Organic farming avoids the use of synthetic fertilizers and promises food production with minimal environmental impact, however this farming practice does not often result in the same productivity as conventional farming. In recent years, biochar has received increasing attention as an agricultural amendment and by coating it with minerals to form biochar–mineral complex (BMC) carbon retention and nutrient availability can be improved. However, little is known about the potential of BMC in improving organic farming. We therefore investigated here how soil, bacterial and plant properties respond to a combined treatment of BMC and an organic fertilizer, i.e., a compost based on poultry manure. In a pakchoi pot trial, BMC and compost showed synergistic effects on soil properties, and specifically by increasing nitrate content. Soil nitrate has been previously observed to increase leaf size and we correspondingly saw an increase in the surface area of pakchoi leaves under the combined treatment of BMC and composted chicken manure. The increase in soil nitrate was also correlated with an enrichment of bacterial nitrifiers due to BMC. Additionally, we observed that the bacteria present in the compost treatment had a high turnover, which likely facilitated organic matter degradation and a reduction of potential pathogens derived from the manure. Overall our results demonstrate that a combination of BMC and compost can stimulate microbial process in organic farming that result in better vegetable production and improved soil properties for sustainable farming. In order to balance conservation of natural resources and food production, contemporary agriculture and horticulture rely heavily on soil amendments to ensure and improve soil quality and plant yields  to alter the bacterial communities in contemporary soils when compared to traditional fertilization , we hypothesized here that BMC can be used to alter nutrient conversion and availability from compost and that this can lead to better plant growth. To test this hypothesis we analyzed plant yields, soil properties, and bacterial communities under treatments of either BMC or compost alone or in their combination as well as in comparison to an unamended control treatment. As a study system, we chose pakchoi, a major vegetable grown in China in intensive horticultural settings.The soil used in this experiment was collected at an experimental station of Shanghai Jiao Tong University located in Chongming Island, Shanghai, China . This collection site had vegetable farming from 2008 to 2011. In 2011 the soil was amended with compost with no further activity until the sampling for the experiment. Bulk soil (0–20 cm depth) was collected in July 2014 after removing the top vegetation, which was mainly grass. The soil is characterized as light loam. The soil was sieved through 5 mm mesh and homogenized in the laboratory.Fresh chicken manure provided by a suburban poultry farm was composted for 30 days and used as compost in this study. Biochar was produced from jarrah wood  in a vertical retort with a residence time of ~12 h and a maximum temperature of 600°C. This biochar was activated with phosphoric acid and mixed with high iron bearing kaolinitic clay , chicken manure , rock phosphate, basalt dust illmenite, and dolomite. This mixture was torrefied at 220°C for 3 h to produce BMC. After pyrolysis the BMC was stored aseptically in ziplock bags. The basic physicochemical properties of soil, compost, and BMC are shown in Table −1), (3) 1.9% (w/w) compost , and (4) 0.1% (w/w) BMC and 1.9% (w/w) compost (treatment BMCO). Soil and amendments for each treatment were homogenized on a sterile working bench and three random samples were taken from each mixture for microbial community analysis at day 1. Treatment mixtures were then divided into 15 replicate pots .A pot trial was carried out with a full factorial design including the following four treatments: (1) no additions (CK), (2) 0.1% (w/w) BMC  named “Huawang” were sown into each pot. The pots were then randomly placed in an array  within a climate-controlled chamber. The chamber was configured to a day temperature of 20°C, a night temperature of 18°C and a photoperiod of 14 h with a light intensity of 300 μmol m−2 s−1 photosynthetically active radiation. Irrigation was applied twice per week. To achieve a realistic planting density, only six uniform seedlings were kept in each pot after seedlings reached the stage of one leaf and one bud (about 6 days). No additional fertilizer was added during the cultivation.Twenty aseptic seeds of the commercial variety of pakchoi  and the resulting image was processed using the WinRhizo software  to determine the leaf length and surface area. Fresh pakchoi was used for chlorophyll, soluble protein, soluble saccharides, and nitrate measurement with standard methods  and the end (Day 40) of the cultivation period were subjected to total DNA extraction and sequencing of the 16S rRNA gene. Total DNA was extracted from 0.5 g sieved soil using the PowerSoil DNA Isolation kit  according to the manufacturer's instruction. DNA quality and concentrations were determined using a Nanodrop 1000 (Thermo Fisher Scientific Inc. USA). The V1–V3 regions of the 16S rRNA gene were amplified using barcoded primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 519R (5′-GWATTACCGCGGCKGCTG-3′) that target conserved sequences found in bacteria. Amplicons from each PCR sample were normalized to equimolar amounts and sequenced on a MiSeq platform  at the Ramaciotti Centre for Genomics . The sequencing data has been deposited at the National Centre for Biotechnology Information under BioProject accession no. 16S rRNA gene sequencing data was processed using the Mothur pipeline , compost (CO), and BMC in the following analysis. As plants grew in small pots, any temporal variation seen in the soil could be due to time and/or the introduction of the root system. Hence, the factor of CT includes the effect of both time and root input. All factors had two levels, that is day 1 and day 40 for CT, with and without additives for CO and BMC.To analyze the overall effects of CO and BMC, we compared edaphic and plant variables using both multivariate and univariate methods. For the multivariate analysis, unconstrained principal coordinates analysis (PCoA) was applied to the Euclidean distance matrix generated from normalized soil and plant data. Two-way permutational analysis of variance (PERMANOVA) was used to test the effect of each factor. For the univariate analysis, edaphic variables, and plant properties were subjected to a two-way analysis of variance (ANOVA). In addition, the net increments of TC and TN in each treatment were calculated as the average difference between treatments and control and considering the inherent carbon and nitrogen introduced through the treatments . The Bray–Curtis similarity coefficient matrix was visualized using unconstrained PCoA. Three-way PERMANOVA was applied to the Bray–Curtis similarity matrix to test the significance levels of differences between experimental factors and to explain the contribution of each factor to the total variance. Since the discriminative power of permutation-based analyses for pairwise comparisons of three and five replicates was limited by 10 and 126 unique permutations, respectively, we obtained pairwise comparisons of within-group to between-group Bray–Curtis similarities using the non-parametric Wilcoxon rank sum test  with multiple partial correlations was performed to visualize the model given by BEST selection.Distance-based linear models (DISTLM) were used to explore the relationship between edaphic variables and bacterial β-diversity  within STAMP  and plant (P < 0.001) parameters  were clearly grouped after 40 days of pakchoi cultivation Figure , whereasrs Table . HoweverP = 0.025, Table Univariate analysis showed that CO had a significant impact on all variables, except soil pH, while BMC displayed effects on humidity, TN, TSN, AK, and leaf area Table . Pairwis2, which was not achieved by adding BMC or compost alone  for pakchoi relative to the CK and BMC treatments. Surprisingly though, the combination of BMC and compost (treatment BMCO) resulted in a further increase in leaf area reaching values of up to 4.81 ± 0.98 cmne Table . The addGiven the results above, the addition of compost and BMC  had clear additive and synergistic effects on the nutrient content of the soil. We hypothesized that this might be due to changes in metabolic processes occurring in the soil and therefore investigated the composition of its bacterial community. At total of 1,174,380 high-quality 16S rRNA sequences were generated from 32 samples covering three replicates of day 1 and five replicates of day 40 soil samples for each of the four treatments. After subsampling and clustering, 10,509 operational taxonomic units (OTUs) at 97% identity (roughly corresponding to species) were obtained, with the number of OTUs ranging from 519 to 1940 per sample. The Good's coverage for the observed OTUs was 89.2 ± 5.5% at a sequencing depth of 5793 reads, which indicates good sampling of the community.P < 0.001, Table P = 0.604, Table P < 0.001).The addition of compost (treatments CO and BMCO) significantly decreased the bacterial evenness in samples of day 1  and a smaller effect associated with the interaction between the factors BMC and CT . Pairwise comparison within levels of CT showed that CO preserved a significant effect over time , humidity and C:N explained an additional 5.4% (P = 0.02) and 5.2% (P = 0.05), respectively. These variables constituted the BEST selection solution (i.e. the best fitted model). dbRDA analysis illustrated the adequacy of the model comprised of these three variables to predict patterns of bacterial β-diversity , and we therefore explored correlations between soil variables and microbial β-diversity using a distance-based linear regression method. Marginal tests showed that all variables (except pH) had significant correlations with bacterial communities Table . Furthery Figure . DiffereGiven the clear distinction of bacterial communities between treatments and their correlation with edaphic parameters, we next explored which specific microbial taxa were present and responsive in each treatment. Proteobacteria, Actinobacteria, and Bacteroidetes were the three most dominant phyla Figure , which aNocardiopsaceae (phylum Actinobacteria) made up between 9 and 15% of the bacterial communities in treatments receiving compost, but was virtually absent in soil without amendment. After 40 days, however, other OTUs contributed most strongly to the difference between treatments, including those belonging to the genera Pseudoxanthomonas, Microbulbifer, Mesorhizobium, and Devosia , Proteobacteria (22.8%), Bacteroidetes (16.5%), and Firmicutes (9.5%). This introduction of new bacteria was further illustrated when only the 10 most abundant OTUs were considered  have on the bacterial communities in soil and on the cultivation of pakchoi. Overall, the results show that soil, plants, and bacterial communities were clearly influenced by the amendment of compost or biochar–mineral complex, either alone or in combination Figure . Compost−1; Wong et al., The typical application rate , which are known to degrade complex carbohydrates  of relatively recalcitrant BMC maintained bacterial communities that are oligotrophic in nature. This indicates that the natural soil used for pakchoi cultivation was nutrient-limited, which was also reflected in the poor plant yields and high soluble saccharides . A representative sequence for this Nocardiopsaceae OTU showed a 100% identity to the 16S rRNA gene sequence of an isolate from cattle skin lesions (Genbank accession JQ031766) and 96% identity to the 16S rRNA gene of Nocardiopsis nikkonensis, which was isolated from matured compost  than what was obtained by the CO treatment (4.02 mg ± 1.25 kg−1) and could not be explained by simply accounting for the effect of BMC alone (3.08 ± 0.75 mg kg−1) and the soil control (1.96 ± 0.53 mg kg−1). Soil nitrate is an important factor for plant growth and has been shown to stimulate the growth of leaves  or nitrogen-fixing  taxa (see above). While this increase in the BMCO treatment had no immediate benefits for the plant growth in the short time frame of our growth experiment, we expect that longer trials, in particular those with multiple growing seasons, will see a continued