Burkholderia fungorum FM-2 (GenBank accession no. KM263605) was isolated from oil-contaminated soil in Xinjiang and characterized morphologically, physiologically, and phylogenetically. Environmental parameters including PHE concentration, pH, temperature, and salinity were optimized, and heavy metal tolerance was investigated. The MIC of strain FM-2 tolerant to Pb(II) and Cd(II) was 50 and 400 mg L−1, respectively, while the MIC of Zn(II) was >1,200 mg L−1. Atypically for a B. fungorum strain, FM-2 utilized PHE (300 mg L−1) as a sole carbon source over a wide pH range (between pH 3 and 9). PHE and heavy metal metabolism were assessed using gas chromatography (GC), inductively coupled plasma optical emission spectroscopy (ICP-OES), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), Fourier-transform infrared (FTIR) spectroscopy and ultraviolet (UV) absorption spectrometry. The effects of heavy metals on the bioremediation of PHE in soil were investigated, and the findings suggest that FM-2 has potential for combined bioremediation of soils co-contaminated with PHE and heavy metals.Phenanthrene (PHE) is a common pollutant of acidic and non-acidic environments that is recalcitrant to biodegradation. Herein,  Polycyclic aromatic hydrocarbons (PAHs) fused-ring aromatic compounds consisting of two or more fused benzene and/or pentacyclic rings  Burkholderia cluster are able to exploit diverse aromatic compounds as sources of energy and carbon, and some have considerable biotechnological potential due to their ability to degrade chemical pollutants . Bacterial strains capable of degrading PHE were isolated using the selective enrichment method. PHE (purity ≥97%) and other reagents were purchased from Energy Chemical Technology Co. Ltd.4, 2.04 g KH2PO4, 12.5 g Na2HPO4·12H2O, and 0.4 g (NH4)2SO4 (per L of distilled water) was used for isolating microorganisms capable of utilizing PHE as a sole source of carbon and energy source.Minimal medium (pH 7 ± 0.2) consisting of 0.1 g MgSO4, 3.48 g KH2PO4, 1.5 g Na2HPO4·12H2O, 3.96 g (NH4)2SO4, and 0.01 g yeast per L of distilled water.Enrichment of the strain before screening was performed using mineral salt medium (pH 7 ± 0.2) containing 0.7 g MgSO2PO4, 0.1 g Na2HPO4, 0.5 g NH4NO3, 0.5 g (NH4)2SO4, 0.2 g MgSO4, 20 mg CaCl2, 2 mg FeCl2, and 2 mg MnSO4 per L of distilled water  contained 0.1 g KHScreening of conditions for PHE-degrading strains was performed as described previously . PHE was dissolved in n-hexane, and then added into 100 ml flask at final concentration of 300–600 mg L−1. The flasks were shaken for 2 h at 200 rpm and 25°C. A thin film of PHE was left at the bottom of the flask after removing n-hexane, and then MM was added. The enrichment culture was sub-cultured aerobically (shaking at 200 rpm) at 25°C to facilitate degradation of PHE. Bacterial cell growth was monitored by measuring the absorbance (Abs) at 600 nm, and degradation of PHE was confirmed by both color change and gas chromatography (GC) analyses. The remaining PHE was extracted from the whole culture (30 mL) in a erlenmeyer flask twice with 30 ml of n-hexane . The organic phases were combined and dried with anhydrous Na2SO4. The solvents were removed under reduced pressure and the residues were dissolved in n-hexane. Gas chromatograph equipment  was applied to detect the variation of PHE in the n-hexane. Ten microliter of the organic phase was analyzed by using a HP-5 type capillary column . During the analytical process, the column temperature was kept at 80°C for 1 min, then programmed to 290°C at a rate of 10°C min−1 which was maintained for 10 min. Media not inoculated with cells were used as sterile control. Control tests with different PHE concentrations were performed to evaluate the actual PHE recovery efficiency after extraction, which was over 87.2% at 300–600 mg L−1 PHE. The above mentioned methods are based on the approach reported by Tao et al.  agar plates several times to guarantee culture purity. A single colony was picked from the final LB agar plate and inoculated in 30 mL mineral salt medium with 2% glycerol. After culturing for 48 h at 200 rpm and 25°C, a 600 μL aliquot of the fermentation broth was transferred to 30 mL minimal medium containing PHE , and the initial pH was adjusted to 7.0. A control culture inoculated with boiled FM-2 was also included, and PHE degradation was evaluated by measuring the change in absorbance at 600 nm, and also by GC analysis  supplemented with 300, 400, 500, and 600 mg L−1 PHE. The pH was tested at intervals of 1.0 between 2.0 and 11.0. NaCl was tested between 0 and 20 g L−1 in minimal medium at pH 7.0, the temperature was assessed between 15 and 40°C, cultivation was performed for 1, 2, and 3 days, and the inoculation volume and sampling operations were as described above.The pH, salinity, temperature, and cultivation duration were optimized using an appropriate amount of biomass in a 100 mL conical flask containing 30 mL minimal medium and 300 mg L−1 ZnSO4·7H2O, Pb(NO3)2, and CdCl2·5/2H2O based on minimum inhibitory concentration (MIC) tests, and working test solutions were sterilized filtration trough a 0.45 μm membrane. LMM medium was separately supplemented with each of the heavy metals and 300 mg L−1 PHE, inoculated with 2% (v/v) FM-2, and cultured at 25°C with shaking at 200 rpm for 7 days. Cultures containing LMM, FM-2, and each heavy metal but without PHE were included as control I, and LMM supplemented with FM-2 and 300 mg L−1 PHE but without heavy metals were included as control II. Finally, a sterile control culture without FM-2 was included to assess abiotic loss of PHE.Heavy metal resistance of the PHE-degrading FM-2 strain was assessed using 20 mg LB. fungorum FM-2 into LMM medium containing 200 mg L−1 Zn (II), 50 mg L−1 Pb (II), or 200 mg L−1 Cd(II) as well as 300 mg L−1 PHE. Cultures were incubated at 25°C with shaking at 200 rpm for 1, 3, 5, and 7 days. As described previously  were separated in different 50 mL centrifuge tubes, pellets were washed three times with sterile distilled water, disrupted using an ultrasonic cell disrupter, and the resulting supernatant (S2) was collected by centrifugation . S1 and S2 were analyzed using a Vista MPX ICP-OES instrument  to determine the heavy metal content of cell walls (S1) and the intracellular space (S2) , 50 mg L−1 Pb (II), or 200 mg L−1 Cd(II) as well as 300 mg L−1 PHE. Cultures were incubated at 25°C with shaking at 200 rpm for 0, 1/2, 1, 2, 3, 4, 5, 6, and 7 days. Specific experiment procedures were carried out according to the literature reported by Vishan et al. , 50 mg L−1 Pb (II), or 200 mg L−1 Cd(II) at 25°C with shaking at 200 rpm for 7 days as described previously  and Adsorption Isotherms, cell pellets were obtained by centrifugation , washed three times with deionised water, and lyophilized. Bacterial cells grown in LMM without metal ions served as a control. FTIR spectra of KBr pellets of dried cells associated with or without Zn (II), Pb (II), and Cd (II) were obtained using a Vector 33 FTIR instrument .−1 zinc sulfate or 6 mg kg−1 cadmium chloride into soils, respectively. All cadmium and zinc amendments were achieved by dissolving 200 mg ZnSO4·7H2O or CdCl2·5/2H2O in 10 mL of sterile deionised water prior to adding to the appropriate soil treatments. Then PHE was mixed with soil at 150 mg kg−1 soil dry weight, and experimental procedures, which lasted 15 days, were performed as previously described  were prepared for calibration. GC-MS operating conditions were set as follows: initial oven temperature was held at 40°C for 1 min, followed by an increase to 250°C at 10°C min−1. This temperature was held for 3 min and the total run time was 35 min. PHE analysis was performed on days 0, 1, 3, 5, 7, 9, 11, 13, and 15. These researchers adopt similar methods for the determination of PHE degradation rate  was prepared based on previous work  of Burkholderia species has classified them into two main groups; Paraburkholderia  and pathogenic species  and detrimental (bad) categories purely based on taxonomy has been questioned  turned from transparent to dark yellow /mL. PHE degradation by strain FM-2 was investigated using minimal medium supplemented with PHE ranging from 300 to 600 mg L−1. As shown in −1 PHE was almost completely degraded within 3 days, while the bacterial cell density remained almost unchanged. These results indicate that B. fungorum FM-2 is a more powerful biodegrader of PHE than Sphingobium chlorophenolicum C3R that metabolizes <65% PHE within 3 days at an initial PHE concentration of 300 mg L−1 in a liquid culture comparable to the present work  was only 78.4% after 15 days of culture, which was isolated from a petroleum-contaminated site at Karamay in Xinjiang in China  could degrade 250 mg L−1 of PHE, and its degradation efficiency was 56.9% after 14 days of culture  NaCl was found to be optimal . A relat−1 over a pH range from 6.0 to 9.0. The highest degradation rate occurred at pH 7.0, and degradation was slightly lower at pH 8.0. Furthermore, the bacterial cell density (OD600) was comparable at pH 7.0 and 8.0, in accordance with the PHE degradation rate, but the cell density was decreased markedly at pH 2.0, 10.0, and 11.0, indicating some limitations for cell growth. However, when the pH of the culture medium was three, the OD600 value of strain FM-2 was 0.512, and the PHE-degrading rate was 50.23%, demonstrating that the strain is strongly resistant to acidic conditions. This finding is important because only very few PAH-degrading bacteria such as Mycobacterium  and 50 mg L−1 for Pb (II). However, an MIC value for Zn (II) was not obtained because strain FM-2 still displayed potent degradation of PHE at a Zn concentration of 1,200 mg L−1 (data not shown) after 7 days. Addition of Zn at 1,200 mg L−1 did not inhibit bacterial growth, possibly due to complexation of Zn (II) by bacterial biosurfactants. As shown in As shown in −1), it had little effect on PHE degradation or FM-2 cell growth. However, when the Cd (II) concentration was increased to 60 mg L−1, cell growth was significantly diminished, and PHE degradation was also decreased to 49.25%. As shown in −1) of Pb (II) in LMM medium lowered the PHE degradation rate after 7 days. Our results indicate that Pb (II) was the most toxic heavy metal and potent inhibitor of PHE degradation by strain FM-2, whereas low concentrations of Zn (II) promoted cell growth. Therefore, resistance to heavy metals was ordered Zn (II) >Cd (II) >Pb (II). This could be due to different responses to heavy metals in PHE degraders  was added to LMM medium at low concentrations  or Pb(II), the result of Cd(II) fitted better in Freundlich adsorption model. The Langmuir isotherm suggests that the biosorption of metal ions can be regareded as a single-layer adsorption with no interaction between metal ions , Pb (II), and Cd (II) biosorption (−1 due to complexation of -OH groups with Zn (II), Pb (II), and Cd (II) ions. Similar results