In the original article, there was an error. The molar concentrations of LPS and C-SVMP, used in the assays, were incorrect.Materials and Methods section, subsection Human Whole Blood Model, paragraph two:A correction has been made to the 2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), LPS  or C-SVMP  for 30 minutes in a water bath at 37°C under agitation. Under these conditions, C-SVMP did not promote blood coagulation (data not shown), allowing for subsequent analyses of plasma and leukocytes, as described below”.“The human whole blood model described by Mollnes et al. (14) was used. Blood samples from healthy consenting donors were collected by venepuncture into tubes containing 50 μg/mL of lepirudin , the recombinant form of hirudin, a thrombin-inhibitor anticoagulant that does not interfere with the complement cascade. Immediately after collection, 720 μL of the blood samples were incubated with 280 μL of PBS , compstatin  or control peptide  for 30 min at 37°C. Then, 140 μL of PBS, LPS  or C-SVMP  were added and incubated for 30 min in a water bath at 37°C under agitation. Plasma and leukocytes were processed for analysis, as described below”.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


AbstractIsoetesamazonica and I.gardneriana were the first two species of the genus to be collected from Brazil. Isoetesamazonica was gathered by Richard Spruce in the Amazon basin near Santarém in the state of Pará in 1850. Isoetesgardneriana was collected by George Gardner in the current Dianópolis in Tocantins State in 1843. Despite being known for a long time by botanists, these species have not been recollected since then, which raised questions about their taxonomic recognition, current distribution ranges and conservation status. Fieldwork efforts led to the rediscovery of I.amazonica and I.gardneriana after 167 years. These collections enrich our understanding of their habitats and morphologies. We provide here re-descriptions for these species. Based on IUCN criteria, Isoetesamazonica and I.gardneriana should be assigned as data deficient (DD) and endangered (EN), respectively. The rediscovery of these species raises hopes that other areas in Amazon and Cerrado biomes harbour I.amazonica and I.gardneriana, respectively. This study will serve as a basis towards the conservation of these species. Brazil presents the greatest diversity of plants in the world , which pIsoetes L. is globally distributed with an estimated 250 species  in January 2018. Additional efforts to find this species took place in other Brazilian Cerrado areas: Ondas river, Barreiras (Bahia) ‒ ca. 200 km away from the type location ‒ in January 2018; Parque Nacional Serra da Mesa (Maranhão) ‒ 500 km away from the type location ‒ in November 2017; Parque Nacional da Serra do Cipó ‒ 900 km away from the type location ‒ in June 2018; Fazenda Modelo, Campo Experimental da Embrapa, Terenos (Mato Grosso do Sul) ‒ 1200 km away from the type location ‒ in November 2017.For I.amazonica  and I.gardneriana .Besides field trips, specimens from the following herbaria were consulted to check for previous records of these species  and made available by the “Centro de Monitoramento do Tempo e Clima, MS” . Images of the spores were made by transferring the spores to aluminium stubs coated with a carbon adhesive. The stubs were then coated with gold-palladium-alloy in a sputter-coater for 180 s and then digitally imaged using a Zeiss SIGMA VP.I.gardneriana being misused for several collections of I.panamensis Maxon & C.V.Morton sensu lato. We consulted these materials to check whether the identification was correct or not in these cases. Amongst these materials were collections from: Paraguay in 1878 ; municipality of Barreiras in Bahia, Brazil, in 1971 ; an area next to type location of I.gardneriana in the municipality of Formosa do Rio Preto in Bahia, Brazil, in 2015 . In this step, megaspores of these materials were removed, images were taken using SEM and then compared with the type of I.gardneriana. We used both qualitative and quantitative characters to identify the species. The terminology used for the description of the spores follows that of Since megaspore ornamentations are essential for the correct species identification, the absence of detailed images of spores during the determination process may have potentially led to the name Taxon classificationPlantaeIsoetalesIsoetaceaeA. Braun, J. Bot. 18: 109. 1880.60F1455B-6783-5663-AE6E-519272C77725Stems globose, 0.35‒0.7 cm wide, 3-lobate. Leaves 0.45‒1 mm wide at mid length, 4‒17 cm long, 9‒23 per individual, filiform, straight, ascending, apex acute; alae 0.8‒4.5 cm long, extending from the base 1/10 ‒ 1/4 of total leaf length, hyaline, membranaceous, attenuate. Subula present, olive green, trigonal. Labium present, 0.5‒0.7 × 0.9‒1.1 mm long, cordate. Ligule 2.5‒3 × 1‒1.2 mm, hyaline, triangular. Velum 0.1‒0.2 mm along the lateral edges of the sporangium, rudimentary. Sclerified phyllopodia absent. Sporangium at the base of the leaf, 2‒2.5 × 1.8‒2.5mm, elliptic, hyaline, light brown, brown dots present or absent. Megaspores white, 420‒512 (‒590) µm in equatorial diameter (average = 490 µm), trilete; laesurae as wide as high or higher than wide, 40‒53 × 35‒47 µm; proximal surface verrucate, projections 24‒41.4 × 24‒46 µm; equatorial ridges arched, slightly sinuous; distal surface verrucate, macroscuptural projections 25‒45 × 25‒46 µm. Microspores 28‒32 µm long (average = 30 µm), proximal surface echinate, distal surface sparsely echinate.Brazil. Province of Pará: inundated places near Santarém, Sept 1850, Spruce 1081, .Isoetesamazonica was rediscovered at its type location in July 2017 after 167 years . This species was found in a single area at approximately 2.5 km from the left bank of the Tapajós river at the geographical coordinates 2°24'15.15"S, 55°3'1.89"W , trilete; laesures higher than wide, 40‒50 × 11‒16 µm; proximal surface tuberculate, macrosculptural projections 20‒39 × 13‒24 µm; equatorial ridges arched, straight; distal surface tuberculate, projections 24‒44 × 17‒34 µm. Microspores 33‒40 µm long (average = 37 µm), proximal and distal surface smooth or sparsely microechinate.Brazil. Province of Goyaz: Missiones Duro, Sept 1841, Gardner 3563, .I.gardneriana was only rediscovered in Terenos in the state of Mato Grosso do Sul at the geographical coordinates 20°33'32"S, 54°47'23"W. This area is located at about 1200 km away from its type location  Britton, Pontederiacordata L. and Xyris spp.  that grow over a dense herbaceous stratum. The life form of I.gardneriana was of a partially submerged aquatic in September, although in November, plants were found both partially and totally submerged . The megaspores are brown, sparsely verrucate, 490‒650 µm diameter (vs. 548‒615 µm), with knife-like laesurae Fig. . The micI.gardneriana. The megaspores of these collections are both qualitatively and quantitatively distinct from the type of I.gardneriana. The Balansa and Labiak collections have baculate-tuberculate megaspores and Irwin’s collection revealed baculate-clavate megaspores  to be Although fieldwork investigation is fundamental to improve our understanding about how human impacts on biological systems can be recognised, mitigated or averted, fieldwork has considerably decreased in the past decades with negative implications for global biodiversity conservation . The redIsoetes is one of the most threatened groups  in north-eastern Brazil was tentatively identified as I.amazonica . However, despite its resemblance to I.amazonica by size of megaspores and number and size of leaves, the presence of brown sporangium  and its occurrence in Caatinga (vs. Amazon) leads us to believe that this population is either a variant of I.luetzelburgii U. Weber or an undescribed species.Additionally, an Isoetes, the proportion of species with narrow-range distributions is remarkably high , according to IUCN criteria , according to IUCN criteria (criteria . On the ing area . This hacriteria .Isoetesamazonica and I.gardneriana, respectively. We hope that these rediscoveries spark research towards a deeper understanding of the life history of Isoetes and provide information for any future efforts to protect Isoetesamazonica and I.gardneriana from extinction.In conclusion, the rediscovering of these species raises hopes that other areas in Amazon and Cerrado biomes still harbour


The designed probe, RT, was readily synthesized by the one-step reaction of resorufin with phenyl thionochloroformate. The RT probe displayed a prominent color change from yellow to pink and a marked turn-on fluorescence signaling behavior exclusively toward the Hg2+ ion. The signaling of RT was due to Hg2+-induced hydrolysis of the phenyl thionocarbonate moiety to form the parent resorufin dye, which restored its spectroscopic properties. In addition, RT exhibited the Hg2+-selective signaling behavior without interference by coexisting environmentally relevant metal ions. The detection limit for Hg2+ in simulated wastewater samples was estimated to be 5.8 × 10−8 M. In particular, an RT-equipped μPAD prepared using a wax printing technique enabled simple and convenient determination of Hg2+ ions in simulated wastewater samples, with a detection limit of 5.9 × 10−6 M.In this study, a novel resorufin thionocarbonate-based Hg It has harmful impacts on the respiratory system, causing chest pain, cough, dyspnea, and hemoptysis2, and damages the nervous system, leading to e.g., severe behavioral and personality changes, increased excitability, loss of memory, and insomnia3. Hence, many environmental and health-related departments, such as the United States Environmental Protection Agency (US EPA) and the World Health Organization (WHO), have designated mercury one of the most dangerous metal species4. Therefore, the development of a fast and convenient method for mercury determination has become an important issue in analytical and environmental sciences.Mercury, which is released into the environment as vapor or inorganic and organic mercurial species, has been reported to cause serious damage to human health2+ determination methods based on analytical instrumental techniques, such as atomic absorption spectroscopy (AAS)5, X-ray fluorescence spectroscopy (XRF)6, differential pulse anodic stripping voltammetry (DPASV)7, and capillary electrophoresis (CE)8 have been reported. Nevertheless, due to their high costs and technical difficulties in operation, the development of selective and sensitive signaling methods employing simple colorimetric or fluorescence changes is highly desirable. In this context, many UV–vis and/or fluorescence signaling methods using reaction-based probes have been developed14.Several quantitative Hg2+ ions. Following the pioneering research of Czarnik’s anthracene thioamide-based Hg2+ signaling system15, many Hg2+-selective signaling probes using Hg2+-induced desulfurization of sulfur-based compounds, such as thiocarbonyl17, thioamide18, thioimide20, and thiourea derivatives23, have been reported. Furthermore, a number of probes exist which use the Hg2+-induced desulfurization-based cyclization of thioureas26, and cleavage of thioacetals29, thioethers30, thiophosphinates31, and thionocarbonates32. Sulfur-based functional groups in many of these probes effectively suppress the fluorescence of signaling subunits, and desulfurization results in the restoration of the absorption and fluorescence properties of the parent dyes33.In particular, a large number of elaborately designed probes containing sulfur-based moieties have been designed to exploit the highly thiophilic nature of mercury in the determination of HgμPADs)34, many paper-based kits for the determination of biologically and environmentally important materials have been reported. Paper-based devices manifest many features such as desirable mechanical properties , economic feasibility, high surface-to-volume ratio, and capillary action37. In particular, using fabrication techniques such as drawing, stamping, cutting, and photolithography38, a number of μPADs have been developed for the determination of important species including nitrites39, Hg2+ 40, Cu2+ 41, and Fe3+ 42. However, some of these methods suffer from high instrument costs and technical difficulties, and mass production is difficult in some cases. To overcome the drawbacks of μPAD fabrication methods, the wax printing fabrication technique has been developed44, and successfully used for the preparation of μPADs for the determination of biologically and environmentally important species including Cu2+ 45, Ni2+ 