Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs. Typical donor cells used in MMCT are Chinese hamster ovary (CHO) and mouse A9 cells. Unlike most cell lines, which die under prolong exposure to microtubule inhibitors, the A9 and CHO cells undergo repetitive hyperploidization in the presence of Colcemid with micronucleation occurring during the transition from metaphase to pseudo-G1.2 Micronuclei formed by A9 and CHO are thus smaller and more numerous. These micronuclei can subsequently be extruded from the cell as microcells by centrifugation in the presence of cytoskeleton disruptor, like an actin inhibitor.3 Several groups constructed mouse A9 or CHO-microcell hybrid libraries containing individual human chromosomes that provided valuable resources for mapping and functional studies of human genes.4–7Microcell-mediated chromosome transfer (MMCT) technique was developed in 1977 by Fournier and Ruddle.8 in 1975. Like MMCT, this method also aimed to transfer chromosomes between cell lines. However, it differs by the manner it generates chromosome containing membrane bound particles. Unlike MMCT, the method described by Mullinger and colleagues induces mitotic cells to generate mini-segregants, cluster of small daughter cells. It induces these abnormal mitotic chromosome segregations by storing mitotic cells at 4 °C, followed by resumption of growth upon return to 37 °C incubation.Alternatives to MMCT include a method described by Mullinger’s groupi.e., genes for tumor suppression,9 DNA repair,10–12 metastasis, telomerase regulation, genomic instability,13 mitochondrial disorders,14 and lysosomal storage diseases.15 MMCT technique has been also applied for analysis of specific chromosome status such as aneuploidy and epigenetics.17 A relatively new MMCT application is the transfer of mammalian artificial chromosomes  and human artificial chromosome (HAC) vectors) into recipient cells for gene function studies and other goals.18–25 The advantage of artificial chromosomes over entire chromosomes transfer is that MAC and HAC vectors can carry an individual gene of interest, while natural chromosomes contain numerous genes, which would complicate the study a single specific gene.Although the MMCT method was developed nearly 40 years ago, two main limitations make the method tedious. First, the frequency of the chromosome transfer from donor cells into recipient cells is very low. Second, MMCT is not universally applicable, particularly in cell lines where fusion with microcells is very inefficient. Yet despite these limitations, MMCT technique has been applied to various studies over the years. For example, MMCT has contributed to mapping the genes through functional complementation, de novo creation) or a top-down approach .18–25 MACs and HACs are maintained stably as additional chromosomes in mammalian cells over multiple generations due to the presence of a functional kinetochore. Some modified MACs and HACs contain a single gene-loading site that allows insertion a therapeutic gene up to several Mb in size.26–28 Recently, MACs/HACs were constructed with multiple gene-loading sites29–31 to carry several genes or assemble a single large gene along with its native gene regulatory elements. Many publications have described the application of MMCT to transfer MACs/HACs carrying certain genes to gene-deficient cells for gene function studies, humanized animal transgenesis, and high-yield protein production.32–37 As MMCT has numerous applications, a more efficient procedure for chromosomes or MACs/HACs transfer is a worthy goal.Mammalian artificial chromosomes have been generated by either a bottom-up approach  to stop the cells at the metaphase phase stage of cell cycle for 48–96 hours   .24,38,39romosome .33,34,38ll types  to gauge44Colcemid is a well-known microtubule inhibitor. It binds to plus ends of microtubules and suppresses microtubule dynamics. This suppression prevents formation of the mitotic spindle, allowing cells to be captured at metaphase.2(1) = 91.02, P < 0.0001).From our observations, hamster CHO cells are very resilient to Colcemid treatment . Only 3–F = 80.35, P = 0.0001).We changed step 1 in the protocol and incubated donor cells with combination of TN-16 + Griseofulvin instead of Colcemid . After t46The second key drug component of the original MMCT protocol is Cytochalasin B, which disassembles the actin cytoskeleton and allows extrusion of micronuclei to form microcells. Cytochalasin B is an old drug, and it inhibits actin filament polymerization through binding to the fast-growing (barbed) end of F actin filaments.47 From actin inhibition test, we identified three potential actin cytoskeleton disarrangement inhibitors that were better or equal to Cytochalasin B—Cytochalasin D, Chaetoglobosin A, and Latrunculin B  = 12.83, P < 0.0001).We then compared MMCT efficiency between Cytochalasin B and these three inhibitors. We changed step 2 in the standard MMCT protocol and incubated donor cells with one of candidate inhibitors using Cytochalasin B as a control . We counobtained . The preobtained ,d. Latruobtained  As Latrunculin B works at a concentration of 0.2 µmol/l, 100 times lower than Cytochalasin B (20 µmol/l), it represents a significant reduction in the cost of MMCT experiments. As an example, in 2016, Santa Cruz Biotechnology sold 5 mg of Cytochalasin B  at $104 and 1 mg of Latrunculin B  at $216. Thus, the working solutions of 20 µmol/l Cytochalasin B cost $199.52/l, while 0.2 µmol/l Latrunculin B cost $17.09/liter. We have reused 0.2 µmol/l Latrunculin B solution twice and 20 µmol/l Cytochalasin B solution eight times with no loss of MMCT efficiency. It is also worth noting that the Cytochalasin B solution has been reported to be reusable up to 30 times. However, we filter our working solutions to prevent microcell cross contamination between experiment, so the cost of the filter system should be considered. A case of twelve 500 ml Corning Disposable Sterile Filter Systems  cost $200.21 or $16.68 per filter. Hence, it is cost competitive to use new solutions of 0.2 µmol/l Latrunculin B for every new experiment.It has long been observed that cells in metaphase round up and are easily detached from the plate surface. There is even a method to isolate metaphase cells by shaking culture plates. As formation of microcells is believed to be caused by extrusion of micronuclei from donor cells by centrifugation, we guessed that adhering more metaphase cells to the culture flask surface would improve the microcells production. Therefore, we compared the percentage of metaphase cells obtained from surfaces coated by four different cell adhesion proteins  singularly and in combinations against the tissue culture plastics that is normally used.P = 0.0020; two tailed) increase (1.3-fold) of attached metaphase cells was detected from plates coated by Collagen and Laminin  = 721.5, P < 0.0001). In mouse embryonic stem cells (mES), no HAC transfer events were detected using the original MMCT protocol. In contrast, seven HAC transfer events, averaging 2.3 colonies per experiment repeat, were detected when the new MMCT protocol was used  , but no 48 and at the concentrations of Cytochalasin B (20 µmol/l) used in MMCT49 could potentially induce damage to the HAC. Hence, we decided to examine if there is a difference in the level of HAC damage when the original MMCT protocol with Cytochalasin B versus the new protocol with Latrunculin B was applied. We performed Southern blot analysis of the structure of the alphoid array within the alphoidtetO-HAC after SpeI digestion43 . A total of 36 HAC-containing clones, obtained from the original (18 clones) and new protocol (18 clones), were analyzed  for microcell–cell fusion.52 The HN protein of the hemagglutinating virus of Japan recognizes acetyl type sialic acid as a receptor. Another modification involves the expression of H and F measles virus envelope proteins in the donor cells .53 The H (hemagglutinin) and F (fusion) proteins mediate both viral attachment and membrane fusion. They bind to human CD46 surface receptor. The HAC transfer efficiency of this approach in HT1080 is comparable with the standard MMCT protocol using inactivated hemagglutinating virus of Japan viral envelope. This modified protocol is limited to recipient cell lines that express the CD46 receptor. A more recent work overcomes this limitation by fusing a targeting protein such as single-chain antibodies (scFv), peptides, growth factors, or cytokines to the extracellular C terminus of the H protein.49 So in principle, any cell type may be targeted as a recipient cell for MMCT. Oshimura’s group has also established a cryopreservation method to store microcells at −80 °C.54 This innovation significantly aids the MMCT protocol as preparation of microcells is time-consuming and not easily scaled up.The main work on optimizing the MMCT protocol has thus far been carried out by Mitsuo Oshimura’s lab at Tottori University. This group has mainly focused on the last step of MMCT, In our study, we also attempted to improve the efficiency of the MMCT transfer technology. However, our focus has been on the sections of the MMCT protocol that has thus far been neglected. We demonstrated that the replacement of key chemicals, Colcemid and Cytochalasim B for TN-16 + Griseofulvin and Latrunculin B in combination with a surface coating (Collage/Laminin), significantly increased the efficiency of HAC transfer to recipient cells. The modified MMCT protocol was successfully applied to four recipient cell lines including human fibrocarcoma HT1080, cervical cancer HeLa, human mesenchymal stem cells (hiMSC), and mouse embryonic stem (mES) cells. Southern blot analysis suggests that the modified MMCT protocol is less damaging compared with the standard protocol. However, to make a definitive conclusion, more HAC-containing clones have to be characterized.The maximum increase in MMCT efficiency that we have observed in our experiments was about sixfold. It is obvious that further work to optimization of the MMCT protocols is required. The next step in further developing the MMCT protocol may lie in the improvement of purification of microcells and the combination of our protocol for microcell generation with the improved fusion and storage protocols developed by Oshimura’s group. To conclude, the improved MMCT technique for gene transfer via HAC vectors will facilitate the functional studies of genes and cell engineering such as reprograming of cells.2 atmosphere in F12 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mmol/l l-glutamine. HT1080, HeLa, and hiMSC cell lines were routinely maintained in 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mmol/l l-glutamine. Murine ES cells  were cultured on gelatin-coated dishes in Knockout-DMEM supplemented with 15% ES cell-qualified fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mmol/l l-glutamine, nonessential amino acids, 50 μmol/l β-mercaptoethanol, and 1,000 U/ml leukemia inhibitory factor (LIF). For routine passaging, cells were washed in phosphate-buffered saline (PBS), treated with 0.25% Tripsine solution, and split at 1:4 ratio. All cell culture media and components were purchased from Life Technologies,  and Sigma , unless other indicated. CHO cells were routinely maintained in 5% CO40 with minor modifications. Exponentially growing (80% confluent) HAC-carrying cells were treated for 2–4 hours or overnight at 37 °C with 0.1 μg/ml Colcemid  in 5% CO2 atmosphere. Cells were harvested by trypsinization and incubated in hypotonic (0.56% KCl) solution for 20 minutes. After that, cells were fixed in a fixative solution , washed three times in the fixative solution, and stored, if necessary, in fixative solution at −20 °C. For metaphase spreads, cells suspension was placed dropwise on precleaned fat-free microscope glass slides , air-dried, and aged at least overnight or longer at room temperature (RT) in dust-free place. Metaphase spreads were prepared as described previouslySlides with metaphases were rehydrated with 1× PBS for 15 minutes at RT, fixed with 4% paraformaldehyde, prepared on 1× PBS for 2 minutes, and washed three times with 1× PBS for 5 minutes. Then, slides were gradually dehydrated at RT for 5 minutes each: 70% ethanol (EtOH), 90% EtOH, 100% EtOH and dried up. Hybridization mix  was applied onto each dehydrated slide in 20 μl of volume and covered with cover glass. After, slides were denaturated at 80 °C on heating table for 3 minutes (covered from light). Slides were incubated in darkness 2–6 hours at RT. After indicated period of time, cover glasses were removed, and slides were washed two times in Washing Solution I  for 15 minutes, three times