Leishmania martiniquensis cases in Thailand have dramatically increased in the recent years. L. martiniquensis infection primarily occurs in immunocompromised patients, especially AIDS patients. In Thailand, amphotericin B is the only drug available for leishmaniasis treatment, and some patients relapse after amphotericin B therapy. Moreover, the efficacy of anti-leishmanial drugs against L. martiniquensis has not been evaluated to date. In this study, we determined the efficacy of various anti-leishmanial drugs against the promastigote and intracellular amastigote stages of L. martiniquensis using a colorimetric assay. Two strains (CU1 and CU1R1) were isolated from leishmaniasis HIV co-infected patient from Songkhla province, southern Thailand. The CU1 strain was isolated from the patient in 2011, and CU1R1 was isolated from the same patient in 2013, when he was diagnosed as relapse leishmaniasis. The third strain (LSCM1) used in this study has been isolated from immunocompetent patient from Lamphun province, northern Thailand. All strains were identified as L. martiniquensis by sequencing of ribosomal RNA ITS-1 and large subunit of RNA polymerase II gene. Bioassays have been conducted both with promastigote and intracellular amastigote stages of the parasite. All L. martiniquensis strains have been tested against amphotericin B, miltefosine and pentamidine to determine the efficacy of the drugs against the parasite by using a PrestoBlue. The efficacy of miltefosine and pentamidine exhibit no significant difference between each stage of L. martiniquensis among all strains. Surprisingly, the promastigote and intracellular amastigote of the CU1R1 isolate, which was isolated from a relapsed patient after amphotericin B treatment, exhibited a two-fold increased inhibitory concentration (IC50) against amphotericin B compared with other strains, and the difference was statistically significant (p < 0.05). Moreover, intracellular amastigotes isolated from CU1R1 exhibited slightly increased susceptibility to amphotericin B compared with the promastigote (p < 0.05). The result of this experiment is a scientific evident to support that in case of relapsed leishmaniasis caused by L. martiniquensis, increasing dosage of amphotericin B is essential. Moreover, this study also determined efficacy of other anti-leishmanial drugs for treatment the leishmaniasis in Thailand in case of these drugs are available in the country and the clinicians should have alternative drugs for treatment leishmaniasis in Thailand apart from amphotericin B.Autochthonous leishmaniasis caused by  Current species . Howeverreatment . Moreove22.1Leishmania martiniquensis strains were used for drug sensitivity tests, including Leishmania co-infected patient from Songkhla province, southern Thailand. The culture originated from bone marrow aspirate. The patient was treated with intravenous amphotericin B deoxycholate . For continuous maintenance, the cultures were passaged every 2–3 days by diluting the original culture with Schneider's media at 1:2 dilutions.Three ycholate . The secycholate . The finycholate . 50–100 2.22 in a humidified atmosphere. The cultures were inspected for the parasites every 24 h under an inverted microscope .Murine macrophages J774A.1 cells were purchased from American Type Culture Collection and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in 5% CO2.3Twenty microliters of promastigote cells or macrophage cells were obtained from 1 ml of cultures and gently mixed with 20 μl of trypan blue solution stock at a dilution factor of 2. Then, 10 μl of the stained cell mixtures was transferred onto the hemocytometer and incubated at room temperature for 5 min. Viable cells, which were unstained, were counted in 5 squares under a microscope at 400× magnification. The number of cells was calculated using the following equation:2.46:105 cells/ml/well and incubated at 37 °C in 5% CO2 for 24 h. The cells were washed with DMEM medium to remove non-phagocytosed parasites. Twenty-four hours after the experiment, Leishmania infections were confirm by structural changes in host cells by Giemsa staining. The intracellular amastigotes was demonstrated using an inverted, light microscope .J774A.1 cells were infected with stationary phase promastigotes in a 24-well plate at a ratio of 10:1 (parasites/macrophage) or 102.5L. martiniquensis (1 × 106 cells/ml/well) were transferred into 96-well culture plates or intracellular amastigotes (1 × 106 cells/ml/well) in 96-well culture plates. Both of stages were exposed to different concentrations of anti-leishmanial drugs. The experiments were performed in triplicate amphotericin B from Streptomyces sp.  was used at concentrations of 2, 1.5, 1, 0.5, 0.3, 0.25, 0.2, and 0.15 μM miltefosine  was used at concentrations of 60, 50, 40, 30, 20, 15, 10, and 5 μM. Pentamidine  was used at concentrations of 60, 50, 30, 20, 10, 8, 5, and 3 μM. Cultures treated with diluents without anti-Leishmania agents were used as controls. Seventy-two hours after the treatment, living cells were quantified, and IC50 values were calculated.The promastigotes of 2.66 cells/ml PrestoBlue™ is a resazurin-based solution reagent that functions as a cell viability indicator, utilizing the reducing power of living cells to quantitatively measure the proliferation of cells. The PrestoBlue™ reagent is a cell-permeable compound that is blue in color and virtually non-fluorescent. When added to cells, PrestoBlue™ is modified by the reducing environment of the viable cell and becomes red and highly fluorescent. The method was performed as follow. First, 90 μl of 1 × 10The percent inhibition of different drug concentrations and calculations for the IC50 value were performed using GraphPad Prism version 6 program .2.7p < 0.05 was considered to be statistically significant for differences and correlations. Differences in promastigote and intracellular amastigote forms for each drug were compared for statistical analysis using an independent t-test. A p-value < 0.05 was regarded as indicating statistical significance. The IC50 values of each anti-leishmanial drug and each Leishmania stage are presented in the table.IC 50 value of promastigotes and intracellular amastigotes were calculated for the mean and standard deviation (SD) from triplicate experiments. Statistical analysis was performed using the statistical package for social sciences (SPSS) software, version 16.0 for Windows . The correlations between drugs and isolations were determined using one-way ANOVA. 33.1In vitro means ‘in glass’, the experiment were conducted outside organisms, in this study is in vitro was refer to promastigote form. IC50 values were calculated from % inhibition values of anti-leishmanial drugs against the parasites, which was determined by the change in the color of PrestoBlue  (estoBlue . IC50s oestoBlue . IC50 va < 0.05) , whereas3.2Ex vivo means ‘out of living’, it refers to experiment done in or on living tissue in an artificial environment outside the organism, in this study is refer to intracellular amastigote form. The efficacy of amphotericin B against CU1, CU1R1, and LSCM1 as indicated by IC50 values was 0.483 ± 0.217 μM, 0.856 ± 0.172 μM, and 0.486 ± 0.207 μM, respectively. Interestingly, the IC50 of the CU1R1 isolate exhibited a significant two-fold increase compared with other strains , whereas no significant difference were noted between CU1 and LSCM1. The IC50 of miltefosine were 18.933 ± 0.737 μM, 19.267 ± 0.404 μM, and 17.433 ± 0.416 μM for CU1, CU1R1 and LSCM1, respectively. The LSCM1 isolate exhibited increased efficiency compared with CU1 and CU1R1 strains. However, mean IC50 against CU1 and CU1R1 were not significantly different. The IC50 of pentamidine were 12.0 ± 0.7 μM, 12.8 ± 1.1 μM, and 12.7 ± 0.346 μM for CU1, CU1R1 and LSCM1, respectively. The susceptibility of all strains exhibited no significant differences. The efficacy of each anti-leishmanial agent  was determined against the intracellular amastigote and promastigote forms. The IC50s of each drug were summarized in 4Leishmania martiniquensis infection is an emerging disease in Thailand , luciferase, and β-galactosidase -2,5-diphenyl-tetrazolium bromide (MTT)  is reduced to resorufin (pink and highly fluorescent). in vitro drug sensitivity of African trypanosomes (Trypanosoma brucei gambiense and T. b. rhodesiense) using the Alamar Blue® rezasurin-based assay, which is a non-toxic cellular assay that can be used with long incubation periods . The IC50 results revealed that extracellular promastigotes and intracellular amastigotes of CU1R1 isolated from a relapsed patient after amphotericin B treatment exhibited two-fold increased IC50 values against amphotericin B compared with the other strains; moreover, the amastigote form  exhibits slightly increased susceptibility to amphotericin B compared with the promastigote form . No difference in susceptibility to miltefosine and pentamidine was observed for the intracellular amastigote and promastigote forms of the three strains. Information from clinicians stated that some patients were relapsed after received amphotericin B treatment , L. donovani strain AG83 , L. tropica, K27 , and L. braziliensis strain L280  in all stages of Leishmania infections as assessed using an MTS assay based on the bio-reduction of the tetrazolium salt MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt) into soluble formazan by mitochondrial dehydrogenase enzymes , L. infantum , L. tropica , L. braziliensis , L. mexicana , L. amazonensis , and L. major  as assessed with the MTS assay  . Howeverde (MTT) . In this periods . Mikus aar Blue® . The resnditions . The difin media . Severalreatment . The resectively . In cont greater . The IC5exposure . The IC5exposure . The IC5 enzymes . The leiTS assay . Based of action . However5L. martiniquensis. This assay could be applied for preliminary screening of new anti-leishmanial agents and as an alternative tool in Leishmania research in the future. This report showed that IC50 against amphotericin B was two-fold increased for L. martiniquensis in the CU1R1 isolate from a relapsed patient compared with the other strains. Other antileishmanial drugs may be considered for use in Thailand. This study showed that miltefosine and pentamidine are effective in vitro and ex vivo. However, the efficacy of other drugs against L. martiniquensis should be determined in the future. This study also determined efficacy of other anti-leishmanial drugs for treatment the leishmaniasis in Thailand in case of these drugs are available and the clinicians should have alternative anti-leishmanial drugs apart from amphotericin B. Data presented in this study are valuable to improve treatment, reveal drug efficacy, and control drug-resistant L. martiniquensis in Thailand.The PrestoBlue® Rezasusin-based assay was to evaluate drug activity against CU1Promastigote was isolated from the patient in 2011CU1R1Promastigote was isolated from the same patient (CU1) in 2013, when he was diagnosed as relapse leishmaniasis.LSCM1Promastigote was isolated from immunocompetent patient.In vitroin vitro was refer to promastigote form.‘in glass’, the experiment were conducted outside organisms, in this study Ex vivo‘out of living’, it refers to experiment done in or on living tissue or cell line in an artificial environment outside the organism, in this study refer to intracellular amastigote form.


