Salmonella, similarly to phytobacteria, appears to be capable of producing the plant hormone auxin via an indole-3-pyruvate decarboxylase (IpdC), a key enzyme of the IPyA pathway. A deletion of the Salmonella ipdC significantly reduced auxin synthesis in laboratory culture. The Salmonella ipdC gene was expressed on root surfaces of Medicago truncatula. M. truncatula auxin-responsive GH3::GUS reporter was activated by the wild type Salmonella, and not but the ipdC mutant, implying that the bacterially produced IAA (Indole Acetic Acid) was detected by the seedlings. Seedling infections with the wild type Salmonella caused an increase in secondary root formation, which was not observed in the ipdC mutant. The wild type Salmonella cells were detected as aggregates at the sites of lateral root emergence, whereas the ipdC mutant cells were evenly distributed in the rhizosphere. However, both strains appeared to colonize seedlings well in growth pouch experiments. The ipdC mutant was also less virulent in a murine model of infection. When mice were infected by oral gavage, the ipdC mutant was as proficient as the wild type strain in colonization of the intestine, but it was defective in the ability to cross the intestinal barrier. Fewer cells of the ipdC mutant, compared with the wild type strain, were detected in Peyer's patches, spleen and in the liver. Orthologs of ipdC are found in all Salmonella genomes and are distributed among many animal pathogens and plant-associated bacteria of the Enterobacteriaceae, suggesting a broad ecological role of the IpdC-catalyzed pathway.The ability of human enteric pathogens to colonize plants and use them as alternate hosts is now well established.  Salmonella and shigatoxigenic E. coli and linked to the consumption of sprouts, vegetables, fruits and nuts led to the hypothesis that these enteric pathogens use plants as alternate hosts , is one of the mechanisms by which plant-associated bacteria and fungi manipulate plant growth and development. Microbial production of IAA alters root architecture to increase availability of nutrients to microbes capable of producing this plant hormone. Microbially derived auxins also contribute to the formation of new organs , suppress plant defenses and regulate virulence in phytobacteria  into IAM, and then to IAA by IAM hydrolase (encoded by iaaH)   cultures were grown overnight from glycerol stocks at 37°C. 1 mL was washed 3x in PBS and was diluted 1,000x in 5 mL Minimal A medium with or without 1 mM tryptophan. Cultures were incubated at 22°C for 72 h. Supernatant was harvested from 1 mL of culture by centrifugation for 15 min at 7,000 × g. The supernatant was transferred to a 12 mm × 75 mm glass tube and 2 mL of fresh Salkowski reagent R1  were subject to reverse phase (C18) liquid chromatography using Waters Atlantis dC18-5 μM column using isocratic elution (72 water 7.2% acetic acid 20.8% methanol), as before  and transgenic line containing the GH3::GUS reporter  or CEC1002. Plants were watered as needed with the N-free Hoagland solution. To detect activation of the GH3::GUS reporter, seedlings were withdrawn after 48 hrs and 6 days past infection, and stained with X-gluc (10 mg/ml) in 0.2 mM Phosphate buffer (pH 7.2) overnight. They were then washed 3 times with cold phosphate buffer to remove excess substrate and the seedlings were kept in 50% ethanol/phosphate buffer at 4C. They were imaged under a dissecting microscope Olympus MVX10 fit with a MicroFIRE camera (Optronics).Seeds of M. truncatula seedlings as described in Supplemental Information. At indicated time intervals, seedlings were excised from pouches, blended with a glass mortar and pestle and dilution plated onto XLD with kanamycin. Bacteria were patched from these onto LB with tetracycline.For the reporter assays, washed RIVET reporter suspensions  were inoculated onto M. truncatula A17 were surface sterilized, imbibed and then transferred into growth pouches. Seedlings were inoculated with 100 μl of the washed bacterial suspension containing ~5,000–10,000 CFU of either CEC1000  or CEC1002 (ipdC::kan).To detect changes in lateral root formation, seeds of S. enterica sv. Typhimurium 14028 and CEC1002 were streaked on LB agar and incubated at 37°C overnight, colonies were harvested and re-suspended in 1 ml of sterile PBS. Each suspension was adjusted to a final dilution of ~108 CFUs/ml. Groups (n = 3) of female BALB/c mice received three doses of Salmonella in 0.2 ml of PBS. Inoculum doses were confirmed by serial titration, and were found to be 106, 104, and 102 CFU per injection dose for the wild type, and 2.2 × 106, 2.2 × 104, and 2.2 × 102 CFU per injection for the ipdC mutant. Animals were observed daily for 7 days after infection and distressed animals presenting signs of morbidity were euthanized, their organs were not sampled. The experiment was terminated at 7 days, and all surviving animals were sacrificed. Liver, spleen, large intestine, and Peyer's patches were harvested. Tissues were weighed, and homogenized in sterile PBS in TissueLyser II (Qiagen). Bacterial homogenates were serially diluted in PBS and plated onto Xylose-Lysine Deoxycholate (XLD) agar  plates, followed by incubation at 37°C overnight for bacterial CFU counts. All animal care and procedures were in accordance with institutional policies for animal health and well-being and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC).BALB/c mice were inoculated by oral gavage, which results in a systemic infection. Prior to the infections, Synteny analysis was carried out using default parameters in the SyntTax web server Oberto, . SequencipdC is conserved in salmonellae. ipdC, the gene encoding indole-3-pyruvate decarboxylase, a key enzyme in the IPyA pathway, is common in many members of Enterobacteriaceae; including organisms that are commonly associated with animals as well as with plants. Genomic analysis showed that all available Salmonella genomes harbor ipdC, but that it is absent in E. coli, Shigella, or Erwinia genomes and in some strains of Enterobacter. The presence or absence from the enterobacterial genomes does not seem to correlate with the source of the isolates, and ipdC was as likely to be found in the genomes of animal as of plant isolates of this genus. Synteny analysis revealed a distribution of orthologs in the genomes of many strains belonging to the Enterobacteriaceae where it is generally found within similar genomic context  was present between ipdC and ypeC except in E. coli and Pectobacterium. These results suggest that the Salmonella ipdC may encode an ortholog of a well-characterized indolepyruvate decarboxylase, responsible for IAA synthesis in other bacteria. Therefore, we tested whether ipdC is capable of directing IAA production in S. enterica sv Typhimurium.t Figure . In SalmSalmonella to produce IAA was first tested in culture in Minimal A medium using a colorimetric assay based on the Salkowski reagent. While there was no auxin production by either the wild type or the isogenic ipdC mutant grown in unammended Minimal A medium, production of IAA was detected in the medium supplemented with 1 mM L-tryptophan, the precursor for the IPyA pathway  in the isogenic strain lacking ipdC  reporter was low (less than 10%) after 24 h of incubation, however, it increased by 72 h  reporter in ipdC was expressed in a laboratory medium at 45–55%  family are among the regulators that control the dynamic process of endogenous auxin homeostasis . Seedlings inoculated with the S. Typhimurium wild type strain showed 5–12 secondary roots, while seedlings inoculated with the ipdC mutant had 2–7 secondary roots per plant. When the wild type and the ipdC mutant were tagged with a plasmid constitutively expressing GFP, the ipdC mutant was found to be evenly distributed throughout the root surface and on root hairs, while the wild type tended to form large aggregates at the sites of the lateral root emergence , alter plant tissue development, and induce leakage of nutrients  have evolved to thrive. Studies suggest that in some bacteria, IAA aids in stress adaptation and protection against stressors such as UV, salt and acidity  to the handling Editor. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While the effects of OA on coral calcification are well documented, the response of reproduction is still poorly understood since no information are reported for temperate corals. Here we investigate for the first time the influence of OA on sexual reproduction of the temperate azooxanthellate solitary scleractinian Leptopsammia pruvoti transplanted along a natural pCO2 gradient at a Mediterranean CO2 vent. After 3 months, future projection of pH levels did not influence the germ cell production, gametogenesis and embryogenesis in this azooxanthellate coral. These findings suggest that reproductive potential may be quite tolerant to decreasing pH, with implications for ecosystem function and services in a changing ocean.Anthropogenic carbon dioxide (CO Seawater pH has already decreased by 0.1 pH units since the industrial revolution3 and if CO2 emissions continue under the “business-as-usual” scenario, oceanic pH levels will drop to 7.8 by the end of the century and may reach 7.4 units by 25003.Anthropogenic CO5, corals7 and coral reef communities, including fish8, show reduced calcification rates under low seawater pH, due to depleted carbonate saturation. Moreover, many biological processes and physiological functions independent of calcification may be impacted by decreasing pH11. Ocean acidification negatively influences the metabolism of marine invertebrates that have a low ability to compensate for disturbances to the extracellular ion and acid-base status, leading to negative effects on reproduction and behaviour12. Sexual reproduction represents a crucial process in the development and persistence of populations and its reduction threatens the resilience of species, leading to shifts in size and abundance of populations13. Early life history stages of echinoderms, mollusks and crustaceans including larval availability19 , larval development20 and growth20, larval settlement20, post-settlement growth11 and survival11 seem to be very sensitive to increasing pCO2. The few available studies on the effects of ocean acidification on coral sexual reproduction were conducted in tropical species and only under laboratory conditions24, thus lacking information on the complex natural environment25.Several studies reveal how calcifying algae2 vents) mimic future ocean conditions and can be used as natural laboratories to investigate the effects of increasing pCO2 on marine ecosystems in