in Washing Solution II  for 5 minutes, and briefly rinsed once in PBS. Then, slides were gradually dehydrated at RT for 5 minutes each: 70% EtOH, 90% EtOH, 100% EtOH and dried up. Slides were mounted in Vectashield mounting media, containing 4′,6-diamidino-2-phenylindole . Images were captured and analyzed using a DeltaVision microscopy imaging system and software in the CRC, LRBGE Fluorescence Imaging Facility (NIH). The PNA probes used for FISH were provided by Panagen Company .About 5 × 104 CHO cells were plated onto gelatin-covered cover slips in 24-well plate and left overnight. At the next day, cells were washed with DMEM and treated with DMEM-diluted inhibitors of actin cytoskeleton formation; Cytochalasin D, Chaetoglobosin A, and Latrunculin B were diluted in concentrations 0.2, 2, and 20 μmol/l; Swinholide A was diluted in concentrations 0.1, 1, and 10 μmol/l; and Wiskostatin was diluted in concentrations 5, 50, and 500 μmol/l also see . Cells wAfter MMCT, colony of cells were stained with crystal violet for counting. Dishes with cells were washed twice with PBS. Cells were fixed with 4% paraformaldehyde on PBS 15 minutes at RT. After fixation, cells were stained with crystal violet (prepared in 10% ethanol) for 15 minutes at RT. After staining, dishes were gently washed with deionized water until the water no longer runs blue. Images of stained colonies were captured and analyzed using Gel-Doc Documentation System and Quantity One Software .About 10 µg/ml Colcemid was purchased from Thermo Fisher Scientific. All other microtubule inhibitors were purchased from Santa Cruz Biotechnology. The following stock solutions of microtubule inhibitors were prepared and kept at −20 °C; TN-16 80 mmol/l in dimethyl sulfoxide (DMSO), Griseofulvin 25 mmol/l in DMSO, Nocodazole 25 mmol/l in DMSO, Vinorelbine ditartarate 25 mmol/l in DMSO, Noscapine hydrochloride 25 mmol/l in water, and Myoseverin B 2.5 mmol/l in DMSO. CHO cells were plated onto 100-mm treated cell culture dishes  at 40% confluence and allowed to grow to 70% confluence. The cell cultures were then washed once with PBS and 10 ml of fully supplemented F12 culture medium and the appropriate concentration of microtubule inhibitors added. The plate was then left to incubate for 18 hours. Next, the spent medium was removed and the number of floating cells within was counted using a hemocytometer. The plate was then washed with 10 ml of PBS, and adherent cell was removed by incubation with 1 ml of trypsin. Nine milliliter of F12 media was added, and the cells were washed off. Cell count of adhere cell was made using a cell counter  and resuspended in 50 mmol/l of KCl solution for 20 minutes at 37 °C. After that, cells were fixed by three washes of fixative solution . Fixed cells were dropped onto microscope slides and mounted in Vectashield mounting media, containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). Images of cells were obtained using a DeltaVision microscopy imaging system at the CRC, LRBGE Fluorescence Imaging Facility (NIH), and the percentage of metaphase in total cell population was counted using ImageJ.About 3,090 µg/ml Collagen I, bovine (sc-29009), 1,960 µg/ml Laminin (sc-29012), and 500 µg/ml Fibronectin (sc-29011) were purchased from Santa Cruz Biotechnology. About 0.01% solution of Polylysine (P4707) was purchased from Sigma–Aldrich. To each 60-mm dish , cell adhesion factors diluted in the appropriate buffers were added; 51 µl of 3,090 µg/ml Collagen in 2,950 µl of 20 mmol/l acetic acid; 39 µl of 1,960 µg/ml Laminin in 2,960 µl of PBS, pH 7.4; 300 µl of 500 µg/ml Fibronectin in 2,700 µl of PBS, pH 7.4; 1,500 µl of 0.01% Polylysine in 1,500 µl of PBS, pH 7.4. The plates were left overnight at RT and later washed three times with 3 ml of PBS. CHO cells were then added. Once 70% cell confluence was achieved the culture was blocked at metaphase by overnight incubation with 50 µmol/l Griseofulvin and 160 µmol/l TN-16. The spent media were then removed, and the plate was washed once with PBS. The culture was then trypsinized, resuspended in 50 mmol/l of KCl solution for 20 minutes at 37 °C, fixed by three washes of fixative solution , spotted onto microscope slides, and mounted in Vectashield mounting media, containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). Slides were analyzed using a DeltaVision microscopy imaging system , and the percentage of metaphase in total cell population was counted using ImageJ.32P-labeled probe as described previously43 with minor changes. Genomic DNA was prepared in agarose plugs and restriction-digested either by SpeI in the buffer recommended by the manufacturer. The digested DNA was CHEF  separated , transferred to membrane (Amersham Hybond-N+), and blot-hybridized with a 201-bp probe specific for the YAC/BAC vector sequence in the alphoidtetO-HAC. This sequence is present in multiple copies in the HAC. DNA sequence for the probe was amplified by PCR using the primers indicated here . Blot was incubated for 2 hours at 65 °C in prehybridization Church’s buffer (0.5 mol/l Na-phosphate buffer containing 7% sodium dodecyl sulfate (SDS) and 100 µg/ml of unlabeled salmon sperm carrier DNA). The labeled probe was heat denatured in boiling water for 5 minutes and snap cooled on ice. The probe was added to the hybridization buffer and allowed to hybridize overnight at 65 °C. Blot was washed twice in 2× saline sodium citrate buffer (SSC) , 0.05% SDS for 10 minutes at RT; then twice in 2× SSC, 0.05% SDS for 5 minutes at 60 °C; twice in 0.5× SSC, 0.05% SDS for 5 minutes at 60 °C; and twice in 0.25× SSC, 0.05% SDS for 5 minutes at 60 °C. Blot was exposed to X-ray film for 24–72 hours at −80 °C.Southern-blot hybridization was performed with a


Lung weight characterises severity of pulmonary oedema and predicts response to mechanical ventilation. The aim of this study was to evaluate the accuracy of quantitative analysis of thorax computed tomography (CT) for measuring lung weight in pigs with or without pulmonary oedema.in vivo) and were then sacrificed and exsanguinated. Fourteen pigs underwent a second thorax CT (CTpost-exsang.) after exsanguination. Lungs were excised and weighed with a balance . Agreement between lung weights measured with the balance (considered as reference) and those estimated by quantitative analysis of CT was assessed with Bland-Altman plots.Thirty-six pigs were mechanically ventilated with different tidal volumes and positive end-expiratory pressures that did or did not induce pulmonary oedema. After 54 h, they underwent thorax CT . Bias between methods was −111 g (−35%) and limits of agreement were −176 (−63%) and −46 g (−8%). Measurement error was similar in animals with  or without  pulmonary oedema (p = 0.88). In 14 pigs with thorax CT after exsanguination, lung weight measured with balancepost-exsang. was 342 ± 165 g and that estimated with CTpost-exsang. was 352 ± 160 g (p = 0.02). Bias between methods was −9 g (−4%) and limits of agreement were −36 (−11%) and 17 g (3%). Measurement errors were similar in pigs with  or without  pulmonary oedema (p = 0.12).One animal unexpectedly died before CTin vivo constantly overestimated the lung weight, as it included pulmonary blood . By contrast, CT obtained after exsanguination provided accurate and reproducible results.Compared to the balance, CT obtained  During ventilator-induced lung injury and acute respiratory distress syndrome, lung weight increases as a result of inflammatory pulmonary oedema ,2. Exactin vivo is quantitative analysis of thorax computed tomography (CT). Lungs are selected on CT images and their total (gas and tissue) volume and physical density (that presumably reflects relative content of gas and tissue) are measured. Lung tissue volume is derived from these two parameters. Lung tissue weight is computed assuming that the density is 1 g/ml  and medetomidine [2.5 to 10.0 μg/kg/h IV]) and paralysis (pancuronium bromide [0.3 to 0.5 mg/kg/h IV]). After 54 h, thorax CT .One animal unexpectedly died before we could obtain thorax CTpost-exsang.) once exsanguinated. The volume of blood removed was always quantified, whereas density (g/ml) was measured in three cases. Lungs were then excised and weighted with the balance  as above.Fourteen (out of 36) animals underwent a second thorax CT . Quality controls were performed every month using standard phantoms.CTs were obtained at 0 cmHExperienced operators manually countered the lung profile excluding proximal airways, large vessels and lymph nodes, mediastinum, muscles and bones and pleural effusions .For each voxel of interest, tissue weight was3.Voxel density was expressed in Hounsfield units (HU), with values of −1,000, 0 and +1,000 HU assigned to air, lung tissue  and bone, respectively. Voxel volume was 1.8 mmLung tissue weight was the sum of the weight of all selected voxels .in vivo of one animal with and one animal without pulmonary oedema were analysed thrice by the same operator (repeatability) and once by three different operators (reproducibility).Thorax CTst test or Mann-Whitney rank sum test. Agreement between reference  and test  methods was studied with Bland-Altman plots and analysis of correlation [p < 0.05 .Normality of data distribution was verified with the Shapiro-Wilk test. Results are reported as means ± standard deviations. Difference between groups was assessed with Student's relation . Percentpost-exsang. and 481 ± 189 g (p < 0.001) when estimated with quantitative analysis of CTin vivo. Bias was −111 g (−35%) and limits of agreement were −176 (−63%) and −46 g (−8%)  , lung weight was 371 ± 184 g when measured with balancepost-exsang. lung weight was 606 ± 140 g in animals with pulmonary oedema (n = 11) and 263 ± 55 g in those without pulmonary oedema (n = 24). Absolute measurement errors were not associated with (mean) lung weight (r = 0.16) (p = 0.36) and did not differ between animals with (−112 ± 45 g) or without (−110 ± 27 g) pulmonary oedema (p = 0.88). Diversely, relative measurement errors were smaller in animals with  than in those without  pulmonary oedema (p < 0.01).On average, balancepost-exsang. and 352 ± 160 g when estimated with quantitative analysis of CTpost-exsang. (p = 0.02). Bias was −9 g (−4%) and limits of agreement were −36 (−11%) and 17 g (3%)  , lung weight was 342 ± 165 g when measured with balancepost-exsang. lung weight was 640 ± 11 g in animals with pulmonary oedema (n = 3) and 261 ± 42 g in those without pulmonary oedema (n = 11). Absolute measurement errors were not associated with (mean) lung weight (r = 0.41) (p = 0.15) and did not differ between animals with (−1 ± 26 g) or without (−12 ± 7 g) pulmonary oedema (p = 0.12).On average, balanceThe volume of blood aspirated during exsanguination was 1,049 ± 283 ml. The density of blood  was 1.01 g/ml.in vivo overestimated lung weight by 105 ± 32 g compared to CTpost-exsang. and by 117 ± 32 g compared to the balance (p = 0.23).In 13 animals that underwent both CTs, CTCoefficients of intra- and inter-subject variability were 2.4% and 4.2%, respectively, for healthy lungs and 2.2% and 0.3%, respectively, for oedematous lungs.post-exsang.: bias −4%; percentage error 15%). However, it largely overestimates the weight of lungs of living animals  as it includes pulmonary blood.Compared to gravimetry, quantitative analysis of thorax CT accurately measures the weight of lungs of exsanguinated animals  or intra-operatively with a balance (ex vivo). One plausible explanation for this discrepancy is blood volume: measurements taken before surgery included blood, whereas those obtained after graft procurement did not [in vivo (before exsanguination) exceeded by around 100 g that obtained with CTpost-exsang. or the balance (after exsanguination). Pulmonary blood is around 5 ml/kg of body weight in pigs [2 of body surface area [Other authors have shown that CT properly quantifies the weight of inanimate objects , artific did not ,18. In o in pigs  and reprace area ). Thereface area . As a copost-exsang. vs. balancepost-exsang.). To further corroborate this finding, two other animals underwent thorax CTin vivo and were then sacrificed but not exsanguinated to allow intravascular blood clotting. Two hours later, lungs were excised with minimal blood loss. As expected, results of quantitative analysis of thorax CTin vivo differed by only 24 g (5%) and 11 g (3%) from those obtained with the balance as both methods measured the weight of blood-filled lungs.In line with the model described so far, differences between computed tomography and gravimetry virtually disappeared when they both measured blood-free lung weights . In bloodless conditions, it provided accurate, reproducible and repeatable results.


