Grateloupia imbricata is an intertidal marine seaweed and candidate model organism for both industry and academic research, owing to its ability to produce raw materials such as carrageenan. Here we report on the transcriptome of G. imbricata with the aim of providing new insights into the metabolic pathways and other functional pathways related to the reproduction of Grateloupia species. Next-generation sequencing was carried out with subsequent de novo assembly and annotation using state-of-the-art bioinformatic protocols. The results show the presence of transcripts required for the uptake of glycerol, which is a specific carbon source for in vitro culture of G. imbricata and nucleotide sequences that are involved in polyamine-based biosynthesis, polyamine degradation, and metabolism of jasmonates and ethylene. Polyamines, ethylene and methyl jasmonate are plant growth regulators that elicit the development and maturation of cystocarps and the release of spores from seaweeds. Our results will inform studies of the mechanisms that control polysaccharide accumulation, cystocarp formation and spore release. Moreover, our transcriptome information clarifies aspects of red seaweed carposporogenesis with potential benefits for enhancing reproduction. Pyropia, Gracilaria and Chondrus [Next-generation sequencing has been used to obtain complete genome sequences of a species and transcriptome profiling. Altogether these two aspects allow to address the unknown aspects of algae physiology. In red seaweeds, sequencing approaches have been carried out on species of commercial value such as those of the genera Chondrus ,2, reporGrateloupia imbricata is a red algal taxon of major interest for the biotechnology industry because it produces raw materials such as carrageenan. G. imbricata is an intertidal macroalga inhabiting the rocky pools of the Canary Islands; it is fully exposed to light, evaporation and an increase of temperature during low tide. Reproduction in G. imbricata occurs by both sexual and asexual processes. Fertilization involves the fusion of a non-flagellated male gamete, the spermatium, with a sessile egg-containing cell, the carpogonium on the female gametangial thalli. After fertilization, the zygote is retained on the female gametangial thalli where it develops into the third generation, the carposporophyte. Carposporophyte of the red algae may bear a macroscopically visible reproductive structure, the cystocarp. Cystocarps appear as small dark spots (1–5 mm diameter) embedded in the thalli, and eventually release the carpospores which serve as the agents of recruitment for the tetrasporophyte generation, a tetraspore producing phase. Tetraspores originate from male and female gametangial thalli, completing the life cycle.G. imbricata in glycerol-containing media can increase polymer biosynthesis [G. imbricata toward the goal of establishing a global functional profile for this species with particular focus on transcripts related to the synthesis of biopolymers, growth, development, and reproduction.Culture of ynthesis ,4, and tynthesis ,6,7,8,9.ynthesis ,11,12. OG. imbricata spans 11.6 Mb and was de novo assembled into 19,284 contigs.The transcriptome of −15) to their subjects, 25% were significant with e-values between 1 × 10−14 and 1 × 10−5, and the remainder were annotated based on e-values > 1 × 10−5. The greatest number of annotation top hits per subject species was provided by the red macroalga C. crispus, with ~43.7% of all subjects, followed by the unicellular red alga G. sulphuraria, with 7.1%. Of the entire set of sequences annotated as “coding”, 56% were assigned GO annotations and ECs and later were used to identify 99 metabolic pathways  that could be associated with the G. imbricata transcriptome. As for associations with known orthologs, the G. imbricata transcriptome could be classified into 2304 KOG clusters comprising 5394 transcripts annotated according to a distribution of e-values similar to that indicated for the annotation based on the NR/NT databases. See G. imbricata, the identification of ECs  that participate in 99 metabolic pathways allowed us to approximate a metabolic profile for this organism  or in O. tauria , information processing, energy production, the metabolism of carbohydrates, lipids, amino acids, cofactors, vitamins, and including enzymes involved in the chlorophyll-mediated biosynthesis of components related to both photosystems (I and II) and light-harvesting complexes. The Venn diagram also shows that G. imbricata generates transcripts for enzymes related to four pathways present in C. crispus and G. sulphuraria  and to one pathway of C. crispus (atrazine degradation). These pathways highlight certain compounds that may be of interest for studies of the processes related to the metabolism of terpenoids and polyketides, cofactors and vitamins, and xenobiotics and/or the biosynthesis of glycans and other secondary metabolites in red algae. The G. imbricata transcriptome also includes a transcript for an EC related to steroid degradation that also is related to transcripts encoding ECs of G. sulphuraria. To evaluate the G. imbricata transcriptome at the functional level, we carried out an analysis to determine the distribution of the abundance of the GO terms annotated to Molecular Function, Biological Process and Cellular Component. For subsequent analysis will only be taken into account those GO terms supported by at least 75 transcripts. According to this analysis, 91 of the 99 pathways are present in G. imbricata. First, G. imbricata and C. crispus share more metabolic pathways than other algal taxa, i.e., given that C. crispus was the top-hit species during our annotation of the G. imbricata transcriptome. Second, these two red seaweeds are the only red multicellular algae with commercial interest belonging to class Florideophyceae that have been sequenced so far [G. imbricata transcripts with annotations related to sugar transport and uptake; ion/cation transport; floridoside and carrageenan synthesis; and the metabolism of polyamines, ethylene, and jasmonate as well as annotations related to stress pathways. Finally, annotations were interpreted and pathways were reconstructed based on information for C. crispus metabolism. Several benchmarks were considered when inferring metabolic information from our transcriptome evaluation of d so far ,15 and tG. imbricata transcriptome regarding sugar transport and uptake, polysaccharide biogenesis, phosphorylation mechanisms, and transmembrane transport. These annotations were in fact anticipated based on the predicted protein trafficking patterns for G. imbricata during growth and development , which catalyzes the formation of glycerol 3-phosphate that condenses with UDP-galactose to produce floridoside phosphate [Concerning carbon sources, a number of annotations could be obtained for the elopment . Althoughosphate  , and from arginine via the action of arginine decarboxylase [ODC is an indicator of cystocarp maturation and sporulation in G. imbricata [Studies of in vitro carposporogenesis in ed algae ,8,9,12. ed algae ,24,25,26ed algae . The synmbricata ,11,12,26Other groups of plant growth regulators meriting attention are those related to the metabolism of each of the jasmonates and ethylene, for which the main precursor is SAM, which is the substrate for 1-aminocyclopropane-1-carboxylate (ACC) synthase in the ethylene biosynthesis pathway and the main donor of methyl groups for jasmonate synthesis . Figure Methyl jasmonate induces both cystocarp maturation and sporulation . In high-d-xylulose 5-phosphate synthase, 1-deoxy-d-xylulose 5-phosphate-reductosisomerase, farnesyl diphosphate synthase, geranyl diphosphate synthase, glutathione S-transferase, cytochrome P450, and tyrosine aminotransferase. Interestingly, genes that govern the synthesis of jasmonate derivatives may also be linked to oxidative stress because they lead to the generation of reactive oxygen species, including hydrogen peroxide, superoxide anions and hydroxyl free radicals [G. imbricata reproduction were recently described [Also noteworthy is that our selection of transcripts includes those encoding jasmonate-responsive factors, which are not directly related to jasmonate biosynthesis but coevolved with signaling mechanisms and stress responses as demonstrated in higher plants . The genradicals . Hence, Finally, certain transcripts related to ethylene signaling and synthesis were selected according to their descriptions and annotations, as summarized in G. imbricata transcriptome, but we did not detect transcripts with annotations for ACC oxidase and DMSP lyase , ACC synthase, ACC oxidase, and dimethyl sulfopropionate (DMSP) lyase ,36. Of tSP lyase . In the z et al.  demonstrz et al. , DMSP syz et al. , gene-enG. imbricata ethylene receptor differs from those of other organisms, and indeed among higher plants there is great variation in the structure [G. imbricata, additional gene expression information is necessary to clarify their roles in carposporogenesis in response to growth regulators. Therefore, these annotated sequences of the G. imbricata transcriptome can also be used as a potential starting point for further work on the pathways that guide reproductive processes in this red seaweed.Likewise, the ethylene receptor was also not annotated. Garcia-Jimenez and Robaina  reportedtructure . DespiteIn conclusion, consideration of the genes identified in our transcriptome analysis with the subsequent annotation of gene functions may help identify deficits in our current knowledge of many aspects of seaweed reproduction and shed light on the genes that govern carposporogenesis. The BUSCO results support our transcriptome as a tool to understand the biology of G. imbricata. Unlike the mere annotation of a transcriptome, this work will allow future studies to focus on reproduction control. Biotechnology may also benefit from these data because certain aspects of reproduction, such as reproductive stages, may inform technological innovation—most prominently by controlling the synthesis of cell-wall polysaccharides.Grateloupia imbricata (Florideophyceae) was sampled as whole individuals