46, Hg2+ 47, Ca2+/Mg2+ 48, and organophosphate pesticides49.Following the introduction of microfluidic paper-based analytical devices   mixture of acetonitrile and citrate buffer solution .The designed probe, 7%) Fig. 32. The cRT in the presence of representative metal ions. In an acetonitrile/citrate buffer solution , RT exhibited a broad absorption band centered around 448 nm  at 578 nm clearly showed the prominent Hg2+-selective signaling behavior (A/A0 at 578 nm = 320 for RT in the presence of Hg2+), while the response to other metal ions was negligible (A/A0 varied between 0.1 for K+ and 1.4 for Ag+).First, we investigated the UV–vis signaling behavior of RT in the presence of environmentally relevant metal ions was measured . Other tested metal ions caused minor changes in fluorescence emission  /AHg(II)) at 578 nm only varied in a narrow range between 0.84 (Fe3+) and 1.0 (Li+). The pH-dependency of Hg2+ signaling by RT showed that signaling became more pronounced as the solution pH increased, up to pH 6.2, and was subsequently not influenced significantly by pH variation between pH 6.2 and 9.4  was nearly identical to that of the postulated signaling product resorufin. Furthermore, the mass spectrum of the signaling product of RT with the Hg2+ ion revealed a diagnostic peak at m/z = 213, which is consistent with the proposed signaling product, resorufin  53. In addition, because the determination of mercury pollution is one of the most critical challenges in environmental and industrial fields, we attempted to determine Hg2+ in simulated wastewater samples54. The titration of RT with Hg2+ showed a satisfactory calibration plot up to 1.0 × 10−5 M. From these concentration-dependent experiments, the detection limit for Hg2+ in simulated wastewater was estimated to be 5.8 × 10−8 M , clearly revealed the Hg2+-selective signaling behavior. For instance, the control ΔLRG value of the μPAD kit was 11.83 for distilled water, whereas the value increased to 85.47 on exposure to Hg2+ ions. In the presence of thiophilic Ag+, noticeable interference was observed due to the reactivity of the thionocarbonate moiety of probe RT with Ag+ ions. However, interference from Ag+ was dramatically suppressed by use of a tris-HCl buffer as an Ag+ masking agent, due to the formation of an insoluble AgCl precipitate (Ksp = 1.8 × 10−10) by interaction of Ag+ with chloride ions in the tris-HCl buffer solution (ΔLRG = 21.35 for Ag+). However, considerable interference was still observed, as the residual AgCl in the measurement solutions released a small amount of Ag+ which induced a considerable colorimetric response in the μPAD. We believed that Ag+ ions released from AgCl subsequently react with RT to form much more insoluble Ag2S (Ksp of Ag2S = 8.0 × 10−51) which induced a colorimetric response in the RT-equipped μPAD. Thus, we removed the residual AgCl precipitate using a syringe filter and found that the interference from Ag+ decreased significantly  . Finally, the Hg2+ signaling by the μPAD was evaluated quantitatively, by plotting ΔLRG values as a function of [Hg2+]  for Hg2+ in the concentration range between 0 and 50 μM was obtained . UV–vis and fluorescence spectra were measured using a Scinco S-3100 spectrophotometer and a Scinco FS-2 fluorescence spectrophotometer, respectively. High-resolution mass spectrometry (HRMS) results were obtained on a JEOL JMS-700 mass spectrometer using fast atom bombardment (FAB) ionization. Column chromatography was performed using silica gel .Resorufin, phenyl thionochloroformate, and triethylamine were purchased from Aldrich Chemical Co. All solvents were of spectroscopic grade and obtained from Aldrich Chemical Co. RT  as a scarlet powder. 1H NMR : δ 7.87 , 7.48 , 7.44 , 7.35 , 7.27–7.20 , 6.87 , 6.34 ; 13C NMR : δ 193.5, 186.2, 155.4, 153.4, 149.1, 148.9, 144.4, 135.4, 134.8, 131.9, 131.4, 129.8, 127.1, 121.6, 119.7, 110.5, 107.4; HRMS : calcd for C19H12NO4S+ [M + H]+: 350.0482, found 350.0483.Resorufin  was dissolved in dichloromethane (30 mL), and triethylamine  was added to the solution. After 30 min of stirring, phenyl thionochloroformate  was added dropwise, and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was washed with distilled water and brine several times. The solution was evaporated under reduced pressure and the remaining residue was purified by column chromatography  to yield RT (5.0 × 10−4 M) was prepared in spectroscopic grade acetonitrile. Stock solutions of the metal ions (10 mM) were prepared by dissolving the metal perchlorate salts in distilled water. Caution: metal perchlorates are highly explosive, thus should be handled carefully and used in small quantities. The simulated wastewater was prepared by following the reported literature procedure54. Composition of the simulated wastewater: [NaClO4] = 2.39 mM, [NaHCO3] = 1.23 mM, [NaCl] = 987 μM, [NaNO3] = 484 μM, [Mg(ClO4)2] = 288 μM, [KClO4] = 281 μM, [Ca(ClO4)2] = 250 μM, [Na2SO4] = 208 μM, [NaH2PO4] = 105 μM, [NaF] = 16 μM.A stock solution of RT  in 10 mL of methanol was slowly added to mercury perchlorate  in methanol. The signaling progress was monitored by TLC. Due to the low solubility of resorufin in methanol, a simple filtration was sufficient for the separation of the primary signaling product resorufin and side product phenol in the filtrate. 1H NMR and mass spectral measurements of the solid product (resorufin) were performed after column chromatography. The 1H NMR and 13C NMR of phenol were obtained after the evaporation of the filtrate.A solution of μPAD was designed using Microsoft Office Power Point. The μPAD was printed on Whatman® cellulose chromatography paper using a commercial wax printer 43. After printing, the paper was baked in a 150 °C oven for 90 s and cut into separate μPADs. The RT was dissolved in dichloromethane (8.0 mM) and loaded onto the middle of the circle in the prepared μPAD using a capillary tube  (applied volume = 1.6 μL). Varying concentrations of Hg2+ in simulated wastewater sample were added to the RT-equipped μPAD kit, which resulted in a color change from yellow to pink. The analyte solution was prepared by adding 1% (v/v) of tris buffer solution  to the wastewater stock solution. Synthetic wastewater samples with varying concentrations of Hg2+ were prepared, and each sample was added to a separate vial. The bottom of an RT-equipped μPAD kit was placed in each vial and the analyte solution was eluted to the top of the μPAD. The μPAD was removed and dried under ambient conditions for 10 min. The results were recorded using a flatbed scanner , and part of the RT spot on the μPAD, encircling 4.0 mm from the center, was analyzed using the Photoshop program . We chose to analyze a subsection of the spot due to the change in the size of the spot to approximately 4.2–4.3 mm in diameter, caused by broadening of the wax barrier during the baking step, and to the absence of a clear boundary line between the RT spot and the wax barrier of the μPAD.The Supplementary Information


Dihydralazine and hydrochlorothiazide were stored at high temperature and humidity, under UV/Vis light and different pH, as individual drugs and the mixture. Then, a sensitive and selective HPLC-UV method was developed for simultaneous determination of dihydralazine and hydrochlorothiazide in presence of their degradation products. Finally, the degradation products were characterized through LC-DAD and LC–MS methods. Dihydralazine was sensitive to high temperature and humidity, UV/Vis light and pH ≥ 7. At the same time, it was resistant to acidic conditions. Hydrochlorothiazide was sensitive to high temperature and humidity, UV/Vis light and changes in pH. Its highest level of degradation was observed in 1 M HCl. Degradation of the drugs was higher when they were stressed in the mixture. In the case of dihydralazine, the percentage degradation was 5–15 times higher. What is more, dihydralazine became sensitive to acidic conditions. Hydrochlorothiazide was shown to be more sensitive to UV/Vis light and pH > 4. Degradation of dihydralazine and hydrochlorothiazide followed first-order kinetics. The quickest degradation of dihydralazine was found to be in 1 M NaOH while of hydrochlorothiazide was in 1 M HCl  or at pH 7–10 (hydrochlorothiazide in the mixture). A number of new degradation products were detected and some of them were identified by our LC-DAD and LC–MS methods. In the stressed individual samples, (phenylmethyl)hydrazine and 1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide were observed for the first time. Interactions between dihydralazine and hydrochlorothiazide in the mixture were confirmed by additional degradation products, e.g., 2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1,4-trioxide. Hydralazine and its derivative dihydralazine are used in the treatment of hypertension in Europe, the USA and China . BecauseAlthough two-component tablets present many advantages, they introduce the problem of interactions between the active ingredients. For example, degradation of one drug may be accelerated by another one. Interactions can also lead to generate new degradation products. Therefore, the main goal of the present study was to examine stability of dihydralazine and hydrochlorothiazide, as individuals and in the mixture, to detect such interactions using HPLC-UV, LC-DAD and LC–MS methods.Only few chromatographic procedures (HPLC) have been reported so far for determination of dihydralazine alone  or in prMore HPLC methods were elaborated for determination of hydrochlorothiazide as an individual analyte , 7 or inIn the study of Che , determiStability of hydrochlorothiazide in a solid state was tested at high temperature in the range 60–110 °C –25, 27. Thus, a new quantitative HPLC method for simultaneous determination of dihydralazine and hydrochlorothiazide in presence of their degradation products was elaborated and validated. Then, the concentration of non-degraded dihydralazine and hydrochlorothiazide as a function of degradation time was investigated, to determine kinetics of degradation. The next step was to elucidate the possible degradation pattern of these drugs using LC-DAD and LC–MS methods.The results reported here can be useful in development of a new combined formulation of dihydralazine and hydrochlorothiazide, by knowing dangerous conditions for both constituents. In addition, these data may be the starting point for further studies on new degradation products in terms of their potential toxicity.3COOH), sodium acetate (CH3COONa), hydrochloric acid, sodium chloride (NaCl), sodium tetraborate (Na2B4O7), sulphuric acid, sodium hydrogen phosphate (NaHPO4), sodium hydroxide (NaOH), kalium dihydrogen phosphate (KH2PO4) and kalium hydroxide for analysis from POCh , acetonitrile and water for LC–MS from J.T. Baker , Dihydralazinum® tablets 25 mg from Pabianickie Zakłady Farmaceutyczne  and Hydrochlorothiazidum® tablets 25 mg from Polpharma  were used. Acetate buffer was prepared with 0.2 M CH3COOH and 0.2 M CH3COONa. Phosphate buffer was prepared with 0.067 M KH2PO4 and 0.067 M Na2HPO4. Borate buffer was prepared with 0.05 M Na2B4O7 and 0.1 M NaOH. Buffers were prepared as described in Eur. Ph -3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide) l et al.  and Mahal et al.  where thm/z values 293.94 and 259.98 were detected . In addition, further products occurred after degradation in 1 M HCl (m/z 199.07), 1 M NaOH, buffers (m/z 211.07) and under UV/Vis light (m/z 169.05). Unfortunately, they were not identified by us. However, quite a new product of photodegradation of hydrochlorothiazide of m/z 275.98 was detected in the mixture hydrazine and 1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide were observed for the first time.Percentage degradation of drugs in the mixture was greater than of individual drugs under the same stress conditions. An interesting effect was observed for dihydralazine which became sensitive to acidic degradation. Increased sensitivity of both active substances to stress and potent interactions between them were confirmed by new degradation products detected, e.g., 2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1,4-trioxide.The results presented here complemented the current knowledge about degradation processes of dihydralazine and hydrochlorothiazide. These data may be the starting point for further studies on new degradation products in terms of their potential toxicity and then, for qualifying them as new related substances in pharmacopoeial monographs. These data may also serve as a starting point for designing new two-component formulations.


