B. burgdorferi determined to a r.m.s. deviation of 0.4 Å over the protein backbone is reported. The NMR structure was determined using multidimensional NMR spectroscopy and consists of four α-helices and two 310-helices. Structural comparison reveals that this protein is highly similar to the acyl-carrier protein from A. aeolicus.The high-resolution NMR structure of the acyl-carrier protein from the pathogen  Borrelia burgdorferi, a target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure-determination pipeline, are reported. This protein was chosen as a potential target for drug-discovery efforts because of its involvement in fatty-acid biosynthesis, an essential metabolic pathway, in bacteria. It was possible to assign >98% of backbone resonances and >92% of side-chain resonances using multidimensional NMR spectroscopy. The NMR structure was determined to a backbone r.m.s.d. of 0.4 Å and contained four α-helices and two 310-helices. A structure-homology search revealed that this protein is highly similar to the acyl-carrier protein from Aquifex aeolicus.Nearly complete resonance assignment and the high-resolution NMR structure of the acyl-carrier protein from  It was isolated and cultured in the 1980s and was named after its discoverer . After reaching an OD600 of 0.4–0.5, the temperature was lowered to 295 K and the cells were induced at an OD600 of 0.6–0.7 by the addition of 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 16–18 h. The protein was purified using an Ni–NTA column followed by Protease 3C cleavage and gel filtration. The protein eluted as a single peak corresponding to a monomer and was confirmed by SDS–PAGE to be >95% pure. The fractions from the gel filtration containing protein were pooled, concentrated and quantitated by absorption at 280 nm using an absorbance ∊280 = 2980 M               −1 cm−1. The final NMR sample contained ∼1.4 mM protein, 100 mM KCl, 20 mM potassium phosphate pH 7.0 in 93% H2O plus 7% 2H2O or in 99.9% 2H2O for other experiments.The gene coding for the acyl-carrier protein  was amplified from the genomic DNA of 2.2.15N–1H]-HSQC, 3D HNCO, HNCA, HN(CO)CA, CBCACONH, CBCANH, 3D 15N-edited TOCSY-HSQC (mixing time 68 ms) and 15N/13C-edited NOESY-HSQC (mixing times 60 and 120 ms). We also recorded a 2D GFT HNHA /(13C–1H)]-HSQC and 2D [1H–1H]-NOESY spectra were integrated to obtain distance constraints. The calibration of cross-peaks was performed using the macro within CYANA with the minimum distance set to 2.4 Å and the maximum distance set to 6.2 Å. Cross-peaks in the NOESY spectra were classified based on their intensities as 1.8–2.7 Å (strong), 1.8–3.7 Å (medium), 1.8–5.0 Å (weak) or 1.8–6.2 Å (very weak). GFT D HNHA . Hydrogen-bond constraints were only added for residues that are involved in α-helices as characterized by initial structures and based on the protection observed in H/D-exchange experiments at a later stage of structure calculation. An upper limit of 2.0 Å was used for the H—O distance in all hydrogen bonds. A total of 1113 distance constraints (an average of ∼15 constraints per residue), which include 345 intraresidue, 308 interresidue , 245 medium-range and 177 long-range distance constraints and 38 hydrogen-bonding constraints were used in the structure calculations of BobuA.00658.a (Table 1Cross-peaks from 3D [a Table 1.2.4.3               J(HN—Hα) .The NMR structure of BobuA.00658.a was determined using distance constraints and dihedral constraints as detailed in Table 1Symbiobacterium thermophilum, Aquifex aeolicus, Prochlorococcus marinus and Clostridium thermocellum revealed that BobuA.00658.a has 48% sequence similarity to the enzyme from S. thermophilum, 44% to that from A. aeolicus, 52% to that from P. marinus and 58% to that from C. thermocellum  revealed that this protein has 37% similarity to the acyl-carrier protein from A. aeolicus, which is structurally closest to it. The backbone r.m.s.d. between the structures from A. aeolicus and B. burgdorferi was 3.1 Å, whereas alignments of BobuA.00658.a with structures from other organisms had higher values. Only residues 8–82 of BobuA.00658.a were selected for r.m.s.d. comparison. Based on the UniProt and homology search, Ser39 seems to be a central residue involved in fatty-acid binding. This residue is close to the core involving three downfield-shifted residues . Finally, a proper study including mutation in vivo will provide a clearer picture regarding fatty-acid binding.Sequence alignment with the enzymes from um Fig. 3. A struc4.B. burgdorferi. Since this protein was selected as a potential target for drug-discovery efforts (Myler et al., 2009We report here the structure of an acyl-carrier protein from BobuA.00658.a, 2kwl            PDB reference:


Pseudomonas mandelii. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. 1H-nuclear magnetic resonance analysis revealed that the granules were composed of PHB. P. mandelii CBS-1 grew optimally at 20°C. When cultured aerobically for 48 h with sucrose as the sole carbon source, strain CBS-1 yielded a maximum cell density of 29.3 g/L cell dry weight and synthesized 22.3 g/L of PHB. The ability of strain CBS-1 to grow at a low temperature and rapidly synthesize high levels of PHB may reduce the costs of industrial PHB production.A novel facultative psychrotroph (strain CBS-1), which accumulates poly-β-hydroxybutyrate (PHB), was isolated from soil samples taken from Changbai Mountain, China. Phylogenetic analysis based on 16S rRNA sequence data and Biolog analysis identified strain CBS-1 as  Alcaligenes sp.  are biodegradable polyesters composed of (R)-3HA monomers, which are ubiquitously synthesized by microorganisms. Many bacteria store carbon as PHAs to protect themselves from nutrient limitation, including lack of nitrogen, phosphorus, and magnesium , the least complex PHA biopolyester, offers distinct advantages for polyester production because of its biodegradability, lighter mass, thermoplasticity, and resistance to abrasion  from the soil of Changbai Mountain, China, and its identification as 2O, vortexed thoroughly, and cultured in an enriched medium containing 5 g/L yeast extract, 10 g/L tryptone, and 2.5 g/L NaCl at 20°C for 2 days. A 50-μL aliquot was spread on PHB detection agar 2SO4, 13.3 g/L KH2PO4, 1.2 g/L MgSO4.7H2O, 1.7 g/L citric acid, 1.7 g/L trace elements solution, 15 g/L agar, and 0.5 μg/mL Nile blue A , Jilin Province, China, was suspended in sterile H600 55–80) was fixed with 4% glutaraldehyde and 1% osmium tetroxide and dehydrated by sequential treatment with 30%, 50%, 70%, 80%, 90%, and 100% acetone. The dehydrated cells were immersed in epoxy resin and then transferred into sample boats. The resin was polymerized for 24 h at 30, 45, and 60°C. Ultra-thin sections were prepared using an ultramicrotome, stained sequentially with uranyl acetate and lead citrate for 22 min and 5 min, respectively, and observed using an H-7650 transmission electron microscope .Cell morphology was examined using transmission electron microscopy as follows: a 2-mL aliquot of cells in late exponential phase  using the BLAST algorithm  and 1492R (5′-ggytaccttgttacgactt-3′) Lane, , and the2O, dried at 40°C, and extracted with chloroform for 1 h at 60°C. Insoluble material was removed by filtration, and the soluble PHB was separated from the chloroform phase by evaporation, washed twice with methanol, filtered, dried at 60–70°C, and weighed using an electronic balance .Cells were harvested and treated with 10% sodium dodecyl sulfate at 100°C for 15 min. After centrifugation at 10,000 rpm for 20 min, the pellets were washed twice with H1H-probe, and deuterated chloroform (CDCl3) was used as a solvent at a final concentration of 10 g/L. The 1H-NMR spectrum for PHB was recorded at 400 MHz. A PHB standard was purchased from Sigma-Aldrich .The NMR spectra of 1-mL samples were recorded using a BRUAK AV-400 spectrometer with a 5-mm 4Cl. All media contained 1.0 g/L KH2PO4, 2.5 g/L Na2HPO4, pH 7.0, and cells were cultured at 20°C, 200 rpm. To test the effect of the C/N ratio, sucrose and monosodium glutamate served as the sole carbon and nitrogen sources and were added at the C/N ratios as follows: 1, 5, 10, 15, 20, 25, and 30. Other conditions were the same as those described above. The optimum C/N ratio was used to test the conditions described next. NaCl concentrations were varied from 0–50 g/L , and at initial pH values from 5.0 to 9.0 . Cultures were incubated from 5°C to 40°C . After 3 days, cells were collected by centrifugation, and the pellets were dried at 70°C to a constant weight.The effects on cell growth of carbon and nitrogen sources, C/N ratio, temperature, initial pH, and NaCl concentration were investigated using single-factor tests  sucrose as the carbon source and monosodium glutamate as the nitrogen source. The pH was adjusted to 7.0 with 0.5 M NaOH. A 10-mL inoculum from a CBS-1 starter culture (OD600 0.5) was added to a 150-mL minimal medium. The 250-mL flasks were incubated at 20°C for at least 3 days and shaken at 200 rpm. Samples were collected at 8-h intervals from 0–72 h, centrifuged, and dried at 70°C to a constant weight.Cells was cultured at 20°C in medium containing 1.0 g/L KHt-test, analysis of variance (ANOVA), and Duncan’s multiple range test were used to determine the statistical significance of differences in the nitrite-to-nitrogen conversion rate. Statistical significance was defined as P < 0.05. All analyses were performed using SPSS for Windows version 11.0.All results are shown as the average and standard deviation of at least three independent experiments. Student’s Oxazine dye Nile blue A was used to develop a simple and highly sensitive staining method to directly detect poly-3-hydroxybutyric acid and other PHAs in growing bacterial colonies  to t Figure .Figure 2P. mandelii was not present in the database. The GN MicroPlate performance characteristics indicated that strain CBS-1 was similar to P. fluorescens biovar C. The similarity index was 0.81 at 24 h  identifies 564 common species in accordance with classical identification methods and current taxonomic nomenclature. Unfortunately, Sucrose and monosodium glutamate were the optimum carbon and nitrogen sources for culturing CBS-1 Table  at a C/N1H-NMR analysis , and the peaks at δ = 0.0 ppm and δ = 0.8 ppm represent the internal standard (CH3)4Si. A small peak at δ = 1.6 ppm was attributed to minor contamination of the solvent with H2O. These results were identical to those for the PHB standard. Therefore, we concluded that the only PHA synthesized by strain CBS-1 grown in the presence of sucrose as the carbon source was PHB.After extracting cells with SDS and isolating PHB as a dried residue as described in Materials and methods, we conducted s Figure . PHB peaStrain CBS-1 was cultured in 150-mL sucrose-containing medium, pH 7.0, at 20°C in a 250-mL flask, which was shaken as described in Materials and methods. Cells were harvested upon reaching the stationary phase (48 h). The CDW was 29.3 g/L, and the PHB concentration reached 22.3 g/L  and robustly produced PHB (76.1% CDW) at 20°C. The yields of PHB produced at 15, 20, and 25°C were significantly different. The yield of PHB produced at 20°C was significantly higher than at 15°C (P < 0.001) and 25°C (P < 0.05).Regions of high mountains perennially covered with snow are another abundant source of psychrotrophs. The mean temperature above 2000 m on Changbai Mountain is 3.3°C Liu . We repoPseudomonas strains that produce PHB at a low temperature, strain CBS-1 is more promising for industrial use. For example, strain CBS-1 uses sucrose as its best carbon source; therefore, PHB can be produced more economically than by strain 14-3T, which requires the more costly carbon source octanoate (Lopez et al., T (Lopez et al., T and A2a5.Compared with a few other Rongpeng Li and Yuji Jiang co-first author.


