Fruits of persimmon plants are traditional healthy food in China, Korea and Japan. However, due to the shortage of morphological and DNA markers, the development of persimmon industry has been heavily inhibited.Diospyros cathayensis, D. virginiana, D. rhombifolia and D. deyangensis were newly sequenced. Comparative analyses of ten chloroplast genomes including six previously published chloroplast genomes of Diospyros provided new insights into the genome sequence diversity and genomic resources of the genus. Eight hyper-variable regions, trnH-psbA, rps16-trnQ, rpoB-trnC, rps4-trnT-trnL, ndhF, ndhF-rpl32-trnL, ycf1a, and ycf1b, were discovered and can be used as chloroplast DNA markers at/above species levels. The complete chloroplast genome sequences provided the best resolution at inter-specific level in comparison with different chloroplast DNA sequence datasets.Chloroplast genomes of Diospyros oleifera, D. deyangensis, D. virginiana, D. glaucifolia, D. lotus and D. jinzaoshi are important wild species closely related to the cultivated persimmon D. kaki. The hyper-variable regions can be used as DNA markers for global genetic diversity detection of Diospyros. Deeper study on these taxa would be helpful for elucidating the origin of D. kaki.The online version of this article (10.1186/s12870-018-1421-3) contains supplementary material, which is available to authorized users. Diospyros is the largest genus in the family Ebenaceae, including more than 400 species all over the world, with a wide distribution and cultivation from tropical to temperate zones. Their fruits have been traditional healthy food source in China, Korea and Japan. Ebony trees are valued for their hard, heavy and dark timber. In addition, the bark, leaves, wood, fruits and seeds of several species are the main sources of medicines. Diospyros kaki is an important economic tree crop and widely cultivated species of the genus [he genus , 2.Diospyros kaki) for edible fruits and Chinese medicine is commonly one of the best choices for regional development of characteristic fruit tree industry, with different regions having different cultivars. At present, there are about 1000 persimmon cultivars in China. Most of the extant persimmon cultivars are elite plants from natural seedlings or bud mutation. Among these cultivars, a majority is of the pollination- constant and astringent (PCA) type, pollination-constant and non-astringent (PCNA) type of premature persimmon and disease-resistant cultivars are rarer and are highly desirable. In general, closely related wild species possess elite genetic background for qualitative improvement and high-yield breeding of the persimmon crops [In China, cultivation of persimmon plants  according to their chromosome number [Diospyros plants worldwide is still a global challenge [The germplasm resources of e number . Howeverhallenge –12.Diospyros, molecular resources have been developed in recent years for species identification. Some chloroplast genome markers  and nuclear DNA markers  were used to discriminate plants in Diospyros. However, these markers are low in variability, or have limited resolution and cannot reach the goals [Diospyros plants.In he goals , 13–18. The angiosperm chloroplast genomes are uniparentally inherited with relatively stable structure , 17. AccD. cathayensis, D. deyangensis, D. rhombifolia and D. virginiana. The aims of our study are: (1) to evaluate the variation in Diospyros; and (2) to develop new and efficient cpDNA markers for species identification in Diospyros.In this study, we report newly sequenced complete chloroplast genomes of Diospyros taxa were collected in spring 2016 from the Beijing Botanical Garden of Chinese Academy of Sciences and the National Field Genbank for Persimmons of College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China and were dried immediately using silica gels for DNA extraction. Total genomic DNAs were extracted following the procedure of Plant Genomic DNA Kit (DP305) from Tiangen Biotech (Beijing) Co., Ltd., China.The fresh leaves of the Diospyros kaki (GenBank accession No. KT223565) as a reference sequence, we selected chloroplast genome contigs using the BLAST method. The contigs of the chloroplast genomes were assembled using Sequencher (v5.4) with default parameters. Ambiguous nucleotides or gaps and the four junction regions between the IRs and SSC/LSC in the chloroplast genome sequences were further confirmed by PCR amplification and Sanger sequencing with specific primers or the universal primers [DNA was sheared to fragments of 400–600 bp with an ultrasonic disruptor. An Illumina paired-end library was constructed with the NEBNext® Ultra™ DNA Library Prep Kit according to the manufacturer’s protocol. Paired-end sequencing (2 × 150 bp) was conducted on an Illumina HiSeq 4000 platform. The high-throughput sequencing data were qualitatively assessed and assembled using SPAdes 3.10.1 . Using t primers . After t primers  to avoid primers . The chl primers .http://pgrc.ipk-gatersleben.de/misa) was used to detect simple sequence repeats (SSR) within the chloroplast genomes. Thresholds for a minimum number of repeat units were established as follows: > 10 for mono-nucleotide, > 5 for di-nucleotide, > 4 for tri-nucleotide, and > 3 for tetra-nucleotide, penta-nucleotide, or hexa-nucleotide SSR. Repeating sequences were scanned over the complete chloroplast DNA sequences, taxon by taxon, using the REPuter program.MISA , Maximum Likelihood (ML) and Bayesian Inference (BI) using the entire chloroplast genome.MP analyses were performed using PAUP v4b10  describeDiospyros  using an Illumina Hiseq 4000 system, 20,675,288 to 33,584,779 paired-end raw reads were obtained (average read length was 150 bp). After mapping the paired-end reads of each Diospyros taxon, 357,721 to 1,338,624 reads were extracted, yielding 340 × to 1271 × coverage .Previous experiments indicated that chloroplast genome sequences are identical among individual plants of the same species. Therefore, we sequenced four taxa of Diospyros investigated in this study ranged from 157,300 base pairs (D. jinzaoshi) to 157,934 base pairs (D. deyangensis) in length. All the chloroplast genomes possessed the typical quadripartite structure of angiosperms, consisting of a pair of the inverted repeat region  separated by a large single-copy region  and a small single-copy region   to 82 (D. jinzaoshi) among the ten Diospyros taxa. The homopolymer repeat number with the highest variability, ranged from 29 (D. cathayensis) to 70 (D. jinzaoshi), while the number of di-nucleotide, tri-nucleotide or penta-nucleotide repeats had no significant difference among the ten Diospyros taxa  ranged from 48 (D. kaki) to 22 (D. deyangensis), which were found to have no significant difference among the ten chloroplast genomes. Tandem repeat number ranged from 20 (D. jinzaoshi) to 29 (D. virginiana). Palindromic repeat number ranged from 18 (D. discolor or D. virginiana) to 33 (D. kaki). The highest value of palindromic repeat number which occurred in D. kaki is significantly different from that of other taxa. The highest tandem repeat number was observed in D. virginiana  of nucleotide substitution number occurred between D. cathayensis and D. virginiana, showing a wider range of variability according to the sequence alignment of the ten chloroplast genomes  of nucleotide substitution number was observed between 1 of nuclDiospyros taxa ranged from 0.0002 to 0.0092. The smallest sequence distance occurred between D. lotus and D. glaucifolia, and the largest sequence distance occurred between D. virginiana and D. cathayensis  were able to discriminate all the taxa investigated in this study.According to the chloroplast genome sequence alignment of the ten red Fig. . These erbcL and matK) achieved only 40% and 80% of discrimination success respectively  are considered to be the primary mechanisms affecting length variation of angiosperm chloroplast genomes. Only minor variations were detected at the SC/IR boundaries of the ten Diospyros chloroplast genomes. Occurrence of indels was the main factor effecting the variation of the length in Diospyros chloroplast genomes. Similar to previously published chloroplast genomes of the angiosperms, the Diospyros chloroplast genomes contained more AT content than GC content.In this study, we sequenced four chloroplast genomes of Diospyros chloroplast genomes were identified. The number of SSRs ranged from 48 to 82 in Diospyros, similar to those in Lagerstroemia [Simple sequence repeats (SSRs) are genetically variable molecular markers and are used widely in population genetics , 42, polstroemia . SSRs wistroemia , 45. HomDiospyros is the largest genus in its family, including more than 400 species all over the world. For effective exploration, conservation, and domestication, accurately identified wild species would provide a clear genetic background of this genus. However, the taxonomic inventory of Diospyros still has a long way to go, because of the vast amount of species with extensive global distribution and the plasticity of the morphological characteristics. DNA barcoding has been largely used as a new biological tool to facilitate accurate species identification [rbcL and matK, are recommended as core universal DNA barcodes in plants. Therefore, genomic comparative researches of more complete chloroplast genome sequences have become necessary for developing variable DNA barcodes. These mutation “hotspot” regions could provide adequate genetic information for species identification, and can be used to develop novel DNA barcodes [trnH-psbA, rps16-trnQ, rpoB-trnC, rps4-trnT-trnL, ndhF, ndhF-rpl32-trnL, ycf1a, and ycf1b) identified in this study could be suitable barcodes for plant classification in Diospyros. In previous study, ycf1 gene showed high divergence in chloroplast genome and was recommended as core DNA barcode for plants [. Ycf1 gene has been more and more widely applied in plant phylogeny and DNA barcoding studies [. TrnH-psbA, ndhF and rps16-trnQ were popular candidates for phylogenetic studies [. Rps4-trnT-trnL and ndhF-rpl32-trnL were newly identified in this study.fication . The twobarcodes . The eigr plants . Ycf1 ge studies –51. TrnH studies , 53. RpsDiospyros  is equal to the sum of the chromosome numbers of D. deyangensis (2n = 4× = 60) and D. oleifera (2n = 2× = 30). This strongly implied that cultivated D. kaki might be resulted from a superposition event of genetic materials between D. deyangensis and D. oleifera through certain genetic mechanism during the evolutionary history of Diospyros plants. D. oleifera is naturally distributed south of the Changjiang River in China, has strong adaptability and resistance to wet condition, and has been used as rootstocks of D. kaki in southern China. Its fruits can also be used for extraction of persimmon paint. D. deyangensis, naturally distributed in Sichuan Province, China, produces red flowers and has fine hairs on the surface of the leaves, shoots, fruits, and some parts of the flowers [The DNA evidences obtained in this study clearly support the previous taxonomic proposals that  numbers , 9–12. I flowers .Diospyros rhombifolia and D. cathayensis are genetically remote from D. kaki, being identical with the classification based on phenotypic characteristics. D. rhombifolia could not be used as rootstocks or inter-stocks because of its dwarfing effect [D. glaucifolia and D. lotus were clustered together based on cpDNA sequences. They are very similar in phenotypic characteristics [D. lotus has made great contributions as rootstocks in persimmon production areas of northern China because of its highest cold resistance in the genus. The small fruits of D. lotus have been used as food and Chinese medicine with a long history. In southern China, the dioecious species D. glaucifolia is used to produce wood and could also be used as rootstocks. The species boundary between D. lotus and D. glaucifolia should be further studied by sampling more individual plants in future [g effect . D. glaueristics , 6, 55. n future .Diospyros, and further accelerating directive breeding of Diospyros plants.Elucidation of the questions mentioned above would definitely improve our understanding on phylogeny, relationship, and the origin of the cultivated persimmons in Diospyros plants at inter-specific level. The results obtained in this study implied that D. oleifera, D. deyangensis, D. virginiana, D. glaucifolia, D. lotus and D. jinzaoshi are important wild species closely related to the cultivated persimmon D. kaki and deeper studies on these taxa would be helpful for understanding the origin of D. kaki. Our results have significant value for global genetic diversity assessment, phylogeny, and population genetics in Diospyros in future.Chloroplast DNA sequences can be used for classification of Additional file 1:Figure S1. NJ trees of Diospyros taxa based on each of the ten chloroplast barcodes, showing the resolutions of each different locus for revealing the phylogeny. The figures above the lines are the bootstrap support values for the clades. (PDF 506 kb)


