable when using the recommended and tested doses.The present study has demonstrated that the intake of allium extract containing PTSO as a feed supplement has a positive effect on the productivity of laying hens, increasing egg size and weight.Lactobacillus spp. and Bifidobacterium spp. populations. In addition, by monitoring possible residues of PTSO or its derivatives in egg samples by UHPLC-MS/MS, this study has proven that the use of allium extract does not alter egg biochemical composition.In addition, evaluation of intestinal microbiota has shown that allium extract reduced Enterobacteriaceae and increased


Accumulative studies showed that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) was up-regulated in the blood and urine from patients diagnosed with lupus nephritis (LN) and that it might be used as a novel biomarker for active LN. This meta-analysis aimed to determine the diagnostic value of TWEAK in active LN. We searched the Cochrane Library, Embase, PubMed, Springer, Wanfang and CNKI databases for articles published up to 20 August 2020. The diagnostic capacity of TWEAK for active LN was assessed using pooled sensitivity and specificity, positive and negative likelihood ratios (PLR and NLR), diagnostic odds ratio (DOR), and area under the receiver operating characteristic curve (AUC). Quality assessment and publication bias were also evaluated. STATA 11.0 and Meta-Disc 1.4 were used to perform these analyses.p = .32). Nine cross-sectional studies were included in this meta-analysis. The overall pooled sensitivity of TWEAK for the diagnosis of active LN was 0.69 , and specificity was 0.77 . The overall pooled PLR and NLR were 3.31  and 0.38 , respectively, with a DOR of 10.89  and AUC (SE) of 0.8276 (0.0289). Deeks’ funnel plot revealed that the publication bias was insignificant in the study ( Our results suggest that TWEAK might be a potential biomarker for patients with active LN. Future cross-sectional and longitudinal studies are needed to confirm its diagnostic value, as well as to establish more definite cutoff for active LN. As a common clinical manifestation of systemic lupus erythematosus (SLE), lupus nephritis (LN) is characterized by immune complex deposition, renal microvascular lesions, inflammation, proteinuria, hematuria and progressive renal dysfunction ,2. It afTumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily ligands, is a multi-functional cytokine which binds to its receptor known as fibroblast growth factor-inducible 14 kDa protein (FN14) . TWEAK iEmerging evidence showed that TWEAK was up-regulated in the blood and urine from patients diagnosed with LN and that it might be used as a novel biomarker for active LN ,19. HoweA comprehensive literature search was performed in the Cochrane Library, Embase, PubMed, Springer, Chinese National Knowledge Infrastructure (CNKI) and Wanfang databases up to 20 August 2020, using one or a combination of the following terms: TNF-like weak inducer of apoptosis, TWEAK, systemic lupus erythematosus, SLE, lupus nephritis. There was no language limitation in the literature searching. In addition, the relevant references and cited papers were searched manually to identify additional studies meeting the inclusion criteria.The study inclusion criteria were as follows: (1) the study participants were human; (2) patients diagnosed with SLE according to the 1982 and 1997 American College of Rheumatology criteria  and the 2012 Systemic Lupus International Collaborating Clinics (SLICC-2012) ; (3) SLEThe study exclusion criteria were as follows: (1) studies not focusing on the diagnostic performance of TWEAK in predicting active LN in patients with SLE; (2) case reports and reviews; (3) studies without mandatory predictive variables including the area under the receiver operating characteristic curve (AUC), sensitivity and specificity.All data were extracted independently by two authors using a paper data extraction form. The accuracy of the extracted data was further confirmed by a third author. The extracted information included: (1) characteristics of the included studies: author and year of publication, country, study design, participants, baseline serum creatinine (Scr) level, definition of active LN and key findings; (2) diagnostic accuracies of the included studies for TWEAK to predict active LN: sample type (urine or serum), detection method, TWEAK cutoff value, sensitivity, specificity and AUC in each study. If a study lacked basic data, we calculated the TP/FP/FN/TN according to the following formulas: sensitivity = TP/(TP + FN), specificity = TN/(FP + TN), (SLE with active LN) + (SLE without active LN) = TP + FP + TN + FN and filled in the 2 × 2 table. The risk of bias was assessed using guidelines proposed in Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) .2) statistics test. If the heterogeneity was statistically significant (p < .05 or I2 > 50%), the random-effects model