without visible epiphytes from the northeastern coast of Gran Canaria island . At 1 h post-collection, thalli were selected, brushed, removed of associated biota, and acclimatized for 1 day in sterilized seawater. Additionally, thalli were frozen in liquid nitrogen before storage at −80 °C. RNA isolation was carried out on six different days and with four independent replicates for each one. Then, 12 of the 24 samples were chosen randomly and pooled for assays. The pooling samples enabled to obtain a more reliable transcriptome and to overcome possible inter-individual variations of gene expression. The pooling samples enabled to obtain a more complete transcriptome and to overcome possible inter-individual variations of gene expression. Total RNA was isolated from thalli (100 mg) using a NucleoSpin RNA plant kit . The quality of the total RNA was checked using 2% agarose denaturing gel electrophoresis by inspection of the integrity of 28S and 18S rRNA bands. Then, samples were further purified with a RNA clean-up XS kit (Machery Nagel). As assessed with the Bioanalyzer System of Agilent Technologies , samples with a 28S/18S rRNA ratio between 1.7–2.0 and a RIN (RNA Integrity Number) 9–10 were selected for poly(A)-RNA enrichment in two successive rounds using a Dynabeads mRNA direct microkit  following the manufacturer’s instructions with some modifications. In short, 8 μg total RNA in a microtube was diluted to 150 μL with nuclease-free water and heated at 70 °C for 2 min. After the addition of an equivalent volume of lysis/binding buffer from the kit, samples were mixed with 30 µl of oligo-d(T) magnetic beads (10 times) and incubated for 5 min at room temperature. The resultant enriched poly(A)-RNA samples were resuspended in 50 µL elution buffer from the kit, divided into three aliquots, and stored at −80 °C. The concentration of poly(A)-RNA was measured using a Qubit 2.0 fluorometer  as well as the Agilent Bioanalyzer System.Two cDNA libraries (1 and 2) were constructed and sequenced using a PGM Ion Torrent platform (Life Technologies) for next-generation sequencing. Both libraries were sequenced independently to increase the sequencing deep and overcome the Ion Torrent limitations; in addition, both libraries can be used as technical replicates. Poly (A)-RNA (300 ng) was used as starting material. The first step consisted of a partial digestion with RNase III at 37 °C for 3 min. The length, size distribution, and concentration of the fragmented RNA was assessed using the Agilent Bioanalyzer with an RNA 6000 Pico kit (Agilent Technologies). Then, 50 ng of material was used in the AB Library Builder system (Applied Biosystems) with the Ion Total RNA-Seq kit (Life Technologies) to automatically construct each library. Double-stranded cDNA, obtained by consecutive reverse transcription and PCR amplification, was also analyzed with the Agilent Bioanalyzer using a high-sensitivity DNA kit (Agilent Technologies). Samples were then serially diluted such that the smallest concentration was 23 pM and subjected to emulsion PCR and enrichment using the OneTouch system . Finally, each sample was loaded on a single Ion 316 chip and sequenced in two consecutive runs using the same initialization procedure on the PGM platform. The sequencing data for both cDNA libraries was outputted as two fastq files, one for library 1, with 3,431,580 raw reads, and the other for library 2, with 2,946,415 raw reads, for a total of 6,377,995 raw reads. The average and maximum sequence lengths were 250 and 308 bp, respectively. The quality of the two fastq files was assessed using FastQC tools . Prinseq  was usedG. imbricata with that of other algae, the KEGG database website was browsed to obtain information on the metabolic pathways of the red multicellular alga Chondrus crispus, the unicellular red alga Galdieria sulphuraria, and the green alga Ostreococcus tauri. To complement the information provided by KEGG, different searches of the Entrez site at NCBI with the Ensembl Biomart platform [G. imbricata transcriptome. Subsequently, GPRO software (GPRO suite by Biotechvana) was used to perform statistical analyses, for which Venn diagrams were generated based on the metabolic pathways of G. imbricata and those of the aforementioned taxa. To compare the metabolic profile of platform  were perG. imbricata transcriptome are presented in the Raw data were deposited at the NCBI Sequence Read Archive (SRA) with BioProject record PRJNA309128 and BioSample record SAMN04420758. Contig sequences, protein predictions of at least 100 amino acid residues in size, and all annotations performed for the


FHT and CYP86A33, indicator genes for skin suberization, was significantly upregulated. Earlier applications of the polyhalite mineral did not alter mineral elements concentrations in the tuber peel compared to control plants. Overall, polyhalite fertilization positively affected tuber skin appearance and skin-related gene expression. However, the effect was moderate, and the mineral did not fully mitigate skin imperfections. The effect of polyhalite may be dependent on local conditions and cultivar type.The protective peel of potato tuber consists of periderm tissue, the outmost cell layers of which contain corky cell walls and are termed “skin”. The skin protects the tuber from water loss and pathogen invasion, and its visual appearance is a highly important marketing factor. Physiological skin blemishes are of great concern, mainly russeting disorder and skinning injuries. We previously showed that application of calcium (Ca) reduces the rate and severity of skin russeting. Here, polyhalite fertilization was tested as an alternative source of Ca. The polyhalite mineral is a hydrated sulfate of potassium (K), Ca, and magnesium (Mg), and thus contains additional important nutrients that may contribute to skin quality. Furthermore, in view of the direct interaction of soil mineral elements with the tuber skin, we tested application of polyhalite at the end of the growth period, assuming that providing the mineral at the last stages of skin development may enhance its quality. Accordingly, polyhalite was applied at three time points: preplanting, in-season at around 3–4 weeks prior to haulm desiccation, and 2 days post-haulm desiccation. The experiments included several cultivars and locations. Data indicated that late application of polyhalite, after haulm desiccation, results in reduced concentrations of Ca and Mg and increased concentration of K in the tuber peel of fertilized plants compared to controls. Tuber appearance was improved, and the expression of  The marketability and price of fresh market potatoes are determined by the tuber’s visual appearance, which is largely a function of tuber shape, color, and skin. Smooth-skinned varieties are characterized by a clean, shiny appearance compared to the earthy look of russeted potatoes, where soil often adheres to the rough skin surface. The rough skin of russeted varieties  is a desired, genetically inherited character . HoweverPhysiological skin blemishes that are not caused by pathogens, mainly the russeting phenomenon and skinning injuries, are therefore of great concern . In potaThe potato skin is the outermost layer of the tuber periderm, a protective tissue of secondary origin that replaces the epidermis when the latter is damaged. The periderm is made up of three cell types: phellem, phellogen, and phelloderm . The pheSuberin, which accumulates in the phellem cell walls, is a macromolecule containing both polyaromatic and polyaliphatic domains that is found between the primary cell wall and the plasma membrane. The aromatic domain is composed of monolignols and hydroxycinnamic (ferulic) acids and is covalently bound to the primary cell wall . The aliThe suberin-biosynthesis pathway has not been completely resolved; it includes β-ketoacyl-coenzyme A (CoA) synthases (KCS), fatty acyl reductases, long-chain acyl-CoA synthetases, cytochrome P450 monooxygenases, glycerol 3-phosphate acyltransferases, and phenolic acyltransferases 4·2H2O] is a hydrated sulfate of potassium (K), Ca, and magnesium (Mg) in the relative proportions: 48% sulfur trioxide (SO3), 17% calcium oxide (CaO), 14% potassium oxide (K2O), and 6% magnesium oxide (MgO), and as such contains additional important nutrients that may contribute to skin quality. Furthermore, in view of the direct interaction between soil minerals and the tuber skin, we tested polyhalite application at the end of the growth period, assuming that providing the mineral at the last stages of skin development might enhance the tuber’s market value.There are various formulas and modes of application for Ca fertilization. Here, we made use of a fertilizer produced from the mineral polyhalite. Polyhalite [KThe experiments were carried out in the Western Negev region of Israel which is characterized by a semiarid Mediterranean climate, with average annual rainfall of 200–300 mm during the winter (November–March), and dry summers.Experiments were conducted in two soil types: sandy clay loam , and fine sand . Essential elements composition of water extract of the soils (1:1 w/w): Ca, 0.7–1 mM; Mg, 0.1–0.3 mM; and K, 1.5–2.0 mM.3/ha. Total amount of water was around 4,000–4,800 m3/ha, depending on growth period and occasional rain. Following haulm desiccation, irrigation was applied at 70 m3/ha every 5–7 days – that is to keep the soil moist for prevention of surface cracking and penetration of pests into tuber region, and to allow optimal skin-set. When polyhalite was applied after haulm desiccation, it was followed by water irrigation of 250 m3/ha.Irrigation (sprinklers) was with reclaimed water, with the following major components (mg/L): chloride, 182; sodium, 133.5; Ca, 83.6; sulfur, 23.88; Mg, 18.62; K, 11.1; phosphorus, 0.155; and pH 7.69. Irrigation was applied every 3–4 days, 200 m3/ha compost, followed by sprinkler irrigation of 250 m3/ha. From 40 to 80 days after seed-tuber planting (DATP), nitrogen (N) was applied at 320 kg/ha as ammonium nitrate by fertigation; 20–30 units of N every irrigation. N concentration in the plant was monitored by petiole test. Only for fine sand soil, phosphate