have been reported for Bacillus cereus after treatment with As (III) , Cr (VI), Fe (III), and Ni (II) , consistent with primary and secondary amide groups of amide and protein peptide bonds. This peak was shifted to a higher frequency  following complexation of amide groups with Zn (II), Pb (II), and Cd (II) ions. The observed absorption band around 1,398 cm−1 could be assigned to the C = O stretching of carboxyl groups  and 6 mg L−1 for Cd (II), as summarized in −1 Cd (II) clearly inhibited degradation of PHE in soil, strain FM-2 still enhanced PHE degradation under these conditions, suggesting that strain FM-2 is a candidate strain for bioremediation of PHE and heavy metals. The capacity of strain FM-2 to tolerate Cd (II) and Zn (II) while degrading PHE demonstrates its feasibility for the bioremediation of soils and sediments co-contaminated with PAHs and heavy metals.In a previous time-course metagenomic study , which was initially determined the gene as PAHs dioxygenase gene. We also obtained the Protocatechuate 3,4-dioxygenase gene related to PHE degradation, which had 100% homology of the pcaH gene  sequence from Paraburkholderia fungorum strain ATCC BAA-463 (CP010027.1). The heavy metal removal genes of strain FM-2 were detected, and it has an identical function with the TonB dependent receptor family protein of Paraburkholderia xenovorans LB400 (CP008760.1), but their homology is only about 64%. TonB gene has been reported broardly, which generally involves in the transportation of many important substances such as iron, heme, vitamin B12, carbohydrates, and various metal elements transition  that enhanced PHE degradation, which revealed the effects of different minerals on the microbial degradation for PAHs and the underlying mechanism  under both sufficient oxygen and microaerobic conditions. After 3 days, the degradation efficiencies of PHE were 100 and 69.7%, respectively. However, strain FM-2 cannot grow under anaerobic conditions and did not degrade PHE and adsorb heavy metals. We would further explore the removal efficiencies of strain FM-2 on heavy metals under different culture conditions . We also would use mini-bioreactor to simulate different external environments, and determine the optimal growth conditions of strain FM-2 for removing heavy metals, which aims to improve its efficiencies for practical applications.Potential environmental issues have been considered into microbial bioremediation process. Minerals in the environment, including Kaolinite and quartz, were found to be useful for PAHs degradation  had a positive effect on PHE degradation, while low concentrations of Cd (II) had the opposite effect. However, the presence of Pb (II) strongly inhibited the PHE-degrading capability of strain FM-2. ICP-OES, SEM-EDS, and FTIR spectroscopy analyses following metal accumulation further confirmed that metal ions were associated with the cell wall and intracellular space of strain FM-2. Importantly, strain FM-2 displayed versatile catabolic activity and the ability to tolerate high concentrations of toxic metals while degrading PHE. Additionally, FM-2 exhibited high tolerance to acidic pH and very few PAH-degrading bacteria capable of oxidizing PAHs at acidic pH have been reported to date. These findings suggest that the multifunctional B. fungorum FM-2 strain may offer a cost-effective bioremediation strategy for soils and sediments contaminated with multiple pollutants, most notably in acidic environments suffering long-term pollution.In the present study, strain FM-2 was enriched from oil-contaminated soil collected from an oilfield in Xinjiang, and found to degrades PHE as a sole energy and carbon source when supplied at 300 mg LLH made substantial contributions to the experimental design and revised the article critically for important intellectual content. XL made substantial contributions to the acquisition of data, and the analysis and interpretation of data as well as drafting the article. XH conducted the Fourier-transform infrared spectroscopy test. YC tested the soil. WP was responsible for scanning the electron microscopy-energy and undertaking the dispersive X-ray spectroscopy test. JH was responsible for inductively coupled plasma optical emission spectroscopy test. PG conducted the phenanthrene biodegradation test.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.


Given the impact of ROS on Ca2+ homeostasis and Ca2+-dependent signaling, cytosolic Ca2+ was measured to monitor cellular perturbations. We found that H2O2 disrupted Ca2+ homeostasis in Se0 pollen only, while Se1 samples were preserved. The same trend was observed when Se0 samples were treated with sodium selenate or Se-methionine, which recapitulated in vitro the protective capacity of Se-fertigation. Furthermore, we found that germination rates were much better retained in Se1 as compared to Se0  after exposure to 20 mM H2O2. The same was observed with Se0 pollen treated with Se-methionine, which is the organic form of Se into which most fertigated sodium selenate converts in the plant. These results, together, show a close correlation between ROS, Ca2+ homeostasis and pollen fertility, and provide strong evidence that Se-fertigation is an excellent approach to preserve or enhance agricultural productivity.Selenium (Se) displays antioxidant properties that can be exploited, in plants, to counteract abiotic stresses caused by overly-produced reactive oxygen species (ROS). Here, we show that fertigation of maize crops with sodium selenate effectively protects pollen against oxidative stress. Pollen isolated from Se-treated plants (Se1) and untreated controls (Se0) was incubated  However, studies also indicated that, as a dietary supplement, Se should be taken with caution based on toxic effects that may appear at higher concentrations3. Se-intake from the diet can largely vary based on differences in soil composition of the land where plants are grown and animals raised. In this regard, a critical factor is the particular capacity of a given plant to metabolically engage, and subsequently accumulate, Se in various forms5. Frequently, foliar or soil Se-fertilization is undertaken in agricultural crops in order to improve Se concentration in edible plants10.Selenium (Se) is a microelement that, over the last decade, has gained much attention in disease prevention in light of its antioxidant properties12 and, for this reason, can be considered a protective agent against abiotic perturbations that may be caused by changes of temperature, sources of nutrients, and concentration of salts, which, ultimately, can cause ROS levels to rise13. In plants, ROS are commonly produced by the aerobic energy system in cellular organelles such as chloroplasts, mitochondria and peroxisomes14. At low concentrations, ROS act as signal molecules that become involved in numerous biological processes such as vegetative growth and seed production in plants19. However, increased levels of ROS, in any of its chemical varieties, result in oxidative stress as a consequence of inadequate scavenging ability of the cell20.Se is known as a powerful antioxidant capable of counteracting ROS-induced oxidative stress22. To this end, ion exchanges mediate cell signaling events that guide the male microgametophyte towards the female egg19.Higher plants have a specialized, sexual reproduction system based on transport and delivery over greater distances of large amounts of male microgametophytes (i.e. pollen grain). In Angiosperms, upon landing on the right stigma, pollen granules quickly hydrate and begin to germinate. Timely germination of the male microgametophyte followed by rapid elongation of the pollen tube are both essential to ensure the sexual reproduction of plants2+ as a secondary messenger in plant cells27. In the plasma membrane of the plant cell, several channels regulate Ca2+entry, however, despite the ion’s permeability, none of these channels appeared to be Ca2+ specific17. Ca2+ enters the cell through these channels and activates vertical cell growth19. Additionally, the formation of the pollen tube is Ca2+-dependent in the pollination process29.In particular, a number of studies pointed to the physiological role of Ca14. High concentrations of ROS can alter the molecular signals of the cell, including that mediated by cytosolic Ca2+. ROS act like agonists by either stimulating the mobilization of the ion from internal Ca2+ stores such as endoplasmic reticulum, Golgi vesicles and vacuoles30, or activating the Ca2+ entry from the extracellular medium, thereby disrupting the thousands-fold gradient between nanomolar and micromolar concentrations that the cell tightly maintains in the cytosolic and the extracellular space, respectively36. A large body of literature describes the interaction between ROS and Ca2+, however only scant information exists about the effect of either inorganic or organic Se in the relationship between ROS, Ca2+ signals and pollen germination37.ROS-mediated oxidative stress can cause infertility of pollen with obvious negative repercussions on plant reproduction and, in turn, agricultural production2+ and preserve germination of corn pollen grains, suggesting that Se should be included in soil fertilizers to enhance the plants’ response to ROS formation. Furthermore, results point to a strong correlation between levels of cytosolic Ca2+ and the mechanistic events that underlie the germination process.Here, we provide evidence that Se, under oxidative stress, can maintain cytosolic Ca38 however, Se1 samples displayed an especially rough, grainy appearance, which differed from a much smoother outer wall surface found in Se0 counterparts.Initially, to assess any morphological alterations of pollen following treatment with Se, we generated images (500X) by Field Emission Scanning Electron Microscopy, as shown in Fig. 9. Thus, we decided to investigate abundance and chemical fate of Se-treated pollen. In untreated maize pollen, Se mainly accumulated in organic form such as Se-methionine (SeMet), which represented more than 95% of the organic pool, while Se(IV) accounted for about 2/3 of the total inorganic fraction , respectively than in Se0.2O2 was subsequently employed to challenge pollens with oxidative stress in order to test the antioxidant potential of Se treatment, we also analysed the supernatant obtained from pollen treated with 10 mM H2O2. In this regard, virtually identical results were found in both Se1 and Se0 samples . As shown in Fig. 2+ entry was essentially negligible in the absence of H2O2 and up to concentrations of 1 mM H2O2. Instead, Ca2+ entry, ultimately leading to higher [Ca2+]cp, was evidently increased, in a dose-dependent fashion, with higher doses (5–20 mM) of H2O2  maize pollen in the presence of increasing doses (1–15 μM) of sodium-selenate (SeO4-2) and Se-methionine (SeMet) in pollen incubation medium supplemented with 10 mM H2O2.To assess the effect of inorganic and organic Se under oxidative