Spectroscopic studies were supported by gauge including projector augmented wave (GIPAW) calculations of carbon chemical shielding constants. The main goal of our research was to find salts with better physicochemical properties and to make an attempt to associate them with both the anion structure and the most prominent interactions exhibited by the protonated trazodone cation. The dissolution profiles of trazodone from tablets prepared from various salts with lactose monohydrate were investigated. The studies revealed that salts with simple anions show a fast release of the drug while the presence of more complex anion, more strongly interacting with the cation, effects a slow-release profile of the active substance and can be used for the preparation of the tables with a delay or prolonged mode of action.New pharmaceutically acceptable salts of trazodone (trazodone hydrogen bromide and trazodone 1-hydroxy-2-naphthonic acid) for the treatment of central nervous system disorders are synthesized and described. Although trazodone salts are poorly crystalline, single-crystal X-ray diffraction data for trazodone 1-hydroxy-2-naphthonic acid were collected and analyzed as well as compared to the previously described crystal structure of commercially available trazodone hydrochloride. The powder samples of all new salts were characterized by Fourier transform infrared spectroscopy, X-ray diffraction and  H)-one, known as “trazodone”, is used to treat mood disorders, such as depression -propyl}--triazolo--pyridin-3-(2pression . Trazodone (NET) . In the H)-one and 1-(3-chlorophenyl) piperazine hydrochloride under microwave radiation in the presence of potassium carbonate has been developed -pyridin-3-. Although synthesis of several trazodone salts has been described in the literature  is commercially produced -Neuraxpharm, Tritticoulfonate , benzoatulfonate , acetateulfonate , benzilaulfonate , 2,4-dinulfonate , nitrateulfonate , iodide,ulfonate ) the sysTrazodone was obtained in a solvent-free method of synthesis in the field of microwave radiation under PTC  conditions ,21. The ®).Among all pharmaceutical dosage forms, tablets represent the most popular API delivery system, exhibiting numerous advantages compared to other drug forms. However, only some drugs can be tableted directly without the addition of any excipients, mostly due to diversified active pharmaceutical ingredients (API) stability, which can change, for example upon pressure. On the other hand, the addition of even a small amount of excipients may also change the stability or solubility of the active substances in tablets. In the case of the tested trazodone compounds, we were unable to prepare pure API tablets. During the compression process, the API melted or transformed into another, unstable polymorphic form with a color change. To compare the properties of different trazodone salts from tablets, we prepared tablets with the addition of lactose monohydrate. It is a frequently used excipient, often significantly improving the quality of tablets obtained, as it was also observed with the tablets we prepared  addition. Profiles of the trazodone release from T:HCl + LA, T:HBr + LA, T:OHN + LA, T:EM + LA tablets are shown in The maximum trazodone release in 0.01 M HCl from reference T:HCl + LA tablets is achieved in the first two hours, whereas for all other compounds different time was needed—about 10 min for T:HBr + LA and more than 4 h for T:EM + LA and T:OHN + LA. The best dissolution profile from the pharmaceutical point of view has the T:HBr + LA tablet. In this case, 100% of the API is released during the first 10 min, so a fast mode of action may be assumed. In contrast, in the case of T:EM and T:OHN, the slow dissolution of the drug can be an advantage in the manufacture of prolonged-release tablets. From a therapeutic perspective, this retarded release might be very promising in curing the depression, presumably with a lower or less frequent dosage than usually taken by a patient .One may notice that the dissolution profiles of simple anions (chloride and bromide) differ substantially from the profiles obtained for bigger anions (1-hydroxy-2-naphthoate and embonate). Hence, it might be interesting to compare the trazodone cation structure and its interactions in crystals of these salts and relate it to the dissolution properties. Below, we present the scrutinized analysis of crystal structures for the candidates for tablets of fast (chloride) and slow (1-hydroxy-2-naphthoate) modes of action. It should be emphasized that trazodone salts are weakly crystalline, as evidenced by a small number of structures deposited in the Cambridge Structural Database (CSD) . So far,P21/c and P21/n, respectively. In the case of T:OHN, except for the anion and cation pair one water molecule is present in the asymmetric part of the unit cell  [p1c1 rod symmetry group [Previous studies showed that even such small differences in chemical shifts of chemically identical moieties may be attributed to the presence of weak intermolecular interactions, i.e., various hydrogen bonds or aromatic interactions ,32,33 InC22(6)  and the ry group . Therefo21 screw axis and the anions resides in the middle of the cross-section of these chains. Hence, N–H···Cl and C–H···Cl interactions join these chains into a 3D structure. In T:OHN chains formed by strong O–H···O H-bonds between anion and water molecules are further organized into layers mostly by C–H···π and π···π interactions between anion and chlorophenyl ring of trazodone  %C = 29.31, %H = 3.86, %N = 8.31. 1H-NMR , δ (ppm): 7.75 , 7.21 , 7.18–7.06 , 6.97 , 6.84 , 6.58–6.50 , 4.17 , 3.88 , 3.66 , 3.21 , 3.08 , 2.59 . FT-IR (cm−1), 2983 (C–H), 2937; 2860 (C–H), 1694 (C=O), 1640 (C=N), 1594; 1491 (C=C), 1356 (C–N), 742 (C–Cl).T:HBr. UPLC-MS, t3 of methanol was prepared. In a beaker, 0.372 g of trazodone free base was dissolved in 20 cm3 of methanol by heating the solution to reflux. The 1-hydroxy-2-naphthoic acid solution was then added portionwise while stirring. The solution was cooled to 4 °C and allowed to crystallize. The resulting crystals were filtered off. Y = 57%.A solution of 0.190 g of 1-hydroxy-2-naphthoic acid in 15 cm3 of acetone was prepared. In a beaker, 0.372 g of trazodone free base was dissolved in 20 cm3 of acetone by heating the solution to boiling. The 1-hydroxy-2-naphthoic acid solution was then added portionwise to the trazodone solution with continued stirring. The solution was cooled to 4 °C and allowed to crystallize. The resulting crystals were filtered off. Y = 39%.A solution of 0.190 g of 1-hydroxy-2-naphthoic acid in 20 cm3 of ethyl acetate is prepared. In a beaker, 0.372 g of trazodone free base was dissolved in 30 cm3 of ethyl acetate by heating the solution to boiling. The 1-hydroxy-2-naphthoic acid solution was then added portion-wise to the trazodone solution with continued stirring. The solution was cooled to 4 °C and allowed to crystallize. The resulting crystals were filtered off. Y = 31%.A solution of 0.190 g of 1-hydroxy-2-naphthoic acid in 30 cmM = 3.89 min. Elemental analysis, (C30H30ClN5O4) %C = 62.14, %H = 5.53, %N = 12.05. 1H-NMR , δ (ppm): 8.37 , 7.83 , 7.80–7.67 , 7.59–7.43 , 7.18 , 7.04–6.93 , 6.85 , 6.80–6.73 , 6.45 , 4.12 , 3.48–3.30 , 3.13–3.01 , 3.02–2.89 , 2.40–2.29 , FT-IR (cm−1), 3456 (O–H), 3042 (C–H Ar), 2942; 2843 (C–H), 1716 (C=O), 1638 (C=N), 1592; 1488 (C=C), 1375; 1262 (C–O), 1346 (C–N), 749 (C–Cl).T:OHN. UPLC-MS, t3 of 0.1 M NaOH was prepared. In a beaker, 0.372 g of trazodone free base was dissolved in 20 cm3 of 0.1M HCl. The embonic acid solution was added portionwise to the trazodone solution while stirring. The resulting mixture was adjusted to pH = 7.0. After cooling to 4 °C, a precipitate formed which was filtered off. Y = 84%.A solution of 0.388 g of embonic acid in 20 cm3 of 1M NaOH was prepared at room temperature. In a beaker, 0.372 g of trazodone free base was dissolved in 5 cm3 of 1M HCl. The embonic acid solution was added portionwise to the trazodone solution while stirring. The resulting mixture was adjusted to pH = 7.0. After cooling to 4 °C, the resulting precipitate was filtered off. Y = 63%.A solution of 0.388 g of embonic acid in 5 cmM 3.87 min. Elemental analysis, %C = 61.28, %H = 4.39, %N = 3.19).1H-NMR  δ 8.39 , 8.15 , 7.84 , 7.34–7.12 , 7.03 , 6.95 , 6.85 , 6.66–6.58 , 4.76 , 4.02 , 3.17 , 2.18 , 1.30 . FT-IR (cm−1), 3280 (O–H), 2970 (C–H Ar), 2949; 2840 (C–H), 1657(C=O), 1633 (C=N), 1594; 1453 (C=C), 1353; 1205 (C–O), 1339 (C–N), 738 (C–Cl).T:EM. UPLC-MS tPowder X-ray diffraction patterns for a bulk sample of T:OHN  were recorded at room temperature on a Bruker Advance D8 diffractometer  equipped with a LYNXEYE position sensitive detector using CuKα radiation (λ = 0.15406 nm). The data were collected in the Bragg−Brentano (θ/θ) horizontal geometry (flat reflection mode) between 4° and 50° (2θ) in a continuous scan using 0.03° steps and 384 s/step. The diffractometer incident beam path was equipped with a 2.5° Soller slit and a 1.14° fixed divergence slit, while the diffracted beam path was equipped with a programmable anti-scatter slit (fixed at 2.20°), a Ni β-filter, and a 2.5° Soller slit. The diffraction pattern is presented in 3). H atoms of hydroxy and amino groups were located from a Fourier map and refined with a fixed isotropic parameter of the parent atom equal to 1.2 × Ueq and 1.5U × eq for NH and OH, respectively. Water molecule was refined as a rigid group (AFIX 6). Molecular diagrams were generated using ORTEP-3 for Windows [30H32ClN5O5: (M =578.05 g/mol): monoclinic, space group P21/n (no. 14), a = 8.17642(17) Å, b = 36.3453(5) Å, c = 10.12988(19) Å, β = 109.651(2)°, V = 2835.01(10) Å3, Z = 4, T = 298.15 K, μ(Cu Kα) = 1.600 mm−1, Dcalc = 1.354 g/cm3, 60220 reflections measured (9.586° ≤ 2Θ ≤ 134.566°), 5065 unique  which were used in all calculations. The final R1 was 0.0438 (I > 2σ(I)) and 2wR was 0.1163 . CCDC deposition number: 2051183.X-ray reflections for T:OHN were measured at room temperature on a Rigaku Oxford Diffraction Gemini A Ultra diffractometer  using mirror monochromated CuKα radiation λ = 1.54184 Å). Data collection, cell refinement, and data reduction were performed with CrysAlisPro software . The empirical absorption corrections using spherical harmonics, implemented in the multiscan algorithm were performed. The structure was solved with the SHELXT  using Le84 Å. Dat Windows  Figure . H atoms Windows   utilizing a 13C resonant frequency of 100.61 MHz (magnetic field strength of 9.39 T). Approximately 100 mg of crystalline sample was packed into a zirconium rotor with a Kel-F cap. The cross‑polarization, magic angle spinning (CP-MAS) pulse sequence was used for spectral acquisition. Each sample was spun at a frequency of 8 kHz, and the magic angle setting was calibrated by the KBr method. Each data set was subjected to an 8 Hz line broadening factor and subsequently, Fourier