situ29. The Panarea CO2 vent  is shallow, and characterized by almost pure CO2 emissions at ambient temperature, with no toxic compounds, making this an ideal site for ocean acidification studies in a natural setting29. The pCO2 gradient is characterized by extremely low pH values on the rim of the crater that exceed the most pessimistic scenarios for the coming century, representing a more distant scenario (25003) and by progressively higher values that match near future scenarios (2100) all the way to the periphery where normal pH conditions are re-established.Naturally acidified areas , were found in both, the gonadal development and fertilization periods , shows a significant decrease in gonadal development17.During both, gonadal development and fertilization period, the development of male and female germ cells did not follow trends associated with increasing CO2 Fig. , and allCO2 Fig. . These rL. pruvoti were unaffected by future pCO2 levels.During the fertilization period, the detection of embryos at all sites suggested that the fertilization process had started at all sites. Moreover, no differences were found in the embryo production and embryo size, indicating that after three months under experimental conditions, also the fertilization process and embryogenesis of 39. Fertilization success of the tropical coral Acropora palmata decreases with increasing pCO2, with a reduction  of 13% at the CO2 level expected for the end of this century23. At the contrary, the fertilization process of A. tenuis and A. millepora is unaffected by elevated pCO2 projected for the end of this century, alone or in combination with temperature40, but only if sperm concentration is high41.Influence of decreasing pH on fertilization has been well studied through aquarium experiments in marine invertebrates, such as echinoderms, mollusks, crustaceans and tropical corals. Many experiments show negative responses, especially if sperm concentration is low and limitedpCO2 negatively affects embryo growth and development in mollusks14, echinoderms42 and crustaceans43, but not significant effect was detected on the timing of embryonic development in the tropical corals A. palmata under seawater pH levels expected for the end of this century (pH 7.744).Moreover, experimental laboratory studies show that increasing L. pruvoti seemed quite tolerant to future pH levels, showing normal reproduction along the gradient. The lack of effects on gametogenesis and embryogenesis in L. pruvoti under decreasing pH may be explained in different ways.The solitary non-zooxanthellate L. pruvoti extends over the year, in fact, male germ cells takes approximately 12 months to mature while female germ cells need ~24 months32. Considering the short-term exposure to experimental conditions in this study (3 months), spermatogenesis was exposed to high pCO2 levels for a quarter of its time, while oogenesis was exposed for one eighth of its length, possibly explaining the low sensitivity observed with future pH levels.The gametogenesis of L. pruvoti places to keep the gametogenesis constant along the pH gradient may leave little energy for the coral to sustain net calcification in the face of ocean acidification45. Confirming this hypothesis, a parallel study on L. pruvoti transplanted along the same natural pCO2 gradient shows decreased net calcification rate with decreasing pH38, even under pH values projected for the end of this century. Thus, under future pH conditions, L. pruvoti may allocate more energy to maintain constant the reproduction process, at the expense of net calcification, which significantly decreases under acidified conditions.Another explanation concern the channeling of available energy into reproduction at the expense of other metabolic processes. The high investment of energy that L. pruvoti seems that will be fine in coming decades, showing no effects on gametogenesis, spermatogenesis and embryogenesis along the pH gradient. However, this study did not consider the post-fertilization process, including settlement success, larval survival and development and juvenile growth, even if other studies have reported significant impact of ocean acidification on tropical corals.In conclusion, the reproductive process of L. pruvoti is capable of maintaining a constant reproductive output, fertilization success and embryo growth under a long-term exposure to future pH levels.This study considered the short-term effect of pH (~3 months), but further investigations are needed to understand if et al.28, is located near Panarea Island , where an underwater crater (20 × 14 m) at 10 m depth, generates a stable and continuous column of bubbles (98–99% CO246) at ambient temperature, creating a natural pH gradient. Along this gradient, four sampling sites were selected: the control site  pHTS 8.07), located about 34 m away from the center of the crater, two intermediate pH sites (site 2 and 3: mean pHTS respectively 7.87 and 7.74), representing the intermediate and the most pessimistic IPCC scenario for the end of this century and the extreme pH site (site 4: mean pHTS 7.40), situated in proximity of the vents, representing the projection for 2500. The experimental site has stable hydrothermal–chemical properties and only pCO2 concentration differed significantly among the four sites (see Supplementary information of 38 for detailed seawater carbonate chemistry in each transplantation site).The experimental site, which has been previously described in detail by Goffredo L. pruvoti). The species is not protected in Italy, nor is it subject to any regulations. Thus, no permit was needed to sample specimens. For this study, sampling was limited strictly to the number necessary for the described analyses and performed where the species has high population density to minimize the impact of removing individuals and preserve both the demographic and genetic structure of the natural populations.This study was carried out following the fundamental ethical principles. According to the European normative, there is no active conservation measure for the Mediterranean scleractinian species under study  of similar size (average length 7 mm) were sampled at ~6 m depth by SCUBA diving in an area ~2 km away from the vent area and transplanted to the four sites in six experimental periods.Sexually mature 32. At the end of each transplantation period, samples were fixed in a formaldehyde fixative solution  to preserve the sample tissue for further histological analysis.The same number of corals was randomly assigned to each of the four sites and each polyp was considered as a replicate , width , and height  were measured for each specimen with a pair of calipers (±0.05 mm). The body volume (V) was estimated using the equation:Biometric measurements were used to calculate the reproductive parameters (see paragraph below).32.Polyps were post-fixed in Bouin solution. After decalcification in EDTA and dehydration in a graded alcohol series from 80 to 100%, polyps were embedded in paraffin, and serial transverse sections were cut at 7 µm intervals along the oral-aboral axis, from the oral to the aboral poles. Tissues were stained with Mayer’s hematoxylin and eosin37, were measured. The maximum and minimum diameters of embryos found in the gastrovascular cavity were measured.Cytometric analyses were performed with a light microscope NIKON Eclipse 80i using an image analysis software: NIKON NIS-Elements D 3.1. The maximum and minimum diameters of oocytes in nucleated sections, and spermaries, classified in five maturation stages in accordance with previous studies on gametogenesis of this speciesoocyte and spermary abundance, both defined as the number of reproductive elements per body volume unit (100 mm3); 2) gonadal index, defined as the percentage of body volume occupied by oocytes and spermaries; 3) reproductive element size, defined as the average of the maximum and minimum diameter of spermaries and oocytes in nucleated section; 4) fertility, defined as the number of embryos per body volume unit (100 mm3); 5) embryonal index, defined as the percentage of body volume occupied by embryos; 6) embryo size, defined as the average of the maximum and minimum diameter of embryos.Reproductive output was defined by six parameters: 1) gonadal development period, characterized by the beginning of spermary and oocytes development, and 2) fertilization period, characterized by a little stock of big mature oocytes not yet fertilized, a bigger stock of oocytes that will be fertilized in the following reproductive year, advanced maturation stages of spermaries and presences of embryos in the coelenteric cavity of females.Based on the developing stages of oocytes and spermaries found in the analyzed samples and on the presence/absence of embryos in the coelenteric cavity of female polyps, samples from the six transplantation periods were grouped in two gonadal activity periods: 1) 47 solves problems in the non-parametric test for small samples, estimating the P-value by taking a random sample from the reference set and studies its permutations48 . This method was used to estimate the significance of the Kruskal-Wallis test when comparing the reproductive parameters  among sites.Levene’s test was used to test homogeneity of variance and one-sample Kolmogorov-Smirnov’s test was used to test normality of data distribution. When the sample size was lower than 2000, the Shapiro-Wilk test was used. The two-sample Kolmogorov-Smirnov’s test was used to do a pairwise comparison of the distribution of oocyte size and spermary maturation stages between the two periods. The Monte Carlo methodThe analyses were computed using PASW Statistics 22.0.49 based on Euclidean distances was performed with 999 permutation, to test differences in oocyte size distribution, spermary maturation stage distribution and in oocyte, spermary and embryo diameter among sites, using the software Primer 6 (Quest Research Limited).A one-way permutation multivariate analysis of variance (PERMANOVA)The datasets analysed during the current study are available from the corresponding author on reasonable request.Supplementary information