increase in soil nutrients and thus likely sustained productivity.BMC on its own was not sufficient to achieve the same plant yields as the CO treatment, but we found a number of additive and synergistic effects when BMC and compost were combined. Of particular interest is that the BMCO treatment had increased values for TC, TN, OC, TSN, Ellin6067, MND1, and Ellin6075 were significantly more abundant in BMCO treatments when compared with the CO at day 40  with the BMCO treatment having the highest number of nitrifying bacteria (1.10 ± 0.07%) followed by the BMC (1.04 ± 0.04%), CK (0.95 ± 0.09%), and CO (0.79 ± 0.05%) treatments. Increased nitrifying activities, as indicated by a relative higher number in nitrifying bacteria, would result in higher nitrate levels  on BMC  by the addition of BMC, whose intricate chemical properties and surface structures are known to influence the chemico-physical properties of the soil environment (e.g., humidity, Figure JY, RZ, SN, SDJ, DH, and TT conceived and designed the research; JY and RZ performed the research; JY, RZ, SN, DH, and TT analyzed data; JY and TT wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Infections are still the most common complications of cerebral shunt procedures. Even though fungal etiologies are considered to be rare, they are associated with significant morbidity and mortality. Due to their uncommonness, diagnostic procedures and optimal therapy are poorly defined. We report a case of Candida tropicalis infection of ventriculo-peritoneal cerebrospinal fluid (CSF) shunt in a 49-year-old immune competent male treated with voriconazole (VOR).Microbiological and CSF markers  of fungal infection, biofilm production capacity, sensitivity of serial isolates of the pathogen, and the concentration of the antifungal drug have been monitored and related to the clinical course of this infection.Despite appropriate treatment with VOR, in terms of adequate achieved CSF drug concentrations and initial effective therapeutic response, loss of VOR susceptibility of the C tropicalis and treatment failure were observed.Biofilm production of the C. tropicalis isolate might have had a significant role in treatment failure. Of interest, clinical and microbiological unfavorable outcome was anticipated by persistence of BDG in CSF. Rising titers of this marker were associated with relapse of fungal infection. Until now, few cases of Candida tropicalis infections of CNS have been reported in the medical literature.,3 Therefore, risk factors, prognosis, diagnostic procedures, and optimal therapy are poorly defined. Despite appropriate therapy, the referred mortality of patients affected by C tropicalis meningitis approached 30%.,2 We describe a case of an infection of the cerebrospinal fluid (CSF) shunt device in a middle-aged patient sustained by C tropicalis. Peculiarities of this report of such a rare infection are that measurement of -β-d-Glucan (BDG) in the CSF, biofilm production capacity and modifications of the azole susceptibility of serial isolates of the pathogen, as well as antifungal drugs CSF concentration have been monitored and related to the clinical outcome.2pontocerebellar acoustic neuroma with placement of a ventriculo-peritoneal (VP) shunt and received an successful 10-day Fluconazole (FLU) treatment course for a catheter-related C tropicalis urinary tract infection. CSF analysis, performed at the admission, revealed glucose concentration of 34.2 mg/dL, protein level of 332 mg/dL, and leukocytes cell count of 471 cells/μL (75% polymorphonucleocytes). No signs of persistent Candida urinary tract infection were found in serial urine analysis and cultures performed during this second hospitalization. Fungal meningitis was diagnosed on the basis of C tropicalis isolation from the CSF culture. The same pathogen was also cultured from the removed VP shunt, while blood cultures were negative. Intravenous voriconazole (VOR) was started  and an external ventricular drainage (EVD) was inserted. The clinical course and the main laboratory findings, expressed in function of time, are shown in Fig. A 49-year-old immune-competent male was admitted to the Neurosurgical Intensive Care for fever (up to 38°C) associated with ideomotor slowdown, severe confusional state, and meningism. Two months prior hospitalization, the patient underwent surgical removal of a C tropicalis was still cultured from CSF and consequently EVD was changed. On the day 31 of VOR treatment, CSF cell count and glucose concentration achieved normal values and CSF culture yielded no microbial growth. With apparent C tropicalis microbial eradication from CSF, a clinical improvement of patient's condition in terms of Glascow Coma Score  (3 mg/kg/daily) along with FLU  and EVD was changed again. On the day 66 of antifungal therapy, the patient died for a concurrent bacterial sepsis, showing no further significant clinical improvement of his meningitis after the replacement of VOR by LAMB along with FLU. CSF cultures remained positive for C tropicalis. Given that interventions and all investigations described in this case report were performed as part of the standard health care whose informed consent was obtained, no ethical approval was either sought or obliged.33.1susceptibility of C tropicalis in CSF samples was tested by means of the minimal inhibitory concentrations (MICs) measurement , Italy), according to CLSI M27A3 guidelines. The susceptibility of the C tropicalis isolates was monitored and the changes recorded over the time (Table 2 dilution factor) of the VOR MICs was observed in serial CSF C tropicalis isolates.The antifungal 3.2C tropicalis strains was measured through XTT-reduction dehydrogenase assay and expressed as the ratio of the mean sample absorbance performed in triplicate experiments, over the 50% mean absorbance of a reference C. albicans strain , in terms of biofilm index (BFI).,6 Clinical isolates with BFI ≥0.5 were considered biofilm-producing strains. The aim of this experiment was to determine eventual time-related changes in biofilm-forming ability of strains of C tropicalis isolated from the CSF samples. All the isolates of C tropicalis were biofilm producers. The BFI fell from 2.1 to 1.1, with a 52% reduction at day 40. The recurrence of the isolation of C tropicalis on the day 48 was accompanied by a rise in the BFI .Biofilm production by 3.3Limulus amebocyte lysate pathway, using a commercially available diagnostic kit , according to the manufacturer instructions. Glucan-free dilution tubes and pipette tips were used, and all assays were performed in a biosafety cabinet that had not been used to manipulate fungal cultures. Individuals performing the assay were blinded to all diagnostic and clinical information. This analysis was performed posthumously on CSF samples stored in the biobank. Our results showed a progressive decrease in the levels of CSF BDG (from 5230 to 1233 pg/mL), even though the values were constantly high and the negativity of the marker was never reached. Of note was a new gradual rise in the BDG in the CSF samples some days before the new isolation of C tropicalis  over the concentration of 0.1 to 50 mg/L. Validation data for accuracy and precision were coefficient of variation between 2.4% and 7.3%; intra-day accuracy was in the range of 95% to 106.5 %.VOR plasma concentration was determined by high-performance liquid chromatography (HPLC), using Perkin Elmer chromatograph  with wavelength ultraviolet (UV) detector set at 255 nm. An aliquot (0.5 mL) was pipetted into a 2 mL polypropylene tube  and acetonitrile (1.0 mL) added. The mixture was vortex mixed briefly and after standing for 10 minutes at room temperature the mixture was centrifuged at 1200C tropicalis.A median CSF VOR concentration of 2 mg/L (range 0.27–3.82 mg/L) was registered, while the mean blood level of VOR was 2.48 mg/L (range 1.91–3.05 mg/L). The CSF/plasma VOR concentration ratio was 0.8. The CSF VOR concentrations observed were always higher than the coeval MIC of 4VOR should be used as a first-line treatment for CNS Candida infections. The rationale for the use of this drug in this setting is linked to its ability to reach high concentrations in the CSF.–11 Lutsar et al reported that in patients treated with VOR for CNS fungal infections, the median CSF/plasma ratio was 0.46 (range 0.22–1.0). Wiederhold et al, testing 173 CSF samples, confirmed a similar result (CSF/plasma ratio 0.52) and described that the median quantifiable CSF level of VOR was 2.47 mg/L (range 0–15.3 mg/L) with CSF concentrations more heavily distributed toward the lower end of the range. However, the CSF concentration and the ratio may vary depending on the serum drug concentrations and is influenced by several variables such as liver disease, age, genetic polymorphism of the cytochrome CYP2C19, and comedications. These variables may lead to large inter- and intra-patient differences in VOR pharmacokinetic, perhaps sometimes associated with a decreased efficacy. Therefore, therapeutic drug monitoring (TDM) is required to improve the efficacy and the safety of VOR therapy in patients with invasive mycoses.The Italian Consensus for invasive candidiasis management (ITALIC) has suggested that C tropicalis isolates, ranging from 0.06 to 0.25 mg/L, were always below the resistance breakpoint that is >0.5 mg/l, according to the CLSI interpretation. However, a progressive increase of the MIC values was observed during the course of VOR treatment and a value of 0.25 mg/L was observed on day 48 of VOR therapy. To date, according to new CLSI VOR clinical breakpoints for C tropicalis (available only after the occurrence of the described case), MICs values for VOR of 0.25 to 0.5 mg/L should be interpreted as intermediate sensitivity. Of interest, a recent Italian study reported that 99% of the isolates of C tropicalis tested were highly susceptible to VOR  with only 1 out of 102 isolates showing dose-dependent susceptibility. These data were confirmed by the results of a large multicenter study conducted in Europe and North and South America where only 2.2% and 3.5% of 3127 C tropicalis isolates showed MICs for VOR of 0.25 and ≥0.5 mg/L, respectively. As a matter of fact, in our case, we cannot exclude that the above-mentioned loss in susceptibility to VOR might have affected the efficacy of therapy.VOR was used in our case and the drug achieved plasma and CSF levels that should have provided a clinical efficacy (CSF/plasma concentration ratio of 0.8). Furthermore, the MICs of VOR treatment failure, LAMB as well as FLU therapy was attempted on the basis of previous apparently favorable in vivo and in vitro observations of this antifungal agents combination.,16 However, the efficacy of this salvage therapy could not be fully evaluated, as the patient finally died for bacterial superinfection.Following C tropicalis to form biofilm on devices is considered a potential virulence factor and might have represented another limitation for therapy with VOR (or LAMB) in our case.,18 Such a phenomenon might be strongly implicated in the final VOR treatment failure despite susceptibility of fungal isolates and prompt removal and substitution of EVDs.Further insights might be gained by the observation of biofilm production by all consecutive fungal isolates. Indeed, the ability of BFI has been observed in serial isolates during antifungal therapy. To this end, recent studies showed that VOR fails to eradicate Candida spp. in mature biofilm, but seems to display in vitro efficacy in the reduction of biofilm formation.–21 Of course, knowledge of the clinical significance of these observations requires for further investigations.Of interest, as shown in Fig.  Therefore, due to these possible bias, serum and/or BDG seem to be a suitable marker in this setting.The therapeutic efficacy of VOR was initially assumed by the progressive restoration of the normal physical-chemical and microbiological CSF parameters. These findings were also indirectly confirmed by the gradual decline of CSF BDG observed until a few days before the recurrence of positive CSF culture under VOR therapy. Erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and BDG might be used for monitoring efficacy of antifungal therapy. Nevertheless, ESR and CRP are nonspecific inflammatory markers and can be affected by concomitant conditions such as concurrent bacterial infections that compromise their usefulness in monitoring the fungal disease in critically ill patients, as it occurred in our patients.CSF BDG as a marker for the detection of fungal infections in the CNS and for clinical therapy monitoring.