stress, we carried out analyses 4-2 or SeMet mitigated the effect of 10 mM H2O2, which could not be detected any longer when SeO4-2 and SeMet doses achieved 3.4 μM and 1 μM, respectively .For comparison purposes, we tested SeO40. As shown in Fig. 2O2 reduced rates in a dose-dependent fashion. However, the inhibitory effect of H2O2 was much more pronounced in Se0 than Se1 pollen, as germination curves at increasing H2O2 concentration progressively diverged. At the highest H2O2 concentration employed (20 mM), Se-treated pollen germinated at 18% ± 0.5 rate, which represented 46% efficiency in relation to normal conditions. Instead, the same amount of H2O2 almost abolished  germination in untreated pollen. Interestingly, Se0 pollen exposed in vitro to 1 μM SeMet exhibited germination rates similar to Se1  of SeO4−2 or SeMet, caused a dose-dependent reduction of germination ability. Figure 4−2 form was slightly stronger than that displayed by organic SeMet.In the absence of oxidative stress, incubation of Se0 samples Here, we show that pollen obtained from maize crops exposed to a Se-supplemented fertilization regimen has a superior capacity to counteract ROS-mediated effects that may negatively impact germination rates.2O2 in pollen preparations to raise oxidative stress in vitro. Conditions related to fertigation, and the setup of the cellular oxidative model were previously established .Our experimental setup implied field maize fertigation with sodium selenate, isolation of pollen from plants and, subsequently, application of H2+ in the presence and absence of H2O2. Cytosolic Ca2+ plays a major role as a second messenger, and alterations of Ca2+ homeostasis may prompt molecular switches in the regulation and signalling networks of the pollen tube29. Furthermore, germinated pollen grains and elongated pollen tubes require an internal Ca2+ gradient that is maintained by an extracellular ion supply25. Thus, in light of the well-documented, two-way relationship between ROS, which can modulate calcium-dependent cellular networks, and calcium signaling, which plays a key role in ROS assembly36, we decided to use cytosolic Ca2+ as the end-point for monitoring perturbations caused by oxidative stress.Pollen samples, from plants either treated (Se1) or untreated (Se0) with sodium selenate, were used to measure cytosolic levels of Ca2O2-induced oxidative stress caused a significant alteration of Ca2+ homeostasis, shown by an increase in cytosolic Ca2+ and a markedly enhanced Ca2+ entry when 1 mM CaCl2 was exogenously added to the pollen resuspension medium. By contrast, Ca2+ homeostasis was fully maintained in both Se1 pollen and Se0 pollen  exposed to either sodium selenate or Se-methionine in vitro for 50 s prior to the addition of H2O2 and, immediately after, the start of measurements. Given the short incubation times, it is possible to rule out, at least in vitro, the implication of any metabolic mechanism and conclude that Se, in either chemical form, acts basically as a mere ROS scavenger that ultimately prevents ROS-mediated dysfunction of Ca2+ channels. Our results, however, indicate that the pollen obtained from Se-fertilized maize plants, as compared to Se0 controls, consisted of >95% organic Se, thereby suggesting that the inorganic form fertigated into crops was metabolically, and almost quantitatively, converted in Se-methionine (88%) and other organic forms such as SeCys and MetSeCys.We found that H2O2) is especially important for agricultural productivity, based on multiple abiotic factors that can ultimately lead to an excess of ROS production20, and the observation made by other groups showing that abiotic stresses of different nature may inevitably lead to excessive ROS accumulation and, consequently, pollen sterility14.The capacity of Se1 pollen to tolerate oxidative stress , that were found to be within a range previously reported in the literatureBiochemical elucidation of the effects of Se in pollen are likely required in order to optimize conditions and address possible unexpected events or, perhaps, uncover mechanistic events that could further improve germination rates. For example, it will be interesting to understand why the surface of Se1 pollen is morphologically different compared to normal Se0 samples, as shown by Field Emission Scanning Electron Microscopy. The rough aspect of Se1 pollen may relate to a higher permeability of the outer wall to proteins and Se itself , selenium methionine, hydrogen peroxide (H2O2), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), glucose, Hepes, and dimethyl sulfoxide (DMSO), were acquired from SigmaAldrich Italy. Any other chemicals and reagents were of the highest quality, and obtained from reputable, commercial sources.FURA 2-AM (CAS# 108964-32-5), PBS , Triton X-100, EGTA, sodium selenate  was grown in 2018 at the Field-Lab of the University of Perugia located in Papiano , in a clay-loam Fluventic Haplustepts soil41 featuring 10.3 g kg−1 of total organic C, 250 µg kg−1 of total Se and a pH of 7.75.Maize . To prevent crops from the occurrence of weeds, we employed pre-emergence herbicides such as Terbuthylazine 17.4% + S-Metolachlor 28.9% (4.0 L ha−1) and hand hoeing.At the time of seeding, fertilizers were applied with broadcast distribution of urea  placed on soil surface beside each seeding row.−1 (1449 mg plant−1) was applied by fertigation on June 25th 2018. Elementary plots consisted of 24 maize rows, each of which was 20 m long.Experimental procedures, aimed at evaluating fertilization conditions, implied block randomization with four replicates. Se treatments included i) no external Se input , and ii) Se fertilization (Se1). In Se1, 100 g sodium selenate hard 2018. Male inflorescences were shaken and pollen rain harvested with a steel funnel (20 cm diameter) outfitted with a steel filter (0.5 mm) to reduce debris (chaff and stamens) and keep insects out.Pollen was sampled at maize silk (R1 Stage) on June 3Pollen harvesting was carried out early in the morning with stable weather conditions .To minimize cross contamination, sampling of each replicate was performed by a dedicated group of technicians, and harvesting devices were thoroughly and consistently cleaned after each sampling procedure.Within ten minutes from collection, pollen from each plot was cleaned up by hand, placed in polypropylene tubes wrapped with aluminum foil, and immediately transferred into a refrigerated container (5 °C).43, with minor modifications related to the biological material used, as previously described. Briefly, pollen (0.5 g/sample) was microwave-digested  with 8 mL of nitric acid (65%w/w) and 2 mL of hydrogen peroxide (30%w/w), after which digests were diluted with 20 mL water, passed through 0.45 μm filters, and tested by graphite furnace atomic absorption spectroscopy in a Shimadzu AA-6800 apparatus  equipped with deuterium lamp background correction and a matrix modifier (Pd(NO3)2, 0.5 mol L−1 in HNO3. Determinations were carried out in triplicate.Fresh pollen (200 mg/sample) was resuspended in PBS (8 ml) for 48 h in the dark, after which pollen supernatant was obtained by centrifugation . Both pollen grains and the correspondent supernatant were used to determine total selenium content. To this end, we employed a previously described protocol44.Total protein content in supernatant was assessed by the Bradford’s method, with bovine serum albumin used as the standardet al. in43. Briefly, fresh pollen (0.25 g/sample in 10 mL PBS) was treated with pronase (20 mg), sonicated for 2 min, and stirred in a waterbath at 37 °C for 4 h. Samples were then centrifuged, and the supernatant 0.22 μm- filtered. Se analyses were conducted by anion-exchange HPLC using an Agilent 1100 instrument equipped with a Hamilton PRP-X100, 250 × 4.6 mm column. Standard solutions  of inorganic  and organic  forms of Se were prepared using ultrapure (18.2 MΩ cm) water43.The procedure employed to breakdown Selenium content in its different chemical forms was already described by Fontanella The morphology of the pollen samples was examined at 500x by Field Emission Gun Electron Scanning Microscopy using a LEO 1525 Gemini workstation (ZEISS) following Chromium metallization.2+-free HBSS buffer composed of 120 mM NaCl, 5.0 mM KCl, MgCl2 1 mM, 5 mM glucose, 25 mM Hepes, pH 7.4.Pollen samples (100 mg), stored in the dark at 5 °C, were initially resuspended in 10 ml PBS, and hydrated for 2 days at 25 °C. Hydrated pollen was then collected by centrifugation , and resuspended in 2 mL Ca2O2, which was directly added to pollen suspensions immediately prior to measurements.Oxidative stress was induced with variable doses  of H37. Samples were incubated with FURA-2AM (2 μL of a 2 mM solution in DMSO) for 120 min, then centrifuged . Pollens were then resuspended in ~10 mL of Ca2+-free HBSS, supplemented with 0.1 mM EGTA to reduce background signal caused by contaminating ions, to obtain a suspension containing 106 pollen grains/mL.Intracellular levels of calcium were measured using the fluorometric FURA-2AM indicator dye according to a protocol previously described by our group37. The signal ratio 340–380/510 nm was finally used to determine concentrations.Fluorometric measurements were performed in a Perkin-Elmer LS 50 B spectrofluorometer  with a 10 nm and a 7.5 nm slit width in the excitation and emission windows, respectively. Signals were taken after 300–350 s, a time range previously found to be optimal2 to evaluate the accumulation in the cell of the extracellular ion36. Cytosolic calcium concentrations were calculated as reported by Grynkiewicz45.To this end, measurements were performed in two steps: initially, without calcium in the incubation medium and, 250 s later, in the presence of 1 mM CaCl46, and then transferred to 6-well culture Corning plates (1 mg of pollen per plate) containing 3 mL of an agar-solidified growing medium composed of 1.2% agar, 10%, sucrose, 0.03% boric acid and 0.15% calcium chloride (pH 5.5)39.Fresh pollen samples from each plot were hydrated in a humid chamber at room temperature for 30 min2O2 was directly dispensed in the culture wells.Pollen suspensions were incubated for 24–48 h in a growth chamber at 27 °C with gentle shaking to ensure homogeneous distribution of the samples in the wells. To induce oxidative stress, H39.Germinated and non-germinated pollen grains were counted under a 10X magnification microscope. Germination rates were calculated based on three replicates, each of which consisted of one hundred grains. Germination of grains was confirmed when the pollen tube had grown longer than the grain’s diameterStatistical evaluations were performed using the GraphPad Prism 6.03 software. Variance assessments included homogeneity analysis by the Levene’s test, and the normality test by D’Agostino-Pearson. Significance of differences was assessed by the Fisher’s least significant differences test. Differences with p < 0.05 were considered statistically significant.


Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231 Cancer cell fusion can generate new populations of cancer hybrid cells, either by homotypic (autofusion—the combination of cells from the same population) or heterotypic (heterofusion—the hybrid formation of different cell types) processes ,2,3. ThePrevious work has demonstrated that cell fusion in vitro requires a hypoxic environment, together with an optimized pH and proper alignment of certain glycoproteins, as well as a permissive lipid composition of the involved parts of the cell membranes to facilitate the initiation of a fusion event . Indeed,Although the molecular triggers and precise mechanism of hybrid cell formation remain unclear to date , previouWhile the actin-based cytoskeleton was previously suggested to be involved, e.g., in myoblast fusion , the preWhile heterofusion between MSC and neoplastic breast cells represents a rare event that occurs in 0.2% to about 1% of the co-cultured population, treatment with actin polymerization inhibitor cytochalasin D significantly reduces the rate of hybrid cell formation A. ExperiGFP populations, together with either MCF10Acherry , after 24 h exposure to 0.05 µM cytochalasin D. No significant differences in cell adhesion molecule expression were detectable in the three control versus cytochalasin D-treated cell cultures , supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine . The benign breast carcinoma cell line MCF10A was cultured in phenol red-free mammary epithelial cell basal medium (MECBM) with an appropriate growth medium supplement mix .The usage of primary human MSC was approved by the Ethics Committee of Hannover Medical School, Project #443 on February 26th, 2009, and informed written consent was obtained from each patient. MSC were isolated from umbilical cord tissue explant cultures, as described previously , and culAll cells were tested for mycoplasma by the luminometric MycoAlert Plus mycoplasma detection kit , according to the manufacturer’s recommendations. Authentication of the human breast carcinoma cell lines was performed by short tandem repeat (STR) fragment analysis, using the GenomeLab human STR primer set .mCherry gene (MDA-MB-231cherry or MCF10Acherry) or the enhanced green fluorescent protein (eGFP) gene (MSCGFP). Co-cultures of MSCGFP and MDA-MB-231cherry were cultivated in αMEM, whereas co-cultures of MSC with MCF10A were grown in 60% MECBM and 40% αMEM.For discrimination of MSC and breast cancer cell lines in co-culture, cell populations were stably transduced with a third-generation lentiviral SIN vector carrying either the The quantification of hybrid cells was performed as previously described . BrieflyFor flow cytometric analysis, the samples were first blocked with 2% FCS in PBS for 15 min at room temperature. After a PBS washing step, the cells were stained with appropriate PE- or FITC-labeled antibodies at 4 °C for 15 min. The following antibodies were used: mouse monoclonal CD11b-PE ; mouse monoclonal CD18-PE ; rat CD49f ; mouse monoclonal CD54-PE ; CD56-PE ; mouse monoclonal CD106-PE ; rat CD146-PE ; mouse monoclonal CD166-PE ; mouse monoclonal CD324-PE ; CD326-PE ; and mouse monoclonal CD31-FITC . PE- or FITC-labeled antibodies of the corresponding IgG subclass  served as a control. Thereafter, the cells were washed again with PBS and subsequently analyzed by a flow cytometer using FACSCalibur  and FlowJo V10 software.5 cells were fixed in 70% ice-cold EtOH at 4 °C for 24 h. The fixed cells were then stained with propidium iodide (PI) staining solution containing 12.5 µg/mL PI, 0.5% Triton-X-100, and 100 U/mL DNase-free RNase in PBS for 30 min at room temperature. Thereafter, the samples were measured by flow cytometry  and analyzed by FlowJo V10 software.The cell cycle analysis was performed as described previously . Briefly-FSCN -GAPDH The total cellular RNA was isolated using an RNeasy Mini Kit  according to the manufacturer’s protocol. Briefly, 1 µg RNA was reverse transcribed into cDNA using 500 µM of dNTP, 5 µM of Random Hexan primer, 5 µM of Oligo(dT) primer, 1 U of RiboLockTM RNase Inhibitor, and 5 U of RevertAidTM Reverse Transcriptase in the supplied reaction buffer . The cDNA reaction was performed for 10 min at 25 °C, followed by 1 h at 42 °C, and stopped at 72 °C for 10 min. Then, 2.5 µL of cDNA were amplified by polymerase chain reaction (PCR) with the following specific primers :The PCR reaction included 200 nM of each primer, 200 µM of dNTP, and 0.03 U One Taq Hot Start DNA polymerase  in the appropriate buffer. The PCR cycling conditions included 30 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C, with an initial 5 min denaturation step at 95 °C and a final 5 min extension step at 72 °C (35 cycles). Aliquots of 25 µL of PCR products were separated on a 2% agarose gel using the standard GeneRuler 100 bp DNA Ladder (Thermo Scientific). Visualization was performed by GelRed staining .v/v) Ethanol and 10% (v/v) acetic acid in water for 60 min, and stained with Coomassie Brilliant Blue overnight. Background staining was reduced with 25% (v/v) methanol in water. Each lane was cut into four pieces, which were further minced to 1 mm³ gel pieces. Subsequent sample processing was performed as described [g, aliquots of 12.5 µL each were stored at −20 °C.Protein from co-cultures in the absence (control) or presence of cytochalasin D was mixed with 5× loading buffer  and incubated for 5 min at 95 °C. Proteins were then alkylated by addition of acrylamide up to a concentration of 2%, and incubation at room temperature (RT) for 30 min. SDS-PAGE was performed on 10% gels. After electrophoresis, the proteins were fixed with 40% , equipped with a trapping column  and a 50 cm-long separation column . Peptide mixtures were injected, enriched, and desalted on the trapping column at a flow rate of 6 µL/min with 0.1% TFA for 5 min. The trapping column was switched online with the separating column, and peptides were eluted with a multi-step binary gradient: a linear gradient of buffer B  in buffer A (0.1% formic acid) from 4% to 25% in 30 min, 25% to 50% in 10 min, 50% to 90% in 5 min, and 10 min at 90% B. The column was reconditioned to 4% B in 15 min. The flow rate was 250 nL/min, and the column temperature was set to 45 °C. The RSLC system was coupled online, via a Nano Spray Flex Ion Soure II (Thermo Scientific), to an LTQ-Orbitrap Velos mass spectrometer. Metal-coated fused-silica emitters  and a voltage of 1.3 kV were used for the electrospray. Overview scans were acquired at a resolution of 60 k in a mass range of Raw data were processed using Proteome Discoverer (Thermo Scientific), as well as human and virus uniprot/swissprot databases containing common contaminants. The stated proteins were identified by a false discovery rate of 0.01 at the protein and peptide level, and were quantified by extracted ion chromatograms of all peptides.While the present work confirms previous findings of hybrid formation between benign neoplastic breast epithelial cells and different MSC cultures, as well as fusion between malignant breast cancer cells and MSC populations, evidence is provided for an important role of actin filament structures in the fusion process. Thus, cytochalasin D and latrunculin B, which both block actin polymerization via different mechanisms, were capable of selectively inhibiting cell fusion, while no significant effects were observed in other cellular pathways, including cell cycle progression or the expression of various cell adhesion molecules. Moreover, the identification of a panel of actin-associated proteins by comparative proteome analysis further substantiates a predominant involvement of actin cytoskeletal components to provide a fusion-permissive environment. Although a detailed molecular interplay for the regulation of cancer cell fusion processes remains unclear, further evaluation may also unravel the heterogenic properties of resulting cancer hybrid cells, displaying enhanced or reduced tumorigenic and metastatic potential.


Postural stability deficits have been proposed to influence the onset and progression of adolescent idiopathic scoliosis (AIS). This study aimed to systematically identify, critically evaluate and meta-analyse studies assessing postural stability during unperturbed stance with posturography in AIS compared to typically developed adolescents.d effect size (ES) and homogeneity estimates.Studies from four electronic databases  were searched and case-control methodological quality assessed using a risk-of-bias assessment tool and a posturography methodological quality checklist. Pooled data regarding centre of pressure (COP) parameters such as sway area, Mediolateral (ML) and Anteroposterior (AP) position and range were compared for AIS and typically developed adolescents using Cohen’s d showed a medium ES difference in sway area 0.65, 95% CI (0.49–0.63), whereas ML showed no  and large (MLR) ES differences; MLP1 0.15, 95% CI (0.08–0.22); MLP2 0.14, 95% CI (0.08–0.19); and MLR 0.94, 95% CI (0.83–1.04). Cohen’s d for AP showed small ES (APP1) and large ES difference (APP2 and APR); APP1 0.43, 95% CI (0.31–0.54); APP2 0.85, 95% CI (0.72–0.97); and APR 0.98, 95% CI (0.87–1.09). Cochran’s Q and Higgins I2 showed homogeneity between studies.Eighteen studies for quality analysis and 9 of these for meta-analysis were identified from 971 records. Risk-of-bias assessment identified 6 high, 10 moderate and 2 low risk-of-bias studies. The posturography methodological quality checklist identified 4 low, 7 moderate and 7 high-quality studies. Meta-analysis was performed for sway area whereas ML and AP are presented in three different meta-analyses due to divergent measurement units used in the studies: ML position 1 (MLP1), ML position 2 (MLP2) and ML range (MLR); AP position 1 (APP1), AP position 2 (APP2) and AP range (APR). Cohen’s There is moderate quality evidence for decreased postural stability in AIS measured as COP parameters sway area, ML and AP range with a positional shift posteriorly in the sagittal plane. The findings support studying postural stability in early stage AIS and also prospectively identify cause and effect of the curvature as well as effectiveness of postural control interventions in the prevention of scoliosis progression. Postural control involves the body’s position in space for dual purposes of stability and orientation . PosturaAll neuromuscular disorders that act on the growing body, particularly during the rapid pubertal growth period, can lead to the development of scoliosis . AdolescThe pathogenesis of AIS remains unknown but is considered to be multifactorial –16. A diThere are a variety of methods to assess postural control during different conditions and ages –24. PostVarious COP parameters calculated as sway area, mediolateral (ML), and anteroposterior (AP) change of position and range have been derived from COP data , 24, 26.The purpose of this review was to identify, critically evaluate and meta-analyse studies assessing postural stability during unperturbed stance with posturography in AIS compared to CON. We hypothesised that AIS would have decreased postural stability compared to typically developed adolescents measured as COP parameters sway area, ML and AP position and/or range.Studies published until the end of 2016 was retrieved from a search of four electronic databases: PubMed, Scopus, CINAHL and PEDro in April–May 2017 to identify eligible studies. The authors in consultation with an academic librarian designed the search strategy to identify relevant studies comparing postural stability within AIS compared to a control group with typically developed adolescents (CON). The following main search terms were used: , appropriate to each database (Table The inclusion criteria were as follows: (1) adolescents aged 10–18 years; (2) use of force plate to measure postural stability during stance; (3) inclusion of one or more reported parameters: mean sway area, mean position and/or range in ML and AP direction; (4) AIS and typically developed adolescents; (5) case-control studies; and (6) studies published in English. The exclusion criteria were as follows: (1) non-idiopathic scoliosis; (2) non-healthy subjects ; (3) patients who have undergone scoliosis correctional surgery