Haemoproteus, Plasmodium, and Leucocytozoon are well-known avian haematozoa and can cause declined productivity and high mortality in wild birds. The objective of the study was to record the prevalence of haematozoan parasites in a wide range of wetland birds in Bangladesh. Six species of Haemoproteus, seven species of Plasmodium, one unidentified species of Leucocytozoon, and one unidentified microfilaria of the genus Paronchocerca were found. Data on the morphology, size, hosts, prevalence, and infection intensity of the parasites are provided. The overall prevalence among the birds was 29.5% (95 out of 322 birds). Of those, 13.2% (42 of 319) of birds were infected with Haemoproteus spp., 15.1% with Plasmodium spp. (48 of 319) and 0.6% with Leucocytozoon spp. (2 of 319). Two birds were positive for both Haemoproteus sp. and Plasmodium sp. A single resident bird, Ardeola grayii, was found positive for an unidentified microfilaria. Prevalence of infection varied significantly among different bird families. Wild birds of Bangladesh carry several types of haematozoan parasites. Further investigation with a larger sample size is necessary to estimate more accurately the prevalence of haematozoan parasites among wild birds as well as domestic ducks for better understanding of the disease ecology.The parasites of genera  Haemoproteus, Plasmodium, and Leucocytozoon are well-known genera of avian haematozoa. [Haemoproteus produce subclinical infections. Liver, spleen, kidneys, and gizzards become enlarged [Haemoproteus can also parasitize in the lungs [Plasmodium genus cause avian malaria which has sublethal effects on host fitness. The most significant impact is long-term effect on the reproductive system of the host causing population decrease [Leucocytozoon typically causes anemia and enlargement of liver and spleen [Plasmodium genus, one of Haemoproteus genus, and two unidentified microfilariae of different birds were reported from Pakistan [Haemoproteus spp. and Plasmodium spp. in rock pigeons (Columba livia) from different areas of India have been described with their prevalence and seasonal variations [matozoa. –3. Bitinmatozoa. . Though enlarged –8. Haemohe lungs . In somedecrease . Leucocyd spleen . Earlierd spleen –15. In 2Pakistan . Distribriations .Leucocytozoon spp. from domestic ducks [Haemoproteus spp. in domestic pigeons [Of 690 known bird species of Bangladesh, 380 are resident and 310 are migratory  . To date pigeons  have beeA total of 322 wild birds were studied belonging to 48 species (15 families and four orders) from January 2011 to March 2011. The birds were sampled from two wetland sites in Bangladesh: Hakaluki haor  in Sylhet and Moulvibazar districts (212 birds) and Tanguar haor  in Sunamganj district (110 birds) . Hakalukhttp://dx.doi.org/10.1155/2014/493754).From each bird, typically three (but sometimes two) thin blood smears were prepared on clean, grease-free slides. All slides were fixed in absolute methanol for one minute in the field. The fixed smears were then stained with 20% Giemsa stain and were observed under 400x and 1000x magnification by skilled parasitologists. Identification of the haematozoans was performed using the taxonomic description of Valkiunas  and BartP value less than 0.05 was used. The birds were divided into 2 groups: migratory and resident birds. All measurements in the text and the tables are presented in micrometers and given as mean ± standard deviation. Intensity of invasion was calculated per 100 microscopic fields. Statistical Package for the Social Sciences (SPSS) v 17  was used to analyze the data. A two-tailed Fisher's exact test was used to find the correlation of prevalence within bird families and parasite genus. To indicate statistical significance, a Haemoproteus spp., 15.1% for Plasmodium spp. (48/319), and 0.6% for Leucocytozoon spp. (2/319). Two birds were positive for parasites of both Haemoproteus and Plasmodium genera. A single slide was positive for unidentified microfilariae of genus Paronchocerca from Ardeola grayii, a resident bird . Among those, 13.2% (42/319) prevalence of infection was recorded for ent bird .n > 20), the highest prevalence was found in the family Laridae: 44.1% (15 of 44 birds) for genus Haemoproteus; in the family Scolopacidae: 20.8% (5 of 24) for Plasmodium genus; in the family Dendrocygnidae: 2.3% (1 of 43) for Leucocytozoon genus. The prevalence of infection for the three genera varied considerably amid different families of birds . Among tHaemoproteus genus it ranged from 2 to 43, mostly being below 20 parasites per 100 microscopic fields. The lowest intensity of invasion for parasites of Plasmodium genus was 2, while the highest was 18 parasites per 100 microscopic fields, mostly being less than 10. Parasites of Leucocytozoon genus had 4-5 parasites per 100 microscopic fields, while the only bird infected with microfilariae had only 3 parasites per 100 microscopic fields.The intensity of invasion varied across different parasite genera. For parasites of n = 233) of the birds studied were migrant and 27.6% (n = 89) were resident; among these 33.5% (n = 78) of migrant birds and 19.1% (n = 17) of resident birds were positive for any of the parasites of genus Haemoproteus, Plasmodium, or Leucocytozoon. These parasites were more commonly found in migratory birds (P < 0.05). 15.9% (n = 37) of migrant birds and 5.7% (n = 5) of resident birds were positive for Haemoproteus genus, while 16.8% (n = 39) of migrant birds were positive for Plasmodium genus in contrast to 10.3% (n = 9) of resident birds. Migratory birds were significantly preferred by parasites of Haemoproteus (P < 0.05), but for parasites of Plasmodium, there was no significant preference. One migrant bird and one resident bird were found positive for parasites of genus Leucocytozoon spp. One of each migrant and resident bird had mixed infection by parasites of genera Haemoproteus and Plasmodium. There was no significant difference in the infection rates among the study sites.In the present study 72.4%  parasites were identified up to genus level. A single bird was found to be infected with unidentified microfilarae species of genus Paronchocerca. Six species of (1) Haemoproteus (Parahaemoproteus) pastoris, Mello, 1935Morphology. A single bird was infected with the macrogametocyte of H. pastoris. The outline of the gametocytes was amoeboid, adhering to the erythrocyte nucleus and envelope filling the erythrocyte up to their poles. They displaced the erythrocyte nucleus slightly. The measurement of the macrogametocytes was 14.575 ± 0.4 μm ×  4.12 ± 0.3 μm. The nucleus of the macrogametocyte was compact and in subcentral position measuring 3.15 ± 0.02 μm ×  2.575 ± 0.1 μm. 9 to 17 small roundish pigment granules were seen randomly scattered in the cytoplasm. The NDR was 0.5 ± 0.2. Invasion intensity was found to be 17 parasites per 100 microscopic fields.Host. Sturnus contra (Asian Pied Starling). Locality. Hakaluki haor (n = 1).(2) Haemoproteus (Parahaemoproteus) scolopaci, Galli-Valerio, 1929Morphology. A single bird was found to be infected with the macrogametocyte of H. scolopaci. The gametocyte was oval with amoeboid outline and was slightly appressed with the host erythrocyte envelope and nucleus, completely encircling the erythrocyte nucleus and occupying all available cytoplasmic space in the erythrocytes. The nucleus was slightly displaced laterally and the host cell was hypertrophied. The NDR was 0.66 ± 0.03. The measurement of the gametocyte was 14.575 ± 1.47 μM ×  3.575 ± 0.515 μM. Parasite nucleus was centrally situated with the measurement of 3.09 ± 0.3 μM ×  2.57 ± 0.03 μM. The cytoplasm of the gametocyte was homogenous in appearance and contained small roundish pigment granules. The intensity of invasion was found to be 13 parasites per 100 microscopic fields.Host. Charadrius dubius (Little Ringed Plover).Locality. Hakaluki haor (n = 1).(3) Haemoproteus (Parahaemoproteus) plataleae, Mello, 1935Morphology. Both macro- and microgametocytes of H. plataleae were found in 17 birds of two species. Two birds were infected with both gametes. Both the gametocytes markedly encircled the erythrocyte nucleus and appressed closely with the host cell envelope.μM × 4.83 ± 0.88 μM  small roundish pigment granules scattered randomly in the cytoplasm. Infected erythrocytes were slightly hypertrophied. Invasion intensity per 100 microscopic fields was 4 to 28. The NDR was 0.4 ± 0.25.The macrogametocytes were dumbbell-shaped with a measurement of 15.5 ± 1.65  0.88 μM . The nucμM ×  4.50 ± 0.78 μM. The nucleus of the parasite was in submedian position and was also laid free in the cytoplasm like macrogametocyte and measured 3.60 ± 0.73 μM ×  2.83 ± 0.7 μM. The cytoplasm contained more pigment granules than macrogametocytes. 10 to 41 microgametocytes were seen per 100 microscopic fields. The NDR was 0.4 ± 0.01.The microgametocytes were dumbbell-shaped with amoeboid outline and measured 12.45 ± 2.50 Hosts. Larus ridibundus (Black headed gull) and Ardeola grayii (Indian pond heron).Locality. Hakaluki haor (n = 12), Tanguar haor (n = 5).(4) Haemoproteus (Parahaemoproteus) greineri, Bennett, Turner and Whiteway, 1984Morphology. A total of 15 birds of seven species were found to be infected with H. greineri by both macro- and microgametocytes. Both gametocytes were dumbbell-shaped. The nucleus of the host erythrocytes was enclosed slightly by the gametocytes. Some host cells were hypertrophied.μM ×  4.47 ± 0.63 μM. The nucleus was compact, comparatively small, and submedian in position measuring 4.74 ± 1.54 μM ×  3.14 ± 0.92 μM. 18–32 (24 ± 5.4) small roundish pigment granules were randomly scattered throughout the parasite cytoplasm. The NDR was 0.66 ± 0.14. The invasion intensity ranged from 11 to 39 parasites per 100 microscopic fields.The macrogametocytes were closely appressed with both host erythrocyte nucleus and envelope. The macrogametocytes measured 15.10 ± 1.02 μM ×  3.74 ± 0.82 μM in measurement and were loosely appressed with erythrocyte nucleus and envelope. The nucleus of the microgametocyte was submedian or median in position and measured 2.72 ± 0.63 μM ×  1.71 ± 0.38 μM. 16 to 19 small (17.85 ± 2.54 μM) oval pigment granules were seen scattered randomly on the cytoplasm making it granular in appearance or sometimes were aggregated in dense clumps at the end of the parasite. The NDR was 0.7 ± 0.13. 