Aristeus antennatus, is a commercially important crustacean, in the Mediterranean Sea, which has been listed as a priority species for fishery management. Hypervariable microsatellite markers could be a useful tool to identify genetic stocks among geographically close fishing grounds. Potential microsatellite markers (97) identified from next-generation sequencing of an individual shrimp using a 454 GS Junior Pyrosequencer were tested on a preliminary panel of 15 individuals representing the four worldwide genetic stocks of the species from which 35 polymorphic loci were identified and used to characterize an additional 20 individuals from the Western Mediterranean Sea. In the Western Mediterranean sample, 32 out of 35 were polymorphic loci and the number of alleles per locus ranged from 2 to 14 and expected heterozygosity ranged from 0.050 to 0.968. No linkage disequilibrium was detected, indicating the independence of the loci. These novel microsatellites provide additional tools to address questions relating to genetic diversity, parentage studies and connectivity patterns of A. antennatus populations and help develop effective strategies to ensure long-term sustainability of this resource.The blue and red shrimp,  Aristeus antennatus   is distributed in the Mediterranean Sea, eastern Atlantic waters adjacent to Gibraltar Strait and the Indian Ocean from Mozambique to Zanzibar  (60 ng/µl) with a Paradigm Detection Platform (Beckman Coulter) (CEGEN-UPF Services).Genomic DNA isolation was performed from 10 mg of muscle tissue stored in 95% ethanol of an individual  by means of two series of A. antennatus is a diploid (heterozygoteMode: true), eukaryotic organism (largeGenome: true).Two shotgun whole-genome sequencing was performed on a Roche 454 GS Junior Pyrosequencer by Pompeu Fabra University’s Genomics Service . The reads obtained were assembled into contigs using the GS De Novo Assembler program included in the Newbler software package v.2.7.  using the default settings with two modifications to take into account that e-values <10−10 was performed against all published A. antennatus microsatellites , EM (n = 3), AO (n = 4) and IO (n = 3) (Putative microsatellites were preliminarily screened for amplification success and polymorphism by genotyping a panel of 15  (n = 3) , 2011. F (n = 3) , 1989. T (n = 3) , which i2 (50 mM), 2 µl dNTP (10 mM), 0.2 µM of both reverse and 6-FAM-labeled M13 primers, 0.04 µM forward primer and 1µl DNA template (30–50 ng) plus ddH2O. PCR reactions were performed on a MJ Research PTC-200 thermal cycler  under the following conditions: an initial denaturation at 92°C for 2 min; followed by 35 cycles of denaturation at 94°C for 30 s, annealing at the optimal temperature at 50°C or 60°C  PCR reactions containing 0.5 U of EcoTaq DNA polymerase , 2 µl 10× PCR buffer, 0.6 µl MgCl or 60°C  for 90 sSubsequently, 2 µl of amplified product was added to 10 µl of Hi-Di™ formamide with 0.1 µl of GeneScan™ 500 LIZ size standard and were loaded on to an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Finally, allele scoring, after manually checking, was performed using Geneious v7.1.9 , 2012.O), expected heterozygosity (HE) and inbreeding coefficient (FIS) were estimated with Genepop v4.2.2  used in the preliminary screening. For each locus, observed heterozygosity . Comparatively, the number of reads and total bp sequenced conforms to the values obtained by A. antennatus genome and the number of singletons was 128,835. These contigs had an average length of 873 bp  and N50 contig size was 858 bp.During the last decade, 454 pyrosequencing technology has been used widely to develop microsatellites in non-model organisms , 2013, aA. antennatus microsatellites was found in the BLASTn search, except for our contig00595 that matched the microsatellite Ant93 described in EU417952).No homology with already available In 247 contigs, a total of 317 tandem repeats were identified with Tandem Repeats Finder of which 241 presented motifs with a period size of di-(31%), tri-(27%), tetra-(24%) or penta-nucleotides (18%), repeated at least 5 times. The di-, tri- and tetra-nucleotides are the most common repeat motifs for microsatellite loci described in decapod shrimps , 2002, tKU195267 –KU195299)  when stringent filters were used on massive 454 sequencing datasets , 2012. FU195299) . The proU195299) , 2013, iU195299) , 2011 anU195299) , 2015).Of the 35 initial loci, 32 were polymorphic in the Western Mediterranean sample analysed with a mean number of alleles per locus of 4.8 (2–14) and a mean observed and expected heterozygosity of 0.350 (0.000–1.000) and 0.521 (0.050–0.968), respectively . The numP = 0.0001), suggesting their independent segregation. In 28 out of 32 polymorphic loci, proper amplification was obtained for more than 70% of the analysed specimens (N = 20). In 21 out of 28 the polymorphic loci, genotype proportions were consistent with HWE expectations after Bonferroni correction (P = 0.0018), with a mean number of alleles per locus of 4.4 and a mean expected heterozygosity of 0.455, which are in line with those obtained by IS values, see IS values  contained the 35 polymorphic microsatellites of Aristeus antennatus.Click here for additional data file.