was used to calculate the pooled effect sizes such as sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR); otherwise, the fixed-effects model was employed. The AUC and Cochrane indices (Q*) were calculated. An AUC ≥ 0.70 defines a useful risk predictor. We also tested the publication bias using Deeks’ funnel plot method.We performed the analysis using Stata 11.0 software  and Meta-Disc version 1.4 . Heterogeneity among the studies was assessed with the Cochran Q test and I-squared (IA flowchart of the selection process is shown in Characteristics of the included studies are summarized in p = .32).The results of the QUADAS-2 tool are illustrated in Pooled sensitivityExtracted data from the included studies are shown in p < .0001, I2 = 84.3%), so the random-effects model was used for the meta-analysis. The overall pooled sensitivity of TWEAK for the diagnosis of active LN was 0.69  (There was a significant heterogeneity among the studies (63–0.75) .Pooled specificityp < .0001, I2 = 77.7%), so the random-effects model was used for the meta-analysis. The overall pooled specificity of TWEAK for the diagnosis of active LN was 0.77  (There was a significant heterogeneity among the studies (71–0.82) .Pooled positive likelihood ratio (PLR)p = .003, I2 = 66.2%), so the random-effects model was used for the meta-analysis. The overall pooled PLR of TWEAK for the diagnosis of active LN was 3.31  (There was a significant heterogeneity among the studies (05–5.35) .Pooled negative likelihood ratio (NLR)p < .0001, I2 = 72.2%), so the random-effects model was used for the meta-analysis. The overall pooled NLR of TWEAK for the diagnosis of active LN was 0.38  (There was a significant heterogeneity among the studies (26–0.55) .Pooled diagnostic odds ratio (DOR)p = .360, I2 = 9.0%), so the fixed-effects model was used for the meta-analysis. The overall pooled DOR of TWEAK for the diagnosis of active LN was 10.89  (There was no significant heterogeneity among the studies (3–17.63) .Summarized receiver operating characteristic (SROC) curveThe SROC curve was calculated by sensitivity against (1-specificity). Figure 5.Diagnostic sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) of TWEAK in predicting active LN across all included studies. CI: confidence interval; df: degree of freedom; LR: likelihood ratio; OR: odds ratio.Next, we performed subgroup analysis of the included studies . As showAs LN is one of the most serious manifestations of SLE leading to significant morbidity and mortality among patients, early recognition of the disease is of vital importance. Conventional laboratory parameters cannot ideally differentiate renal activity from renal damage in LN . TherefoAs a member of the TNF superfamily, TWEAK and its specific receptor FN14 can regulate a number of biological processes such as cell proliferation, migration, differentiation, tissue regeneration, angiogenesis and induction of inflammatory cytokines . TransieRecently, TWEAK has been proposed as a promising biomarker of active LN in patients with SLE ,21,30–36p = .32). Further subgroup analysis revealed that TWEAK had a higher diagnostic value to predict active LN in the subgroup of patients with a rSLEDAI score of more than 4. Urinary TWEAK seemed to be a more promising biomarker as compared to serum TWEAK based on its higher DOR.Current studies focusing on the diagnostic performance of TWEAK were inconsistent because of single-center design or small sample size. In this context, we therefore conducted the present study. To our knowledge, this might be a novel meta-analysis which assessed the diagnostic value of TWEAK for active LN. Our findings suggested that TWEAK was elevated in patients with LN and might serve as a promising predictor, with DOR of 10.89  and AUC (SE) of 0.8276 (0.0289). The pooled PLR and NLR of the included studies were 3.31  and 0.38 , indicating a satisfactory diagnostic performance. There was no publication bias of the included studies (Biostatistics and Epidemiology: A Primer for Health Professionals, the choice of cutoff point is influenced by many factors [However, there were some limitations in this meta-analysis. First, the number of the included studies was limited and most of the studies had a small sample size. Second, a majority of the enrolled studies used rSLEDAI score in the diagnosis of active LN; however, due to the unreliable nature of urinalysis, this could have constituted an inherent limitation of this study. For instance, false positive proteinuria might be encountered in the setting of SLE, which is not necessarily indicative of renal disease . Besides factors .In conclusion, the present meta-analysis provided evidence that TWEAK might be a potential biomarker for patients with active LN. Future cross-sectional and longitudinal studies are needed to confirm its diagnostic value, as well as to establish more definite cutoff for active LN in the clinical practice.