Satureja hortensis L. (summer savory) is an annual herbaceous crop, native to Europe and in our days spread and used all over the world. Although its use as spice and medicinal plant is known since ancient times, peer-reviewed studies presenting the scientific data are scarce. The natural products obtained from summer savory  are dominated by polyphenols and flavonoids, responsible for their antioxidant, antimicrobial, antiparasitic, pesticidal, anti-inflammatory, analgesic, hepatoprotective and anticancer properties, among others. The current study presents the progress made in the last decade regarding the potential applications of summer savory, being the first review study focused on S. hortensis, in the same time suggesting future research opportunities, as they appear from the properties of other Satureja species. The available data presenting the properties of summer savory represents a scientific support for application in industry, for developing “clean label” food products. Satureja hortensis L.) represents an annual herbaceous crop species, strongly branched, with linear leaves belonging to the Lamiaceae family. The flowers, purple or violet white with red peeled on the inside, are grouped at the upper nodes of the branches [Summer savory  matched the keyword search “Satureja hortensis effect” , protein (4.2%), fat (1.65%), sugar (4.45%), fibre (8.60%) and ash (2.11%) [The chemical composition of summer savory was revealed in several studies performed on oisture 7%, protei2%), fat .65%, sugp-cymene (3.5–19.6%) [The volatile oil isolated from summer savory has as major components carvacrol, thymol, phenols, and flavonoids . Severalp-cymene .5–19.6% 5–19.6%) ,11,12. D5–19.6%) . Thus, wS. hortensis represents the subject of significantly fewer studies, when compared with the essential oils. The methanolic extract obtained by maceration was proven to be dominated by rosmarinic acid (24.9 mg/g), caffeic acid (1.3 mg/g), naringenin (1.1 mg/g), isoferulic acid (220 µg/g) and apigenin (165 µg/g). Other flavones (luteolin) and their glycosides (apigetrin and vitexin), as well as flavonol (quercetin), flavonol glycosides  and coumarin derivatives (aesculin and aesculetin) were also detected [p-cymene, β-phellandrene, γ-terpinene and carvacrol. Their conclusions were that due to several mechanisms presented by the authors, the quality of the essential oil increased with storage time . The conclusion is of particular interest for industrial applications, in which the storage times are extensive.The extracts obtained from aerial parts of detected . A compadetected . Rosmariapigenin 5 µg/g. Odetected  evaluateS. hortensis plants revealed that the phenolic compounds increased upon salinity stress, a non-enzymatic defense mechanisms against salinity-induced oxidative stress; a similar increase being observed for the essential oil quantity and quality  to chitosan nanoparticles offered the nanoparticles antioxidant properties , in direct correlation with the EO content [S. hortensis samples, Samadi et al. [50 values obtained by the DPPH assay to the value resulted using the positive control BHT) between 0.17 and 0.46. The authors assign the antioxidant activity registered to the presence of p-cymene, carvacrol and β-bisabolene.Due to the presence of polyphenolic compounds,  studies . Commerc studies . The add content . A simil content  upon thei et al.  obtainedS. hortensis were also proven to have antioxidant properties: Mašković et al. [S. hortensis was evaluated for antioxidant properties, by comparison with Artemisia dracunculus extract [S. hortensis extract, in good correlation with the higher total phenolic and total flavonoid content registered.The extracts obtained from ć et al.  compared extract . ResultsS. hortensis aerial part against cisplatin-induced oxidative damage in liver, kidney, and testes in rats, regulating levels of superoxide dismutase enzyme (SOD), catalase enzyme (CAT), glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) affected by the administration of cisplatin.The in vivo study published by Boroja et al.  revealedS. hortensis  are also well-known for their antioxidant potential [Considering the main components identified in summer savory essential oil and literature survey, the SHEO antioxidant activity could be attributed to the high content in carvacrol, γ-terpinene, p-cymene and thymol compounds with known antioxidant activity ,25. In totential ,27.S. hortensis are currently considered for use in meat industry (the water leaf extract increased the shelf-life of ground beef) [Due to the antioxidant potential shown, natural extracts obtained from nd beef)  or as annd beef) .Candida glabrata to 8 µL/mL for Pseudomonas aeruginosa and minimal lethal concentration (MLC) values ranging from 0.06 µL/mL for Candida glabrata to 16 µL/mL for Pseudomonas aeruginosa [P. aeruginosa [p-cymene indirectly increasing their effect. The antimicrobial mechanism is presented , in the same time evaluating the individual EO’s components antimicrobial properties.The presence of monoterpenes such as carvacrol, cymene and thymol in the essential oil indicates the high possibility of antimicrobial activities against food, plants and human pathogens . The evaruginosa . The resruginosa . The autS. aureus, E. coli, B. cereus, S. typhimurium and P. aeruginosa (in decreasing order of the inhibition zone diameter), the films being more effective in direct contact [E. coli, L. monocytogenes and S. aureus) obtaining positive results for materials having different EO content, in a concentration dependent manner. The authors avoid assigning the antimicrobial effect to a particular compound, but rather assign the effect to the synergistic effect of different components in the EO. The SHEO exhibited a notable inhibitory effect against several bacteria  with controlled release when incorporated in alginate microparticles [The addition of SHEO to κ-carrageenan films conferred on the materials antimicrobial properties against  contact . The sam contact  regardinarticles .L. innocua, P. fluorescens and A. hydrophila/caviae (in disc diffusion experiments) and against L. innocua and A. hydrophila/caviae (in volatile diffusion experiments) [Spraying SHEO obtained from Iranian plants  had a positive effect on the preservation of kiwi fruits in the post-harvest stage, by reducing fungal rot . Belgianriments) .p-cymene (12.3%), α-terpinene (2.0%), and thymol (2.0%). The exposure to SHEO vapors reduced the decay of strawberry and grape fruit in post-harvest stage, especially at low temperatures (5 and 10 °C), in a dose-dependent manner. Similar results were obtained on grapes at post-harvest stage [Penicillium digitatum and Rhizopus stolonifer) [R. stolonifer, P. digitatum, Aspergillus niger and Botrytis cinerea [Aspergillus species [Rhizopus stolonifera [SHEO was obtained by Dikbas et al.  from wilst stage , as welllonifer) , R. stol cinerea , Aspergi species  and Rhizlonifera . The obtSaharkhiz et al.  studied Salmonella enterica suggested the potential of S. hortensis natural products for the application in liquid whole eggs preservation [E. coli inoculated on freshly cut cucumber and tomato suggested its potential application for fresh-cut product sanitation [SHEO obtained from Algerian plants tested on ervation , while tnitation .S. hortensis plants  showed antimicrobial activity against 10 Pseudomonas species isolated from freshwater fish, with MIC values ranging from 6.25 µL/mL against P. antarctica to 25 µL/mL for P. koreensis, P. mandelii and P. proteolytica [Essential oil obtained using an industrial method from Slovakian eolytica .Candida albicans strains  isolated from HIV-positive patients, with MIC values ranging from 200 to 400 µL/mL.Evaluation of the antimicrobial potential of SHEO on human pathogens revealed a strong antimicrobial activity of oils obtained from Turkish plants against 15 periodontal pathogens  also exhS. hortensis. The antifungal efficiency tested against two aflatoxigenic moulds Aspergillus parasiticus, revealed an increase with the exposure of the extract to low radiation doses (1.2 kGy), followed by the decrease with higher doses (3 and 5.1 kGy). The authors assign the decrease of the antifungal activity with the high irradiation doses to decreases or structural changes in the phenols, carvacrol and thymol contents of the irradiated extracts.Gumus  reportedS. hortensis against fourteen plant pathogenic bacteria by comparison with pure compounds (carvacrol and thymol). The EO showed a good antimicrobial activity against all tested strains , while the extracts had a weak activity against a limited number of pathogens. The pure compounds had MIC values ranging from 15.63 to 31.25 µL/mL. In the same time, the experiments performed on lettuce seeds showed that the hexane–methanol extract mixture can be used as a seed disinfectant and as potential control agents for management of bacterial diseases.Kotan et al.  determinThe extracts obtained by Mašković et al.  from SerSatureja family possesses antiparasitic properties [S. hortensis is very scarce. Hezarjaribi et al. [S. hortensis alcoholic extracts on Giardia cysts, with a mortality of 84.3%. Similar results were obtained by Soosaraei et al. [S. hortensis natural products as an antiparasitic agent against Leishmania major.Although several plants from the operties , the liti et al.   presenti et al. , citing i et al.  regardinCulex quinquefasciatus Say (Diptera: Culicidae). From the study, covering twenty-two aromatic plant species, resulted that the SHEO had a good larvicidal activity , with a total mortality of 75% and an 100% deterrence of female oviposition for a concentration of 0.02% EO [The SHEO obtained by hydrodistillation using plants collected from the Czech Republic (with a composition dominated by carvacrol and γ-terpinene) was studied for larvicidal activity against 0.02% EO .p-cymene and thymol) was evaluated as a natural insecticide against broadbean weevil (Bruchus dentipes) [S. hortensis and Origanum acutidens correlated with their composition, the authors assign the toxic effect to carvacrol, in the same dime drawing attention to the potential synergistic and antagonistic effects of other compounds.SHEO obtained from Turkish vegetal material  . The resSitophilus granaries of eleven plant species from Turkey, at four different concentration, obtaining 31.00–33.00% mortality, after 24 h, at concentrations between 1–20 μL oil.Yildirim et al. , evaluatp-cymene) were tested in terms of acute toxicity, by measuring the mortality after 24 h, by application to the larvae of Spodoptera littoralis, Musca domestica, Culex quinquefasciatus, and Leptinotarsa decemlineata and to the adults of M. domestica and L. decemlineata. Summer savory natural products  were active against all tested insects, the most sensitive being the larvae of Colorado potato beetle, with a LD50 of 22 µg.Sajfrtova et al.  studied S. hortensis Iranian plants  was successfully encapsulated in chitosan/TPP (tripolyphosphate) nanoparticles by Ahmadi et al. [Tetranychus urticae Koch. The results indicated that encapsulation enhanced the SHEO fumigant toxicity after 24 h with the LC50 values significantly higher than the pure EO. This approach increased the longevity of