Objective. TLR7 ligation in plasmacytoid dendritic cells is promising for the treatment of cancer, allergy, and infectious diseases; however, high doses of ligands are required. We hypothesized that the combination of structurally different TLR7 ligands exponentiates the resulting immune response. Methods. CAL-1 (human pDC line) cells were incubated with the TLR7-specific adenine analog CL264 and single-stranded 9.2s RNA. Protein secretion was measured by ELISA. Microarray technique was used to detect modified gene expression patterns upon synergistic stimulation, revealing underlying functional groups and networks. Cell surface binding properties were studied using FACS analysis. Results. CL264 in combination with 9.2s RNA significantly enhanced cytokine and interferon secretion to supra-additive levels. This effect was due to a stronger stimulation of already regulated genes (by monostimulation) as well as to recruitment of thus far unregulated genes. Top scoring canonical pathways referred to immune-related processes. Network analysis revealed IL-1β, IL-6, TNF, and IFN-β as major regulatory nodes, while several minor regulatory nodes were also identified. Binding of CL264 to the cell surface was enhanced by 9.2s RNA. Conclusion. Structurally different TLR7 ligands act synergistically on gene expression patterns and on the resulting inflammatory response. These data could impact future strategies optimizing TLR7-targeted drug design. Plasmacectively , 7. Howeectively . Thus, p47H73N13O7S, molecular weight 413.43) is a 9-benzyl-8-hydroxyadenine derivative containing a glycine on the benzyl group (in para). According to the manufacturer specification, the substance we used is highly pure (endotoxin level < 0.001 EU/µg). The oligoribonucleotides (ORNs) 9.2s RNA and the nonstimulatory control Poly-A RNA were individually designed and obtained from Biomers  and also dissolved in sterile, RNAse-free water [PBS (pH 7.4) and RPMI 1640 were purchased from Life Technologies . The latter was supplemented with 10% heat inactivated fetal bovine serum  and 2 mmol/L Glutamine (Life Technologies). Cell culture flasks and 24- and 96-well flat-bottom plates were obtained from Greiner Bio-One . CL264 (tlrl-c264e) and FITC-labeled CL264 (tlrl-fc264) were purchased from InvivoGen  and dissolved in sterile, RNAse-free water  containing 10% DMSO (Sigma-Aldrich) and further diluted in culture medium. CL264 (formula CReagents for Fluorescent Activated Cell Sorting (FACS) were purchased from Miltenyi Biotec  and BD Biosciences , respectively. Assay buffer for FACS was prepared by adding 0.5% FBS and 2 mM EDTA  to PBS. Paraformaldehyde (PFA) was obtained from Sigma-Aldrich.The human pDC line CAL-1 (kindly provided by Drs. Maeda and Kamihira) was grown in complete medium (RPMI 1640) supplemented with 2 mmol/L Glutamine and 10% heat inactivated FBS and cultured as previously described .6 cells in 3 mL per well), and, for ELISA studies, cells were plated in a 96-well plate (2 × 105 cells in 0.2 mL per well). After overnight resting in serum reduced (1% FBS) medium, CAL-1 cells were stimulated with the TLR7 ligand CL264 (5 µg/mL), with 9.2s RNA (2 µg/mL), or with the combination of both. The reagents were diluted in culture medium and added to the cell suspension in the wells of the 96- or 24-well plates, respectively. Vehicle and nonstimulatory Poly-A RNA (2 µg/mL) were used as control. Before stimulation, ORNs were complexed with the polycationic polypeptide poly-L-arginine hydrochloride  to facilitate RNA delivery. ORNs were incubated with PLarg for 2 minutes at room temperature before adjusting to the desired concentration by adding culture medium and incubating for another 15 minutes [6 cells/mL by adding fresh, prewarmed cell culture medium.For gene expression studies, cells were plated in a 24-well plate  and PBL Assay Science , resp.) according to manufacturer instructions.TNF-µL chloroform was added and the suspension was shaken vigorously, followed by incubation at room temperature for 3 minutes. After centrifugation for 15 minutes at 12.000 rpm and +4°C, the upper aqueous phase was carefully aspired and transferred into an RNAse-free tube. The RNA was precipitated using 1 mL isopropanol and incubating at room temperature for 10 minutes. After another centrifugation step, the pellet was washed using 75% ethanol. The air-dried RNA pellet was resuspended in RNAse-free water, and the concentration was determined spectrophotometrically .After thawing cells suspended in TRIzol reagent, they were left at room temperature for another 5 minutes and well pipetted up and down to lyse the cell membrane. 400 µL each) were combined, denatured by heating for 2 minutes at 98°C, and mixed with 36 µL of hybridization solution at +42°C . Murine microarrays  were overlaid with this solution and hybridized for 18 hours at +42°C using an actively mixing MAUI hybridization system . After hybridization, the arrays were washed in 1x SSC/0.05% SDS and 0.1x SSC, centrifuged to remove remaining liquid with unbound cDNA, and air-dried. Intensity values were generated using an array scanner (NimbleGen). Data were uploaded to the mAdb database  and formatted via the export function for use with BRB ArrayTools .Total RNA was incubated with anchored oligo-dT primers at +70°C and subsequently chilled on ice. CDNA synthesis was performed using a mastermix containing first-strand buffer, 20x aminoallyl-dNTP labeling mix, dithiothreitol (DTT), RNAse inhibitor, and reverse transcriptase enzyme . Following incubation, residual RNA was degraded by NaOH and HCl incubation. Resulting cDNA was purified using the Qiagen MinElute kit  and was coupled to Cy3 and Cy5 dyes, respectively, by incubation for 90 min. Cy3-labeled reference and Cy5-labeled sample cDNAs (10 µM), PLarg-complexed 9.2s RNA (2 µg/mL), or FITC-labeled CL264  or a combination, respectively  and analyzed using FlowJo (version 10.0.5) software.CAL-1 cells were seeded in 24-well plates one day prior to the experiment and incubated under standard conditions as described above. Fresh cold medium was added together with FITC . The alpha level was set at 5%.Data are presented as mean of biological triplicates ± SEM. Each experiment was repeated at least three times. Statistical analysis was performed by two-sided unpaired Student's p value cutoff of 0.001 was used to identify genes whose expression was significantly upregulated after TLR7 ligand stimulation when compared to controls. Data were evaluated using Ingenuity Pathway Analysis . IPA maps each gene within a global molecular network, pathways, and functional groups developed from information contained in the Ingenuity Pathways Knowledge Base ).For the analysis of microarray experiments, data from three independent experiments and three untreated controls was used for statistical analyses. Expression analyses were performed using BRB ArrayTools. Data were background corrected, flagged values were removed, spots in which both signals were <100 were filtered out, ratios were log base 2-transformed, and lowest intensity-dependent normalization was used to adjust for differences in labeling intensities of Cy3 and Cy5 dyes [µg/mL, stimulation with CL264 resulted in a dose-dependent secretion of TNF-α after 6 hours (µg/mL). Using this concentration, stimulation with CL264 for up to 12 hours led to a robust, time-dependent release of TNF-α reaching absolute levels of 1347 pg/mL TNF-α into the supernatant (α with lower absolute cytokine levels (526 pg/mL) after stimulation for 12 hours (µg/mL), no notable secretion of proinflammatory cytokines was measured. However, the addition of 9.2s RNA to CL264 considerably increased the secretion of TNF-α and IL-6 compared to the monostimulation only with CL264 .CAL-1 cells were stimulated with the specific TLR7 ligand CL264  and the TLR7/8 ligand 9.2s RNA complexed to PLarg. CAL-1 cells do not express TLR8, so response to 9.2s RNA is limited to TLR7 stimulation . Dose ti 6 hours . In conternatant . The kin12 hours . In contβ release under the same experimental conditions using CL264 and 9.2s RNA. Again, costimulation with CL264 and 9.2s RNA for 12 hours resulted in a marked and significant increase of IFN-β protein compared to monostimulatory conditions (p < 0.001) (We previously demonstrated that activation of CAL-1 cells with TLR9 ligands induces detectable amounts of type 1 IFN . Accordi< 0.001) .Of note, the synergistic effect of CL264 and 9.2s RNA on CAL-1 cells could be abolished by stimulating the cells sequentially instead of simultaneously. More specifically, no enhanced cytokine secretion could be detected when CL264 was the first stimulus, followed by washing and then ligation with 9.2s RNA. On the other hand, switching the order of stimulation preserved the supra-additive activation even when cells were washed between the ligation steps .For control experiments, nonstimulatory Poly-A RNA was used instead of 9.2s RNA and had no enhancing effect. Similarly, the use of PLarg alone or 9.2s RNA not complexed with PLarg prior to stimulation had no additive effect .To gain more insights into the present findings, microarray experiments were performed on CAL-1 cells after stimulation either with CL264, 9.2s RNA, or the combination of both. An early time point for this analysis (4 hrs) was chosen to minimize secondary effects, such as autocrine/paracrine cytokine stimulation. All treatment groups were normalized to untreated controls. Beside the characterization of genes significantly upregulated by either treatment, main goal was the identification of underlying regulatory genes that play a central role for synergistic effects.p < 0.001, treatment with 9.2s RNA significantly increased expression of 17 genes in CAL-1 cells, while treatment with CL264 resulted in an upregulation of 111 genes  were also present in the costimulatory group. Furthermore, we addressed the question whether the unique genes significantly upregulated solely by costimulation  were also increased in the monostimulatory groups when the statistical cutoff was lowered by one magnitude (p < 0.01). In this case, 96 additional genes could be ascribed to either of the monostimulatory groups. Altogether, 208 genes  in the costimulatory group were significantly upregulated in either of the monostimulatory groups. Overall, these results indicate that the synergistic effect found in the costimulatory group is partially due to a stronger stimulation of already regulated genes but furthermore an effect of the upregulation of so far unregulated genes.Using a statistical cutoff of 11 genes . HoweverWe next addressed the question whether costimulation with CL264 and 9.2s RNA also resulted in a significant increase of gene activation in terms of magnitude. Therefore, we analyzed the expression levels of the 50 most significantly upregulated genes in each treatment group. Results show that the average fold change increased from 1.11 (9.2s RNA) and 7.17 (CL264) to 19.6 (9.2s RNA/CL264)  comparedactivation of IRF by cytosolic pattern recognition receptors” (p < 4.31E − 13), “dendritic cell maturation” (p < 1.08E − 10), and “interferon signaling” (p < 5.94E − 10). Top scoring functional groups were, for example, “cellular development, growth, and proliferation” (p < 7.26E − 34), “cell death and survival” (p < 5.72E − 29), “infectious disease” (p < 1.6E − 24), and “inflammatory response” (p < 1.2E − 20)   impacted the expression of around 95% of all genes within the network of the costimulatory group, while the minor regulators only had modulatory properties on the induced gene expression network.Furthermore, IPA was used to identify the pattern of regulatory interactions underlying synergistic gene activation after costimulation with 9.2s RNA and CL264. The network analysis enabled us to define the regulatory nodes of this synergistic regulation. Major regulators were previously defined as regulatory nodes that trigger more than 30% of the costimulatory network, while minor regulators were defined as regulating 10% to <30% of the network , 14. Appand JUN) . Togetheimmune response” regulation and 60% were related to “cellular growth and proliferation.” Furthermore, three out of four major regulators  as well as type 1 IFN . The immunostimulatory properties of the single-stranded RNA 9.2s were first described in 2005, when Hornung et al. originally intended to design different siRNAs (named 9.1 to 9.4) downregulating the human TLR9 to avoid effects of non-target-related IFN induction in human PDCs [5′-GUCCUUCAA-3′) of the sense strand of siRNA9.2  was responsible for the immunostimulatory activity. Injection of 9.2 RNA into TLR7-deficient mice elicited, in contrast to the injection into wild-type mice, no detectable IFN serum levels.We stimulated our cells simultaneously with two different ligands for TLR7. Similarly to the substituted 8-hydroxyadenine derivative SM360320, CL264 induces the activation of NF-ng cells . CL264 lβ . Taken together, this indicates a stronger stimulation of genes yet upregulated upon stimulation by either ligand. On the other hand, this finding also delineates the upregulation of new or rather so far unregulated genes by the combination of CL264/9.2s RNA (46% of the genes). These synergistic effects primarily broaden the existing immune response, rather than creating a new one. This observation is supported by the fact that the functional groups  induced by the combinatory versus single stimulation did not alter qualitatively .Finally, exclusive IPA analysis of the 62 synergistically upregulated genes . Since CAL-1 cells do not express the TLR8, costimulatory effects of TLR7 and TLR8 by the use of ssRNA and CL264 are highly improbable . HoweverAlthough various synthetic TLR7/8 agonists have been used as adjuvants during preclinical trials, not many of them are approved for use in humans. The TLR7/8 agonist R837 is used for the topical treatment of genital warts, basal cell carcinoma, and bladder cancer . Howeverα secretion in response to TLR stimulation [β  resulted in a strong feedforward regulation of their TLR-triggered type 1 IFN production in vitro and concluded that the in vitro activity of a TLR ligand on primary pDCs does not necessarily correspond to its in vivo activity [β (instead of IFN-α) since we studied early time point effects up to 12 hours to minimize the mentioned auto- and paracrine cytokine effects, which is essential when studying synergism of different TLR ligands. We previously demonstrated that, in contrast to IFN-β, IFN-α expression peaks rather late (>24 hours) in response to TLR stimulation, which is likewise influenced by a type 1 IFN feedback loop [Our study has some limitations. Despite the fact that CAL-1 cells share many of the phenotypic and functional properties of human pDCs, Maeda et al. have described some significant differences between this cell line and freshly isolated primary human pDCs including little IFN-mulation . It is wmulation , 34. Kimactivity . Howeveractivity , 36–38. A number of studies analyzed synergistic effects activating different TLRs –29. Howe


A low-dose cosyntropin test was done before and after L-T4 therapy. Thirty and sixty minutes after the low-dose cosyntropin, the mean cortisol values were lower in the cases group (612.6 ± 133.1 and 603.4 ± 153.7 nmol/L) when compared to the control group (677.0 ± 82.4 and 669.9 ± 83.7 nmol/L) (P = 0.001 and 0.003), respectively. After L-T4 therapy, this difference was lost. Four out of 60 cases (6.7%) taking a cortisol cutoff value of 500 mmol/L and 11 out of 60 (18.3%) having 550 mmol/L as the cutoff had adrenal insufficiency before L-T4 therapy. After L-T4 therapy, 50% and 81% of the cases had normal cortisol response. In conclusion, patients with different degrees of intensity of primary hypothyroidism had improved cortisol response after reaching euthyroidism. The incidence of adrenal insufficiency was 6.7–18.3% and more than 50% of the cases had a normal cortisol response after L-T4 therapy. Adrenal function might be impaired in patients with primary hypothyroidism. The objective was to evaluate adrenocortical function using the low-dose cosyntropin test in patients with primary hypothyroidism. Consequently a prospective, longitudinal, controlled study of sixty adult patients with primary hypothyroidism was carried out. Patients Thyroid gland dysfunction is a highly prevalent disorder in day-to-day clinical practice. In fact, one of the most common reasons for primary care consult is primary hypothyroidism. Recent studies have found up to 25% prevalence in women aged 60 and over –5. The mSome nondistinctive clinical manifestations of hypothyroidism and adrenocortical insufficiency may overlap, particularlly in the musculoskeletal and neurological systems . After sAccordingly, a prospective, longitudinal, and controlled study was carried out to evaluate adrenocortical glucocorticoid function using the low-dose cosyntropin test, in patients with subclinical, mild-moderate, and severe primary hypothyroidism. The primary endpoint was to contrast the cortisol response to low-dose cosyntropin before L-T4 replacement and after normalization of thyroid function tests, between groups and in each category and, furthermore, to compare the cortisol values before and after L-T4 replacement in cases versus their controls. A secondary endpoint was to identify the prevalence of transient or permanent adrenocortical insufficiency. naïve to L-T4 treatment, were included. Primary hypothyroidism was documented by the results of the total and free thyroxin levels , thyrotropin (TSH) values, and positive antibodies to thyroid peroxidase. Cases were classified into three groups: subclinical primary hypothyroidism ((group #1) (n = 30) ), mild/moderated hypothyroidism ((group #2) (n = 15) ), and severe hypothyroidism ((group #3) (n = 15) ). A complete clinical history and physical examination were performed in all participants. We excluded individuals with other endocrinopathies, glucocorticoid use in the last 24 months, and current use of oral/nonoral contraceptives or drugs that may interfere with cortisol metabolism, transport, or the hypothalamo-pituitary-adrenal axis. An age and gender matched control group (n = 60) of healthy subjects was recruited from the primary care clinic.After obtaining approval from the Research and Ethics Committee and signed informed consent from each subject, we began the study. Patients were recruited through the internal medicine, primary care, and endocrinology clinics of the “Dr. José E. González” University Hospital. A total of 60 men or women, aged 18 to 70 years, with a diagnosis of primary hypothyroidism andμg synthetic ACTH , first to 50 μg/mL, then to 5 μg/mL, and finally to the 1 μg/mL concentration. The vials were maintained refrigerated at 4°C for a 3-month period. All participants, cases and controls, underwent a 1 μg cosyntropin dose test. After overnight fasting, between 8 and 9 h, an intravenous plastic catheter was inserted into a forearm vein and a baseline blood sample was drawn for measurement of basal cortisol one minute before each test. Subsequently, 1 μg ACTH was injected as a bolus through the catheter and flushed afterwards with 10 mL of normal saline. Blood samples for serum cortisol were taken at 30 and 60 min. All samples were centrifuged, separated immediately, and frozen at −20°C. A serum cortisol value equal to or greater than 500 nmol/L at 30 or 60 min after stimulation was defined as a normal glucocorticoid response [To perform a low-dose cosyntropin test, a solution was prepared from serial dilutions in normal saline of one vial of 250 response –20. Fiveresponse .A low-dose cosyntropin test was carried out at baseline in all cases (before starting L-T4 therapy) and repeated 16 weeks later during follow-up, once thyroid function tests  were documented to be within normal values. All cases received L-T4 replacement therapy as recommended . In the Thyroid function testing included measurement of TT4, FT4, and TSH. Total and free thyroxin were measured using a commercial kit for electrochemiluminescence . Intra-assay variation coefficients were 3.7, 3.4, and 4.2% and 2.7, 2.6, and 3.6% for low, medium, and high TT4 and FT4 values, respectively. TSH was measured using a commercial electrochemiluminescence kit . Intra-assay variation coefficients were 7.2, 3.2, and 3.3% for low, medium, and high TSH values, respectively. Serum cortisol was measured using a commercial RIA kit . Intra-assay variation coefficients were 1.6, 1.5, and 1.6% for low, medium, and high cortisol values, respectively. All samples were assessed twice.P value equal to or less than 0.05 was considered statistically significant. Descriptive statistical analysis was used for quantitative variables, measures of central tendency, and dispersion. In the case of qualitative variables, frequencies were obtained. To compare differences between basal and follow-up cortisol responses within each patient group and between patients and controls, paired and unpaired Student's t tests were used, respectively. The statistical analysis was performed with IBM SPSS Statistics 20.0 .All results are reported as mean ± standard deviation unless otherwise stated. A P = 0.83). All cases and controls completed the study.The demographic characteristics of the study participants are shown in P = 0.58). See P = 0.001 and 0.003), respectively.Mean baseline (0 min) cortisol value between all cases (357.3 ± 144.8 nmol/L) and controls (369.2 ± 91.4 nmol/L) was not statistically significant (P = 0.03). Group 3 had a lower cortisol value at 30 minutes (540.9 ± 178.7 nmol/L) when compared with controls (687.9 ± 61.6 nmol/L). All other cortisol values, even though lower in cases than in controls, were not statistically different. See Comparison of the cortisol values  between group 1 and their controls was not statistically different. When group 2 was compared with controls a lower mean cortisol value at 60-minute time  was observed in the case group  due to a lower mean cortisol value in group 3 participants. This significant difference disappeared at 60-minute time comparison between groups 1 versus 2, 1 versus 3, and 2 versus 3 . See Mean baseline cortisol values were not different between all three groups of cases . Groups 1 versus 2, 2 versus 3, and 1 versus 3 resulted in a Once levothyroxine replacement reached normalization of the thyrotropin values in all cases, comparison of the mean serum cortisol values at baseline, 30, and 60 minutes after stimulation against the control group did not show any difference. See Mean serum cortisol values at baseline, 30, and 60 minutes after cosyntropin stimulation did not reach statistical differences in groups 1, 2, and 3 when compared with their controls. See P = 0.009. The rest of the comparisons between groups at baseline and at 30 and 60 minutes were no different. See Mean cortisol values were different between group 2 and group 3 at baselines 302.3 ± 87.4 and 442.5 ± 198.4 nmol/L; P = 0.04. See When cases were compared before and after L-T4 therapy, mean serum cortisol values were lower before thyroid hormone normalization. Nevertheless, statistical significance was found only at 30-minute time (612.6 ± 133.1 versus 653.2 ± 138.3 nmol/L). P = 0.03). In group 2 there was no statistically significant difference between mean cortisol values before and after L-T4 therapy. In group 3 the mean cortisol values at baseline and 30-minute time were statistically lower before L-T4 . See The mean cortisol values at baseline and after stimulation with cosyntropin when compared before and after levothyroxine in group 1 showed lower values before L-T4 replacement but statistical significance was reached only at 60-minute time  had insufficient glucocorticoid reserve before L-T4 treatment, one of them in group 1 and three in group 3. After L-T4 therapy 2 out of 4 cases reached a normal cortisol response and the other two cases (both in group 3) stayed with glucocorticoid insufficiency. They had an elevated plasma adrenocorticotropic level  and glucocorticoid replacement therapy was initiated. Adrenal cortex antibodies were not carried out. When a cortisol value of 550 nmol/L or more was taken as the normal cortisol response, 11 out of 60 cases (18.3%) had an abnormal cortisol response to cosyntropin at 30 and 60 min, six of them in group 3, three in group 2, and two in group 1. After L-T4 replacement 9 out of 11 cases (81.8%) showed a normal cortisol response. The two persistent abnormal cases were in group 3. In the control group, all of the participants had an adequate cortisol response after cosyntropin stimulation at 30 or 60 minutes with both 500 nmol/L and 550 nmol/L cutoff values.In this prospective study of patients with different degrees of intensity of primary hypothyroidism we found with the low-dose cosyntropin test that their serum cortisol response before L-T4 replacement therapy was significantly lower than controls. After thyroid hormone level normalization, an increase in their cortisol response was observed and this difference was lost. This change was primarily due to an increase in the cortisol response in the severe hypothyroidism group. The analysis by group of cases  after L-T4 therapy showed a modest but significant increase in cortisol response at 0 and 30 minutes in the severe hypothyroidism group and at 60 minutes in the subclinical hypothyroidism group. Remarkably, 4 out of 60 cases (6.7%) with 500 nmol/L as the cortisol cutoff value and 11 out of 60 cases (18.3%) with the 550 nmol/L as a cutoff had an abnormal cortisol response  that reverted after L-T4 therapy in 2 (50%) and 9 cases (81.8%), respectively, without any other treatment.