3 to 42 parasites were seen per 100 microscopic fields. The microgametocytes were amoeboid in outline and 12.45 ± 2.58 Hosts. Aythya fuligula (Tufted Duck), Aythya nyroca (Ferruginous Pochard), Netta rufina (Red-Crested Pochard), Aythya ferina (Common Pochard), Anas strepera , Tadorna ferruginea (Ruddy Shelduck), and Anas acuta (Northern Pintail).Locality. Hakaluki haor (n = 9), Tanguar haor (n = 6).(5) Haemoproteus (Parahaemoproteus) nettionis, Johnston and Cleland, 1909Morphology. A total of seven birds belonging to three species were infected with both macro- and microgametocytes of H. nettionis. The cytoplasm of both gametes was homogenous in form, with occasionally some minute vacuoles. The gametocytes almost completely encircled the erythrocyte nucleus and markedly displaced laterally. Host cell was hypertrophied.μM ×  4.85 ± 0.64 μM (μM × 3.33 ± 0.28 μM. Pigment granules were roundish and ranged from 13 to 28 (20.25 ± 6.18). The NDR was 0.38 ± 0.05. Invasion intensity ranged from 2 to 35 parasites per 100 microscopic fields.The macrogametocyte was dumbbellshaped with measurement of 15.75 ± 2.68  0.64 μM . The macμM × 4.3 ± 0.26 μM measurement. The parasite nucleus was submedian in position with 2.7 ± 0.58 μM × 2.07 ± 0.5 μM measurement. The microgametocytes have 17 to 19 (16 ± 1) small roundish pigment granules. The NDR was 0.4 ± 0.08. The invasion intensity was 4 to 16 parasites per 100 microscopic fields.The microgametocytes were also dumbbell-shaped with 12.745 ± 4.8 Hosts. Aythya ferina (Common Pochard), Aythya nyroca (Ferruginous Pochard), and Anas acuta (Northern Pintail).Locality. Hakaluki haor (n = 5), Tanguar haor (n = 2).(6) Haemoproteus (Parahaemoproteus) sp.Morphology. Parasites of genus Haemoproteus of four birds of two species were identified up to subgenus level only. All of the parasites were macrogametocytes and were of oval shape. The gametes were laid closely with the host erythrocyte envelope. The dimension was 12.785 ± 1.47 μM ×  3.595 ± 0.515 μM. The nucleus was oval and measured 4.63 ± 0.9 μM ×  2.56 ± 0.5 μM. It was laid in the outer periphery of the macrogametocyte. The cytoplasm was coarsely granular with 25 to 29 roundish pigment granules. Host erythrocyte cell was slightly hypertrophied and the erythrocyte nucleus was slightly displaced laterally. The NDR was 0.9 ± 0.3. The invasion intensity was 3 to 9 per 100 microscopic fields.Hosts. Dendrocygna bicolor (Fulvous Whistling Duck) and Netta rufina (Red Crested Pochard).Locality. Hakaluki haor (n = 3), Tanguar haor (n = 1).(1) Plasmodium (Haemoamoeba) relictum, Grassi and Feletti, 1891Morphology. A total of 25 birds belonging to 17 species were infected with P. relictum. One was found to be infected with the microgamete and trophozoite of P. relictum and others were infected with only trophozoites. The microgamete was lobulated with oval pigment granules and markedly displaced the erythrocyte nucleus. The pigment granules were loosely clumped in the cytoplasm and the granules were 3.58 ± 0.05 μM ×  2.06 ± 0.03 μM in measurement. The invasion intensity was 5 parasites per 100 microscopic fields for the microgamete.μM ×  2.37 ± 0.7 μM. Invasion intensity ranged from 2 to 19 per 100 microscopic fields.Some trophozoites were oval in shape and some were lobulated . The troHosts. Rostratula benghalensis (Greater Painted Snipes), Anas clypeata (Northern Shoveler), Anas penelope (Eurasian Wigeon), Aythya fuligula (Tufted Duck), Gallinura chloropus (Common Moorhen), Larus brunnicephalus (Brown Headed Gull), Anas querquedula (Gargeney), Tringa stagnatilis (Marsh Sandpiper), Fulica atra (Common Coot), Tringa totanus (Common Redshank), Dendrocygna bicolor (Fulvous Whistling Duck), Anas acuta (Northern Pintail), Todorna ferruginea (Ruddy Shelduck), Nycticorax nycticorax (Black-Crowned Night Heron), Vanellus cinereus (Grey-headed Lapwing), Netta rufina (Red-Crested Pochard), and Anas strepera .Locality. Hakaluki Haor (n = 13), Tanguar Haor (n = 12).(2) Plasmodium (Haemoamoeba) lutzi, Lucena, 1939Morphology. A total of nine birds belonging to seven species were found to be infected with P. lutzi. Trophozoites, situated in the subpolar or in polar position of the erythrocyte, were somewhat oval in shape with measurements of 3.67 ± 1.34 μM × 2.5 ± 0.7 μM (μM. Infected erythrocyte was deformed and the erythrocyte nuclei were displaced markedly. Invasion intensity ranged from 2 to 12 parasites per 100 microscopic fields.± 0.7 μM . The nucHosts. Fulica atra (Common Coot), Gallinura chloropus (Common Moorhen), Anas strepera , Ardeola grayii (Indian Pond Heron), Larus ridibundus (Black-Headed Gull), Porphyrio porphyrio (Purple swamp hen), and Anas acuta (Northern Pintail).Locality. Hakaluki haor (n = 4), Tanguar haor (n = 5).(3) Plasmodium (Giovannolaia) polare, Manwell, 1934Morphology. Three birds belonging to two species were found to be infected with P. polarae. In two birds somewhat oval shaped trophozoites were observed with a measurement of 3.3 ± 0.34 μM × 3.35 ± 1.1 μM. Several oval pigment granules were seen clumped in the cytoplasm. Vacuoles in the cytoplasm, when present, were plentiful. The trophozoites were in subpolar or polar position, occupying less than half of the erythrocyte. Invasion intensity was 4 for one bird and 7 parasites per 100 microscopic fields for the other bird.μM ×  4.12 ± 0.1 μM. Invasion intensity was 6 parasites per 100 microscopic fields. Microgamete was elongated in shape. It has oval pigment granules dispersed in the cytoplasm. The microgamete measured 7.21 ± 0.04 μM ×  3.09 ± 0.23 μM.Both types of gametes were seen in one bird and were lateral to the erythrocyte nucleus in position. Oval macrogamete with coarse oval pigment granules was seen. No vacuoles were seen. The macrogamete measured 3.575 ± 0.24 Hosts. Mesophoyx intermedia (Intermediate Egret) and Dendrocygna bicolor (Fulvous Whistling Duck).Locality. Hakaluki haor (n = 3).(4) Plasmodium (Giovannolaia) circumflexum, Kikuth, 1931Morphology. A total of seven birds of five species were infected with P. circumflexum. Trophozoites were oval in shape measuring 3.5 ± 1.63 μM × 2.84 ± 0.8 μM. Pigment granules somewhat roundish to oval in shape were seen clumped at the edge of the trophozoites and sometimes dispersed in the cytoplasm. Several vacuoles were seen with an average diameter of 1.03 ± 0.01 μM. Invasion intensity ranged from 5 to 18 parasites per 100 microscopic fields.Hosts. Nettapus coromandelianus (Cotton Pygmy Goose), Aythya fuligula (Tufted Duck), Aythya ferina (Common Pochard), Anas acuta (Northern Pintail), and Dendrocygna javanica (Lesser Whistling Duck).Locality. Hakaluki haor (n = 5), Tanguar haor (n = 2).(5) Plasmodium (Giovannolaia) lophurae, Manwell, 1934Morphology. A single bird was found to be infected with P. lophurae. Trophozoites were highly amoeboid in outline measuring 3.09 ± 0.02 μM × 2.06 ± 0.04 μM. Parasite nucleus was large with plentiful cytoplasm. Oval pigment granules are clumped in the cytoplasm. Several vacuoles were seen in trophozoites with average diameter of 1.03 ± 0.01 μM. Intensity of invasion was 7 parasites per 100 microscopic field.Host. Larus ridibundus (Black-Headed Gull).Locality. Hakaluki haor (n = 1).(6) Plasmodium (Haemoamoeba) sp., Grassi and Feletti, 1890Morphology. Four birds of four different species were infected with trophozoites of Plasmodium genus. They were not identified up to species level due to the small number of parasites, but they were identified up to subgenus Haemoamoeba. In general, the trophozoites were elongated with oval pigment granules and dispersed in the cytoplasm, measuring 3.3 ± 0.8 μM × 2.6 ± 0.2 μM. They contained several vacuoles with a diameter of 1.03 ± 0.01 μM.Hosts. Anas acuta (Northern Pintail), Aythya nyroca (Ferruginous Pochard), Anas querquedula (Garganey), and Anas strepera .Locality. Hakaluki haor (n = 2), Tanguar haor (n = 2).(7) Plasmodium (Giovannolaia) sp., Corradetti, Garnham and Laird, 1963Morphology. A single bird was infected with trophozoites of Plasmodium genus. This was identified up to Subgenus Giovannolaia. The merozoites were elongated with oval pigment granules, measuring 5.15 ± 0.02 μM ×  2.575 ± 0.2 μM. It did not contain vacuoles.Host. Podiceps cristatus (Great Crested Grebe).Locality. Tanguar haor (n = 1).Morphology. Only two birds of two species were positive for Leucocytozoon sp.  and Dendrocygna bicolor (Fulvous Whistling Duck).Locality. Hakaluki haor (n = 2).Ardeola grayii (Indian Pond Heron). It was caught from Hakaluki haor. The microfilaria is of Paronchocerca genus , Plasmodium (15.1%) and Leucocytozoon (0.6%) genera was observed here than in studies in India and Myanmar, which are neighboring countries of Bangladesh. In India 18% prevalence was reported for Haemoproteus and 28% for Plasmodium in wild birds and in Myanmar 40% for Haemoproteus and 60% for Plasmodium in wild birds [A lower prevalence of ld birds . Their rld birds  and therld birds , 22.Haemoproteus were below 20 parasites per 100 microscopic fields and invasions for Plasmodium were below 10. This verifies the proposition of Valkiunas [Haemoproteus than in Plasmodium and Leucocytozoon. Haemoproteus gametocytes persist in the peripheral blood for a long time [Leucocytozoon prefer visceral circulation [Leucocytozoon may have escaped our attention. In contrast to Haemoproteus and Leucocytozoon, for parasites of Plasmodium genus, though they prefer peripheral blood circulation, the schizogonic cycle in the erythrocytes lasts for only few days [The intensity of invasion for parasites varied remarkably. Most of the invasions for alkiunas  that it alkiunas . The intong time , while sculation ; therefofew days . As a reVariations in the prevalence of infection in different bird families have been reported in this study, a finding supported by different studies around the globe , 25–28.The report of microfilarial infection in birds in Bangladesh is perhaps the first. The prevalence of microfilariae in Bangladeshi birds appears to be low. The intensity of infection was also low, which may be an effect of the absence of microfilariae in the peripheral blood stream in the daytime due to their periodicity (sampling was done in the daytime) , 30. It In our study, more migratory birds were infected than resident birds. This finding is supported by some other studies , 27. In The pathogenic effects of these parasites can play a vital role in declining wildlife population . Wild biAccession numbers of the Parasite slides deposited in U.S. National Parasite Collection (USNPC), Beltsville, MD 20705, USAClick here for additional data file.