transformed and phase corrected to produce a frequency domain spectrum. The chemical shifts were referenced to TMS using adamantane as an external secondary standard. The optimized recycle delay for T:HCl and T:OHN was 170 s and 50 s, respectively. The NMR spectra were processed with the ACD/SpecManager NMR program .The solid-state iso) were converted to chemical shifts (δiso), using the following equation: δiso = (σGly + δGly) − σiso, where σGly and δGly stand for the shielding constant and the experimental chemical shift, respectively, of the glycine carbonyl carbon atom (176.50 ppm).The quantum-chemical calculations of geometry optimization and NMR shielding constants were carried out with the CASTEP ,29 progrCylindrical tablets were prepared by direct compression of the appropriate trazodone compound with lactose monohydrate . All tablets were prepared using a laboratory press fitted with a 14 mm flat-faced punch and die set, applying 5 t force for 30 s. Each tablet contained 150 mg lactose monohydrate and an appropriate trazodone compound equivalent to 75 mg of trazodone . The amount of the active substance in the tablet: 75 mg ± 5%. As a reference tablets with T:HCl + LA were used.3 of acceptor fluid. For 240 min at appropriate intervals, 5 cm3 of the sample were taken. Samples were taken after 1, 2, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min. To maintain the constant volume of the dissolution medium after each sampling the fresh portion of the medium was added. All samples were filtered with cellulose syringe filters with a membrane diameter of 0.45 µm Millipore filter and diluted.Dissolution experiments were carried out using Erweka DT-80  equipment with controlled temperature (at 36 ± 1 °C) for 4 h. In this method, hydrochloric acid solution (concentration 0.01M) was used as dissolution medium. The rate of stirring for the whole experiment time was 50 ± 2 rpm. In all formulations, the amount of trazodone was 75 mg. Prepared tablets were placed in 900 cm2 > 0.994) the mean of 3 determinations was used  was used to analyze all samples. To minimize possible errors because of overlapping peaks, the maximum in trazodone UV-Vis spectrum at 311 nm was used to calculate drug concentrations without any interferences. The calibration curve used in these studies was presented in was used .v/v). Ionization method: electrospray. Detector: quadrupole. Mass spectra were recorded with positive mode. 1H-NMR: Bruker 400 MHz instrument  with TMS (trimethylsilane) as the internal standard.UPLC-MS analysis conditions: Waters UPLC with PDA detector. Column: 1.7 μm Aquity UPLC BEH C18 . Mobile phase: methanol: water + formic acid  different ways. To determine their therapeutic usefulness, we evaluated the dissolution profiles of trazodone from tablets containing tested salts with added lactose monohydrate. It turned out that the trazodone salt T:HBr showed a much faster release time of the active substance compared to the reference T:HCl salt. On the other hand, T:OHN showed a slow-release profile of the active substance concerning the commercially available hydrochloric salt. Changing the dissolution profile of the drug (indicating slower release) may be useful when designing new formulations containing this drug. Based on these preliminary studies, it may be tempted to state that the salts of trazodone with small anions , due to only weak interactions occurring in the crystal lattice, are released from the tablet faster than salts containing much larger anions . Spacious anions, especially those with aromatic substituents or H-bond active groups, create more significant interactions with the cation, making them more difficult to break, which could explain the retarded release time of the drug.


T, was isolated from the unidentified sponge (SO4) in the deep sea collected of the North Atlantic Ocean. Study of 16S rRNA gene sequences indicated that strain ASO4wetT is a member of the genus Streptomyces and showed the closest similarities to Streptomyces karpasiensis K413T (98.87 %), Streptomyces glycovorans YIM M 10366T (98.38 %), and Streptomyces abyssalis YIM M 10400T (97.53 %). Strain ASO4wetT contained MK-9(H8) as the predominant menaquinone and the major fatty acids are iso-C16:0, anteiso-C15:0, and iso-C15:0. Polyphasic taxonomy was carried out between strain ASO4wetT and its phylogenetically closely related Streptomyces strains, which further elucidated their relatedness and revealed that strain ASO4wetT could be distinguished from currently known Streptomyces species. Strain ASO4wetT clearly represents a novel species in genus Streptomyces. We propose the name Streptomyces bathyalis sp. nov., with the type strain ASO4wetT (= DSM 106605T = NCCB 100657T). Analysis of the whole-genome sequence of S. bathyalis revealed that genome size is 7,377,472 bp with 6332 coding sequences.A novel actinobacterium, designated ASO4wet Streptomyces, some studies recently are focused on the neglected and unexplored regions in order to enlarge the successful isolation of new species  Waksman and Henrici 1948 (Approved Lists 1980)  collected by ROV (remotely operated vehicle) from the North Atlantic Ocean  at 1092 m water depth.In the course of our investigation of Actinobacteria from the deep sea in the extended Continental shelf of Portugal, near Madeira Islands, strain ASO4wet2HPO4 0.5 g, MgSO4.7H2O 5.0 g, artificial seawater (ASW) 1000 ml, agar 20.0 g, pH 7.3) and incubated at 30 °C. Artificial seawater (ASW) contained 3.9 % (w/v) of sea salt from ATI Coral Ocean. Morphological observations of spores and mycelia on International Streptomyces Project 2 or ISP2 agar , ISP3 agar , ISP4 agar  agar, ISP5 agar (glycerol-asparagine), ISP6 agar (peptone-yeast extract-iron), and ISP7 agar (tyrosine)   on GYM medium  and pH range  on ISP2 medium were evaluated after incubation for 14 days. Utilisation of carbohydrate was examined on ISP9 medium supplemented with 1 % carbon sources (Shirling and Gottlieb Growth of strain ASO4wethttp://www.mbio.ncsu.edu/BioEdit/bioedit.html). The almost-complete 16S rRNA gene sequence  of strain ASO4wetT was obtained and submitted for BLAST analysis . The 16S rRNA gene sequence was deposited in GenBank as MT036271. The similarity and homology of the 16S rRNA gene sequence were examined for sequence homology with the database of 16S rRNA gene sequences from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). The 16S rRNA gene sequences of strain ASO4wetT and some related type strains were aligned using the CLUSTAL W algorithm .Extraction of DNA, PCR amplification, and purification of the 16S rRNA gene sequence was performed as described by Landwehr et al. . PCR ampBiomass for the chemical analyses was collected by cultivation in glucose-yeast-malt extract (GYM) medium in flasks on a rotary shaker (160 revolutions per minute) at 30 °C for 3–7 days. The freeze-dried cells from biomass were used for chemical analysis. The whole-cell diaminopimelic acid isomers and sugars were evaluated based on the method of Staneck and Roberts . MenaquiT was incubated at 30 °C for 6–8 days. The samples were prepared using ethanol/formic acid extraction, as described by Schumann and Maier  system and the ACTIN version 6 database Sasser . For matnd Maier .T and the strain Streptomyces karpasiensis DSM 42068T, Streptomyces glycovorans DSM 42021T, and Streptomyces abyssalis DSM 42024T. Standardised and automated ribotyping analysis was conducted using the RiboPrinter system (Hygiena) involving PvuII as a restriction enzyme  according to the instructions of the manufacturer. SMRTbell™ template library was prepared according to the instructions from PacificBiosciences, Menlo Park, CA, USA, following the Procedure & Checklist – Greater Than 10 kb Template Preparation. Briefly, for preparation of 15 kb libraries 8µg genomic DNA was sheared using g-tubes™ from Covaris, Woburn, MA, USA according to the manufacturer´s instructions. DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosciences, Menlo Park, CA, USA. Reactions were carried out according to the manufacturer´s instructions. For the bacterial DNAs, BluePippin™ Size-Selection to greater than 4 kb was performed according to the manufacturer´s instructions . Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell™ template were assessed with the Calculator in RS Remote, PacificBiosciences, Menlo Park, CA, USA. 1 SMRT cell was sequenced on the PacBio RSII  taking one 240-minutes movie.The complete genome sequence of strain ASO4wetBacterial DNAs libraries for sequencing on Illumina platform were prepared to apply Nextera XT DNA Library Preparation Kit  with modifications according to Baym et al. . Sampleshttps://rast.nmpdr.org/)  .Genome assembly performed applying the RS_HGAP_Assembly.3 protocol included in SMRT Portal version 2.3.0 using default parameters. The assembly revealed a single linear chromosome with a coverage value of 117x. Error-correction was performed by a mapping of Illumina short reads onto finished genome using Burrows-Wheeler Alignment bwa 0.6.2  and at pH 6–9 (optimum at pH 7). The strain grew on CYE medium  supplemented with up to 10 % NaCl. Antibiotic susceptibility test indicated that the strain was sensitive to ampicillin, erythromycin, gentamycin, penicillin G, tetracycline, vancomycin, and rifampicin. However, it was resistant to cefotaxime (30 µg/disc).Strain ASO4wetT related to the genus Streptomyces. The strain closely related to Streptomyces karpasiensis K413T (98.87 %), Streptomyces glycovorans YIM M 10366T (98.38 %), and Streptomyces abyssalis YIM M 10400T (97.53 %). Strain ASO4wetT formed a stable clade with Streptomyces karpasiensis K413T that was supported by 82 % bootstrap value in the maximum-likelihood tree based on the 16S rRNA gene sequence , anteiso-C15:0 (22 %), iso-C15:0 (13.8 %), anteiso-C17:0 (8.8 %), and iso-C14:0 (6.3 %). The menaquinone composition was identified as MK-9(H8) and MK-9(H6) in a ratio of 12:1. The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidyl-N-methyl-ethanolamine, phosphatidylinositol mannoside, and four unidentified polar lipids  was conducted to further delineate the relatedness between strain ASO4wetT and its closely related type strains, i.e., Streptomyces karpasiensis DSM 42068T, Streptomyces glycovorans DSM 42021T, and Streptomyces abyssalis DSM 42024T. The levels of DNA-DNA relatedness between strain ASO4wetT and Streptomyces karpasiensis DSM 42068T, Streptomyces glycovorans DSM 42021T, and Streptomyces abyssalis DSM 42024T were 40.4/54.7 %, 40.5/44.4 %, and 35.8/28.2 % respectively. These values are below the threshold value of 70 %, as suggested by Wayne et al.  the part of the pelagic zone between 1,000 and 4,000 m).T (= DSM 106605T = NCCB 100657 T), isolated from a sponge collected from the North Atlantic Ocean at 1092 m depth. The genomic DNA G + C content of the type strain is 70.24 mol%. The genome size is 7,377,472 bp with 6,332 coding sequences, 59 tRNA genes, and six rRNA operons. The complete genome and the 16S rRNA sequence of strain ASO4wetT were deposited at NCBI GenBank with accession number CP048882 and MT036271, respectively.Aerobic, Gram-positive actinomycete that forms branched substrate mycelium. Aerial hyphae can be seen only in ISP3 and ISP7. Spores are not detected in any medium tested even after 4 weeks of incubation at 30 °C. It grows well on ISP2, ISP3, ISP4, and ISP5 after 2 weeks incubation at 30 °C. Optimum growth occurs at 25–30 °C and at pH 7. The NaCl tolerance is 0–10 % (w/v) NaCl. Positive for esterase (C4), trypsin, chymotrypsin, β-galactosidase, and α-glucosidase. Negative for lipase (C14), α-galactosidase, β-glucuronidase, and β-glucosidase. Glucose, arabinose, sucrose, xylose, inositol, mannose, fructose, rhamnose, raffinose are used as sole carbon sources, but not cellulose. Major fatty acids are iso-C16:0 (35.0 %), anteiso-C15:0 (22.0 %), and iso-C15:0 (13.8 %). The major menaquinone is MK-9(H8). The diagnostic amino acid in the peptidoglycan is LL-diaminopimelic acid. Glucose and xylose are present in whole-cell hydrolysates. The type strain is ASO4wet