Pistacia vera), one of the most important commercial nut crops worldwide, is highly adaptable to abiotic stresses and is tolerant to drought and salt stresses.Pistachio  biosynthetic pathway plays an important role in salt tolerance in pistachio. Moreover, we resequence 93 cultivars and 14 wild Pistacia genome sequences should facilitate future studies to understand the genetic basis of agronomically and environmentally related traits of desert crops.Our study provides insight into genetic underpinning of local adaptation and domestication of pistachio. The The online version of this article (10.1186/s13059-019-1686-3) contains supplementary material, which is available to authorized users. With reducing agricultural acreage and human population growth, feeding the world is becoming an increasing problem. Deserts take up about one third of the land surface area of Earth, are extreme environments that are barren landscapes where little precipitation occurs, and often have dry and alkaline soils, thus have hostile living conditions for most plant and animal life . HoweverP. vera, 2n = 30, Fig. http://faostat.fao.org/). Iran and the USA were the major producers of pistachios, together accounting for 72.65% of the total world production in 2015, with the USA overtaking Iran in 2016 to become the country with the biggest pistachio production (FAOSTAT). In addition to its economic, nutritional, and medicinal values, pistachio is highly adaptable to abiotic stresses and is considered to be a species that tolerates drought and salt stresses, making it ideal for reforestation of arid and salinized zones [Pistachio  and the jasmonic acid (JA) biosynthetic pathway that are likely involved in stress adaptation. Comparative population genomic analyses revealed that pistachio was domesticated ~ 8000 years ago and that likely key genes for domestication are those involved in tree and seed size, which experienced artificial selection  and six long-insert mate-pair libraries . A draft genome of 569.12 Mb was assembled, with contig and scaffold N50 sizes of 20.69 kb and 768.39 kb, respectively. , we investigated the evolution of gene families by identifying unique and shared gene families among different plants using OrthoMCL . Among tPistacia and Citrus, rather than a very short-term evolution of pistachio domestication from the wild. Therefore, we propose that the expansion of genes in the above categories is probably related to the metabolism of organic compounds found in wild Pistacia species. Phytochemical screening of wild Pistacia species found many phytochemicals such as alkaloids, flavonoids, coumarins, sterols, tannins, terpenoids, and sesquiterpene [Next, we examined the expansion and contraction of gene families in pistachio Fig. c. As it iterpene –14.P ≪ 0.001) contains many cytochrome P450 genes, which encode proteins involved in multiple metabolic pathways with complex functions and playing important roles in multiple processes, particularly roles in stress responses. To assess the function of these genes, we used BLASTP to search the A. thaliana proteome and identified the best-hit genes  , which encodes a member of the cytochrome P450 CYP74 gene family that functions as an allene oxide synthase (AOS). This enzyme catalyzes the first committed step in the synthesis of jasmonates ) [AOS increased from nearly 0 in the control to 2163.75 under saline conditions in the leaf, and from 1.87 in the control to 87.74 for the saline-treated root  were enriched for “response to jasmonic acid” ) . The exp species . Jasmona species . Here, eP = 0.03 after correction, Additional file CHIB, EP3, ChiC, AT2G43590). Plant chitinases are involved in diverse biological systems. Some chitinases in plants are expressed in response to environmental stresses  and can be upregulated by phytohormones such as ethylene, jasmonic acid, and salicylic acid [ChiC encodes a class V chitinase, and its expression can be induced by the jasmonic acid and the stress resulting from salinity in Arabidopsis thaliana [CHIB encodes a basic chitinase involved in jasmonic acid-mediated signaling pathway [Differentially expressed genes were also found to be enriched in “chitin binding”  indicated lower diversity among the domestic accessions compared with the wild  between the cultivated and wild samples . Multiple pair-end libraries, including two types of small-insert libraries (270 bp and 500 bp) and six types of long-insert mate-pair libraries , were constructed using the Illumina paired-end and mate-pair kits according to the manufacturer’s instructions.An individual of N); (2) reads with > 10 nucleotides aligned to the adapter, allowing ≤ 10% mismatch; and (3) reads with > 50% bases having Phred quality < 5. Finally, we generated a total of 98.08 Gb and 55.87 Gb clean reads for the paired-end and the mate-pair libraries, respectively.The libraries were sequenced on the Illumina Hiseq 2500 platform. For the raw reads, sequencing adaptors were removed and contaminated reads  were screened by alignment to the NCBI NR database using BWA v0.7.13  with defP. vera. Finally, only equal or longer than 500 bp PacBio subreads were performed the gap filling. Based on the ALLPATH-LG assembly, we utilized PBJelly [https://github.com/broadinstitute/pilon) using Illumina data with the parameters “--mindepth 10 --changes --threads 4 --fix bases.”Single-molecule sequencing was performed by the PacBio Sequel System, which yielded a total of 4,038,150 filtered subreads with average lengths of 14,568 bp for  PBJelly  to do gakmer showed a major peak at 84×. Based on the total number kmers and the corresponding kmer depth of 84, the pistachio genome size was estimated to be ~ 519.17 Mb using the formula: Genome size = kmer_Number/Peak_Depth.A total of 58.80 Gb Illumina reads from the 270 bp library were selected to perform the genome size estimation. The distribution of 21-The whole genome was de novo assembled into longer contigs using ALLPATH-LG  with theAfter detecting the variation using the corresponding sequencing data, the SNP rate besides the genome heterozygosity was estimated as the error rate of the genome assembly. Completeness of the assembly was assessed by mapping 458 conserved core eukaryotic genes (CGEs) and 248 highly conserved CGEs to the genome using CEGMA v2.5 .The amount of the assembly composed of repeats was estimated by building a repeat library employing the de novo prediction programs LTR-FINDER , MITE-HuA. thaliana (TAIR 10), Oryza sativa , Theobroma cacao (Phytozome v12.1), and C. sinensis (Phytozome v12.1) genomes were aligned to our assembly to identify the homologous genes with GeMoMa v1.4.2 [E value cutoff of 1e−5 used to assess orthology.Protein-coding genes were predicted using de novo and protein homology-based approaches. Genscan v1.0 , Augustua v1.4.2 ; the RNAa v1.4.2 , and thea v1.4.2 , and suba v1.4.2 ; then, ta v1.4.2  and Genea v1.4.2 , respecta v1.4.2 . Annotata v1.4.2 , KEGG [6a v1.4.2 , NCBI-NRa v1.4.2  with an E value of 1e−10 and an identity cutoff of 95% or more. The tRNAScan-SE v2.0 [rDNA genes were identified by aligning with rRNA template sequences , and V. vinifera (Phytozome v12.1) proteomes filtered for transposable elements and alternative splicing. An all-against-all comparison was performed using BLASTP  followed by clustering with OrthoMCL v2.0.9 [P < 0.0001) on the Pfam domains.To identify homologous relationships among pistachio and related plants, the pistachio proteome was globally compared with the V. vinifera, Eucalyptus grandis (Phytozome v12.1), G. raimondii, Medicago sativa (Phytozome v12.1), Brassica rapa (Phytozome v12.1), A. thaliana, P. trichocarpa , C. sinensis, and pistachio—was constructed using PhyML software v3.0 [V. vinifera as the outgroup) was estimated using the MCMCtree program implemented in the PAML package v4.9 [http://www.timetree.org/). Expansion and contraction of OrthoMCL-derived gene clusters was determined by a CAFÉ v2.1 [E value of 1e−5, respectively. BLAST2GO [A phylogenetic tree of 9 species—are v3.0  based onage v4.9 . CalibraAFÉ v2.1  calculatBLAST2GO  was usedBLAST2GO .E value < 1e−5) was used to detect paralogous genes in pistachio, C. sinensis, V. vinifera, and P. trichocarpa as well as orthologous genes in pistachio C. sinensis and pistachio V. vinifera. Homologous blocks were then detected using Mcscan v1.1 [The all-against-all BLASTP method  and P. trichocarpa  is syntenic to the regions in another genome . We perfa (ε-WGD ) (http:/nd β-WGD ) genomesP. vera L. cv. Ohadi, and the wild type (Sarakhs) were surface sterilized with 5% solution of sodium hypochlorite in distilled water and then incubated at 30 °C on a sterile moist cloth for 1 week. Ten seeds that germinated were sown in each pot filled with about 7 kg sandy-loam soil at a depth of about 2 cm. The number of seedlings per pot was reduced to three uniform seedlings when the emergence period was completed about 3 weeks of planting.A greenhouse experiment was conducted in 2016 at the Pistachio Research Center, Rafsanjan, Iran: 30° 24′ 49.0″ N, 55° 59′ 20.8″ E, at 1528 masl. Seeds of cultivar pistachio rootstocks, −1) at field capacity. Two months after planting, when seedlings were well established, salinity treatment was imposed on half of the pots  by irrigation water with an electrical conductivity of 4.5 dS m−1 while the other half of the pots (control group) were irrigated using the Rafsanjan normal urban water throughout the entire experiment. Salinity stress continued for 1 month when the symptoms of salinity toxicity in the leaves  were observed. Leaf samples were taken before applying the salinity stress on the seedlings at 2 months of age. Both the leaf and root samples from all replicates were taken at the end of the experiment when the seedlings were 3 months of age. The fresh leaf and root tissue samples were immediately dissected and submerged in five volumes of RNAlater® solution and stored in a − 80 freezer for subsequent RNA extraction. During the experiment, the maximum temperature was 34 ± 5 °C; the minimum temperature was 21 ± 4 °C, and the relative humidity was 40 ± 5%.The experiment consisted of a completely randomized design with three replications for a total of six pots. Irrigation of all pots was carried out for 2 months using Rafsanjan normal urban water . Before library construction, we evaluated the degradation of the RNA on a 1% agarose gel and checked the purity using the Qubit® 3.0 Fluorometer , with integrity and concentration assessed using an RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system . Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations and then sequenced on the Illumina HiSeq X Ten platform to generate 150 bp paired-end reads.Pistacia vera L. using TopHat  and the (v2.1.1 . Differe (v2.1.1 .P. vera L.), International Plant Genetic Resources Institute, Rome, Italy). The following phenotypes were recorded: dried pistachio fruit weight (gr) , fresh fruit weight with green skin (gr) , dried pistachio fruit length (mm) , dried pistachio fruit diameter (mm) , dried pistachio fruit width (mm) , dried pistachio fruit and kernel shape , dried kernel weight (gr) , kernel diameter (mm) , kernel width (mm) , kernel length (mm) .Fruit size-related traits for pistachio were measured based on the pistachio descriptor (IPGRI. 