lethal activity, with a mortality of 82% after 14 days, compared with the lethality of pure SHEO, reduced to 12% after the same time period.Essential oil obtained from i et al. . Having S. hortensis essential oil as a natural herbicide was recently presented by Hazrati et al. [Amaranthus retroflexus and Chenopodium album (at a nanoemulsion concentration of 1 mL/L in laboratory and 4 mL/L in greenhouse conditions), two weeds with world-wide spread.The potential application of i et al. . The SHES. hortensis represents the subject of several studies presenting its anti-inflammatory and antinociceptive potential.Inflammation represents a defence appearing as a response to pathophysiological problems . The seaS. hortensis roots . Using acetic acid-induced writhing test, formalin licking test and carrageenan-induced paw edema, the authors observed the inhibition of the abdominal writhes of all products, reduction of the licking time in the acute formalin test by the hydroalcoholic extract and by all materials in the chronic phase, as well as the anti-inflammatory potential of the EO (400 µL/kg), hydroalcoholic extract (100 and 200 mg/kg) and polyphenolic extract (400 mg/kg). Vafaei et al. [Hajhashemi et al.  presentei et al.  obtainedS. hortensis determined by formalin and tail flick test; the anti-inflammatory potential of summer savory are also presented by other review papers [The review paper of Bahmani et al.  presentsw papers ,5,6,61.S. hortensis extract was presented by Boroja et al. [S. hortensis extract were similar regarding the regulation of the hepatic serum parameters similar to silymarin . Although good anti-diabetic and anticholesterolemic properties were reported for other Satureja species [S. hortensis products in the time range reviewed. This could be an opportunity for future studies, as the EO and extracts are rich in carvacrol and thymol (responsible for lowering the serum cholesterol levels) and in flavonoids (possessing antioxidant and anti-hyperlipidemic properties) [The hepatoprotective role of a et al.  for trea species ,6,30, thSatureja species exhibited anticancer potential: S. intermedia EO on oesophageal squamous cell carcinoma and human bladder carcinoma cell lines, S. spicigera on HT29/219 (Rectosigmoid adenocarcinoma cells), Caco2 , NIH-3T3 (mouse embryo fibroblast cell) and T47D  lines, S. sahendica EO on MCF7 (breast cancer cells), Vero (fibroblast-like kidney cells), SW480 (colon adenocarcinoma cells) and JET 3 (choriocarcinoma cells) lines, S. montana on HT29 (colon adenocarcinoma cells) line, etc. [The study of Misharina et al.  presentene, etc. .Ceker et al.  studied S. hortensis natural products such as antiviral properties  [Several review papers from the last decade present other activities of V virus) ,64,65, iV virus) ,30, antiV virus) , anti-asV virus) , expectoV virus) , emmenagV virus)  or for sV virus) .S. hortensis on tracheal smooth muscles contracted by methacholine and KCl were reviewed by Shakeri et al. [The muscle relaxant properties of i et al. , the relAlthough the original research articles do not match our search criteria (studies published in the last decade), we consider that the properties should be mentioned, to provide a better image regarding the potential applications of this understudied source.Satureja species, such as anti-Alzheimer disease agent, anti-leishmanial, anti-protozoal, trypanocidal, etc. [Other potential applications remain to be studied, considering the known effects of other al, etc. ,72.S. hortensis use were discovered in a Roman colony in Northern Italy established in 183 BC [Summer savory represents an aromatic plant known and safely-used since ancient times. For example, evidences regarding n 183 BC . When diHerbes de Provence ; the extensive use as a spice, with no reported harmful effects suggests that summer savory it is safe for consumption as a spice.As spice, the distinctive taste of summer savory led to its world-wide use and cultivation. The leaves are used as seasoning in stuffing and meat dishes, especially in Europe, while the sprigs can be boiled with peas or cabbage for improving digestion . It is oAs a medicinal plant, summer savory can be consumed either as hot tea or cold infusion and, more recently, in tablet form. As infusion, it can be prepared from two teaspoons of herb to a cup of water, 2–3 cups a day . As a reSummer savory essential oil is considered safe for human consumption by the U.S. Food and Drug Administration . As everS. hortensis found application in meat industry [p-cymene and thymol), SHEO could offer an alternative for increasing the shelf life of meat products or vegetables [More recently, based on the properties presented in the previous chapters, natural products obtained from industry , food anindustry , food paindustry  and fresindustry . Thus, dgetables ,82,83,84getables ,82,83,84S. hortensis leaf extract found application in nanotechnology. Using aqueous leaf extracts obtained from plants cultured under different salinity conditions, Rasaee et al. [Bacillus subtilis, Bacillus vallismortis and Escherichia coli [More recently, e et al.  phytosyne et al. . This wae et al. . The obthia coli .Satureja hortensis L.) is known and used since ancient time as herbal remedy, spice and food flavoring. Original from Europe, it is now grown and used in most parts of the world, in several industries. With a composition dominated by polyphenols and flavonoids, the natural products obtained from summer savory were proven to have antioxidant, antimicrobial, antiparasitic, pesticidal, anti-inflammatory, analgesic, hepatoprotective and anticancer properties, among others. The literature survey demonstrates that the chemical composition of the natural products is changed significantly with the cultivation and geographical conditions, as well as with the stages of development at harvesting time.Summer savory (S. hortensis, although the different uses demonstrated for other Satureja species would suggest the exploration of new applications. This, in turn, would be a promising research area for future studies.The current study aimed to present the progress made in the study of this understudied plant in the last decade. According to our findings, most of the literature data focuses on antioxidant and antimicrobial potentials of Exploration of new application could result in the development of new natural alternatives for serious illnesses, such as cancer, Alzheimer, cardiovascular diseases and many others. In the same time, application of already established properties at industry scale would provide alternatives for “clean label” food products.


Acinetobacter species have emerged as one of the most clinically important pathogens. The phenotypic techniques which are currently available are insufficient in accurately identifying and differentiating the closely related and clinically important Acinetobacter species. Here, we discuss the advantages and limitations of the conventional phenotypic methods, automated identification systems, molecular methods and MALDI-TOF in the precise identification and differentiation of Acinetobacter species. More specifically, several species of this genus are increasingly reported to be of high clinical importance. Molecular characterization such as of blaOXA-51-like PCR together with rpoB sequencing has high discriminatory power over the conventional methods for Acinetobacter species identification, especially within the Acinetobacter calcoaceticus–Acinetobacter baumannii complex. Acinetobacter species are considered to be one of the most important pathogens and associated with increased mortality. The species within the Acinetobacter calcoaceticus–Acinetobacter baumannii complex have emerged as high priority pathogens, especially in intensive care units, thereby posing a challenge to infection management practices. However, identification of Acinetobacter to the species level is difficult. Clear differentiation among various Acinetobacter species with available standard biochemical methods and automated systems is challenging. Although various molecular methods are available, they are not regularly used in diagnostic laboratories. The advantages and disadvantages of different methods useful in the accurate identification of Acinetobacter species are discussed in this review. Acinetobacter includes a heterogeneous group of bacteria that have been considered ‘the most troublesome’ by clinicians and scientists ). The following sections briefly describe each of the methods.For the molecular identification of Acinetobacter strains to five distinct DNA groups with four strains ungrouped [A. radioresistens [DDH is a gold standard method where the whole genome of the organisms is compared for delineation of bacterial species. Johnson and colleagues assigned ngrouped . Bouvet ngrouped . Bouvet ngrouped . A paralngrouped . In 1988esistens .Although DDH has been considered a gold standard for bacterial species differentiation over the past 50 years, due to several disadvantages like a labor-intensive and error prone nature, genotype-based technology was needed . As wholThe OGRI is digital DDH-like similarity indices based on computational comparisons of two genome sequences . Variouset al., using the genus Acinetobacter, examined whether bacterial taxonomy could rely upon genome-based sequence data using ANI [A. nosocomialis NCTC 10304 exhibits ANI values more than 95% with 14 sequenced A. baumannii, thereby confirming the designation as A. baumannii NCTC 10304. Similarly, A. calcoaceticus PHEA-2 was designated as A. pittii PHEA-2 [Acinetobacter [Chan sing ANI . For exai PHEA-2 . Since ttobacter ,40.rpoB, 16S–23S intergenic spacer (ITS) have been studied in classification and identification of Acinetobacter.DNA sequence-based analysis is used to understand the relatedness of organisms from their DNA sequence similarity. Various DNA sequences like 16S rRNA, Acinetobacter at the genus level [et al. amplified 1500 bp 16S rRNA region using three primer pairs to identify clinical isolates of Acinetobacter species and all were identified as A. baumannii [A. baumannii is 99.9–100%, A. nosocomialis 99.7–100%, A. pittii 99.8–100%, A. calcoaceticus 97.6–99.3%, A. seifertii 97.4–97.8%, A. dijkshoorniae 99–100%. The interspecies similarity of 16S rRNA of A. baumannii with A. nosocomialis, A. pittii, A. calcoaceticus, A. seifertii and A. dijkshoorniae were 97.2, 97.1, 96.9, 98.5 and 97.1%, respectively. Although 16S rRNA sequencing was reported to be polymorphic for the identification of other bacterial genera, it is not effective in differentiating Acinetobacter at the species level due to its extremely low polymorphic nature of the variable region [rpoB, gyrB and recA, which evolve much faster, were investigated as diagnostic biomarkers. The rpoB gene is 4089 base pair (bp) long and only one copy is present [rpoB gene has four variable regions which includes two polymorphic regions and two flanking spacers enclosed by conserved sequences [rpoB for A. baumannii was 98.7–100%, A. nosocomialis 98.7–100%, A. pittii 98.6–100%, A. seifertii 98.5–100%, A. dijkshoorniae 98.6–100% and A. calcoaceticus 97.7–99.7% [rpoB for A. baumannii with A. pittii, A.nosocomialis, A. calcoaceticus, A. seifertii and A, dijkshoorniae were 92–93%, 94–95%, 91–92%, 94–95% and 93–94%, respectively [16S rRNA sequencing is the most commonly used gold standard technique for unknown identification of bacterial species . The 16Sus level . Misbah aumannii . The inte region . Due to  present . The rpoequences . The intAcinetobacter by 16S rRNA and rpoB sequencing, respectively [A. grimontii and A. junii, was ambiguous due to high similarity between these two species, whereas the rpoB gene sequencing showed 100 and 99% similarity to A. grimontii and A. junii respectively, [et al. proposed that A. grimontii as a heterotypic synonym of A. junii by DDH and AFLP [et al. reported that the similarity of rpoB sequences of A. grimontii and A. junii is greater than 99%, thereby suggesting their combination into one species [Recently, Lee and colleagues reported, 93.4 and 98.2% accuracy for the species-level identification of ectively . Similarctively, . Initialand