NKY-312 is a highly active insecticide candidate with a simple structure. In order to carry out field trials and toxicity tests, its scale preparation is urgently needed, but the final step of the original synthetic route is a low-yielding sulfonylation reaction that generates a high proportion of a bissulfonylated by-product, its foliar contact activities against bean aphid (80% at 100 mg/kg) is significantly lower than that of NKY-312 (100% at 5 mg/kg), and uses pyridine as the solvent. In this work, we developed a highly selective (4-dimethylaminopyridine)-catalyzed monosulfonylation reaction that avoids the use of pyridine as a solvent and shows a much higher yield (98% yield with 98% HPLC purity) than the original reaction (68%). Then, we carried out the field trials and toxicity tests. In field experiments, the activities of NKY-312 against rice planthopper and wheat aphid were equal to pymetrozine and imidacloprid respectively. Aphids suck juice out of plant tissues through their oral needles, which retards plant growth; in addition, they can spread plant viruses, which actually cause more damage to crops than the aphids themselves2. Insecticidal chemicals are currently the main tool used for controlling aphids. Pymetrozine, a pyridine azomethine compound that was developed by Syngenta in the 1990s3, as a chordotonal organ TRPV channel modulators, shows excellent activity against homopteran insects, especially aphids, whiteflies, and black tail leafhoppers. In addition, it has long-lasting efficacy and low mammalian toxicity, and organophosphorus and carbamate insecticides do not confer cross-resistance to it. Attempts to develop related compounds by modifying the structure of pymetrozine have been the focus of considerable research14. However, only two candidates have been generated to date, R-76815 and pyrifluquinazon16, both which are far less effective than pymetrozine . The penultimate intermediate in the industrial route to NKY-312 is 4-amino-6-methyl-4,5-dihydro-1,2,4-triazin-3(2H)-one27, which is subjected to a sulfonylation reaction to afford NKY-312 ; and unsuitability for scale-up. Meanwhile, the foliar contact activities against bean aphid of by-product (80% at 100 mg/kg) is significantly lower than that of NKY-312 (100% at 5 mg/kg)26. In order to carry out field trials and toxicity tests, large amount sample is needed; the development of a new, clean process for selective monosulfonylation is urgently needed.Because sulfonyl compounds have been reported to display insecticidal28. DMAP reacts with acylation reagents to form N-acyl-4-dimethylaminopyridine salts29, which exhibit charge delocalization that leads to the formation of compact ion pairs  might be useful for this purposeirs Fig. . As a re1 and p-toluenesulfonyl chloride (2) in DCM gave the highest yield of the desired monosulfonylation product , and the NKY-312:3 ratio was > 50:1. Using DCM as the solvent, we then screened various organic and inorganic bases (entries 6–10), but no improvements were observed. Specifically, reactions with K2CO3, Na2CO3, and triethylamine as the base gave mainly the bissulfonylated by-product, the structure of which was confirmed by single crystal X-ray analysis . When both the sulfonyl chloride addition and the subsequent reaction were carried out at room temperature, the target product was obtained in 51% yield (entry 12).In the previously reported sulfonylation step, pyridine is used as both the base and the solvent. Here, we reduced the amount of pyridine to 1.5 equiv, used 10 mol % DMAP as the catalyst, and screened several solvents . Increasing the concentration turned out to be beneficial: the yield of NKY-312 was highest (89%) when the concentration of 1 was 0.5 mol/L (entry 5). Then we varied the amount of sulfonyl chloride 2 (entries 5 and 9–11) and found that increasing the amount was deleterious; it is possible that the excess sulfonyl chloride increased formation of the bissulfonylated by-product, leading to a corresponding decrease in the yield of NKY-312. Finally, we screened the amount of pyridine (entries 5 and 12–14) and found that 2.5 equiv was optimal, affording NKY-312 in 98% yield (entry 13).Having optimized the solvent, base, and temperature, we explored various other reaction parameters Table . First, 1  in deuterium chloroform at room temperature, 1-tosyl-4-dimethylaminopyridinium chloride can be generated in situ . When the synthesized salt was allowed to react with NKY-312 under the standard conditions, no bissulfonylated by-product was obtained, which indicates that the salt did not react with the monosulfonylation product . With the help of pyridine, DMAP∙HCl reacts with p-toluene sulfonyl chloride to regenerate 4 (fast reaction) and complete the catalytic cycle. The steric hindrance prevents 4 from further reacting with NKY-312 to form bissulfonylated by-products 3.On the basis of control experiments and literature precedentsAfter the completion of process optimization and sample preparation, NKY-312 was employed to evaluate its insecticidal activities against rice planthopper and wheat aphid in field trials using pymetrozine and imidacloprid as controls respectively. The results exhibited that NKY-312 showed the same efficacy as the controls. Tables 1 to obtain insecticide candidate NKY-312. This process, which afforded NKY-312 in 98% yield, with 98% HPLC purity, was highly selective for the monosulfonylation product, did not use pyridine as a solvent, and afforded a higher yield than the previously reported synthesis of this compound. In addition, the process could be used to prepare more than 60 g of NKY-312. After completing the process optimization, we conducted the field trials and toxicity tests. In field experiments, the activities of NKY-312 against rice planthopper and wheat aphid were equal to pymetrozine and imidacloprid respectively. This compound has a very good prospect in commercial development.In summary, we have developed a process for DMAP-catalyzed monosulfonylation of N--yl)acetamide (CAS. no. 136738-23-3) was purchased from Chemieliva Pharmaceutical Co. Reaction progress was monitored by thin-layer chromatography (TLC) on silica gel GF254 with UV detection. Melting points (mp) were obtained with an X-4 binocular microscope melting point apparatus and are uncorrected. 1H and 13C NMR spectra of samples in CDCl3 or d6-DMSO were recorded with a Bruker AV400 spectrometer; tetramethylsilane was used as an internal standard. Chemical shifts (δ) are given in parts per million (ppm). Mass spectra were obtained with a Fourier transform ion cyclotron resonance mass spectrometer . HPLC was performed on an Agilent 1260 Infinity II chromatograph with a VP-ODS column  under the following conditions: mobile phase, 80:20 MeCN/H2O; flow rate, 1.0 mL/min; column temperature, 40 °C; UV detection wavelength, 220 nm; detection time, 20 min. An external standard curve method (quantitative method) was used to determine the purity of the final product (NKY-312). Double-recrystallized NKY-312 (HPLC purity > 99.5%) was used as a reference substance.All reagents were obtained from commercial suppliers and used as received, assuming 100% purity. N--yl)acetamide  in methanol (500 mL) at room temperature, and the resulting mixture was heated at reflux until the reaction was complete, as indicated by TLC (15:1 DCM/methanol). After the reaction solution cooled to room temperature, the pH was adjusted to 7 with 50% sodium NaOH solution, and the solvent was removed by evaporation in vacuo. The residue was taken up in 125 mL of ethanol, and the solvent was again removed in vacuo. Finally, the residue was taken up in 700 mL of acetonitrile, undissolved precipitates  were removed by filtration, and the filtrate was concentrated in vacuo to give 1  as a light yellow solid. Mp 120–122 °C. 1H NMR  δ 9.54 , 4.60 , 3.93 , 1.83 ; 13C NMR  δ 153.6, 144.8, 52.5, 20.3.Concentrated HCl  was added dropwise to a solution of H)-one , pyridine , and DMAP (0.285 g) were dissolved in 225 mL of DCM, and the resulting white suspension was stirred at room temperature for 15 min. Then p-toluenesulfonyl chloride  dissolved in 195 mL of DCM was added dropwise over the course of about 180 min. About 20 min after the start of the addition of 2, the reaction mixture gradually became a yellow solution. After the addition was completed, the reaction was allowed to continue for 10 h at room temperature, at which point TLC (60:1 DCM/methanol) indicated that the reaction was complete. Note that a white suspension formed during the course of the 10 h reaction. DCM (360 mL) and dilute HCl  were added, and the resulting two phases were separated in a separatory funnel. The aqueous phase was extracted with DCM (250 mL × 3), and the combined organic phases were washed with water (600 mL) and saturated brine (600 mL), dried with anhydrous sodium sulfate (about 10 g), and filtered. Evaporation of the filtrate gave NKY-312 . Recrystallization of the crude product from methanol (1.0 L) afforded 60.2 g of the desired product (91% yield) as a white solid .1H NMR  δ 10.09 , 9.78 , 7.69 , 7.37–7.35 , 4.05 , 2.37 , 1.83 ; 13C NMR  δ 151.1, 146.0, 143.9, 136.3, 129.7, 128.2, 53.0, 21.4, 20.2. ESI-HRMS (m/z): Calcd. for C11H15N4O3S [M + H]+ 283.0859; found 283.0863.Amino-6-methyl-4,5-dihydro-1,2,4-triazin-3(23 was obtained as a white solid (mp 219–220 °C) by means of recrystallization from a 50:1 mixture of methanol and acetonitrile. 1H NMR  δ 10.21 , 7.78 , 7.47 , 4.20 , 2.44 , 1.87 ; 13C NMR  δ 149.9, 146.6, 146.3, 135.2, 130.2, 129.1, 53.4, 21.6, 20.3. ESI-HRMS (m/z): Calcd. for C18H21N4O5S2[M + H]+ 437.0948; found 437.0947.Dissulfonylated by-product 32. Briefly, DMAP  was dissolved in dry EtOAc (40 mL), and the solution was cooled with an ice-water bath. p-Toluenesulfonyl chloride  in dry EtOAc (12.5 mL) was added by means of a syringe. The reaction mixture was allowed to warm to room temperature and kept at that temperature for at least 22 h. The product was filtered from the solution, washed thoroughly with diethyl ether (50 mL × 3), and dried in vacuo to afford 1.30 g (79%) of a bright white solid (mp 128–130 °C). The spectral data is consistent with the literature data.1-Tosyl-4-dimethylaminopyridinium chloride was prepared according to a literature procedureSupplementary Information