was applied preplanting together with the compost, at 200 units/ha. Haulms were desiccated, as specified in each experiment below, with Reglone® .Beds were of two ridges with total width of 1.93 m. Each experiment described below was done in 5–6 replicates, 20 m bed length for each replicate. Replicate blocks were organized in random. Growth practices included preplanting application of 20 mExperiment 1: The experiment was performed with the red-skinned potato cv. Rosanna which is prone to severe russeting and skinning injuries. Tubers were planted in January 2016 in a commercial field with sandy clay loam soil. Polyhalite in the form of PolysulphateTM product (http://www.polysulphate.com/fertilizing-potatoes-with-polysulphate/)  was applied preplanting at 1,500 kg/ha, followed by sprinkler irrigation (PolyH plants). Control plants were not fertilized with the polyhalite (C plants). Late application of Polysulphate  was performed manually on bed tops at the end of growth , when the foliage was partially wilted, followed by sprinkler irrigation. This late polyhalite treatment (PolyH” plants) was applied to half of the beds of PolyH plants, and to half of the beds of their controls. Accordingly, at harvest, sampling included tubers from four types of plants: the C plants that were not fertilized with polyhalite at all, C+PolyH” plants that were fertilized only at the late application, PolyH plants that were fertilized only at the beginning of growth, and PolyH+PolyH” plants that received polyhalite at the beginning and end of the growth period. Experimental design included six replicates arranged in randomized blocks for each polyhalite treatment. Tubers were collected at the late tuber-bulking stage (100 DATP), at tuber maturation, when haulm desiccation was applied (118 DATP), and at harvest (135 DATP). At each time point, treatment, and replicate, tubers were collected from 1–1.5 m of bed , and the 10 biggest tubers were selected for further analyses. Tuber skin was sampled from all 10 tubers, hence each sample  represented several plants. Skin samples were used for mineral elements analysis, and extraction of RNA for molecular analysis of skin reporter genes. Visual evaluation of tuber appearance was performed at harvest and after 1 month of storage.Experiment 2: This experiment tested the effect of polyhalite application only at the end of growth on tuber visual appearance. It was performed in different plots, in sandy clay loam soil. Polysulphate powder was applied at 1,500 kg/ha 2 days after haulm desiccation, followed by sprinkler irrigation, as in Experiment 1 for the PolyH” plants. The experiment was performed with the early (winter growing) white-skinned potato cv. Arizona that is prone to russeting and skinning injuries, and three late potato cultivars (spring growing)—two white-skinned cultivars, Sifra and Panamera, and red-skinned cv. Mozart. Experimental design included five replicates arranged in randomized blocks for each cultivar. Visual evaluation of tuber appearance was performed after 1 month of storage.Experiment 3: The experiment was performed with two potato cultivars at two locations. The red-skinned cv. Rosanna in a sandy clay loam soil, and the white-skinned cv. Vivaldi in a fine sand soil. The latter also exhibits skin russeting, albeit to a lesser extent than Rosanna. Polysulphate  was applied preplanting of seed tubers at the end of October 2016. In-season application of Polysulphate powder  was performed manually on bed tops at 85 DATP for Rosanna and 95 DATP for Vivaldi, followed by sprinkler irrigation. Haulm desiccation was applied around 3–4 weeks later. Tubers were harvested in mid-March 2017, 140 DATP. Experimental design included five replicates arranged in randomized blocks. Potatoes were collected at early tuber-bulking stages, 60 and 75 DATP, and at the late tuber-bulking stage, 95 DATP for Rosanna, and 105 DATP for Vivaldi. Tubers were also collected at harvest (140 DATP) for visual evaluation. For each cultivar, at each time point, treatment and replicate, tubers were collected from 1–1.5 m of bed , and the 10 biggest tubers were selected for further analyses. Tuber skin was sampled from all 10 tubers, hence each sample  represented several plants. Skin samples were used for mineral elements analysis, anatomical study, and the extraction of RNA for molecular analysis of skin reporter genes. Visual evaluation of tuber appearance was performed after 2 months of storage.At the end of the growth, after harvest, tubers were stored at 14°C for 2 weeks (curing), followed by a gradual decline in temperature to 2°C, and were stored at 2°C for 1–2 months, as practiced in the potato industry. After storage, tubers were acclimated to 6–8°C for a week, then washed, allowed to dry for 24 h at room temperature, and evaluated for skin appearance. Twelve tubers for each treatment and biological replicate were compared visually to a reference cultivar, using a visual index of 1–5, with 5 indicating high skin quality and 1, poor skin quality. The evaluation panel included four people who rate commercial products routinely.Samples of tuber skin were dried in an oven at 60°C and pulverized. Total concentrations of K, Ca, and Mg were determined by atomic absorption spectrophotometry (Perkin-Elmer Model 460) after digestion with nitric acid and perchlorate for Ca and Mg, and with sulfuric acid and peroxide for K . MineralSamples of the tuber surface (blocks of 4 x 3 x 3 mm) were fixed in FAA , dehydrated in an ethanol/Histoclear  series and embedded in paraplast  according to standard methods . Tissue TM Blue QPCR SYBR® Green ROX Mix  was used for qPCR according to the manufacturer’s protocol with specific primers  as the reference gene . Significant difference was determined at P < 0.05.Data were analyzed for statistical significance by two-factorial analysis of variance, or by Student’s The level of mineral elements making up the polyhalite—Mg, Ca, and K—was monitored in tuber peel samples collected from Rosanna plants that were fertilized preplanting or following haulm desiccation, and their controls. The objective was to test whether polyhalite fertilization altered skin Mg, Ca, and K concentrations Figure 1Tuber appearance was examined at two time points: 1 day after harvest ; therefore, an additional experiment was conducted to test this approach with other potato cultivars. Experiment 2 was performed with the early (winter growing) white-skinned cv. Arizona which is prone to russeting and skinning injuries, and three late (spring growing) cultivars: white-skinned cvs. Sifra and Panamera, and red-skinned cv. Mozart Figure 4To maximize the positive effect of polyhalite on skin appearance, we hypothesized that its application a few weeks before haulm desiccation, while skin development is highly active, would enhance its effect. The experiment was performed with the red-skinned cv. Rosanna and the white-skinned cv. Vivaldi. The experimental design was similar to that in experiment 1, except that the second application of polyhalite was done in-season, 3–4 weeks before haulm desiccation. Accordingly, tuber peel was sampled at early time points in tuber development: during early tuber bulking, 60 and 75 DATP, as well as 10 days after the second polyhalite application, 95 DATP for Rosanna, and 105 DATP for Vivaldi.Following preplanting application of polyhalite, mineral elements analysis of tuber peel from Rosanna indicated significant increases in the concentrations of Ca and K between 60 and 75 DATP, with no significant change afterwards ; but it was not significant.For Rosanna  differs from that of maturing tubers (up to around 120 DATP), and that of tubers that are undergoing skin-set (after application of haulm desiccation). This was true for tuber peel from polyhalite-treated plants and the untreated controls. Combining the data from experiments 1 and 3, it is suggested that the peel of young developing tubers accumulates Ca and K at a high rate, which slows toward the end of the growth period 6A, E, aThe effect of polyhalite application on mineral elements acquisition by the peel is not clear. When applied at the beginning of growth or in-season (3–4 weeks prior to haulm desiccation), there was no difference in mineral elements concentrations in the tuber peel between polyhalite-treated and control plants 6A, C, EIt can be argued that significant changes are not detected due to the low solubility of the polyhalite mineral, and thus a longer incubation time might reveal such changes. Alternatively, changes may be significant, but below detection levels. In addition, it may be assumed that polyhalite application affects the interactions between tuber peel and soil extract and thus affects tuber skin indirectly.Although the motivation for this work was to increase available Ca, it is highly possible that Mg contributes to skin quality , and posExternal tuber skin cells are continuously sloughed off as the tuber expands. In parallel, new skin tissue is formed to cover the increasing surface area of the tuber. At the end of the growth period, tuber expansion stops, as well as skin formation. Practically speaking, the skin that is formed toward the end of tuber growth is the skin that covers the final potato product for marketing. Moreover, at the end of the growth period, the plant is aging and may not optimally support the final stages of skin maturation; this suggestion is backed by our observation of increasing incidence of skin imperfections when the growth period was extended beyond 120 DATP and plants seemed “exhausted”. Based on these considerations, we hypothesized that application of polyhalite to strengthen the skin of the final potato product should be performed at the end of growth. First, polyhalite was applied 2 days after haulm desiccation, assuming that the polyhalite mineral elements would support skin quality  through Similar experiments with additional potato cultivars: Arizona, Sifra, Panamera, and Mozart, indicated improved tuber appearance of the first two, but reduced visual appearance for the latter two Figure 4Results were less conclusive when the polyhalite was applied 3–4 weeks prior to haulm desiccation Figure 7KCS6, CYP86A33, and FHT. For the red-skinned cv. Rosanna, expression of CYP86A33 and FHT was upregulated, albeit not significantly, in the periderm of young tubers following preplanting polyhalite fertilization  was conducted. The analyses were conducted for two experimental years and included ongation , and of ongation , suggestKCS6, CYP86A33, and FHT was significantly downregulated in 105-DATP periderm of control tubers when compared to young 75-DATP periderm. Preplanting polyhalite fertilization moderated this downregulation , and is a contribution of ARO, the Volcani Center.