In the complex scenario of cancer, treatment with compounds targeting multiple cell pathways has been emerging. In Glioblastoma Multiforme (GBM), p53 and Translocator Protein (TSPO), both acting as apoptosis inducers, represent two attractive intracellular targets. On this basis, novel indolylglyoxylyldipeptides, rationally designed to activate TSPO and p53, were synthesized and biologically characterized. The new compounds were able to bind TSPO and to reactivate p53 functionality, through the dissociation from its physiological inhibitor, murine double minute 2 (MDM2). In GBM cells, the new molecules caused Δψm dissipation and inhibition of cell viability. These effects resulted significantly higher with respect to those elicited by the single target reference standards applied alone, and coherent with the synergism resulting from the simultaneous activation of TSPO and p53. Taken together, these results suggest that TSPO/MDM2 dual-target ligands could represent a new attractive multi-modal opportunity for anti-cancer strategy in GBM. Combination therapy has been the standard of care in several diseases, such as diabetes and immunoinflammatory disordersThe advantages of multi-target action are well known in cancer, as oncogenesis is a multigenic, multifactorial process, characterized by the misregulation of more than one proteinhttp://clinicaltrials.gov), offer a limited improvement in progression free survival45in vitro and in vivo, both as single agents or combined with the chemotherapeutic agents etoposide or ifosfamide911The multi-target strategy may be of great importance against glioblastoma multiforme (GBM), a particularly aggressive form of brain malignancy. Despite massive research efforts, the surgical resection and the currently approved anti-GBM agents, such as Temozolamide, and Bevacizumab, or Cilengitide, which is in clinical trials  dissipation. These events resulted in cell-cycle arrest, apoptosis and a final anti-proliferative effect on human GBM cells.Thus, while agents targeting either the TSPO or MDM2/p53 interaction have been already investigated and provided to have some survival benefit in cancers, molecules targeting both proteins are not known so far. In this work, with the perspective that co-targeting TSPO and MDM2/p53 with one molecule could maximize the anti-tumor efficacy, dual binders were rationally designed, synthesized and biologically characterized in human GBM cells. Specifically, molecular modeling studies were firstly conducted to identify, among our in-house database of TSPO ligands11121718These effects were similar to those obtained by coincubation with a typical TSPO ligand and a MDM2/p53 inhibitor, namely PK11195 and Nutlin-3, respectively.To the best of our knowledge, this is the first work on TSPO-MDM2 dual-target ligands. These agents represent an attractive multi-modal opportunity for enhancing anti-cancer activity in a tumor with poor prognosis, such as GBM.1 and 7  docking results showed that the carboxylic group flipped toward the Leu26 pocket H-bonding the Nδ of the H96 imidazole ring, thus compensating the loss of the hydrophobic interactions established by the ethyl ester 1. In both cases, the found binding modes definitely suggested that the design hypothesis was sound and that the synthesis of 1 and 7 was worthwhile.In particular, in the first one  in anhydrous dimethylformamide to obtain compound 3, and then deprotected by treatment with trifluoroacetic acid in dichloromethane. Acylation of the commercially available 2-phenylindole 5 with oxalyl chloride, in anhydrous ethyl ether at 0°C, yielded the corresponding 2-phenylindolylglyoxylyl chloride 6176 with compound 4, in the presence of triethylamine in anhydrous toluene at 0°C, gave compound 1 which was purified by flash chromatography. The ester moiety of 1 was finally hydrolyzed with lithium hydroxide monohydrate in a MeOH/H2O (3:1) solution under reflux overnight to yield the corresponding carboxylic acid 7.The convergent procedure applied fort the synthesis of the target indole derivative 1 and 7 to bind TSPO was tested. For this purpose, radioligand binding assays with the TSPO-selective radioligand [3H]PK11195 were performed. Compounds 1 and 7 were able to displace specific [3H]PK11195 binding, in a concentration-dependent manner, with Ki values of 438 ± 35 nM and 759 ± 56 nM  for 15 min lead to a significant reduction in tetrachloro-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) accumulation compared to control untreated mitochondria, demonstrating the collapse of Δψm . When compound 1 or 7 are added  to the MDM2/p53 complex, the W23 peak appears .To confirm the qualitative results obtained in the NMR experiments, a quantitative sandwich immune-enzymatic assay technique, on crude cell lysates obtained from U87MG cells was developed. As a first step, protein dependence of the assay was revealed by adding increased aliquots of U87MG cell lysate. As shown in 16ar range . Noticea1 and 7 on p53 protein levels in U87MG cells, a cell line widely used as a representative model of GBM. These cells maintain wild-type p53, and show reduced p53 functions due to the overexpression of MDM2The cellular parameters commonly studied to support the reactivation of p53 pathway, following cell exposure with a MDM2 inhibitor15161 or 7 for 8 h caused an increase in p53 protein levels   .1 and 7 (5 μM), suggesting a cell cycle block in G2 phase  was able to induce a significant phosphatidylserine externalization, both in the absence (early apoptosis), or in the presence of 7-amino-actinomysin binding to DNA (late apoptosis/death)   . Finallys/death) . All togt (24 h) , in agre1 and 7 (5 μM) for 24 h, and then Δψm was measured. In mitochondria isolated from compound-treated cells, JC-1 accumulation was markedly reduced, demonstrating collapse of Δψm . These results suggest that amplification in Δψm collapse might be ascribable to the co-activation of TSPO and p53 pathways. To confirm these data, the effects of the standard MDM2 inhibitor, Nutlin-3, and of the TSPO ligand, PK11195, alone or in combination, were tested . Also these experiments were repeated using Nutlin-3 and PK11195. Nutlin-3 caused a dose-dependent reduction of U87MG cell viability . The indole is indeed considered a “privileged scaffold” for drug discovery, and PIGAs are prone to be easily substituted. Taking into account available SARs on TSPO, and the crystallographic data about the p53/MDM2 interaction, the 2-phenylindolglyoxylyldipeptide thesized . Dockingthesized .1 and 7 started with the evaluation of their ability to bind TSPO, and to induce, through TSPO activation, Δψm collapse in mitochondria isolated from GBM cells 367 was confirmed by means of Marvin Sketch (ChemAxon). The target MDM2 structure was prepared through the Protein Preparation Wizard within the Maestro 9.0.2112 package using the OPLS-2001 force field. After removing water molecules and adding hydrogen atoms, the protein structures were converted to AD4 format files using ADT. The docking box has been centered on the H96 residue and AutoGrid4 was used to calculate grids points for all the ligand atom types (60 × 60 × 60 with 0.375 Å spacing). 100 separate docking calculations were executed for each ligand. Lamarckian genetic algorithm local search (GALS) method has been used for each docking calculation consisted of 25 × 106 energy evaluations. For each docking run the following parameters were used: population size of 150; 300 rounds of Solis and Wets local search were applied with a probability of 0.06; the mutation rate of 0.02; crossover rate of 0.8. The docking outcomes from each of the 100 calculations were clustered on the basis of the root-mean square deviation (RMSD 2.0 Å) between the Cartesian coordinates of the binder atoms and were ordered based on the free energy of binding. All figures were rendered using Chimera software packageMDM2 structure selection. Several 3D structures of MDM2 can be found in the Protein Data Bank (PDB). Besides the apo-protein NMR structure (PDB code: 1Z1M) and the X-ray structure of MDM2 bound to the transactivation domain of p53 (PDB code: 1YCR), several complexes of MDM2 with different small molecules are reported. A superposition of all these structures on the alpha carbon atoms, using 1YCR as reference, shows that several of them, including 1YCR, lack the N-terminus residues 16–24 while others possess this region which forms  an ordered helix that not only modifies shape and size of the catalytic pocket but provides further points of interactions. Thus, among the non-truncated X-ray structures containing an organic compound, the one with the highest resolution  cocrystallized with a spirooxindole derivative was selected for docking studies. The binding modes of compounds d6 and a Varian Gemini 200 spectrometer operating at 200 MHz was utilized. Analytical TLC was carried out on Merck 0.2 mm precoated silica gel aluminum sheets (60 F-254). Optical rotatory powers ([α]D) were determined using a Perkin Elmer model 343 polarimeter, at a temperature of 22°C. The ≥95% purity of tested compounds was confirmed by combustion analysis. 2-Phenylindol-3-ylglyoxylyl chloride 6 was prepared according with reported procedures17A Reichert Köfler hot-stage apparatus was used to determine the melting points, that are uncorrected. For routine nuclear magnetic resonance spectra, compounds were dissolved in DMSO-2  was solubilized in dry DMF (10 ml) in a nitrogen atmosphere; after addition of CDI , the solution was stirred at room temperature for 2 h. Then, a solution of L-phenylalanine ethyl ester hydrochloride  in 5 ml of the same solvent was added dropwise. The dipeptide 3 formed almost quantitatively within 4–5 h . The solvent was evaporated under reduced pressure and the residue was triturated with saturated aqueous NaHCO3 solution, washed with water and filtered, yielding compound 3 in the desired purity degree. Yield 93%; mp = 78–80°C; [α]22D = −19° (c = 2.0 mg/ml in MeOH).1H NMR  δ (ppm): 0.79–0.84 ; 1.07 ; 1.34–1.55 ; 2.89–2.98 ; 3.97–4.03 ; 4.40–4.46 ; 6.81 ; 7.21 ; 8.13 . Anal. Calcd. for C22H34N2O5: C, 65.00; H, 8.43; N, 6.89. Found: C, 64.82; H, 8.22; N, 6.67.N-Boc-L-Leu 3  in 10 ml of CH2Cl2 and the mixture was maintained at room temperature for 6 h . Evaporation of the solvent under reduced pressure yielded a solid residue that was taken up with water. The solution obtained was cooled in an ice bath, made alkaline (pH = 10) by adding solid K2CO3, and extracted with CH2Cl2. After drying with MgSO4, the organic solvent was eliminated under vacuum and product 4 was isolated in the desired purity degree. Yield 89%; mp = 268–270°C; [α]22D = −7° (c = 2.0 mg/ml in MeOH).1H NMR  δ (ppm): 0.86–0.89 ; 1.07 ; 1.53–1.66 ; 3.01 ; 3.76 ; 4.02 ; 4.50 ; 7.26 ; 8.25 ; 9.04 . Anal. Calcd. for C17H26N2O3: C, 66.64; H, 8.55; N, 9.14. Found: C, 66.44; H, 8.26; N, 8.90.Trifluoroacetic acid  was added to a stirred solution of derivative 5 (3.0 mmol) in freshly distilled diethyl ether (10 ml). The mixture was maintained at room temperature for 2–4 h. The generated precipitate was collected by vacuum filtration to give the acyl chloride 6 that was directly used in the subsequent reaction. A solution of compound 4  in 5 ml of dry toluene was added dropwise to a stirred suspension, cooled at 0°C, of the 2-phenylindol-3-ylglyoxylyl chloride 6 (2.0 mmol) in 15 ml of the same solvent, followed by the addition of a solution of triethylamine  in 1.5 ml of dry toluene. The reaction mixture was left under stirring for 12–24 h at room temperature  and then filtered. The collected precipitate was washed with a 5% NaHCO3 aqueous solution and collected again to give a first