or in brace; (4) an absence of CON; and (5) measurements during perturbed stance. Further exclusion criteria for meta-analysis were (6) divergent measurement units or discrete values and graphs missing, (7) use of two force plates, (8) procedure with feet or heels together, (9) and high severity scoliosis group, Cobb > 60°.The study followed the PRISMA reporting guidelines and a flow diagram was used to document the study screening and selection process  Fig. 1)1). From Methodological quality was assessed using two critical appraisal tools. A risk-of-bias assessment tool for case-control methodology Table  was chosKey data regarding the study settings, participant demographics, study protocol, COP parameters, results and conclusions were extracted and tabulated. If discrete values were not reported, estimated values were extracted from graphs or figures. A critical qualitative synthesis was conducted with a consideration of risk-of-bias assessment and posturography methodology Tables  and 3.d) [d = 0.20 is considered as a small, d = 0.50 medium and d = 0.80 a large ES difference.To evaluate the quantitative differences in postural stability in AIS compared with CON, meta-analysis was conducted. Increased values for one of the groups expressed decreased postural stability. Means and standard deviations (SD) for posturography data were collected from each study; if multiple AIS subgroups were presented, combined means were calculated. All measures were converted to square millimetres and millimetres. Confidence interval (CI) and standard error/of measurements (SE and SEM) were converted to SD . Effect sizes (ES) (Cohen’s d)  for COP d) , 36. Thed) . The metd) . Accordid) , d = 0.2I2 statistics were calculated to assess homogeneity across studies using the random-effects model [χ2), alpha level p < 0.05, where degrees of freedom are the number of studies in the analysis minus one. If the Cochran’s Q value is under the chi-square value, we accept the null hypothesis that all studies are homogeneous [I2 is an estimate of the percentage of total variability that can be attributed to the variation between studies [I2 around 25% (I2 = 25), 50% (I2 = 50) and 75% (I2 = 75) were classified as low, medium and high heterogeneity, respectively. In this review, I2 below 25% suggests homogeneity [Cochran’s Q and Higgins’s ts model , 39. Cocogeneous . Higgin’ studies . Thus, pogeneity , 41.In this review, seven meta-analyses were calculated from COP parameters. One represents sway area while ML and AP are each presented as three different meta-analyses due to divergent measurement units used in the included studies: ML position 1 (MLP1), ML position 2 (MLP2) and ML range (MLR); AP position 1 (APP1), AP position 2 (APP2) and AP range (APR), where APP2 describes the change in position from a location from the back of the heels and leaning forward in opposite to APP1 where the location is estimated from the centre point of support and backwards. The three different categorisations were performed likewise for MLP1 and MLP 2 to minimise the risk of heterogeneity in the meta-analyses.Electronic database searches yielded 971 potential studies . The OB showed 15% higher values than the CON; however, this was not a significant difference (NS).Of 18 included studies, 9 investigated sway area , 57–59 (p < 0.05) difference between AIS and CON. Eight studies reported higher values in the ML range for the AIS group compared to the CON with 7 studies reporting significant differences [Of 18 included studies, 13 reported COP in ML position and range , 54–59 . Two stuferences , 58, 59 ferences , 57, 59.p < 0.05) located posteriorly towards heels for AIS compared to CON in 5 of the included studies [Of 18 included studies, 14 reported COP in AP position and range , 53–59 . Two stu studies , 55, 57.To ensure consistency across the quantitative meta-analysis, a further 9 studies were removed due to divergent measurement units , 52, 56,d (pooled) showed a medium ES 0.65, 95% CI (0.49–0.63) (Table d pooled effect size 0.65 (CI 0.49–0.63). Cochran’s Q 5.93 when compared to a chi-squares critical values table the χ2 of heterogeneity test was NS . Indicating that the variation between studies was homogeneous therefore accepting the null hypothesis that all studies are equal have a common ES. Further, the I2 statistic was 15.71% indicating low variability across studies, due to heterogeneity rather than chance. This suggests no observed heterogeneity across studies.The overall ES for sway area, Cohen’s 3) Table  displayed (pooled) for MLP1 and MLP2 showed no ES difference but MLR showed large ES difference: MLP1 0.15, 95% CI (0.08–0.22); MLP2 0.14, 95% CI (0.08–0.19); and MLR 0.94, 95% CI (0.83–1.04) (Table d pooled effect size 0.15 (CI 0.08–0.22), Fig. d pooled effect size 0.14 (CI 0.08–0.19), Fig. d pooled effect size 0.94 (CI 0.83–1.04). Cochran’s Q showed that the variation between studies were homogeneous for each of the parameters MLP1 1.00 , MLP2 1.48 , and MLR 4.84 , consistent with Higgins I2 results of low variability, 6.66, 0, and 0%, respectively. This suggests no observed heterogeneity across studies, therefore accepting the null hypothesis that all studies are equal.Cohen’s 5% CI 0.0–0.22; ML5% CI 0.8–1.04 (Tad (pooled) for APP1 showed small ES difference but a large ES difference for APP2 and APR; APP1 0.43, 95% CI (0.31–0.54), APP2 0.85, 95% CI (0.72–0.97) and APR 0.98, 95% CI (0.87–1.09) (Table d pooled effect size 0.43 (CI 0.31–0.54), Fig. d pooled effect size 0.85 (CI 0.72–0.97), Fig. d pooled effect size 0.98 (CI 0.87–1.09). Cochran’s Q showed that the variation between studies were homogeneous for each of the parameters; APP1 1.00 , APP2 2.04 , and APR 6.35 , consistent with Higgins I2 results of low variability across studies: APP1 4.42%, APP2 1.74% and APR 21.30%. This suggests no observed heterogeneity across studies, therefore accepting the null hypothesis that all studies are equal.Cohen’s 95% CI 0.2–0.97 anThis systematic literature review aimed to evaluate the current literature assessing COP parameters in AIS compared with CON to better understand and determine if AIS affects postural stability. According to the results from 7 different meta-analyses of COP parameters, AIS affects postural stability. In APP2, APR and MLR, a large ES difference was evident while sway area had medium ES difference for AIS compared to CON. This means larger postural instability (range and sway) for AIS with a COP positional shift posteriorly in the sagittal plane.Although AIS is a three-dimensional spinal deformity, the lateral deviation of the spine in the coronal plane is often considered the most distinct deformity. Gruber et al.  reasonedAn underlying aetiology of AIS is not well established within the current literature, as this condition is thought to be multifactorial in nature and no conclusive evidence exists to support any one theory . PosturaThe sensory integration hypothesis indicates individuals with AIS have impaired dynamic regulation of sensorimotor integration due to an inaccurate weighting of sensory inputs. This sensory deprivation has been linked with balance dysfunction reflected in an inability to recalibrate the position of the COP in relation to the body’s COM, and thus, exaggerated body sway oscillations are evident , 42. A sThe volume of studies included for analysis was a major strength of the present review. The total number of participants involved provided a greater power to the conclusions drawn from the literature. Furthermore, study participants were representative of the AIS female dominant gender distributions apparent in the community. Comparisons and conclusions are however limited by posturography methodological differences between studies and a need for standardisation of study protocols for future research. A review by Ruhe et al.  concludeIt is also important to consider potential limitations of the current review. For example, 2 of the studies in the meta-analyses showed a high risk of bias , 57, 5 mThere is moderate quality evidence for decreased postural stability in AIS measured as COP parameters sway area, ML and AP range with a positional shift posteriorly in the sagittal plane. The findings support studying postural stability in early stage AIS and also prospectively identify cause and effect of the curvature as well as effectiveness of postural control interventions in the prevention of scoliosis progression.


Neutrophils can combat the invasion of pathogens by the formation of neutrophil extracellular traps (NETs). The NET mechanism is not only an effective tool for combating pathogens, but is also associated with diseases. Therefore, NETs are a potential target for combating pathologies, such as cystic fibrosis and thrombosis. We investigated the potential of nanoparticles, which were modified with α2,8-linked sialic acid chains, to modulate NET release during phorbol myristate acetate stimulation. Interestingly, when these nanoparticles were applied, the formation of reactive oxygen species was partly inhibited and the release of NET was counteracted. However, although the release of NET fibers was prevented, the nuclei still lost their characteristic segmented structure and became swollen, indicating that only the release, and not complete activation was suppressed. Intriguingly, coincubation of α2,8-sialylated particles with free sialic acid chains prevented the outlined inhibitory effects. Thus, the sialic acid chains must be attached to a linker molecule to generate an active bioconjugate that is able to inhibit the release of NET. Neutrophil granulocytes are the most abundant leukocytes of the innate immune system representing the first line of defense against invading pathogens ,2,3. In Moreover, in 2004, Brinkmann and colleagues discovered that neutrophils can undergo beneficial suicide, resulting in the release of neutrophil extracellular traps (NETs) . The DNAIn addition to beneficial effects, the formation of NET is associated with diseases, such as infertility, small-vessel vasculitis, rheumatoid arthritis, preeclampsia, ulcerative colitis, Crohn’s disease, systemic lupus erythematous, thrombosis, and cystic fibrosis ,21,22,23In mammals, several biomolecules have the capacity to modulate immunological mechanisms. Since all our cells are surrounded by a glycocalyx, consisting of glycolipids and glycoproteins, glycan-dependent mechanisms frequently take place during processes of innate and adaptive immunity . In mammThus, we hypothesized that α2,8-linked sialic acid residues can be used to modulate the activation of neutrophils and NETosis. The obtained results demonstrate that the release of NETs is inhibited by the application of particles containing α2,8-linked sialic acid chains, indicating that α2,8-sialylated nanoparticles are a tool for manipulating the formation of NET.Endoneuraminidase (endoN) was kindly provided by Martina Mühlenhoff  . All reaAll volunteers provided written informed consent and all samples were anonymized. The use of human neutrophils was approved by the local ethics office of the University of Giessen, School of Medicine (05/00).