–28 In our case, during VOR treatment, the retrospective analysis in serial samples of CSF demonstrated persistence of BDG, even when cultures yielded no microbial growth, albeit with a progressive reduction of titers. On the basis of these observations, persistence of BDG in the CSF might suggest the need for maintaining the antifungal treatment despite negative cultures. On the contrary, clearance of this marker might suggest the optimal timing to reposition a VP shunt. Not less important, a sudden increase of CSF BDG values during antifungal therapy, as that observed in our case before the C tropicalis recurrence, might represent a warning sign for the treatment failure.Recent studies, performed in animal models and on human beings, proposed  According with the results of the fungal meningitis rabbit model proposed by Petraitiene et al, plasmatic levels of β-glucan lower than CSF levels hint a compartmentalization of this polymeric carbohydrate, with higher concentrations within the CNS not readily crossing the blood–brain barrier. Taken together, these data could indicate that serum BDG values may not be helpful in determining invasion of the CNS by a fungal pathogen.On the contrary, serum BDG levels do not seem to be concordant with CSF BDG levels: in fact in our case, BDG values were significantly higher in CSF than in blood. As previously suggested, this gap could suggest isolated CNS infection without significant spillover into venous blood or that BDG may be more rapidly cleared in blood than in CSF.Candida infections, as proposed by ITALIC, is not shared by the most recent guidelines for the “Management of Candidiasis,” updated by the Infectious Diseases Society of America (IDSA) in 2016. In these recommendations, VOR is not cited as a possible choice in the treatment of CSN Candida infections, although it is recognized that it can achieve significant CSF concentration, potentially active against susceptible isolates, and it has been shown to be efficacious in small case series.–10,12 Wide variability in serum levels, need of the TDM, common drug–drug interactions with potential toxicity might have discouraged the use of VOR in these Guidelines.Finally, despite the data supporting the efficacy of VOR in CNS fungal infections, use of this azole as first-line therapy in CSF 5C tropicalis meningitis wherein appropriate treatment with VOR, in terms of adequate achieved CSF drug concentrations, was associated with loss of VOR susceptibility of the offending pathogen and treatment failure. Of interest, clinical and microbiological unfavorable outcome was anticipated by persistence of BDG in CSF. Eventually, rising titers of this marker were associated with clinical and microbiological relapse of fungal infection. Biofilm production of the C tropicalis isolate might have had a significant role in treatment failure. The importance of all these data should be confirmed by further studies.We described a rare case of


In separate studies, the accumulation of advanced glycation end-products has been associated with joint inflammation in RA. Objective of this study was to assess the association between EFF beverages intake and non-age, non-wear and tear-associated arthritis in US young adults.There is a link between joint and gut inflammation of unknown etiology in arthritis. Existing research indicates that regular consumption of high-fructose corn syrup sweetened (HFCS) soft drinks, but not diet soft drinks, may be associated with increased risk of seropositive rheumatoid arthritis (RA) in women, independent of other dietary and lifestyle factors. One unexplored hypothesis for this association is that fructose malabsorption, due to regular consumption of excess free fructose (EFF) and HFCS, contributes to fructose reactivity in the gastrointestinal tract and intestinal 2 was used for prevalence differences and logistic regression for associations, adjusted for confounders.In this cross sectional study of 1209 adults aged 20–30y,  exposure variables were high EFF beverages, including HFCS sweetened soft drinks, and any combination of HFCS sweetened soft drinks, fruit drinks (FD) and apple juice, referred to as tEFF. Analyses of diet soda and diet FD were included for comparison. The outcome was self-reported arthritis. Rao Scott Ҳp⩽0.021; 95% confidence intervals=1.20–7.59), independent of all covariates, including physical activity, other dietary factors, blood glucose and smoking.Young adults consuming any combination of high EFF beverages (tEFF) ⩾5 times/week (but not diet soda) were three times as likely to have arthritis as non/low consumers (odds ratios=3.01; in situ formation of enFruAGEs.EFF beverage intake is significantly associated with arthritis in US adults aged 20–30 years, possibly due to the intestinal  Arthritis is the leading cause of disability in the US.2Arthritis encompasses musculoskeletal disorders consisting of >100 different conditions that destroy joints, bones, muscles, cartilage and other connective tissues, hampering or halting physical movement. With the exception of ‘wear and tear' age-associated osteoarthritis, most forms of arthritis are auto-immune disorders that are characterized by inflammation wherein the exact etiologies are unknown.3 rheumatoid arthritis (RA),5 lupus erythematosus,7 psoriatic arthritis8 and undifferentiated arthritis.10Although the risk of developing most types of arthritis increases with age, arthritis also occurs in young people. Types of arthritis most known to affect young adults include ankylosing spondylitis,12 and lupus erythematosus.13 Coincident gut and joint inflammation is also common in ankylosing spondylitis and psoriatic arthritis.14 While adverse gastrointestinal symptoms are known side effects of glucocorticoids and excessive nonsteroidal anti-inflammatory drug use, existing research provides evidence that gastrointestinal symptoms of unknown etiology are independent of medication use in a subset of patients. There is evidence that there may be an immunological link between gut immunity and arthritis of dietary origin.18 Some researchers have suggested that gut bacteria may be involved in the coincident gut and joint inflammation, but the ‘gut bacteria model' does not explain the co-localization of inflammation to the synovium and gut, or identify the specific bacterial antigens that may incite inflammation.14Notably, in many arthritis patients there is a link between joint and gut inflammation. For example, gastrointestinal disease is one of the most common comorbidities in patients with RA4Interestingly, a recent large scale prospective epidemiology study of nearly 200,000 women  found that regular consumption of high-fructose corn syrup (HFCS) sweetened soft drinks, but not diet soft drinks, was associated with increased risk of seropositive RA in adult women, independent of weight and lifestyle factors. Researchers concluded that the deleterious effect of non-diet soda on RA development might not be through weight gain and obesity, as the risk of RA was not attenuated after adjustment for weight change and body mass index (BMI). Researchers suggested that non-diet soda intake might increase the risk of RA by upregulating inflammatory cytokines and decreasing insulin sensitivity due to the glycemic load of non-diet soft drinks (ndSD), and possibly due to the advanced glycosylation (AGEs) end-products present in the caramel coloring of soda.19 Second, diet soft drinks contain the same caramel coloring as ndSD, but were not found to be associated with RA.4 Third, their results were independent of diabetes history,4 suggesting that mechanisms other than glycemic load may underlie the ndSD association with RA. Notably, HFCS, and not sucrose, is the main sweetener is US soft drinks.However, this hypothesis is unclear for three reasons. First, existing research indicates that ndSD are not a significant source of AGEs.in situ formation of advanced glycation end-products (enFruAGEs) that may be an overlooked source of pro-inflammatory AGEs that travel beyond the intestinal boundaries to other tissues and may play a role in the etiology of auto-immune arthritis.21 It is noteworthy that adult FM is not associated with consumption of sucrose or equal monomers of fructose and glucose.27Another unresearched and overlooked source of AGEs that may be arthrogenic is intestinally generated AGEs. According to the intestinal enFruAGE ‘fructositis' hypothesis, underlying fructose malabsorption (FM) and unabsorbed EFF intake, contributes to the intestinal 32 This possibility is increased because of evidence that more EFF is being consumed than has been assumed; independent lab results indicate that popular soda beverages are sweetened with an HFCS variant that is 60% fructose and 40% glucose, rather than the 55/45 formula that is generally recognized as safe.35 Different varieties of fruit drinks (FD) with high apple juice (AJ) content were found to contain a 67/33% combination of fructose and glucose monomers.35 This is because AJ, unlike orange juice, is also a high-excess-free fructose beverage.36It is possible that enFruAGEs may play a role in idiopathic arthritis, as existing research provides evidence that AGEs are elevated in connective tissues including the synovium, sub-lining and cartilage of arthritis sufferers.36 FD and AJ36 in a large database containing information about beverages intake and arthritis prevalence in adults ages 20 and older  2003–2006). Analysis of diet soda (a non-EFF beverage) was conducted for comparison. For this analysis the focus was on young adults, aged 20–30 year, as the interest was in a cohort more likely to be diagnosed with an autoimmune variant of arthritis and less likely to be diagnosed with ‘wear and tear' age-associated osteoarthritis. In addition, young adults consume significantly more ndSD than older adults.37 There were 1084 study participants.This study, examined a potential role for enFruAGEs in arthritis pathogenesis by studying the association between young adult arthritis prevalence and intake frequency of high EFF beverages, including HFCS sweetened soft drinks,38 NHANES for the years of this analysis included a food frequency questionnaire (FFQ). A consistent relationship between reported FFQ frequency of food and food-group consumption and probability of consumption on 24-h recalls has been demonstrated.39 Data from FFQ are used as a reliable measure of dietary patterns and food intake information in epidemiological research.41Cross-sectional data were used to assess the association between high EFF beverages intake and arthritis using a nationally representative survey. NHANES are administered by the US Center for Disease Control and are part of their ongoing mission to produce vital and health statistics for the US.38NHANES for the years 2003 through 2006 included an FFQ with frequency of intake for the beverages of interest as potential predictor variables. The medical questionnaire component included arthritis status information. Because NHANES uses a complex sampling design and constructs sample weights to produce nationally representative data, sample weights provided in the food frequency data files were used to perform statistical analysis for this study.42 Therefore all statistics and summary tables are appropriately weighted.The weights account for oversampling of various groups and reflect the fact that, of the 20,470 people who participated in the overall (4 year) study, not all respondents  participated in the food frequency part of the survey.38The outcome variable was self-reported arthritis or history of arthritis. On the NHANES questionnaire, this was asked as ‘Has a doctor or other health professional ever told you that you have arthritis?' In the NHANES, the target age group for this question was adults, aged 20 and older. Beverage intake exposure variables were obtained from the FFQ questions: How often did you drink (1) AJ? (2) orange or grapefruit juice? (3) other FD  (4) soft drinks, soda or pop in the summer? and (5) soft drinks, soda or pop the rest of the year? Additional questions clarified how often FD or soft drinks were diet or sugar-free or caffeine free.38 Intake data were combined to establish new intervals for analysis purposes as follows: ⩽2–3 times per month as the reference group; 1–4 times