Benefiting from the coordination, carbonized bimetallic Ni-Co-MOF maintained its initial skeleton and transformed into magnetic-carbon composites with tunable nano-micro structure. During the thermal decomposition, generated magnetic particles/clusters acted as a catalyst to promote the carbon sp2 arrangement, forming special core-shell architecture. Therefore, pure Ni@C microspheres displayed strong MA behaviors than other Nix1−Cox@Carbon composites. Surprisingly, magnetic-dielectric Ni@C composites possessed the strongest reflection loss value − 59.5 dB and the effective absorption frequency covered as wide as 4.7 GHz. Meanwhile, the MA capacity also can be boosted by adjusting the absorber content from 25% to 40%. Magnetic–dielectric synergy effect of MOF-derived Nix1−Cox@Carbon microspheres was confirmed by the off-axis electron holography technology making a thorough inquiry in the MA mechanism.Intrinsic electric-magnetic property and special nano-micro architecture of functional materials have a significant effect on its electromagnetic wave energy conversion, especially in the microwave absorption (MA) field. Herein, porous NiThe online version of this article (10.1007/s40820-020-00488-0) contains supplementary material, which is available to authorized users. As a result, MOF-derived materials have drawn wide research as functional materials because of fine structure regulation and selective preparation. At the same time, building a composite structure with other active materials and constructing a special morphology can further strengthen its role in practical applications.Metal–organic frameworks (MOF), constructing by inorganic metal ion and organic linkers via strong chemical bonds, have gained rapid development and huge influence in the past 2 decades. As functional materials, MOF possess versatile advantages including multi-dimension morphology, specific structure, high surface area, controllable pore size, and so on –4. BasedFor microwave absorption (MA) materials, the complex permittivity and permeability occupy a vital position, which determines material capacity to store and loss electromagnetic waves energy. Generally, the MA performance is mightily governed by the overall effect of the intrinsic electrical and magnetic characteristics modulating by component, dimensional, electron conductivity, and electromagnetic balance –15. Due 2–C composites for enhanced polarization behaviors and boosted conductivity loss starting from MXene/MOF hybrids. In adding, benefiting from the diversity of coordination between the organic and inorganic units, MOFs can be fabricated into changeable dimensional and sculptured into specific structure. Zhang et al. [Secondly, to prove the dielectric loss ability and impedance matching, hybridizing the MOF derivatives and high conductive substrate proved an effective solution. Yu et al.  used mulg et al.  achievedg et al.  obtainedg et al. –29. The x1−Cox@Carbon (Ni@C) composites with tuning nano–microstructure are successfully synthesized via combining the solvothermal reaction and carbonization treatment under Ar atmosphere. Considering the coordination effect between metal elements and inorganic linkers, we project an investigation on the MA ability of a series of Ni@C composites, which construct with different Ni/Co adding ratios. Reduced Ni/Co particles/clusters acted as a catalyst further to facilitate the formation of graphitized carbon layers, which wrap the inner magnetic and build a special core–shell unit. The related results demonstrated that Ni@C composites exhibit increased saturation magnetization (Ms) value, higher conductivity, and enhanced RL ability with the nickel increased nickel content. Therefore, strong magnetic Ni@C microspheres show strongest MA capacity than other Ni@C absorbers. Surprisingly, special Ni@C microspheres have optimized RL value up to − 59.6 dB at only 25% adding. Meanwhile, the effective absorption frequency of magnetic–dielectric Ni@C covers as wide as 4.7 GHz ranging from 9.9 to 14.6 GHz. To probe the intrinsic electromagnetic property and magnetic loss mechanism, off-axis electron holography technology was applied to prove the polarization behavior at the interfaces and magnetic coupling between particles.Herein, using the Ni–Co–MOF as the template, large-sized MOF-derived Ni3)2·6H2O, Ni (NO3)2·6H2O, 0.15 g p-benzenedicarboxylic acid, and 1.0 g PVP K-30 were dissolved in the mixture solution, which contains deionized water (10 mL), ethanol (10 mL), and N, N-dimethylformamide (10 mL). The obtained solution was intensely magnetic stirred for 30 min and transferred to a 50-mL autoclave and keep at 150 °C for 12 h. The gained Co–Ni–MOF products are collected via centrifugation and washing. As-prepared dried products are heated at 600 °C under Ar flow for 5 h with a heating rate of 2 °C min−1. To investigate the effect of different Co2+/Ni2+ addition ratios on the nano–microstructure and electromagnetic properties, Co(NO3)2·6H2O and Ni (NO3)2·6H2O were added into the solution following millimole content: MNi/MCo = 1.5/0, 1.2/0.3, 0.75/0.75, 0.3/1.2, and 0/1.5, respectively. The final magnetic Nix1−Cox@Carbon powders were marked as Ni@C, Ni0.8Co0.2@C, Ni0.5Co0.5@C, Ni0.2Co0.8@C, and CoO@C, respectively.All of the chemicals used were of analytical grade without further purification and were purchased from Sinopharm Chemical Reagent Co., Ltd. In a typical synthesis, Co(NOx1−Cox@Carbon microspheres are measured by vibrating sample magnetometer (MPMS (SQUID) VSM, Quantum Design, USA). The morphology and microstructure of the magnetic powders are examined by a field-emission scanning electron microscope  and a field-emission transmission electron microscope . The absorber samples are prepared by uniformly mixing the magnetic powders with paraffin matrix in different mass ratios 25% and 40%. The sample is compacted into a coaxial ring of 7.00 mm outer diameter and 3.04 mm inner diameter. Related electromagnetic parameters were measured via a vector network analyzer  over the 2–18 GHz range. Related reflection loss curves are calculated by the following equations [Zin and Z0 are the input characteristic impedance and the free space impedance, εr is the complex permittivity and μr is the complex permeability, ƒ is the tested frequency at 2–18 GHz, d is the simulation thickness, c is the velocity of microwave in free space, respectively.The chemical composition of the final products is characterized via powder X-ray diffractometer . Raman data are obtained on a Ramoscope with a He–Ne laser . The magnetic prosperities of Niquations , 31:1\do2O/DMF/EtOH solution. Due to the space coordination effect between metal ions and linkers, Ni–Co–MOF precursors show changeable nano–microstructure and increased particle size. MOF-derived Ni@C exhibited a similar increase size distribution (Table S1). Before obtaining the Ni@C microspheres, the Ni–Co–MOF templates were carbonized in the given Ar atmosphere. Reduced metal/alloy particles can be work as the catalysis to encourage the rearrangement of carbon element in the bridged organic ligands .Porous Ni@C microspheres are fabricated via a facile two-step process as displayed in Fig. n Table S. Before −1 (D-band) and 1592.1 cm−1 (G-band), respectively. Generally, the D-band represents the disorder carbon structure and the atomic crystal defects, and the other G-band means the in-plane stretching vibration stemming from the sp2 hybrid carbon atom. Therefore, the intensity ratio of ID/IG is used as a referenced standard to evaluate the graphitized degree of carbon component. As Cobalt adding content increases, the values of ID/IG of obtained magnetic powders reflect a downward trend from the Ni@C microspheres to the CoO@C composites. The intensity data of Ni@C, Ni0.8Co0.2@C, Ni0.5Co0.5@C, and Ni0.2Co0.8@C composites are 0.92, 1.06, 0.95, and 0.98, respectively. Due to the poor catalytic ability in the thermal treatment process, as-synthesized CoO@C material exhibits weak D-band and G-band signal.Then, the Ni@C composites exhibited controllable electromagnetic properties accompanied by the enhanced saturation magnetization and increased graphitized carbon content. In Fig. M vs H) loop, the saturation magnetization (Ms) value of Ni@C, Ni0.8Co0.2@C, Ni0.5Co0.5@C, Ni0.2Co0.8@C, and CoO@C is 138.5, 131.4, 121.1, 100.7, and 47.2 emu g-1, respectively. Magnetic Ni@C composite has the highest Ms value, which indicates strongest storage for the microwave energy in terms of magnetic component. Compared with the CoO@C microspheres, the other three samples still exhibit better magnetic response capacity, which is necessary to the enhanced magnetic loss ability. Among the magnetic features, the coercivity force (Hc) plays a significant factor to determine the hysteresis and magnetic permeability. The Hc values of five composites are 195, 248, 250, 305, and 356 Oe, respectively. Increased Hc values from Ni@C to CoO@C microsphere are highly associated with the magnetic particles size, space distribution, and integrated responding behavior. According to the latest published literatures, promoted magnetic permeability is benefit to the electromagnetic wave energy conversion because of the higher Ms and suitable Hc values [Simultaneously, magnetic properties of micro-size Ni@C composites are tested at room temperature and related results are pictured in Fig. c values –34.0.8Co0.2@C microspheres have a special hollow structure and uniform size distribution about ~ 3 μm  and the relative complex permeability (μr = μ′ − jμ″) from 2 to 18 GHz. As the MA theory said, the real parts  imply the storage electrical and magnetic capacity toward the incident microwave energy, while the imaginary parts (ε″) represent the matched dissipation ability to the electrical and magnetic components [ε′ values range from 10.4 to 6.8 for Ni@C, from 9.4 to 6.1 for Ni0.8Co0.2@C, from the 4.2 to 3.0 for Ni0.5Co0.5@C, and from 3.6 to 2.8 for Ni0.2Co0.8@C, respectively. Among those MA composites, pure Ni@C composite has the highest ε′ value meaning the strongest ability for storing the electromagnetic wave energy. Similarly, the imaginary part (ε″) values exhibit a decreasing curve, falling from 4.4 to 2.8 for Ni@C, from 3.8 to 2.6 for Ni0.8Co0.2@C, from 0.7 to 0.3 for Ni0.5Co0.5@C, and from 0.4 to 0.2 for Ni0.2Co0.8@C, respectively. The real part (μ′) and