−1, 2 h, 37 °C, 250 rpm). Resulting cleavage products were separated and collected according to the degree of polymerization (DP) by anion exchange chromatography, as described earlier, to get sialic acid chains with chain lengths consisting of <9 N-acetylneuraminic acid (Neu5Ac) residues [–1. The following gradient was used for the separation, 0 min = 0% (v/v) E2, 20 min = 26% (v/v) E2, 30 min = 34% (v/v) E2, 45 min = 38% (v/v) E2, 85 min = 88% (v/v) E2, 110 min = 100% (v/v) E2, and 141 min = 0% (v/v) E2. DMB-labeled sialic acid chains were detected using a fluorescence detector at 372 nm for excitation and 456 nm for emission. In order to proof the degree of polymerization, aliquots of dried samples were mildly DMB labeled, as described above. Sample separation took place on an analytical DNAPac PAc-100 column . MilliQ water (E1) and 2 M ammonium acetate buffer (E2) were used as eluents at a flow rate of 1 mL min–1 using the following gradient, 0 min = 100% (v/v) E1, 5 min = 100% (v/v) E1, 15 min = 92% (v/v) E1, 20 min = 89% (v/v) E1, 30 min = 86% (v/v) E1, 55 min = 84% (v/v) E1, 100 min = 80% (v/v) E1, and 130 min = 77% (v/v) E1.10 mg colominic acid  was digested via the use of endoN  were washed twice prior usage. A total of 150 µL latex particles were resuspended in 123 µL PBS and homogenization was performed by using ultrasonic. A total of 200 µg of dried sugars were dissolved in 25 µL PBS and then added to the latex particles. A total of 1.5 µL 5 M NaCNBH3 was added and the coupling reaction took place for 2 h at 65 °C and 250 rpm. After this, sialic acid–coupled particles were washed twice and resuspended in 100 µL PBS. An aliquot was taken to quantify the coupling reaction, as described in the previous chapter. The used nanoparticles showed no cytotoxic characteristics as tested previously in [The separated sialic acid chains, as well as Neu5Ac  , were used for coupling reaction. Aliphatic amine latex particles  cells were washed with PBS. The erylysis was performed two times. As a final step, the received pellet was washed and resuspended in RPMI 1640 (Thermo Fisher Scientific) with 1% penicillin/streptomycin  and 1% fetal bovine serum . A total of 30,000 cells/well were dissolved in RPMI 1640 with 1% PenStrep, and 1% FBS and incubated 1 h at 37 °C and 5% CO2 before the NETs were induced.Human neutrophils were isolated as described previously by Saffarzadeh et al. . Therefol-Lysine coated 12-well chamber slides for 2 h/4 h at 37 °C and 5% CO2. To inhibit NETosis cells were incubated with PMA and a final concentration of 10 µg mL−1 sialic acid was coupled on latex particles. Coincubations of α2,8-sialylated particles with 30 µg mL−1 free sialic acid chains of an inherent chain length were performed under equal conditions.The stimulation of neutrophils with 20 nM PMA took place in Poly-–1). Fixation was performed with 2% PFA for 20 min at RT. Samples were then mounted and analyzed using fluorescence microscopy . The determination of the NET area per cell was based on the release of decondensed DNA fibers during NETosis (DAPI staining) and was performed using Cellprofiler 2.2.0. Three pictures were randomly taken of each biological sample.After 2 h or 4 h of PMA stimulation cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 °C. After washing, cells were incubated with 0.5% Triton X-100 for 1 min, followed by three further washing steps. Blocking was performed for 30 min at 37 °C with 2% IgG free bovine serum albumin (BSA)  and anti-Neutrophil Elastase  was incubated overnight at 4 °C. After further washing steps, the secondary antibody ) was added before the nuclei staining with 4′,6-Diamidin-2-phenylindol (DAPI) was done . Therefore, 35,000 cells/well were seeded and NETosis was induced with 20 nM PMA for 3.5 h and cells were further incubated 30 min with 250 nM DiBAC4. Measuring took place with a Gallios, Beckman Coulter, at an excitation of 488 nm and with the emission at 525 nm ± 25 nm (argon laser), as described previously by Löhrke et al. [The determination of the membrane potential was performed with DiBACl-Lysine -coated 12-well chamber slide. To de-stress cells from the isolation procedure, the following step was an incubation in RPMI 1640 for 1 h at 37 °C and 5% carbon dioxide. Afterwards, the cells were washed twice with RPMI 1640 and stained with 1 µg mL–1 DAPI (Carl Roth) for 30 min before a further washing step was done. To stain the membrane, Deep Red Cell Membrane Stain  was added 15 min before NETosis induction, with 50 nM PMA. Pictures were taken at an interval of 2 per min.After the isolation of human neutrophils, 50,000 cells/well were seeded in a Poly-–1. After removing PI, nuclei were stained with Hoechst  in a final concentration of 1 µg mL–1. Cells were directly analyzed using fluorescence microscopy . Three pictures were randomly taken from three independently performed experiments. The total cell number and PI positive cells were counted.Membrane integrity was analyzed using PI  in a final concentration of 1 µg mLt-test with Welch’s correction, when the calculated values passed the Shapiro–Wilk normality test. Otherwise, the Kruskal–Wallis test in combination with Dunn’s test for multiple comparisons was applied. Differences were considered statistically significant at p ≤ 0.05. Statistically significant differences are given the labels * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001.Data sets were analyzed with Graph Pad Prism 7.0 software using ANOVA and a multiple-comparison Tukey test or a NADPH oxidase represents a key enzyme in the signaling cascade during the formation of NET induced by PMA . To test4, a substance that can enter depolarized cells. Here, DiBAC4 can bind intracellular proteins exhibiting enhanced fluorescence and a red spectral shift. In The occurrence of membrane depolarization after activation of the NADPH oxidase complex was described in the 1980s ,53,54. EIn sum, the outlined results of the cell assays suggested that the essential steps during PMA stimulation, which trigger the formation of NET, are impaired by α2,8-sialylated nanoparticles.The formation of ROS is directly linked to the translocation of neutrophil elastase into the nucleus because ROS serves as a substrate for myeloperoxidase. Subsequently, the increase in HOCl initiates the detachment of neutrophil elastase from the myeloperoxidase/neutrophil elastase complex, enabling the transfer of neutrophil elastase into the nucleus and triggering decondensation of DNA ,9. The rWhen we compared the nuclear structure of unstimulated neutrophils with that of the activated neutrophils in the presence of α2,8-sialylated nanoparticles, we observed that nearly all nuclei lost their segmented structure and the nuclei were swollen 4 h after the beginning of the PMA stimulation . Thus, tThe translocation of neutrophil elastase to the nuclei is an important step during the decondensation of DNA and the formation of NETs . To testIn contrast to the unstimulated neutrophils, neutrophil elastase was transferred into the nucleus after stimulation . In addiIn addition, the release of NET was examined. The stimulation of NETosis leads to the formation of the DNA meshwork, which can be visualized with DAPI. As shown in The same experimental test setup was also performed with monosialylated beads, which showed no impact on the activation by PMA . In addiFor the release of NETs, cell membrane rupture is necessary . Using DIn order to test whether the outer membranes were still intact we used PI, a substance that can pass only perforated, not intact, biomembranes. Once PI enters the cell, it interacts with DNA, resulting in a red fluorescent staining. The calculation was performed by counting the total number of cells compared to the number of PI-positive cells. The results indicate that, compared to untreated neutrophils, PMA led to an increasing number of perforated cells . SimilarIn 2015, Spence et al. discovered that nanoparticles coated with the murine siglec-E ligand, α2,8-linked sialic acid residues, decrease macrophage-driven inflammation . Based oNevertheless, the release of NETs was retained and the α2,8-sialylated particles prevented the perforation of the outer membrane . InteresIn addition to lactoferrin, α2,8-linked polymers of sialic acids are known to induce membrane interactions, because these polymers can interact with phospholipids . This inHowever, in contrast to lactoferrin, α2,8-sialylated beads influence ROS production , which iAlthough the exact mechanism of action has been unknown until now, α2,8-sialylated nanoparticles are a promising option for modulating NETosis during NET-related pathologies. Furthermore, for application as a pharmacological tool, the α2,8-sialylated nanoparticles must be tested in different in vivo models where NETs are the main cause that induces pathology.


Urinary concentrations of four mycotoxins, , zearalenone (ZEA), fumonisin B1 (FB1), and ochratoxin A (OTA)), and four key metabolites, , aflatoxin M1 , α-zearalenol (α-ZOL), and β-zearalenol (β-ZOL)) were identified and measured by UPLC-MS/MS. Statistically significant regional differences were detected for both total DON (DON + DOM-1) and total ZEA (ZEA + α-ZOL + β-ZOL) concentrations in pig urine from the three regions. These regional differences were in good agreement with the occurrence of Fusarium graminearum mycotoxins (DON + ZEA) in cereal grains harvested in 2011 in Sweden. There were no statistically significant differences in FB1, AFM1 and OTA urinary concentrations in pigs from the three regions. The overall incidence of positive samples was high for total ZEA (99–100%), total DON (96–100%) and OTA (85–95%), medium for FB1 (30–61%) and low for AFM1 (0–13%) in the three regions. Urinary mycotoxin biomarker concentrations were used to estimate mycotoxin intake and the level of mycotoxins in feeds consumed by the monitored pigs. The back-calculated levels of mycotoxins in feeds were low with the exception of seven samples that were higher the European limits.The determination of mycotoxin and metabolite concentrations in human and animal urine is currently used for risk assessment and mycotoxin intake measurement. In this study, pig urine ( In deveand feed . Subsequand feed . More reand feed , were deand feed . Urinary in pigs . Few stu in pigs ,11,12,131 that gave a mean recovery of 64% but a good repeatability of results (7%). In The UPLC-MS/MS method and MS/MS parameters used in this study to analyze pig urine were previously reported ,7. As a In 1 + total ZEA + OTA (34.9% of samples) (The occurrence of multiple mycotoxins and metabolites in pig urine is reported in samples) . Each of1 in Swedish pigs was comparable to those reported for Belgian pigs and French piglets [1 results of Swedish and Vietnamese pigs. As reported in 1 concentration 8–11 and 4–27 times lower compared to Vietnamese pig urine, respectively [1 which is mainly excreted in urine as AFM1. Taken together, the results reported in In  piglets ,12. A reectively . These rThe urinary mycotoxin and metabolite concentrations measured in this study and the urinary excretion rate of the five target mycotoxins and their metabolites in pigs  were use1 and AFB1. The calculated PDI values, mean pig weight, mean daily urine volume and mean weight of feed consumed daily by pigs were used to calculate the level of each mycotoxin in the feed consumed by each pig monitored in this study according to Formula (2), reported in the experimental section. The results of this evaluation are reported in 1 [1 and AFB1, they ranged from 16.1–0.6 µg/kg. These values are largely below the limits for DON (900 µg/kg), ZEA (250 µg/kg), OTA (50 µg/kg), AFB1 (20 µg/kg) and FB1 (5000 µg/kg). However, the estimated mycotoxin levels of the feeds consumed by seven pigs were higher the limits of DON, OTA or AFB1. Five out of the seven pigs were bred in the West region and three of them consumed feed with high DON levels, whereas two pigs consumed feeds with high levels of OTA  was observed for total DON between West (56.8 ± 88.7 ng/mL) and South (19.3 ± 22.8 ng/mL) and between West and East (12.8 ± 13.6 ng/mL). The statistical analyses of crea adjusted urine concentrations confirmed the significant difference (p < 0.009) of total DON between West (45.7 ± 106 ng/mg crea) and South (12.9 ± 15.1 ng/mg crea), but no differences were observed between West and East and between South and East. Total ZEA was significantly higher (p < 0.009) in the West (7.8 ± 10.4 ng/mL) compared to South (4.3 ± 9.5 ng/mL) but no significant difference was found to the East. Similar results were obtained for crea adjusted concentrations, although a significant difference (p < 0.05) was also observed between East (12.5 ± 42.2 ng/mg crea) and South (3.7 ± 10.5 ng/mg crea).These results prompted us to statistically compare the mycotoxin concentrations in the urine of pigs bred in the West, South and East regions of Sweden . The resFusarium graminearum mycotoxins (DON and ZEA) in cereal grains harvested in 2011 in different Swedish regions [n = 1300) and South . Despite the fact that pigs are not fed oats, the data reflect the situation of a high infection rate with F. graminearum in the fields of the West region, which was particularly troublesome in oats this year.These regional differences were in good agreement with the differing occurrence of  regions ,16. Morep > 0.05) in FB1, AFM1 and OTA between the three