per week and ⩾5 times per week.The average daily frequency of ndSD and diet soft drinks over the past year was calculated by summing individual values for caffeinated and caffeine-free non-diet soda-in-the-summer and rest-of-year. The NHANES utilized specialized software  to assign frequencies to responses from the FFQ using algorithms as follows: never=0; 1 time per month or less=0.03; 2–3 times per month=0.08; 1–2 times per week=0.21; 3–4 times per week=0.5; 5–6 times per week=0.79; 1 time per day=1; 2–3 times per day=2.5.To analyze the combined effects of EFF, the following algorithm was used. Intake frequencies for ndSD, FD and AJ were assigned a zero for once a month or less; 0.117 for 2–3 times a month; 0.357 for 1–4 times per week and 1 for >5 times per week. These values were summed to calculate average daily intake of total EFF beverages. The same approach was used to analyze the combined effects of diet soft drinks and diet FD.38 Adjustment variables were selected for use in this study based on existing research.40 Total energy intake and total fruit and vegetable intake were the only variables obtained from the 24-h dietary recall and were calculated using either the average of two 24-h recalls or one 24-h recall if the second recall was missing.Adjustment variables included sex, race/ethnicity, age, BMI, total energy intake, smoking, family income, head of household education level, physical activity, total fruits and vegetables intake, a measure often used as a barometer of healthy lifestyle, and glycated hemoglobin (A1c), a measure of the average level of blood glucose over the previous three months used in the diagnosis and assessment of control of diabetes.38Smoking and history of smoking was self-reported. This was asked as a series of questions including, ‘Do you now smoke cigarettes? During the past five days, did you use cigarettes?' Have you smoked at least 100 cigarettes in your entire life? For analysis purposes these were used to distinguish between non-smokers, non-smokers with a history of smoking and current smokers.38 For analysis purposes these were reduced to 0–$19,999; $20,000–$34,999; $35,000–$54,999; $55,000 and over. Head of household education level was obtained by asking, ‘What is the highest grade or school level you have or the head of household has received?' Categories were <9th grade; 9th–11th grade; HS/ GED; some college; and college graduate.38 For analysis purposes these were reduced to HS/GED and below, and some college and above.Socioeconomic status (SES) was included as a potential confounding variable using data obtained for family income and the head of household education level. NHANES used the Family Interview Income Questionnaire to obtain combined family income for 13 income ranges.38The NHANES asked several questions to obtain information of physical activity and provided recommended metabolic equivalent scores for each activity. For analysis purposes, metabolic equivalent scores were summed and divided into three equal quantiles. The physical activity questions and corresponding metabolic equivalent scores used in this study are as follows: which of four sentences best describes your usual daily activities? You sit during the day and do not walk about very much (1.4). You stand or walk about a lot during the day, but do not have to carry or lift things very often (1.5). You lift light loads or climb stairs or hills often (1.6). You do heavy work or carry heavy loads (1.8). Have you over the past 30 days, walked or bicycled to get to/from work (4.0); done tasks in or around home or yard that required moderate or greater physical effort (4.5); done moderate activities that caused light sweating or moderate increases in breathing or heart rate including brisk walking, bicycling for pleasure, golf, and dancing, and so on (3.5); done vigorous activities that caused heavy sweating or large increases in breathing or heart rate including exercise, sports and other active hobbies (7.0).2 analysis was used to test for significance of differences in arthritis prevalence by intake frequency. Analysis was conducted individually for intake frequencies of diet soft drinks; HFCS soft drinks; any combination of HFCS sweetened soft drinks, FD and AJ (tEFF); and any combination of diet soft drinks and diet FD. A p-value of ⩽0.05 was considered significant, with values <0.10 approaching significance.Analysis was performed utilizing statistical software from STATA Corporation , revision 18. Appropriate procedures were used to account for complex sample design. As previously described, weight variables were used to account for non-response and oversampling of various age groups and ethnic groups. Rao Scott Ҳ2. Two multivariate logistic regression models were used to analyze adjusted odds ratios (OR) as follows: the first model adjusted for age, sex, race/ ethnicity, BMI, SES, total energy intake, total fruit and vegetable intake, physical activity level, and smoking. The second model also adjusted for glycated hemoglobin (A1c) – a measure of the average level of blood glucose over the previous three months. During individual analysis of ndSD, an adjustment for AJ and FD was done to assess the association between HFCS sweetened soft drinks and arthritis, independent of the other high EFF beverages. In logistic regression analysis, confidence intervals (CI) that did not include 1 and p-values ⩽0.05 were considered statistically significant.Logistic regression was used to assess the adjusted odds between exposure variables and arthritis, independent of confounding variables, for the same beverages and beverage groups analyzed using Rao Scott Ҳ2 However, analyses based on NHANES are generalizable to the US non-institutionalized civilian population.38 Descriptive characteristics of the sample are presented in p⩽0.05), but not with diet drinks.44Consumption of beverages high in excess-free fructose was significantly correlated with arthritis in US young adults aged 20–30 years. Overall, 4.6% of 1084 adults, aged 20–30 years were reported to have arthritis (or history of arthritis). A comparison of arthritis prevalence with national statistics was not possible for this age sub-group, as available US Centers for Disease Control and prevention data only include adults aged 18–44 years (7.3%).2 comparisons with arthritis prevalence showed that intake of non-diet soda and any combination of high EFF beverages (tEFF) was highly significant, but not intake of diet soda and diet FD. Arthritis prevalence was significantly higher among young adults who regularly consumed ndSD (3.8%), or any combination of high EFF beverages  (3.5%), relative to non/low consumers (0.7%) and (0.5%). p=0.0021, p=0.0133. Arthritis prevalence was low among regular consumers of diet soft drinks and diet FD (1.1%), relative to non/ low consumers (0%), p=0.2159. Unadjusted Rao Scott Ҳp⩽0.021; 95% CI=1.20–7.59). p⩽0.102; 95% CI=0.81–8.84). In logistic regression models adjusted for age, sex, race/ ethnicity, BMI, SES, physical activity, total energy intake, total fruit and vegetable intake , smoking, history of smoking, and glycated hemoglobin (A1c), the cumulative effect of HFCS sweetened soft drinks, FD, and AJ (tEFF) was significant. Young adults reporting tEFF intake ⩾5 times per week had three times higher odds of arthritis as ⩽1–3 times per month consumers, after adjusting for potentially confounding variables , nor was any combination of diet soft drinks and diet FD  In logistic regression analysis of diet beverages, the reference group was set to ⩽1–4 times per week as there was no arthritis prevalence among non/ low (⩽1–3 times per month) diet drink consumers. After adjustment for all potential confounders, consumption of diet soft drinks ⩾5 times per week was not associated with arthritis , but there was no association with diet drinks .Consumption of a combination of HFCS sweetened soft drinks, FD and AJ (tEFF) was significantly associated with arthritis in young adults aged 20–30 years, independent of known co-morbidities, including other dietary lifestyle factors, physical activity, BMI, smoking, and glycated hemoglobin concentration  that may be an overlooked source of pro-inflammatory AGEs that travel beyond the intestinal boundaries to sites of arthritis tissue inflammation.Arthritis prevalence among regular consumers (⩾5 times per week) of tEFF beverages (3.5%) and HFCS sweetened soft drinks (3.8%) was significantly higher than among non/low consumers (0.5/0.7%). In comparison, arthritis prevalence among regular consumers of diet soft drinks and diet FD (1.1%) was considerably lower than arthritis prevalence among regular consumers of HFCS sweetened soft drinks (3.5%). This difference provides further evidence and support for the hypothesis that it may be the cumulative effect of excess free fructose that is underlying the association with young adult arthritis.27 possibly because they are transported and absorbed differently by the body during digestion.47 An analysis of the sugar composition of HFCS sweetened soft drinks, AJ and orange juice helps to illustrate how HFCS sweetened soft drinks and FD have more in common with AJ, than AJ has with orange juice.Existing FM research provides insights into how beverages with similar levels of total sugars, but different fructose composition could have different effects on the body. Research indicates that adult FM occurs after consumption of EFF, but not after consumption of sucrose or equal amounts of fructose and glucose monomers,36 Similarly, the total fructose amounts in AJ and orange juice are comparable; per 100 g, AJ contains 6.4 g and orange juice contains 4.5 g of fructose.36 Whereas analysis of their EFF content indicates that these juices are quite different from one another. Specifically, the excess free fructose content in an 8 oz cup of AJ is 7.7 g (NDB no. 09400) and in orange juice it is 0.4 g (NDB no. 09207). Per (8 oz.) cup, AJ contains 19 times more EFF than orange juice.36 Therefore, AJ is a high EFF beverage, whereas orange juice is not.According to the USDA National Nutrient Database for Standard Reference (NDB) release 26 per 100 g, the amount of total sugars in AJ and orange juice are comparable; AJ contains 9.6 g and orange juice contains 8.8 g of total sugars.36 in the US, HFCS sweetened soft drinks are the most significant source of EFF in adult diets,49 even though many other foods are sweetened with HFCS.49 Present estimates indicate that the US average per capita consumption of HFCS is just under a lb. per week, or 65 grams per day,48 which is the same amount of HFCS in a 20 ounce bottle of non-diet cola.50 When sweetened with the 60/40 variant of HFCS, a 20 ounce bottle of cola contains 13 g of EFF. Therefore, EFF intakes may exceed dosages associated with adult FM in subsets of the population, as FM research indicates that 30% of healthy adults are FM positive after a 25 g EFF dose, and 10% are FM positive after a 12 g EFF dose, but not after consuming sucrose or equal amounts of fructose and glucose monomers.22Although AJ is a higher EFF beverage than HFCS sweetened soft drinks, and contributes to total excess free fructose load,51 As there was no correlation between IgG-AGE and glucose, researchers hypothesized that it was the inflammation and not hyperglycemia that exerts the major influence on AGEs formation in patients with early synovitis.51 This supports the possibility that intestinally formed enFruAGEs, due to underlying fructose malabsorption, may contribute to arthritis-associated inflammation.It is also noteworthy that regular consumption of tEFF beverages remained significantly associated with young adult arthritis even after adjustment for glycated hemoglobin concentration. This is consistent with research of glycation in arthritis, wherein early onset arthritis patients with elevated IgG-AGE had significantly higher concentrations of the inflammatory markers C-reactive protein and erythrocyte sedimentation rate, but there was no correlation with blood glucose.4 This suggests that the link with RA may be driven, at least in part, by factors other than glycemic load, β cell malfunction, and insulin insensitivity. This largescale longitudinal study provides further evidence that excess free fructose in HFCS sweetened soft drinks may be responsible for the statistically significant association between regular consumption of ndSD and rheumatoid arthritis in women.This conclusion is further supported