Labyrinthula, is an important stressor of the dominant macrophyte in Florida Bay (FB), United States, Thalassia testudinum. FB exhibits countervailing gradients in plant morphology and resource availability. A synoptic picture of the Thalassia-Labyrinthula relationship was obtained by assessing the activity of four immune biomarkers in conjunction with pathogen prevalence and load [via quantitative PCR (qPCR)] at 15 sites across FB. We found downregulated immune status paired with moderate pathogen load among larger-bodied host phenotypes in western FB and upregulated immunity for smaller-bodied phenotypes in eastern FB. Among the highest immune response sites, a distinct inshore-offshore loading pattern was observed, where coastal basins exposed to freshwater runoff and riverine inputs had the highest pathogen loads, while adjacent offshore locations had the lowest. To explain this, we propose a simple, conceptual model that defines a framework for testable hypotheses based on recent advances in resistance-tolerance theory. We suggest that resource availability has the potential to drive not only plant size, but also tolerance to pathogen load by reducing investment in immunity. Where resources are more scarce, plants may adopt a resistance strategy, upregulating immunity; however, when physiologically challenged, this strategy appears to fail, resulting in high pathogen load. While evidence remains correlative, we argue that hyposalinity stress, at one or more temporal scales, may represent one of many potential drivers of disease dynamics in FB. Together, these data highlight the complexity of the wasting disease pathosystem and raise questions about how climate change and ongoing Everglades restoration might impact this foundational seagrass species.Seagrass wasting disease, caused by protists of the genus  Seagrass meadows form the benthic habitat of some of the most globally significant and ecologically diverse communities in the aquatic realm . As autoAs the impacts of climate change intensify, predictions can be made regarding the persistence of seagrass-dominated communities across time and space . For exaLabyrinthula include a set of opportunistic pathogens that infect seagrasses worldwide with a lesion-forming “seagrass wasting disease” (SWD) that reduces productivity  and immunoassay techniques , morphological , and stress  gradients in Florida Bay (FB), Florida, United States.Protistan stramenopiles of the genus uctivity  and contchniques , we examFlorida Bay is a semi-enclosed estuary bordered by the Everglades and Florida Keys, opening westward into the Gulf of Mexico and eastward through a series of cuts into the Atlantic Ocean . Within T. testudinum monocultures, even within coastal basins.The resultant salinity regime reflects a long history of upstream changes to the Everglades watershed, resulting in altered freshwater, nutrient, and carbon flows into FB . ReductiThalassia testudinum, Halodule wrightii, and Syringodium filiforme  and other physiological challenges presented to FB seagrasses  and pe of FB . Our colpe of FB . HoweverT. testudinum beds was conducted across FB in late May, 2015  for the scope of our investigation as it is considered an important aspect of disease ecology , in coordination with the end of the dry and wet seasons of FB by FHAP, a component of the Monitoring and Assessment Plan (MAP) of Restoration, Coordination, and Verification (RECOVER) within the CERP . Startin ecology . AlthougLabyrinthula sp. in vitro and in planta dynamics . Ideally, all salinity measurements would be obtained from the SFWMD database, as the monitored regions coordinate perfectly with 13 of our 15 study sites . However, as this survey records only monthly values, we substituted in daily salinity data from the SFNRC DataForEver monitoring stations at locations matching 10 of our 15 sample sites to analyze the short-term temporal scale as follows: six of the ENP stations  matched perfectly or were very close to our sampling locations ; another four sites  were located close enough that we considered them a proxy for our sites  10 cm beneath the surface. Our investigation led us to consider whether more local and/or recent historical salinity conditions helped explain an intriguing dichotomy we observed in pathogen loading among a group of sites of the “East” immune class . One site, MAN, is not included in the SFWMD survey, so we extracted values from the SFNRC data (which does sample at this location) for all dates when other sites were sampled.The long-term scale spanned ~4.5 years antecedent to our sampling point (January 2010 to May 2015), and was measured at roughly 1-month intervals. FHAP permanent transects are co-located with the SFWMD stations, so we relied on the SFWMD-DBHYDRO database to capture salinity (psu) measures at the permanent transect sites on a monthly basis, weather permitting. The protocol measures a suite of water quality parameters including salinity Labyrinthula spp. loading in seagrass tissue . Our initial specificity test surveyed 37 Labyrinthula sp. isolates, reliably amplifying 100% of putatively pathogenic strains and only 15% of non-pathogenic strains  relative Cq values of pathogenic vs. non-pathogenic isolates; (2) lower DNA concentrations (per reaction) reflective of field sample pathogen loading; (3) presence of “background” DNA; and, (4) simulation of a scenario where a non-pathogenic phylotype , to better match realistic concentrations in the field. This reflects a cell count greater than the load present in >95% (and within 1 SD of the highest load detected) of our FB-collected T. testudinum samples with detectable