1997. Descriptors for pistachio (Fresh leaves (4–5 g) were sampled from the germplasm collections of the Pistachio Research Institute in Rafsanjan, Iran: 30° 24′ 49.0″ N, 55° 59′ 20.8″ E, at 1528 masl; the pistachio germplasm of Ardakan, Iran: 32° 18′ 36″ N, 54° 1′ 3″ E, at 1040 masl; and Jiroft, Iran: 28° 40′ 41″ N, 57° 44′ 26″ E, at 650 masl. Leaf tissues were harvested during the 2015–2017 period and were transported on ice and stored at − 80 °C in the Biotechnology Laboratory, Animal Science Department, Shahid Bahonar University of Kerman, Iran, until subjected to DNA extraction.Total genomic DNA was extracted from 1 g fresh leaves using hexadecyl trimethyl ammonium bromide (CTAB) protocol with some modifications. The quantity and quality of isolated DNA were assessed by NanoDrop spectrophotometer and 1% agarose gel electrophoresis, looking for a single absorbance peak at 260 nm, a 260/280 absorbance ratio of 1.8–2.0, and no evidence of substantial band shearing or contamination. Extracted DNA was dissolved in 20 μl TE buffer  and stored at − 20 °C for subsequent NGS analysis.P. mutica, 13 P. khinjuk, 4 P. integerrima, and 5 P. palaestina were chosen for genome resequencing. Ten micrograms of genomic DNA, prepared by the standard CTAB extraction protocol, was used to construct libraries with 350 bp insert size. Sequence libraries were constructed according to the Illumina library preparation pipeline and were sequenced on the Illumina Hiseq 4000 platform to generate 150 bp paired-end reads.Among the above samples, 93 domestic and 14 wild pistachio individuals and another 13 http://picard.sourceforge.net), we called SNPs using Genome Analysis Toolkit (GATK) [We mapped the reads to our reference genome (version 1) with BWA-MEM . After st (GATK) . The crip-distance matrix by TreeBeST (http://treesoft.sourceforge.net/treebest.shtml) with 100 bootstrap replications. Principal component analysis (PCA) was performed using the toolset SNPRelate from the R package. We ran frappe to estimate the genetic ancestry of each sample, specifying a range of 2–5 hypothetical ancestral populations. The maximum number of interaction was set to 10,000 in the frappe analysis.We constructed a neighbor-joining tree using a FST [θπ and Watterson’s estimator θw ratios (windows with a number of variants < 20 were ignored) [θπ and FST. Our approach was to identify genomic regions with high differentiation between cultivated and wild Pistacia vera .We calculated the genome-wide distribution of population fixation statistics FST  and nuclignored)  for eachD statistic (ABBA-BABA test) [We inferred gene flow between the diverged populations using the maximum likelihood method implemented in TreeMix . First, BA test) .P. vera, we only considered SNPs with more than 40-fold sequencing coverage at the population level in intergenic regions to ensure their neutrality. Missing genotypes were imputed using the program BEAGLE [P. vera. The model with the maximum log-likelihood value was chosen as the optimal one.To uncover the recent demographic history of the cultivated and wild m BEAGLE . After iPistacia species were inferred using the PSMC program [Dynamic changes in the effective population size of  program , with a  program .https://david.ncifcrf.gov/) and g: profiler (https://biit.cs.ut.ee/gprofiler/).Gene Ontology (GO) enrichment analyses were performed using the DAVID program Additional file 2:Tables S1-S24. Supplementary tables supporting the manuscript. (XLSX 281 kb)


Meloidogyne aegracyperi n. sp. is described from roots of purple nutsedge in southern New Mexico, USA. Mature females are small (310–460 µm), pearly white, with their egg masses completely contained inside root galls. The neck is often at a 90 to 130° angle to the protruding posterior end with the perineal pattern. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet is relatively long (4.0–6.1 µm), and the excretory pore is level with the base of the stylet. The anterior portion of the rounded lumen lining of the metacorpus contains 3 to 10 small vesicles. The perineal pattern has a rounded dorsal arch with a tail terminal area that is smooth or marked with rope-like striae. Only two males were found. The body twists 90° throughout its length. The DGO to the base of the stylet is long (3.0–3.3) µm. The cephalic framework of the second-stage juvenile is weak, and the stylet is short (10.1–11.8 µm). The DGO to the base of the stylet is long (3–5 µm). The tail is very long (64–89 µm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Eggs are typical for the genus and vary in length (85.2–99.8 µm) and width (37.1–48.1 µm), having a L/W ratio of (2.1–2.6). Maximum likelihood phylogenetic analyses of the different molecular loci  rRNA, cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA gene fragments and partial Hsp90 gene) placed this nematode on an independent branch in between M. graminicola and M. naasi and a cluster of species containing M. chitwoodi. M. fallax, and M. minor. Greenhouse tests showed that yellow and purple nutsedge were the best hosts, but perennial ryegrass, wheat, bentgrass, and barley were also hosts. Cyperus esculentus L.) and purple nutsedge (C. rotundus L.) are perennial weeds of global importance that can enhance survival and population densities of Meloidogyne incognita  Chitwood, 1949 and result in injury to crops . Subsequent dissection of the galls revealed small, mature Meloidogyne females and egg masses that were primarily contained inside the root tissue. Eggs were recovered from roots of purple nutsedge, but not that of tomato following extraction with sodium hypochlorite. Additional pots of purple nutsedge and Rutgers tomato that were established in pasteurized soil using surface-sterilized nutsedge tubers and inoculated with eggs recovered from galled purple nutsedge roots showed similar results: root galling and egg recovery from nutsedge, but no galling or egg recovery from tomato.Yellow nutsedge  were extracted from washed galled roots incubated in a moist chamber. Light microscopy (LM) observations were made from specimens mounted on 5% water agar pads and paralyzed with 0.1 m sodium azide . FemalesMedicago sativa L.) cv. Doña Ana, chile pepper (Capsicum annuum L.) cv. NM 6-4, corn (Zea mays L.) DynaGro 58UP30, cotton (Gossypium hirsutum L.) cv. Deltapine 393, onion (Allium cepa L.) cv. nuMex Dulce, sorghum (Sorghum bicolor (L.) Moench.) cv. Sordan Headless, common oats (Avena sativa L.), winter rye , perennial ryegrass (Lolium perenne L.) cv. Barlennium, wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) cv. Robust, tomato (Solanum lycopersicum L.) cv. Rutgers, purple nutsedge (Cyperus rotundus L.), yellow nutsedge (Cyperus esculentus L.) were transplanted as single plants into 11 cm diameter clay pots containing 500 cm3 of sterilized sandy loam soil. They were inoculated with a suspension of 3,000 freshly hatched juveniles in 50 ml of water in holes around the root system. Each treatment was replicated 10 times and the plants were kept in a greenhouse at 22 to 28°C. Roots were washed with tap water and stained with phloxine B  tubes containing 20 μl of lysis buffer  and stored at −20°C.Lysis was performed using a rapid single tube lysis procedure . Briefly, samples were removed from −20°C and incubated at 90°C for 10 min. After heating, 30 μl of Proteinase K digestion mixture was added to each sample , 3 μl of 10× Platinum Taq DNA Polymerase PCR Buffer , and 22 μl water per sample). Samples were then sonicated for 8 min in a Branson 2510 ultrasonic cleaner then incubated for 30 min at 60°C. After Proteinase K digestion samples were frozen at −80°C for 10 min then incubated at 90°C for 10 min. After heating, 50 μl of water was added to each tube and samples were well mixed then stored at −20°C prior to PCR.Hsp90) markers with primers used for amplification and sequencing listed in Hsp90 gene  and the reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers were used . For ampSystematicsMeloidogyne aegracyperi n. sp.; Table 2Mature females with their egg masses are usually contained completely inside galled root tissues. They are very small (373 µm long) and pearly white. Their body shape is unique from many other species because the neck is often at a 90 to 130° angle to the protruding posterior end that contains the perineal pattern. Lip region low, cephalic framework weakly developed, with one head annule. The cone of the stylet slightly curved dorsally, posterior edges of the knobs angular, and tapering onto the shaft. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet relatively long (4–6 µm). Excretory pore level, with the base of the stylet, is present. The lining of the metacorpus triradiate, with the posterior and anterior portions rounded. Numerous (3–10) small vesicles present in the anterior metacorpus. Two, small, rounded esophago-intestinal cells at the base of the metacorpus, followed by a large nucleated dorsal esophageal gland lobe with two smaller nucleated subventral esophageal gland cells. The didelphic ovary is typical for the genus. Six, large rectal gland cells connect to the rectum and produce the gelatinous matrix forming the egg mass. The perineal pattern is raised on a protuberance at the posterior end of the body. It contains a rounded dorsal arch with a tail terminal area that is usually smooth, but may be marked with thick lines and many horizontal, rope-like striae. Phasmids are typical for the genus. The vulval lips are usually flattened, but they may be rounded and slightly protruding. Smooth, regular striae surround the vulva and tail terminal area and give the appearance of a dorso-ventrally elongated oval pattern.The characteristics are as follows: anterior end tapering, labial disc slightly concave around the stoma, one distinct head annule, cephalic framework slight, stylet shaft tapering posteriorly. Body twisting 90° throughout its length. Stylet knobs rounded and set-off from the shaft. The distance of the DGO to the base of the stylet is long (3–3.3 µm). Esophageal glands overlapping the intestine ventrally. Four lines in the lateral field. Paired spicules with gubernaculum are typical for the genus. Tail tip slightly set-off from the remainder of the body.It has a body with a very long tail and tail terminus. Cephalic framework is weak, stylet is small, with a constriction near the junction of the shaft and knobs. In SEM, the head has a slit-like oral opening placed on the rounded labial disc and surrounded by six small pit-like openings of the inner labial sensilla. Small depressions in the cuticle on the dorsal and ventral lip pairs mark the outer labial sensilla. Rounded knobs, tapering onto the shaft, are present. The distance of the DGO to the base of the stylet is long (3–5 µm). Esophageal glands overlap the intestine ventrally. Four lines in the lateral field are present. The tail is very long (64–89 µm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Phasmids are located midway between anus and tail tip.Eggs are typical in shape for the genus and vary in length [91.6 ± 2.3 (85.2–99.8 µm)] and width [39.7 ± 0.1 (37.1–48.1 µm)], having a L/W ratio of .Comparisons of males of these three species reveal that the DGO is very similar (3–3.1 µm), but the length of the stylet is quite dissimilar [si=18 µm ; and M. M. aegracyperi n. sp., M. naasi, and M. graminicola are very different (M. aegracyperi n. sp., and M. naasi as reported by M. naasi, but M. aegracyperi n. sp., unlike the ambiguous results reported for M. naasi. The questions remain: Were Radewald and Allen working with the same species, or does M. naasi occurs as several different host races (The host ranges of ifferent . The comst races ?M. aegracyperi n. sp. and M. graminicola include yellow and purple nutsedge and wheat; however, M. graminicola parasitizes onion, common oats, and sorghum (M. aegracyperi n. sp. cannot.The common hosts of  sorghum , but M. Meloidogyne aegracyperi n. sp. is morphologically similar to M. naasi, but it can be distinguished as a unique species based on features of the female, male, and second-stage juvenile. Superficially, the gross morphology of the second-stage juvenile and similar appearance of the perineal pattern of the female could cause a wrong identification of M. aegracyperi n. sp. as M. naasi. However, measurements of the stylets, DGO, body length, tail length, and hyaline tail terminus easily separate these two species. Meloidogyne aegracyperi n. sp. appear to be closely related phylogenetically to M. graminicola and M. naasi according to the trees that were drawn based on similarities of DNA sequences of the 18s rRNA, D2-D3 region of rRNA, ITS region, COII-16S, and Hsp90 genes. Likewise, their host ranges feature some common species including oats, barley, bentgrass, and wheat (M. naasi from M. aegracyperi n. sp.nd wheat , but hos