After infection of the porcine dam at about 90 days of gestation, porcine reproductive and respiratory syndrome virus (PRRSV) crosses the placenta and begins to infect fetuses. Outcomes of include abortion, fetal death and respiratory disease in newborn piglets. CD163 is the receptor for the virus. In this study, CD163-positive fetuses, recovered between 109 days of gestation or 20 days after maternal infection, were completely protected from PRRSV in dams possessing a complete knockout of the CD163 receptor. The results demonstrate a practical means to eliminate PRRSV-associated reproductive disease, a major source of economic hardship to agriculture. Losses are the result of respiratory disease in young pigs, poor growth performance, reproductive failure, and in utero infection2. The reproductive form of the disease accounts for an estimated 45% of losses, the result of abortions, dead fetuses, and respiratory disease in newborns. In its severest form, reproductive PRRS can result in 90% mortality of fetuses/neonates, along with increased mortality for the dams. Pigs that survive in utero infection become continuous sources of virus in downstream production phases, resulting in endemically infected herds3. The severest form of reproductive disease is associated with a group of highly virulent isolates referred to as atypical PRRSV5. Interestingly, many of the atypical PRRSV isolates emerged from PRRS-vaccinated farms6. In 2006, an atypical virus, called high pathogenic PRRSV (HP-PRRSV), appeared in China and continues to decimate pig populations in that country7. Since the standard commercial breeding facility contains about 5,000 sows, an outbreak of high mortality reproductive PRRS can have a devastating impact. To ensure sustainability of pork production and food security, solutions for the control of reproductive PRRS remain a priority. Vaccines have been unable to control the disease, largely because of genetic diversity within the structural proteins of the virus8. In practice, intensive biosecurity measures provide the only means of protecting the reproductive herd.Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease of swine in North America, Europe and Asia, costing North American producers approximately $600 million annuallyArterviridae. Structurally, the arteriviruses resemble togaviruses, but similar to coronaviruses, replicate via a nested 3′-co-terminal set of subgenomic mRNAs, which possess a common leader and a poly-A tail. Arteriviruses exhibit a tropism for macrophages and possess the capacity to establish subclinical persistent infections, as well as cause severe and fatal disease9.Along with lactate dehydrogenase-elevating virus (LDV) of mice, equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV), PRRSV belongs to the family, 11. After an initial phase of replication in maternal macrophages, the virus crosses the placenta and begins to productively infect fetuses. The virus initially infects only a small number of fetuses, followed by horizontal transmission of virus from fetus to fetus12. The exact mechanism of how the virus crosses the placenta remains unknown, but could be similar to the infected “Trojan Horse” macrophage, previously described for LDV13. Unlike the alveolar macrophages in adult animals, the primary site of PRRSV replication in the fetus is the thymus3. Since the pig fetus becomes immunocompetent at about 70 days of gestation, PRRSV infection occurs in a fetal immune environment containing functional B and T cells11.The reproductive form of PRRS occurs following the infection of pregnant gilts or sows at about 90 days of the 114 day gestation period13. The resulting heme degradation products regulate the associated inflammatory response14. CD163 as a receptor for PRRSV was first described by Calvert et al.15. Transfection of non-permissive cell lines with CD163 cDNA from a variety of species, including simian, human, canine, and mouse can make cells permissive for infection. We recently showed that pigs with a complete knockout (KO) of the CD163 gene lack CD163 expression on macrophages and fail to support PRRSV infection17. Since CD163 expression is a dominant trait and inherited in a classic Mendelian fashion, offspring possessing normal CD163 expression and function can be derived by crossing a KO CD163−/− female pig with a wildtype (WT) CD163+/+ male. For this study, CD163 KO gilts were bred with WT boars producing heterozygous, CD163+/− fetuses. The hypothesis to be tested was that the presence of the CD163 KO genotype of the dam would be sufficient to protect fetuses following maternal infection with PRRSV.CD163 is a 130 kDa type 1 membrane protein composed of nine scavenger receptor cysteine-rich (SRCR) domains and two spacer domains along with a transmembrane domain and a short cytoplasmic tail. In addition to functioning as a virus receptor, CD163 exhibits several important functions related to maintaining normal homeostasis. For instance, following infection or tissue damage, CD163 functions as a scavenger molecule, removing haptoglobin-hemoglobin complexes from the bloodCD163 KO parents are summarized in Table CD163+/+ dams carrying CD163+/+ fetuses (++/++ group). A second group (−/+−) were CD163−/− dams carrying CD163+/− fetuses. In this group, the CD163−/− dams are unable to support PRRS replication, while the CD163+/− fetuses retain susceptibility to PRRS infection. And finally, a third group (−/−) consisted of CD163−/− dams carrying CD163−/− fetuses. For the last group, both dams and fetuses should be resistant to infection.A detailed description of the knockout alleles used in this study is shown in Table 10 templates per reaction, demonstrating the presence of a productive PRRSV infection. Between 15 and 20 dpi, all remaining dams were euthanized and uterine horns immediately removed. Beginning at the tip of each horn, fetuses and placentas were removed, assessed for the presence of anatomic pathology. A blood sample was obtained from each fetus. If blood was not obtainable, a sample of fluid was collected from the abdominal cavity. The number of fetuses recovered from each dam is listed in Table CD163 WT group (++/++) (including the dam that aborted), the number of fetuses were 16, 14 and 12 (mean = 14.0). The CD163 KO dams carrying the CD163+/− fetuses (−/+− group) yielded 14, 16 and 11 fetuses (mean = 13.6). For the CD163 KO dams carrying CD163 KO fetuses (−−/−−group), the numbers of fetuses were 7 and 9. The results for fetal viremia and gross pathology are summarized in Fig. CD163 WT (++/++) dams, No. 138 and No. 140, showed some degree of pathology, including smaller than normal fetuses , fetuses with detached or necrotic placentas (14%), meconium staining (7%), and fetuses that were dead and necrotic (25%). The pathology observations are typical of reproductive PRRS. The same litters showed a high rate of PRRSV infection, with 92% of the fetuses testing positive for the presence of PRRSV nucleic acid. The PCR results for the fetuses from the WT dams illustrate two important properties of fetal PRRSV infection. First, there was a wide variation between fetuses in the concentration of virus detected in serum, the result of fetuses becoming infected at different times. Secondly, the level of viremia was not always correlated with pathology. For example, Fetus No. 5 from Dam No. 138 possessed a high level of viremia (7.3 log10 templates per reaction) and yet the fetus appeared unaffected. The reason for the discrepancy between viremia and the pathology is unclear. One possibility is that fetal pathology is the result of tissue damage that occurs on the maternal side and not related to the level of fetal viremia. In the field, these normal, but infected newborn piglets can function as “supershedders”, which facilitate the rapid dissemination of PRRSV throughout a production system. For the −−/+− group , all fetuses appeared normal, with the minor exception of two fetuses that were smaller than the other littermates. The smaller than normal size is likely a consequence of crowding within the uterine horn that decreases the surface area of the placenta, thus restricting the growth of the developing fetus. All dams and fetuses in the −−/+− group were negative for the presence of PRRSV nucleic acid. For the last group, −–/−−, there was no visible pathology, and all dams (No. 86 and 121) and fetuses were negative for PRRSV nucleic acid.Clinical signs in the infected WT dams included lethargy and transient inappetance. The KO dams showed no clinical signs. During the study period, one WT dam, No. 139, aborted on day 106 of gestation (15 dpi). PRRSV nucleic acid, measured at 7 dpi, showed a viremia level for Dam No. 139 of 5.5 logCD163 KO dams are susceptible to virus immediately after birth, the protection from PRRSV in utero provides a means to eliminate a major source of economic loss and animal suffering.The results from this study clearly demonstrate that the absence of CD163 in the dam is sufficient to protect the PRRSV-susceptible fetus. Although CD163-positive offspring derived from et al.18. The specific edits for alleles A, B, D and E are described in Whitworth et al.18. The specific edit in Allele C (2 bp insertion) is described in Whitworth et al.16. The alleles, described in Table et al.17The CRISPR/Cas9 methods used to generate all of the KO alleles are described in detail in Whitworth 4. The virus, maintained as a low passage isolate, was propagated and titered on MARC-145 cells. At 89 to 91 days of gestation, gilts were inoculated with 105 TCID50 of virus diluted in 5 ml of culture medium. One half of the inoculum was administered by intramuscular injection and the remainder administered intranasally. All gilts were maintained in an environment that allowed for the continuous exposure to virus shed by infected pen mates. Blood samples were taken from the gilts prior to infection, 7 days post-inoculation (dpi), and at the time of euthanasia. PRRSV nucleic acid was measured by isolation of total RNA from serum followed by reverse transcriptase real-time PRRSV PCR . A standard curve was generated using the quantification standards supplied in the RT-PCR kit. Results were reported as log10 templates per 25 µl reaction, which approximates the number of viral RNA templates per ml of blood.The PRRSV strain used in this study, NVSL 97–7895 (NVSL), is a laboratory strain isolated in 1997 from a herd in Southeast Iowa, USA that was experiencing a PRRS abortion stormExperiments involving animals and virus were performed in accordance with the Federation of Animal Science Societies Guide for the Care and Use of Agricultural Animals in Research and Teaching, the USDA Animal Welfare Act and Animal Welfare Regulations, or according to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, and were approved by the Kansas State University and University of Missouri institutional animal care and use committees and institutional biosafety committees. Animals were humanely euthanized by pentobarbital overdose following the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals, and all efforts were made to minimize suffering.