levels of the pathogen (this study). Third, we accounted for the possibility that non-specific binding may occur when using pure non-pathogenic Labyrinthula sp. cultures as the only template, so we introduced UltraPure™ Salmon Sperm DNA Solution (Invitrogen™) as background DNA in our diagnostic qPCR assays . Fourth, we evaluated bias for the case of pathogenic types being outnumbered by non-pathogenic. Specifically, we tested pathogenic isolate “8b” and non-pathogenic isolate “98b,” both of which reliably amplified in Labyrinthula sp. culture at 1x concentration ; (2) Labyrinthula sp. DNA equivalent of 25 cells per reaction with salmon sperm DNA to bring template volume to 20 ng total ; and (3) both Labyrinthula sp. DNA loaded in a single reaction at a 1:70 ratio , brought to 20 ng total with salmon sperm DNA. Each reaction was run in duplicate. First, summary information was compiled for the average cycle quantification value  of amplified strains across previous qPCR runs . The resulting eluent was purified using a OneStep™ PCR Inhibitor Removal Kit . Prior to assaying, DNA concentration was quantified Labyrinthula sp. “E” isolate 8b, a strain isolated from and known to readily infect T. testudinum  for use in the standard curve for all qPCR runs.We utilized the same standard curve protocol and template samples as in studinum . Briefly−1 of DNA template, 0.025μM of each primer, 2.7 ng μl−1 of BSA, 1X of iTaq SYBR Green Supermix , and nuclease free water up to 20 μl. Reactions were run in triplicate on a CFX Connect thermal cycler (Bio-Rad) with the following cycle parameters: 5min at 95°C, followed by 45 rounds of 30 s at 95°C and 60 s at 63°C. Reactions were terminated with a melting curve analysis . Results are reported as the number of Labyrinthula spp. cells per mg starting seagrass tissue . See Primers  targeted the ITS region of the ribosomal RNA gene complex . QuantitLabyrinthula spp. cells) and pathogen severity as the number of cells present in a sample (averaged across three qPCR assay replicates) or group of samples (arithmetic mean across samples) in cells per mg dry weight of seagrass tissue.We defined pathogen prevalence as the proportion of individuals infected at a given site  was homogenized and assayed. POX activity was measured using a modified guaiacol assay and presented as the change in absorbance at 470 nm min−1 μg−1 of total crude protein. EXOC activity was quantified using the enzymatic conversion of 4-methylumbelliferyl N-acetyl-β-D-glucosaminide into 4-methylumbelliferone (MU) and expressed as nmol of MU released per μg of total crude protein. PPO activity followed the conversion of L-DOPA to an oxidized dopachrome product and was quantified as a change in absorbance at 490 nm min−1 μg−1 of total crude protein. Finally, LYS activity was measured as the degree to which crude sample extract inhibited growth of Micrococcus luteus, given as percent inhibition per μg of total crude protein added as compared to uninhibited growth of the bacteria.Several common, well-studied immune activity enzymes were recently adapted for use with turtlegrass, including peroxidase (POX), exochitinase (EXOC), polyphenol oxidase (PPO), and a group of enzymes with lysozyme-like activity (LYS). The reader is referred to Activity levels at each of the four biomarkers  were compared among the 15 FB sites using a series of bubble plots. More specifically, bubbles were scaled following a linear relationship between mean sitewide biomarker level and bubble diameter, and then superimposed over a base map of the region to examine spatial patterns with respect to the geography of the bay.Multivariate analyses performed in PRIMER (v6) were utilized to group sites according to their overall immune status . First, we performed a hierarchical cluster analysis (CLUSTER routine), producing a dendrogram of similarity among sites based on immune status. This was followed by a similarity profile routine (SIMPROF), which is a permutation procedure intended to prevent over-dissection of subgroups and assess significance . We also2) per shoot for each of three immune classes. While the original FHAP data set surveyed sites biannually , we detected no significant patterns between seasonal changes in leaf area and our factor of interest (immune grouping), so the results represent yearly averages of the two sampling points. The main box in the plot represents the interquartile range (IRQ) of the data, and the whiskers signify minimum and maximum spread of the data outside of the IRQ.We characterized turtlegrass morphology as a function of immune status by generating box plots to visually summarize mean leaf area (cmα = 0.05) on data that were tested for normality and computed using RStudio software (Z-score conversion in Microsoft Excel (v16.16.19). All error bars represent SEM, unless otherwise stated. Differences between groups were globally tested using a one-way ANOVA, followed by Tukey’s test for post-hoc analysis; alternatively, the non-parametric Kruskal-Wallis test was used when the data could not be transformed to meet assumptions of normality.All statistical tests were performed with 95% confidence intervals , the average (±SEM) Cq value for pathogenic strains  was significantly lower (p < 0.001) than that of non-pathogenic strains .A series of experiments were performed to further demonstrate that our qPCR assay  preferenprovided . HoweverLabyrinthula cells per reaction) and supplemented with background DNA  to deter non-specific binding, the representative non-pathogenic strain failed to amplify in either replicate, while the pathogenic strain amplified readily (results not