by the 2014 prospective epidemiologic study of nearly 200,000 women , which found that the association between regular consumption of ndSD and seropositive rheumatoid arthritis in women (but not with diet soda) was independent of diabetes history.32 Connective tissues including synovial tissues and cartilage contain relatively high levels of membrane bound receptor of advanced glycation end-products (RAGE) that are associated with pro-inflammatory signaling.52 Existing research provides evidence that RAGE is overexpressed in tissues with increased ligands of RAGE.53 RAGE overexpression has been implicated in the pathogenesis of arthritis.54 In contrast, soluble RAGE (sRAGE) acts as a decoy receptor and down regulates inflammation. Decreased sRAGE is associated with increased arthritis prevalence.56This study's results and the results of the 2014 Nurses Health Studies should be considered in the context of research that has focused on glycation in arthritis. By-products of glycation that have been identified in synovial tissues, sub-lining and endothelium of arthritis patients correlate with inflammation.14 It should be considered along with research that has provided evidence of inflammatory bowel disease, or early Crohn's disease, in the majority of ankylosing spondylitis patients evaluated, regardless of genetic marker HLAB27.57 Additional consideration should be given to research indicating that there may be an immunological link between gut immunity and arthritis of dietary origin.18 The link between regular consumption of high EFF beverages and arthritis prevalence may explain, at least in part, the unexplained gut and joint inflammation prevalence in idiopathic arthritis.These results should also be considered in the context of research that has provided evidence that lymphocytes and macrophages traffic from the gut to synovial tissues, but the inciting antigen or [gut] immune trigger has remained unclear.16 It is possible that dietary AGEs play a role in the association between red meat intake and arthritis, as cooked red meat contains high levels of dietary AGEs, relative to other foods.19Interestingly, results of a large scale longitudinal study (the European Prospective Investigation of Cancer in Norfolk (EPICNorfolk)) provide support for a role for AGEs in polyarthritis. After adjusting for total energy intake, smoking, and other possible dietary confounders, participants with the highest intake of red meat , meat and meat products combined , or total protein  were at an increased risk for inflammatory polyarthritis.14 However, the gut immune trigger has remained elusive. EnFruAGEs could account for21 the postulated relationship between observed intestinal abnormalities and the pathogenesis of some forms of arthritis, independent of glucocorticoids and nonsteroidal anti-inflammatory drug use.18This study's results, along with existing research, provide support for the hypothesis that gastrointestinal generated enFruAGEs may be inciting antigens and gut immune triggers that cause lymphocytes and macrophages to traffic from the gut to synovial tissues.60 and adult chronic bronchitis, a subcomponent of COPD.61 It is noteworthy that chronic respiratory disease is a common co-morbidity of arthritis, as 19% of people with arthritis report a history of chronic respiratory conditions, according to the US Centers for Disease Control and Prevention.2Lastly, these results should also be considered in the context of recently published epidemiologic studies which indicate that excess free fructose intake is associated with other pro-inflammatory diseases known to be associated with elevated levels of advanced glycation end-products and the RAGE including childhood asthma,62 - was not possible, as this data was unavailable. Third, it is possible that in adults over age 30, there is an association between EFF beverages consumption and autoimmune arthritis. However, this was not assessed, as in the NHANES, unlike the Nurses Health Study, there were no questions which sought to distinguish between types of arthritis. Therefore, in older adults, analysis of autoimmune arthritis, as distinguished from wear and tear, age associated osteoarthritis was not possible. Fourth, arthritis status in NHANES is based on self-report, so there is potential for reporting bias. Fifth, high EFF beverages are only one food category that could contribute to daily EFF load. Numerous other food categories contain HFCS as an added sweetener, and therefore, also contribute to daily EFF load. However, among US adults, particularly young adults, soft drinks are the most significant source of HFCS in the American diet.63 Sixth, it is possible that the prevalence of arthritis is not raised among high EFF beverages consumers, but is instead reduced among non/low consumers due to unknown lifestyle or protective factors that may be conferred by diets low in EFF beverages, although our results remained significant after adjusting for lifestyle and other dietary factors.This study is subject to limitations. First, the associations are cross-sectional; therefore, the exposures and outcome are simultaneously assessed. For this reason, cross-sectional studies do not provide evidence of a temporal relationship between exposures and outcome. Follow-up longitudinal studies are needed. Second, it is possible that an association between EFF beverages consumption and arthritis exists in teens. However, this could not be assessed, as in the NHANES, the target age group for questions of arthritis status was adults ages 20 and older. Therefore, analysis of teens aged 15–19 – a group that consumes more ndSD than other age groupsRegular consumption of high EFF beverages is associated with young adult arthritis. Results support the possibility that there exists a nexus between fructose malabsorption, regular EFF consumption, and increased odds and arthritis prevalence in US adults aged 20–30 years. Unabsorbed excess free fructose may interact with dietary proteins resulting in the in situ formation of enFruAGEs that may play a role in the idiopathic link between gut and joint inflammation. Study results are consistent with the possibility that enFruAGEs are an overlooked source of AGEs that may contribute to arthritis pathogenesis in young adults. Longitudinal and biochemical research are needed to confirm and clarify the mechanisms involved.


Toxoplasma infection and its risk association among people having close contact with animals. A total of 312 blood samples were collected from veterinary personnel  and pet owners from veterinary clinics and hospitals in the area of Klang Valley, Malaysia. About 4 cc of blood samples drawn from agreed participants were processed for measurement of anti-Toxoplasma IgG and IgM antibodies as well as avidity test of Toxoplasma IgG by ELISA I, II, and III kits. Meanwhile, the demographic profiles and possible risk factors of these participants were also recorded in the standardized data collection sheets. Overall seroprevalence of toxoplasmosis was observed in 62 (19.9%) participants being 7 (18.4%) in veterinarians, 15 (33.3%) in veterinary technicians, 29 (14.9%) in veterinary students, and 11 (31.4%) in pet owners. Of 19.9% Toxoplasma seropositive samples, 18.3% was positive for IgG antibody, 1.0% for IgM antibody, and 0.6% for both IgG and IgM antibodies. Of three different IgG avidity ELISA kits, ELISA III showed high avidity in all five seropositive samples (IgM and IgG/IgM antibodies) indicating chronic Toxoplasma infection which is consistent with no evidence of clinical toxoplasmosis diagnosed during the time of this study. Univariate analysis showed that age group, gender, study population, gardening, task performance, and working duration were significantly associated with Toxoplasma seropositivity. Further analysis by multivariate analysis using logistic regression showed that age group of ≥30 years old  and working or study duration of >10 years having close contact with animals  were identified as significant risks for Toxoplasma infection. Based on the results obtained, a comprehensive Toxoplasma screening and health surveillance program on toxoplasmosis should be implemented among people having close contact with animals in general and confirmed Toxoplasma seronegative individuals in particular to prevent seroconversion.A cross-sectional study was conducted to determine the seroepidemiology of  Toxoplasma gondii (T. gondii), an obligate intracellular protozoan parasite (a zoonotic pathogen) is capable of causing both the infection rate that affects approximately one-third of human populations worldwide and the disease burden of clinical toxoplasmosis in human. Toxoplasma infection can be transmitted via several routes in different host species , MEC Ref. No. 1024.6 in accordance with the Helsinky Declaration for the inclusion of human subjects in research. The purpose and procedures of this study were explained to all the participants. Informed consents were obtained from agreed participants prior to samples and data collection.Approximately 5 mL venous blood was drawn, sera were processed and were kept at −20°C until further testing.Toxoplasma IgG and IgM antibodies by using standard ELISA commercial kit  in accordance with the manufacturer’s instruction. A positive sample for the anti-Toxoplasma IgG and/or anti-Toxoplasma IgM antibody was also tested for its avidity using three standard ELISA commercial kits, namely, ELISA-I, II, and III for comparison and according to its manufacturer instruction. The interpreted results for ELISA-I was that avidity of >40% suggest chronic/past infection and of <40% suggest acute/recent infection, for ELISA-II was that avidity of <15% (low avidity), indicates acute or primary infection and avidity between 15 and 30% (borderline activity) indicates possibility of primary infection during the last 6 months is possible and >30% (high avidity) excludes primary infection within last 3 months and ELISA-III’s avidity of <50% (low avidity) indicates primary infection.The collected serum was screened primarily for anti-2 test were used to investigate the association between Toxoplasma seropositivity as a dependent variable and possible demographic and risk factors as independent variables; p < 0.05 was regarded as being statistically significant. However, to retain all possible significant association, variables that showed an association with p ≤ 0.20 were used to apply to a multivariate logistic regression model (stepwise forward). Each dependent factor was modeled as dichotomous variables.The data collected in the questionnaires and the serology results were analyzed by using statistical software SPSS version 17.0 . The qualitative variables were estimated and presented as frequencies and percentages. Univariate analyses and the χDuring this study period, a total of 312 people were recruited as studied subjects. The age range was 17–64 with a mean 27 ± 9.08 years. Majority of the subjects were in the age group of 21 and 30 years , female , Malay , veterinary students , and city dwellers .Toxoplasma seropositive samples, one sample was recently acquired and four other samples were past infections as detected from ELISA-I, all five samples were regarded as recently acquired infection, as demonstrated by ELISA-II, while ELISA-III showed past infection from all five seropositive samples (data were not shown). At the end of this study, there was, however, no clinical evidence of toxoplasmosis diagnosed in the Toxoplasma seropositive with low avidity.The overall seroprevalence of toxoplasmosis in this study was 62 19.9%) in which 57 (18.3%) samples were positive for IgG, 3 (1.0%) samples were positive for IgM, and 2 (0.7%) samples were positive for both IgG and IgM antibodies (Table 9.9% in wToxoplasma seropositivity (p < 0.05) (Table Toxoplasma infection was found among veterinary technicians (33.3%) followed by pet owners (31.4%), veterinarians (18.4%), and veterinary students (14.9%). Interestingly, gardening  was significantly associated with Toxoplasma infection found among these subjects  Table . The reshnicians 3.3% follwners 31.