portion of crude product. The toluene was removed under reduced pressure and the residue dissolved with CH2Cl2. The organic solution was washed with diluted HCl, a 5% NaHCO3 aqueous solution and water, dried (MgSO4), and evaporated to dryness to yield an additional amount of crude products. Compound 1 was finally purified by flash-chromatography (AcOEt/Ether petrol 60–80°C = 4/6 as eluent). Yield 74%; mp = 86–88°C; [α]22D = −20° (c = 1.0 mg/ml in MeOH).1H NMR  δ (ppm): 0.75–0.82 ; 1.01–1.32 ; 2.91–2.96 ; 3.92–4.03 ; 4.41–4.47 ; 7.18–7.23 ; 7.41–7.57 ; 7.99 ; 8.39 ; 8.57 ; 12.34 . Anal. Calcd. for C33H35N3O5: C, 71.59; H, 6.37; N, 7.59. Found: C, 71.81; H, 6.11; N, 7.42.Oxalyl chloride  was added dropwise, at 0°C to a well-stirred mixture of the 2-phenylindole 1  in 20 ml of a MeOH/H2O (3:1) solution. The mixture was stirred under reflux overnight . Subsequently, the solid precipitate was eliminated through vacuum filtration, and the solution was acidified with 10% HCl to pH = 5. Compound 7 was isolated directly in a pure state by filtration. Yield 70%; mp = 107–109°C, [α]22D = −13.5° (c = 2.0 mg/ml in MeOH).1H NMR  δ (ppm): 0.71–0.83 ; 1.23–1.45 ; 2.89–3.03 : 4.12–4.14 ; 4.43–4.48 ; 7.19–7.59 ; 8.01 ; 8.20 ; 8.59 ; 12.36 ; 12.79 . Anal. Calcd. for C31H31N3O5: C, 70.84; H, 5.94; N, 7.99. Found: C, 70.71; H, 6.05; N, 7.53.Lithium hydroxide monohydrate  was added to a suspension of (2-phenylindol-3-yl)glyoxyl-L-Leucine-L-Phenylalanine ethyl ester 3H]PK 11195 in the presence of new synthesized compounds (0.1 nM–10 μM) in a final volume of 0.5 ml for 90 min at 4°C. Incubations were terminated by rapid filtration through GF/C glass fiber filters and washed three times with 4 ml of cold buffer. The radioactivity was measured by liquid scintillation counter. Non-specific binding was estimated in the presence of unlabeled 1 μM PK 11195. IC50 value was determined using Graph-Pad Prism computer program .Binding studies were performed as previously described2. The U87MG cells were plated at 5,000 cells/cm2. After 24 h, the culture medium was replaced with fresh medium containing newly synthesized compounds solubilized in DMSO for the indicated incubation times. DMSO was added to control cells (<1% v/v).The human glioblastoma multiforme U87MG cells were obtained from the National Institute for Cancer Research of Genova  and monitored for DNA profiling. The U87MG cells were cultured in RPMI medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 1% non-essential amino acids at 37°C in 5% CO1 and 7 (5 μM) were added to the mitochondrial suspension for 15. Then, in a 96 well plate with black bottom, 90 μl of the JWS and 10 μl  of untreated or treated isolated mitochondria were added. The fluorescence  of the sample was read in a spectrofluorimeter using a time-drive method , with settings excitation wavelength at 490 nm and emission wavelength at 590 nm. For evaluation of Δψm in whole cell, U87MG cells were treated with DMSO or compounds (new compounds at 5 μM and standard MDM2 and TSPO ligands at 10 μM), alone or in combination, for 24 h. After incubation time, cells were collected, mitochondria were isolated, and the JC-1 staining procedure was performed using the above-described method.The Δψm dissipation was assessed using the fluorescent dye 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), that has been considered a more reliable and sensitive fluorescent probe for detecting differences in Δψm600, induced for 3 h with 0.4 mM IPTG and collected by centrifugation. Cells were suspended with 5 ml of 20 mM TrisHCl pH 7.2, 20 mM β-mercaptoethanol, 1 mM PMSF and 1 mM EDTA (buffer A) and disrupted by a French press. The cell extract was then centrifuged at 13,000 rpm for 1.5 h to obtain the inclusion bodies as a pellet. This fraction was solubilised with 5 ml of 8 M urea in buffer A and centrifuged at 13,000 rpm for 1.5 h to remove insoluble material. MDM2 was purified under denaturing conditions by affinity chromatography, and to this aim, the supernatant was incubated over night at 4°C with 1 ml of NiNTA Agarose (Qiagen). After washing of the packed resin with 8 M urea in buffer A, pure MDM2 was eluted with 250 mM imidazole in the washing buffer. Fractions containing MDM2 were pooled together (4 ml) and refolded through three dialysis steps. The protein sample was firstly dialysed against 1 L of 2 M urea in 20 mM TrisHCl pH 7.2, 20 mM β-mercaptoethanol and 1 mM EDTA for 2 h at room temperature. A second dialysis step was carried out against 1 M urea in 20 mM TrisHCl pH 7.2 and 20 mM β-mercaptoethanol for 2 h at 4°C, and finally, the protein sample was dialysed over night at 4°C against 20 mM TrisHCl pH 7.2 containing 20 mM β-mercaptoethanol. The recombinant human His-tagged p53 protein (residues 1–312) was expressed and purified using the same protocol above mentioned for MDM2, except that the buffer used in the final dialysis contained also 0.2 mM ZnCl2. Purified proteins appeared homogeneous on 12% SDS-polyacrylamide gel electrophoresis, and the protein concentration was derived from absorbance readings at 280 nm, using a molar absorption coefficient (1 cm) of 0.54 and 0.69 calculated for MDM2 and p53, respectively, on the basis of their amino acid sequence.The recombinant human His-tagged N-terminal region of MDM2 (residues 1–118) was obtained using an Escherichia coli BL21(DE3) RIL expression system based on a pET-46Ek/LIC (Novagen) derivative vector in which the coding region of the MDM2 sequence was cloned. In particular, transformed cells were grown at 37°C in 1 L of LB medium up to 0.6 A3. Water suppression was carried out by gradient echo2O, at a concentration of about 0.1 mM, and a 10 mM stock solution of compounds 7 and 1 in DMSO-d6 were used. The formal final molar ratio protein:inhibitor was 1:2. The addition of 1 from a stock DMSO solution  to the MDM2/p53 complex caused sample precipitation indicating that the actual 1 concentration was lower than 0.2 mM.NMR spectra were acquired at 25°C on a Varian Unity INOVA 700 MHz spectrometer equipped with a cryoprobe. NMR samples contained 0.1 mM of proteins in 20 mM TrisHCl, 150 mM KCl, pH 7.4, 5 mM β-mercaptoethanol, 0.02% NaN50 values of MDM2/p53 complex were derived. As standard controls, two characterized MDM2 inhibitors, Nutlin-3 and ISA27, were employed. The assay was also validated using different concentration of human recombinant p53 /MDM2  proteins. P53 and MDM2 standards were reconstituted and the MDM2/p53 complex was formed following the manufacturer's instructions. A semi-quantitative curve of the MDM2/p53 complex was obtained using different complex concentrations, ranging from 12.5/4 ng to 50/16 ng (p53/MDM2 ratio)To test the ability of new compound to dissociate the native MDM2/p53 complex, a quantitative sandwich immune-enzymatic assay, on crude cell lysates obtained from U87 MG cells was developed. Cells were washed twice in ice-cold phosphate-buffered saline, collected by centrifugation, and suspended in lysis buffer  cells versus control set to 100%. U87MG cells were treated with different concentrations (10 nM–10 μM) of each compound and after 24 h of incubation viable cells were counted. Sigmoid dose-response curves were generated from which the ICThe western blot analysis for the evaluation of p53 protein levels was performed as previously describedThe amount of MDM2/p53 complex was determined using co-immunoprecipitation experiments; U87MG cells were treated with DMSO (control) or 5 μM new compound for 8 h. One milligram of cell lysates, obtained as described above, was pre-cleared to precipitate and eliminate IgG. Samples were then incubated with an anti-MDM2 antibody (5 μg/sample) overnight, and then immunoprecipitated with protein A-Sepharose (2 h at 4°C). After washing, the immunocomplexes were resolved by SDS-PAGE (8.5%), transferred to PVDF membranes and probed overnight at 4°C with primary antibodies to p53  or MDM2  as described above.2O. All reactions were performed for 40 cycles using 55°C as annealing temperature. The p53 target gene mRNA levels for each sample were normalized against β-actin mRNA levels, and relative expression was calculated by using Ct value. PCR specificity was verified by both the melting curve analysis and gel electrophoresis.The assessment of p53 target gene mRNA levels was performed by real-time reverse transcription polymerase chain reaction 5 cells) was centrifuged at 300 × g for 5 minutes, washed once with PBS and suspended in the fluorescent reagent (Muse™ Cell Cycle reagent). After incubation for 30 minutes at room temperature in the dark, the measurements of the percentage of cells in the different phases was acquired.The measurement of the percentage of cells in the different cell phases was performed using the Muse™ Cell Analyzer, Merck KGaA, Darmstadt, Germany). Briefly, U87MG cells were treated with DMSO or compounds (5 μM) for 24 h. Adherent cells were collected and centrifuged at 300 × g for 5 minutes. The pellet was washed with PBS and suspended in 100 μl of PBS; finally cells were slowly added to 1 ml of ice cold 70% ethanol and maintained o/n at –20°C. Then, a cell suspension aliquot  was added to 100 μl of fluorescent reagent and incubated for 10 minutes at room temperature. After incubation, the percentages of living, apoptotic and dead cells were acquired and analyzed by Muse™ Cell Analyzer in accordance to the manufacture's guidelines. In cells undergoing apoptosis, annexin V binds to phosphatidylserine, which translocates to the outer leaflet of the cytoplasmatic membrane. Double staining is used to distinguish between viable, early apoptotic, and necrotic or late apoptotic cellsDual staining with Annexin V coniugated to fluorescein-isothiocyanate (FITC) and 7-amino-actinomysin (7-AAD) was performed using the commercially available kit . U87MG cells were treated with DMSO (control), Nutlin-3 (10 μM) or compounds (5 μM) for 4-16-24 hours. Both floating and adherent cells were collected, centrifuged at 300 × g for 5 minutes and suspended in cell culture medium. Then, a 100 μl aliquot of cell suspension  was used for data analysis and graphic presentations. All data are presented as the mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni's corrected t-test for post-hoc pair-wise comparisons. P < 0.05 was considered statistically significant.S.D., E.D.P., C.G., B.C. and M.L.T. performed most of the biological work. V.L.P., T.S. and L.M. conceived the idea and conducted the design and the synthesis. A.L. was in charge of expressing the MDM2 protein, while A.C. and A.L. performed the NMR experiments. E.B. and L.R. worked at the expression and purification of the MDM2 protein. L.M. and C.M. coordinated the project. L.M., V.L.P., C.M., F.D.S., A.C. and E.N. wrote the main manuscript text. All the authors reviewed the manuscript.supplementary information