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cannabis sativa L. is a plant long used for its textile fibers, seed oil, and oleoresin with medicinal and psychoactive properties. It is the main source of phytocannabinoids, with over 100 compounds detected so far. In recent years, a lot of attention has been given to the main phytochemicals present in Cannabis sativa L., namely, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). Compared to THC, CBD has non-psychoactive effects, an advantage for clinical applications of anti-tumor benefits. The review is designed to provide an update regarding the multi-target effects of CBD in different types of cancer. The main focus is on the latest in vitro and in vivo studies that present data regarding the anti-proliferative, pro-apoptotic, cytotoxic, anti-invasive, anti-antiangiogenic, anti-inflammatory, and immunomodulatory properties of CBD together with their mechanisms of action. The latest clinical evidence of the anticancer effects of CBD is also outlined. Moreover, the main aspects of the pharmacological and toxicological profiles are given. Cannabis sativa L. is a plant long used for its textile fibers, seed oil, and oleoresin, with medicinal and psychoactive properties [Cannabis genus being monotypic, that is, including only one highly variable species, Cannabis sativa L., or polyspecific—enclosing four  species with distinct geographical, chemotypic, and morphological features [Cannabis hybrids are absent. On the other hand, the common use of the appellation “strains” is considered improper, as this designation only applies to bacteria and viruses [Cannabis  can be made [operties . It is coperties . Taxonomfeatures ,4. The c viruses . From a  be made ,7.th century great variability in opinion on the therapeutic effects and preparations and also worries about drug abuse emerged [In Europe, great attention has been paid to the medical use of cannabis since 1840, and this was due to William O’Shaughnessy, an Irish physician who traveled to India and noticed the medicinal properties of Indian cannabis . Later,  emerged . The dec emerged , and to  emerged .The identification of the major cannabinoids, THC and CBD, has been an important step for further research. Numerous studies have been conducted on THC after its isolation and characterization in the 1960s . The canCannabis sativa L. is the main source of phytocannabinoids, with over 100 compounds detected so far [Phytocannabinoids are a type of cannabimimetic compound which can interact with the endocannabinoid system . Cannabid so far . The comd so far . The capd so far ,17.Cannabis occur due to non-enzymatic modifications [Cannabinoids are terpenophenolics comprising a diphenol and a monoterpene moiety. The synthesis of the former part occurs via the polyketide pathway by the stepwise condensation of three malonyl-Coenzyme A molecules with hexanoyl-Coenzyme A, in order to yield olivetolic acid . The monications . Studiesications . The exaCannabidiol is the second most abundant type of cannabinoid, showing higher concentrations than THC in many cannabis strains . CompareThe pharmacokinetics of CBD depends on its pharmaceutical formulation and on its routes of administration . It is amax (time taken to reach the maximum concentration) of between 1 and 4h [CBD is rapidly distributed in the lungs, heart, liver, and brain, and to the less vascularised tissues. It has a high distribution volume and can accumulate in adipose tissue in patients under chronic treatment, having the risk of a prolonged activity  ,35. Thus1 and 4h .Further, CBD is metabolised mainly in the liver, by the main isoenzymes CYP2C19 and CYP3A4, and to a lesser extent by CYP1A1, CYP1A2, CYP2C9, and CYP2D6 . After iCannabidiol is a non-psychoactive cannabinoid, compared with THC, the psychoactive compound which produces the main side effects of cannabis ,39. Comp1a (serotonin receptor 1A) receptor, PPARγ (nuclear peroxisome proliferation activated receptor γ) and glycine receptor subtypes [The main physiological function of the endogenous cannabinoid system consists of inhibiting the release of other neurotransmitters  in the nervous system, usually by stimulating CB1receptors . Cannabisubtypes ,31  impairment, as well as to increase anxiety, psychotic symptoms, heart rate, and blood pressure, and to alter perception. By mixing CBD with THC, the side effects of THC were reduced ,68.There are few data regarding its safety profile in children (limited pharmacokinetic information) . Due to Caution should be taken when CBD is administered in patients with hepatic impairment (risk of accumulation) or when taken with other drugs, because inducers/inhibitors of CYP3A4 or CYP2C19 can decrease/increase its plasmatic concentration ,69. MoreBoth cannabis and tobacco smoking can induce CYP1A2 metabolism (with addictive effects when they are smoked together), causing significant interactions for substances that are substrates of this enzyme .There were case reports of delirium and hypomania when CBD was associated with dysulfiram (unknown mechanism) and mania after association with fluoxetine (possibly CYP2D6 mediated) ,68.®, containing nabiximols , Epidiolex® (pure CBD), currently in Phase III trials, and Arvisol® , currently in Phase I trials for the treatment of schizophrenia and epilepsy [Cannabidiol is present for the moment in three pharmaceutical formulations, such as: Sativexepilepsy .® is an oromucosal spray, 100 µL = 2.7 mg THC + 2.5 mg CBD, obtained from Cannabis sativa L. It is suggestedto be used as a symptomatic treatment (in combination with other current anti-spasticity medicine) in adults with moderate-to-severe spasticity caused by multiple sclerosis, in case of failure of previous treatments. It requires up to two weeks of titration period to achieve an optimal dose and its administration should be associated with food intake. Nabiximols can be used once (afternoon or evening) or twice a day (morning and evening), the average dose being 8 sprays/day (maximum 12 sprays/day), with a minimum 15 min gap between administrations. After 4 weeks of treatment, the specialist physician should evaluate the patient’s response to the treatment. It is not recommended to be used in patients less than 18 years old and with caution in the elderly. It is contraindicated in case of hypersensitivity to cannabinoids, known/suspected history/familial history of schizophrenia or other psychotic illnesses, or severe cardiovascular diseases [Sativexdiseases .® is an oral solution containing 100 mg/mL of CBD, which is suggested for the treatment of seizures associated with Lennox–Gastaut syndrome or Dravet syndrome in patients older than 2 years. The starting oral dose is 2.5 mg/kg, administered twice daily and which can be increased after one week at 10 mg/kg/day, the maximum dose being 20 mg/kg/day (divided into 2 administrations). Side effects of somnolence, sedation, hepatotoxicity, suicidal behavior, ideation (being recommended to monitor the patients), decreased appetite, diarrhea, fatigue, asthenia, sleep disorder, insomnia, and an increased risk of infections were reported [Epidiolexreported .The literature presents controversial studies regarding the hepatotoxic potential of CBD. On the one hand, Mallat et al. have shown that activation of the body’s endocannabinoid system, specifically the CB2 receptor, is therapeutically beneficial in the treatment of many liver diseases. Moreover, studies have shown that CBD can help fight cirrhosis by kill hepatic stellate cells . Other sFrom a different viewpoint, Ewing et al. have demonstrated that despite the many beneficial effects of CBD, it may possess a risk for liver toxicity . This reAn increased number of studies have demonstrated that CBD and other structurally related cannabinoids present anticancer potential both in vitro on various cell lines  as well Lung cancer is one of the most common causes of cancer deaths. According to the World Cancer Research Fund International, in 2018 there were two million new cases of lung cancer detected worldwide [orldwide . In Euroorldwide . Ramer eorldwide . In anotorldwide ,89. Inteorldwide . Ramer eorldwide .melanoma cases has permanently increased over the past few years compared to other types of cancer [The number of f cancer . A recenf cancer ,93,94.Breast cancer is the primary cause of death among women and is the second most common cancer overall [50 value of about 6 µM and exhibits significantly lower potency in non-cancer cells [50 values were 2 µM (MDA-MB-231 cells) and 5 µM (T-47D cells). At the same time, both cell lines showed improved nuclear localization of PPARγ following treatment with 1–7 µM CBD for 24 h. Moreover, treatment with CBD led to an interaction between PPARγ, mTOR, and cyclin D1 to the advantage of apoptosis induction [ overall . There a overall . Using M overall . Other ser cells . Recent nduction . In 2014nduction . McAllisnduction .Colon cancer is the second most common cause of cancer patient mortality all over the world. Moreover, colorectal cancer is the third deadliest cancer in the United States [d States ,102. Avid States . Kargl ed States . Anotherd States . A recend States .Prostate cancer is one of the most common types of cancer in men [−1) significantly inhibited prostate cancer cell viability in an animal model using male MF-1 nude mice [r in men . A recenr in men . Petroceude mice . Sharma ude mice . Anotherude mice .Brain cancer is identified as one of the most terrible forms of cancer due to several impediments [2+ influx in U87MG cells with an IC50 of 22.2 μM. Thereby, CBD could