AFLP . Wang et species .rpoB gene sequencing for the differentiation and identification of 17 reference strains of Acinetobacter spp. and seven unnamed genome species. The percentage similarities of rpoB gene sequences of different strains varied between 83 and 99% [rpoB sequence analysis (PRBS) on a collection of Acinetobacter spp. by analyzing residues spanning the 350 base pair (bp) hypervariable zone of the rpoB gene [rpoB sequence analysis, it was found that while interspecies similarity were well separated (ranging from 88.3 to 96.9%), the intraspecies similarity of the rpoB gene sequences was found to be 98–100% for A. baumannii, 98–100% for A. nosocomialis, 98–100% for A. pitti and 98.6–100% for A. calcoaceticus [La Scola’s group investigated the efficacy of  and 99% . FurtherrpoB genes among Acinetobacter spp. are highly variable and considered to be appropriate for species identification [In summary, while 16S rRNA sequencing is highly reliable at the genus level, it has poor discriminatory ability at the species level. In contrast, fication .recA are widely used [This method involves amplification and restriction of conserved gene sequences using various restriction enzymes. Several gene sequences such as 16S rRNA, 16S–23S ITS and ely used .Acinetobacter species except A. haemolyticus (genomic species 4) and A. johnsonii (genomic species 7) [et al. used the 16S–23S ITS region, identical PCR and RFLP patterns were observed [recA and 16S rDNA genes followed by RFLP yielded reliable identification of Acinetobacter geno species five, seven and ten including the closely related species within ACB complex [In the amplified ribosomal DNA restriction analysis (ARDRA) method, amplification of the 16S rRNA gene followed by RFLP using different restriction enzymes enabled identification of all observed . Multipl complex .Acinetobacter species, they are too laborious for day-to-day application in the laboratory or field setting. As these techniques involve skilled labor, they are restricted only to reference laboratories.Although the above methods are helpful in understanding the clinical relevance and epidemiology of Acinetobacter species are summarized in recA and 16S–23S rRNA ITS region for the identification of Acinetobacter species within the ACB complex was developed using 22 reference strains and 138 clinical isolates of Acinetobacter spp. The multiplex PCR was found to be 100% accurate in identification of Acinetobacter at the species level, which was later confirmed by sequence analysis [A. baumannii using the 16S–23S rRNA ITS region. The sensitivity of the LAMP assay was found to be 100-fold superior to the simple multiplex PCR method in identifying Acinetobacter species [The other sequence targets available for the identification and discrimination of analysis . In 2013 species . HoweverA. baumannii is the blaOXA-51-like gene, which is intrinsic to A. baumannii. The utility of blaOXA-51-like gene-specific PCR has been studied by Turton and colleagues on 106 A. baumannii isolates and 23 different Acinetobacter spp, which were previously identified by amplified 16S rDNA restriction analysis (ARDRA). All 106 A. baumannii clinical isolates, as well as the ATCC 19606 strain, flagged positive for blaOXA-51-like gene, whereas isolates that showed negative were identified as another species of Acinetobacter. So far, 95 variants of blaOXA-51-like gene have been described and the possible limitation of this PCR method could be due to its inability to detect all the described variants of the blaOXA-51 gene [Another reliable marker for identification of -51 gene ,57.blaOXA-51 gene were reported by a few studies, as mentioned below. False-positive results were observed by Chen’s group in two different studies involving 676 and 74 isolates of non-baumannii Acinetobacter species, which showed 0.9 and 5.5% prevalence of the blaOXA-51-like gene, respectively. The blaOXA-51-like gene has weak carbapenem hydrolyzing activity. Insertion sequences (ISs) such as ISAba1 could be inserted upstream, leading to overexpression of the blaOXA-51-like genes [blaOXA-51-like gene in non-baumannii species including A. nosocomialis and other genomic species close to A. nosocomialis. All these isolates contain the upstream insertion of ISAba1 on plasmids, which might have been transferred from A. baumannii by the horizontal gene transfer method [However, false-positive and false-negative results for detection of the ke genes . It was r method .blaOXA-51-like gene by the insertion element ISAba19, thereby suggesting the blaOXA-51-like gene to be an unsuitable molecular marker for A. baumannii identification [blaOXA-51-like gene by insertion elements like ISAba15 and ISAba19 [The false negative issue was also observed during an outbreak situation, where approximately 14 isolates generated a 1.7-kb PCR product instead of 353 bp for this marker. Sequencing of the 1.7-kb PCR band revealed disruption of the fication . A separ ISAba19 .blaOXA-51-like gene confers high level carbapenem resistance, thereby limiting the use of blaOXA-51-like gene as a target for differentiating A. baumannii from other Acinetobacter species. Therefore, it is essential to include additional gene targets like rpoB sequencing along with the blaOXA-51-like PCR for the differentiation of A. baumannii from ACB complex.Notably, presence of such plasmid-borne insertion elements along with gyrB primers was proposed for the identification of A. baumannii and A. nosocomialis [A. pittii and A. calcoaceticus [Acinetobacter were subjected to gyrB multiplex PCR and VITEK2 analysis. The accuracy rates were found to be 90.5 and 76.6% for gyrB multiplex PCR and VITEK2, respectively [gyrB multiplex PCR showed 100% concordance in identifying species within the ACB complex whereas VITEK2 was able to identify only A. baumannii and failed to identify other species within the ACB complex [Multiplex PCR based on species-specific genes is considered simple, rapid and reliable for identification of different species within the ACB complex. This multiplex PCR is robust, reproducible and cost effective and yields results in less than 2 h. A multiplex PCR based on comialis . This muaceticus . A totalectively . The gyr complex .recA and gyrB are available for the identification and differentiation of Acinetobacter species within the ACB complex, none of them has been validated for the recently included novel species A. seifertii and A. dijkshoorniae, which can be considered as a limitation.Although various target regions like Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is increasingly used in diagnostic microbiology for the routine identification of bacteria at the genus, species or subspecies level ,65. GeneAcinetobacter spp. using MALDI-TOF.Bacterial cultures subjected to ethanol-formic acid extraction as well as directly from bacterial colonies were used for identification . Table 3Acinetobacter isolates  and seven reference strains of different Acinetobacter species using MALDI-TOF with Bruker database. It was found that 85% (n = 6) accuracy in identification at species level for the reference strains. Species-level identification was accurate and achieved for all A. baumannii  isolates and all A. pittii , except one. None of the A. nosocomialis were identified by MALDI-TOF as the Bruker database lacks a signature specific for A. nosocomialis. A set of reference spectra was created using three strains each of A. baumannii, A. pittii and A. nosocomialis to supplement the Bruker database. Reanalysis of spectra from all 60 isolates using a local database incorporated to MALDI-TOF was performed with reference signatures of A. nosocomialis, which provided unequivocal identification for all the tested strains [Espinal’s group tested the previously characterized 60  strains .et al. found an inability of MALDI-TOF to discriminate between A. baumannii and A. nosocomialis within the closely related ACB complex. In their study with a taxonomically clearly defined set of 105 strains representing the four species of the ACB complex, they evaluated a protocol with change in the matrix composition without changing the other steps of the sample processing. With the modified protocol, differentiation between A. baumannii and A. nosocomialis was improved and 13 A. nosocomialis strains which were wrongly assigned were correctly identified [Sedo entified .et al. evaluated the ability of MALDI-TOF (Bruker Biotyper) in the correct identification of species within the ACB complex as well as species other than the ACB complex. The overall accurate identification rate of species within the ACB complex was only 85.3%.Hsueh A. nosocomialis, the correct identification rate was 72.4%. Additionally, accurate identification of non-ACB complex species like A. junii, A. ursingii, A. johnsonii and A. radioresistens was achieved using the Bruker Biotyper [Although the current database included Biotyper .Acinetobacter to the species level by comparison with sequence analysis of rpoB using 123 isolates of Acinetobacter species [Acinetobacter isolates to species level and 13% of the isolates to genus level. With rpoB sequence analysis, 84% were confirmed to the correct species level and discordant results were obtained for 16% isolates. The rpoB sequencing, which is better for species delineation, was able to assign 123 isolates to 13 species whereas MALDI-TOF assigns only to 9 species indicating the less discriminatory power of MALDI-TOF [Kishii and colleagues evaluated MALDI-TOF for the identification of  species . MALDI-TALDI-TOF .A. baumannii.MALDI-TOF combined with chemometric tools like partial least squares discriminant analysis and hierarchical cluster analysis was reported to have (100%) accuracy in the identification within the ACB complex . OverallA. seifertii and A. dijkshoorniae and several studies have shown that the estimated identification rate for A. nosocomialis range at 70% suggesting to update and improve the database for accurate identification of species within the ACB complex [Acinetobacter isolates representing A. baumannii, A. nosocomialis, A. pittii, A. dijkshoorniae and A. seifertii were used to calculate the species-specific average spectra. Mean spectra derived from A. seifertii, A. dijkshoorniae and A. nososcomialis were added to the database [Acinetobacter species in the default taxonomy with the new reference signatures of A. nosocomialis, A. seifertii and A. dijkshoorniae allowed the identification of Acinetobacter to the correct species level within the ACB complex [MALDI-TOF technique was unable to identify the two novel species,  complex . A totaldatabase . Finally complex .Variations in the sensitivity and specificity in identifying ACB complex by MALDI-TOF have been reported . This isA. baumannii within the ACB complex. PCR targets such as the blaOXA-51-like gene, 16S–23S rRNA ITS, recA and gyrB; and PCR–RFLP of 16S rRNA, 16S–23S ITS and recA were considered as suitable markers for Acinetobacter speciation. However, these methods are mostly performed by reference laboratories. With an accurate database, MALDI-TOF can be helpful for species-level identification. To conclude, molecular characterization using blaOXA-51-like PCR together with rpoB sequencing has high discriminatory power over the conventional methods for Acinetobacter species identification, especially within the ACB complex.Species within the ACB complex are difficult to distinguish. The differences in clinical relevance of the ACB complex and their antimicrobial susceptibility profile warrant species-level identification. The traditional phenotypic methods such as culture and biochemical identification are slow, unreliable and less efficient at differentiating the most clinically important Acinetobacter species within the ACB complex is still challenging. Currently, available phenotypic methods are insufficient. Although various commercially available automated identification systems are widely used in clinical laboratories, such systems are still not effective for identification. Incorporation of more diverse mass spectra of different species within the ACB complex will improve the MALDI-TOF database, which eventually will be useful in accurate identification of the ACB complex at the species level. For molecular characterization, a single gene target as a biomarker is not sufficient for accurate identification of ACB complex. Whole-genome sequencing can be considered as a promising tool; however, its usage is limited in lower resource settings. Furthermore, comparative genomic studies will help us to identify the species-specific targets that can be used as a marker for molecular identification.Identification of Acinetobacter species within the closely related Acinetobacter calcoaceticus–Acinetobacter baumannii complex is crucial.Identification of Acinetobacter.The existing phenotypic methods and commercial automated systems are not reliable for the species-level identification of rpoB sequencing is considered to be appropriate for species level identification.At the genus level, 16S rRNA sequencing is reliable whereas blaOXA-51-like gene is considered to be the species-specific biomarker for Acinetobacter baumannii.blaOXA-51-like gene confers carbapenem resistance, thereby limiting the use of blaOXA-51-like gene as a target for differentiating Acinetobacter baumannii from other Acinetobacter species.Presence of plasmid-borne insertion elements along with A. calcoaceticus–A. baumannii complex.MALDI-TOF with an accurate database will be useful in the species-level identification within the