shown). Finally, both isolate DNA templates were analyzed simultaneously at a 1:70 ratio favoring the non-pathogenic strain and in the presence of background DNA. The amplified product featured a strong melt peak at 76°C, matching the expected peak temperature of 8b, with no indication of a secondary peak corresponding to 98b at 78.5°C . Site prevalence (n = 10 per site) ranged from 0% (DUC) to 100% , was analyzed at both the individual level  loading values among all individuals were 1009.08 and 4.89 × 10−9 cells mg−1, from JOE and CAR, respectively. Further, five of the top 10 most infected samples were collected from JOE; the remaining were from TER (n = 2), LON (n = 2), and BLK (n = 1). Site-wide severity averages ranged from 0.00 to 280.55 cells mg−1  harbored 143.14 ± 36.13 Labyrinthula spp. cells mg−1 tissue; plants in Class 2  had an average of 4.03 ± 1.60 cells mg−1; and finally, individuals belonging to severity Class 1  harbored 0.0017 ± 0.0013 cells mg−1. The overall differences between pathogen loading values across severity groups were highly significant .We further categorized sites into three classes according to their average (relative) pathogen severity for downstream analyses. Four sites  fell under the “high” pathogen loading class, coded as “3”; seven sites  were characterized by “moderate” pathogen loading, coded as “2”; the remaining four sites  had “low” (or zero) pathogen loading and were coded as the number “1” . The aven = 10 per site). The size of the bubble represents relative activity at a given biomarker. Sites significantly differed from one another, overall, at the EXOC, POX, and PPO biomarkers at α = 0.01 (p < 0.0001), but the differences between sites were not significant at the LYS biomarker (p = 0.159). Generally, enzymatic trends followed an east-to-west gradient with low activity in the west and higher activity in the east , and at the LYS biomarker at α = 0.05 (p = 0.042). The three distinct immune classes generated through these analyses  also distributed geographically, and thus further designated with the FB region they grouped in, where: A = “West” sites JON, TWN, LIL, and RKB; B = “Central” sites GAR, RAN, TER, and WHP; and C = “East” sites BLK, EAG, CAR, LON, JOE, DUC, and MAN  across sites clustered into the three immune classes (p < 0.0001); further, pairwise differences were significant between the East class and both the Central and West classes (Tukey’s post-hoc: p < 0.0001), but not between the West and Central classes  and median (20.5 cm2) values pooled across FB , which decreased along the same gradient  activities: lowest in western and central FB and highest in the east  with a lTolerance and resistance are two basic strategies plants have when facing disease: the term “tolerance” describes a host’s ability to minimize the impact of infection on fitness without specifically reducing pathogen load in host tissue, while “resistance” characterizes the plant’s ability to mount a response that actively limits pathogen reproduction . There cLabyrinthula sp. infection, and all four markers were often associated with pathogen defenses , while theory predicts the two may coevolve with one another, and with pathogen virulence, in complex ways . Since tdefenses . We, thedefenses  and consy-axis; y-axis; post-hoc look at several fitness metrics yielded the same basic pattern, consistent with our conceptual model  ends of a resource availability gradient y-axis; , with thal model –D. This al model , reinforLabyrinthula sp. evolution has the potential to predict where and when more virulent strains might emerge, provide a mechanistic explanation for outbreaks, or inform conservation efforts that might target tolerance over resistance  and periods of extreme hyposalinity (Labyrinthula spp. infection (Salinity stress might also play a role in the relative success of resistance vs. tolerance strategies adopted by different turtlegrass populations of FB over longer temporal scales. The 5-year view of monthly salinity trends among sites of the East immune response zone revealed that locations with highest pathogen loads also experienced dramatic fluctuations between moderate saline conditions (i.e., within turtlegrass and salinity . Thus, cnfection . From thLabyrinthula sp., yet, we struggle to predict or describe such outbreaks (Labyrinthula spp. – a term that may reflect more about our lack of knowledge regarding the specifics and/or variety of mechanisms affecting hosts and pathogens (e.g., emergent properties: It has been nearly a century since the massive 1930’s north Atlantic eelgrass wasting events attributed to utbreaks , 2018. TThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Labyrinthula culture collections were carried out by DM. PD acquired samples, curated sample data, and led the formal analysis and investigation, with help from DM, BF, and CR. Environmental data acquisition and multivariate analyses relied on the expertise of BF. Visualization of data was primarily achieved by PD and BF, with input from DM and CR. All authors contributed to the article and approved the submitted version.CR initiated and acquired funding for this project. PD, DM, and CR conceptualized the ideas that drove the manuscript. PD and DM led methodological advancements in the qPCR assay. Pure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is still controversy about the effect of early hypothermia on the outcome of spinal cord injury (SCI). The aim of this review article is to investigate the effect of local or general hypothermia on improving the locomotion after traumatic SCI.Electronic databases (Medline and Embase) were searched