Extracellular acidosis is a prominent feature of multiple pathological conditions, correlating with pain sensation. Acid-sensing ion channels (ASICs), a family of proton-gated cation channels, are distributed throughout the central and peripheral nervous systems. Activation of ASICs, particularly ASIC3 and ASIC1a channels, by acidic pH and the resultant depolarization of nociceptive primary sensory neurons, participates in nociception. Agents that inhibit the activation of ASICs are thus expected to be analgesic. Here, we studied the effect of local anesthetic tetracaine on ASIC currents.50 of 9.96 ± 1.88 mM. The degree of inhibition by tetracaine was dependent on the extracellular pH but independent of the membrane potential. Furthermore, 3 mM tetracaine also inhibited 29.83% of the sustained ASIC3 current. In addition to ASIC3, tetracaine inhibited the ASIC1a and ASIC1β currents. The inhibition of the ASIC1a current was influenced by the frequency of channel activation. In contrast to ASIC3, ASIC1a, and ASIC1β currents, ASIC2a current was not inhibited by tetracaine. In cultured mouse dorsal root ganglion neurons, 1–3 mM tetracaine inhibited both the transient and sustained ASIC currents. At pH4.5, 3 mM tetracaine reduced the peak ASIC current to 60.06 ± 4.51%, and the sustained current to 48.24 ± 7.02% of the control values in dorsal root ganglion neurons. In contrast to ASICs, voltage-gated sodium channels were inhibited by acid, with 55.15% inhibition at pH6.0 and complete inhibition at pH5.0.Tetracaine inhibited the peak ASIC3 current in a concentration-dependent manner with an IC+ channel) has been suppressed by protons.These findings disclose a potential new mechanism underlying the analgesic effects of local anesthetics, particularly in acidic conditions where their primary target (i.e. voltage-gated Na For example, ASIC1a and ASIC3 are very sensitive to protons with an activation threshold close to pH7.0. ASIC1a has a pH0.5 of ~6.2 and mediates fast decaying, transient currents [0.5 of ~6.2 and a sustained current component with a pH0.5 of ~4.3 [0.5 of ~6.0 and mediates a transient current [0.5 of ~4.4 [Acid-Sensing Ion Channels (ASICs), members of the degenerin/epithelial sodium channel (Deg/ENaC) superfamily, are abundantly distributed in the central and peripheral neurons ,2. All A domains . In rode domains . ASIC1a  domains -7. ASICscurrents . ASIC3 h of ~4.3 . Similar current . ASIC2a  of ~4.4 . ASIC2b  of ~4.4 -14. Acti of ~4.4 -19, syna of ~4.4 , and in  of ~4.4 -24, seiz of ~4.4 , multipl of ~4.4 , and tum of ~4.4 ,28.In painful conditions such as ischemia, skin and muscle incision, arthritis and inflammation, protons are produced or released by the injured tissues, resulting in tissue acidosis ,29,30. FLocal anesthetics have multiple effects including antinociception and analgesia, antiarrhythmia, and neuroprotection -38. Bloco from 7.4 to 4.5 for 1 second evoked a large transient inward current  neurons. As reported previously ,51, a bi+ current by acidosis in DRG neurons in our recording condition, we compared voltage-gated Na+ currents recorded in normal solution (pH7.4) with that in acidic solution (pH6.0 and 5.0). Voltage-gated Na+ current was induced by step depolarization of the membrane potential from −60 mV to −30, -20 and −10 mV. We found that the amplitude of the inward Na+ currents was significantly suppressed at pH6.0, with a 55.15% inhibition observed at −30 mV (n = 6). Surprisingly, no inward Na+ current was recorded at pH5.0  was used to elicit complete functional block for withdrawal response to pinch [Our previous study found that lidocaine inhibits the ASIC1a current in mouse cortical neurons at 1 mM concentration. Since ASIC1a is implicated in neurological disorders such as brain ischemia while lidocaine can be used systemically and showed neuroprotection in some studies, inhibition of the ASIC1a current could be a potential and alternative mechanism for its neuroprotective effect. However, the concentration of lidocaine required to inhibit the ASIC1a current is unlikely to be tolerable for systemic use owing to the potential neuronal toxicity reported even at a much lower concentration range . On the protocol . Howeverto pinch . This di+ channels) has already been suppressed by H+.ASICs are implicated in pain sensation, and local anesthetics have analgesic effects. In the present study, we demonstrated that tetracaine can frequency-dependently inhibit the ASIC1a current and concentration-dependently inhibits the ASIC3 current. Inhibition of ASIC1a and ASIC3 activity by tetracaine discloses a potential novel mechanism for the analgesic effects of tetracaine, particularly in acidic conditions where its conventional target  containing 10% fetal bovine serum , 50 units/ml penicillin, and 50 μg/ml streptomycin. At ~50% confluence, cells were transfected with complimentary DNA for rat ASIC1a fused with a green fluorescence protein (GFP) at the c-terminal , or cotr2 atmosphere incubator. After 24 h, culture medium was replaced with Neurobasal medium supplemented with B27 and glutamax. The cultures were fed twice a week and used for electrophysiological recordings 6 ~ 8 days after plating.Dorsal root ganglia were dissected from embryonic Swiss mice at 16 days of gestation, enzymatically dissociated with 0.25% trypsin for 10 min, and plated in poly-D-lysine coated dishes. Cells were initially cultured in DMEM containing 10% fetal bovine serum (FBS) and 10% horse serum and maintained at 37°C in a humidified 5% CO+ current were used for ASICs recording. For fast-perfusion, a multibarrel perfusion system  was used. Patch pipettes were pulled from borosilicate glass. Pipettes had a resistance of 2–4 MΩ when filled with the intracellular solution. Whole-cell currents were recorded using Axopatch 200B amplifiers . All data were filtered at 2 kHz and digitized at 5 Hz using Digidata 1320 DAC units (Axon Instruments). The on-line acquisition was done using pCLAMP software . The recordings with an access resistance of less than 10 MΩ and a leak current less than 100 pA at −60 mV were included for data analysis [ASICs currents were recorded with the whole-cell patch-clamp and fast perfusion techniques, as previously described . GFP-posanalysis . Extrace2, 1 MgCl2, pH 7.4 - 4.5, adjusted with NaOH and HCl, 320–330 mOsm. Intracellular solution contained (mM): 140 CsF, 1 CaCl2, 10 HEPES, 11 EGTA, 2 TEA, 4 MgCl2, pH 7.3, adjusted with CsOH, 290–300 mOsm [Extracellular solution contained (mM): 140 NaCl, 5.4 KCl, 20 HEPES, 10 Glucose, 2 CaCl300 mOsm . Tetracat test were used to examine the statistical significance. The criterion for significance was set at p < 0.05. The dose–response curve was fitted with 3 parameter logistic nonlinear regression model: y = a/(1 + (x/x0)^b), where a is the relative maximal current, x0 is C50 and b is the Hill coefficient.All data were expressed as mean ± SD. GraphPad Prism 4 and Sigma Plot were used for statistical analysis. ANOVA followed by Bonferroni posttests or student’s The authors declare that they have no competing interests.TL carried out the electrophysiological recording, data analysis and drafting the manuscript. ZX designed, supervised the project and revised the manuscript. JL and JEC participated in the design of experiments and revised the manuscript. All authors contributed to data interpretation, and approved the final manuscript.


Zerumbone also produced the same antitumor activity in pancreatic carcinoma cell lines SW1990 and AsPC-1. In summary, we found that zerumbone was able to induce apoptosis of pancreatic carcinoma cell lines, indicating to be a promising treatment for pancreatic cancer.Pancreatic carcinoma is one common cancer with gradually increasing incidence during the past several decades. However, currently the candidate drugs to suppress pancreatic cancer remain lacking. This research was carried out to investigate if zerumbone, a natural cyclic sesquiterpene isolated from Zingiber zerumbet Smith, will produce the anticancer effects on pancreatic carcinoma cell lines. The results showed that zerumbone concentration, and time, dependently produced inhibitory actions on cell viability of PANC-1 cells. In addition, Hoechst 33342, AO/EB, TUNEL staining, and caspase-3 activity assay further showed that zerumbone induced apoptosis of PANC-1 cells. The expression of p53 protein was markedly upregulated, and the p21 level was also obviously elevated in zerumbone-treated PANC-1 cells. Moreover, ROS production was increased by about 149% in PANC-1 cells treated by zerumbone 30  As a crucial part of the digestive system, the incidence of pancreatic cancer is gradually increasing during the past decades all over the world. It was reported that approximately 37,000 individuals were diagnosed with pancreatic cancer in the United States . The 5-yZingiber zerumbet Smith is one kind of plant growing mainly in Southeast Asia, which has been demonstrated to possess antinociceptive, anti-inflammatory, antiulcer, antihyperglycemic, and antiplatelet activities –7. As a μg/mL), and L-glutamine (300 μg/mL) supplemented with 10% fetal bovine serum (FBS). AsPC-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. The culture condition for these cell lines was at 37°C in a humidified atmosphere of 5% CO2 and 95% air in a plastic flask. All cultured medium was changed twice every week.Human pancreatic carcinoma cell lines PANC-1 and SW1990 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 units/mL), streptomycin -2,5-Diphenyl Tetrazolium Bromide) kit, and other reagents, if not otherwise specified, were purchased from Sigma-Aldrich, St. Louis, MO, USA. Hoechst 33342 dye and Trizol were obtained from Invitrogen, Carlsbad, CA. In Situ Cell Death Detection Kit was bought from Roche, Penzberg, Germany . Caspase-3 activity colorimetric kit was purchased from R&D Systems Inc. . PA Lysis Buffer was obtained from Beyotime, Shanghai. The p53, p21 monoclonal, and PUMA polyclonal antibody were bought from Santa Cruz Biotechnology . RNeasy Mini Kit and RNase-free DNase Set were obtained from Qiagen, Valencia, CA. TaqMan Reverse Transcription Reagents were purchased from Applied Biosystems, Foster City, CA. For all experiments of this study, DMSO was used to dissolve zerumbone. In order to avoid possible effects to these cells by DMSO, the volume of DMSO should not exceed 0.1% of the total volume (v/v).μM for 24 h or were cultured for 24, 48, and 72 h in the presence of 30 μM, respectively. Then, the culture media were washed out and the fresh media containing 5 mg/mL MTT were added. The cells were continuously incubated at 37°C for an additional four hours. After this time, the media were washed out, and reduced MTT product (blue formazan product) was solubilized by adding 100 μM DMSO to each wells. After agitation of these plates for 15 min, the optical density of the solubilized formazan product in each well was measured using a microplate reader at 570 nm with background subtraction at 650 nm. The experiment to observe different concentration of zerumbone on cellular viability of pancreatic cancer cells was carried out six times, and the experiment to study different incubation times of zerumbone on cellular viability was performed five times.The cellular viability of pancreatic cancer cells was determined by MTT -2,5-Diphenyl Tetrazolium Bromide) assay. Briefly, the cells were collected and seeded in 96-well plates to attach overnight in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Human pancreatic cancer cells were rendered quiescent by incubation in serum-free media for 24 h. Then pancreatic cancer cells were incubated with zerumbone 3, 10, 30, and 100 μL prepared AO/EB working solution (100 μg/mL AO and 100 μg/mL EB in PBS) was added to each well for 5 min. Then the pancreatic cancer cells were harvested and the apoptotic cells were counted under an inverted fluorescence microscope .Morphological signs of apoptosis were detected by using acridine orange-ethidium bromide (AO/EB) staining in pancreatic cancer cells. The cells were incubated with zerumbone for 24 h. The procedure to perform AO/EB staining is just as described below. In order to staining the apoptotic cells, 10 μg/mL Hoechst 33342 for 15 min at room temperature. The cells were imaged with fluorescence microscope.Morphological changes of apoptotic pancreatic cancer cells were evaluated by Hoechst 33342 staining. In brief, the cultured cells were planted in 6-well plates and then exposed to zerumbone treatment for 24 h. After being