regions. Considering that AFB1 and FB1 were not found in Swedish grains, the positive samples were most likely due to other feed components or the farmer was not producing their own feed and used commercial feed; in that case, we should not expect regional differences. Protein concentrates were found to contain AFB1 from 5–500 μg/kg and the more heavily contaminated samples were destined for pig rations [According to the Kruskal–Wallis equality of population rank test, there was no difference ( rations .in vivo studies [p < 0.05) than those measured in in vivo studies where pig diets contained higher levels of DON and/or ZEA , Skåne county (n = 83) and Kalmar county (n = 38), respectively , ZEA (100 µg/mL), α-ZOL (10 µg/mL), β-ZOL (10 µg/mL) and OTA (10 µg/mL) were prepared in acetonitrile (ACN), whereas FB1 solution (50 µg/mL) was prepared in acetonitrile–water (50:50 v/v). The enzymatic solution β-glucuronidase/sulfatase type H-2 from Helix pomatia  was purchased by Sigma Aldrich . Chromatography-grade methanol (MeOH) and glacial acetic acid were obtained from Carlo Erba . Ultrapure water was obtained from a Milli-Q system . Myco6in1+TM immunoaffinity columns were purchased from Vicam L.P . OASIS HLB® columns  were purchased from Waters  and regenerated cellulose filters (0.45 µm) were purchased from Sartorius Stedim Biotech .Standard solutions of mycotoxins and their key metabolites were purchased from Romer Labs Diagnostic . In particular, solutions of DON (100 µg/mL), DOM-1 (50 µg/mL), AFM1, FB1, ZEA, α-ZOL, β-ZOL and OTA) were performed using an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method reported elsewhere [Helix pomatia to hydrolyze glucuronide- and sulfate- conjugates of mycotoxins and their key metabolites. The digested sample was diluted with water  and purified on a Myco6in1+TM multi-antibody immunoaffinity column and OASIS HLB® column connected in tandem. The purified urine was dried down and reconstituted in 200 µl of mobile phase  and analyzed by UPLC-MS/MS with a triple quadrupole API 5000 mass spectrometer , equipped with an ESI interfaced to an Acquity UPLC system comprising a binary pump and a microautosampler . Data acquisition and processing was performed with Analyst version 1.5.1 software . Detailed chromatographic and mass spectrometric operating conditions are reported elsewhere [The analyses of pig urinary mycotoxin biomarkers , matrix-matched calibration solutions were prepared for 6 purified urinary extracts: one aliquot was analyzed as a control and 5 aliquots were used to prepare calibration samples. In particular, aliquots of urine from 5 pigs were pooled and mixed, then 6 aliquots (5 mL each) were purified according to the protocol reported above. After purification, adequate and increasing amounts of standard solutions of DON, DOM-1, AFM1, FB1, ZEA, α-ZOL, β-ZOL and OTA. Triplicate experiments were performed. The spiking concentration of each analyte was reported in A mixture of 3 blank pig urine samples was used for the recovery experiment of DON, DOM-1, AFMLeft-censored analytical results, i.e.; values below the limit of detection (LOD) and the limit of quantification (LOQ), were evaluated with the substitution method as suggested by the European Food Safety Authority (EFSA 2010) . Within 1, OTA and AFB1 in pigs using urinary biomarker concentrations measured in this study according Formula (1), reported by [Calculation of intake estimation was performed for DON, ZEA, FBorted by :PDI = (CPDI: probable daily intake of each mycotoxin (µg/kg body weight);C: pig urinary biomarker concentration (µg/L);V: mean 24 h pig urine volume (2.5 L);W: mean pig body weight (110 kg);E: mean urinary excretion rate of each mycotoxin in 24 h post dose in piglets .The DON, ZEA, FBML: mycotoxin level in the feed (µg/kg);PDI: probable daily intake of the mycotoxin (μg/kg body weight);W: mean pig body weight (110 kg)V: mean 24 h pig urine volume (2.5 L)p value). Values were judged to be significantly different if p values were < 0.05.Mean, median and standard deviation of the results were calculated using Microsoft Excel 2013 software . Statistical analyses were performed using GraphPad Instat software version 3.00 . Data were subjected to the unpaired t-test . A


Chlamydia trachomatis infections in women continue to be a major public health concern due to their high prevalence and consequent reproductive morbidities. While antibiotics are usually efficient to clear the Chlamydia, repeat infections are common and may contribute to pathological outcomes. Interferon-gamma (IFN-γ)-mediated immunity has been suggested to be protective against reinfection, and represent an important anti-chlamydial agent, primarily via the induction of indoleamine-2,3 dioxygenase 1 (IDO1) enzyme. IDO1 catalyzes the degradation of tryptophan, which can eliminate C. trachomatis infection in vitro. Here, we sought to measure IDO1 expression levels and related immune markers during different C. trachomatis infection statuses (repeated vs single infection vs post antibiotic treatment), in vitro and in vivo.C. trachomatis-infected women, with either single or repeated infection. In addition, we used an in vitro co-culture model of endometrial carcinoma cell-line and peripheral blood mononuclear cells (PBMCs) to measure the same immune markers.In this study, we measured the expression levels of IDO1 and immune regulatory markers, transforming growth factor β1 (TGF-β1) and forkhead box P3 (FoxP3), in vaginal swab samples of C. trachomatis infections vaginal IDO1 and TGF-β1 expression levels were significantly increased. Whereas, women who cleared their infection post antibiotic treatment, had increased levels of IDO1 and TGF-β1, as well as FoxP3. Similarly, using the in vitro model, we found significant upregulation of IDO1 and TGF-β1 levels in the co-culture infected with C. trachomatis. Furthermore, we found that in PBMCs infected with C. trachomatis there was a significant upregulation in IDO1 levels, which was independent of IFN-γ. In fact, C. trachomatis infection in PBMCs failed to induce IFN-γ levels in comparison to the uninfected culture.We found that in women with repeated C. trachomatis infection, both in vivo and in vitro, without elevated IFN-γ levels. This study implicates IDO1 and TGF-β1 as part of the immune response to repeated C. trachomatis infections, independently of IFN-γ.Our data provide evidence for a regulatory immune response comprised of IDO1, TGF-β1 and FoxP3 in women post antibiotic treatment. In this study, we demonstrated a significant increase in IDO1 expression levels in response to The online version of this article (10.1186/s12879-019-3843-4) contains supplementary material, which is available to authorized users. Chlamydia trachomatis infections continue to be a major public health concern due to high infection rates and long-term reproductive morbidities [C. trachomatis reinfections has been associated with interferon gamma (IFN-γ) production in several human studies [C. trachomatis-infected cell lines have also shown the important role of IFN-γ in eliminating infection [C. trachomatis have an inhibitory effect on infection in vitro. While high IFN-γ levels were shown to eradicate the infection, lower levels can drive urogenital C. trachomatis to enter their persistence form, characterized by in vitro aberrant, non-infectious bodies [C. trachomatis aberrant bodies were also identified in infected women, and were associated with low IFN-γ levels in the genital tract [C. trachomatis infections in women [C. trachomatis (single or repeated infection) versus uninfected women [C. trachomatis infections. Specifically, it was found that women with repeated C. trachomatis infection had significantly higher kynurenine levels in their vaginal secretions. High kyn/trp ratios however, did not correlate with the low IFN-γ levels measured from vaginal secretions of the same women. IDO1-IFN-γ axis, which is a well-known important antimicrobial mechanism [C. trachomatis was shown to have several immune evasion mechanisms that allow it to establish infection, which can often persist for long periods of time without any symptoms [C. trachomatis single versus repeated infection and post antibiotic treatment, in a cohort of a previously published study [C. trachomatis infected endometrium cell line (ECC1) co-cultured with PBMCs, with or without azithromycin treatment. We found that in women with repeated infections and in women who have cleared their infection after antibiotic treatment, immune-regulatory markers, IDO1, TGF-β1 and FoxP3 were significantly upregulated in comparison to C. trachomatis negative women and in those with single current infection. We also show that IDO1 induction was not accompanied by elevated IFN-γ levels. Although IFN-γ-induced IDO1 activity and tryptophan depletion was previously proposed to be an anti-chlamydial mechanism, our data suggest that repeated C. trachomatis infections, as well as antibiotic treatment, can induce IDO1 in an IFN-γ independent manner, that contribute to downregulation of the immune response. These mechanisms may assist the pathogen to avoid immunity and establish an infection.Sexually transmitted bidities , 2. Whilbidities , treatmebidities . Protect studies –7. Varionfection , 9, due nfection , 11. IDOnfection . Deprivis bodies . C. tracal tract . Furtherin women . Althougin women . In a reed women . It was echanism , 19, is echanism –30. The echanism . This isechanism . Tryptopechanism . Anotherechanism , 27. Othechanism , throughechanism . IDO1 isechanism –30. In aechanism . The LPSechanism . TGF-β-cechanism , whereasechanism , may be symptoms . These msymptoms ). Here, ed study . In addiC. trachomatis infection using Cobas® 4800 CT/NG Test  , and were either, C. trachomatis negative , C. trachomatis positive with single infection  or C. trachomatis positive with a recent repeated infection (up to 1 year) . Other sexually transmitted infections were excluded from this study. Women with single C. trachomatis infection were treated with a single dose of azithromycin and were invited back to second and third visits for follow up  , Caucasian female donor, . Cryopreserved PBMCs were shipped in liquid nitrogen. Upon arrival, cells were placed immediately in liquid nitrogen for one week before use. PBMCs were quickly thawed in 37 °C water bath for 2 min, and were suspended in RPMI-1640 medium , containing 10% heat inactivated fetal calf serum (FCS) , treated with 20 U/ml DNase I . Cells were then left to rest for 12 h prior to the experimental assay [7 cell/vial, using hemocytometer.Human PBMCs were received from a 61 years old  was routinely cultured in ECC1 cell line (ATCC CRL-2923) with DMEM  containing 5% heat inactivated fetal calf serum (FCS) , 120 μg/ml streptomycin , 50 μg/ml Gentamycin , 37 °C, 5% CO2. ECC1 is an endometrial primary cell line, responsive to hormones and to C. trachomatis infections, was previously used in infectivity assays and similar co-culture models [5 female human PBMCs  cells/well were seeded with RPMI-1640 medium  containing 10% heat inactivated FCS , 120 μg/ml streptomycin , 50 μg/ml Gentamycin , 37 °C, 5% CO2. In addition, control plates with either ECC1 cells alone or human PBMCs alone were used. Female PBMCs donor was reported negative for C. trachomatis. Co-cultures were infected with C. trachomatis at a multiplicity of infection (MOI) of 0.1. At the time of infection, the ECC1 monolayer was at 90% confluence. At 20 h PI infected cells were treated with 1 μg/ml of azithromycin . For evaluation of transcript levels, cells were harvested at 20 h post seeding, 44 h and 68 h PI. Infectious yields were measured at 44 h PI. Infected cells and culture supernatants were then sonicated and used to infect a new ECC1 cell monolayer in three replicates, for enumeration of recoverable inclusion forming units (IFUs). After staining with an in-house anti-HtrA and goat anti rabbit IgG (H + L) Alexa Flour 488 , wells were visualized for inclusion presence using fluorescence microscopy (Nikon Eclipse TiS Fluorescent Microscope). The chlamydial-HtrA is a virulence and stress response serine protease and molecular chaperone, which is specific for unfolded proteins [e models , 37. Expproteins , 39. Thelater  in − 80 °C until processed. Total RNA was extracted from the swabs using RNeasy micro kit , according to the manufacturer’s instructions. The RNA concentration and purity were determined using Nano-drop Spectrophotometer. 