μU/mL) [It has been generally accepted that adrenal function might be impaired in patients with primary hypothyroidism. In 1957, Peterson in seven patients with severe hypothyroidism described that cortisol secretion and synthesis were reduced, primarily due to an impairment of cortisol metabolism by the liver. After L-T4 therapy was initiated cortisol production returned to normal. However, not every patient was evaluated after L-T4 therapy and adrenal function was not assessed using a stimulation test . Later, μU/mL) . The corμU/mL) . In the The exact site and how the HPA axis is impaired by hypothyroidism are still not well elucidated. It has been described that thyroidectomy decreases plasma ACTH levels (even though the pituitary content of ACTH might be increased) due to a reduction in corticotropin releasing hormone (CRH) gene expression in the hypothalamus , 23. AddInterestingly, cortisol response to cosyntropin was not impaired in all hypothyroid patients. This suggests that even though low serum thyroid hormones play an important role in adrenal dysfunction, there are other associated factors that still have to be recognized. Two of our cases remained with adrenal insufficiency despite adequate L-T4 therapy. Both were from the severe hypothyroidism group and even though the possibility of a permanent adrenal dysfunction due to low thyroid hormone levels is plausible, it is important to remember the link between hypothyroidism and adrenal insufficiency. It is likely that these cases could have an underlying autoimmune etiology.It is worthy to mention that even though cortisol levels improved after L-T4 therapy mean basal and stimulated cortisol levels in the case group were within normal range in all subgroups. When this data is seen as a whole, it suggests that in primary hypothyroidism there is an impaired HPA axis that improves with L-T4 therapy. Nonetheless, this condition will probably have a discrete, if at all, clinical significance as cortisol levels at baseline and after cosyntropin stimulation before and after L-T4 therapy remained within normal range. However, when each case is viewed separately, 4 out of 60 cases (6.7%) with a cortisol cutoff value of 500 mmol/L and 11 out of 60 (18.3%) with a cutoff of 550 mmol/L had adrenal insufficiency before L-T4 therapy. Remarkably, 50% and 81% of the cases had a normal cortisol response after L-T4 therapy without any other treatment. Most of the cases (>50%) were in the severe hypothyroidism group, suggesting, as previously described in rats, that a prolonged state of hypothyroidism might deeply impair cortisol secretion . Neverth3 levels were the same before and after treatment suggesting that TSH could be increased as a direct effect of adrenal deficiency without thyroid dysfunction. Thyroid antibodies in all six patients were negative. The patients in whom TSH remained high had positive thyroid antibodies and higher TSH levels. The proposed mechanism was that low cortisol levels cause a rise in pro-TRH mRNA in the hypothalamic paraventricular nucleus with a consequent increase of TSH levels [A direct TSH elevation secondary to adrenal insufficiency with reversal of thyroid function after glucocorticoid replacement has been described , 24–26. H levels . BarnettH levels . In our μg of cosyntropin is a pharmacologic rather that a physiologic stimulus that may stimulate partial atrophied or mildly dysfunctional adrenal glands, multiple studies have demonstrated that the low-dose cosyntropin test has better or equal results than the standard test. Particularly, in the setting of chronic partial pituitary ACTH deficiency and recent onset ACTH deficiency, low-dose stimulation has demonstrated a better performance [μg) had a near-normal/partial abnormal cortisol response that could have been otherwise overestimated if we had used the standard 250 μg cosyntropin test.It has been debated for a long time which test (low or standard dose) cortisol stimulation has better performance. Based on the concept that 250 formance , 27–33. In conclusion, this prospective study of patients with different severities of primary hypothyroidism showed, using the low-dose cosyntropin test, an improved cortisol response after normalization of thyroid hormone levels. The mechanism of impaired cortisol secretion in cases with hypothyroidism still has to be elucidated but our data support an adrenal rather than a pituitary affection. The incidence of adrenal insufficiency was 6.7–18.3% and more than 50% of the cases had a normal cortisol response after L-T4 therapy. Remarkably, 25–50% of the cases were in the subclinical/mild/moderate hypothyroidism groups. This finding could have important clinical implications especially in the setting of acute stress situations occurring during the period while a euthyroid state is still not achieved.


Populus yunnanensis Dode were examined in a greenhouse for 48 h to analyze their physiological and proteomic responses to sustained heat, drought, and combined heat and drought. Compared with the application of a single stress, simultaneous treatment with both stresses damaged the plantlets more heavily. The plantlets experienced two apparent response stages under sustained heat and drought. During the first stage, malondialdehyde and reactive oxygen species (ROS) contents were induced by heat, but many protective substances, including antioxidant enzymes, proline, abscisic acid (ABA), dehydrin, and small heat shock proteins (sHSPs), were also stimulated. The plants thus actively defended themselves against stress and exhibited few pathological morphological features, most likely because a new cellular homeostasis was established through the collaborative operation of physiological and proteomic responses. During the second stage, ROS homeostasis was overwhelmed by substantial ROS production and a sharp decline in antioxidant enzyme activities, while the synthesis of some protective elements, such as proline and ABA, was suppressed. As a result, photosynthetic levels in P. yunnanensis decreased sharply and buds began to die, despite continued accumulation of sHSPs and dehydrin. This study supplies important information about the effects of extreme abiotic environments on woody plants.Plantlets of  Global warming, the most typical manifestation of worldwide climate change, is a focus of increasing attention. Although warming experiments have often been used to simulate future climate conditions, this approach is limited by the unproven assumption that plant responses to experimental warming match their long-term responses to global warming Research on plant responses to heat, drought, or their combination has mainly focused on model plants and crops that are herbaceous, such as wheat Populus yunnanensis Dode), native to high altitude areas of southwestern China, is one of the woody plants most commonly used in stress resistance studies P. yunnanensis populations have recently experienced climate warming and continuous drought stress in southwestern China, an understanding of the combined effects of heat and drought on this species is a research priority. Previous studies of P. yunnanensis have involved greenhouse experiments to determine the effects of abiotic stresses on its growth and physiology. The applied stresses have included heavy metals, salinity, acid rain, elevated CO2, warming, drought, UV-B, and their various combinations P. yunnanensis are still largely unknown. In the present study, we performed experiments to explore the response of P. yunnanensis to sustained high temperature and drought using comparative proteomic and physiological analyses. To precisely determine the effect of combined heat and drought stress, we also performed comparison experiments involving the application of separate high temperature and drought treatments. We aimed to understand how global climate change may affect woody plants, with P. yunnanensis used as a model.Yunnan poplar  and electron transport rates (ETRs) of photosystem II (PSII), which can indicate plant photosynthetic capacity Fv to Fm of plantlets exposed to high temperature changed significantly after 24 h of stress   , and thels (0 h) .P. yunnanensis rose gradually over time during individual heat or drought stress treatments  of samples subjected to high temperature, drought, and combined heat and drought stress. The 2-DE was repeated three times on each sample with similar results; therefore, one set of representative gels per treatment was visualized by Coomassie Brilliant Blue (CBB) staining (P<0.05) among samples subjected to heat, drought, and combined heat and drought treatment, respectively. Fifty-seven of these differentially expressed proteins , defense-related , cell structure and division , nucleic acid metabolism , redox metabolism , photosynthesis , signal transduction , energy metabolism , and other proteins  , implyinP. yunnanensis, we performed protein immunoblot analysis with specific antibodies against plant mitogen-activated protein kinase 6 (MAPK6), heat shock protein 18.2 (HSP18.2), abscisic acid (ABA) synthase 9-cis-epoxycarotenoid dioxygenase (NCED), and dehydrin  under three different stress regimes  and the expression of related proteins. Small amounts of H2O2 and O2− were detected under individual heat and drought stress conditions  Several other defense-related proteins, including desiccation-related protein LbLEA3_3–06 (spot 3), resistance protein (spot 6), late embryogenesis abundant protein D-113 (spot 18), and 3-isopropylmalate dehydratase small subunit (spot 82) were observed to be differentially expressed under different stress conditions . Under sP. yunnanensis plantlets exposed to different stresses , a common broadleaved deciduous tree of southwestern China, respond to extreme high temperature accompanied by drought, with individual treatments of heat and drought used for comparison. Our results provide insight into how woody plants may respond to excessive heat, as is expected with global warming. When exposed to individual heat or drought stress, plantlets exhibited different levels of resistance, similar to results reported from many previous studies P. yunnanensis, with females usually experiencing greater negative effects than males In this study, we performed comparative physiological and proteomic profiling to investigate how plantlets of poplar , Yunnan Province, China. No specific collecting permits were required for this location, as it was located adjacent to our institute, the Kunming Institute of Botany of the Chinese Academy of Sciences. Plant administration was under the auspices of the Kunming City Forestry Bureau, with any studies beneficial or non-damaging to plants permitted and supported by relevant departments. We confirm that the plant we used is a common native species that is neither endangered nor protected.