Deinagkistrodon acutus (D. acutus) and Daboia siamensis (D. siamensis), can cause significant coagulopathy. However, a significant proportion of patients with snakebites cannot identify the correct snake species after envenomation, which hampers the application of antivenom. Hence, the differential diagnosis between the two snakebites by clinical presentations is important. This study aims to compare their clinical and laboratory features for the purpose of differential diagnosis between the two snakebites.There are 6 species of venomous snakes in Taiwan. Two of them, D. acutus or D. siamensis envenomation, between 2003 and 2016, in one medical center in eastern Taiwan. Since these snakebites are rare, we also included 3 cases reported from another hospital in central Taiwan.We retrospectively reviewed the medical records of patients who arrived at the emergency department due to D. acutus and 12 patients by D. siamensis were analyzed. Hemorrhagic bulla formation and the need for surgical intervention only presented for D. acutus envenomation cases . As to laboratory features, lower platelet counts , lower D-dimer level , higher proportion of patients with moderate-to-severe thrombocytopenia (platelet count < 100 × 103/μL)  = 20.0, 95% CI, 2.77–144.31; P = 0.002), and lower proportion of patients with extremely high D-dimer (> 5000 ng/mL)  were found among cases of D. acutus envenomation compared to D. siamensis envenomation. The combination of hemorrhagic bulla, thrombocytopenia, and a lack of extremely high D-dimer had good discriminatory power (area under the curve (AUC) = 0.965; 95% CI, 0.904–1.00) for distinguishing D. acutus from D. siamensis envenomation.In total, 15 patients bitten by 3/μL) and hemorrhagic bulla formation may indicate D. acutus envenomation. However, the envenomed patient with extremely high D-dimer levels may indicate a D. siamensis envenomation. These findings may help diagnose and select the right antivenom in patients with unknown snakebites who present significant coagulopathy.The presentation of moderate to severe thrombocytopenia (platelet count < 100 × 10 Protobothrops mucrosquamatus (Taiwan habu), Trimeresurus stejnegeri (Taiwan bamboo viper), Deinagkistrodon acutus (D. acutus), Daboia siamensis (D. siamensis), Bungarus multicinctus (banded krait), and Naja atra (Taiwan cobra) . All statistical tests were two-tailed while a P value of < 0.05 was considered statistically significant. Odds ratios (ORs) were calculated using logistic regression analysis. In addition, receiver operating characteristic (ROC) curves for different combination of the significant variables were calculated to determine which clinical manifestations can distinguish these two types of snakebite. All data were analyzed through the software SPSS, version 12.0 .The normality of the quantitative variables’ distribution was tested by the Kolmogorov-Smirnov test .Respective totals of 15 and 12 patients with D. acutus envenomation received surgical intervention  due to suspicion of compartment syndrome by clinical symptoms (4 patients), or tissue infection or necrosis according to local findings (4 patients), but no D. siamensis envenomation patient underwent surgery . Morganella morganii and enterococcus faecalis were the leading isolated pathogens  and D-dimer levels (P = 0.008), but higher PT (P = 0.025) and DIC scores (P = 0.036) were found in patients with D. acutus envenomation  compared to patients with D. siamensis envenomation. However, a significantly lower proportion of patients with D. acutus envenomation presented with extremely high D-dimer levels compared to patients with D. siamensis envenomation  . Moreover, the presentation of both thrombocytopenia and hemorrhagic bulla was also a more apt predictor for D. acutus envenomation   and D. siamensis (58.3%) envenomation . Combining the two features of thrombocytopenia and hemorrhagic bulla formation is also an acceptable diagnostic marker in distinguishing these two types of snakebites .In order to apply our findings to the clinical practice of EDs, we focused our analysis on simply defined abnormalities of coagulation profiles, such as extremely high D-dimer levels and noncoagulation in PT or aPTT, but we did not measure the optimal cutoff point via the ROC curve from individual laboratory data. Moreover, when considering that a single clinical feature may not be acceptable to accurately differentiate between these two types of snakebites, we combined different clinical and laboratory features to optimize the ROC curve. The combined model using thrombocytopenia, hemorrhagic bulla formation, and a lack of extremely high D-dimer levels had the best discriminatory power in distinguishing D. acutus and D. siamensis envenomation over the last 20 years in Taiwan, the sample size was still small due to the rarity of both types of snakebites. Third, due to the lack of a definitive guideline to manage these two snakebites in Taiwan, different treatment strategies in clinical practice might influence clinical outcomes and result in the missing values of some laboratory tests. Fourth, although we tried our best to discriminate snake species according to patient’s identification, clinical symptoms or defined criteria, there is still probable misidentification owing to non-visible real snake in ED. A prospective study conducted to validate our findings should be considered. In addition, the time-dependent change in coagulation profiles, the quantification of specific clotting factors, such as factor X, and development of the severity grading system should be considered in further studies.There are several limitations in our study. First, this is a 13-year retrospective study and all patient data were collected from patient charts or electronic medical records. Non-uniform descriptions of signs or symptoms recorded by different physicians may influence and induce some bias. Second, although this is the largest native study regarding D. acutus or D. siamensis envenomation. In the clinical differential diagnosis between these two types of snakebites, the presence of hemorrhagic bulla and moderate-to-severe thrombocytopenia are clinical features uniquely associated with D. acutus envenomation. However, extremely high D-dimer levels are indicative of D. siamensis envenomation.Among the 6 most common venomous snakes in Taiwan, life-threatening coagulopathy is frequently attributed to either


Pineapple is one of the most economically important tropical or subtropical fruit trees. However, few studies focus on the development of its unique collective fruit. In this study, we generated a genome-wide developmental transcriptomic profile of 14 different tissues of the collective fruit of the pineapple covering each of the three major fruit developmental stages. In total, 273 tissue-specific and 1,051 constitutively expressed genes were detected. We also performed gene co-expression analysis and 18 gene modules were classified. Among these, we found three interesting gene modules; one was preferentially expressed in bracts and sepals and was likely involved in plant defense; one was highly expressed at the beginning of fruit expansion and faded afterward and was probably involved in endocytosis; Another gene module increased expression level with pineapple fruit development and was involved in terpenoid and polyketide metabolism. In addition, we built a pineapple electronic fluorescent pictograph (eFP) browser to facilitate exploration of gene expression during pineapple fruit development. With this tool, users can visualize expression data in this study in an intuitive way. Together, the transcriptome profile generated in this work and the corresponding eFP browser will facilitate further study of fruit development in pineapple. Pineapple is an economically important fruit herb widely cultivated in tropical and subtropical regions . PineappRecently, with the popularization of high-throughput sequencing technologies, several genomic studies of pineapple fruit have been performed . Moyle ehttp://www.bar.utoronto.ca/). In addition, eFP browsers have also been developed for several fruit-bearing horticultural crops, including tomato (http://bar.utoronto.ca/efp_tomato/cgi-bin/efpWeb.cgi), grape (http://bar.utoronto.ca/efp_grape/cgi-bin/efpWeb.cgi), and strawberry (The electronic fluorescent pictograph (eFP) browser is a powerful web-based tool for exploring gene expression . It disprawberry .http://www.tbtools.xyz/efp/cgi-bin/efpWeb.cgi), aiming to provide an important resource for researchers interested in pineapple breeding and crop improvement.Databases make bioinformation accessible to research community. All plants of pineapple cultivar “Shenwan” used for this study were cultivated in Shenwan Town, Zhongshan City, Guangdong, China. Three distinct plant stages were defined according to fruit development. Stage 1 begins when flower buds begin to bloom and the collective fruit begins to expand in size. Stage 2 begins when flowering ceases, and the collective fruit expands at its fastest rate. Stage 3 corresponds to the time when collective fruit stops expanding and begins to ripen. In total, 11 tissues were manually separated from pineapple. These included bract, core, flower disk, leaf, ovary wall, ovule, petal, placenta, receptacle, sepal, stamen, and style tissues. All samples were collected and stored immediately in liquid nitrogen before RNA extraction. We extracted total RNA from all samples using TRIzol  and determined RNA quality of samples using Agilent 2100. RNA samples with RIN numbers lower than eight were re-extracted. All RNA samples were then forwarded to a sequencing provider for RNA sequencing.