Seed nutrients reserves have direct relationship with seed functional traits and influence offspring performance. Effects of plant density, foliage boron (B) nutrition and mepiquat chloride (MC) growth regulation on seed nutrients reserves, seed mass and production, and emergence and offspring growth traits of cotton were studied in two years field experiment. Seed nutrients reserves and seed mass were decreased at higher maternal plant density relative to lower plant density with concomitant decrease in emergence and offspring seedling growth. However, maternal foliage B nutrition and MC growth regulation enhanced seed nutrients reserves, seed mass, emergence and offspring seedling growth performance. There was a significant positive relationship between seed mass and seed nutrients reserves indicating that changes in nutrient availability/uptake in response to maternal ecological factors determine variation in seed functional traits. Nonetheless, seed mass was positively correlated with emergence percentage and negatively with emergence timing. Furthermore, variation in offspring seedling growth traits with seed mass indicated the significance of initial seed nutrients reserves for early seedling vigour and establishment. In conclusion, lower maternal plant density, B nutrition and MC growth regulation ensued in higher emergence and offspring seedling growth of cotton because of higher seed nutrient reserves and seed mass. Seed size is correlated with various seed functional traits, and often affects the timing of germination, germination capacity, offspring seedling establishment, and diversity and composition of community4. Although seed mass has a great importance in natural ecosystems, its significance cannot be overwhelmed in agro-ecosystems where seed size is the primary determinant of ecosystem productivity and successful establishment of succeeding generation5. However, studies on the effects of ecological factors  on seed mass, subsequent germination and seedling establishment of cultivated annual crops in agro-ecosystems are sparse and not well understood7.Effects of seed size (mass) on germination and seedling establishment of perennial plants in grasslandGossypium hirsutum L.) is cultivated as an annual crop, its perennial nature exists predominantly under environmental factors  that favour the vegetative growth8. This often consequences in greater amounts of photosynthates and nutrients to be partitioned to vegetative parts while the decreased partitioning and remobilization of reserves to developing fruits become the source of variation in seed development and size9. Studies on natural ecosystems and perennial plants have revealed that a significant correlation exists between maternal environment and seed development and seed mass12.Cotton  contents, and seed mass and seed macronutrients contents indicating that the positive effects of seed size are more often dependent on nutrient availability and seed nutrient reserves2.Plant competitiveness, soil and plant nutrient status, and alteration in the ability of plants to acquire nutrients and their translocation to seeds can alter the pattern of seed development, seed mass, productivity and subsequent germination and seedling establishment in succeeding generation18. An agro-ecosystem is characterized by inclusion of intensive external inputs. Thus manipulation in planting density can alter management decisions to acquire potential benefits from ecosystem services. Increasing the plant density enhances the inter-plant competition owing to limited plant available resources. Plant density modifies the plant canopy structure, plant micro environment 19 and capability of plants to acquire mineral nutrients from the immediate rhizosphere owing to modulation in root growth due to competition for space20. Moreover, the seed mass is influenced due to variation in plant density. Zhi et al.5 observed higher seed number per boll with concomitant increase in seed size and vigour index at lower plant density of cotton while there was a progressive decrease in seed area (density) and vigour index with increasing plant density.Plant density induces changes in the pattern of plant establishment, performance and productivity of an ecosystem, and success in the establishment of next generation because of complex relationship of inter-plant competition within a population and community21. A short term B deficiency during microsporogenesis hinders the development of anthers, and adversely affects the pollen viability and growth of pollen tube consequently leading to male sterility and poor seed set23. Boron nutrition affects the seed nutrients contents through modulation in uptake and translocation of other nutrients as well24. For example, B has positive interactions with N, P, K, Cu, Zn and Fe, whereas, negative interactions with Ca, Mg27, and Mn28. Moreover, it is involved in photo-assimilation and assimilate partitioning, and directly or indirectly affects the seed development and seed size29. Dordas7 and Dordas et al.30 observed that seed mass, yield and germination quality of cotton and sugar beets was superior in B treated plants and it was correlated with enhanced B concentration in reproductive tissues.Boron is an essential micronutrient that significantly affects the seed development and quality33. Some studies have provided the ground basis that exogenously applied PGRs improve the nutrient uptake and accumulation within the plant body35. Mepiquat chloride  is a gibberellic acid (GA) inhibitor36 and is used worldwide to suppress excessive vegetative growth of cotton by decreasing the plant height, internodes length, leaf area and length of fruiting and vegetative branches, and to hasten crop maturity and avoid the yield losses45. Apart from manipulation of plant canopy structure, MC enhances the cotton root growth by increasing number of lateral roots and increases the root vigor through increased reducibility and respiratory rate thereby improving the nutrient uptake and partitioning within the plant body47. Sawan et al.6 and Yang et al.14 noticed an increase in uptake and translocation of N and K to cotton seed in MC treated plants.Exogenous application of plant growth regulators (PGRs) is a well-known strategy to improve the crop physiological performance, nutrient dynamics within plant body and crop yield under normal and stressed conditions in agro-ecosystemEffects of plant density, foliage B nutrition and MC growth regulation on seed nutrients reserves, seed traits and offspring growth performance were studied in two years multi-factor field experiment in agro-ecosystem. The objectives of the study were to (1) quantify the single and interaction effects of plant density, B nutrition and plant growth regulation on degree of variation in cotton seed nutrients reserves, seed mass and production, emergence and offspring plasticity in seedling growth (2) to elucidate relative changes in seed nutrients reserves and seed mass in response to maternal ecological factors and draw possible relationship (3) determine the quantitative relationship between seed mass and cotton seed production (4) examine the seedling emergence, emergence time and growth plasticity of cotton in next generation in relation to seed mass.The lower plant density produced greater individual seed mass of cotton as compared to higher plant density, being 3.5 and 4.6% higher during 2014 and 2015, respectively  and micronutrients  reserves significantly differed between plant densities. Higher seed N (2.1 and 2.2%), P (5.1 and 8.2%), K (9.9 and 8.4%), B (19.9 and 20.5%), Zn (5.4 and 6.7%), Fe 4 and 3.6%) and Mn (8.6 and 7%) contents were produced at lower plant density as compared to higher plant density during 2014 and 2015, respectively ; except B for which the interaction effects of B with MC and B with plant density were significant, during both years while interaction of MC with plant density was significant during 2015 Tables . Seed B Seed nutrients contents  and individual seed mass were positively correlated in response to plant density, foliar B nutrition and MC growth regulation, during both years Table . This poFinal emergence and seedling vigour index were significantly greater from seed produced at lower plant density with concomitant decrease in mean emergence time. Higher final emergence  occurred at lower plant density than higher plant density  of cotton in succeeding generation were significantly greater at lower plant density as compared to higher plant density Table . Boron tThe results of this study have revealed new information that maternal plant density, foliage B nutrition and MC growth regulation influence the offspring performance in terms of emergence and seedling growth plasticity in annual cultivated cotton.48. The root growth is depressed at higher plant density due to competition20 while increased by B49 and MC47 influencing the nutrient dynamics within plants. Moreover, B and MC alters the ability of plants for nutrient uptake and translocation through improved nutrient metabolism and assimilation52. However, decreased seed Mn content by B may be attributed to their antagonistic effect with each other that led to decreased uptake and translocation53. Previous studies have reported similar results that plant and seed nutrients contents decreased at high plant density20, while increased in response to B nutrition27 and MC application14.Individual seed mass of cotton was decreased at higher plant density while increased by foliage B nutrition and MC growth regulation Table . This mi2, competition among plants for available resources4 and/or nutrient enrichment1. Plants often growing in nutrient rich environments produce large sized seeds containing greater nutrient contents54. In this study, the strength of relationship between concentration of nutrients and seed mass  and seed mass Table  manifestss Table  that theStipa krylovii and Artemisia frigida produced heavier seeds in temperate steppe ecosystem. Furthermore, a significant positive relationship existed between seed mass and seed production1.In this study, individual seed mass was linearly related to seed production per plant of cotton Fig. . Therefo55. This indicates that bigger seeds having greater seed nutrients reserves are advantageous pertaining to emergence and emergence timing, and have more time for the seedling establishment. Moreover, lower seedling vigour index of seed from plants at higher plant density and higher seedling vigour index of seed from B and MC treated plants  may be the result of increased mass of seed structures  rather than seed embryo or endosperm58. Moreover, in this study a significant negative relationship between seed mass and mean emergence time . The positive linear relationship between seed nutrients reserves and seed mass indicated that nutrient availability and/or uptake influence the seed phenotypic traits in response to maternal ecological factors. The variation in seed mass ultimately influenced the cotton seed production, offspring emergence and seedling growth plasticity. The results suggest that lower maternal plant density, B nutrition and MC growth regulation results in higher emergence and offspring seedling growth due to higher seed mass and nutrients reserves. This improved emergence and competitive ability of offspring seedlings of cotton through better management practices could be exploited to improve the agro-ecosystem productivity while reducing the input cost .−1), while lower in organic matter . The soil was medium in nutrient status with lower total N , available P , exchangeable K , B , Zn  and Fe  during both years, respectively. The soil of experimental site belongs to Lyallpur soil series , Haplarged in the USDA classification and Haplic Yermosols in the FAO classification. The meteorological data during both experimental seasons are given in Table A 2-years field experiment was conducted at Agronomic Research Area, University of Agriculture, Faisalabad , Pakistan, during 2014 and 2015. Soil samples were collected prior to sowing the crop using auger from a depth of 0–30 cm from different locations of field to determine the physico-chemical properties of soil, during both years. The soil was alkaline-calcareous, B deficient and sandy loam in nature with high pH , calcium carbonate  and electrical conductivity  and higher plant density (88888 plants ha−1)], foliage applied B  and single foliar applications of MC solution with 70 mg/L concentration at two different growth stages viz. squaring and flowering. Control plants received water spray. The B was foliage applied five weeks after sowing using boric acid (17% B); while, MC  was foliar applied using a Knapsack hand sprayer at a pressure of 207 kPa. The spray volume for MC application at squaring was 300 L ha−1 while for B and MC application at flowering stage was 350 L ha−1.This study was accomplished in two phases. In first phase, 2-year field experiment was conducted. The experiment was carried out using randomized complete block design with three-factor factorial arrangement including three replications. Net plot size was 6 m × 3 m. The treatment variables were two plant densities [lower plant density  and treated with fungicide (dynasty CST 125 FS @ 3 g kg−1 seed). The sowing was carried out at beds using dibbler and keeping the inter row spacing of 75 cm while intra row spacing was varied according to the treatments i.e. 25 and 15 cm to maintain plant densities of 53333 and 88888 plants ha−1, respectively. Seed rates of 15 and 25 kg ha−1 was used for attaining plant densities of 53333 and 88888 plants ha−1, respectively and 2–3 seeds were sown per hill. Thinning was carried out 25 DAS to keep the desired plant densities. Fertilization was carried out at the rate of 200-120-110 kg NPK ha−1 using urea (46% N), diammonium phosphate (18% N: 46% P2O5) and