be used as a promising therapeutic agent against GBM cancer cell lines [ediments . Massi eediments . Recentlediments . Furtherediments . The rolll lines . Solinasll lines . Anotherll lines . Alharrill lines .One of the struggles of cancer treatment consists in the possibility of activation of the immune system against the tumor. In recent years scientists’ efforts were focused on developing therapies that target tumor immunity ,121. ImmThe stimulation of cannabinoid receptors (CBR) can lead immune cells to regulate the DNA binding of various nuclear factors, an effect mainly triggered by down-regulation of cyclic adenosine monophosphate (cAMP) formation . Cyclic Both in vitro and in vivo models have been used in order to evaluate the CBD effects on T-cells and macrophages. Results indicated that CBD has the ability to alter the reactivity of the immune system’s cells .Cannabidiol has been reported to decrease the production of T-helper 2 cytokines such as IL-10, which is well known to play an important role in humoral immunity . FurtherMacrophages have an important role in innate and adaptive immunity and are one of the main producers of IL-12 . Sacerdo−/−/CB2−/− mice showed that CBD administration caused suppression of IL-2 and IFN-γ expression and proliferation, suggesting an inhibition of T-cell function [Another study performed on splenocytes derived from CB1function .According to Carrier et al., CBD possesses immuno-suppressive effects through the enhancement of endogenous adenosine signaling . The autIn a study performed on splenocytes, CBD treatment induced a reduction of IL-2, IL-4, and IFN-γ production. Moreover, when tested on mice prior to ovalbumin sensitization, CBD induced a significant inhibition of antigen-specific antibody production, indicating an effect on the suppression of humoral immunity .Cannabis sativa L., CBD, proved to have a modulatory effect on tumor immunity, suggesting its therapeutic potential in cancer treatment. The compound is considered to be a component of the formulation known as “medical cannabis,” which is currently used in some countries [In a recently published paper, Jensen et al. evaluated the immune gene expression following treatment with CBD using a zebrafish model that resemblances the human genome by around 70%. The authors concluded that CBD modulated the immune genes differently, up-regulating IL1B and IL17A/F2 and down-regulating transforming growth factor beta, alpha (TGFBA), S100A10B , immunoglobulin heavy constant Mu (IGHM) and CD4-1 . The nonountries . FurtherAlthough for all higher organisms inflammation is the most competent defensive response of the innate and adaptive immune system, when it becomes chronic it can eventually cause organ dysfunction and structural impairment. Various studies have shown that sphingolipids participate in the structural preservation of cell membranes and mediate cellular functions specifically: migration, proliferation, and apoptosis. Therefore, they are prone to regulate the fate of the cell and consequent onset of inflammation and cancer . SphingoSchwiebs et al. have shown that, based on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer and cancer-induced inflammation evolve through separate, observable molecular stages. The presence of two different mechanisms of carcinogenesis production was observed in a model of compartment-specific SGPL1 depletion in immune cell compartment and tissue cell compartment. In the tissue cell section, they noted fast tumor growth with particular modulation of the tumor microenvironment, and chronic, complex inflammation with succeeding, but relatively delayed carcinogenesis, in the immune cell section . The theSummarizing these aspects, chronic inflammation increases the probability of different types of cancer, suggesting that abolishing inflammation may represent a well-founded strategy for cancer prevention and therapy . These fAs already described above, the multi-target effect of CBD includes anti-proliferative and pro-apoptotic activities, and as more recently described, anti-angiogenic properties . AngiogeCannabinoids were found to be responsible for down-regulating VEGF receptors in different cancer types. Apoptosis of endothelial cells was observed by activating CB receptors , and a rSo far, few studies have investigated the effects of CBD as an angiogenesis modulator. Solinas et al. discovered strong anti-angiogenic effects of CBD, both in vitro, reducing growth, migration, and invasion of HUVEC cells, and in vivo, using the Matrigel sponge assay in mice. Multiple mechanisms in modulating angiogenic factors relatingto impaired angiogenesis were observed .An interesting difference compared to the results obtained for cannabinoids in general was that CBD did not induce either apoptosis or necrosis on HUVEC cells at high concentrations (12 µM), but it did induce endothelial cell migration. The anti-migration activity was supported by the molecular modulation of several factors. Cannabidiol inhibits matrix metalloproteinase-2, 9  and tissue inhibitor of metalloproteinases 1 (TIMP1), thus impairing cell motility and invasion, suppresses the activity of urokinase-type plasminogen activator (uPA), and serpinE1/plasminogen activator inhibitor-1 (PAI1), which are involved in extracellular matrix degradation. By inhibiting chemokineligand 16 (CXCL16) and IL-8, cell motility and network formation are impaired, whereas down-regulating endothelin-1 (ET-1), platelet-derived growth factor subunit A (PDGF-AA), and VEGF expression, micro vessel density is reduced . VasculaA more recent publication explored how CBD could inhibit angiogenesis in colon cancer, and it was shown that it significantly decreased the level of proinflammatory cytokines IL-6 and IL-8 and increased the activity of malonaldehyde (MDA), an antioxidant enzyme. Others found that CBD reduces the levels of an mTOR substrate and STAT5, inducing vasorelaxation, thus contributing to the underlying mechanism of the anti-angiogenic effect in human endothelial cells ,139.Another recent study evaluated the effect of CBD in breast cancer and found that it can modulate the tumor microenvironment by reducing the recruitment of macrophages, which leads to angiogenic inhibition .Next to its low toxicity and the non-psychoactive activity, the anti-angiogenic properties and the multi-target anti-tumor effects indicate that CBD is an interesting candidate for anticancer clinical applications .Today, CBD has become extraordinarily popular around the world. It is now accessible in a growing number of products with different administration modes . There aCurrently, there are hundreds of active manufacturers and sellers of CBD, and their number is rapidly growing, because CBD is capable, as shown by important studies in the field, of inhibiting the development of an increased number of cancer cells both in vitro and in vivo .Information collected to date in relation to the anticancer effects of CBD are nearly completely limited to preclinical studies conducted on cell lines and also on animal models . AlthougIn terms of clinical trials, CBD was most studied for glioblastoma. A clinical trial that analyzed the effect of CBD as a single agent for solid tumor was conducted in 2014 . Another placebo-controlled phase II clinical trial analyzed the effect of the combination of THC and CBD with adjuvant chemotherapy with temozolomide for patients with glioblastoma, and reported positive results using this approach  .An outstanding example is the use of CBD products for the self-medication of cancer with the aimof thoroughly curing it. In 2018, Sulé-Suso et al. reported an important case: an 81-year-old man, who was diagnosed with lung adenocarcinoma in 2016, refused chemotherapy and radiotherapy and began self-administration of CBD oil. He started first with two drops twice daily for a week and then nine drops twice daily for a month. One month later, a significant reduction on the number and size of mediastinal lymph nodes was observed on CT scan .In 2018, a clinical trial with 119 cancer patients was conducted over a four-year framed period. Patients were given CBD oil three days on and three days off, with an average dose of 10 mg twice a day (max 30 mg for increased tumor mass). In this study, patients with different types of cancer  were included. From 119 cancer patients, the most stunning case was a 5-year-old male patient with anaplastic ependymoma, a very rare brain tumor. The patient had all the standard treatments . He started the treatment with CBD oil in February 2016, and in December 2016 the relevant scans showed that tumor volume had decreased by approximately 60%. The other patients from this study have reported that the side effects completely disappeared, and although the duration of treatment was six months, many continued the treatment . Stella All these clinical cases prove that CBD demonstrates promising results, as it has undeniably helped different types of cancer patients. Although the literature describes the results of some clinical trials that include the pharmacokinetics and pharmacodynamics of CBD, there is an urgent need for further clinical trials to gain a complete picture of the effect of this molecule within the human body.This review provides an update on the phytochemistry of CBD. Moreover, it highlights important aspects of the pharmacokinetics, pharmacodynamics, toxicology, route of administration, and dosage of this bioactive molecule. An in-depth screening of the literature led to the conclusion that preclinical studies (in vitro and in vivo) have shown that CBD possesses a very complex mechanism of action through which it can inhibit tumor formation and propagation in different types of cancer. However, despite the large number of preclinical studies, further research is needed because at this point there is not yet enough clinical evidence to prove that CBD can safely and effectively treat any or one particular type of cancer in humans. To date, most clinical positive outcomes following treatment with CBD have been reported for glioblastoma. However, all the data described above suggest that CBD is a promising candidate for an anticancer agent.