from inception until May 7, 2018. Two independent reviewers screened and summarized the relevant experimental studies on hypothermia efficacy in traumatic SCI. The data were analyzed and the findings were presented as pooled standardized mean difference (SMD) and 95% confidence interval (95% CI).20 papers containing 30 separate experiments were included in meta-analysis. The onset of hypothermia varied between 0 and 240 minutes after SCI. Administration of hypothermia has a positive effect on locomotion following SCI . Subgroup analysis showed that general hypothermia improves locomotion recovery , while local hypothermia does not have a significant effect on motor recovery . In addition, general hypothermia was found to affect motor recovery only if its duration was between 2 and 8 hours  and the target temperature for induction of hypothermia was between 32 and 35° C .We found that general hypothermia improves locomotion after SCI in rats. Duration of induction and the target temperature are two essential considerations for general therapeutic hypothermia. Current treatments that are considered to improve spinal cord injury (SCI) outcome include medicinal therapy (e.g. methylprednisolone), surgery, and rehabilitation -3. HowevHypothermia can be classified into three groups, including severe hypothermia below 28°C), moderate hypothermia 28 to 32°C), and mild hypothermia 33-35°C). Early studies have shown the efficacy of local hypothermia in improving neurological complications in animal models -27, and °C, moder°C, and m-35°C. EaStudy design and search strategyThe method of searching databases and performing analyses was the same as the previously published Meta-analyzes by the present researchers -43. Foll1. Spinal Cord Injury / OR Quadriplegia / OR Paraplegia / OR  OR ) OR Quadripleg * OR Paraplegic * OR Tetraplegi * OR Quadripares' s OR ((Trauma * OR Posttrauma *) adj Myelopath *)) ti.ab.2. Cold Temperature / OR Hypothermia / OR Ex Cryotherapy / OR Cryoanesthesia / OR Ex Hypothermia, Induced / OR Body Temperature Changes / OR Gastric Hypothermia / OR (Cryoan? Esthe * OR Cryogen * OR Cryotherap * OR Cryotherm * OR Cryotreat * OR Cold * OR Cool * OR Chill * OR Hypotherm * OR (Temperature adj4 (Decreas * OR Reduc * OR Low * OR Minim * OR Taper *)) OR ((Decreas * OR Reduc * OR Low * OR Minim * OR Taper *) adj4 Temperature) OR "Artificial Hybernation" OR (Refrigerat * adj An? Esthe *)) ti.ab.3. 1 AND 2Eligibility criteriaAll rat studies that examined the effects of general or local hypothermia on locomotion recovery following traumatic SCI  were included. Exclusion criteria consisted of in vitro studies, lack of functional assessment, lack of control group, non-traumatic SCI (aortic cross-clamping model of SCI), mild severity of SCI model, combination therapy of hypothermia with other treatments, not reporting details of hypothermia administration and reviews.Quality assessment and Data ExtractionTwo independent reviewers initially screened titles and abstracts, identified potentially relevant articles, and screened their full texts based on inclusion and exclusion criteria. They independently recorded animals’ age/weight, strain, species and sex, mechanism of the induction of SCI, details of hypothermia induction, number of samples and locomotion score. In animal studies, examination for locomotion recovery is usually conducted in several time sessions, so we only included the last session of follow-up. If the results were presented in the charts, data were extracted using the WebPlotDigitizer software. WebPlotDigitizer is a reliable software for extracting data and its accuracy has been proven in a previous study . SeveritQualitative assessment of papers was performed based on the method suggested in Hassannejad et al. study . DisagreStatistics2 tests were used to evaluate heterogeneity between the studies. In cases with high levels of heterogeneity (I2≥ 50%), subgroup analysis was performed to determine the cause of heterogeneity. Funnel Plot was used to identify publication bias using Egger's test  and 95% confidence interval (95% CI). Ir's test .Studies’ characteristicsThe titles and abstracts of 1677 non-duplicate articles were screened and then full text of 136 articles were selected for in-depth assessment. Finally, the data of the 20 included studies were pooled in a meta-analysis , 48-62 . These sQuality assessment of articles and publication biasThe effect of hypothermia on locomotion recovery2 = 75.4%, p <0.0001). Subgroup analysis showed that general hypothermia improves locomotion , while local hypothermia does not have any effect on motor recovery . The findings of this meta-analysis showed that hypothermia  has a positive effect on locomotion following SCI  , whereas in severe injuries, does not have a significant effect . Level of injury was another factor affecting the influence of general hypothermia. The effect of general hypothermia on locomotion recovery was significant only in case of thoracic injuries . In addition, it was found that general hypothermia could only improve locomotion if its duration was between 2 and 8 hours , but when the duration was less than 2 hours (p = 0.102) or more than 8 hours (p = 0.572), its effect was not significant.for 4 to 7 weeks follow-up = 1.00, p < 0.0001; SMD for 8 weeks and more follow-up = 0.85, p = 0.003)  resulted in motor recovery improvement, while moderate (p = 0.380) and severe (p = 0.571) general hypothermia had no effect on locomotion recovery. Another factor influencing locomotion recovery was the duration of follow-up. Analyses showed that the role of general hypothermia in improving locomotion