sulfate of potash (50% K2O), during both years. All P, K and 1/3rd N was applied at sowing; while, remaining N was applied at squaring and boll formation stages in equal splits. The crop was irrigated eight times during 2014 and five times during 2015 besides irrigation applied at sowing keeping the crop and weather conditions in consideration. During second year, occurrence of heavy rainfall decreased the irrigation requirements  was sown on May 22−1) was determined after ginning. Individual cotton seed mass was determined by taking the mean mass of 100 seeds collected randomly after ginning. The delinted seed samples were assayed to determine the concentration of macro and micronutrients. The delinted seed samples were dried under sun and electric oven at 70 °C for 24 h, ground to powder form  to pass through a 30-mesh screen. The samples were digested using sulfuric acid and catalyst mixture and the N was determined by Kjeldhal distillation method as described by Estefan et al.59. The P was analyzed by ammonium-vanadomolybdate colorimetric method at 410 nm using the same extract59. For determination of K and micronutrients viz. Zn, Fe and Mn the samples were soaked over-nightly in di-acid mixture and digested on block digester. The K was determined by flame photometer using the method of Chapman and Pratt60; while, Zn, Fe and Mn were assayed using the atomic absorption spectrometer according to the method described by Estefan et al.59. Boron was determined by dry ashing the ground seed material in muffle furnace at 550 °C for 6 h60. The ash was taken in 0.36N H2SO4 and the B concentration was determined by spectrophotometer at 420 nm wavelength using azomethine-H colorimetric method61. The contents of seed macronutrients  was expressed as mg g−1 dry weight (DW) and micronutrients  as µg g−1 DW.The crop was harvested in two manual pickings. The seed cotton obtained from all pickings was mixed and ginned using roller type, hand-fed laboratory gin. Cotton seed production  to avoid effects of dormancy and poor seed germination. The bioassay was performed in subsequent year every time after collection of seed i.e. 2015 and 2016 . The experiment was conducted by using completely randomized design with factorial arrangement and three replications. Ten seeds were sown in soil filled pots (25 cm diameter and 20 cm height) following scarification treatment with sulfuric acid (1:10 ratio of seed and acid). At the start 150 mL of deionized water was applied to each pot and then according to the requirement. The experiments were observed for 30 days. The average maximum temperature was 31.1 and 33.0 °C, and minimum temperature was 19.3 and 19.7 °C during first and second year, respectively, during the period of experiment.62. Final emergence percentage was calculated by using following the formula;The seedlings emerged were counted daily up to the end of experiment as per method of Association of Official Seed Analysts63 was used to calculate the mean emergence time;n is the number of emerged seedlings on day D and N is the total number of germinated/emerged seeds.Equation of Ellis and Roberts64:At the end of experiment, seedlings were uprooted and their shoot and root lengths were measured. Shoots and roots were separated and dried in oven at 70 °C till constant weight to measure the dry biomass accumulation. Seedling vigour index (SVI) was calculated using the equation of Abdul-Baki and AndersonRainfall, air temperature and accumulation of heat units were different in 2014 and 2015 from May through November at field experimental site, therefore data of each year was analyzed separately. All the data were analyzed using the Univariate General Linear Models (GLM) procedure of SPSS 19. The plant density, B and MC were entered as fixed factors while replication (block) was entered as random factor. The treatments’ means were compared by using the Tukey’s honest significance difference (HSD) test at 5% probability. Pearson’s coefficients were calculated to determine the correlation between seed mass and seed nutrients contents  to test the effect of plant density, B nutrition and MC growth regulation on seed nutrients and seed mass. Linear regression analyses were performed to determine the quantitative relationship of individual seed mass with seed production, final emergence, mean emergence time and seedling growth traits.


No correlation was found between MUIC (median urinary iodine concentration) and the incidence rate of CH. Linear regression analysis showed a 0.1% increase in the recall rate for a one unit decrease in MUIC . MUIC, at a cut-off point of 144.7 µg/L, was predictive for a recall rate < 3% (p = 0.05). Conclusion: Frequencies of TSH ≥ 5 mU/L may be a more sensitive indicator for iodine status during pregnancy rather than in the general population. As higher recall rates reflect inadequate iodine nutrition, sufficient iodine supplementation is needed to reduce the recall rate in such communities.Back ground: Iodine deficiency is one of the important factors in increasing the recall rate in congenital hypothyroidism (CH) screening programs. The present study assessed whether the iodine status of the general population may predict the recall rate or vice versa. Methods: In the current national study, among 1,382,229 live births delivered between March 2010 and March 2011, 1,288,237 neonates were screened for detecting CH by TSH (thyroid stimulating hormone) measurement via heel prick sampling. Simultaneously, a total of 11,280 school-aged children, aged 7–8 years, were selected using random multi-cluster sampling for measurement of urinary iodine. Results: A negative correlation was found between median urinary iodine (MUI) and the recall rate ( Iodine is an essential micronutrient for thyroid hormone synthesis . AdequatIodine deficiency is one of the important factors in the increasing recall rate (the percentage of screening tests with elevated TSH) and the incidence of transient congenital hypothyroidism and infants born in iodine depleted areas tend to have higher thyroid stimulating hormone (TSH) concentrations as an adaptive mechanism to maintain serum thyroxin (T4) within normal or low normal ranges (128.7 to 205.9 nmol/L) ,7. BasedData shows that in the past two decades, the prevalence of congenital hypothyroidism (CH) has been increasing worldwide ,12, beinConsidering the high prevalence of congenital hypothyroidism and the recall rate in Iran, and also considering reports of iodine deficiency in pregnant women, this study assessed the association of iodine status based on the median urinary iodine and recall rate and the CH incidence in all provinces in Iran based on the results of two national studies conducted in 2011.In Iran, medical education, research, and health services are all integrated and supervised by the Ministry of Health and Medical Education and health care in all provinces is under coverage of the universities of medical sciences in each province. All 46 universities from 31 provinces were included in this study. Among 1,382,229 live births delivered between March 2010 and March 2011, 1,288,237 neonates were screened for detecting CH. Screening was performed by TSH measurement on a filter paper blood spot by heel prick sampling, obtained 3–5 days after birth. Neonates with a Guthrie TSH test ≥ 5 mU/L, were recalled for confirmatory tests (with venous blood samples) and the final diagnosis was made by a pediatric endocrinologist, appointed as the focal point of the CH program in each university region. Neonates with TSH > 10 mU/L and T4 < 6.5 µg/dL in venous samples were diagnosed as having hypothyroidism  and promSimultaneously (2010–2011), data on population iodine status were extracted from the national monitoring program for elimination of iodine deficiency under the supervision of the Nutrition Office, Ministry of Health and Medical Education. A total of 11,280 school-aged children (240 from each region) aged 7–8 years were selected from different schools in each region using random multi-cluster sampling . BrieflySpot urine samples (10 mL) were collected between 08.00 and 11.00 am, while children attended school and examined in the reference laboratory in each province for iodine measurement, based on the WHO protocol. Since morning fasting urine generally tends to underestimate the iodine status, non-fasting spot urine samples can be taken randomly at any time during the day . UrinaryThe ethics committee of the Research Institute for Endocrine Sciences of Shahid Beheshti University of Medical Sciences approved the protocol for this study (ethic code: IR.SBMU.ENDORINE.REC.1395.341) and written informed consent was obtained from the parents. The recall rate is the percentage of screening tests with elevated TSH (≥5 mU/L), based on which the physician notifies the authorities to contact the parents in order to arrange another test ,27. Accop = 0.304). In order to investigate the association between MUIC and indices of congenital hypothyroidism, assuming normality, a Pearson product-moment correlation was run between MUIC, recall rate, and incidence rate. All statistical assumptions were confirmed using scatter plots. An independent t-test was also used to determine whether there was any statistical difference in means of MUIC in provinces with recall rates > 3%, in comparison to those with recall rates < 3%. Assumption of homogeneity was confirmed using Levene’s Test (p = 0.163). The receiver operating characteristic (ROC) curve was calculated in order to find the optimal threshold (best cut-off point) of MUIC in prediction of recall rate. p values < 0.05 were considered significant. All statistical analyses were performed using SPSS 15.0 statistical software package .Median urinary iodine concentration, recall rate, and incidence rate of congenital hypothyroidism in each province were tested for normality using Shapiro-Wilk tests . MUIC was correlated with TSH screening values of 5–9.9  and ≥10 mU/L . Also, MUIC was significantly lower in provinces having recall rates ≥ 3% compared to those with recall rates <3% . There was a high correlation between recall rate and incidence rate of CH ; however, the nonlinear quadratic correlation showed a significant improvement in R2 with a coefficient of −0.48 for X and +0.01 for X2. There was no correlation between MUIC and incidence rate of congenital hypothyroidism. A negative linear correlation was found between median urinary iodine levels and recall rate (p = 0.05). Our results indicate that 11 out of 46 regions (23.9%) had a recall rate ≥ 3%, while, MUIC < 100 µg/L was observed in 3 out of 46 regions (6%). Linear regression analysis also showed a 0.1% increase in recall rate for each unit decrease in MUIC .Recall rate was higher in regions with MUIC <100 µg/L compared to regions with MUIC ≥ 100 µg/L, .As shown in To the best of knowledge, this national study is one of the first to investigate whether iodine status in a general population , in an area with iodine sufficiency, can be correlated to the recall rates and incidence rates of congenital hypothyroidism in newborns or vice versa. Findings of the current study indicate a weak negative association between median urinary iodine concentrations in school-aged children and the recall rate in the newborn screening program for CH, demonstrating that higher recall rates reflect inadequate iodine status; however, no association was found between urinary iodine levels and incidence of CH. The distribution of neonatal TSH concentration, along with median urinary iodine of school-aged children, are reported to be sensitive indicators of population iodine status ,7,27. MeBased on previous reports, in Iran the rate of recall and incidence of CH are higher than those of other countries . High reIn addition, in our iodine-sufficient area, no association was observed between MUIC and incidence of CH, which may be due to the difference between MUIC in school-age children and MUIC in pregnant women (not measured). Other factors which affect the incidence of CH should also be considered, e.g., maternal TSH receptor-blocking autoantibodies, exposure to excess iodine, use of goitrogenic agents or antithyroid drugs during the perinatal period, very low birth weight, and prematurity and genetic factors, not just inadequate iodine status per se ,40.The main strengths of this study are assessment of the impact of iodine status at population level on the recall rate and incidence of congenital hypothyroidism, for the first time in an area of iodine sufficiency. The results derived from two national surveys facilitated data analysis based on population indicators and not individual data. The limitations, however, include small sample size (areas), especially a few areas with MUIC < 100 µg/L and using a single urine sample due to significant day to day variations in salt intake (the main source of dietary iodine in Iran), and also variations in hydration in individuals ,42; on tIn conclusion, although both MUIC and the neonatal TSH distribution have been introduced as indicators of the iodine nutritional status of population; moderate association and discrepancies between neonatal TSH levels and MUICs in school-aged children were found in our study, suggesting that the percentage of TSH > 5 mU/L may be a more sensitive indicator for maternal iodine status during pregnancy than iodine status of the general population, specifically due to evidence of iodine deficiency among pregnant Iranian women; hence, prescribing prenatal supplements containing 150 µg iodine is warranted to reduce the recall rate in screening program for congenital hypothyroidism in Iran. More community-based studies with larger sample sizes need to be conducted to define more sensitive cut-points for MUIC of the population to precisely predict recall rate for the congenital hypothyroidism screening program.