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All sequencing adapters and low quality reads were filtered using Trimmomatic  to provide service for all users.We used a docker container to construct a pineapple fruit eFP browser that could be used over the long term. The CentOS 7 image was pulled from docker.io, and dependent software packages for the eFP browser were installed according to the manual. The eFP browser installation package was downloaded from Pineapple forms a collective fruit that is developed from approximately 100 individual florets. This fruit formation is similar to some berries  and 1B. Pineapple fruits contain several main tissue types: bracts, sepals, flower disks, receptacles, ovaries walls, placentas, ovules, and cores . In addiTo characterize expression dynamics throughout pineapple fruit development, RNAseq libraries were constructed and sequenced for 14 different tissues collected at three major developmental stages ; Fig. S1The pineapple genome was expected to contain 27,024 genes . We founGenes can be expressed constitutively or preferentially according to their different roles in plant development. To study the mechanisms underlying the development of pineapple collective fruit, we utilized TAU methods to detect housekeeping (TAU scores lower than 0.2) and tissue-specific (TAU scores higher than 0.99) genes. In total, 273 genes were found to be expressed constitutively with varying expression levels –S3. In cUsing the sequencing data generated earlier, we used WGCNA to detect gene co-expression during pineapple fruit development. In total, 18 gene modules were classified, with the number of genes involved ranging from 155 (light green module) to 4,522 (turquoise module) . Among thttp://www.tbtools.xyz/efp/cgi-bin/efpWeb.cgi). The browser provides a user-friendly web-based interface. Users can enter a specific pineapple gene id and obtain information on its expression in a short time. To ensure that the pineapple eFP browser was working correctly, we manually checked the gene expression pattern graphs of three different genes. Three genes  were used for this test. Aco031614) is expressed at the highest levels in bracts, followed by sepals, petals, core, and others (Aco023419) was shown in an image in which only stamens were colored red; all other tissues were colored bright yellow (Aco004566)  .Although extensive RNA sequencing data for pineapple exists on public repositories such as NCBI SRA, only a few of these were produced from fruit . No dataTissue-specific genes play fundamental roles in tissue differentiation and maintenance . DetectiAco002250 coding a copper ion binding protein possessed the highest expression level. Its homology, LOC_Os01g32790, in rice is preferentially expressed in inflorescence according to the records in the MSU Rice database . Pineapple flower-disk decides the depth of the fruit-eye. Controlling expression pattern of pineapple ARF6 might be a possible way to breeding pineapples for shallow fruit-eyes. A total of 34 genes were found to be ovary-wall specific genes. It is interesting that, out of them, two shikimate kinase gene, Aco004125 and Aco029719, possessed the highest expression levels. Shikimate kinase is the key component of the shikimate pathway, from where a vast number of aromatic compounds originated (Aco006346 coding a homology of sugar transporter SWEET17. In Arabidopsis, SWEET17 mediates fructose transport across the tonoplast (In contrast, only a few genes were preferentially expressed in tissues of the pineapple fruit. a total of 29 genes were core-specific genes. Among them, database . A Chalc Content . The piniginated . Pineapponoplast . PineappGenes function as networks in all biological processes. To uncover the mechanisms underlying different traits, network-level analyses are required. Here, we classified all expressed pineapple genes into 18 gene modules. Among these, genes of the red module were preferentially expressed in bracts and sepals. According to the KEGG pathway enrichment result, most members were associated with plant defense, including plant-pathogen interactions and MAPK signaling. As the pineapple fruit develops, it faces various kinds of bio-stress like pest binding. Thus, it is possible that these resistance-associated genes are expressed with higher levels in the outer part of pineapple fruit to defense the adversity. Genes of salmon module might have similar functions but function in a different way. These genes, functionally related to cutin, suberine, and wax biosynthesis, are only highly expressed at the beginning of fruit expansion. They may be important for all reproductive tissues of pineapple but only functions at the specific stage. In contrast, genes in the light cyan module seems to be linked to fruit quality. Most members of this group are involved in terpenoid and polyketide metabolism. It is likely that this module contains key genes related to pineapple ripening and fragrance biogenesis. In addition, other modules also seem to have distinct expression profiles and apparent functions. Together, these modules can be used for gene screening and further experimentation.Studies of fruit are important for fruit tree breeding and research, since different crops form different fruit types. Tomato, the most famous fruit study model species , bears sIn the present study, we provide a comprehensive and high quality pineapple fruit transcriptome. We have characterized thousands of tissue-specific genes as well as 18 gene co-expression modules. Moreover, a pineapple fruit eFP browser has been constructed to explore gene expression in pineapple fruit and to facilitate further study of pineapple fruit development.10.7717/peerj.6028/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.6028/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.6028/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.6028/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.6028/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.6028/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.6028/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.6028/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.6028/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.6028/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.6028/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.6028/supp-12Supplemental Information 12Click here for additional data file.


Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 colonise geese and ducks, and could be associated with infections of avian respiratory and nervous systems, cause mild to severe inflammation of cloaca and genital tracts, and embryo lethality. Co-occurrence of these Mycoplasma species in waterfowl is frequently detected and the identification of these mycoplasmas to the species level at a regular microbiology laboratory is difficult due to their similar morphological, cultural and biochemical properties. Moreover, species differentiation is only possible based on the sequence analysis of the product of a genus-specific PCR assay. Therefore, the aim of the current study was to develop an effective and robust method for the identification of these species in avian clinical specimens. Polymerase chain reaction (PCR) assays using species-specific primers, which target housekeeping genes in order to identify these species, were designed in the present study. The developed PCR assays can precisely identify these four mycoplasmas to the species level directly from DNA samples extracted from clinical specimens, and no cross-amplification was observed among these species and with other well-known avian mycoplasmas. The average sensitivity of the assays was 101−102 genomic equivalents per reaction. These conventional PCR assays can be run simultaneously at the same PCR cycling program, and the species can be differentiated directly  by gel electrophoresis due to the specific sizes of the amplicons. In conclusion, the presented species-specific assays were found to be suitable for routine use at regular veterinary diagnostic laboratories and promote the rapid, simple and cost-effective differentiation of these waterfowl Mycoplasma species. Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 are well-known waterfowl mycoplasmas. M. anatis, M. anseris, and M. cloacale are well-characterized, validly published species [M. sp. 1220 has been isolated in 1983 in Hungary from a goose with clinical inflammation of the phallus [M. sp. 1220 were comprehensively studied [Mycoplasma species, with a proposed name M. anserisalpingitis [Mycoplasma species are also associated with some pathological conditions in waterfowl. Clinically manifested mycoplasmosis caused by these Mycoplasma species can occur in waterfowl during excessive stress, such as inadequate housing, crowding or extensive egg production [M. anatis could cause infection associated with neurological symptoms in ducks under stress conditions [M. anatis [M. anatis was also isolated from healthy geese [M. anseris was associated with airsacculitis, peritonitis and embryo lethality in geese [M. cloacale was associated with egg infertility in geese [M. sp. 1220 cause the most significant production and economical losses in the goose farming business, especially in Central and Eastern European countries where the goose production is particularly popular [Mycoplasma species are well-known to be mainly vertically transmitted [M. anseris, M. cloacale and M. sp. 1220 in geese has been frequently observed [ species , while M phallus  but not  studied ,5, whichpingitis –8. All toduction , or due . anatis . Howeverhy geese . M. ansein geese  and mighin geese . M. cloain geese  but coulin geese ,14,15. I popular ,8,16,17. popular ,16,18,19nsmitted ,20,21. Cobserved .M. anatis and M. sp. 1220, and M. anseris and M. cloacale have undistinguishable biochemical properties  and all isolates/strains of these species have typical “fried-egg” colony morphology on solid media. Practically, these species could be identified using serological testing via growth inhibition test (GIT) and immunofluorescent antibody test (IFA); however, these tests are time-consuming, require pure culture of isolates and the qualified reference antisera, which are not available at regular veterinary laboratories in most countries either due to absence of national veterinary mycoplasma reference centres or due to a limited use of animals for production of antibody reagents [M. sp. 1220 in geese [The species pairs, reagents ,9,14. Threagents . Howeverin geese ) and assMycoplasma genus-specific PCR assay, which targets the 16S-23S rRNA intergenic transcribed spacer region (ITS) [M. anatis-, M. anseris- or M. cloacale-specific PCR assays have been published, and only one study on the species-specific PCR detection and phylogenetic identification of M. sp. 1220 isolates in geese from the Russian Federation and Ukraine has been published [The identification of these mycoplasmas to the species level is possible with the use of the on (ITS) ; howeverublished . The aimM. anatis, M. anseris, M. cloacale and M. sp. 1220, several housekeeping genes were randomly selected from the mycoplasma minimal genome set [M. anatis, M. anseris and M. cloacale type strains [M. sp. 1220 type strain (ATCC BAA-2147) were obtained from the whole-genome sequence determined by de novo sequencing in our laboratory using the Illumina  next generation sequencing platform (unpublished data). Also, the rpoB gene sequences of M. sp. 1220 type strain and M. sp. 1220 field isolates were previously determined by our group and can be found under GenBank accession numbers EU596576 and MH003302—MH003317, respectively. The genome annotation for these species was performed using the RAST software [ackA), cytidine deaminase (cdd), DNA polymerase III subunit gamma/tau (dnaX), DNA-directed RNA polymerase subunit beta (rpoB), glucose-6-phosphate isomerase (pgi), ribulose-phosphate 3-epimerase (rpe), ATP-dependent DNA helicase (pcrA), and transcriptional regulator (mraZ) genes. Based on our genome analysis, these genes are presented in genomes of all these four Mycoplasma species. In addition, the genes that were found to be unique only for some studied Mycoplasma species were also included in this analysis. Thus, the 1-phosphofructokinase (pfkB), beta-phosphoglucomutase (pgmB), phosphoenolpyruvate-protein phosphotransferase (ptsP) and UTP-glucose-1-phosphate uridylyltransferase (gtaB) genes are presented only in the genomes of M. anatis and M. sp. 1220 type strains. The arginine deiminase (arcA) and 5'-methylthioadenosine nucleosidase (mtn) genes appeared only in the genomes of M. anseris and M. cloacale type strains. The thymidylate synthase (thyA) and deoxycytidylate deaminase (comEB) genes were only found in genomes of M. anatis and M. anseris type strains, respectively. Based on the genome analysis, all these genes are presented as a single copy in the genomes of these four mycoplasmas.In order to design species-specific primers for detection of nome set ,27, whic strains . The samsoftware . The folhttp://www.premierbiosoft.com/netprimer). The specificity of the primers was analysed in silico using BLAST NT algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi), as well as in vitro to demonstrate a possibility for any cross-amplification among the tested