recovery is significant only when the animals were followed up for at least 4 weeks (SMD = 0.003) .The present study showed that general hypothermia has a positive effect on locomotion recovery following SCI, but local hypothermia did not have any effect on locomotion recovery. General hypothermia's effectiveness in improving motor recovery can be attributed to its angiogenic, neurogenic, and anti-inflammatory effects . FollowiIn a meta-analysis, Batchelor et al. (2013) have demonstrated that general and local hypothermia can improve animal locomotion by about 24.5% and 26.2%, respectively, . HoweverOne of the most important findings of the present study is the effectiveness of mild general hypothermia in improving locomotion recovery. This finding is completely in discrepancy with the Batchelor review, which showed that the highest efficacy was observed when hypothermia was induced at 4 to 19°C. The reason for this controversy can be the difference in the type of analyses. Analyses of Batchelor et al. were limited to the local hypothermia , while wTreatment duration was another factor influencing the effectiveness of general hypothermia. Our findings showed that if the treatment duration was less than 2 hours or more than 8 hours, general hypothermia had no effect on the outcome of SCI, which perhaps suggested a role of a therapeutic window in the effectiveness of an intervention. It is likely that less than 2 hours of induction of hypothermia is not sufficient for the researchers to observe its effects on locomotion recovery, whereas treatment for more than 8 hours may increase the destructive effects of hypothermia on motor function.One of the limitations of the present study was the existence of significant heterogeneity between studies. Although subgroup analysis was performed, in some cases the source of heterogeneity was not recognized. Also, the duration of follow-up varied between studies, which could have had affected the findings. Subgroup analysis showed that the role of hypothermia in recovery was significant only when the animals were followed for at least 4 weeks.We found that general hypothermia improves locomotion after SCI in rat models. Duration of general hypothermia being between 2 and 8 hours, and hypothermia being mild (the target temperature being 32 to 35°C) are two essential considerations for best results in general therapeutic hypothermia.


Accurate diagnosis of early lung cancer from small pulmonary nodules (SPN) is challenging in clinical setting. We aimed to develop a radiomic nomogram to differentiate lung adenocarcinoma from benign SPN.n = 147; malignant nodules, n = 94) and a validation dataset . Radiomic features were extracted from the cancerous volumes of interest on contrast-enhanced CT images. The least absolute shrinkage and selection operator (LASSO) regression was used for data dimension reduction, feature selection, and radiomic signature building. Using multivariable logistic regression analysis, a radiomic nomogram was developed incorporating the radiomic signature and the conventional CT signs observed by radiologists. Discrimination and calibration of the radiomic nomogram were evaluated.This retrospective study included a total of 210 pathologically confirmed SPN (≤ 10 mm) from 197 patients, which were randomly divided into a training dataset , accuracy of 81.0%, sensitivity of 82.9%, and specificity of 77.3%. The two conventional CT signs achieved an AUC of 0.833 (95% CI: 0.707–0.958), accuracy of 65.1%, sensitivity of 53.7%, and specificity of 86.4%. The radiomic nomogram incorporating the radiomic signature and conventional CT signs showed an improved AUC of 0.857 (95% CI: 0.723–0.991), accuracy of 84.1%, sensitivity of 85.4%, and specificity of 81.8%. The radiomic nomogram had good calibration power.The radiomic nomogram might has the potential to be used as a non-invasive tool for individual prediction of SPN preoperatively. It might facilitate decision-making and improve the management of SPN in the clinical setting. The most common cause of cancer death around the world is the lung and bronchus according to the 2017 cancer statistics . PatientRecently, by high throughput extracting quantitative imaging features from standard-of-care medical images, radiomics provides us a promising and non-invasive tool in cancer research , 10. TheTherefore, the aim of this study was develop and validate a radiomic nomogram for the individual preoperative prediction of lung adenocarcinoma from benign SPN, which would improve the decision-making of SPN in clinical practice.Our institutional review board approved this retrospective study and waived the need for informed consent from patients. A total of 197 patients with 210 SPN treated with surgical resection were included from January 2011 to March 2017. Inclusion criteria were as follows: (1) Patients had histopathologically-confirmed SPN ≤10 mm; (2) Patients had available clinical data; (3) Patients underwent baseline lung CT scan with the same imaging parameters and reconstruction slice thickness; and (4) Patients’ lung CT performed within 1 month before surgery. The patients were excluded if: (1) Patients received surgery before CT scans; and (2) Patients’ lung CT images have breathing artifacts. The patients were randomly divided into training and validation sets by a computer algorithm at a ratio of 7:3. Figure A total of 11 CT findings of each nodule were collected from the last CT scan before surgery, including the maximum diameter, location, involvement of pleura , nodule consistency , shape , margins , cavity (presence or absence), calcification (presence or absence), intranodular