Background: The pangolin is a Pholidota mammal with large keratin scales protecting its skin. Two pangolin species (Manis pentadactyla and Manis javanica) have been recorded as critically endangered on the International Union for Conservation of Nature Red List of Threatened Species. Optical mapping constructs high-resolution restriction maps from single DNA molecules for genome analysis at the megabase scale and to assist genome assembly. Here, we constructed restriction maps of M. pentadactyla and M. javanica using optical mapping to assist with genome assembly and analysis of these species.Findings: Genomic DNA was nicked with Nt.BspQI and then labeled using fluorescently labeled bases that were detected by the Irys optical mapping system. In total, 3,313,734 DNA molecules (517.847 Gb) for M. pentadactyla and 3,439,885 DNA molecules (504.743 Gb) for M. javanica were obtained, which corresponded to approximately 178X and 177X genome coverage, respectively. Qualified molecules (≥150 kb with a label density of >6 sites per 100 kb) were analyzed using the de novo assembly program embedded in the IrysView pipeline. We obtained two maps that were 2.91 Gb and 2.85 Gb in size with N50s of 1.88 Mb and 1.97 Mb, respectively.Conclusions: Optical mapping reveals large-scale structural information that is especially important for non-model genomes that lack a good reference. The approach has the potential to guide de novo assembly of genomes sequenced using next-generation sequencing. Our data provide a resource for Manidae genome analysis and references for de novo assembly. This note also provides new insights into Manidae evolutionary analysis at the genome structure level. Manis pentadactyla and Manis javanica belong to the Asian Pangolin subfamily and have long been used as an ingredient in traditional medicine in China and southeast Asia, and their colonies and habitats have thus largely been destroyed by poaching and deforestation. These two species are on the verge of extinction; they are currently recorded as critically endangered on the International Union for Conservation of Nature Red List of Threatened Species.Pangolins are the sole representatives of order Pholidota. They are a group of nocturnal mammals that are well-known for their full armor of scales. Only eight pangolin species exist worldwide, which have been distributed into three genera  according to Gaudin et al. . Manis pde novo map assembly or sequencing contig anchoring. To date, optical mapping has facilitated or improved assembly arrays of large genomes, including humans [Oropetium thomaeum [Ganoderm lucidum [Manis maps, and we reveal and compare their genetic structures using pairwise sequence alignments to identify their interspecific variations.Optical mapping is a molecular tool for chromosome-wide restriction map production . During g humans , 5, Oropthomaeum , and Gan lucidum . In addi lucidum . Here, wM. pentadactyla and M. javanica blood samples. A total of 3 ml of blood from an orbital sample was anticoagulated with EDTA and then shipped on ice. A total of 9 ml of red blood cell lysis solution was mixed with each 3 ml sample and rocked gently at room temperature for 10 min. The mixture was spun at 2000 × g at 4 °C for 2 min, and the supernatant was discarded. The pellet was suspended in 3 ml of phosphate-buffered saline. After removing the insoluble particulates, the mixture was spun again. The supernatant was discarded and the pellet was resuspended in 563 μl of refrigerated cell suspension buffer at a density of ∼0.5 × 107 cells/ml. For gel casting, 75 μl of resuspension buffer was mixed with 25 μl of preheated 2% agarose, and the gels were solidified at 4 °C for 45 min. The gel casts were immersed in 5 ml of lysis buffer  at 50 °C for 2 days for cell lysis. The cell-lysed gels were washed with 1× Tris-EDTA; then, the immobilized DNA was recovered by melting the gels at 70 °C for 10 min, followed by incubation with GELase  at 43 °C for 45 min. The recovered DNA was drop dialyzed against Tris-EDTA for 4 h using a 0.1-μm membrane. The dialyzed DNA was quantified and stored for nicking.All animal studies were reviewed and approved by the animal ethics review committee of Guangdong Provincial Hospital of Chinese Medicine. High molecular weight DNA was isolated from M. pentadactyla and M. javanica, representing 178X and 177X coverage of their predicted genomes, respectively. Further methodological details available from protocols.io [Prior to molecular nicking, the DNA was equilibrated at room temperature for 30 min and gently mixed with wide bore tips. In a 10-μl reaction system, 300 ng of equilibrated DNA was added to 7 units of Nt.BspQI  nickase and 1 μl of nicking buffer and mixed. The nicking process was conducted in a thermal cycler at 37 °C for 2 h. The nicked DNA was incubated with 5 μl of labeling master mix containing 1.5 μl of labeling buffer , 1.5 μl of labeling mix , and 1 μl of Taq polymerase  to flag specific motifs. The labeling process was conducted at 72 °C for 1 h. Each labeled DNA solution was mixed with 15 μl of Repair Master Mix containing 0.5 μl of 10 Thermo polymerase buffer , 0.4 μl of 50× repair mix , 0.4 μl of 50 mM NAD+ , 1.0 μl of Taq DNA polymerase , and 2.7 μl of ultrapure water for nick repair. The repair reaction was conducted at 37 °C for 30 min, followed by the addition of 1 μl of stop solution  to stop the reaction. After ligating the nicks, the backbone of the labeled DNA was stained with IrysPrep DNA stain solution  at 4 °C overnight. The prepared samples were loaded onto Irys chips  and then applied to the chip nano-channels. The concentration time was 400 s to avoid over-staining or over-loading. The fluorescently labeled DNA was illuminated by the corresponding laser, and the signal was captured by an onboard electron microscope charge-coupled device camera .P-value thresholds were 1 × 10−9 for the pairwise assembly, 1 × 10−10 for the extension and refinement steps, and 1 × 10−11 for the final merging. The false positive and false negative parameters were set to 1.5/100 kb and 0.15/100 kb. Finally, 2202 maps spanning 2.91 Gb of the genome were assembled for M. pentadactyla and 2096 maps spanning 2.85 Gb of the genome were assembled for M. javanica, with N50 lengths of 1.884 Mb and 1.972 Mb, respectively , Guangdong Provincial Hospital of Chinese Medicine Special Fund (2015KT1817), and China Academy of Chinese Medical Sciences Special Fund for Health Service Development of Chinese Medicine (ZZ0908067).CSL, HZH and XJ designed the project. HZH, XSM, and GY prepared the samples. XJ, LBS, and ZCC performed the experiments. LBS, XJ, XSM, and HZH analyzed the data. CSL, XJ, HZH, LBS, and QXH wrote the manuscript.GIGA-D-16-00053_Original Submission.pdfClick here for additional data file.GIGA-D-16-00053_Revision 1.pdfClick here for additional data file.GIGA-D-16-00053_Revision 2.pdfClick here for additional data file.Response To Reviewer Comments Original Submission.pdfClick here for additional data file.Response To Reviewer Comments Revision 1.pdfClick here for additional data file.Reviewer 1 Report .pdfClick here for additional data file.Reviewer 1 Report (Revision 1).pdfClick here for additional data file.Reviewer 2 Report .pdfClick here for additional data file.


Candidatus Neoehrlichia mikurensis” is a gram-negative bacterium belonging to the family Anaplasmataceae that, in Europe, is transmitted by Ixodes ricinus ticks. “Candidatus N. mikurensis” can cause a severe systemic inflammatory syndrome, neoehrlichiosis, mostly in persons with other underlying diseases. To date, “Ca. N. mikurensis” has been found in ticks in different countries in Asia and Europe, but never as far north as at the Arctic Circle.“I. ricinus ticks collected from vegetation and from animals in northern Norway (64–68°N) were analysed for the prevalence of “Ca. N. mikurensis”. Of them, 495 ticks were collected from vegetation by flagging and 609 ticks were collected from dogs and cats. Total nucleic acid extracted from the ticks were converted to cDNA and analyzed with real-time PCR targeting the 16S rRNA gene of “Ca. N. mikurensis”. Positive samples were further analysed by nested PCR and sequencing.A total of 1104 Candidatus N. mikurensis” was detected in 11.2% of all collected I. ricinus ticks in northern Norway. The prevalence differed between ticks collected from vegetation  compared to ticks collected from dogs and cats . The ticks from dogs and cats were collected in Brønnøy area and seven additional districts further north. The prevalence of “Ca. N. mikurensis” in these ticks differed between geographical localities, with the highest prevalence in the Brønnøy area.“Ca. N. mikurensis” in I. ricinus ticks from the Arctic Circle in northern Norway indicates potential risk for tick-bitten humans at this latitude to be infected with “Ca. N. mikurensis”.The detection of “ Candidatus Neoehrlichia mikurensis” is a relatively recently discovered tick-borne pathogen that has been shown to cause a severe systemic inflammatory syndrome, neoehrlichiosis, mostly in persons with other underlying diseases. The first description of the bacterium was published as late as 1999 , Pearson’s Chi-square test was used, and P < 0.05 was regarded as significant.Statistical analyses were performed with IBS software SPSS Statistics version 24. For comparison of “Ca. N. mikurensis”. Of them, 124 (11.2%) were positive for “Ca. N. mikurensis” using the real-time PCR assay  were positive for “Ca. N. mikurensis”. The percentage of positive samples was slightly higher among those from dogs, 7.0% (22/314), compared to 4.1% (12/295) of the ticks from cats, but the difference was not statistically significant .A total of 609 samples from Ca. N. mikurensis”-infected ticks was highest, with 10.3% of samples positive. The higher prevalence in Brønnøy compared to all the other geographical areas taken together were significant . After Brønnøy, ticks collected in Fauske had the second highest proportion of positive samples, 6.2% (2/32). “Candidatus N. mikurensis”-positive samples were also found in ticks collected in Sandnessjøen, Bodø and Mosjøen, but with a lower prevalence . In ticks from dogs and cats collected in the other places in the study, all test results were negative (Table Most of the ticks (261/609) from dogs and cats were collected in the Brønnøy area and it was also in Brønnøy that the prevalence of “I. ricinus ticks collected from dogs and cats, 84.6% (515/609) were adult females, 11.7% (71/609) were adult males, 0.7% (4/609) nymphs and 3.1% (19/609) ticks were of uncertain life stage and sex. The prevalence of “Ca. N. mikurensis” was lowest in adult females, 4.5% (23/515), followed by unidentified stages 10.5% (2/19) and then adult males 11.3% (8/71). Of the four nymphs, one was positive for “Ca. N. mikurensis”  had a simultaneous infection with B. burgdorferi (s.l.). Of the total 609 ticks collected from dogs and cats the proportion of coinfected was 1.6% , followed by adult males, 16.0% (79/495), adult females, 13.3% (66/495) and larvae 6.5% (32/495). The total proportion of “Ca. N. mikurensis”-positive ticks collected by flagging amounted to 18.2% (90/495).From the nøy 2011 , a total: χ2 = 7.032, df = 3, P = 0.071) in the proportion of “Ca. N. mikurensis” positive ticks collected from vegetation between different life stages and sexes. Of the adult female ticks, 19.7% (13/66) were positive for “Ca. N. mikurensis”, of the adult male ticks 13.9% (11/79) and of the nymphs 20.4% (65/318). Of the samples derived from larvae, one sample  was positive for “Ca. N. mikurensis” in the real-time PCR assay, but it could not be confirmed by sequencing (Table There was no significant difference (Chi-square testB. burgdorferi (s.l.). and “Ca. N. mikurensis”  [Ca. N. mikurensis”-positive female ticks had a simultaneous B. burgdorferi (s.l.) infection, while 15.4% (2 of 13) did not. For male ticks, the corresponding proportion was 81.8% (9/11) coinfected and 18.2% (2/11) with only a “Ca. N. mikurensis” infection.Of the total 495 ticks collected by flagging from the vegetation in Brønnøy, 8.5% 42/495) had a co-infection with both /495 had i (s.l.) . Among tCa. N. mikurensis” infection occurs in one of the world’s northernmost populations of I. ricinus ticks, collected in the area surrounding the Arctic Circle in northern Norway. The study has shown that “Ca. N. mikurensis” is found in ticks from northern Norway and that the prevalence is higher in ticks collected from the vegetation compared to ticks that have fed from dogs and cats. The study has also shown that co-infection with B. burgdorferi (s.l.) and “Ca. N. mikurensis” occurs and that it is more common in ticks collected from the vegetation.The purpose of this study was to investigate whether “Ca. N. mikurensis” prevalence was significantly higher in ticks collected in Brønnøy compared to ticks from the more northern districts, as has been shown for the Borrelia prevalence in a previous study [I. ricinus ticks north of 65°N latitude, and that these ticks are believed to have been spread recently with migratory birds [Ca. N. mikurensis” prevalence is higher in ticks collected from the vegetation than in those collected from dogs and cats. The two separate groups differ in terms of the distribution between life stages and sex, which may be a confounding factor, but it is still hard to determine a single reason that completely explains the difference. One possible explanation may be that the number of Borrelia bacteria in ticks is influenced by the duration of tick feeding [Borrelia bacteria compared to ticks with shorter duration of feeding. A possible reason for this is that the Borrelia pathogens migrate from the tick’s gut to its salivary glands and thereafter the pathogen is transferred from the tick to its host. We do not know if the same applies to “Ca. N. mikurensis”, but if it does it could explain the difference in prevalence between unfed ticks collected from the vegetation compared to fed ticks from dogs and cats. Previous studies from