imaginary part (μ″) of complex permeability of obtained Ni@C composites display a similar value, which keeps at ~ 1.1 of μ′ values and at ~ 0.08 of μ″, respectively  according the transmitted line theory –40. As smponents , 42. Wheε′ values drop by half and the ε″ data reach very low level, which is not benefit to the energy conversion [ε′ values start from 11.5 to 8.0 for Ni0.8Co0.2@C, from 9.4 to 6.2 for Ni0.5Co0.5@C, and from 7.5 to 5.2 for Ni0.2Co0.8@C  and imaginary part (ε″), encouraging the amelioration of electromagnetic energy conversion. At the same time, the same upward trend of electromagnetic parameters is also seen in porous Ni0.5Co0.5@C and Ni0.2Co0.8@C composites. In adding, related electromagnetic properties of MOF-derived CoO@C composites are studied and discussed at different adding mass conditions. Unfortunately, no matter how the content of CoO@C powders is adjusted, there is no change in its fundamental electromagnetic properties. Large-sized CoO@C microspheres assembled by CoO particles and amorphous carbon cannot absorb the incident microwave energy reflected by the responding parameters . As a result, the derivative CoO@C from carbonized original Co-MOF precursor is unsuitable for applications in the MA field. As above discussed, adjusted electromagnetic behavior of MOF-derived magnetic carbon absorbers is contributed to the changeable magnetic components, spatial distribution, and induced dielectric properties.To adjust the MA performance, the mass adding content increases from 25 to 40% for the better understanding mass factor. Obviously, the electromagnetic parameters make a significant difference, especially in the complex permittivity. As displayed in Fig. nversion . InspiriRL) curves of as-synthesized MOF-derived Nix1−Cox@Carbon composites are calculated and displayed in Fig. RL value of Ni@C can reach up to − 59.5 dB at the C-band, implying the strongest loss ability . As the adding mass is 25%, the EAB of porous Ni@C composite covers as wide as 4.7 GHz ranging at 2.5 mm and 4.9 GHz ranging at 2.2 mm  when traveling in free space. For the Ni@C absorber, the matching behavior is charge by the complex permittivity and complex permeability reflecting the input characteristic impedance (Zin). As displayed in Fig. To fully understand the intrinsic MA mechanism, unique morphology, electromagnetic parameters, and dissipation behaviors are analyzed in detail to discover electromagnetic wave energy conversion. Taking Ni@C as an example, the MA mechanism mainly involves the impedance matching, dielectric loss, magnetic coupling, and the synergy effect in this magnetic carbon system. It is widely accepted that the impedance matching feature is the principal condition, which determines whether electromagnetic waves can enter the material system . Aiming As a magnetic–dielectric synergy MA composite, the dielectric loss of Ni@C composite mainly consists of two parts: one is the polarization loss, and the other is the conductive loss –51. In tσ) of the Ni@C absorber, which plays a key role to affect the storage and loss ability toward the electromagnetic wave energy. According to the Debye theory, the relationship between the complex permittivity  and intrinsic conductivity (σ) can be explained as follows: [ε′ and ε″ are the real and imaginary part of complex permittivity, εs and ε∞ represent the static and infinite permittivity, ω denotes the angular frequency, τ is the temperature-dependent relaxation time, σ is the conductivity. Based on the mentioned equations, the storage ability (ε″) is proportional to the conductivity (σ). The higher electron migration rate is favor to the strengthen microwave energy conversion [ε″), which is necessary to microwave energy dissipation. Meanwhile, the formed Ni clusters work as the catalyst further encouraging the graphitized conversion of the carbon-containing organic ligands. MOF-derived Ni@C composites are assembled by the metal nickel particles and the high conductivity carbon matrix. Graphitized carbon layers with sp2 arrangement can offer ballistic electron transport path, facilitating rapid electron migration. Therefore, both the carbon shell and derived carbon substrate not only can construct the unique conductive network but also offer powerful conduction loss  and lowest coercivity value, which is benefit to the promotion of imaginary part (μ″) and magnetic responding ability. In order to observe the intrinsic magnetic characteristic of the Ni@C microsphere, an off-axis electronic holography is used to discover the magnetic field line distribution, which offers the powerful evidence for the understanding of the magnetic loss mechanism [Equally important, the Ni@C composite can provide excellent magnetic properties, special magnetic coupling effect, and boosted magnetic loss behavior . As dispechanism , 59. Cleechanism –62. The RL of MOF-derived Ni@C composite up to − 59.5 dB at 6.0 GHz at low adding mass of 25% and the EAB can cover 4.7 GHz from 9.9 to 14.6 GHz. Different with the Ni@C absorber, other porous Ni@C composites need to increase the content of effective materials to achieve same excellent MA performance. When the adding mass up to 40%, the maximum RL of Ni0.8Co0.2@C composite reaches − 39.3 dB at 5.7 GHz and the EAB up to 4.8 GHz from 12.8 to 17.6 GHz. MOF-derived Ni@C absorber possess the high MA intensity and wider EAB, showing its potential as MA material in the practical application. As discussed above, the outstanding MA performance of MOF-derived Ni@C can be attributed to the good impedance, strong magnetic and dielectric loss, involving enhanced polarization dissipation, boosted conduction loss, and advanced magnetic coupling effect. As a result, MOF-derived magnetic–dielectric composites provide eye-catching inspiration for fabricating high effective microwave absorption materials.Considering the impedance matching feature and microwave dissipation ability, magnetic–dielectric Ni@C absorber exhibits excellent matching property and high-performance MA. The maximum x1−Cox@Carbon composites. The maximum RL of Ni0.8Co0.2@C composite reaches − 39.3 dB at 5.7 GHz and the EAB up to 4.8 GHz from 12.8 to 17.6 GHz. Benefiting from the coordination, carbonized bimetallic Ni–Co–MOF maintains its initial skeleton and transforms into magnetic carbon MA material with good impedance matching and multiple loss mechanism, involving interfacial polarization, conduction loss, and magnetic dissipation. Intrinsic dielectric loss ability and magnetic responding capacity of the Ni@C absorbers are significantly promoted to promote the electromagnetic wave energy conversion. Therefore, this study highlights that the MOF-derived magnetic/dielectric synergy Ni@C composites with unique nano–microstructure can be a lightweight, wideband, and highly efficient MA materials.In summary, MOF-derived tunable Ni@C composites with unique nano–microstructure are successfully fabricated via solvothermal reaction and carbothermal reduction process. As expected, magnetic–dielectric synergy Ni@C composites, as MA materials, possess the strongest RL value − 59.5 dB  and the effective absorption frequency covered as wide as 4.7 GHz ranging from 9.9 to 14.6 GHz at 2.5 mm. By increasing the absorber content, the MA capacity also can be boosted for other NiSupplementary material 1 (PDF 1011 kb)Below is the link to the electronic supplementary material.


Microcystis aeruginosa UTEX LB 2385, by monitoring cells with  ZnCl2 treatment. Flow cytometry and phase contrast microscopy were used to evaluate physiological responses in cultures. Molecular assays and an immunosorbent assay were used to characterize the expression of MT and MC under zinc stress. The results showed that the half maximal inhibitory concentration (IC50) was 0.25 mg/L ZnCl2. Flow cytometry and phase contrast microscopy showed morphological changes occurred in cultures exposed to 0.25 and 0.5 mg/L ZnCl2. Quantitative PCR (qPCR) analysis of selected cDNA samples showed significant upregulation of Mmt through all time points, significant upregulation of mcyC at a later time point. ELISA MC-LR analysis showed extracellular MC-LR (µg/L) and intracellular MC-LR (µg/cell) quota measurements persisted through 15 days, although 0.25 mg/L ZnCl2 treatment produced half the normal cell biomass and 0.5 mg/L treatment largely inhibited growth. The 0.25 and 0.5 mg/L ZnCl2 treated cells demonstrated a ~40% and 33% increase of extracellular MC-LR(µg/L) equivalents, respectively, as early as Day 5 compared to control cells. The 0.5 mg/L ZnCl2 treated cells showed higher total MC-LR (µg/cell) quota yield by Day 8 than both 0 mg/L ZnCl2 control cells and 0.1 mg/L ZnCl2 treated cells, indicating release of MCs upon cell lysis. This study showed this Microcystis aeruginosa strain is able to survive in 0.25 mg/L ZnCl2 concentration. Certain morphological zinc stress responses and the upregulation of mt and mcy genes, as well as periodical increased extracellular MC-LR concentration with ZnCl2 treatment were observed.Cyanobacteria harmful algal blooms (CHABs) are primarily caused by man-made eutrophication and increasing climate-change conditions. The presence of heavy metal runoff in affected water systems may result in CHABs alteration to their ecological interactions. Certain CHABs produce by-products, such as microcystin (MC) cyanotoxins, that have detrimentally affected humans through contact via recreation activities within implicated water bodies, directly drinking contaminated water, ingesting biomagnified cyanotoxins in seafood, and/or contact through miscellaneous water treatment. Metallothionein (MT) is a small, metal-sequestration cysteine rich protein often upregulated within the stress response mechanism. This study focused on zinc metal resistance and stress response in a toxigenic cyanobacterium,  Cyanobacteria harmful algal blooms (CHABs) are described as toxigenic or irritating biomasses of mostly oxygenic, photosynthetic bacteria on the rise worldwide due to anthropogenic eutrophication (via excessive P and N loading) and increasing climate-change conditions ,3,4,5,6.The rising occurrence of global CHABs may lead to a greater probability of human exposure and animal detrimental effects through environmental interactions ,13. GeneAside from escalating incidences of detrimental effects to organisms, CHABs will most likely play an increasing role in global economic dynamics. The greater occurrence of global CHABs have reported some monitoring and contingency plan estimate costs of 10,000 s–1,000,000 s USD per country per year . While tMicrocystis spp. and the hepatotoxins microcystins (MCs), have been identified as several of the most commonly encountered freshwater CHABs species and cyanotoxins, respectively, on a global scale and within the U.S.  and evaluated as a combined [log2(RQ)] heatmap within RStudio statistical software. The genes expressed in log2 > 0 were represented