washed with PBS, the cancer cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After being washed again with PBS, the fixed cells were stained with 20 μM for 24 h. The staining of apoptotic cells was carried out using an In Situ Cell Death Detection Kit. In brief, after being washed twice with PBS, human pancreatic cancer cells were then fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h at room temperature. The fixed cancer cells were permeabilised by incubation with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. The cells were rinsed again with PBS and incubated with TUNEL reaction mixture for 1 h at 37°C in the dark. TUNEL staining of apoptotic cells was viewed under a fluorescence microscopy .TUNEL assay was used to identify the apoptosis of pancreatic cancer cells. The cells were seeded in dishes, grown overnight, and subjected to zerumbone 3, 10, 30, and 100 2DCF-DA) probe. The procedure for ROS measurement is as previously described [2DCF-DA 10 μM by incubation for 30 min at 37°C. Fluorescence was measured by flow cytometry. The experiment with ROS assay was repeated four times.To quantify intracellular ROS level, we used 2,7-dichlorodihydrofluorescein diacetate  in 100 mL reaction buffer. The cell lysate was incubated at 37°C for 2 h. Thereafter, the absorbance at 450 nm was measured to represent the release of chromophore p-nitroanilide (pNA). The experiment with caspase-3 assay was repeated three times.For immunolabeling, the lysates were prepared after the cancer cells were subjected to their respective treatment with zerumbone. One hundred micrograms of each lysate were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). After electrophoresis, the proteins were transferred onto a nitrocellulose membrane. After blocking with 5% nonfat dried milk and 0.05% Tween 20 in Tris-buffered saline , the membranes were incubated overnight with the relevant primary antibody followed by the incubation with horseradish peroxidase-conjugated immunoglobulin G (IgG). The blots were then visualized by using Odyssey v1.2 software. The experiments were repeated three times.According to the guideline of the manufacturer, the total RNA from pancreatic cancer cells was isolated by Trizol and purified by RNeasy Mini Kit and RNase-free DNase Set. Total RNA from pancreatic cancer cells was subjected to first-strand cDNA synthesis using TaqMan Reverse Transcription Reagents. The method to determine miR-34 mRNA level in cancer cells is just as described previously . RelativP < 0.05.All data was presented as mean ± S.E.M. Statistical analysis was performed to determine the significance of differences among groups using ANOVA. All statistical analysis was performed using the SPSS 13.0 software for Windows. Statistical significance was initially set at μM, 10 μM, 30 μM, and 100 μM for 24 h resulted in a significant reduction of cellular viability, compared with untreated cells (P < 0.05). Zerumbone 3 μM, 10 μM, 30 μM, and 100 μM decreased the viability of PANC-1 cells from 97.9 ± 5.3 to 78.2 ± 6.4, 70.1 ± 6.0, 55.6 ± 7.2, and 49.1 ± 8.1, respectively (P < 0.05). μM for 24 h, 48 h, and 72 h was decreased from 95.3 ± 3.8 to 57.8 ± 6.2, 47.4 ± 7.5, and 33.6 ± 7.9. The results suggest that zerumbone reduces the proliferation of PANC-1 in a concentration- and time-dependent manner (P < 0.05).The effects of zerumbone on the proliferation of PANC-1 cells were measured by the MTT assay. As displayed in μM, 10 μM, 30 μM, and 100 μM significantly increased the number of TUNEL-positive PANC-1 cells from 7.1 ± 1.9% to 21.3 ± 3.6, 33.8 ± 4.0, 47.1 ± 6.6 and 52.3 ± 5.9 after 24 h incubation (P < 0.05).We further used Hoechst 33342 and AO/EB staining to determine the effects of zerumbone on the apoptosis of PANC-1 cells. As demonstrated in Figures μM, 10 μM, 30 μM, and 100 μM markedly increased the activity of caspase-3 by approximately 56%, 147%, 149%, and 197%, respectively (P < 0.05). These results further confirmed that zerumbone induced apoptosis of PANC-1 cells. Then, we explored the influences of zerumbone on the production of ROS. PANC-1 cells were exposed to zerumbone 3 μM, 10 μM, 30 μM, and 100 μM for 24 h and analyzed for the production of ROS by fluorescence microscopy. P < 0.05).The effect of zerumbone on the activity of caspase-3 in PANC-1 cells was further investigated. As illuminated in μM significantly increased the expression of p53 protein in PANC-1 cells (P < 0.05). In agreement, miR-34 level was also augmented in zerumbone-treated PANC-1 cells (P < 0.05) (P < 0.05). We further investigate the effects of the p53-specific inhibitor pifithrin-α on zerumbone-induced decrease of cellular viability in PANC-1  . These r < 0.05)  (P < 0.0n PANC-1 . The resμM markedly inhibited cellular viability of SW1990 and AsPC-1 after 24 h incubation (P < 0.05). Furthermore, Hoechst 33342 staining displayed that the exposure to zerumbone 30 μM for 24 h induced obvious apoptotic morphological changes in the nuclear chromatin in SW1990 and AsPC-1  are a variety of molecules and free radicals derived from molecular oxygen, which was constantly generated and eliminated in the biological system, and have important roles in cell signaling and homeostasis . ExcessiIn summary, it was uncovered in our study that zerumbone induced apoptosis in pancreatic carcinoma cells through p53 signal pathway. This finding indicates zerumbone, a sesquiterpene in subtropical ginger, as a new therapeutic candidate for pancreatic cancer.


Macrophages represent important cellular imaging targets for assessing vascular inflammation. We evaluated FeCo/GC for vascular macrophage uptake and imaging In situ and ex vivo fluorescence imaging were performed at 48 hours after intravenous injection of FeCo/GC conjugated to Cy5.5 . Significant fluorescence signal from FeCo/GC-Cy5.5 was present in the ligated left carotid arteries, but not in the control (non-ligated) right carotid arteries or sham-operated carotid arteries (p = 0.03 for ligated vs. non-ligated). Serial in vivo 3T MRI was performed at 48 and 72 hours after intravenous FeCo/GC . Significant T2* signal loss from FeCo/GC was seen in ligated left carotid arteries, not in non-ligated controls (p = 0.03). Immunofluorescence staining showed colocalization of FeCo/GC and macrophages in ligated carotid arteries.Hyperlipidemic and diabetic mice underwent carotid ligation to produce a macrophage-rich vascular lesion. in vivo, allowing fluorescence and MR imaging. This multi-functional high-relaxivity nanoparticle platform provides a promising approach for cellular imaging of vascular inflammation.FeCo/GC accumulates in vascular macrophages  Inflammation is a major contributor to atherosclerosis, a leading cause of death worldwide in vitro as well as high-resolution vascular angiography in vivoin vivo cellular imaging of FeCo/GC has not been studied.Cellular and molecular imaging of atherosclerosis and vascular inflammation has made significant progress, but remains challenging in vivo macrophage uptake and imaging in a murine model of vascular inflammation. We show carotid artery macrophages take up FeCo/GC and can be imaged by in situ fluorescence and in vivo high-field MRI.The aim of the current study was to investigate FeCo/GC for in situ and ex vivo fluorescence imaging at 48 hours showed high signal from the ligated left carotid arteries, confirming FeCo/GC uptake in the vascular lesions  right carotid arteries (in situ (left vs. right): 2.3±0.4 vs. 0.7±0.1, p = 0.03, ex vivo (left vs. right): 59.2±17.2 vs. 0.3±0.3, p = 0.03). Also, the ligated left carotids had significantly higher fluorescence than sham left carotids (in situ (ligated vs. sham): 2.3±0.4 vs. 0.9±0.1, p = 0.046, ex vivo (ligated vs. sham): 59.2±17.2 vs. 0.3±0.3, p = 0.046).Quantitative analysis of both prior to FeCo/GC administration  right carotid arteries . All procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.A macrophage-rich murine carotid ligation model was used The 7 nm FeCo/GC were synthesized as previously described 5000-NH2 (NOF corp.). For fluorescent dye attachment, Cy5.5-NHS ester was mixed with FeCo/GCPL-PEG-NH2 in phosphate buffered saline. Dye that was not attached to the nanocrystal was removed by filtration. Samples prepared for in vivo MRI imaging at 3 T were functionalized using DSPE-mPEG5000 5000], Laysan Bio).For fluorescence imaging experiments the PL-PEG used was DSPE-PEGin vivo uptake of Fe/Co-GC in the carotid macrophages, Cy5.5 was conjugated to FeCo/GC, allowing fluorescence imaging and microscopy. FeCo/GC-Cy5.5  was injected intravenously via tail vein into mice two weeks after carotid ligation (n = 6) or sham operation (n = 2). These mice were imaged at 48 hours using the Maestro™ in-vivo imaging system . Under inhalational anesthesia (2% isoflurane), left and right carotid arteries were surgically exposed and in situ florescence imaging was performed. Then, the carotid arteries were carefully removed en bloc followed by ex vivo fluorescence imaging and immunohistochemistry (see below).To verify To study noninvasive imaging of FeCo/GC by MRI, ligated mice (n = 6) were injected with FeCo/GC (270 µg Fe/mouse) and imaged serially at 48 and 72 hours on a whole-body 3T MRI scanner  with a 50mT/m, 150T/m/s gradient system and a custom 3 cm surface radiofrequency coil. Another group of mice (n = 3) were injected with an 8-fold lower dose of FeCo/GC (32 µg Fe/mouse) and imaged at 48 hours on a small-bore 7T system , with a 9 cm gradient insert  and a custom 6-cm RF coil. To detect the T2* effects of FeCo/GC, bright-blood images were acquired using gradient echo sequences (3T: repetition time (TR)  = 100 ms, echo time (TE)  = 10 ms, matrix size  = 256×256, slice thickness  = 1.0 mm, field of view (FOV)  = 3 cm, flip angle  = 60°; 7T: same parameters except TR/TE  = 50 ms/4.2 ms, slice thickness  = 0.5 mm). The slice position was matched using the aortic arch as a reference point.in situ carotid fluorescence imaging, regions-of-interest (ROIs) were placed on the left and right carotid regions and trachea. Average signal intensity divided by exposure time for left and right carotids was calculated and normalized to the trachea signal. For ex vivo carotid imaging, ROIs were placed around the entire left or right carotid artery and total signal intensity divided by exposure time was calculated. For in vivo MRI, FeCo/GC accumulation was assessed by measuring the extent of T2*-induced reduction in carotid lumen size, which was calculated as follows: % reduction of carotid lumen area  =  (1-[post-contrast carotid lumen area]/[pre-contrast carotid lumen area]) x 100% For After euthanasia, carotid arteries were cut into two 3 mm sections. These sections, as well as liver and heart, were embedded immediately in optimum cutting temperature (OCT) compound  and flash frozen in liquid nitrogen. Frozen sections (5 µm) were fixed in acetone for 10 minutes at 4°C Immunofluorescence double staining with confocal microscopy  was performed to examine colocalization of FeCo/GC-Cy5.5 and macrophages. After incubation with anti-mouse Mac-3 antibody overnight at 4°C, sections were stained with Alexa Fluor 488-conjugated anti-rat IgG  at room temperature for 2 hours. Finally, sections were stained with DAPI (Sigma-Aldrich) to visualize cell nuclei.All data are expressed as mean±SEM (standard error of the mean). Comparisons of fluorescence and MRI measures between left (ligated) and right (non-ligated) carotid arteries and between timepoints were analyzed by the Wilcoxon signed-rank test. Comparisons between ligated and sham left carotid arteries were analyzed by the Mann-Whitney U test. P<0.05 was considered statistically significant.