−2 s−1 light intensity). One of three stress treatments was applied to each group: (1) a constant temperature of 40°C with regular watering, (2) the normal pre-treatment temperature regime with no watering, or (3) a constant temperature of 40°C with no watering. Treatments were begun simultaneously during the day time, prior to the scheduled daily watering, with treatment continuing for 0, 6, 12, 24, or 48 h. After plant morphological changes were recorded, the fourth to sixth leaves from the top were harvested to determine their physiological and biochemical properties for each treatment. Five replicates were performed per experiment, and samples from the 0-h treatment were used as controls for the data analysis.Yunnan poplar cuttings were obtained from male plants in March 2012. After survival in the field for 30 d, 150 healthy plantlets with an average of five nodes and a height of 15–20 cm were transplanted into plastic pots (15 cm ×20 cm) containing equal biomass in a greenhouse. The plants were grown in the greenhouse for 30 d under sunlight conditions (23–25°C day and 18–20°C night) and watered daily on a regular schedule with 100 ml of water per pot. The plantlets were then divided into three groups and subjected to different stresses in an incubator with a 12-h photoperiod  were measured, with dry weights (DWs) recorded after drying at 80°C for 48 h P. yunnanensis plantlets after treatment were dark-adapted for 30 min to measure the maximal quantum yield of PSII (Fv/Fm), which was determined for each sample by analyzing a whole leaf. The maximal fluorescence (Fm) was recorded using a 0.8-s pulsed light at 4,000 µmol s−1 m−2, and minimal fluorescence (Fo) was recorded during the weak measuring pulses. ETRs at a given actinic irradiance were calculated according to the instrument manual as follows: (Fm' – Fs)/Fm' × PAR ×0.5×α, where (Fm' − Fs)/Fm' is the quantum yield of PSII (φPSII) in light, PAR is the photosynthetically active irradiance, 0.5 is the assumed proportion of absorbed quanta used by PSII reaction centers, and α is the absorbance for poplar leaves.Chlorophyll fluorescence was analyzed as previously described g for 10 min. Two milliliters each of the extract, acidic ninhydrin, and glacial acetic acid were heated for 1 h in a boiling water bath, with the reaction then terminated in an ice bath. The reaction mixture was extracted with 4 ml toluene, with vigorous mixing using a test-tube stirrer for 15–20 s. The chromophore-containing toluene was aspirated from the aqueous phase and warmed to room temperature, and its absorbance was read at 520 nm using toluene for a blank. The proline concentration was determined from a standard curve and calculated on a DW basis as follows:Proline content was measured as previously reported −1 proline × ml toluene) ×5 (g sample)−1 =  µg proline g−1 DW material.  = 6.45 (A532−A600) −0.56A450.MDA content was determined as described previously In situ detection of H2O2 and O2− were performed using a previously reported method with some modifications 2O2, three leaf discs drilled at specific time points during different treatments were vacuum-infiltrated in 10 ml of 1 mg ml−1 diaminobenzidine solution for 2 h, and were then cleared in boiling ethanol (95%) for 10 min. The samples were subsequently stored and examined in 95% ethanol. The amount of O2− in leaves was monitored by 10−2 M nitro-blue tetrazolium (NBT) reduction at specific time points. Three leaf pieces were vacuum-infiltrated with 10 ml NBT for 2 h, cleared in boiling ethanol (95%) for 10 min, and stored and examined in 95% ethanol.2·6H2O, 1% [w/v] PVP-40, and 20% [v/v] glycerin). The homogenates were centrifuged at 12,000×g for 15 min at 4°C, and the total soluble protein content of the supernatants was measured by the Bradford method Approximately 0.5 g of leaves from each sample was homogenized in 10 ml extraction buffer  DTT at −20°C for 30 min and then centrifuged at 8,000×g for 20 min at 4°C; this step was performed a total of three times. Finally, the pellets were vacuum-dried and then dissolved in lysate  for 2 h at room temperature with intermittent shocking, followed by centrifugation at 12,000×g for 20 min at 20°C. The supernatants were collected for the 2-DE experiments, which were performed in triplicate.Protein extraction and 2-DE were performed as reported previously Extracted proteins  were first separated by isoelectric focusing (IEF) using gel strips to build an immobilized non-linear pH gradient from 4 to 7  and then by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% polyacrylamide. The strips were rehydrated for 14 h in 320 µl of dehydration buffer and then focused at 20°C for a total of 64 kV-h with a PROTEAN IEF Cell system (Bio-Rad). After IEF, the strips were equilibrated for 20 min, first in equilibration buffer I  and then in equilibration buffer II . The equilibrated strips were placed over 12.5% (w/v) SDS-PAGE gels for 2-DE at 25 mA for 5 h. Gels were stained with colloidal CBB. After staining, gels were scanned using PDQuest 2D analysis software (Bio-Rad) on the basis of their relative volumes as described by Bai et al. 4HCO3 for 1 h at 40°C, reduced with 10 mM DTT in 100 mM NH4HCO3 for 1 h at 60°C, and incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min. Gels were then minced, air-dried, and rehydrated in 12.5 ng µl−1 sequencing-grade modified trypsin  in 25 mM NH4HCO3 overnight at 37°C. Tryptic peptides were extracted three times from the gel grains using 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile. Supernatants were concentrated to approximately 10 µl using a SpeedVac  and then desalted using reversed-phase ZipTip pipette tips . Peptides were eluted with 50% acetonitrile and 0.1% TFA. Protein spots that differed in concentration by more than 1.5-fold and differed significantly  compared with the control were analyzed by MS.Protein spots displaying significant changes in abundance following plant exposure to heat, drought, or their combination were excised manually from colloidal CBB-stained 2-DE gels using sterile pipette tips. Spots were transferred to 1.5-ml sterile tubes, destained with 50 mM NHwww.matrixscience.com). The following search parameters were used: no molecular weight restriction, one missed trypsin cleavage allowed, iodoacetamide-treated cysteine, oxidation of methionine, a peptide tolerance of 100 ppm, and an MS/MS tolerance of 0.25 Da. Protein identifications were validated manually based on at least three matching peptides. Keratin contamination was removed, and the MOWSE threshold was set above 20 (P<0.05). Only significant hits in the MASCOT probability analysis were accepted as protein identifications.Lyophilized peptide samples were dissolved in 0.1% TFA, and MS analysis was conducted using a 4800 Plus MALDI-TOF/TOF Proteomics Analyzer . MS acquisition and processing parameters were set to reflector-positive mode and an 800–3,500-Da acquisition mass range, respectively. The laser frequency was 50 Hz, and each sample spectrum was acquired over 700 laser pulses. For secondary MS analysis, four to six ion peaks with signal-to-noise ratios exceeding 100 were selected from each sample as precursors. TOF/TOF signal data for each precursor were then accumulated from 2,000 laser pulses. Primary and secondary mass spectra were transferred to Excel files and compared against a non-redundant NCBI protein database (NCBI-nr 20101014) restricted to Viridiplantae  using the MASCOT search engine Click here for additional data file.Figure S2Representative set of 2-D gels of samples subjected to drought stress. Marked numbers represent differentially expressed proteins in the treatment.(TIF)Click here for additional data file.Figure S3Representative set of 2-D gels of samples subjected to a combination of high temperature and drought. Marked numbers represent differentially expressed proteins in the treatment.(TIF)Click here for additional data file.Table S1Protein spot intensity ratios from different treatments at different treatment times  relative to the control (0 h).(DOCX)Click here for additional data file.


Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as  For nitrogen-source testing, minimal media was supplemented with 20 mM glucose and nitrogen sources were used at a final concentration of 20 mM except for phenylalanine (5 mM), tryptophan (5 mM), and xanthine (0.5 mM). Lastly, minimal media consisting of 20 mM glucose, 20 mM NH4Cl and 0.5 mM MgCl2 (in exchange of MgSO4) was used to measure growth on sulfur compounds, which were provided at a final concentration of 0.5 mM. Compounds used in the experiments are listed in Growth analyses were done in triplicate. Strains were first grown on solid CPG at 30°C for 48 h. Single colonies were inoculated into 1.0 mL of liquid CPG, and the inoculums were grown at 30°C, 200 rpm for 24 h. Next, 10 μL of a CPG-grown seed culture was used to inoculate 1.0 mL of a low-salt minimal media , rpoN2 (pBRL584) or no insert (pBBR1MCS-2) were electroporated into ΔrpoN1 and ΔrpoN1 ΔrpoN2 R. solanacearum GMI1000. Following initial selection, recombinant strains were grown in 1.0 mL of CPG supplemented with Km at 30°C, 200 rpm for 24 h. Minimal media (1.0 mL) was inoculated with 10 μL of CPG-grown seed culture. For complementation experiments relating to carbon utilization, minimal media was supplemented with 20 mM NH4Cl and 20 mM α-KG, ethanol, fumarate, malate, propionate or succinate. For complementation experiments relating to nitrogen utilization, minimal media was supplemented with 20 mM glucose and 20 mM alanine, nitrate, ornithine, proline or serine. Minimal media cultures were grown at 30°C, 200 rpm for 24–96 h.Growth analyses were done in triplicate. Plasmids carrying rpoN E. coli BW25113 was transformed with pBBR1MCS-2, pBRL584 or pBRL587. Recombinant strains were selected on LB supplemented with Km. Individual colonies were inoculated into 1.0 mL of LB supplemented with Km, and the inoculums were grown at 37°C, 200 rpm for 24 h. Minimal media (1.0 mL) was inoculated with 10 μL of LB-grown seed culture. For carbon utilization experiments, minimal media was supplemented with 20 mM NH4Cl and acetoacetate (20 mM) or propionate (20 mM). For nitrogen utilization experiments, minimal media was supplemented with 20 mM glucose and arginine (20 mM), asparagine (20 mM), NH4Cl (0.5 mM) or xanthine (0.5 mM). Minimal media cultures were grown at 37°C, 200 rpm for 24–48 h.ΔSolanum lycopersicum) cultivar (cv.) Bonny Best and cv. Hawaii 7996 were grown in growth chambers (Conviron) with 12 h light-and-dark cycles at 25°C with 70% relative humidity. R. solanacearum strains were grown on solid CPG supplemented with 0.005% (w/v) of 2,3,5-tripheny tetrazolium chloride (TTC) at 30°C for 48 h. Single colonies that displayed a virulent morphology (mucoid and pinkish-white in color) . R. solanacearum NtrC exhibits 60% homology to E. coli AtoC. Expression of rpoN2 restored the growth of ΔrpoN E. coli on acetoacetate, suggesting that RpoN2 can partner with AtoC to regulate transcription from AtoC-controlled genes. R. solanacearum PrpR and E. coli PrpR have ∼50% homology. The differences in sequence between the PrpR EBPs are reflected in the inability of RpoN2 to fully restore the growth of ΔrpoN E. coli on propionate.Region II of RpoN has been implicated in EBP interactions , and NtrR. solanacearum in the host plant environment [rpoN1 gene was needed for the growth of R. solanacearum GMI1000 on these compounds, it was suspected that rpoN1 might also be a necessary factor in the pathogenesis of this pathogen. Indeed, this suspicion was recently confirmed in a study, which found that the rpoN1 gene was necessary for virulence of R. solanacearum GMI1000 on tomato plants (Lycopersicum esculentum cv. Marmande)[rpoN1 mutants generated in our study were also avirulent. To this end, tomato plants consisting of cv. Bonny Best (susceptible) and cv. Hawaii 7996 (resistant) were inoculated with a bacterial suspension comprising of wild-type, ΔrpoN1, ΔrpoN2 or ΔrpoN1 ΔrpoN2 R. solanacearum GMI1000. The inoculated plants were then examined on a daily basis for signs of wilt disease, which was quantified and used to calculate a disease-wilting index (dwi). Wilting was first observed on the older leaves of cv. Bonny Best plants inoculated with wild-type and ΔrpoN2 R. solanacearum GMI1000. The overall pattern of disease was similar for both tomato cultivars but the extent of wilting was severe in cv. Bonny Best compared to cv. Hawaii 7996  . Therefoaii 7996 . Entire ectively . Mock plectively . At 2 dpat 3 dpi . The wili (0–8%) .in planta growth of the rpoN mutants. Plants were inoculated with each strain at a titer of 1 x 106 CFU mL-1 and bacterial growth (CFUs per gram of fresh tissue) was determined at 4 dpi for cv. Bonny Best and 7 dpi for cv. Hawaii 7996. The bacterial growth could not be determined at 7 dpi for the cv. Bonny Best plants, because all plants were dead by this particular dpi. At 4 dpi in cv. Bonny Best, wild-type cells and the ΔrpoN2 mutant produced ~1000-fold more CFUs than that of the ΔrpoN1 and ΔrpoN1 ΔrpoN2 mutants. Similarly, at 7 dpi in cv. Hawaii 7996, wild-type cells and the ΔrpoN2 mutant produced ~100-fold more CFUs than that of the ΔrpoN1 and ΔrpoN1 ΔrpoN2 mutants  was grown in minimal media in which the indicated compound served as either the sole carbon, nitrogen or sulfur source. The mean absorbance value at 600 nm (± standard deviation) is reported for each strain.(DOCX)Click here for additional data file.