within an orange-red color scale and indicate upregulation as a response to ZnCl2 treatment, and the genes in log2 < 0 were represented within a green color scale and indicated downregulation as a response to ZnCl2 treatment. Welch two sample t-tests of 16S rRNA endogenous average Ct values control were found to not be statistically different within each sample condition . The specific sample treatment and days were abbreviated as D1_0 mg/L ZnCl2, D1_0.1 mg/L ZnCl2, D1_0.25 mg/L ZnCl2, etc. Correlating to Day 1 treatments for 0, 0.1, and 0.25 mg/L ZnCl2, respectively  cells (by Day 1 . By Day 2) cells —top row.M. aeruginosa UTEX LB 2385 mcyE gene expression showed the least change of expression between both mcy genes profiles, with an initial slight mcyE down-regulation by Day 1 in 0.1 and 0.25 mg/L ZnCl2 treated cells. For 0.25 mg/L ZnCl2 treated cells, mcyE gene expression showed slight up-regulation from Days 5 to 15 in comparison to the control cells . A standard 2nd-order polynomial equation (y = 7.5536x2 − 54.927x + 96.06) was used to calculate MC-LR equivalents below the 2.5 µg/L standard range. The ELISA analysis was performed on extracted MC-LR intracellular and extracellular samples at time periods . Total MC-LR (µg/cell) quota was calculated by adding intracellular and extracellular MC-LR equivalents and dividing by average cells/L. The Pearson’s r correlation showed that total MC-LR (µg/cell) quota was positively correlated with ZnCl2 concentration .ELISA quantitative analysis of 0, 0.1, 0.25, and 0.5 mg/L ZnClM. aeruginosa UTEX LB 2385 cultures treated with 0.1 mg/L ZnCl2 were comparable to 0 mg/L ZnCl2 control cells  × 100}; 23% increase of MC-LR equivalents for Day 12: {(1.40/1.82) × 100})  equivalents at Day 5 compared to control cells {~ 40% increase for 0.25 mg/L ZnCl2 cells (1.05/1.77) × 100; ~ 30% increase for 0.5 mg/L ZnCl2 cells (1.05/1.57) × 100)  quota yield (student’s t-test p < 0.05) than all other ZnCl2 treatment. However, the cell concentration had decreased from Day 5 to Day 8 for 0.5 mg/L ZnCl2 treated cells as stated above. This would indicate possible release of MCs into the extracellular matrix from lysed cells rather than increased MC production due to ZnCl2 concentration.The data was presented as intracellular-extracellular MC-LR (µg/L) equivalents to better evaluate relative concentrations of MC-LR within each sample portion. The extracellular MC-LR (µg/L) equivalents for Days 8 and 12 of  × 100}) . Day 12 t Day 15 . This saells see a above a) × 100) . From Da2 near man-made sources high in processed Zn [Zn is a classified essential, transitional metal ubiquitously involved in numerous cellular biochemical pathways, and is often found within enzyme cavities and protein infrastructures. Zn is often employed in galvanization processes, vulcanization procedures, automobile applications, coating alloy formulations, and various manufacturing components where it often enters the environment in a sequential fashion after ZnO is formed . Environessed Zn .Synechococcus sp. IU 625 cultures were shown to be highly tolerant to 25 mg/L ZnCl2 treatment, and a similar strain showed comparable growth to control when treated with 0.1 mg/L and 0.5 mg/L HgCl2 [Anabaena flos-aquae isolate showed progressive decrease upon treatment with increasing concentrations of CdCl2 and CuCl2—with the highest concentrations presenting the most considerable biophysiological and biomass damage [The interactions of zinc and heavy metal pollution with organisms in naturally occurring and man-made aquatic environments presents its own immediate and long-term problems in our increasingly industrial societies. As an example, a surface river, estuary, and bay sediment study in highly industrial Jinzhou Bay, China found Cu, Zn, Pb, and Cd heavy metals concentrations to be correlated to man-made industrial activities, and has been identified as a polluted ecological risk . Relativ/L HgCl2 ,50. Relas damage .CHABs-associated species and strains often found in metal polluted environments must be given special attention due to their potential survivability and resultant detrimental effects . Although distinct environmental factors have been observed to contribute to favorable conditions conducive to cyanobacteria growth, the specific combinations of effects that causes heavy metal tolerance or resistance in cyanobacteria population are not well understood, or in fact, remain unknown. M. aeruginosa UTEX LB 2385 is tolerant to ZnCl2 concentrations of 0.1 and 0.25 mg/L, with 0.1 mg/L ZnCl2 treated cell numbers and turbidity measurements being similar to control cells through 15 days (p > 0.05 for 0 mg/L and 0.5 mg/L ZnCl2 treated cells/mL). Further, 0.5 mg/L ZnCl2 concentration was found to largely inhibit cell concentration (cell/mL) by Day 15 {(0.01/8.41) × 100 ~ 99.9% inhibition} and decreased the turbidity of the culture. This result is similar to gradual cell density decrease with increasing Zn2+ concentration [M. aeruginosa Kütz 854 cultures showing chlorophyll and phycobiliprotein decrease at CdCl2 concentrations of 1 and 2 µM, and a bactericidal reaction at 4 µM CdCl2 treatment [M. aeruginosa (FACHB-905) and a non-toxic strain (FACHB-469) were shown to accumulate Zn2+ and Cd2+ intracellularly as an uptake metal concentration vs. four-hour time rate [M. aeruginosa. In a different study, a 10 nM treatment of Zn as a free ion concentration did not decrease microcystin/chlorophyll-a (μg/μg) in comparison to UVR and Cu2+ metal treatments of M. aeruginosa strains (UTEX LB 2385 and LE3), but total added Zn to lake water did affect the final biomass and growth rates [M. aeruginosa chlorophyll concentrations were also found to be more stable to CdNO3 treatment than to Pb(NO3)2, and only showed chlorophyll decrease at 20 mg/L Cd after 24 h [M. aeruginosa strain was not inhibited at those concentrations. Conversely, Fe2+ and Fe3+ was shown to increase M. aeruginosa cell density with increasing concentrations up to 12 mg/L [M. aeruginosa (FACHB-905) cultures were observed to show levels of resistance and recovery to treatment with arsenic (III) concentrations  after 48 h incubation, and only showed marked damaging effects to growth and carotenoids production at 10 mg/L [M. aeruginosa is likely dependent on strain and metal species.Our results showed that  15 days a,b. All ntration . Additioreatment . Both a ime rate . This prth rates . Differeter 24 h . Additio 12 mg/L . Lastly, 10 mg/L . These oM. aeruginosa UTEX LB 2385 treated with 0.25 and 0.5 mg/L ZnCl2 possessed larger amounts of multi-cell aggregates compared to 0 and 0.1 mg/L ZnCl2 treated cells at Day 8  histogram profile showed that ed cells . Future y metals . M. aeruic lakes ,10,58.2 treated M. aeruginosa UTEX LB 2385 showed that Mmt gene expression within this strain was significantly upregulated by ZnCl2 concentration dependent and time period dependent parameters  and was not predictive of ZnCl2 concentration response  (relative stability) for MC-LR was: Zn > Cu ≥ Fe ≥ Mg > Ca [qPCR analysis of 0.1 and 0.25 mg/L ZnClrameters —top row.analysis , and a rreatment . The expapeptide , furthernditions . Molecul Mg > Ca .2 treated intracellular and extracellular M. aeruginosa UTEX LB 2385 culture portions . This correlation was inversely associated with the decreased cell biomass through 15 days, indicating release of MC-LR into the extracellular environment. It was observed that intracellular microcystin production was decreased with increasing multiple metal concentrations, indicating a possible distribution of microcystins (intracellularly to extracellularly) due to cell lysis and/or cellular damage [2 treated cells showed a ~40% and 33% increase of extracellular MC-LR equivalents, respectively, as early as Day 5 (2 cell concentration was largely inhibited by the end of the monitor period (99.9% inhibition), a number of multi-cell aggregates and a very low cell number of individual cells (1 × 105 cells/mL) were present at Day 15. The sum of all measured extracellular and intracellular MC-LR (µg/L) concentration through 15 days was slightly higher for cultures treated with 0, 0.1, and 0.25 mg/L ZnCl2 than for the 0.5 mg/L ZnCl2 treated culture. These results are supported by observations that total MC presence is largely associated with overall cell biomass [2 treated cells fluctuated between Days 8, 12, and 15 , but was decreased from Day 5 (2 treated cells from Days 8 through 15 coincided with this observation. These structures may have possibly affected MC interactions with higher ZnCl2 concentrations (0.25 and 0.5 mg/L ZnCl2) in the extracellular environment for the measured time period. A further study of multi-cell aggregation, Exopolysaccharides (EPS) production, and the MC role in the extracellular environment must be evaluated to determine interaction dynamics and possible association. These observations are supported by the following studies. The application of 0.5 mg⋅L −1 Zn2+ was shown to increase the dissolve organic carbon (DOC) concentration of M. aeruginosa (FACHB 469) into the surrounding media by a 18-day study [−1) significantly enlarged Microcystis colony size, specifically increasing EPS production [ELISA quantitative analysis showed MC-LR (µg/L) were present in all ZnClportions . There wr damage . The 0.2as Day 5 . Althoug biomass . Extraceom Day 5 . Interesay study . Furthera FACHB 4 into theMicrocystis aggregation and the associated multi-cell components to heavy metal stress is necessary to determine potential resistance mechanisms and survivability dynamics of this cyanobacteria. A future transcriptomic evaluation of Zn heavy metal response in M. aeruginosa is necessary to profile which intracellular and extracellular localizing genes are in regulation, and to distinguish this important CHAB-associated cyanobacteria species from other cyanobacteria species. Finally, the role of MCs in the extracellular environment must be further determined to understand cyanobacteria-cyanobacteria environment dynamics.Further associative evaluation of 2 metal stress response and resistance capabilities that a universal CHAB species, Microcystis aeruginosa (in this study with strain M. aeruginosa UTEX LB 2385) possesses, and the potential survivability this species demonstrates in increasing polluted aquatic environments. M. aeruginosa UTEX LB 2385 was observed to survive ZnCl2 concentrations of up to 0.25 mg/L, with increasing biomass through 15 days. Though mostly inhibited, 0.5 mg/L ZnCl2 treated cultures presented multi-cell aggregates and residual populations through 15 days. A persistent yield of the cyanotoxin MC-LR (µg/cell) was observed in all ZnCl2 treated cells by 15 days, indicating that this cyanotoxin remains present in the environment even with low cell concentrations. This finding was supported by qPCR data gene expression profiles of Mmt and mcyC, suggesting that M. aeruginosa UTEX LB 2385 possesses