To investigate the risk factors and drug resistance of MDR Acinetobacter baumannii in pneumonia patients.From January 2013 to February 2016, 98 pneumonia patients with MDR Acinetobacter baumannii in our hospital ICU were selected as the observation group, and 49 pneumonia patients with not-MDR Acinetobacter baumannii in our hospital ICU were selected as the control group in accordance with the proportion of 2:1. Sputum samples were collected from the two groups for drug resistance, and the risk factors and prognosis of MDR Acinetobacter baumannii in pneumonia patients were given survey analysis.The observation group was highly resistant to cefotaxime, piperacillin, imipenem, levofloxacin, gentamicin, tetracycline and ceftazidime, and was only sensitive to polymyxin. In addition to piperacillin, polymyxin B, the other antimicrobial drug resistance rates in the control group was significantly lower than in the observation group (P<0.05). Univariate analysis showed that diabetes, infection before hospitalization, admission 24h score of APACHE II and GCS scores, deep venous catheterization, and mechanical ventilation were related to the MDR Acinetobacter baumannii in pneumonia patients(P<0.05). Non conditional logistic regression analysis showed that diabetes mellitus, infection before hospitalization, admission 24h score of APACHE II and GCS scores were the independent risk factors for the MDR Acinetobacter baumannii in pneumonia patients(P<0.05).MDR Acinetobacter baumannii in pneumonia patients in ICU is common, where diabetes infection before hospitalization, admission 24h score of APACHE II and GCS scores are the main risk factors, and the vast majority of the antibiotics are resistant to the MDR Acinetobacter baumannii that can lead to poorer prognosis and followed-up of patients with increased mortality. Pan-res22.16 cfu/ml and sputum quantitative culture separation pathogens ≥ 106cfu / ml were collected by bronchoscopy or artificial airway, and qualified sputum for two consecutive isolates of the same pathogens was screened. Amino-glycosides antibiotics, pseudomonas cephalosporins, complex preparations containing beta-lactamase inhibitors, anti-Pseudomonas carbapenems antibiotics, fluoroquinolones antibiotics and other five kinds of antimicrobial drugs were used with at least one drug-resistant strain in each category, and only 1-2 kinds of antimicrobial agents  sensitive strains were used.Accepting criteria: 98 patients as an observation group was selected to stay in our hospital ICU ward after diagnosis of pan-resistant Acinetobacter baumannii pneumonia in January 2013 to February 2016, which is in line with hospital diagnostic criteria for pneumonia; lower respiratory tract secretions bacteria was checked for the pan-resistant Acinetobacter baumannii; excluding admission and ICU time less than 48h patients. At the same time in accordance with the ratio of 2:1 49 cases of Acinetobacter baumannii pneumonia as a control group was selected in our hospital ICU ward diagnosis and treatment of non-highly resistant patients with hospital-acquired pneumonia diagnostic criteria. According to to the United States ATS / IDSA hospital acquired pneumonia diagnosis and treatment guidelines, lower respiratory tract secretions of isolated pathogens ≥10This study is in line with the medical ethics standards, and have access to the patient or family informed consent, and approved by the hospital ethics committee.2.2Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853) Quality control bacteria, the use of antimicrobial drugs, including cefotaxime, piperacillin, imipenem, polymyxin, gentamicin, levofloxacin, tetracycline, ceftazidime, drugs are from the Chinese pharmaceutical and biological products. The results were judged by reference to the standards set by the American Clinical Laboratory Standardization Committee (CLSI) to determine drug resistance, respectively. Antibacterial paper was produced by OXOID, UK.In the collection of sputum specimens, the use of natural sputum, bronchoscopy, artificial airway suction method, etc. with reference to the hospital infection prevention, control standard operating procedures to operate, daily collection of patients with sputum specimens for 2-3 days were carried out to ensure that sputum specimens qualified. We selected using phoenixTM-100 automatic bacterial identification and drug sensitivity meter produced by BD company in Japan, and the drug resistance analysis was carried out in our institute’s microbiology laboratory. The standard strain was treated with 2.3Basic information included name, sex, age, disease status, combined underlying disease, hospitalization time, laboratory examination data mainly for admission 24h admission APACHE II score, awareness assessment (GCS score), 7d after treatment APACHE II score and GCS score. Drug use included hormones, immunosuppressive agents or chemotherapy, invasive operations, including deep vein puncture, catheterization and time, mechanical ventilation, etc. Antibiotics use included cephalosporins, fluoroquinolones, polysaccharides, aminoglycosides, and follow-up prognosis followed by 6 months of death.2.4t test  or rank sum test  is used for the comparison, and the number of measurement data is selected by the SPSS19.00 software. χ2 test and the univariate analysis of significant differences in the variables were used unconditionally with logistic regression analysis, with P <0.05 as a significant difference.The count data (X ± s) is expressed as a percentage, the 33.1After treatment, the observation group for cefotaxime, piperacillin, imipenem, gentamicin, levofloxacin, tetracycline, ceftazidime were highly resistant, and only polymyxin sensitive. In addition to piperacillin and polymyxin, the resistance rate of the control group to other antimicrobial agents was significantly lower than that of the observation group (P <0.05). See 3.2We compared the clinical data of all patients with independent variables as a single factor analysis. The results showed that diabetes mellitus, pre-infection hospital stay, admission 24h APACHE II score, GCS score, deep vein catheterization, mechanical ventilation and other factors and ICU Mycobacterium pneumoniae are significantly correlated (P <0.05). See 3.3We analyzed the unconvolable logistic regression analysis of the seven variables with significant differences in univariate analysis. The results showed that diabetes mellitus, hospital stay before admission, APACHE II score within 24 hours of admission, GCS score, resulted in pan-resistant Acinetobacter baumannii infection with independent risk factors (P <0.05) shown in 3.4The APACHE II score and GCS score of the observation group were significantly lower than those of the control group (P <0.05). The mortality rate of the observation group was also lower than that of the control group (P <0.05). See 4Acinetobacter baumannii is a simple conditional pathogen, which is widely found in nature and has a strong ability to adapt to the environment and the ability to quickly obtain resistance to become an important pathogen of hospital infection . Studies，especially pan-resistant Acinetobacter baumannii on penicillins, sulfonamides, aminoglycosides, cephalosporins class, carbapenems and other antimicrobial drugs. This study shows that the observation group is highly resistant to cefotaxime, piperacillin, imipenem, gentamicin, levofloxacin, tetracycline and ceftazidime, and is only sensitive to polymyxin. In addition to piperacillin and polymyxin, the resistance rate of the control group to other antimicrobial agents was significantly lower than that of the observation group (P <0.05). From the mechanism analysis, β-lactamase is the main mechanism that is desirable for resistancing β-lactamase antibiotics, acetinase acts on the β-lactam ring, making β- Lactam bond cleavage and loss of antibacterial activity [Acinetobacter baumannii with natural resistance is treated with a variety of antimicrobial drugs, making the pan-resistant strains increase year by year ，especialactivity . Polymyxactivity .The panoramic resistance to Acinetobacter baumannii can be of adherent or colonized form and exists in different tissues and systems of the human body and organs In patients with low immunity, Acinetobacter baumannii can proliferate and produce pneumonia ,19. In t


Background: Variations in blood glucose levels over a given time interval is termed as glycemic variability (GV). Higher GV is associated with higher diabetes-related complications. The current study was done with the aim of detecting the sensitivity of various GV indices among individuals with type 2 diabetes mellitus of different glycemic control status. Methods: We performed a longitudinal study among individuals with type 2 diabetes mellitus (T2DM) who were participating in a two-week diabetes self-management education (DSME) program. Participants were categorized by their HbA1c as poor (≥8%), acceptable (7%–8%), and optimal control (<7%). Continuous glucose monitoring (CGM) sensors recorded interstitial glucose every 15 min from day 1. The evaluated GV measures include standard deviation (SD), coefficient of variation (CV), mean amplitude of glycemic excursion (MAGE), continuous overlapping net glycemic action (CONGA), mean of daily difference for inter-day variation (MODD), high blood glucose index (HBGI), and low blood glucose index (LBGI). Results: A total of 41 study participants with 46347 CGM values were available for analysis. Of 41 participants, 20 (48.7%) were in the poor, 10 (24.3%) in the acceptable, and 11 (26.8%) in the optimal control group. The GV indices  of poorly controlled  were higher than acceptable  and optimal  control group. Glycemic variability was reduced in the poorly and acceptably controlled groups by the end of the 2-week period. There was a rise in LBGI in the optimally controlled group, indicating pitfalls of tight glycemic control. Conclusion: Indices of glycemic variability are useful complements, and changes in it can be demonstrated within short periods. The prevalence of diabetes mellitus is rising, with 1 in 11 adults worldwide affected, and more than 90% being type 2 diabetes mellitus (T2DM) . Studiesn) [Numerous metrices for glycemic variability have been reported ,14,15,16n) ,17 glycen) ,19,20. Wn) .Diabetes self-management education (DSME) to promote higher physical activity levels, dietary modifications, and optimizing drug therapy targeted at patients with poorly controlled diabetes mellitus are likely to achieve improved short- and intermediate-term glycemic control ,23,24. IWe designed a longitudinal study of two-week duration to understand glycemic variability amongst patients with type 2 diabetes mellitus. All participants provided written informed consent prior to initiation of study procedures. This study was approved by the Institutional Human Ethics Committee of AIIMS Bhopal on 16 December 2016 with the reference number IHEC-LOP/2017/EF0036.All India Institute of Medical Sciences, Bhopal is a tertiary care hospital in central India. Patients with type 2 diabetes mellitus are followed up in the diabetes clinic in the Department of Medicine, which maintains contact details, appointment schedules, and a record of sequential glycosylated hemoglobin (HbA1c) levels. We designed a two-week DSME program to understand the effect of dietary interventions, where we were assessing glycemic control using CGM-based measurements. The current study was conducted in the setting of such a program.We identified adults (aged more than 18 years) having type 2 diabetes and a HbA1c of more than 7% on two previous visits over the past one year from the records of the diabetes clinic. All such 379 participants were first telephonically contacted to identify those who lived within a 5–10 km radius of the hospital and were willing to enroll for a two-week DSME program on pre-specified dates. All potentially