%, veteriToxoplasma seropositivity in the study population and working duration of more than 10 years  was identified as significant predictors of Toxoplasma infection among veterinary personnel (data were not shown).Further analysis by multivariate logistic regression showed that age group ≥30 years  contributes to high Toxoplasma infection among general healthy population in Malaysia was quite low (13.9%) and it has been increasing over the years ranging from 16 to 30% (Toxoplasma infection rate (33.3%). This similar result is shown in the earliest study of toxoplasmosis among veterinary members in the United States (Toxoplasma infection rate (16.4%) compared to other groups  than females 15.9%). This could be normally explained by the fact that males have a higher tendency to be involved in sports activities or other activities at work or outdoor alike that expose them to soil and also they are not really careful about hand hygiene, which leads to increase risk of acquiring the infection . We alsowith age , as alsowith age , 22. As %. This cith cats . Supportith cats . Howeverp = 0.015). The participants who frequently do gardening were highly infected (26.6%) compared with the ones who spent less time (15.4%). This could be explained due to the fact that the buried sporulated oocysts of cats might be contaminating the soil the soil and sand and the oocysts remain infectious for about several months and can last beyond 1 year (Toxoplasma seropositivity (p = 0.019). Of this, veterinarians and veterinary technicians had the higher Toxoplasma infection (26.5%) compared to veterinary students (14.9%). This finding was not surprising since the daily task like animal surgery and cleaning the cat excrement are most probably increasing the chance of Toxoplasma transmission if the necessary precaution was taken lightly. However, a previous study in Canada demonstrated that cleaning the cat excrement was not the significant factor contributing to Toxoplasma infection among veterinary personnel  where the longest working duration of ≥21 years had the highest prevalent rate (60%). This might be due to the higher exposure with the animals especially cats since they have a lot of experience in handling and having close contact with cats.Our univariate analysis showed that gardening was identified as one of significant risk factors in this study (d 1 year . Oocystsinfected .6% compaersonnel . Based oToxoplasma infection. This could be explained due to the fact that primary behavioral practices should be advised among people with increasing age, which is more prone to disease transmission of T. gondii. Also, individual with increasing age may have other co-infections that may lower his immune system, which can increase the susceptibility to Toxoplasma infection. The longer working duration contributed to the risk factor of Toxoplasma acquisition since they had a daily routine of handling with animals for long hours. This suggests that they might have a higher chance of close contact with cats and more likely expose to sporulated oocysts in cat’s feces, which can increase the risk of Toxoplasma transmission (After multivariate logistic regression model was applied, it was interesting to find that only age group of ≥30 years old and working duration of >10 years were identified as significant risks for smission .Toxoplasma infection. Hence, basic personal hygiene and management in working and study areas among veterinary personnel should be taken into consideration to minimize the probability of exposure to Toxoplasma infection. Future similar study is recommended periodically and also to investigate other unidentified risk factors to eliminate the infection rate and to eradicate this parasite from the region.This preliminary study shows the high prevalence of chronic toxoplasmosis in both veterinary personnel and pet owners. Age group of ≥30 years old in overall studied populations and working duration of >10 years among veterinary personnel significantly contributed to This work was presented in part at the ICOPA XIII, Mexico, 10–15 August, 2014.VN and TTC designed the study. NAA, TTC, RSKS, and VN carried out the study. GJBM contributed most on manuscript writing. RSKS, YALL, TTC, HA, and VN helped in manuscript writing and editing. RSKS, YALL, TTC, and VN provided opinions and suggestions about this manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Review Editor Yeng Chen declares that, despite being affiliated to the same institution as authors Guo-Jie Brandon-Mong, Nurul Asma Anati Che Mat Seri, Hemah Andiappan, Tian-Chye Tan, Yvonne Ai-Lian Lim and Veeranoot Nissapatornand, the review process was handled objectively and no conflict of interest exists.


Brassica oleracea var. italica). In this study, hybrids and multiple backcrosses were produced from different cytoplasmic male sterile carpelloid stamen sources and maintainer lines. Carpelloid stamens caused dysplasia of the flower structure and led to hooked or coiled siliques with poor seed setting, which were inherited in a maternal fashion. Using four distinct carpelloid stamens and twelve distinct normal stamens from cytoplasmic male sterile sources and one maintainer, we used 21 mitochondrial simple sequence repeat (mtSSR) primers and 32 chloroplast SSR primers to identify a mitochondrial marker, mtSSR2, that can differentiate between the cytoplasm of carpelloid and normal stamens. Thereafter, mtSSR2 was used to identify another 34 broccoli accessions, with an accuracy rate of 100%. Analysis of the polymorphic sequences revealed that the mtSSR2 open reading frame of carpelloid stamen sterile sources had a deletion of 51 bases (encoding 18 amino acids) compared with normal stamen materials. The open reading frame is located in the coding region of orf125 and orf108 of the mitochondrial genomes in Brassica crops and had the highest similarity with Raphanus sativus and Brassica carinata. The current study has not only identified a useful molecular marker to detect the cytoplasm of carpelloid stamens during broccoli breeding, but it also provides evidence that the mitochondrial genome is maternally inherited and provides a basis for studying the effect of the cytoplasm on flower organ development in plants.We previously discovered carpelloid stamens when breeding cytoplasmic male sterile lines in broccoli ( Brassica oleracea L. var italica), sometimes referred to as Calabrese, is the most important commercial form of Brassica [Broccoli  line and an inbred line to create hybrids can reduce the cost of producing hybrids and improve their purity. However, hybrid production is difficult and produces low yields; therefore, CMS sources are crucial in improving the yield of hybrids. The CMS sources of broccoli have mostly been transferred from cabbage, radish, and other related cruciferous species through backcrossing. However, the floral organs often show extremely complex morphological variations because of the lack of coordination between the nucleus and cytoplasm during the transfer process –13. ThesArabidopsis thaliana and are regulated by class B genes of the classic ABC model. If the MADS-box transcription factors APETALA3 (Ap3) or PISTILLATA (PI), which are class B genes involved in conferring identities of the stamen and petal, are mutated or deleted, homeotic conversions of stamens to carpels and petals to sepals can occur [B. juncea var. tumida [B. rapa subsp. chinensis [B. napus [B. juncea [Daucus carota sativus [Carpelloid stamen phenomena have been studied in an occur –17. Furt. tumida , B. rapahinensis –20, B. nB. napus –23, B. j. juncea , and Dau sativus . Thus faArabidopsis thaliana, B. napus (Nap and Pol), B. rapa (Cam), B. oleracea, B. juncea, and B. carinata have been determined [Arabidopsis thaliana and B. napus have also been determined [Mitochondrial and chloroplast genes are mainly inherited in a maternal pattern, with a slow rate of mutation; therefore, mitochondrial and chloroplast DNA have been widely used in evolutionary and phylogenetic studies –31. The termined –39. The termined –41. Owintermined –48 and vtermined –52 and Ctermined –55. HoweThe plant materials used in this study are detailed in These accessions were grown in experimental greenhouses under standard field conditions at the Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences, Changping, Beijing, China, from 2011 to 2014. When the diameter of the main bouquet on each plant was 8–10 cm, we pruned the bouquet, leaving three lateral balls, and then left the plant to develop branches naturally. Plantlets at the eight to ten leaves stage were randomly chosen from each accession for total genomic DNA isolation, using the modified hexadecyltrimethylammonium bromide method .When the plants produced flowers, pollination was performed by hand or naturally  in 2011–2014. Flower morphologies, the pod shapes of each material, and the grain number per pod of maintainer 93219 B1;  and diffBrassica napus mitochondrial genome sequences (Gen Bank GI: 112253843) and 32 pairs of primers for Arabidopsis thaliana chloroplast sequences (Gen Bank GI: 7525012) developed by Zhang [Twenty-one pairs of primers for by Zhang  were use−1) genomic DNA, 12.5 μL Dream Taq™ Green PCR Master Mix , and 1 μL of each primer (10 pmol μL−1). The following amplification protocol was carried out in an ABI Veriti 96-well PCR thermal cycler . Initial denaturation was performed at 94°C for 4 min; followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 10 min. PCR products were analyzed on 2.0% (w/v) agarose gels in 0.5×TBE buffer and visualized after staining with Goldview . The bands were photographed under ultraviolet light under a Universal Hood II .PCR amplifications were carried out in a reaction volume of 25 μL, which contained 2 μL  database.To determine the mechanism of inheritance of the carpelloid stamen phenomenon, we carried out crosses or successively backcrosses between maintainer lines with normal stamens  primers and 21 pairs of mitochondrial SSR (mtSSR) primers developed by Zhang  Table 2Table 2 tThe polymorphic bands obtained after PCR using mtSSR2 were sequenced. The amplicons were identical in all maintainer lines (471 bp), whereas they were 420 bp in all carpelloid stamen materials. Analyses after aligning the polymorphic sequences from normal and carpelloid materials indicated that the similarity of the sequences were 88.96%, with one single nucleotide polymorphism at 100 bp (C/T), and the sequences from carpelloid stamen materials had 51 nucleotides deleted between 309 and 359 bp compared with the normal sequences . Open reorf125 coding region of B. napus, B. juncea, B. rapa subsp. oleifera, Eruca sativa, B. oleracea, B. oleracea var. botrytis, and B. juncea var. tumida. It is also located in the coding region of orf108c and orf108 in B. carinata and Raphanus sativus, respectively. However, the proteins were annotated as being hypothetical and as having unknown functions. Similarity analysis of the amino acid sequences revealed that the protein sequences related to the carpelloid stamen have the highest similarity with orf108c and orf108 in B. carinata and Raphanus sativus, respectively . Zhang et al. [B. oleracea using cpSSR primers. Cheng et al. [1-2, OguCMSHY, and NigCMS (420 bp) from pol CMS and OguCMSR3 (471 bp) [1-2, or NigCMS and was transferred to broccoli. Alignment analysis of the sequences showed that the deleted sequences in broccoli were not at the same location as in cabbage. This will benefit the selection of CMS types and backcross breeding in broccoli; moreover, it will also provide a basis for evolutionary studies in crucifers.The main advantage of organelle genomes is that no recombination can occur between two alleles, because there is only one allele per cell and per organism . Furtherg et al.  studied g et al.  distingug et al.  and crucg et al. , 48, 55.orf108 or orf125 coding regions of mitochondrial genomes in many Brassica species. Compared with the maintainers, the carpelloid stamen materials showed a deletion of 51 nucleotides, suggesting that the deletion of mitochondrial nucleotides could explain the carpelloid stamen phenotype. Thus, it is likely that orf108 or orf125 encodes genes involved in the development of floral organs. Our findings provide an important reference for further study on the molecular mechanism of the interaction between the nucleus and cytoplasm in floral organ development.Furthermore, the sequences of the products were located in In conclusion, we have identified a useful molecular marker to detect the cytoplasm of carpelloid stamens in broccoli. The results of our study are valuable for improving the efficiency of broccoli breeding and also for forming a solid basis for further study of the molecular mechanisms underlying the CMS carpelloid stamen phenomenon.