Intraventricular hydrodynamics plays an important role in evaluating cardiac function. Relationship between diastolic vortex and left ventricular (LV) filling is still rarely elucidated. The aim of this study was to evaluate the evolution of vortex during diastole in hyperthyroidism (HT) and explore the alteration of hydromechanics characteristics with sensitive indexes.P), in different diastolic phases  were calculated and analyzed.Forty-three patients diagnosed with HT were classified into 2 groups according to whether myocardial damage existed: simple hyperthyroid group  and thyrotoxic cardiomyopathy . Twenty-seven age- and gender-matched healthy volunteers were enrolled as the control group. Offline vector flow mapping (VFM model) was used to analyze the LV diastolic blood flow patterns and fluid dynamics. Hemodynamic parameters, vortex area (A), circulation (C), and intraventricular pressure gradient (ΔP < .05). Compared with the control group, the vortex size and strength, intraventricular pressure gradient during early and mid-diastole were higher in HT1 and lower in HT2 (P < .05). And in late diastole, the vortex size and strength, intraventricular pressure gradient of HT2 became higher than those of the control group (P < .05). Good correlation could be found between CE and E/A (P < .05), CM and ΔPM (P < .01), CL and FT3 (P < .05).HT2, with a lower E/A ratio and left ventricular ejection fraction (LVEF), had a larger left atrium diameter (LAD) compared with those of the control group and HT1 (VFM is proven practical for detecting the relationship between the changes of left ventricular diastolic vortex and the abnormal left ventricular filling. Intraventricular vortex formed by the morphology and function of the cardiovascular system can reveal the global diastolic function of LV and the exceptional adaptability of cardiovascular system for maintaining the blood circulation relatively constant under a wide range of workload.Previous studies on left ventricle (LV) filling dynamics focused on single-point flow velocity measurements by Doppler echocardiography. However, the single-point velocity measurements cannot accurately evaluate the global diastolic function of LV because the intraventricular velocity profile is complex.,5 CMR displays 3-dimensional (3D) chamber flow fields in a variety of visualization methods via electrocardiogram (ECG) and respiratory gating, but it has not yet been routinely used in clinical practice due to long duration of the test, a rather high cost, and a low temporal resolution. Compared with CMR, CE-PIV has superior temporal resolution. It uses moving tracks of ultrasound microbubbles to represent the distribution of the blood flow field, thereby providing a more accurate calculation of the flow velocity. However, the test requires contrast media, and the range of velocities that can be measured is limited. VFM visualizes cardiovascular blood flow in vectors based on color Doppler superimposed on speckle tracking—a method for tracking cardiac wall motion. Improvements have been made in new VFM to allow for more accurate and stable calculation of hemodynamic parameters and flow velocity near the wall. It is considered to be one of the representative methods for visualization of blood flow in vivo that can compete with CMR and CE-PIV.,9Computational fluid dynamics (CFD) and particle image velocimetry (PIV) have been applied in some new technologies for the assessment of cardiac function that have expanded our options to visualize and analyze the complex intracardiac flow. Among them, cardiac magnetic resonance (CMR), CE-PIV technique combined contrast echocardiography (CE) and PIV, vector flow mapping (VFM) based on color Doppler flow imaging (CDFI) and speckle tracking are the principal methods that are currently used in the assessment of patients in the very early stage of cardiovascular disease or for investigating complex geometry after cardiac surgery. The evolution of the LV diastolic vortex in HT at different stages might change with the volume load, cardiac structure, and function. This study attempted to detect the LV hemodynamics in HT using VFM and investigate the relationship between the diastolic vortex field and abnormal LV filling.Excessive secretion of thyroid hormones in patients with hyperthyroidism (HT) produces cardiotoxicity.22.1Sixty patients diagnosed with HT between March and December 2014 in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China were randomly recruited in this study. Nine patients were excluded due to poor image quality, and 8 cases were excluded due to cardiac arrhythmia. Forty-three patients were involved in the study finally. Of them, 22 (8 men and 14 women) who were referred to the cardiologist and identified with dilated cardiomyopathy were assigned to thyrotoxic cardiomyopathy group (HT2), aged between 21 and 63 years (mean 42.00 ± 13.01 years). The remaining 21 patients (9 men and 12 women) were assigned to simple HT group (HT1), aged between 18 and 56 years (mean 35.45 ± 10.34 years). In addition, 27 gender- and age-matched healthy volunteers (15 men and 12 women) during the same period in our hospital were enrolled as the control group, aged between 20 and 56 years (mean 40.62 ± 13.08 years). presence of classical clinical symptoms and physical signs of HT; laboratory criteria: thyroid stimulating hormone (TSH) < 0.5 μU/mL , free Triiodothyronine (FT3) > 9.15 pmol/L , and/or free Thyroxine (FT4) > 25.47 pmol/L ; exception of other organic heart diseases and symptom of thyrotoxic cardiomyopathy. Thyrocardiac disease is defined as the presence of congestive heart failure, angina pectoris, or both, in a patient with hyperthyroidism. The study was approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology. All study subjects provided written informed consent.Diagnostic criteria for HT2.2Transthoracic echocardiography was performed on all subjects using Aloka Prosound F75 scanner equipped with a frequency 2 to 4 MHz transducer (UST5415). ECG was simultaneously recorded to determine the cardiac phase. Left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), transmitral peak E and A velocities  were measured at the parasternal LV long-axis view. LV ejection fraction (LVEF) was calculated by the modified biplane Simpson method. Standard 2D color flow Doppler dynamic images in 3 consecutive beats were recorded from the apical long-axis view. The scanning width, imaging depth, and spatial temporal settings were optimized to achieve the highest possible frame rate. Doppler velocity scale varied from 50 to 60 cm/s in this study, and the frame rate was set at 25 frames per second for all subjects. All recordings were stored digitally and analyzed offline.All the stored raw data were processed on offline VFM workstation . As the boundary of flow in LV was outlined, the system automatically traced the flow signals and calculated the velocity vector. Diastole was determined based on synchronous ECG (from the end of the T-wave to the onset of the Q-wave). Early diastole (rapid filling), mid diastole (slow filling), and late diastole  were defined depending on the flow rate-time graph of mitral valve (MV) and behavior of valve.V) (ΔV = velocity of MV − velocity of cardiac apex). Then intraventricular pressure gradients in different filling phases  can be calculated according to the formula ΔP = 4 × ΔV2.A sampling line from the endocardium of cardiac apex to MV was drawn under the guidance of streamline to measure the intraventricular velocity gradient , only the one near the anterior leaflet was analyzed.P and C for both filling phases were analyzed.Observer repeated the same measurements in randomly selected 12 cases after 1 week, and another independent observer repeatedly performed measurements on these 12 subjects. Then, intra- and interobserver reproducibility of Δ2.3P < .05 was considered statistically significant.SPSS Statistic software  was used to perform statistical analysis in the study. Continuous data were expressed as mean ± SD (x ± s). Clinical characteristics and hemodynamic variables were compared using one-way analysis of variance (ANOVA) followed by Dunnett contrasts against the control group, when appropriate. Relationships between vortex variables and clinical, echocardiographic characteristics were evaluated by Pearson linear regression analysis. Intra- and interobserver variability values were analyzed by Bland–Altman bias plots. The mean and intraobserver and interobserver differences were plotted against the mean of 2 measurements, with 95% of the data points recommended to lie within ± 2SDs of the mean difference. 33.1P > .05). HR, SBP, FT3, and FT4 were higher, and TSH was lower in patients compared with those of the controls (P < .05). Patients in HT1 had the shorter course and faster HR than those in HT2. Compared with controls and HT1, HT2 showed significant increases in LAD, LVEDD, and A peak, along with E peak, E/A ratio, and LVEF reduced (P < .05). LVEF in HT1 was higher than that of controls, and there were no significant differences in LAD, LVEDD, E peak, A peak, and E/A ratio between them.The clinical data and hemodynamic variables are described in Table 3.2PE, ΔPM, and ΔPL). In addition, intraventricular pressure gradients during early and mid-diastole (ΔPE and ΔPM) in HT2 were lower than those in controls and became notably higher than controls at the end of diastole (ΔPL) (P < .05).Table 3.3In the control group, a strong inflow jet directed almost straight to the apex with a minor zigzag pattern and 2 asymmetric vortices appeared behind the MV leaflets after MV opening during early diastole. The one near the anterior side was larger. These 2 vortices continued their development during the period of rapid filling, reaching their full development when the E wave approached its peak (E peak). During slow filling, E wave deceleration occurred, and pressure across the MV decreased. Direction of the LV blood flow changed from apex to LV outflow, and the streamlines of inflow blood became rotated turbulently. These 2 vortices evolved into a single large one that was loosely concentrated, progressing toward the apex with the vortex area and circulation continuously growing. Followed by a