eligible participants were invited in the institute for a pre-screening visit. The purpose of this visit was to appraise them synchronously in details about the DSME schedule and pre-conditions to adhere with this schedule. The persons who were in agreement to follow the schedule for DSME were enrolled, and a venous blood sample was drawn to obtain a baseline HbA1c value. Of the 84 individuals who came for the pre-screening visit, 48 agreed to participate.Multi-modal DSME programs were conducted in two batches for the willing participants between April and June 2017. A key objective of this program was to explore the feasibility, and the effect of intensive education and low glycemic index breakfast (served in some of the sessions) to improve glycemic control. Each program batch had 24 participants and was of a 14-day duration. Participants were engaged all 14 days between 6 am and 8 am in the morning. On the first day, a questionnaire was administered to all participants, and information about their demography, comorbidities, known complications, and drug therapy was obtained. Further, participants also self-reported their engagement in diabetes-related self-care activities and performed an assessment of their quality of life (QoL). Thereafter, the study physicians  reviewed their previous prescriptions. Fasting blood samples and urine samples were collected on the fifth day of the program to test for serum creatinine, lipids, and to obtain urine albumin to creatinine ratio. Participants were engaged in activity sessions on different days relating to knowledge enhancement , physical activity promotion , dietary modification , coping strategies (stress reduction), foot care, and drug optimization. These activity sessions  were conA continuous glucose monitoring (CGM) sensor was placed on the upper arm or back  of all participants on the first day of the workshop, and daily retrospective review of readings was done using a reader. This sensor records interstitial glucose (IG) value after every 15 min, for a total of 14 days. These IG values correspond to the blood glucose (BG) values. Participants were provided individualized feedback based on CGM readings, food diary, and activity records. The sensors were removed after 14 days or earlier if not tolerated. CGM sensors were provided by the institute free of charge to the study participants.All the participants were classified in three glycemic control categories, based on HbA1c value obtained at the pre-screening visit—less than 7% as optimal control, 7%–7.9% as acceptable control, and 8% or higher as poor control ,26. Gluct on x-axis and BG value at the time (t + 1 h) on y-axis. The difference (Δy) of coordinate represented the BG rate of change in 1 h. We plotted 3 such graphs (one for each group) at day 0, day 7, and day 14. A gradual, more concerted graph was perceived as a marker of system stability and the scattered plot was perceived as an irregular system. A subjective comment of the trend of dynamic regularity was made. The data analysis and graphical analysis was done using R version 3.4 [Glycemic variability measures were calculated as per definitions provided in sion 3.4 . DetailsA total of 54,524 such values from 46 participants were available for analysis. Out of these, five participants were excluded as they did not have recordings for the entire duration of the program due to early sensor removal. Of the 41 participants included in the analysis, 20 (48.7%) had poor glycemic control, 10 (24.3%) had acceptable control, and 11 (26.8%) had optimal glycemic control. The baseline characteristics of all participants are provided in As compared to the baseline, there was a fall in the mean BG values from the baseline (B) to the end of intervention (I) in all three groups (poorly controlled group: 203.6 mg/dl (B) to 144.62 mg/dl (I); acceptable control group: 127.2 mg/dl (B) to 102.2 mg/dl (I); and optimal control group: 115.9 mg/dl (B) to 98.21 mg/dl(I)). A reduction in glycemic variability indices like SD, CV, MODD, MAGE, and CONGA was also observed .These indices had a similar declining pattern in poorly controlled and acceptably control sub-groups as the minimal change in optimally controlled group . LBGI inThe subgroup of participants with acceptable glycemic control had a larger decline in variability, as compared to those with poor or optimally controlled . The preIn the current study, we described changes in glycemic variability and glycemic control amongst individuals with T2DM, during a DSME program. As anticipated, we noticed improved glycemic control, as well as reduced glycemic variability in all the subgroups . The performance of glycemic variability indices that are based on measures of central tendency  was similar. Indices that are influenced by extreme values  had a differential pattern.The ability to estimate average blood glucose by a single test such as HbA1c has revolutionized diabetes care, and technologies have now enabled us to obtain this measure at point-of-care. While this measure of central tendency over a period of three months is pivotal, a measure of dispersion of blood glucose values has eluded diabetes care providers. Strides in continuous glucose monitoring technology have the potential to provide a summary measure of dispersion or glycemic variability ,12,14. EIndividuals with T2DM who are optimally controlled at baseline are an acceptable benchmark to define achievable limits of glycemic variability. The range of glycemic variability measures from end to start of the DSME program in this subgroup in our study was 23.14–24.56 for MODD, 111.55–126.15 for MAGE, and 7.61–8.67 for CONGA. Thus, intermediate values of 24 for MODD, 119 for MAGE, and 8 for CONGA could be acceptable benchmarks for glycemic variability. As compared to these benchmarks, the poorly controlled subgroup in our study achieved MODD of 32, MAGE of 154, and CONGA of 8.4 towards the end of the two-week DSME program. In terms of proportion, this subgroup was 25%, 22%, and 4.7% away from benchmark MODD, MAGE, and CONGA.In the family of glycemic variability indices, MODD has its unique place, as it can measure inter-day variations ,17. OrigThe gradients of decline in these three measures  are analogous. The gradient is more in those with poor or acceptable control as compared to optimal control, as the greater the dispersion the more there is opportunity for change. As the educational intervention proceeds, it has a cumulative effect on glycemic fluctuations and dispersion of both inter-day and intra-day values will be reduced. Thus, a mutual comparison of these variability measures shows high mathematical concordance, and any one intuitive measure would be reasonable.Hypoglycemia is a trade-off for optimal glycemic control. Risk of hypoglycemia is reflected by LBGI— the higher the value, the greater the risk ,15. ThisSeveral other structured predominantly non-pharmacological interventions, like an introduction to mobile diabetic self-care system and real-time continuous glucose monitoring system, have also reported the decrement in GV measures following interventions ,29. All Variability is the key feature of all biological systems and is an intricate reflection of endogenous  and exogenous  influences ,20. The To conclude, glycemic variability indices were measured and have shown changes over a short period of time and, thus, could be used in clinical practice to assess glucose control status. Various measures correlate with each other, and hence, a most intuitive measure of change (such as MAGE or MODD and LBGI) may be used for clinical application. The utility of individual measures in glycemic control also needs to be evaluated in terms of its ability to reduce variability.


Intestinimonas, Catenisphaera, and Globicatella in the model group. Further analysis indicated a negative correlation of Allisonella and Odoribacter, as well as a positive correlation of norank_f__Peptococcaceae, Clostridium_sensu_stricto_1, and Coprococcus_2, with claudin5, occludin, α-actin, and ZO-1. Our use of a rodent model to explore the association between compromised intestinal and blood–brain barriers and altered fecal microbiota under psychological stress improves our understanding of the gut–brain axis. Here, cues converge to control basic developmental processes in the intestine and brain such as barrier function. This study provides new directions for investigating the pathogenesis of emotional disorders and the formulation of clinical treatment.Altered gut microbiota has been identified during psychological stress, which causes severe health issues worldwide. The integrity of the intestinal barrier and blood–brain barrier regulates the process of bacterial translocation and can supply the nervous system with real-time information about the environment. However, the association of gut microbiota with psychological stress remains to be fully interpreted. In this study, we established a psychological stress model using an improved communication box and compared the expression of tight junction proteins in multiple regions of the intestinal  and blood–brain  barriers between model and control rats. We also conducted fecal microbiota analysis using 16S rRNA gene sequencing. Expression levels of the stress-related indicators adrenocorticotropic hormone, NR3C1,2, and norepinephrine were increased in the model group compared to control group. Psychological stress reduced brain and intestinal levels of tight junction proteins, including claudin5, occludin, α-actin, and ZO-1. Microbiota analysis revealed elevated microbial diversity and fecal proportions of  Stressful life events play an important role in the occurrence of mental illness; however, the unclear pathogenesis leads to many obstacles when forming a treatment plan . The humThe integrity of the intestinal barrier and blood–brain barrier (BBB) is necessary for protecting the body from external stimulation and disturbance of the internal environment. Repeated exposure to social stress can alter the diversity and composition of gut microbiota, accompanied by changes in microbial metabolites, cytokines, chemokines, and monoamine transmitters, which regulate behavior by stimulating the peripheral and central nervous systems . For insModeling of psychological stress generally includes the simultaneous presence of physiological and psychological stressors, as in the chronic unpredictable mild stress (CUMS) , social In this study, we established a real psychological stress model based on that of Gomita and Ramsey  by emploFour-week-old, female, specific pathogen free SD rats were purchased from Vital River Laboratory Animal Technology Company  and housed in the laboratory animal center of the Henan University of Chinese Medicine . The rats were housed in sterile animal colonies under 25°C ± 5°C, 65% ± 5% humidity, and 12 h light-dark cycle. This study was conducted at Henan University of Chinese Medicine under the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals approved by the Animal Ethics Committee of Henan University of Chinese Medicine (Permit Number: DWLL2018030017).n = 15 each). Rats in the model group received psychological-stress stimulation through the communication box system for 28 days  for serum adrenocorticotropic hormone (ACTH) analysis using an ELISA kit; these rats were then sacrificed for isolating the cortex, amygdala, and hippocampus on ice. (ii) High-performance liquid chromatography (HPLC) was used to determine norepinephrine (NE) content in cortex, amygdala, and hippocampus. Subsequently, another ten  rats were perfused with polyformaldehyde for rapid intestine and brain extraction. (iii) Brains were immersed in polyformaldehyde fixative and cut into parts to observe the expression of NR3C1 and NR3C2 in the cortex by immunohistochemistry. (iv) The remaining brain parts and intestines were immersed in polyformaldehyde fixative