species and with other Mycoplasma/Acholeplasma species that could be found in avian clinical specimens , 0.5 μl dNTP , 2 μl of each primer (10 pmol/μl) and 0.25 μl GoTaq DNA polymerase (5 U/μl). The final primer set was selected based on specificity and sensitivity criteria detailed in section “Assessment of specificity and sensitivity of the developed assays”. The sequences of the designed primers and the sizes of the species-specific amplicons are provided in The species-specific PCR assays were carried out in 25 μl total volume, containing 2 μl target DNA sample, 5 μl 5X Green GoTaq Flexi Buffer , 2 μl MgClMycoplasma and Acholeplasma type strains: M. anatis (NCTC 10156), M. anseris (ATCC 49234), M. cloacale (NCTC 10199), M. columbinasale (ATCC 33549), M. columborale (ATCC 29258), M. gallinaceum (ATCC 33550), M. gallinarum (ATCC 19708), M. gallisepticum (ATCC 19610), M. gallopavonis (ATCC 33551), M. iners (ATCC 19705), M. iowae (ATCC 33552), M. meleagridis (NCTC 10153), M. pullorum (ATCC 33553), M. sp. 1220 (ATCC BAA-2147), M. synoviae (NCTC 10124) and Acholeplasma laidlawii (NCTC 10116).The specificity of the PCR assays was analysed by DNA testing of the following avian M. anatis (NCTC 10156), M. anseris (ATCC 49234), M. cloacale (NCTC 10199) and M. sp. 1220 (ATCC BAA-2147) type strains 10-fold serially diluted (106−100) in nuclease-free water. Template copy numbers  per reaction for these strains were calculated based on the used DNA concentrations using an online dsDNA copy number calculator (https://cels.uri.edu/gsc/cndna.html) and the following genome size information: 956,093 bp for M. anatis (GenBank accession number CP030141), 750,010 bp for M. anseris (CP030140), 659,552 bp for M. cloacale (CP030103) and 908,787 bp for M. sp. 1220.In order to test the sensitivity of the PCR assays, all designed primer pairs were tested with the corresponding DNA of the Mycoplasma/Acholeplasma strains were excluded from further study. The sensitivity of the PCR assays using different primer pairs for the detection of a certain waterfowl Mycoplasma species were compared visually during gel electrophoresis. Primer pairs showing the highest sensitivity and specificity were chosen for the final study.During the selection of the final primer set, primers which demonstrated cross-amplification between the tested Mycoplasma type strains (containing 106 GE) were mixed in a ratio of 1:1:1:1 and tested. Also, DNA mixes were created with one DNA sample containing 103 GE and the other three are represented with 106 GE in a ratio of 1:1:1:1, and submitted to the corresponding waterfowl Mycoplasma-specific assay .In order to evaluate the performance of the designed assays in mixed infections, the DNA of the waterfowl M. anatis-specific PCR assay  were also sequenced to confirm their species identity because the presence of M. anatis in geese is not commonly reported. Randomly selected amplicons were also sequenced from the M. anseris- and M. cloacale-specific PCRs to verify their species identity . In the case of the M. sp. 1220-specific PCR, product of the MYCAV 47 isolate originating from a duck, and a randomly selected amplicon  were sequenced to confirm their species identity. The Mycoplasma genus-specific PCR products of clinical specimens No. 1, 13, 25 and 28, which according to the results of the developed species-specific PCR assays represented mixed mycoplasma DNA samples due to natural mycoplasma co-infections in geese, were also sequenced in order to analyse their DNA sequence chromatograms for the presence of mixed sequences. All analysed amplicons were purified using the QIAquick PCR Purification Kit  and directly sequenced with the same primers used for their PCR amplification. The sequences obtained from the type strains, isolates/strains and clinical specimens are deposited in GenBank under accession numbers MK532897 –MK532910.In order to demonstrate that the obtained PCR products were species-specifically amplified we performed direct DNA sequencing of all amplicons from the type strains and amplicons generated from some selected isolates/strains and clinical specimens. All PCR products amplified from goose specimens using the dnaX, pgmB, ptsP and thyA genes to detect M. anatis; the dnaX, pcrA and mtn genes to detect M. anseris; the dnaX and mtn genes to detect M. cloacale; and the rpoB, pfkB and pgmB genes to detect M. sp. 1220. These primary PCR assays based on these designed primers showed species-specificity in silico; however, in vitro analyses revealed cross-amplification among the species in one case , which was excluded from further examinations. The rest of the omitted primers showed one- to two-fold lower sensitivity in the species-specific PCR amplification based on UV visualization, hence, they were excluded from further analyses. According to the combined results on the primers specificity and sensitivity performed by testing the DNA samples of the mycoplasma type strains, PCR assays, which did not demonstrate any cross-amplification with other tested Mycoplasma/Acholeplasma strains and showed superior sensitivity, were selected as the final assays. The dnaX gene-based PCR assays were the most suitable for precise species identification of M. anatis and M. cloacale (sensitivity of these assays was 102 GE per reaction), the pcrA gene-based PCR assay was accepted for species identification of M. anseris (sensitivity of 101 GE per reaction), and the rpoB gene-based PCR assay was suitable for precise identification of M. sp. 1220 (sensitivity of 102 GE per reaction). As end-point PCR systems were designed in the study, the yield of a PCR product does not provide quantity result after UV visualization. However, the visual detection of the PCR products in the sensitivity tests means that the minimum GEs detectable in the samples are 101 in the case of M. anseris-specific PCR and 102 in the cases of M. anatis-, M. cloacale- and M. sp. 1220-specific assays. The similarity between the dnaX gene of M. anatis and M. cloacale is 51.32% and the highest similarity among the bonding sites of the M. anatis- and M. cloacale-specific primer pairs is 54.55%; therefore, cross-amplification among the two species in the specific PCR assays is unlikely. Species identification was successful in all cases during the tests of the M. anatis, M. anseris, M. cloacale and M. sp. 1220 type strain DNA mixes, the presence of the other waterfowl pathogen mycoplasmas did not have any effect on the assays’ performance.Sixteen mycoplasma housekeeping genes were analysed in the study, out of which eight genes were found suitable for the design of species-specific primers based on the above-mentioned acceptance criteria. Therefore, the species-specific primers were designed for the following genes: the Mycoplasma species, 9 specimens for two species, other 9 specimens for three species and one specimen was positive for all tested Mycoplasma species. Sequences of the amplicons obtained from these species-specific assays on the clinical specimens and isolates/strains showed their 97.7–100% identity with the corresponding sequences of the type strains  a single amplicon was amplified by the genus-specific PCR assay, while the species-specific PCR assays were able to detect mixed infections of M. sp. 1220, along with M. anseris and/or M. cloacale in these geese  by the developed assays, which showed the same amplicon size by the genus-specific PCR assay. The DNA sequence chromatogram analysis performed on the genus-specific PCR amplicons from the clinical samples No. 1 and 25 revealed major sequences of M. sp. 1220 and M. anseris, respectively, with evidence of well-visible secondary peaks indicating a mixed DNA sequence due to the natural co-infections. However, the DNA sequence chromatogram analysis performed on the genus-specific PCR amplicons from the clinical samples No. 13 and 28 also revealed major sequences of M. sp. 1220 and M. anseris, respectively, but did not demonstrate any detectable mixed sequences , providing valuable support for the selection and the use of adequate control and/or feasible treatment of affected birds [ed birds ,32.M. anatis and M. sp. 1220 demonstrate significant similarity  and therefore cannot be used for the unambiguous differentiation between these two species [M. anseris and M. cloacale  [Mycoplasma species. Moreover, these methods almost always require the direct sequencing of amplicons for the species confirmation, which can be difficult or impossible in cases of co-infections with different Mycoplasma species, as well as in laboratories in which DNA sequencing technologies are not available.The nucleotide sequences of the 16S rRNA genes of  species . Also, tctively)  makes thctively) ,34–36 deMycoplasma genus-specific PCR assay [Mycoplasma species  due to the length variation of the 16S-23S rRNA ITS among the species [The CR assay  provides species . Therefo species . The preM. sp. 1220 represents a serious threat to the geese industry, especially in Central and Eastern Europe, therefore the detection of this agent is required for a comprehensive laboratory diagnostic analysis of goose clinical specimens. Previous attempts have been made to design primers for the detection and specific identification of this species based on the sequences of the rpoB, DNA polymerase III subunit alpha (dnaE), elongation factor (fusA), and the pyruvate kinase (pyk) genes [M. sp. 1220 in avian clinical samples. The primers for the M. sp. 1220-specific PCR assay described in this study also target the rpoB gene but the sensitivity of the assay was established to ensure its applicability for the diagnostic use.k) genes ; howeverMycoplasma species in new or rarely-observed waterfowl hosts. According to the available literature M. sp. 1220 has been previously isolated only from geese [Mycoplasma genus-specific PCR [M. sp. 1220-infected geese [M. sp. 1220 (isolate MYCAV 47) from an avian species other than goose. The partial rpoB gene (1997 bp long) was also sequenced for this M. sp. 1220 isolate  using primers and conditions described previously [rpoB gene of the type strain of M. sp. 1220.The PCR assays developed in this study were able to identify these om geese ; howeverific PCR  and thised geese . To our M. anatis normally colonises ducks and it was rarely detected in geese [M. anatis isolates were identified in the tested specimens out of which two isolates originated from geese (MYCAV 314 and 317). The observed pathological changes such as the fibrinous peritonitis and lymphohistiocytic infiltration in the lungs of these geese were similar to the pathological evidences reported for M. anatis-infected ducks [M. anatis according to our results generated using the newly designed species-specific PCR assays. DNA sequence analysis of the dnaX amplicons from the two isolates and the four clinical specimens revealed their 97.7–97.9% sequence identity to the same gene of M. anatis type strain  was also sequenced for these two M. anatis isolates originated from geese  using primers and conditions as mentioned above, and the corresponding sequencing result demonstrated their 99% identity to the rpoB gene of the type strain of M. anatis. Thus, the species identity of M. sp. 1220 isolate from a duck and M. anatis isolates from geese was clearly demonstrated in both cases.in geese ,14. In ted ducks . Moreovee strain . This hiMycoplasma species in the examined samples, isolates and type strains; however, as the majority of the samples originated from a restricted geographic region, further examinations on a wider selection of samples would enable the determination of the robustness of the assays.The developed assays clearly discriminated the targeted waterfowl pathogen Mycoplasma species in waterfowl specimens, and therefore proved to be suitable and cost-effective method for the identification of these mycoplasmas in routine veterinary laboratory diagnostics. In addition, these assays can be run simultaneously using the same thermal cycling PCR conditions on conventional PCR equipment.Finally, the species-specific PCR assays developed in the current study showed high sensitivity and specificity, enabling rapid, precise and reliable identification of these four S1 FilePrimers (grey) and single nucleotide polymorphisms (black) are highlighted in the sequences.