late filling of atria contraction, the early filling vortex was weakened by the A wave onset, causing the late filling jet (A peak) to destroy the relatively apically located large vortex, creating another 2 symmetric and smaller vortices close to the mitral tip Fig. .P < .01)  from early to mid-diastole, but became significantly higher than controls during late filling , and negatively correlated with DBP . CM was closely correlated with ΔPM . CL was positively correlated with FT3 and FT4 . There were positive correlations between ΔPE and E peak , ΔPM and E/A ratio , ΔPL and A peak .Relationships between vortex variables and clinical, echocardiographic characteristics evaluated by Spearman linear regression analyses are shown below. C3.5Results of inter- and intraobserver analysis for the assessment of diastolic intraventricular pressure gradients and circulation are presented in Table 3.6 With long-lasting excessive thyroid hormone, functional and morphological abnormalities occur in the heart, and compensatory cardiac hypertrophy exists which ultimately leads to heart failure. Congestive heart failure is the well-recognized complication of HT. Research showed that diastolic dysfunction appeared earlier in patients with hyperthyroid heart failure than those with hypothyroid heart failure. However, pathologic studies of HT generally believed that thyroid hormone enhances LV diastolic filling.,18 Indices of conventional echocardiography are vulnerable to the geometry of the heart, HR, and load. Thus, they cannot objectively reflect the changes in diastolic function. Hydromechanics research demonstrates that intraventricular vortex reflects the LV functional status. Study about the relationship between LV filling and diastolic vortex in patients with HT from the perspective of fluid mechanics is rare. In this paper, we attempted to observe the characteristics of diastolic vortex in patients with HT using VFM to objectively reflect the dynamic changes in LV diastolic function.HT is a common metabolic disorder with prominent cardiovascular manifestations. The direct toxic effect of thyroid hormone and indirect action via catecholamine on the cardiovascular system can lead to a typical hyper-dynamic circulatory state which manifested as accelerated HR, increased blood volume, increased stroke volume (SV), increased LVEF, and high cardiac output. These changes were mainly due to the excess thyroid hormones that directly or indirectly affected the heart via sympathetic nerve and improved the sensitivity of myocardial β receptors to catecholamine, which resulted in cardiac sympathetic nerve hyperactivity. Therefore, the heart was in a hyperdynamic circulation state with high cardiac output, accelerated HR, and increased contraction function.This study showed that patients in HT1 experienced accelerated HR and increased LVEF, which were consistent with the previous studies. Steatosis and hypertrophy existed, LV geometric configuration and function changed, which manifested as cardiac enlargement, reduced cardiac systolic and diastolic function.In this study, patients with thyrotoxic cardiomyopathy had significantly longer courses than simple HT. LVEDD and LAD were higher, LVEF and E/A ratios were lower in HT2 than those in controls and HT1. These were due to the progression of the HT course. Excess thyroid hormones enhanced the calcium and glucose absorption of cardiomyocytes, accelerated metabolism, and increased the proportion of myocardial oxygen consumption. When overloading exceeded the maximum steady state, a relative myocardial ischemia and myocardial damage occurred.,18 Then the larger, stronger vortex was generated in cardiac chambers. Enhanced diastolic vortex in LV can increase positive pressure and assist filling by eliminating convective deceleration load. And the higher intraventricular pressure gradient in patients with simple HT is helpful to pull blood from left atrium into LV more efficiently for adapting the increased circulating blood volume. Martínez-Legazpi et al found that diastolic vortex can assist LV filling without energy consumption and negative impact on intraventricular diastolic pressure distributions. Such vortex formation may be beneficial in efficient MV closure, efficient diastolic filling, minimizing kinetic energy loss, preventing thrombus formation, and maintaining normal diastolic function.,25 The difference in E/A ratio between HT1 and controls had no statistical significance, suggesting that diastolic vortex might be more sensitive in evaluating LV filling capacity. Mintz et al found the enhanced left ventricle diastolic function appeared in a group of newly diagnosed and untreated HT patients, and believed that it has something to do with the early state of thyrotoxic cardiac damage. Additionally, vortices dissipate flow energy by viscous  effects, the dissipated loss increased when the vortical change speed accelerated (heart rate elevated). Therefore, there is a cascade of energy dissipated as heat in patients with HT, under the state of hyperdynamic circulation, which provide a favorable milieu for the development of heart failure.Analysis of flow field parameters showed that the vortex size and strength in these 3 diastolic phases, intraventricular pressure gradient in early and mid diastole of HT1 were higher compared with the control group and HT2, which might be associated with the improved LV filling capacity and the hyper-dynamic circulatory state. Thyroid hormones’ indirect action on the cardiovascular system accelerates HR, reduces peripheral vascular resistance, and enhances cardiac output. In addition, the direct effect of thyroid hormones on heart enhances myocardial relaxation by upregulating expression of the sarcoplasmic reticulum calcium-activated ATPase (SERCA) and downregulating expression of phospholamban. believed that ventricular volume load increasing could only be manifested as slight reduction in early relaxation and significant increase in late compliance, which corresponded to the results of diastolic function and vortex field in patients with thyrotoxic cardiomyopathy. In addition, the intra- and inter-observer reproducibility of VFM parameters was excellent. VFM could be used for accurate and visual evaluation of LV diastolic function and vortex field distribution in patients with HT.The vortex size and strength, intraventricular pressure gradient in HT2 were lower compared with controls in early and mid diastole and then became higher than those of controls in late diastole. We speculated that the change of hydrodynamics might be linked to diastole dysfunction. The lasting stimulating of thyroid hormones on cardiomyocytes in patients with thyrotoxic cardiomyopathy resulted in myocardial necrosis and myocardial interstitial fibrosis, which weakened LV relaxation in early diastole. LV diastole results from the simultaneous and complementary action of relaxation and compliance respectively generated in early and late filling. The vortex generated in early diastole was too weak to ensure complete intraventricular filling. The compliance and vortex in late diastole then showed a compensatory increase during late diastole. Moreover, accelerated heart rate leads to a relative increase in the atrial contribution to left ventricular filling due to a shortened diastolic filling time. Decreased LV filling volume and increased filling pressure during early diastole may cause the syndrome of congestion in pulmonary and systemic circulation. At this time, atria systole was enhanced in patients with thyrotoxic cardiomyopathy for removing the residual blood volume in atrium and compensating the increased circulating volume, which significantly enlarged and strengthens the late diastolic vortex in LV chamber. PasipoularideE was positively correlated with E values and E/A ratio, indicating that the vortex in LV was related to diastolic function during early diastole. On the other hand, the abnormal vortex during active filling suggested that the diastolic dysfunction exists. CM was positively correlated with ΔPM, and ΔPM was positively correlated with E/A ratio, showing the close correlation between vortex and intracardiac positive pressure during filling. By increasing positive pressure gradient and reducing convective deceleration load, vortices contribute to ventricular filling. As LV was a relatively closed chamber when MV closed during mid-diastole, ventricular pressure was not subject to left atria pressure and only associated with intraventricular vortex. CL was positively correlated with FT3 and FT4, which indicated that the ventricular vortex was closely bound up with the level of thyroid hormones during left atria systole in a certain range. The excess thyroid hormones effected the relaxation of LV, which cause the left atrial pressure and vortex during late diastole rising to compensate for the rising volume load.Correlation analysis showed that CThere were certain limitations in our study. First, manual correction for aliasing was performed in the present study, as double or higher aliasing cannot be corrected by VFM, which may underestimate the velocity. Second, color flow frame rate is slightly low. Thus, there might be deviation in the analysis of diastolic phases. Third, we failed to obtain the left atrium-ventricle pressure difference by cardiac catheterization. Therefore, the relationship between the diastolic vortex and the atrium-ventricle pressure difference could not be analyzed directly. More efforts need to be exerted in the future studies. And finally, the vortex is a 3D entity, and fluid evaluation in 3D would be desirable to ensure accurate determination of the evolution of vortex. At present, however, VFM has not been converted to 3D.In summary, our study showed that LV flow field can give insights into abnormal diastolic filling in HT. For patients with simple HT, the improved LV diastolic function and high-performance vortex contribute to accommodating the hyper-dynamic circulatory state. In patients with obvious thyrotoxic cardiac damage, the reduced vortex during early and mid-diastole is partially related to relaxation dysfunction, and the increased vortex during late diastole is about the compensatory raising of volume load.