to observe the expression of claudin5, occludin, α-actin, and ZO-1 by immunohistochemistry, and to observe the structure of tight junctions by electron microscopy.After 7 days of accommodation, a baseline test of rats was carried out through an open field experiment. Thirty rats with consistent emotional level were randomly divided into two groups  At 8:30 am, the rats to be given electric shock and the model rats were placed in the improved communication box chamber, respectively. (ii) Using a stopwatch for timing, the equipment was electrified every other 6 s to give a momentary stimulation , along with a fire alarm. Modeling lasted for 60 s, followed by a 60-s interval. The operation was conducted 15 times in accordance with the above method, totaling 30 min.After the psychological stress stimulation lasting for 28 days, an open field test was employed to evaluate rat behavior. All procedures were conducted according to previous research . The open = 6, each group) in a super-clean workbench using the following steps: (i) the workbench was sterilized using ultraviolet radiation for 15 min; (ii) one person held the rat and massaged its abdomen to promote excretion of feces. Another person wearing sterile gloves collected the feces using sterile filter paper; (iii) the feces were transferred to a sterile centrifuge tube and stored at −80°C for microbiome determination.After modeling, feces were collected from twelve rats . Total DNA was extracted from feces using an OMEGA-soil DNA kit. PCR amplification was carried out using the V3-V4 variable region sequence of the 16S rRNA gene as target and 338F-806R with barcode sequences as primers. PCR products were sequenced on the Illumina MiSeq PE300 platform to obtain V3-V4 variable region base sequence information of bacterial 16S rRNA genes. Sequencing fragments were clustered by operational taxonomic unit (OTU) using the QIIME2.0 software package. Representative sequences of each OTU were compared with sequences in the Silva database to identify the OTUs and determine their corresponding abundance information. Chao, Sobs, Ace, bootstrap, and jackknife indexes were calculated to evaluate the richness and uniformity of bacterial flora in samples. PCA analysis, LEfSe analysis, and ANOVA were used to identify the characteristic bacteria in each group. A Spearman correlation heatmap was used to analyze the correlation between bacterial abundance and tight junction protein content.The level of ACTH in serum was determined using an ELISA kit. All procedures were conducted following the manufacturer’s instructions: (i) the absorbance of each sample was detected at 450 nm wavelength; (ii) the absorbance was taken as the ordinate, and the corresponding standard concentration was taken as the abscissa to draw a standard curve; (iii) the concentration of ACTH in each sample was calculated using the regression equation of the standard curve. All serum samples were analyzed in triplicate.Expression levels of NR3C1 and NR3C2 in the cortex were detected by immunohistochemistry. Norepinephrine (NE) levels in the brain  were detected by HPLC. To eliminate interference from the researchers and ensure the accuracy of the experimental results, we entrusted the immunohistochemical detection to Zhengzhou Dianjie Technology Co., Ltd., and entrusted the determination of NE in the brain to the Medical Laboratory Center of Chinese Academy of Traditional Chinese Medicine.3 were immobilized with 2.5% glutaraldehyde for 4 h and washed four times with 0.1 mol PBS for 15 min each time; (ii) after rinsing, tissues were placed into 1% osmium acid fixative solution and fixed again for 1.5 h; (iii) tissues were then rinsed four times with 0.1 mol PBS, for 15 min each time. Tissue blocks were fixed twice in 50, 70, 80, and 100% ethanol solutions and twice in 100% acetone, each for 15 min. The dehydrated tissue blocks were embedded in a mixture of epoxy resin 812 and acetone (1:1), epoxy resin 812 and acetone (2:1), and pure epoxy resin 812, respectively, and placed overnight at room temperature; (iiii) after the tissue blocks were polymerized, they were cut into 50–60 nm slices using an ultra-thin slicing machine and dyed in saturated uranium dioxide acetate solution for 20 min. After rinsing and drying, the sections were observed and photographed by transmission electron microscopy.Transmission electron microscope images were prepared by the Electron Microscope Center of Scientific Research and Experiment Center of Henan University of Traditional Chinese Medicine. The samples were processed as follows: (i) tissues less than 1 mmt-test, using IBM SPSS Statistics 22. P-value < 0.05 was considered statistically significant. 16S rRNA gene data were analyzed on the free online Majorbio I-Sanger Cloud Platform1. Sequencing data have been deposited under the number SRP201262 or PRJNA548591 in the NCBI database.Results are presented as mean ±SD. Statistical analysis was performed by independent sample P < 0.05, P < 0.05, P < 0.05, P < 0.05; amygdala, hippocampus: P < 0.01; P < 0.01, From continuous monitoring, we found that psychological stress caused negative effects on rat development, reflected by rats in the model group consuming less food and water, and losing significant weight  and intestinal barrier . Significant differences in expression levels of the four proteins in these areas were observed between the two groups of rats. Reduced expression of the four proteins was found in both the brain and the intestine in the model group. We observed strongly positive immunohistochemical staining in the control group and mildly positive staining in the model group . In addition, we can get the consistency information of samples from the box graph of sample discrete distribution on PC1 axis (P > 0.05).We generated 639245 high-quality V3-V4 region 16S rRNA gene sequences from 12 samples, representing 13 phyla, 23 classes, 32 orders, 53 families, 165 genera, and 897 OTUs. First, we examined alterations in the microbial composition with the principal coordinate analysis (PCoA) clustering. PC1 accounted for 40.13% of the variation and PC2 accounted for 32.11%. PCoA results displayed an obvious clustering of microbiota composition for two groups , whereas Clostridium_ sensu_stricto_1 was only found in the control group. Meanwhile, increased abundance of 19 genera (including unclassified_ f__Prevotellaceae and Phascolarctobacterium) and decreased abundance of 17 genera  was detected in the model group when compared with the control group (P < 0.05).The impact of stimulation on the structure of the gut microbiota was clear at the genus level. We identified 36 genera showing a significant difference in abundance between the control group and the model group . Eight gPhascolarctobacterium, Oribacterium, Desulfovibrio, Allobaculum, and Treponema_2 characterized the model group, while Prevotellaceae_UCG_001, Romboutsia, Ruminiclostridium_6, Ruminococcaceae_UCG_013, Oscillibacter, Bacteroides__pectinophilus_group, and Christensen- ellaceae_R_7_group were important in the control group. Inte- restingly, there were 11 stress-related genera: Romboutsia, Desulfovibrio, Ruminococcaceae_UCG-010, Treponema_2, Tyzze- rella, unclassified_o__Bacteroidales, Intestinimonas, Clostridium_ sensu_stricto_1, Catenisphaera, Gemella, and Globicatella.According to the LEfSe and LDA analysis, 53 taxa distinguished the two groups: 25 for the control group and 28 for the model group . There wP < 0.05). There was also a marked difference between the two groups in the qstat index, which reflects community diversity (P < 0.05). These substantial pieces of evidence indicated that psychological-stress stimulation had a significant impact on the gut microbiome: it changed the structure of the gut microbiome and increased the community richness and diversity.Meanwhile, at the genus level, the α-diversity of Chao, Sobs, Ace, bootstrap, and jackknife indexes in the model group, which represent community richness, were higher than those of the control group  in the duodenum, jejunum, and ileum showed strong relationships with the key gut microbiota at the genus level, which distinguished the two groups. It is notable that the similar results were detected in the BBB including amygdala and hippocampus. The  heatmap . Particuoccaceae  and Clostricto_1 , and thrisonella , Odoribaribacter , and Copcoccus_2 .In this study, we explored the association of defective intestinal and blood–brain barriers with altered fecal microbiota under psychological stress, to improve our understanding of the gut–brain axis. We found that real psychological stress could lead to impaired intestinal and blood–brain barriers, characterized by decreased expression of four tight junction proteins, looser tight junctions, and broken basement membrane. Moreover, there was a notable correlation between disordered microbial composition and compromised intestinal and blood–brain barriers.Previous psychological stress modeling methods [e.g., CUMS , social We found that real psychological stress alone had significant effects on the composition and diversity of gut microbiota in rats. There was no significant difference between the two groups at the phylum level: Bacteroidetes, Firmicutes, and Proteobacteria represented approximately 98% of the total microbiota, consistent with previous studies . HoweverFurthermore, the α-diversity of gut microbiota in the model group was significantly higher than that in the control group after long-term chronic psychological stress, which was different from the decreased gut microbiota diversity triggered by the CUMS , social Previous scholars have proved that the brain and intestine can communicate with each other through the vagus nerve, and neuroendocrine, immune, and metabolic pathways . In the It was a pity that we didn’t evaluate the changes in immune or metabolic substances in intestine or blood circulation. Nevertheless, based on the previous research reports, we could conclude the significant role that the defective intestinal and blood–brain barriers plays in the communication between gut and brain. The barriers are vital for sympathetic motor function , neuroenWe hypothesize that defections of the intestinal barrier and BBB at multiple sites are essential links in brain–gut communication under psychological stress. Our findings provide a favorable research basis and direction for future study of the brain–gut axis and the pathogenesis of psychological diseases: whether the occurrence and development of psychological diseases can be blocked or treated by changing the permeability of the BBB and intestinal barrier.Nevertheless, this study included very limited animals to study the correlation between gut microbiome and tight junction proteins of the intestinal and blood–brain barriers under psychological stress. Experiments employing a larger sample size are expected and urgently needed to verify the association. Also, we call for further research to elucidate the pathways in which stress affects such permeability and the material basis for mediating brain–gut communication, thus laying the foundation for the prevention and treatment of psychological disorders.Sequencing data have been deposited under the number SRP201262 or PRJNA548591 in the NCBI database.The animal study was reviewed and approved by the Animal Ethics Committee of Henan University of Chinese Medicine.SG performed the experiments and wrote the manuscript. LY provided the financial support, guided the design of experiments, and revised the manuscript. FC, ZZ, JL, WL, LC, YC, YB, ZF, YLin, ZL, SZ, HeW, and QZ carried out the experiments. YLi, XL, and JH guided the experiments. FC drew the diagram of the modeling device and the mechanism of brain-intestinal communication. JL and LC carried out the data analysis. HoW, XW, and JZ provided help in collecting literature.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.