changes , bronchial disruption , and vessel convergence sign (presence or absence). Two radiologists with 13 years and 18 years of clinical experience in lung cancer reviewed all of the CT images and reached a consensus.Contrast-enhanced CT images were obtained by a 64-slice CT scanner . The imaging parameters were as follows: 120 kV; 120 mA; rotation time = 0.5 s; detector collimation = 64 × 0.625 mm; the field of view = 500 mm; and matrix size, 512 × 512. All patients received intravenous administration of iodinated contrast agent . The CT images were obtained after a 30 s delay and reconstructed with a slice thickness of 2 mm.www.itk-snap.org) and validated by a senior radiologist. Radiomic features were extracted from contrast-enhanced CT images by using an in-house feature extraction algorithm applied in Artificial Intelligence Kit software that developed by GE Healthcare Life Sciences. It can be combined with ITK-SNAP software to obtain three dimensional images. A total of 385 radiomic features consisting of form factor features, histogram features, and textural features . The description of feature extraction algorithms are presented in Figure We applied the least absolute shrinkage and selection operator (LASSO) regression to select the most significant features suggestive malignancy . We perfTo determine the additional value of radiomic signature to conventional CT features, we developed and compared three models . Conventional CT signature was built based on the results of multivariate logistic regression analysis of 11 conventional CT features. Radiomic signature or radiomic score (Rad-score) was calculated by linearly fitting the selected radiomic features after weighted by their respective coefficients. Finally, radiomic nomogram was constructed by a multiple logistic regression using the selected conventional CT features and Rad-score.The area under the receiver operating characteristic curve (AUC), accuracy, sensitivity, and specificity were used to evaluate the performance of the three models in the validation dataset. Calibration curve and the Hosmer-Lemeshow test were used to assess the calibration and goodness-of-fit of the radiomic nomogram .P < 0.05 was considered significant.All the statistical analyses were performed using R software (version 3.4.2). The packages were used as follows: “glmnet” for LASSO logistic regression, “rms” for nomogram and calibration plots, and “vcdExtra” for Hosmer-Lemeshow test. Differences of patient and nodule characteristics between the training dataset and validation dataset were compared using Chi-square test or Fisher’s exact test or Mann–Whitney U test, if appropriate. The AUC comparison of different models used Delong test. A Table A total of 385 radiomic features were extracted from each volume of interest of the nodules on contrast-enhanced CT images. Five features with non-zero coefficients were selected by LASSO   Fig. .Fig. 4VP < 0.001, Fig. P < 0.001, Fig. The radiomic signature achieved an AUC of 0.878 (95%CI: 0.813 to 0.943), accuracy of 85.0%, sensitivity of 90.1%, and specificity of 76.8% in the training dataset Table  and an AP < 0.001 and P = 0.026, respectively). The two CT features attained an AUC of 0.842 (95%CI: 0.779 to 0.906), accuracy of 73.5%, sensitivity of 62.6%, and specificity of 91.1% in the training dataset and an AUC of 0.833 (95%CI: 0.707 to 0.958), accuracy of 65.1%, sensitivity of 53.7%, and specificity of 86.4% in the validation dataset .After multivariate analysis, only two CT findings (nodule consistency and margins) remained . The calibration curve of the radiomic nomogram is shown in Fig. P values of 0.738 and 0.111 in the training and validation datasets, respectively, which indicated good calibration power.A radiomic nomogram incorporating radiomic signature, internal composition and margins of nodule was constructed Fig. a. The raWe trained and tested a radiomic nomogram based on the radiomic signature and the anatomical CT features for individualized preoperative prediction of lung adenocarcinoma, which showed good discriminative power and calibration. This study indicates that CT-derived radiomic features supplement the CT findings reported by radiologists in the prediction process. Note that, this study provides a non-invasive and effective prediction tool to determine those patients with a high probability of lung adenocarcinoma.Early diagnosis of cancer is associated with prolonged survival , for insTo date, radiologists diagnose lung cancer by largely depending on qualitative features of CT images, such as nodule diameter, evidence of spiculation, upper lobe location, and pleural indentation . Low-dosRecently, the increased training of medical image analysis and tools has driven additional studies investigating the radiomics of lung cancer. Radiomic signatures may help to mining bioinformatics behind lung cancer on medical image, for instance, tumor staging , gene exA number of risk models have been developed, of varying complexity for identifying risk of incident lung cancer among patients with visible lung nodules –38. The In summary, this study showed the potential of radiomic features extracted from unenhanced CT images for predicting lung cancer before surgery. Radiomic features showed the added value to the conventional CT features in differentiating lung adenocarcinoma from benign SPN. This study provides doctors a radiomic nomogram as a non-invasive tool for individualized prediction of lung cancer preoperatively. However, before applying in real-world setting, more studies are needed to validate the performance of the radiomic nomogram.Additional file 1.