(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Arterial stiffness and wave reflection are important components of the ventricular afterload. Therefore, we aimed to assess the arterial wave characteristics and mechanical properties of the proximal pulmonary arteries (PAs) in the hypoxic pulmonary hypertensive rat model. After 21 days in normoxic or hypoxic chambers , animals underwent transthoracic echocardiography and PA catheterization with a dual-tipped pressure and Doppler flow sensor wire. Wave intensity analysis was performed. Artery rings obtained from the pulmonary trunk, right and left PAs, and aorta were subjected to a tensile test to rupture. Collagen and elastin content were determined. In hypoxic rats, proximal PA wall thickness, collagen content, tensile strength per unit collagen, maximal elastic modulus, and wall viscosity increased, whereas the elastin-to-collagen ratio and arterial distensibility decreased. Arterial pulse wave velocity was also increased, and the increase was more prominent in vivo than ex vivo. Wave intensity was similar in hypoxic and normoxic animals with negligible wave reflection. In contrast, the aortic maximal elastic modulus remained unchanged, whereas wall viscosity decreased. In conclusion, there was no evidence of altered arterial wave propagation in proximal PAs of hypoxic rats while the extracellular matrix protein composition was altered and collagen tensile strength increased. This was accompanied by altered mechanical properties in vivo and ex vivo.NEW & NOTEWORTHY In rats exposed to chronic hypoxia, we have shown that pulse wave velocity in the proximal pulmonary arteries increased and pressure dependence of the pulse wave velocity was steeper in vivo than ex vivo leading to a more prominent increase in vivo. Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction, vascular remodeling, inflammation, and thrombosis , resultiCommonly, the proximal pulmonary arteries (PAs), i.e., the pulmonary trunk (PT) and right and left PAs, are considered to be capacitance vessels that determine the pulsatile component of RV afterload . SeveralArterial pulse wave transmission is also an important component of ventricular load. Four types of arterial waves have been described in the pulmonary artery. Forward compression waves (which increase pressure and flow) and forward decompression waves (which decrease pressure and flow) are related to ventricular contraction and relaxation, respectively. Backward compression waves (which increase pressure while decreasing flow) and backward decompression waves (which decrease pressure while increasing flow) are reflected waves due to vascular impedance mismatch between the proximal and distal vasculature . Pulse wIn this study, we performed a comprehensive ex vivo assessment of the mechanical properties of proximal PAs and the aorta in the hypoxic rat model. In addition, we explored the feasibility of invasive pulmonary WIA and investigated how in vivo measurements of arterial stiffness relate to ex vivo measurements.A glossary for the variables and abbreviations used in this report is shown in Experiments were performed in accordance with European Union Directive 2010/63 and approved by the Animal Experiments Inspectorate (permission no. 2012-15-2934-00741). A total of 48 male Sprague-Dawley rats  were randomly assigned to 2 groups . One group was exposed to hypoxia by being placed in hypobaric chambers (560 mbar corresponding to 10% oxygen)  was performed on a spontaneously breathing rat anesthetized with sevoflurane (induction: 7% and maintenance: 4%) in oxygen. B-mode and pulsed wave Doppler images of the PT were acquired  were ventilated at 75 strokes/min with a tidal volume of 10 ml/kg . Right heart catheterization (RHC) was performed through a left lateral thoracotomy. A 0.014-in. combined dual-tipped pressure and Doppler flow sensor wire  was inserted through the RV outflow tract into the PT to acquire pressure and velocity data at a sampling rate of 200 Hz . At the end of the procedure, animals were euthanized by decapitation.ds) obtained by TTE as follows:RV stroke volume (m). Total pulmonary resistance was calculated as PAPm divided by cardiac output, and global pulmonary arterial compliance was calculated as stroke volume divided by pulmonary arterial pulse pressure (PAPp). Using diastolic PT diameter (dd) and arterial distensibility  and backward (WI−) components (f) and backward (Pb) pressure waveforms by integrating the differentials, dPf and dPb (Pulmonary WIA was performed using the ensemble-averaged pressure and velocity obtained by RHC as well as RHC-derived pressure combined with TTE-derived velocity. Given the cardiac cycle duration (CCD), WI  and energy density over a cardiac cycle squared. Wave reflection was quantified as the wave reflection index (WRI), defined as the ratio of backward to forward wave energy and as the PDetails on pressure and velocity data processing can be found in the Frozen aortas and PAs were cut into one to three rings with a nominal longitudinal length of 1 mm. Excess artery tissues were set aside for the determination of extracellular matrix (ECM) protein composition. Subsequently, artery rings were mounted on a tapered glass rod at minimal strain and photographed  using a Once thawed at room temperature, artery rings were subjected to mechanical testing  using a cl; x = distance at starting position + travel distance) and the diameter of the hooks (dh = 0.35 mm). The initial luminal circumference  was calculated at a minimal loading force (0.1 mN) as follows:The luminal circumference (A). Assuming that the vessel wall was incompressible and homogeneous and neglecting the residual stress, strain (ε), stress (σ), and the incremental stiffness (k) and elastic modulus (E) were calculated using −Two sides of the artery ring corresponding to the double vessel wall area were loaded simultaneously . Thus, all), luminal radius (r), and vessel wall thickness (h), the distending pressures (P), wall tension (T), wall stress , and corresponding strains were derived from the load-strain curves by estimating the combination of load and strain that fulfilled Laplace’s law for a cylinder (Ep) at each of the three distending pressures were calculated as described above  of 0.45 , the modFinally, from the last hysteresis cycle, the viscous energy loss was calculated as the area inside the hysteresis loop divided by the area under the load-deformation curve.The hydroxyproline content of each artery ring was quantified by colorimetric determination through a modified Woessner method , 69. ColExcess artery tissues were gathered into several sample pools and defatted with acetone before quantification of the fractional elastin and collagen content. Mature elastin was purified through the hot alkali method . After cThe glycosaminoglycan (GAG) content (sulfated + nonsulfated) in the extract was estimated by quantifying the uronic acid concentration through colorimetric determination as previously described . The amoThe aorta and PAs were fixed at zero transmural pressure in 4% formaldehyde overnight and subsequently preserved in 70% ethanol. Vessels were then embedded in paraffin and sectioned into 4-μm-thick slices. Selected sections from each rat were stained with either hematoxylin and eosin or with resorcin (Hart’s solution), Sirius red F3B, and Weigert’s hematoxylin.t-test. Differences between the normoxic and hypoxic groups were compared using an unpaired Student’s t-test or Welch’s t-test for unequal variances. Parameters derived from the mechanical testing were summarized taking clustered data into account, and comparisons within each group as well as between the two groups were performed using modified rank tests for clustered data .Data are presented as means ± SD  or medians (25–75% quartiles). Statistical comparison of hemodynamic parameters within each group was performed using a paired Student’s red data . The levThree rats died during the invasive procedure. Hence, 22 normoxic rats and 23 hypoxic rats were included in the study. The effect of hypoxia was evidenced by the lower weight gain, shorter tibia length, higher hematocrit, and higher Fulton’s index (RV weight/left ventricle + septum weight) in the hypoxic group .m, and vascular resistance increased, whereas PA compliance and distensibility decreased, in hypoxic animals. Thoracotomy caused cardiovascular depression, as evidenced by the reduced heart rate and flow velocity (Representative pressure and flow velocity traces from the PT are shown in velocity .b-to-Pf ratio was 29.8 ± 7.2% in the normoxic group and similar in the hypoxic group (26.5 ± 6.0%). Semi-invasive WIA using RHC-derived pressure and TTE-derived velocity revealed larger wave energies compared with invasive WIA . Elastin was mainly present as circumferentially arranged lamellae in the medial layer in both vessel types. In the aorta, elastin alternated between thick but nonwavy and thin wavy lamellae. In contrast, there were fewer elastic lamellae in the PT, and all lamellae were thin and wavy. Collagen fibers were mainly confined to the adventitia. After exposure to hypoxia, the histological appearance of the aorta was unchanged (not shown), whereas the medial and adventitial layers of the PT thickened (I−L).The PT was thinner than the aorta , A−H. Elhickened , I−L.Stretch curves and dimensions and mechanical properties of the artery rings are shown in Vessel wall thickness and the maximal tensile strength of proximal PAs, defined as the maximum load that artery rings could bear before rupturing, was increased in hypoxic animals , whereasEp of proximal PAs were lower in hypoxic animals , strain, and  animals . Howeversq and PWVBH showed evidence of pressure dependence . PWVBH was significantly lower than PWVsq. PWVBH increased 2.0-fold in the hypoxic group (1.54 ± 0.22 vs. 3.12 ± 0.72 m/s). PWVMK showed clear evidence of pressure dependence in all three proximal PAs (not shown for left PA) and was significantly higher in the hypoxic group at each of the three distending pressures , and there were no significant differences between the two groups of animals.Both PWVressures E and F. V values E, and thIn the aorta, the absolute collagen content was similar in the two groups of animals . HoweverWe investigated the effect of chronic hypoxia on the mechanical properties and arterial wave propagation in proximal PAs. Collagen content, tensile strength per unit collagen, arterial stiffness, and viscous energy loss increased, whereas the elastin-to-collagen ratio and distensibility decreased. Pulmonary PWV was increased both in vivo and ex vivo, but hypoxia had little effect on arterial wave energies. In contrast to proximal PAs, aortic stiffness remained unchanged, whereas wall viscosity decreased.Hypoxia causes pulmonary vasoconstriction and increased hematocrit, leading to increased vascular resistance, elevated pressures, and arterial remodeling. In the early stages of PAH, a small increase in PVR can be associated with a considerable increase in arterial stiffness . The incThe increase in the maximal elastic modulus observed in proximal PAs suggests that hypoxia not only induced hypertrophic remodeling but also a compositional change. Collagen content, the major determinant of the elastic behavior during supraphysiological loading, increases in proximal PAs in response to hypoxia , 43, 68.Ep) was decreased. Based on the elastin-to-collagen ratio, the absolute elastin content in proximal PAs is predicted to have increased by ∼20%. Indeed, several studies have reported elastin accumulation in PAs of hypoxic animals , lower than that of PAs .J.S., A.D.H., and U.S. conceived and designed research; J.S., C.C.L., and C.C.D. performed experiments; J.S., K.H.P., N.M.D., and C.C.D. analyzed data; J.S., A.D.H., K.H.P., C.C.D., and U.S. interpreted results of experiments; J.S. prepared figures; J.S. drafted manuscript; J.S., C.C.L., A.D.H., K.H.P., N.M.D., C.C.D., and U.S. edited and revised manuscript; J.S., C.C.L., A.D.H., K.H.P., N.M.D., C.C.D., and U.S. approved final version of manuscript.