0.2 μg of total RNA was reverse transcribed using QuantiTect Reverse transcription kit , in accordance with the manufacturer’s instructions. For cell culture samples, ECC1, PBMCs and cell’s supernatant were transferred into 1.7 ml Eppendorf tube and centrifuged at 200×g/10 min/4 °C. Cells were resuspended in RNAlater and stored in − 80 °C until processed. Total RNA was extracted from the culture samples using RNeasy mini kit , according to the manufacturer’s instructions. Prior to the reverse transcription, an additional DNase I step was performed. 0.2 μg of total RNA was reverse transcribed using QuantiTect Reverse transcription kit , in accordance with the manufacturer’s instructions.High vaginal swab samples were stored in 1 ml of RNA-ΔCT.Amplification was carried out according to the manufacturer’s instructions using QuntiNova SYBR Green PCR kit . The cycling program was 95 °C for 2 min followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C. Transcript levels were quantified using Rotor-GeneQ . Each primer set was optimized according to a standard curve, while RNA samples were used to assure no DNA contamination. For each sample, transcripts results were normalized against the mRNA of the housekeeping gene β-actin transcripts. The primers used for transcripts quantification are listed in Table All cell culture experiments were conducted in triplicate. The IFU/ml was determined for each condition by measuring the number of inclusions in multiple wells, and accounting for the dilution and volume from the original culture. Data were analyzed using Graph Pad Prism V. 7.01  and presented as the mean ± standard deviation (SD). Statistical differences were determined using Kruskal-Wallis, with Dunn’s post-test , IDO1 expression levels were significantly higher in comparison to C. trachomatis negative women (CT-N) (p < 0.05)  had a trend of higher IDO1 expression levels in comparison to C. trachomatis negative patients. This suggests that although initial C. trachomatis infection does induce IDO1 expression, repeatedly infected women exhibit a stronger IDO1 response in the vagina. Surprisingly, we found that in women that had a single infection (CT-P), after antibiotic treatment and infection clearance (PAT), IDO1 expression levels increased significantly (p = 0.0148) . When compared to C. trachomatis negative women, post antibiotic treatment group had significantly higher TGF-β1 levels (p = 0.0374), while repeatedly infected women had a trend of higher TGF-β1 although not significant . The regulatory transcription factor FoxP3 expression levels were significantly higher only in women post antibiotic treatment, in comparison to C. trachomatis negative women (p < 0.05)  was co-cultured with PBMCs cells sourced from a C. trachomatis negative female human. In addition, ECC1 and PBMCs were cultured separately as a comparison. Cells were infected with C. trachomatis and treated with 1 μg/ml of azithromycin at 20 h PI in order to clear the infection, as well as to evaluate whether azithromycin might have an effect on the increased expression levels of the immune regulatory genes IDO1, TGF-β1 and FoxP3, observed in women post antibiotic treatment. Cultures were then sonicated and used to infect a new ECC1 monolayer, to test for recoverable C. trachomatis IFUs. No chlamydial inclusions were found in any of the cultures treated with azithromycin  . The number of chlamydial IFUs measured from ECC1 and PBMCs co-culture was significantly reduced in comparison to infected ECC1 culture alone (p < 0.0001) by almost two logs.In order to test the host cell immune regulatory response to C. trachomatis infected co-culture of ECC1 and PBMCs, IDO1 and TGF-β1 expression levels were significantly upregulated (p < 0.0001 and p < 0.05 respectively), when compared to uninfected cultures . However, TGF-β1 transcripts in PBMCs alone were initially high in the absence of infection, and were significantly downregulated upon infection (p < 0.0001). FoxP3 expression levels , followed the same trend as TGF-β1 in all cultures , IFN-γ transcript levels were upregulated in comparison to the control (p < 0.05) . This confirms that C. trachomatis stimulation of ECC1 alone can sufficiently trigger the expression of TGF-β1. Whereas ECC1 cells alone cannot express IDO1 in response to C. trachomatis infection, and may require a secondary stimulation by PBMCs.In ECC1 culture alone, there were no detectable IDO1 or IFN-γ expression levels in response to ion Fig. , a, or iChlamydia has developed mechanisms to avoid the immune response while successfully infecting its target host cell. Downregulation of the immune response by Chlamydia has been suggested to occur during infection [C. trachomatis infections in several human studies [As an intracellular bacterium, nfection –44, and nfection . The cytnfection , paradox studies –7.C. trachomatis inhibition due to IDO1-induced tryptophan depletion has been a major hypothesis in the field [C. trachomatis infected women, either single or repeated infection, had significantly higher IDO1 expression levels. This result, along with previously reported data by Ziklo et al., (2018), of significantly higher kyn/trp ratios in the same cohort of women, suggest that tryptophan metabolism via IDO1 is indeed active in the genital tract during C. trachomatis infection in women , which is dominated by L. iners, and CST IV (18%), which comprised of diverse bacterial species and considered as dysbiosis . When measuring FoxP3 expression levels, only women post antibiotic treatment had significantly higher expression levels.he field , 9, and men Fig. . Surprismen Fig. , a. Thes et al., 018, of sia IL-22 –48, in tia IL-22  ia IL-22 . In addidia spp. , 49, 50.C. trachomatis infection-statuses. During antigen presentation to the immune system, hence, an active C. trachomatis infection, either single or repeated, tryptophan catabolism to kynurenine is active, as previously reported [C. trachomatis infection [C. trachomatis infections. The increased expression levels of IDO1, TGF-β1 and FoxP3, in women who cleared their infection, may suggest that during the combined effect of highly expressed levels of IDO1 and TGF-β1, this induces the expression levels of FoxP3, an indicator of regulatory immune response [These data suggest that IDO1 might have a different role when being expressed during different reported , correlareported . These dnfection . Signifiresponse . In addiresponse , 52, andC. trachomatis infection. Surprisingly, while in the C. trachomatis infected co-culture immune-regulatory TGF-β1 and FoxP3 genes were upregulated, in PBMCs alone, expression levels of these genes were downregulated. This downregulation was significant for TGF-β1 in response to C. trachomatis infection or azithromycin treatment alone in PBMCs. Here, we show that the infectivity of C. trachomatis in PBMCs yielded significantly reduced IFUs/ml, consistent with previous literature indicating that C. trachomatis can infect DCs, monocytes and macrophages [C. trachomatis infection (significant effect was shown for TGF-β1 but not in FoxP3). FoxP3 is a key transcription factor previously thought to be expressed mainly in Tregs and is used to assess immune-regulatory function of the host [C. trachomatis infection. Although IFN-γ is known to be the main inducer of IDO1 expression levels, in PBMCs culture alone we found that the significant upregulation of IDO1 was not accompanied by elevated IFN-γ. In fact, C. trachomatis infection did not induce any upregulation in IFN-γ expression levels when compared to the control (−CT -AZ). This suggests that IFN-γ was not solely responsible for the significant induction of IDO1 levels in PBMCs and there is another mechanism, perhaps the stimulation of TLRs by chlamydial particles and innate recognition, which triggered IDO1 expression. Moreover, these in vitro data also correlate with the discordancy in low IFN-γ levels in vaginal secretions of women from the same cohort, with either single or repeated C. trachomatis infection, and the high kyn/trp ratios [In our in vitro ECC1 and PBMCs co-culture model we were able to show that IDO1 and TGF-β1 expression levels were significantly upregulated in response to rophages , 54. Altrophages . This emthe host . It was the host . Here, wp ratios , along wC. trachomatis negative culture, while the effect of the PBMCs and ECC1 co-culture was measured by comparing it to single ECC1 or PBMCs culture. Furthermore, the donor was a healthy woman who had no previous C. trachomatis infections.Although the PBMCs used in this study were sourced from a single donor, the in vitro model was designed in order to control for variations between donors’ immune response. All treatments were compared with C. trachomatis. Although IFN-γ expression levels were not upregulated in response to C. trachomatis infection in PBMCs alone, the addition of PBMCs to the ECC1 culture may have provided additional secreted cytokines, other than IFN-γ, that elicit the immune response cascade and subsequent induction of IDO1. Recently, a study by Jordan et al. (2017), has shown that TNF-α was the predominant cytokine elicited in stimulated PBMCs from C. trachomatis infected patients, rather than IFN-γ [C. trachomatis, triggering the infiltration of immune cells to the infected area, are far more complex than in vitro co-culture environment. Despite these limitations, we were able to show that there is cross talk between the infected epithelial cell line and the PBMCs in response to C. trachomatis infection, while this response is comprised of a significant increase in IDO1 and TGF-β1 expression levels. In addition, these in vitro results correlate with the significantly increased expression levels of IDO1 and TGF-β1 in vaginal swab samples from women who are repeatedly infected with C. trachomatis.While previous studies have shown that IDO can be expressed by some mucosal epithelial and tumor cells , 59, we an IFN-γ . In addian IFN-γ . PerhapsC. trachomatis vaginal tract infections in women, in which differs in case of a single versus repeated infection. In addition, we were able to show that C. trachomatis infection in PBMCs did not elicit IFN-γ response, in spite the significant expression levels of IDO1. These preliminary data may prompt future research into a different key cytokine to elicit IDO1 response and subsequent tryptophan depletion, in order to eliminate C. trachomatis infection in vitro. In addition, this underscore IDO1 and TGF-β1 as a memory immune response to C. trachomatis and their role in driving immune regulation. Consequently, our research provides evidence to include measurements of these immune factors in future vaccine studies.Our results add weight to the available published data of the regulatory immune response to Additional file 1:Patients’ information summary. (DOCX 12 kb)Additional file 2:Detailed participant’s information. (DOCX 14 kb)Additional file 3:Chlamydia-infected ECC1 host cells. Monolayer of ECC1 alone or a co-culture of ECC1 and Chlamydia-negative female donor PBMCs were infected with C. trachomatis at MOI of 0.1 and were harvested at 44 h PI. Cultures were either treated with azithromycin at 20 h PI, or not. Cells and chlamydial inclusions were stained in Chlamydia CEL LPS . Figure show (A) ECC1 cells infected with C. trachomatis. (B) ECC1 cells infected with C. trachomatis and treated with azithromycin. (C) Co-culture of ECC1 and PBMCs infected with C. trachomatis. (D) Co-culture of ECC1 and PBMCs infected with C. trachomatis and treated with azithromycin. (TIF 35745 kb)Microscopic image of Additional file 4:Chlamydia infection and post antibiotic treatment in follow-up visits. Expression levels of (A) IDO1 and (B) TGF-β1 were measured from vaginal swab samples of women who were Chlamydia positive at first visit (time point 0), cleared their infection after azithromycin treatment, and were invited for follow up visits (1–4 months post infection clearance). Results are presented as 2(− ΔCT). (TIF 49 kb)Longitudinal IDO1 and TGF-β1 expression levels from vaginal swab samples of women from initial