Europe [Ca. N. mikurensis” shows a strong seasonal variation. The two examined tick materials were collected at different times . This may be another explanation for the difference in prevalence. In any case, the different collection periods make it somewhat harder to draw firm conclusions about the reasons for a higher “Ca. N. mikurensis” prevalence in the ticks collected from the vegetation.In the material that came from dogs and cats, the “us study . One posry birds . But eve feeding . Ticks tm Europe  indicateCa. N. mikurensis”-positive samples differed slightly between the different tick stages, with a somewhat lower proportion of positive samples from adult ticks, but the differences were relatively small. The significant difference found between life stages in the tick material collected from dogs and cats was based on a low number of ticks, and no firm conclusions can be drawn. A previous study from Sweden [Ca. N. mikurensis”, while a study from the Czech Republic [Ca. N. mikurensis” infection is twice as common in nymphs removed from humans compared to adult ticks.The prevalence of “m Sweden  indicateRepublic  shows thCa. N. mikurensis” by real-time PCR assay. A positive finding in a larva indicates that “Ca. N. mikurensis” may be transovarially transmitted or that the larva was interrupted during feeding on an infected host. Although unlikely, we cannot exclude the possibility that this tick specimen was incorrectly classified as a larva before analysis. However, our result remains equivocal because “Ca. N. mikurensis” infection in the tick could not be confirmed by sequencing.Among the ticks collected from the vegetation in Brønnøy, one larva was positive for “B. burgdorferi (s.l.) or “Ca. N. mikurensis” or both differed between the ticks collected from dogs and cats and the ticks collected by flagging. A previous study performed on I. ricinus ticks collected in different parts of the Netherlands [Ca. N. mikurensis” and B. afzelii occur significantly more than random; in this study, a co-infection of “Ca. N. mikurensis” and B. burgdorferi (s.l.) was common. In a study [Ca. N. mikurensis” and other simultaneous infections in ticks, it is proposed that a co-infection is probably due to that the original rodent host has several infections at the same time and not that the tick would have different infections from more than one host animal. There are studies suggesting that humans infected with both Borrelia and Anaplasma phagocytophilum can have a risk for more severe disease [B. burgdorferi (s.l.) and “Ca. N. mikurensis”. However, other studies have shown contradictory results [The proportion of ticks that had either an infection with herlands  has show a study  investig disease , 24. Pos results .A large number of samples has been analysed in this study and it is likely that the results are representative for ticks in the examined area in northern Norway. A possible limitation in the study is that all analyses have been conducted on single replicates. Analyses of sample duplicates or triplicates would have increased the certainty of the results. However, this was compensated for by the fact that all samples have undergone two consecutive tests, both real-time PCR and a nested PCR.Ca. N. mikurensis” infection is present in up to 18.2% of I. ricinus ticks in different parts of northern Norway. The result is interesting since “Ca. N. mikurensis” can be transmitted to humans after a tick-bite and can then cause severe disease in immunosuppressed persons. Physicians who meet persons with unexplained fever, skin rashes and thromboembolic events should have neoehrlichiosis in mind and ask for previous tick-bites, especially in cases of underlying autoimmune, haemolytic diseases or immunosuppressive treatment.The present study has shown that “


Goat “sarapatel” is a nutritive food, with each 100 g providing, on average, 72 g of moisture, 2 g of ash, 18 g of protein, 9 g of lipids, 2 g of carbohydrates, 282 mg of cholesterol, and high amounts of unsaturated fatty acids and essential amino acids. The analysis of the “sarapatel” samples shows that none of them contain Salmonella spp. or L. monocytogenes. High counts (>104) of total coliforms, thermotolerant coliforms, and sulfite-reducing Clostridium were detected, and coagulase-positive Staphylococcus was found in 31.25% of samples. The storage conditions evaluated  did not affect the physicochemical quality of the “sarapatel”; however, the unsatisfactory microbiological quality indicates that it is necessary to improve the health-sanitary aspects of the processing and sale of this product.Goat “sarapatel” is a product made from blood and viscera. For the first time, the microbiological and nutritional quality of “sarapatel” samples ( Goat slaughter by-products are divided into two categories, edible and inedible. Edible by-products, such as blood and viscera, are intended for direct human consumption (fresh and semi-processed) or for the manufacture of meat products, whereas inedible products are used in the preparation of animal feed, cosmetics, and pharmaceuticals ,2. In thIn Brazil, sheep and goat viscera and blood are frequently used in the preparation of regional dishes such as “buchada,” which is chopped meat, and “sarapatel,” in which the heart, lung, liver, intestines, and stomach are the main organs used in the preparation.Goat “sarapatel” is a dish prepared with edible non-carcass components, including organs, viscera, and blood. This typical recipe of the Brazilian northeast is widely consumed and recognized as an important economic means of adding value to the by-products generated from goat slaughter. However, the formulation of this product is not standardized, which can lead to wide variation in composition and, consequently, nutritional quality. Goat viscera and blood have protein and lipid contents similar to those found in goat meat and are excellent sources of minerals, particularly iron, phosphorus, and vitamins ,5. HowevFew studies have focused on the evaluation of the health and nutrition quality aspects of processed meat products made with goat blood and viscera, yet such products are often cited as viable alternatives for marketing and for income generation in the goat production chain. Previous studies conducted by our research group have found a high potential for the reuse of goat slaughter by-products in foods such as paté , goat “bSalmonella spp. and L. monocytogenes in all goat “sarapatel” samples evaluated. However, 15 samples (31.25%) showed counts of coagulase-positive Staphylococcus above the limits (5 × 102 CFU/g) established by the Brazilian legislation , with respect to blood-based products and processed derivatives) [4 to > 1.1 × 105 MPN/g), thermotolerant coliforms (2.4 × 104 to > 1.1 × 105 MPN/g) and sulfite-reducing Clostridium (3.2 × 10³ to > 9.8 × 105 MPN/g) were found in all samples , which pose a potential risk to public health. The formation of spores by these bacteria allows survival under processing and storage conditions, if other extrinsic conditions  are conducive. Goat “sarapatel” samples showed high Aw (0.97 and 0.99), a pH close to neutral (4.68 to 6.43), and low acidity (0.49 to 0.86), conditions which favor bacterial contamination [et al. [All samples showed high counts of sulfite-reducing mination . Drosino [et al.  and Guer [et al.  reportedp < 0.05) in their chemical composition parameters  were predominant (58%) in goat “sarapatel” samples, among which acids C18:0 (26.80 to 31.15%), C16:0 (21.88 to 23.76%) and C14:0 (2.15 to 2.70%) stood out. The high SFA concentrations found in goat “sarapatel” samples may be related to biohydrogenation in the rumen of ruminant animals, which is a natural process carried out by ruminal microorganisms that reduces the deleterious effect of lipids in these tissues by promoting the lysis of esterified lipids, with subsequent hydrogenation of free fatty acids .trans fatty acids. Information on the fatty acid profile of goat slaughter by-products is generally scarce and is nonexistent for goat “sarapatel,” but it was observed that goat “sarapatel” had a fatty acid percentage similar to those reported for goat “buchada” [Among the detected fatty acids, monounsaturated fatty acids (MUFA), such as oleic acid—C18:1 ω-9c (24.22% to 33.41%) and vaccenic acid—C18:1 n11t (2.5 to 3.8%), also stood out, with percentages ranging from 30.97 to 40.05. Goat “sarapatel” samples also showed some content (from 4.4% to 8.5%) of polyunsaturated fatty acids (PUFA), especially conjugated linoleic acids—C18:2 ω-6 (2.33 to 5.22%) and rumenic acid—C18:2 9c, 11t-CLA (0.39 to 0.48%), which are considered essential fatty acids for humans. These acids not only play a role in the constitution of tissues of the central nervous system but also act in the prevention of cardiovascular, autoimmune and inflammatory diseases, in addition to their anti-cancer activity . Vaccenibuchada” ,21.et al. [The PUFA/SFA ratio in goat “sarapatel” (0.07 to 0.13) was similar to those reported by Santos et al.  for goatet al. . Howeveret al. , ranged et al. . In addiet al. [These values were similar to those detected for sheep meat. According to Turan et al. , the AI The cholesterol levels in the goat “sarapatel” samples ranged from 218.98 mg/100 g to 332.73 mg/100 g, which can be explained by the fact that cholesterol is synthesized in organs such as the liver and kidneys , which aEdible goat meat by-products are recognized as having high amounts of protein (approximately 20 g/100 g) and balanced amino acid profiles, similar to those of meat protein . The amoGoat “sarapatel” showed no limiting amino acids, and the chemical average scores were above 1.0 for all essential amino acids . Previoui.e., in street markets where it is exposed to temperatures ± 30 °C (PS1); in street market where it is refrigerated at ± 10 °C (PS2); in butcher shops where it is frozen at ± −7 °C (PS3); in butcher shops where it is refrigerated at ± 10 °C (PS4); in supermarkets where it is frozen at ± −7 °C (PS5); and in supermarkets where it is refrigerated at ± 10 °C (PS6). Based on this information, goat “sarapatel” samples were collected on a weekly basis, totaling 48 samples, and microbiological and nutritional studies were performed on eight samples for each selling condition of the product (PS1 to PS6).According to a survey performed in the city of João Pessoa, Paraíba, Brazil, goat “sarapatel” is sold in three types of shops and under different storage conditions, Staphylococcus (CFU/g), sulfite-reducing Clostridium (CFU/g) and the presence of Salmonella spp and Listeria monocytogenes according to methodology described by the APHA [Goat “sarapatel” samples were analyzed for total coliforms (MPN/g) and thermotolerant coliforms (MPN/g), coagulase-positive the APHA . pH, metthe APHA  and watethe APHA  were detThe goat “sarapatel” was analyzed for its proximate composition, cholesterol content, and fatty acid and amino acid profiles. The moisture, ash and protein contents were determined using the methodology described in items 950.46.41, 928.08 and 920 153, respectively, of the AOAC . The carThe fatty acid profile of the ether extract was submitted to methylation according to the methodology described by Hartman and Lago , and theTo quantify the levels of total cholesterol, isocratic elution was performed on a liquid chromatography  coupled with an Inertisil C18 column (4.6 mm × 150 mm × 5 mm); chromatographic separation was performed at constant flow (1 mL/min) at 30 °C with a run time of 10 min according to the methodology described by Bragagnolo and Rodriguez-Amaya .The amino acid profile was determined according to the methodology described by White, Hart and Fry , in whicp ≤ 0.05 [The experiment was conducted with a completely randomized design. Nutritional assessment data were submitted to analysis of variance (ANOVA), and means were compared by the Tukey test with p ≤ 0.05 .The evaluated goat “sarapatel” samples had high nutritional quality, especially when considering the protein content and the amino acid and fatty acid profiles, but there was wide variation in these parameters, revealing a lack of standardization in the preparation process. The results of microbiological analyses revealed inadequate health-sanitary conditions, which pose a potential risk to consumer health and indicate the need for the adoption of measures that will improve the health-sanitary conditions during the processing, storage and marketing of goat “sarapatel.” In conclusion, the development of products based on goat slaughter by-products, particularly goat “sarapatel,” is an interesting opportunity to increase the range of goat products for use in human nutrition and to reuse these usually discarded by-products.