several metal response mechanisms.This study exhibits the ZnClMicrocystis aeruginosa UTEX LB 2385 (NCBI Taxon ID: 1296356) was acquired from UTEX Culture Collection of Algae, TX, USA, and was grown to late exponential growth phase  in sterile 1X Cyanobacteria BG-11 Freshwater Medium , within a sterile 250 mL Borosilicate Erlenmeyer flask at 21 ± 2 °C under constant 24 h cool-white fluorescent light (24 µmol⋅m−2 s−1 photons) at a constant agitation of 100 rpm . The 1X Cyanobacteria BG-11 media was prepared with sterile deionized Milli-Q water  and the pH was adjusted to 8.0 ± 0.1 with 1 M NaOH. Duplicate sets of four 20 mL volumes of M. aeruginosa UTEX LB 2385 culture were centrifuged at 2900× g for 10 min, the supernatants were discarded, and the cell pellets were gently washed with sterile deionized Milli-Q water. These cells were repelleted and constituted in 20 mL fresh sterile 1X Cyanobacteria BG-11 media. The 20 mL volumes were aseptically transferred and diluted with 80 mL fresh sterile 1X BG-11 Medium into eight new sterile 250 mL Borosilicate Erlenmeyer Flasks.2)  solution was added to each M. aeruginosa UTEX LB 2385 culture to yield relative ZnCl2 concentrations: 0 mg/L, 0.1 mg/L, 0.25 mg/L, and 0.5 mg/L , as described previously [2 concentrations were targeted through multiple growth curve analysis from past experiments, with concentrations as high as 10 mg/L ZnCl2 (data not shown). The rationale was predicated on a previously studied cyanobacterium, Synechococcus sp. IU 625 with ZnCl2. All ZnCl2-treated cultures were maintained at the same culture growth parameters described above and were monitored by turbidity observation at optical density (O.D.750 nm) with an UltraSpec III  for a predetermined time course of Days: 0, 1, 5, 8, 12, and 15. All M. aeruginosa UTEX LB 2385 culture cell counts were performed via hemocytometer at 400× magnification using an Olympus BH2 BHS-312 Trinocular Microscope , and all experiments were repeated in triplicates. The standard deviations of duplicate set means were used to generate the growth curves. A sterile 1% zinc chloride  and immediately imaged with a Zeiss AxioLab A1 phase contrast microscope coupled with an AxioCam MrC camera  at 1000× total magnification.To determine the physiological effects of ZnCl2-treated M. aeruginosa UTEX LB 2385 cultures was measured (FCM) in 10,000 events per sample within a calibrated MACSQuant Analyzer 10  containing 405 nm, 488 nm, and 638 nm lasers, using the forward scatter setting (FSC-A) for a set short term and long term course period of 8 days . For each time point, 1 mL of each cell treatment was aseptically transferred into a 1.5 mL microcentrifuge tubes, and the parameters were set to gently resuspend before 100 µL was measured in FCM. Different cyanobacteria populations may exhibit distinct patterns of size, complexity, and autofluorescence in regards to colony formation and to their phycobilisome complex. Furthermore, the light harvesting components and phycobilisome complex has been shown to be affected by heavy metals in concentration dependent kinetics [M. aeruginosa UTEX LB 2385 cells were measured in a calibrated MACSQuant Analyzer 10  for the set time course period of 8 days (data not shown). All flow cytometry histograms and statistical analysis of measurements were generated via FlowJo software analysis  to compare the effects of varying concentrations of ZnCl2 treatment within all cultures. Sample cell size for ZnClkinetics ,72. Allo2 treatment (0.5 mg/L) was found to inhibit M. aeruginosa UTEX LB 2385 cell number and decrease turbidity by Day 8. Therefore, total RNA  was isolated and purified using a modified Ambion® RiboPure™ Kit  approach. Homogenization and sample disruption preparation was prepared by adding a volume of 48 mL of 100% EtOH to 60 mL of Wash Solution Concentrate. One mL TRI Reagent was then aseptically mixed with 250 µL of cultures within sterile 2 mL microcentrifuge tubes. The mixtures were homogenized (by vortexing), sonicated 15 times with 3 s pulses (20% power) using a Branson Sonifier Cell Disruptor 200 , and incubated for 5 min at room temperature. Homogenates were centrifuged at 12,000× g for 10 min at 4 °C, and supernatants transferred to new sterile 2 mL microcentrifuge tubes. RNA extraction was performed by aseptically adding a volume of 200 µL CHCl3 solution to each sample, tightly capping and vortexing the sample tubes at 700× g for 15 s, and then incubating at room temperature for 5 min. Each sample was centrifuged at 12,000× g for 10 min at 4 °C before 400 µL of aqueous phase was aseptically transferred to sterile 2 mL microcentrifuge tubes. Then, 200 µL of 100% EtOH was aseptically added to the 400 µL of aqueous phase and immediately vortexed at 700× g for 5 s. The sample mixtures were aseptically transferred to a filter cartridge placed within a collection tube, capped, and centrifuged at 12,000× g for 30 s at room temperature. Each sample flow-through was discarded and the filter cartridge (with bound RNA) was replaced within the same collection tube. Next, 500 µL of wash solution was aseptically added to the filter cartridges, capped, and centrifuged at 12,000× g for 30 s at room temperature. Each sample flow-through was discarded and the filter cartridge was replaced within the same collection tube. The same wash solution step was repeated. Then, the filter cartridges were transferred to new sterile collection tubes and 100 µL elution buffer was added to each respective filter column. The samples were then incubated at RT for 2 min and centrifuged at 12,000× g for 30 s to elute RNA.The ZnCl2O) within a 200 µL nuclease-free reaction tube. The sample tubes were placed and run in a Veriti 96 well Thermocycler  via incubation at 25 °C for ten minutes, 37 °C for two hours, and RT inactivation at 85 °C for 5 m. cDNA sample concentrations and A260/280 ratios were checked using a BioDrop UV/VIS Spectrophotometer, and stored at −20 °C until prepared to use. The concentration (µg/mL) and A260/280 ratios for total RNA samples were checked using a BioDrop UV/VIS Spectrophotometer . The isolated RNA samples served as templates for cDNA synthesis with an ABI High Capacity cDNA Reverse Transcription Kit , using random hexamers as per the manufacturer’s specification. Briefly for each sample, 10 µL DNase-treated RNA was carefully mixed with 10 µL 2X RT MasterMix I  (data not shown). Microcystis aeruginosa mt gene (M. aeruginosa PCC 7941 (NZ_HE973171), and qPCR mcyC and mcyE oligonucleotides were designed based on M. aeruginosa mcy genes  using IDT qPCR PrimerQuest. The following primer pairs were generated: MmtRT2f: 5ʹ-TTGTGAATCCTGTACGTGTCAA-3ʹ and MmtRT2r: 5ʹ-GTCCACAGCCTTTCCCTTTA-3ʹ; mcyC_RT1f: 5ʹ-GGCTAAACCTGACGGGTATAAA-3ʹ and mcyC_RT1r: 5ʹ-CGCAATATTGAGGGAACACAAG-3ʹ; mcyE_RT1f: 5ʹ-AAGTGGGACCAAGACCAATAC-3ʹ and mcyE_RT1r: 5ʹ-TCTAAGCCACGATTGAGAGAAC-3ʹ. An endogenous control was designed to normalize the relative gene expression of mcyC, mcyE, and mt genes using IDT PrimerQuest with M. aeruginosa UTEX LB 2385 (KF372572) 16S ribosomal RNA {MA8516S_RT1F: 5ʹ-GTAGCAGGAATTCCCAGTGTAG-3ʹ and MA8516S_RT1R: 5ʹ-TTCGTCCCTGAGTGTCAGATA-3ʹ}.Oligonucleotides for qPCR were designed using IDT qPCR PrimerQuest  based on CT relative gene expression method was used with the final equation: The cDNA samples were diluted to a concentration of ~100 µg/mL and measured with a BioDrop UV/VIS Spectrophotometer to determine A260/280 ratios. The comparative ® Universal qPCR Master Mix  (containing Hot Start Taq DNA Polymerase). SYBR green dye chemistry was used for reactions containing: 10 µL qPCR Master Mix, 7 µL nuclease-free H2O, 1 µL of forward and 1 µL reverse primers for a total final 1 µM concentration, and 1 µL < 100 ng final mass of diluted cDNA. The qPCR reactions were performed in triplicates through an initial incubation of 50 °C for two minutes, an initial one-cycle denaturation step at 95 °C for 60 s, and 40 cycles of 95 °C denaturation for 15 s with 60 °C extension for 30 s. Quantitative RT PCR reactions were performed within 96-well plate assays using an Applied Biosystems StepOnePlus™ Real Time PCR System  with a Luna2-treated M. aeruginosa UTEX LB 2385 cultures were collected for a predetermined course of 15 days  by centrifuging at 2900× g for 10 min at RT. The supernatants were aseptically removed from the pellets and filtered into new sterile centrifuge tubes using sterile microfiltration apparatus (with sterile ≤ 0.22 µm membrane filters). All pellet and supernatant samples were frozen at −20 °C and stored for 15 days. Intracellular microcystin samples were extracted via modified extraction methods [M. aeruginosa UTEX LB 2385 pellet samples were thawed, refrozen at −20 °C and thawed again, before 5 mL 75% methanol was added and samples were sonicated for 1 min at 20% power using a Branson Sonifier Cell Disruptor 200 . The homogenized pellet samples were allowed to sit at RT for 5 min before they were transferred to a sterile microfiltration device/apparatus (with a sterile ≤ 0.22 µm membrane filter). For initial supernatant samples, 5 mL 75% methanol was added and mixed, before aseptically transferring to their respective sterile microfiltration device/apparatus. The total filtrates were diluted to final 1 mL 4% methanol working sample solutions.ZnCl methods . BrieflyELISA quantitation for intracellular and extracellular microcystin-LR equivalents was performed using a Microcystin-LR ELISA kit (colorimetric)  as per manufacturer specifications. Final absorbances were read at 450 nm in duplicates using a Varioskan™ LUX multimode microplate reader  with SkanIt Software. t method in the ABI StepOne Software . To ascertain if the endogenous 16S rRNA reference gene varied due to experimental conditions, student’s t-tests were performed for all accumulated averaged Ct values organized as treatment groups [t method equation (1) was used to evaluate relative quantifications of mcyE, mcyC, and mt genes using the MA_UTEX LB 2385-specific 16S rRNA endogenous control for the calculations. A Welch two sample t-test was used to evaluate possible statistical significance between different, pooled ZnCl2 treatment groupings of 16S rRNA endogenous control, using RStudio statistical software . A standard 2nd-order polynomial equation (y = 7.5536x2 −54.927x + 96.06) was used to calculate MC-LR equivalents below the 2.5 µg/L standard range. Total MC-LR (µg/cell) quota was calculated by adding intracellular-extracellular MC-LR equivalents (µg/L) and dividing by average cells/L. A Pearson’s product-moment r correlation was used to evaluate increasing ZnCl2 concentration.The growth analysis experiments were performed in triplicates and the standard deviation of means were used to generate growth curves. The statistical analysis of flow cytometry measurements (FCM) were generated via FlowJo software analysis . The initial raw data was analyzed using the comparative Ct groups . The ful