The results of recent trials have brought some confusion to the treatment strategy for renal artery stenosis (RAS). To evaluate the applicability of percutaneous transluminal renal angioplasty (PTRA) for RAS, we extracted the factors that may affect the effectiveness of PTRA from cases experienced at a hypertension center.p < 0.05), but the estimated glomerular filtration rate (eGFR) did not change significantly. There were no factors associated with the BP lowering effects of PTRA. The baseline resistive index (RI) was negatively correlated with the change in eGFR (p < 0.05). After correction for age, sex, BMI, and the dose of contrast medium, the association of RI with change in eGFR remained significant. We retrospectively assessed the blood pressure (BP) lowering effects and renoprotective effects in 50 consecutive patients that had hemodynamically significant RAS and had undergone PTRA and stenting during 2001–2005. Subjects were diagnosed with atherosclerotic RAS (42), fibromuscular dysplasia (6), or Takayasu disease (2). After PTRA, BP significantly lowered from 152.3/80.3 mmHg to 132.6/73.2 mmHg ( In cases with hemodynamically significant RAS, PTRA lowered BP but was not effective in improving renal function. Higher baseline RI may be a factor for predicting poor clinical course of renal function after PTRA. Renal artery stenosis (RAS) is a cause of renovascular hypertension (RVHT) and ischemic nephropathy and has been demonstrated to be a predictor of future cardiovascular events . PercutaTwo large-scale randomized controlled clinical trials (RCTs), ASTRAL  trial , and CORThe current guidelines for atherosclerotic RAS recommend PTRA for patients with hemodynamically significant RAS or with the following conditions: flash pulmonary edema, rapidly declining renal function or refractory hypertension –15. SuchAfter ASTRAL and CORAL, physicians, such as cardiologists or radiologists, who handle RAS tend to hesitate to perform PTRA. However, recent recommendations and review articles have demonstrated the importance of PTRA , 17. TheThere are few reports on the effectiveness of PTRA, and there are even fewer reports of the Japanese or Asian patients. The aim of this study was to clarify factors associated with outcomes in PTRA for RAS patients with hemodynamically significant stenosis, before ASTRAL and CORAL, to reconsider the effectiveness of PTRA for RAS patients.We retrospectively assessed the influences of several factors on clinical courses of patients who had undergone PTRA. The subjects consisted of 50 consecutive patients who had their first PTRA between January 2001 and September 2005 in the National Cardiovascular Research Center in Osaka, Japan. Patients who had undergone two or more rounds of PTRA for restenosis of the treated lesion or progression of opposite-side RAS were excluded. PTRA was indicated for hemodynamically significant RAS with either of the following: (1) peak systolic velocity ≥ 1.8 m/sec by RDU, (2) diameter or area stenosis rate ≥ 75% by magnetic resonance imaging angiography, and (3) prolonged vascular, functional, and distribution phases by renogram. Cases without viability of the affected kidneys  were excluded. Renal artery stenting was done in all cases at the ostial and proximal lesion without distal protection or filtration device. The clinical courses after PTRA in our institute were good overall. Major complications, such as cholesterol crystal embolism, dissection of the renal artery, or other vascular disease, were not observed in the present study subjects.BP was measured on the days before PTRA (w0) and 1-2 weeks after PTRA (w1). Serum creatinine levels (S-Cre: enzymatic method) were obtained at the day before PTRA (W0), 1-2 weeks after (w1), and 1 year (+/−4 weeks) after PTRA (y1).Primarily, we compared the change in mean blood pressure (MBP), use of antihypertensive drugs, and estimated glomerular filtration rate (eGFR) between before and after PTRA.MBP was calculated as follows:Successful BP reduction was defined as a 5 mmHg or more reduction in w1 mean BP compared with the measurement at w0 or reduction in the dosage of one or more antihypertensive medicines. Course of renal function (eGFR) was stratified into “not changed or improved” (w0 ≥ y1) or “worsened” (w0 < y1).2), and the dose of contrast medium by univariate and multivariate analysis. We adapted common log conversion of eGFR and PRA for analysis because they did not follow a standard normal distribution.Additionally, we assessed influences on the BP lowering and renoprotective effects of PTRA by several confounding factors, including age, sex, body mass index (BMI), presence of bilateral stenosis, RI in the affected kidney, impaired glucose tolerance (IGT), DM , dyslipidemia, RI: (1 – (end diastolic velocity/peak systolic velocity)) , plasma t-test. Between the groups of the BP lowering effects, categorical variables were compared by χ2 analysis, and continuous variables were compared by unpaired t-test. The influence of each parameter on the regression of eGFR was analyzed by unpaired t-test or linear regression analysis. p values < 0.05 were considered as statistical significant.All data were analyzed using JMP 9 software . All continuous values were expressed as the mean +/− SD and categorical variables were reported as percentages. BP and eGFR were examined. The time courses of BP and eGFR were examined using the paired 2, and other characteristics and subgroup characteristics divided by causes of RAS are shown in The characteristics of the 50 patients  were as follows. All patients were East Asians. Flash pulmonary edema or rapidly declining renal function was not observed on or before admission. Refractory hypertension and uncontrolled BP (SBP ≥ 140 and/or DBP ≥ 90) despite taking 3 or more antihypertensive drugs, including diuretics, were seen in 8 cases (1 aortitis case and 7 atherosclerotic stenosis cases) among 47 cases . Causes of RAS were atherosclerosis (42 cases), FMD (6 cases), and Takayasu aortitis (2 cases), as shown in p < 0.05, n = 47) at the period of 1-2 weeks after PTRA, but the reduction did not last until 1 year after PTRA (1.95p = 0.39n = 42). Mean BP reduction of 5 mmHg or more was observed in 29 of 44 patients (66%), and reduced dose of antihypertensive drugs was observed in 23 of 50 (46%). Successful BP reduction was observed in 35 of 48 patients (73%). Limited to cases of atherosclerotic RAS, the reduction in BP remained significant . There was a significant correlation between presence of “IGT or DM” and baseline RI . We performed multivariate analysis for regression of renal function accounting for age, sex, BMI, baseline RI, and the dose of contrast medium. Baseline RI remained significant for the regression of renal function  at y1. Univariate analysis of confounding factors for regression of renal function is shown in function . In the In the present study, BP was improved in approximately 70% of patients, but the average eGFR remained unchanged from the baseline. We also demonstrated that patients with a higher RI were associated with a poor renal course, as reported by Radermacher et al. . Higher We also demonstrated that patients with glucose intolerance tended to have worsened renal function. Hyperglycemia causes increased glomerular vascular resistance . RI is rAlthough prognosis of RAS is known to be different between atherosclerosis and FMD , RI demoOn the other hand, 12 subjects in the present study lacked RI measurements. The main reasons why the data were unavailable were undetectable blood flow signals in the kidneys due to severe renal damage or marked stenosis of the renal arteries. There were no significant differences in eGFR values between the subjects with available RI measurements and the subjects for which we could not obtain the RI value. Prognostic factors among such patients remain unclear in the present study.Indication for PTRA in patients with RAS continues to be still controversial because the results of ASTRAL and CORAL failed to demonstrate beneficial effects of PTRA. It is possible that patients who may have benefited from PTRA were excluded and/or patients with mild stenosis were included in these studies.Although our subjects, who were expected to have better outcomes than the subjects of ASTRAL or CORAL, demonstrated hemodynamically significant RAS, overall regression of renal function was not observed. In the results of ASTRAL, the decline in renal function over time was slightly slower in the revascularization group, but the difference was not significant after 34 months of follow-up , 13. RenIt has been reported that approximately 20–45% of patients with atherosclerotic RAS are affected in the bilateral or single-functioning kidney , 23, 24.The limitations of our study are that it was a retrospective and single center design, with a limited number of subjects, which could not detect the difference in clinical course between causes of the RAS, and lack of control groups. Although we applied PTRA for patients with hemodynamically significant RAS, our criteria for applicability of PTRA were not strict, and therapeutic methods were at each doctor‘s discretion in each clinical situation. Furthermore, although the smoking habits of patients seem to be one of the determining factors of clinical outcome , we wereIn cases with hemodynamically significant RAS, PTRA was able to lower BP but was not effective in improving renal function.Higher baseline RI demonstrated a significant correlation with poor renal outcome after PTRA. The presence of IGT or DM may be associated with poor renal outcome. Caution should be taken when PTRA for RVHT is considered in such patients.