The aim of this study was to investigate bile acids in liver bile of patients with benign and malignant biliary stenosis and controls without stenosis. A total of 73 patients entered the study: 7 subjects with benign biliary stenosis , 21 with malignant biliary stenosis , and 45 patients without biliary stenosis ; out of those, 25 subjects have and 20 do not have choledocholithiasis. Twenty-three different bile acids were investigated by high-performance liquid chromatography/mass spectrometry. Serum total bilirubin was significantly higher in patients with malignant biliary stenosis compared with nonstenotic controls (p = 0.005). Significant relationship (r > 0.7) was found between several pairs of bile acids. Significantly lower bile acid concentrations in malignant biliary stenosis compared to controls without stenosis were found for GLCA (p = 0.032), GUDCA (p = 0.032), GCDCA (p = 0.006), GDCA (p = 0.031), GHCA (p = 0.005), TUDCA (p = 0.044), and TDCA (p = 0.036). Significant difference in cholic acid was found between benign and malignant stenosis (p = 0.022). Analysis of bile acids might be helpful in the differential diagnosis of malignant and benign biliary stenosis. More patients need to be enrolled in further studies so that the real diagnostic yield of bile acids can be determined.Differential diagnosis between benign and malignant biliary stenosis can be difficult in clinical practice. Histology of biopsy specimens is often indeterminate. Laboratory markers (serum bilirubin > 75  Histology of biopsy specimens is often indeterminate. Laboratory markers , 21 with malignant biliary stenosis , and 45 patients without biliary stenosis ; out of those, 25 subjects have and 20 do not have choledocholithiasis. Seven patients received long-term treatment with peroral UDCA .Serum total bilirubin and C-reactive protein were investigated less than 48 hours before ERCP (endoscopic retrograde cholangiopancreatography). Samples of hepatic bile were collected from the common hepatic duct endoscopically (prior to contrast media use) and frozen at -80°C immediately until the analysis was carried out.μm particle size) , protected with a disposable 0.2 μm in-line filter . The separation was performed in a gradient elution mode with a flow rate of 0.35 mL min−1 and with the following composition of mobile phases: Solvent A consisted of ammonium acetate (0.5 mM) and acetic acid (0.001% v/v) in water. Solvent B was composed of methanol : acetonitrile  mixture containing 0.5 mM ammonium acetate and 0.001% (v/v) of acetic acid. The gradient program was as follows: 0-0.2 min, 40% of solvent B; 0.2-7.0 min, 40-70% of solvent B; 7.0-8.0 min, 70-90% of solvent B; 8.0-8.5 min, 90 - 95% of solvent B; and 9.0-11 min, 40% of solvent B.The separation of 23 endogenous bile acids was done using UPLC system Acquity I-Class. Briefly, the separation was carried out on an Acquity BEH C18 column  coupled with the Acquity I-Class UPLC was used for detection of compounds. The spectrometer was operated with an ESI interface in a negative ion mode. Optimized settings of an ion source were as follows: capillary voltage, 2.5 kV; cone voltage, 50 V; source temperature, 150°C, desolvation temperature, 600°C; desolvation gas, 1000 L/h; cone gas, 150 L/h; and nebuliser, 6 L/h.Compounds were monitored using the multiple reaction monitoring (MRM) mode of operation. Since nonconjugated bile acids generally do not offer CID (collision-induced dissociation) fragments of sufficient intensity for quantification, the so-called pseudo-MRM was used for their quantification. The following MRM transitions were used: 375 ⟶ 375 , 391 ⟶ 391 , 407 ⟶ 407 , 432 ⟶ 74 , 448 ⟶ 74 , 464 ⟶ 74 , 482 ⟶ 80 , 498 ⟶ 80 , and 514 ⟶ 80 . Energies involved in CID were as follows: nonconjugated bile acids, CE = 10 eV; glycine-conjugated bile acids, CE = 30 eV; and taurine-conjugated bile acids, CE = 60 eV. MassLynx and TargetLynx software was used for LC/MS data acquisition and evaluation .Due to high concentration of bile acids in bile, the samples were diluted prior to analysis. Two dilution factors, 1000x and 10000x, were used to cover a broad range of concentrations, including major and minor bile acids in the samples.μL of 0.1% HCOOH in 50% (v/v) acetonitrile. In the next step, 900 μL of acetonitrile solution containing 1% (v/v) of formic acid and internal standard IS-CAD5 (c = 5 μM) was added to the same tube.A sample preparation corresponding to the 1000x dilution factor was as follows: Ten microlitres of bile in the 1.5 mL Eppendorf tubes was diluted with 90 μL of 0.1% (v/v) formic acid in 50% (v/v) acetonitrile. One hundred microlitres of this solution was taken and pipetted to a clean 1.5 mL Eppendorf, to which 900 μL of cold acetonitrile solution containing 1% (v/v) of formic acid and internal standard IS-CAD5 (c = 5 μM) was further added.The samples with the dilution factor 10000x were prepared as follows: Ten microlitres of bile in the 1.5 mL Eppendorf tubes was diluted with 990 g . One hundred microlitres of a supernatant was taken and mixed in a clean 1.5 mL Eppendorf tube with 900 μL of 0.1% (v/v) formic acid in 50% (v/v) acetonitrile. Samples were vortexed, and 250 μL is transferred to the AcroPrep filter plate . The filtrate was collected to a 96-well sample collection plate , sealed with a sealing mat , and put into an autosampler for analysis.The samples, with the dilution factor 1000x or 10000x, prepared in the previous step were vortexed for 1 minute, left for 10 minutes at -20°C to achieve sample deproteination, and spun at 14.000 × t-test, and data with nonnormal distribution were tested by the nonparametric Mann-Whitney test. The Pearson test was used for correlation analysis .The obtained data were tested statistically by means of descriptive statistics. Data with normal distribution were further analyzed by the parametric unpaired The study was approved by the Joint University Ethics Committee (Protocol No. 201712S06P). All procedures were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments.All patients signed the written consent. For all data obtained, all personal identification information was deleted in compliance with the laws for the protection of confidentiality of the Czech Republic.p = 0.005; see r > 0.7) was found between several pairs of bile acids . Significantly lower bile acid concentrations in malignant biliary stenosis compared to controls without stenosis were found for GLCA (p = 0.032), GUDCA (p = 0.032), GCDCA (p = 0.006), GDCA (p = 0.031), GHCA (p = 0.005), TUDCA (p = 0.044), and TDCA (p = 0.036). Significant difference between benign and malignant stenosis was found for cholic acid (CA)  and controls without stenosis (p < 0.001).Serum total bilirubin was significantly higher in patients with malignant biliary stenosis compared with nonstenotic controls (μmol/L), yet this difference did not reach statistical significance . Bile UDCA did not correlate tightly enough either with bile GUDCA  or with bile TUDCA . Correlation between bile GUDCA and bile TUDCA was significant . Seven patients treated with peroral UDCA had significantly higher concentrations of bile GUDCA (p < 0.001) and bile TUDCA (p < 0.001) compared to those without treatment with peroral UDCA. Treatment with peroral UDCA did not influence significantly the concentration of other bile acids in primary liver bile.Bile UDCA was detectable in 14/73 persons (19%). Out of those, 7 patients were not treated and 7 subjects were treated with peroral UDCA . Bile UDCA was lower in the nontreated group  was published . Bile saFew other studies analyzed bile acids in patients with cancer of the biliary tree –5. LankiA key question that must be addressed is what is the cause and what is the consequence. Bile salts are potentially cytotoxic , 12 and Although the pharmacokinetics of UDCA have been studied since its approval for its use in primary biliary cholangitis, limited work has been published on modelling the metabolism of UDCA and its two major conjugates, a glycine conjugate, GUDCA, and a taurine conjugate, TUDCA . In our Our current project is a pilot study, and our data must be interpreted with caution. We are not able to provide any final explanation for the difference found. Nevertheless, we can hypothesize that long-standing low concentrations of cholic acid can contribute to biliary mucosal instability as one of initial steps in tumorous biology. Unlike us, Jusakul et al.  investigWe are aware of possible limits of this pilot study. The number of patients was limited, especially those with benign stenosis. The control group was rather heterogenic, with or without previous cholecystectomy, some patients underwent repeated ERCPs with or without papillotomy, common bile duct stones were of different aetiology, and not all bile samples were sterile. Patients with malignant stenosis and subjects without stenosis and without choledocholithiasis have both low levels of cholic acid. This observation does not rule out the possible utility of concentration of cholic acid in differential diagnosis between benign and malignant stenosis.Analysis of bile acids might be helpful in the differential diagnosis of malignant and benign biliary stenosis. More patients must be enrolled in the future studies so that the real diagnostic yield of bile acids can be determined.