Plant growth regulators, such as hormones and their respective biosynthesis inhibitors, are effective tools to elucidate the physiological function of phytohormones in plants. A problem of chemical treatments, however, is the potential for interaction of the active compound with the growth media substrate. We studied the interaction and efficacy of propiconazole, a potent and specific inhibitor of brassinosteroid biosynthesis, with common soilless greenhouse growth media for rice, sorghum, and maize. Many of the tested growth media interacted with propiconazole reducing its efficacy up to a hundred fold. To determine the molecular interaction of inhibitors with media substrates, Fourier Transform Infrared Spectroscopy and sorption isotherm analysis was applied. While mica clay substrates absorbed up to 1.3 mg of propiconazole per g substrate, calcined clays bound up to 12 mg of propiconazole per g substrate. The efficacy of the gibberellic acid biosynthesis inhibitor, uniconazole, and the most active brassinosteroid, brassinolide, was impacted similarly by the respective substrates. Conversely, gibberellic acid showed no distinct growth response in different media. Our results suggest that the reduction in efficacy of propiconazole, uniconazole, and brassinolide in bioassays when grown in calcined clay is caused by hydrophobic interactions between the plant growth regulators and the growth media. This was further confirmed by experiments using methanol-water solvent mixes with higher hydrophobicity values, which reduce the interaction of propiconazole and calcined clay. Gibberella fujikuroi, whereas BRs were first identified in pollen of Brassica napus and are polyhydroxylated steroidal hormones  and brassinosteroids (BRs), benefited greatly from the use of chemical inhibitors hormones  [3]–[4].Arabidopsis thaliana, comprehensive understanding of the biological function of BRs in agronomical crops such as maize is still lacking. One of the limitations that have hindered research progress is availability and cost of some of the PGRs. While Ucz and GA are easily accessible and found use in horticultural greenhouse production Arabidopsis and maize as an alternative BR inhibitor (Sclerotinia homoeocarpa) in turf grasses on golf courses and sod farms. It is also registered for use as fungicide in maize. Global sales of Pcz exceeded US$ 200 million in 1995 et al. A number of triazole compounds have been shown to inhibit cytochrome P450 enzymes, which are key components of both, GA as well as BR biosynthesis. Uniconazole (Ucz) and brassinazole (Brz) are two of the classical inhibitors shown to interact with CYP701 of the GA and CYP90B1 of the BR biosynthetic pathways, respectively. Although well characterized in the model plant nhibitor . Not onlArabidopsis, growing mature crops in MS is often difficult and cost inefficient. To achieve optimal growth under greenhouse conditions, larger crops such as maize, rice, or soybean often require a variety of soilless growth substrates varying from sphagnum peat moss to calcined clay. Furthermore, perlite or vermiculite additives are often used to improve substrate aeration and soil health to increase growth and yield. Whereas agar-based MS media was designed to be neutral in its interactions with chemicals, most of the soilless growth substrates have not been studied for their potential to influence the efficacy of biochemical treatments. Previous work showed that Pcz in different soils is not very mobile and had a high retention time Plant growth experiments often require the use of various growth media substrates to control for water retaining potential, nutrient availability, and anchoring of roots in the media. Murashige and Skoog (MS) agar-based media was developed for tobacco as a suitable substrate that allows rapid growth and shows minimal side effects on bioassays and chemical treatments in tissue culture The interaction of PGR with growth media has been an important research topic for the ornamental plant industry for many years 3. The efficacy of Pcz varied by more than 100-fold between media substrates tested. The ability of calcined clay substrates to interfere with the efficacy of triazole-based PGRs is based on the hydrophobic interactions of PGRs with the substrates. Our data suggests that various media substrates have a significant impact on the efficacy of chemical treatments and the reproducibility of such experiments, which illustrates the importance of appropriate media selection for the given experimental design. We show how differences in interaction of chemical compounds with growth substrates can be explained based on physicochemical properties of both interaction partners.Here, we present the characterization of the four most commonly used greenhouse growth substrates vermiculite, perlite, sphagnum peat moss, calcined clay, or combinations thereof, and their interactions with Pcz, 24-epibrassinolide (eBL), Ucz, or GASamples of various growth media substrates were mounted on aluminum stubs using a carbon tape adhesive. Samples were viewed using a JEOL 6610 LV Scanning Electron Microscope  in standard vacuum mode at 5 kV accelerating voltage. The vermiculate has a layered structure so care was taken to view the surface from different angles. Initial attempts to view the surface of Turface resulted in substantial charging artifacts. To overcome this, Turface was sputter-coated with gold. This procedure resolved the charging issue.Maize B73 seedlings were grown under greenhouse conditions at 27°C (day) and 21°C (night) with 16 h of supplemental lighting. Plants were fertilized with 200 ppm Miracle-Gro Excel  adjusted to pH 6 following manufacturer recommendations. For all treatment experiments seeds were sterilized for 7 min at 60°C in a water bath prior to planting and grown under greenhouse conditions. Seeds were planted 5 cm deep in 24.5 cm wide pots with vermiculite extra-coarse grade , Turface MVP , Perlite coarse grade , Peat germinating mix , or mixtures thereof and watered every fifth day. Pcz  was added at indicated concentrations to the watering solution. After 21 d plants were harvested, photographed, and analyzed using ImageJ 3  dissolved in 0.02% ethanol, B73 seedlings were grown under greenhouse conditions as previously stated. Seeds were planted 5 cm deep in 606 trays with respective growth media and treatment. After 9 d seedlings were harvested, photographed, and analyzed using ImageJ For treatment experiments grown on Turface or vermiculite comparing Pcz, Ucz , eBL  dissolved in 0.02% ethanol, or GAHPLC-MS analysis was performed using an Agilent 1100 HPLC system  with a Shimadzu Shim-pack XR-ODS column (75 × 3.0 mm). Isocratic elution was used, with a mobile phase composition of 40% deionized water and 60% acetonitrile, with a buffer of 0.1% formic acid (v/v). Mobile phase flow rate was 0.6 ml/min. Following separation the column effluent was introduced by positive mode electrospray ionization (ESI) into an Agilent MSD time-of-flight mass spectrometer. ESI capillary voltage was 3.5 kV. Nebulizer gas pressure was set at 55 psig, gas temperature was 350°C, and drying gas flow rate was 11 l/min. The fragmentor voltage was set to 135 V, skimmer 65 V and OCT RF V 250 V. Spectroscopic (UV 200 - 350 nm) and mass (m/z 70–1500) data were collected and analyzed using Agilent MassHunter software.−1 region. A background spectrum was obtained using KBr (only) in the sample cup. FTIR spectra of Pcz and pure Pcz (95%)  were obtained using a Pike GladiATR  on a Thermo Scientific Nicolet 6700 FT-IR spectrometer . A background spectrum was obtained from the clean ATR crystal. FTIR spectrum analysis was collected using OMNIC series software .Turface was combined with 15.4 mg of Pcz in a 50 mL Falcon tube for 500 h to obtain Pcz+ Turface and was dried for 48 h in a drying oven at 50°C. FTIR analysis of Turface and Pcz+Turface was performed using a Thermo Scientific Smart Diffuse Reflectance accessory on a Thermo Scientific Nicole 6700 FT-IR spectrometer . A mixture of 5% Turface or Pcz+Turface in KBr was prepared using oven dried KBr powder (10 mg Turface or Pcz+Turface and 190 mg of ground KBr) were weighed and placed in an agate vial and mized using a Wig-L-Bug for 30 seconds. The prepared mixture was then placed in a sample cup in the diffuse reflectance accessory. FTIR spectra were collected using a medium-band MCT detector in the spectral range of 4000 to 700 cm1 g of respective media was allowed to interact with 15.4 mg/40 ml of Pcz in a 50 mL Falcon tube for up to 265 h at room temperature in water or MeOH solvent mixes. Tubes were agitated on a rocker at 100 rpm. At each time point, a 15 µl aliquot of the supernatant was removed and centrifuged to remove excess media particles. Absorption was measured using a NanoDrop photometer  at 225 nm. The isotherms were then calculated to determine the amount of Pcz adsorbed to the respective substrate over time.Seeds were germinated in silica white sand  with the respective chemical treatment for 6 d. Seedlings were removed from growing media and sand was washed from roots, and seedlings were transferred to aeroponics cultivation device . In aeroX-ray diffraction patterns of Turface samples were obtained from oriented samples on glass slides http://xltoolbox.sourceforge.net) was used to obtain all descriptive and comparative statistics. Analyses of variance (ANOVA) for sets of data groups were performed with “Multiple comparisons/Post-hoc” testing. Once a significant difference (p<0.05) was detected “Post-hoc” tests, using the Holm-Sidak algorithm, were performed to test for significance of the possible multiple comparisons between the data groups The Microsoft Excel add-in XL Toolbox  of Turface and the other three growth substrates, respectively. At a concentration of 20 µM, Pcz-mediated inhibition of plant height was not significantly different between plants grown in Turface and Turface 1∶1 mixtures . Turface2 = 0.9989) with a series of spiked concentrations of Pcz in solution  to confirm the identity and quantify the presence of Pcz in solution. Pcz was identified in the HPLC based on its exact mass and natural isotope distribution, with an overall confidence score of 98.3% (. 