CsABCG36 (CsPDR8) and CsABCG40 (CsPDR12), are most abundant in roots and are significantly affected by phytohormones and auxin herbicide. In this study, we analyzed the structure and phylogeny of all the full-size cucumber ABCG transporters and studied the organ expression profiles of the remaining 14 CsABCG genes. In addition, we investigated the effect of different plant growth regulators and the diterpene sclareolide on CsABCG expression in cucumber roots. Until now, the full-size plant ABCG transporters have been grouped into five different clusters. The new phylogenetic analysis of full-size ABCGs from model plants and cucumber clustered these proteins into six different subgroups. Interestingly, the expression profiles of cucumber ABCG genes assigned to the same clusters were not correlated, suggesting functional diversification or different regulatory mechanisms of the full-size cucumber ABCG proteins.Full-size members of the ABCG  subfamily of ABC transporters have been found only in plants and fungi. The plant genes encoding full-size ABCGs identified so far appeared to be differentially regulated under various environmental constraints, plant growth regulators, and microbial elicitors, indicating a broad functional role of these proteins in plant responses to abiotic and biotic stress. Nevertheless, the structure and physiological function of full-size ABCGs in many plant species are still unknown. We have recently identified 16 genes encoding full-size ABCG proteins in cucumber and found that the transcripts of two of them, The online version of this article (doi:10.1007/s11105-015-0956-9) contains supplementary material, which is available to authorized users. The final concentrations of phytohormones and growth regulators are given in the figure captions. The concentrations of all compounds were chosen based on previous reports  according to the manufacturer’s instructions. The RNA integrity was checked by electrophoresis, whereas RNA quality and quantity were determined spectrophotometrically (NanoDrop). Only the samples with A260/A280 and A260/80 values in the range of 1.9–2.0 or 1.8–2.2 were used for further analysis. RNA samples were treated with DNase I (Fermentas) to remove contaminating genomic DNA. cDNA was synthesized from 2000 ng of RNA by priming with random primers using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. The primers specific for each PDR gene were carefully designed using LightCycler Probe Design Software 2.0 (Roche) , 1× Marathon buffer, 0.5 μM dNTP mix, 1 μl of 10 μM forward primers, 1 μl of 10 μM reverse primers, and 1 μl of cDNA prepared from different cucumber organs in a total reaction volume of 20 μl. The reaction conditions were as follows: initial denaturation for 2 min at 94 °C, followed by 30–34 cycles of 30 s denaturation at 94 °C, annealing for 30 s at the appropriate annealing temperature, and extension for 1 min at 68 °C , which proved to be highly stable under plant growth regulator treatments . The reaction mixture contained 2 μl of cDNA diluted eight times, 10 μl of 2× SYBR Master Mix B (A&A Biotechnology), 2 μl of 10 μM forward primer, and 2 μl of 10 μM reverse primer in a 20 μl solution. Amplifications were carried out in 94-well plates (Roche) after pre-incubation at 94 °C for 30 s, followed by 45 cycles of denaturation at 94 °C for 10 s, annealing at 55 °C for 10 s, and extension at 68 °C for 12 s, with final melting at 70 °C for 30 s. PDR genes have been recently identified , GenBank , and Gramene database (O. sativa)  Fig. . The aliva) Fig.  and maxiCsABCGs (CsPDRs) previously , and sequenced. The EST sequences were submitted to GenBank and annotated according to the nomenclature given to all CsABCG36 (CsPDR8) and CsABCG40 (CsPDR12) , five cucumber ABCGs , two A. thaliana ABCGs (AtABCG38 and AtABCG40), and homologous proteins from N. plumbaginifolia (NpPDR2) and Spirodela polyrrhiza (SpTUR2). Cluster II contains six A. thaliana ABCGs , three cucumber ABCGs , three OsABCGs , and one homologous protein from N. tabacum (NtPDR3). The three A. thaliana PDRs, AtABCG29, AtABCG35, and AtABCG36, together with cucumber CsABCG29 and CsABCG36, and rice OsABCG38 and OsABCG42, form cluster IIIa, whereas the four orthologous proteins AtABCG31, OsABCG51, CsABCG31, and CsABCG35 branch into the separate cluster IIIb . The smallest cluster V is formed by OsABCG31, AtABCG32, and CsABCG32  were clearly expressed in all plant organs. Although slightly higher levels of CsABCG32, CsABCG33, CsABCG37, CsABCG39, and CsABCG41 mRNAs were detected in 8-week-old plants than in young seedlings, the distribution in all organs suggests the ubiquitous expression of these cucumber ABCGs. In contrast, no expression of CsABCG38 was detected in both the vegetative organs and inflorescences of cucumbers grown under control conditions  but only slightly detected in young cucumber seedlings  involved in the plant response to biotic  and abiotic  stresses, as well as the natural and synthetic regulators affecting plant growth and development . The organ expression assay revealed that 11 cucumber ABCGs—CsABCG29, CsABCG30, CsABCG32, CsABCG33, CsABCG34, CsABCG35, CsABCG37, CsABCG39, CsABCG41, CsABCG42, and CsABCG44—were clearly expressed in the roots of 7-day-old cucumbers grown in control nutrition medium  of treatment (data not shown). The expression patterns of genes that were significantly affected by different plant growth regulators and sclareolide are presented in Figs. CsABCGs to various treatments was different and often dependent on the time course of the experiment. The CsABCG29 transcript was differentially affected only by two phytohormones: salicylic acid and jasmonic acid  and 2,4-D (up to 10-fold) treatments  as well as the motifs responsive to gibberellins and auxins  was observed only upon 4- and 8-h-long exposure of plants to the auxinic herbicide 2,4-D, whereas phytohormones and the ethylene precursor decreased gene expression over 2-fold  or even up to 7–8-fold (SA) in different time courses of experiments  and salicylic acid (3-fold decrease) treatments   reduced by SA, but the greatest change in gene expression was observed under JA and GA3 (4-fold increase) , 4-fold (JA), or 6-fold (SA) decrease in CsABCG35 transcript level after the first 8 h of treatments  down-regulated upon sclareolide treatment, and the effect of diterpene increased with the time of the experiment  and ACC (over 7-fold) and up-regulated by GA3 (over 6-fold)  and GA3   and ABA (ABRE-motif) were present in the CsABCG42 promoter , kinetin-responsive , gibberellin-responsive , ABA-responsive , jasmonate-responsive , salicylic acid-responsive , 2,4-D-responsive , ACC(ethylene)-responsive , and sclareolide-responsive  genes 2. Using available sequences from different plant species, it has been previously shown that the full-size ABCGs from plants form five separate phylogenetic clusters (I–V) with cluster I containing the so far studied SpTUR2, AtABCG40/AtPDR12, NpPDR1 and most rice ABCGs  located in cluster II. Similarly to rice ABCGs, the largest number of cucumber ABCGs (5) are present in cluster I, whereas the others are quite equally distributed between the remaining clusters III, IV, and V (1–3 sequences in each cluster). This clustering confirms the previous indication that the divergence between plant full-size ABCGs occurred before the separation between monocots and dicots and that the patterns of gene duplication in different plant ancestors were different  and CsABCG40 (CsPDR12), was strongly affected by some plant growth regulators related to stress response, suggesting the contribution of members of the cucumber ABCG subfamily to the hormone-mediated response to environmental constraints  encodes the transporter for structurally and functionally diverse compounds including IAA precursors  were also affected by salicylic and jasmonic acids  indicates the possible role of these proteins in plant-pathogen interactions. Additionally, the cucumber genes encoding CsABCG33, CsABCG37, and CsABCG41 were clearly expressed in all cucumber organs, suggesting that they encode proteins with essential functions for plant cells. The obtained results suggest that members of cluster II may represent a constitutive defense pathway in cucumber plants.Three cucumber full-size ABCG proteins—CsABCG33, CsABCG37, and CsABCG41—have been classified in cluster II. However, genes encoding these proteins showed different expression patterns under plant growth regulators and sclareolide. AA Moons . Hence, CsABCG36 expression was analyzed previously , a closely related gene from rice, was also strongly expressed in old leaves, and up-regulated by JA, cytokinins, and redox perturbations which accompany the aging process  proteins  of cucumber plants  and leaf derivatives  was not affected by plant growth regulators  import into plant cells , encoding a close homolog of CsABCG32, was also up-regulated by stress-related phytohormone ABA ESM 2(DOC 47 kb)Below is the link to the electronic supplementary material.