For this purpose, different samples from four highly popular brands of mentioned products were randomly collected from Iran's market during 2016–2018. All monitored products had trans fatty acid <1.0%. In the case of FFA and IP110, the ranges of 0.03–0.08 (%) and 9.3–17.2 hr were obtained, respectively, being mostly in conformity with the National Standard of Iran (FFA < 0.1% and IP110 > 15 hr). The ranges of PV of cooking oils, frying oils, and vanaspati samples were 1.2–2.7, 0.93–2, and 0.84–1.6 meq/kg, respectively. Our results revealed that p‐AV of frying oils and cooking oils was mostly outside of legal limits of Iran (p‐AV > 6) with the ranges of 4.2–12.5 and 4.3–12.3, respectively. In terms of TOTOX value, monitored products had a range from 5.2 to 13.0 (mostly <10) being nearly acceptable.In this study, postmarketing surveillance (PMS) was conducted in terms of the parameters which are reliable indicators of the oxidative stability of cooking oils, frying oils, and vanaspati samples. The analyzed parameters were fatty acid composition, peroxide value (PV), free fatty acids (FFA), p‐anisidine value (p‐AV), induction period at 110°C (IP After‐sales monitoring is an important action plan for producers and also administration authorities provided for various food products, cosmetics, and drugs in many countries of the world to address potential concerns that may threaten the consumer's health , they should have an acceptable thermal and oxidative stability because high‐temperature processing of oils leads to adverse reactions such as hydrolysis, oxidation, or polymerization  determined by Rancimat test, a time indicator (hr) representing the thermal degradation of oils determined by Rancimat test  . As described by Codex, these limits for PV, acid value (AV), and p‐AV are 10 meq/kg, 0.6 mg KOH/g oil (0.3% FFA), and 8, respectively  peroxide value (PV), an indicator which shows the amount of fatty acid hydroperoxides produced during the oxidation of oils and fats O'brien, ; (b) freRegarding the quality of different types of cooking oils, vanaspati, and frying oils supplied in various countries, some studies have been performed. For example, Mehmood, Ahmad, Ahmed, and Khalid  investigConsidering high consumption of edible oils and fats, particularly cooking and frying oils in retail markets, food industries, and household applications, food administration authorities always have very precise action plans and monitoring measures for controlling the quality of these products within the market. In order to compare common brands and get knowledge of their claims on the quality of produced products, the present study was defined with the aim of monitoring the oxidative stability indices of cooking oils, vanaspati, and frying oils introduced in Iran markets.2All chemicals including sodium thiosulfate, potassium iodide, sodium hydroxide, acetic acid, chloroform, and p‐anisidine reagent were purchased from Merk . All other chemicals were of analytical grade.2.1In this study, four common and popular brands of cooking oils, vanaspati, and frying oils were monitored, and in order to keep them confidential, they were labeled as A, B, C, and D. For this purpose, nine samples from each product of all brands were collected. Cooking oils with the brands of A, B and D, and C contained SFO and RPO, respectively. Collected frying oils were a mixture of some vegetable oil as follows: brand of A, SFO (58%): POO (42%); brand of B, SFO (26%): SBO (40%): POO (34%); brand of C, SBO (45%): POO (55%); and brand of D, CO (50%): RPO (40%): SFO (10%). The composition of vanaspati samples was as follows: brand of A, POO (12%): SFO (60%): RPO (21%): PS (4%); brand of B, POO (12%): SFO (20%): RPO (64%): PS (4%); brand of C, POO (15%): SFO (28%): RPO (53%): PS (4%); and brand of D, POO (12%): SFO (68%): RPO (15%): PS (5%). Sampling was conducted randomly from Iran's market according to the batch number of these brands during 2016–2018. After sampling, collected products were transferred to the laboratory and kept refrigerated at 5°C until further analyses.2.2Preparation of methyl ester of fatty acids was conducted according to American Oil Chemists’ Society (AOCS) method Ce 2‐66 AOCS, . Then, f2.3110 of collected samples was determined in accordance with AOCS method cd 12b‐92 at a temperature of 110°C, 2.5 g samples, and an air flow rate of 2.5 ml/min  model 743, the IPin AOCS, .2.4B, S, N, and W are ml of sodium thiosulfate titrated for the control, ml of sodium thiosulfate titrated for the sample, sodium thiosulfate normality, and weight of the sample, respectively.Evaluation of the PV was carried out based on the AOCS method cd 8‐23 AOCS, . For thi2.5V, N, and W are ml of NaOH, the normality of NaOH, and weight of the sample, respectively.Free fatty acids was measured based on the oleic acid by titration with sodium hydroxide according to the AOCS method Ca 5a‐40 AOCS, . To this2.6W is the weight of sample.The AOCS official method Cd 18‐90 AOCS,  was used2.7TOTOX value) was determined using the following formula:Total oxidation value . Duncan test was used to determine significant differences among samples at 33.1As shown in Table The major fatty acid in frying oil samples from A to D was linoleic acid. Also, the percentage of SFA in frying oils from A, B, C, and D was 10.28%, 10.70%, 6.72%, and 8.73%, respectively. Linoleic acid was found to be the predominant fatty acid in vanaspati samples from A to D. Also, it was shown that the amounts of SFA in vanaspati samples of A–D were 19.7, 17.66, 11.2, and 10.49, respectively.The permitted limit of TFA content for frying oils and vanaspati according to INSO is 2% . In India, the upper limit of TFA content in vanaspati is 10% Kala, . Also, tTFA content in cooking oils, frying oils, and vanaspati samples was in the range of 0.13%–0.4%, 0.5%–0.8%, and 0.8%–0.9%, respectively. In recent years, INSO by legislating strict regulations has reduced the legal limit of TFA and SFA content for edible oils. It is noteworthy that permitted TFA content in vanaspati by INSO was decreased from 25% to 10%, 10% to 5%, and 5% to 2% in 2007, 2010, and 2015, respectively. Because of such strict standards, TFA content in cooking oils, frying oils, and especially vanaspati was satisfactory. In this regard, Kala  reported3.2Free fatty acids is a determination of fatty acids separated from triacylglycerol molecules. The percentage of FFA is reported given to the prevalent fatty acid of each edible oil, which in our study it was oleic acid (C18:1). It should be mentioned that through multiplying the FFA by 1.99, AV will be obtained.The FFA content (%) of the monitored samples is listed in Figure With respect to Codex standard, all collected products were within the legal limit (FFA < 0.3%). Gunstone  suggestep < 0.05). In this regard, cooking oils from C and D brands, and the frying oil produced by brand A and the brand B of vanaspati samples were found to have the lowest FFA content.As shown in Figure 3.3110 of cooking oils is listed in Table 110 > 9 hr). Also, all frying oil samples produced by the selected brands met the permitted limit of INSO (IP110 > 15 hr) as shown in Table 110 for vanaspati samples . Considering that vanaspati is usually not exposed to high temperatures, observed difference can be ignored. On the whole, the ranges of IP110 for cooking oils, frying oil, and vanaspati samples were 9.2–11, 14.2–16.7, and 14.7–17.2 hr, respectively. As presented in Figure 110 of collected samples was observed (p < 0.05). In this regard, frying oils from brand A with 16.15 hr, cooking oils from brand C with 11.02 hr, and vanaspati from brand C with 16.66 hr were found to have the highest IP110. There is no international standard limit for IP110. However, Roszkowska et al., . Since we used a temperature of 110°C in present study, N = 4. As seen in Table 150 of frying oils ranges between 0.643 and 0.582 hr.It should be noted that some vanaspati samples, which were not in the standard range, had a slightly different IP et al.,  reporteda et al.  and Redoa et al.  determina et al.  who evaland Maza  was used3.4It is noteworthy that permitted limit after production for the PV of cooking oils produced by SFO and RPO is 1.5 and 2 meq/kg. Also, the human consumable limit for PV is 5 meq/kg INSO. As can As can be seen in Table As shown in Table p < 0.05). In this regard, cooking oils from the brand C with 0.97, frying oil from the brand C with 0.67, and vanaspati from the brand C with 0.99 meq/kg were found to have the lowest PV. Based on Codex standard (PV < 10 meq/kg), all collected products met the legal limit.According to Figure Due to the stricter legal limit of frying oils in Iran, although most of the collected products did not meet INSO, they had a lower PV than cooking oils and/or vanaspati samples. On the other hand, since frying oils will be exposed to extreme heat processing, they should have acceptable quality indices. It is necessary to be mention that no international legal limit has been set for PV of frying oils or vanaspati, so far. However, Gunstone  suggeste3.5p < 0.05). In this regard, cooking oils with brand A, frying oils with brand D, and vanaspati with brand B were found to have the lowest p‐AV with amounts of 4.9, 5.2, and 5.3, respectively.As mentioned, p‐AV is more reliable than PV; this is due to the fact that primary oxidation products are unstable (Al‐Kahtani, The upper permitted limit legislated by Codex standard for p‐AV is 8 while this limit according to INSO is equal to 6. Additionally, Shahidi and Zhong  suggeste3.6TOTOX value is an index merging PV and p‐AV. Accordingly, TOTOX value is 2PV plus p‐AV. This index is better in comparison with PV or p‐AV alone because of the fact that fatty acid hydroperoxides are unstable and do not offer a reliable report for the oxidative stability of edible oils. In general, edible oils with a TOTOX value <10 are considered to be fresh and with a high quality. As mentioned in Table TOTOX value <10. All frying oils of brands A and D had a TOTOX value <10. This amount also was observed for 7 and 8 sample of frying oils with the brands of B and C, respectively. Finally, all vanaspati samples owned a TOTOX value of lower than 10. As shown in Figure TOTOX values.TOTOX value by INSO and also, international standards. In general, the ranges of TOTOX value for cooking oils, frying oil, and vanaspati samples were 7.0–12.5, 5.2–12.3, and 6.7–9.3, respectively. In case of monitoring the TOTOX value of edible oils, Sebastian et al. (TOTOX <10 with the range of 2.8–8.3. Also, Maszewska et al. (TOTOX value of 1.7, 13.2, 5.2, and 4.3 for RPO, rice bran oil, CO, and peanut oil, respectively.There is no legislated limit of n et al.  reporteda et al.  reportedThere are some monitorings which have been done on heated oils, and therefore, the generalization of the results to current paper does not seem logical. But overall, these results have indicated that the heated oils used in restaurants and fast‐food restaurants did not have a satisfactory quality. In this regard, Arbabi and Deris  examined4110. In the case of PV for cooking oils and/or vanaspati samples, collected products showed a nearly good condition while the PV of frying oils was mostly out of the standard range. The samples with respect to p‐AV showed a deviation from the standard. In this study, monitorings were carried out on the fresh samples. Considering that the heating process is capable of creating free radicals in edible oils resulted in oxidation, it is necessary to evaluate the oxidative parameters after heating. One point to remember is that satisfactory status of the oxidative stability of vegetable oils can sometimes be attributed to excessive use of synthetic antioxidants which has adverse effects on consumer's health. Therefore, monitoring of the synthetic antioxidant dosages used in fresh edible oils is also mandatory. Finally, given the lack of international standards for some products such as frying oil and vanaspati, it is suggested that specific standards to be legislated for each product.The outcome of present study revealed that fresh oil samples supplied to the Iranian market were acceptable in terms of FFA and IPAll authors declare that there is no conflict of interest.There was no human or animal testing in this study.