4 A). The correct empirical formula was predicted, with an exact mass error of −1.2 ppm. Two local UV maxima in the absorption spectrum of Pcz at 204 and 225 nm were detected . Quantifsolution . The Pczsolution . A one-hsolution . In contsolution . These rOur results suggested that Pcz is depleted from the supernatant after contact with Turface and to a lesser extent vermiculite, but the actual cause remained elusive. Given that the cation exchange capacity (CEC) of vermiculite (100–150 meq/100 g) −1), the HOH bending region (1630 cm−1), and sorbed water (3000–3600 cm−1)  and P-line (purple) (−1 and 1555 cm−1 (pP), 1637 cm−1 and 1722 cm−1 (P), 1555 cm−1, 1643 cm−1, and 1730 cm−1 (TP-T).The spectrum of Pcz sorbed to Turface showed the same features of the Si-O stretch, HOH bending region and sorbed water, however, with additional bands in the range from 1750 to 1350 cm586 cm−1 . The for(purple)  is due tTo further test the hypothesis that Pcz directly binds to Turface and to measure the binding capacity of Pcz to Turface we determined its adsorption isotherm by saturating Turface with Pcz. We found that 3.3 mg Pcz was adsorbed by 1 g Turface in the first hour, and that the binding equilibrium was not reached until 242 h of incubation with a total of 11.9 mg Pcz/g Turface adsorbed. Conversely, vermiculite showed a relatively low capacity to bind Pcz. Binding equilibrium was reached at a total of 1.33 mg Pcz/g vermiculite, and remained stable over the 265 h time course . Not onl3, as well as the growth inhibitor Ucz. B73 seedlings grown in vermiculite treated with eBL, Pcz, or Ucz at concentrations of 1 or 10 µM produced dark green leaves accompanied by a dwarfed plant stature. In particular, 10 µM eBL, Pcz, or Ucz reduced the height of seedlings by 31%, 57%, or 78%, respectively, when compared to controls (3 was not significantly different (p = 0.45) in either Turface or vermiculite. A drench application of 10 µM GA3 increased the height of vermiculite and Turface grown plants by 87% or 90%, respectively . This raises the question whether Pcz efficacy is still reduced due to its interaction with vermiculite. One way to investigate this question is to completely circumvent the use of media substrates through the use of aeroponic culture. Instead of using solid growth media, aeroponic systems rely on the use of mists or aerosols to deliver both nutrients and chemical treatments to the plant root system sd1 in rice) or signal transduction (Rht1 and Rht2 in wheat) Growth promoting hormones, such as GA and BRs, have been a focal point of research due to their potential to increase the harvest index or biomass production of agriculturally important crops Arabidopsis is that it hardly affects biochemical treatments. However, to sustain growth of larger crop species until maturity, MS agar media is often inadequate. Very little is known about how other common growth media impact chemical treatments. In this study we focused on the quantitative impact and mechanism of media interaction with biochemical treatments. Pcz, a triazole compound, is commercially used as a fungistat against a broad range of phytopathogenic fungi. Its fungistatic mode of action is the blocking of lanosterol 14R-demethylase (CYP51A1), similar to that of Ucz Lepidium sativum, we observed Pcz induced BR deficient phenotypes in treated maize B73 plants One key advantage of MS agar media as a preferred media for model species such as Given its widespread use as a fungicide, Pcz has been studied for its potential effects on the environment et al. In conjunction with previous reports we were able to detect Pcz within solution using HPLC-MS To investigate if Pcz is directly bound by Turface we used FTIR. The spectra provide direct evidence that the Pcz is adsorbed to Turface and therefore interferes with uptake of the inhibitor by the plant. The spectral features of Pcz are minimally perturbed by Turface and do not provide clear insight into the binding mode of Pcz to Turface. Based on the low solubility of Pcz, a likely mechanism would involve hydrophobic interactions of Pcz with the calcined siloxane surface of Turface 3 in both growth media. Similar to Pcz, high concentrations of eBL drench treatment were ineffective in Turface but showed an expected reduction in plant height when grown in vermiculite. High concentrations of eBL have a growth inhibiting effect in plants, as exogenous eBL induces a negative feedback mechanism, which down-regulates expression of BR biosynthesis genes 3 in drench applications in either, Turface or vermiculite. In both cases, 10 µM GA3 almost doubled the size of treated plants (3 is a much more hydrophilic compound (solubility 5g/l) compared to Pcz (100 mg/l), Ucz (8.41 mg/l), or eBL (5 mg/l). Pcz has a pKa of 1.09, which would generate its protonated form only at very acidic conditions not present in the tested environment. Therefore, Pcz is mostly neutral in solution, which makes it highly hydrophobic. Turface is made up of kaolinite, illite, and quartz calcined at 650°C . The hydrophobic nature of kaolinite and illite is further enhanced by calcination and thus creates more intense hydrophobic areas on the siloxane surface of Turface The differences in Pcz efficacy observed between Turface and vermiculite raised the question whether this effect was specific to Pcz or is characteristic for many PGRs. Hence we tested the efficacy of Ucz, eBL, and GAd plants . One pos3 with Turface and given the similar negative charge of GA3 and vermiculite, this may also explain the lack of interaction between them. Together our data indicates that Pcz, Ucz, and eBL interacted with Turface through hydrophic interactions but not as readily with the highly charged vermiculite. Although we cannot rule out an influence of surfactants in the case of Pcz and Ucz, both eBL and GA3 were supplied in ethanol without additional surfactants.Vermiculite is high-density charged clay and therefore strongly hydrophilic, which reduces interactions with hydrophobic compounds such as Pcz. This hypothesis is supported by lack of interaction observed for charged GAArabidopsisAlthough the Pcz binding capacity of vermiculite was significantly lower than that of Turface, the question remained whether vermiculite negatively impacts Pcz effectiveness. We elucidated this by testing Pcz applications without media substrates. Concentrations of 1 µM Pcz or higher significantly reduced both plant height and root growth compared to mock treatments . This suThe choice of media substrate is rarely considered an important aspect of the experimental design and is primarily based on optimizing the growth conditions for the plant. However, our results illustrate how drastically the tested media substrates influence the efficacy of various chemical growth regulators. The ability of calcined clay substrates to impair the efficacy of triazole-based PGRs is based on their hydrophobic interactions. This emphasizes the importance of appropriate media selection to balance growth conditions with the effectiveness of biochemical treatment studies. Vermiculite is an applicable medium for chemical treatment bioassays, as it minimally interacts with applied compounds. Turface is a more suitable medium for long-term growth of larger plants, such as maize, however it strongly inhibits the efficacy of hydrophobic soil-drenched PGRs.S1 FigureAbsorbance kinetics of Pcz on Turface. Absorbance values of Pcz in the supernatant at 225 nm when interacting with 2 g (grey bars) or 20 g (black bars) of Turface tested from 0 to 23 h. Error bars represent standard deviation.(TIF)Click here for additional data file.S2 FigureX-ray diffraction of Turface. X-ray diffraction pattern of Turface. Analysis was obtained using a PANalytical X-ray diffractometer  using Co radiation. Software analysis identified that its main components are kaolinite (K), illite (I), and quartz (Q). Major characteristic peaks are indicated.(TIF)Click here for additional data file.S3 FigureAeroponic System. (A) Overview of aeroponics setup in a greenhouse with 24 spray containers in groups of three, bearing up to eight plants each. (B) 20 l steel pressurized tanks with maintained pressure by air compressor. Hoses for input of pressurized air and output into the spray buckets are connected to the tanks. Each tank feeds one group of three spray buckets. (C) Bottom of a 20 l spray bucket with microjet nozzle to spray the root space and run-off outlet to remove excess spray solution. (D) Air conditioning unit to control temperature of the root zone and Chrontrol XT series electronic timer device to control frequency and duration of spray treatment. (E) Maize seedlings mounted into styrofoam tops after two weeks of aeroponics culture.(TIF)Click here for additional data file.S4 FigureChlorophyll content measurements (CCI) of Pcz and Ucz treated seedlings. Chlorophyll content index of B73 seedlings grown in silica sand for 6 d then transplanted to aeroponic culture for 9 d when measurements were taken. CCI was measured directly in the middle of the second leaf between the leaf collar and leaf tip using a CCM-200 chlorophyll content meter . Error bars represent standard deviation and lower class letters indicate significant differences between treatments as determined by “Post-hoc” test (p<0.05). n = 12.(TIF)Click here for additional data file.