To the Editor: An outbreak of rectal lymphogranuloma venereum (LGV) has been detected in the Netherlands among men who have sex with men (http://www.essti.org) and the Centers for Disease Control and Prevention (http://www.cdc.gov) are working on outbreak warning and response systems to increase the awareness and the direct management of the LGV outbreak , laborious, and require specially trained personnel in a sophisticated laboratory setting. Therefore, we developed a real-time PCR approach (TaqMan and Rotorgene) that can easily identify LGV strains in 2 hours with equipment that is available in almost all diagnostic settings.Different approaches have been described to diagnose LGV infections . The firpmp gene) as a PCR target because it has a unique gap in LGV strains of C. trachomatis, compared to other serovars, which makes it highly specific. The following primers and probes were selected: LGV-F 5´ CTG TGC CAA CCT CAT CAT CAA 3´, LGV-R 5´ AGA CCC TTT CCG AGC ATC ACT 3´, and LGV MGB-probe 6-FAM-CCT GCT CCA ACA GT. Real-time PCR conditions (20-μL format) for TaqMan were as follows: 2× TaqMan Universal Mastermix , 18 pmol each primer, 0.2 μmol/L probe, and 2 μL (LGV L2) DNA or clinical sample; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 sec at 95°C and 1 min at 60°C. Conditions for Rotorgene were as follows: 10× buffer , 10 pmol each primer, 0.04 μmol/L probe, 2 μL (LGV L2) DNA or clinical sample; 2 min at 50°C, 10 min at 95°C, and 45 cycles of 15 sec at 95°C and 1 min at 60°C. By using a previously described serial dilution of LGV L2 , C. pneumoniae, C. pecorum, C. psittaci, and 32 other microorganisms that normally reside in the human perianal and urogenital region and in the oropharynx. These organisms included gram-positive and gram-negative bacteria and yeast: Acinetobacter baumannii, Campylobacter jejuni, Candida albicans, other yeast, Enterobacter agglomerans, Enterococcus faecalis, Escherichia coli, Streptococcus spp., Haemophilus influenzae, Klebsiella pneumoniae, Mycoplasma spp., Neisseria meningitidis, Pasteurella spp., Pseudomonas aeruginosa, Salmonella enteritidis, Shigella sonnei, Staphylococcus aureus, and others. Only LGV strains L1, L2, L2b, and L3 tested positive in both the TaqMan and Rotorgene assays, which shows the analytical specificity of real-time PCR.To determine specificity, we tested different C. trachomatis–positive rectal swab . By using the 2 reference standard techniques to type C. trachomatis serovars (PCR-based RFLP of the omp1 gene or sequencing the variable segment 2 [VS-2] of the omp1 gene)  accordip1 gene) ,10 with


Background. Data on the relationship between homocysteine, plasminogen activator inhibitor 1, hs-CRP, fibrinogen, and carotid intima media thickness (CA-IMT) is plenty but contradicting and the majority of the studies investigated this issue in only specificthyroidism groups. The aim of this paper was to investigate these relations in patients with subclinical and clinical hypo- and hyperthyroidism. Methods. In this cross-sectional study, 16 patients from each thyroidism group and 20 healthy cases were enrolled. Fibrinogen levels and plasminogen activator inhibitor 1 (PAI-1) activity were assessed. CA-IMT was determined by gray-scale high-resolution color Doppler ultrasound. Results. Serum homocysteine levels were higher in hypothyroidic patients compared to the control (P = 0.003). Fibrinogen levels were higher in patients with subclinical hypothyroidism compared to other groups (P < 0.05). There was no difference between groups regarding PAI-1. Whereas total cholesterol, homocysteine, and LDL were correlated with CAIMT, hs-CRP, PAI-1, and fibrinogen were not. In the clinical hypothyroidism group, the correlation of homocysteine with CA-IMT was derived from the correlation between CA-IMT and homocysteine. Conclusions. Homocysteine and fibrinogen levels are higher in patients with clinical and subclinical hypothyroidism, respectively. Homocysteine level is associated with CA-IMTonly in patients with clinical hypothyroidism. Thyroid diseases are among the most common endocrinological disorders. Depending on the levels of thyroid hormones, it may present either as normo-, hypo-, or hyperthyroidism. It can also be classified as subclinical  or clinical . Subclinical hypothyroidism is the most common form.Disturbances in thyroid metabolism affect various organs such as the heart, vessels, and kidney –6. With Cardiovascular diseases are the main causes of death in the world. Atherosclerosis is a major contributor to this problem. Carotid intima media thickness (CA-IMT) measurement by Doppler USG is a validated method to determine extent of atherosclerosis , 11. AgeThyroid hormones via blood vessel dilatation, production of vasodilator molecules, inhibition of angiotensinogen II receptor expression, and its signal transduction regulate endothelial function and vascular homeostasis and have anti-atherosclerosis effects –18. RegaWith this background in mind, our aim was to investigate the extent of atherosclerosis in subclinical and clinical hypo- and hyperthyroidism along with potential roles of homocysteine and PAI-1 in an otherwise healthy population.Of the first two patients who applied to the endocrinology outpatient clinic with symptoms and/or signs of thyroid dysfunction each day and met the inclusion and exclusion criteria below were stratified into the corresponding thyroidism groups according to their thyroid function results . Each group was planned to consist of 16 patients. Group 1 included patients with high TSH and normal fT3 and fT4 levels , group 2 high TSH and low fT3 and fT4 levels , group 3 low TSH and normal fT3 and fT4 levels , and group 4 low TSH and now fT3 and fT4 levels . Also, 20 random people without any thyroid hormone abnormality were used as control.2, to have no history of any kind of thyroid dysfunction, and not to be on thyroid hormone replacement. Exclusion criteria were to have history of diabetes, hypertension, coronary artery disease, and familial hypercholesterolemia, to be on lipid lowering drugs, and to have body mass index <20 kg/m2 or >30 kg/m2.Inclusion criteria were to be aged between 18 and 65 years, to have thyroid hormone dysfunction assessed by laboratory findings, to have a body mass index of 20–30 kg/mDemographical, clinical, and laboratory findings were recorded from patients' charts. Routine biochemical parameters were measured at our university's core biochemistry laboratory. fT3, fT4, and TSH were measured by chemiluminescence method by an automated analyzer . Normal ranges for fT3 were 1.8–4.2 pg/mL, for fT4 0.8–1.9 ng/dL, and for TSH 0.4–4 mU/mL. Fibrinogen levels were measured nephelometrically according to the manufacturer's protocol by using a commercially available kit on Behring BCS coagulation analyzer . Plasminogen activator inhibitor 1 (PAI-1) activity was measured according to the manufacturers' protocol with indirect chromogenic assay by using SPECTROLYSE PAI-1 ELISA kit .The study protocol was approved by local ethics committees and informed consent was obtained from each patient. The study was performed according to the recommendations of the Declaration of Helsinki.Ultrasonographic studies on common carotid arteries were carried out by gray-scale high-resolution color Doppler ultrasound  equipped with 12 MHz linear transducer. The same operator performed all procedures on both sides of two longitudinal images of the each common carotid artery on the morning in supine position. Average of the two CA-IMT  values from each side was used to calculate mean CA-IMT on each side. Intraobserver coefficient of variation was 2.21%.P value less than 0.05 was considered as statistically significant. Comparisons between two groups were assessed with the Student's unpaired t-test or Mann-Whitney test, as appropriate. Pearson's and Spearman's rank correlations were used to assess correlations of fibrinogen, homocysteine, hs-CRP, and PAI-1 with left and right CA-IMT. Correlation was analyzed both in the whole population except control and also in the subgroups. All statistical analyses were performed using SPSS, version 15 .All results are reported as mean ± SD. Mean age was 40.8 ± 11.8 years and 81.3% were female. Mean fT3, fT4, and TSH levels were 3.02 ± 0.67 pg/mL, 1.13 ± 0.24 ng/mL, and 6.91 ± 1.74 IU/mL. Mean homocysteine level was 12.76 ± 3.11 mmol/L, hs-CRP 5.98 ± 7.84 mg/L, and PAI-1 52.21 ± 31.93 ng/mL. Mean CA-IMT was 0.61 ± 0.11 mm.Mean age was 38.0 ± 10.2 years and 87.5% were female. Mean fT3, fT4, and TSH levels were 2.52 ± 0.92 pg/mL, 0.76 ± 0.29 ng/mL, and 29.87 ± 23.65 IU/mL. Mean homocysteine level was 15.05 ± 9.87 mmol/L, hs-CRP 5.41 ± 3.48 mg/L, fibrinogen 283.00 ± 81.16 mg/dL, and PAI-1 42.78 ± 33.65 ng/mL. Mean CA-IMT was 0.62 ± 0.13 mm.Mean age was 41.6 ± 16.43 years and 81.3% were female. Mean fT3, fT4, and TSH levels were 3.46 ± 0.53 pg/mL, 1.33 ± 0.18 ng/mL, and 0.12 ± 0.09 IU/mL. Mean homocysteine level was 11.93 ± 3.72 mmol/L, hs-CRP 3.80 ± 1.72 mg/L, fibrinogen 275.37 ± 68.42 mg/dL, and PAI-1 53.83 ± 37.21 ng/mL. Mean CA-IMT was 0.58 ± 0.17 mm.Mean age was 41.6 ± 11.8 years and 68.8% were female. Mean fT3, fT4, and TSH levels were 6.7 ± 3.33 pg/mL, 2.90 ± 1.70 ng/mL, and 0.01 ± 0.02 IU/mL. Mean homocysteine level was 10.23 ± 3.13 mmol/L, hs-CRP 3.93 ± 1.91 mg/L, fibrinogen 275.37 ± 68.42 mg/dL, and PAI-1 53.83 ± 37.21 ng/mL. Mean CA-IMT was 0.59 ± 0.15 mm.P = 0.035). This was more pronounced in patients with clinical hypothyroidism (P = 0.008). A similar trend was true for total cholesterol and LDL levels. Comparison of the clinical and demographical parameters of the groups is shown in As would be expected, patients with subclinical hypothyroidism had higher BMI compared to the control (P = 0.003). There was no difference between hypo- and hyperthyroidic patients. Fibrinogen levels were higher in patients with subclinical hypothyroidism compared to other groups (P < 0.05). There was no difference between groups regarding PAI-1. Comparison of the clinical and demographical parameters of the groups is presented in Serum homocysteine levels were significantly higher in hypothyroidic patients compared to the control  and CA-IMT in patients with clinical/subclinical hypo- and hyperthyroidism. Only homocysteine level was associated with CA-IMT and this was derived from the strong association between homocysteine and CA-IMT in patients with clinical hypothyroidism.Data on PAI-1 in thyroid diseases is controversial. Erem et al. reported higher levels patients with hypothyroidism compared to the controls , 26. Thin = 112) compared to patients with TSH ≤ 4.6 mcu/mL (643) was observed [Hyperhomocysteinemia is proposed as a contributor to atherosclerosis by stimulating oxidation of LDL and causing endothelial dysfunction –35. Bothobserved . Our stuThe association between homocysteine levels and CA-IMT is not clear –43. DataSeveral studies have reported a positive association between CRP levels and cardiovascular events and atherosclerosis –47. HoweIn patients with hypothyroidism data on the level of CRP is contradictory –58. In aThe strongest side of our study is comparison between thyroidism subgroups along with a comparison with a control. The major limitation of our study is its cross-sectional nature and measurement at one time-point. Despite most of the studies in the literature have enrolled less than 30 patients in comparison studies, the number of patients in each group in our study may still be considered relatively small. A further study enrolling higher number of patients and analyzing the value of these parameters in a time-dependent manner may yield more data on this issue.As a conclusion, our results show that homocysteine and fibrinogen levels are higher in patients with clinical and subclinical hypothyroidism, respectively. Homocysteine level was associated with CA-IMT in patients with thyroidism, and this was derived from the strong association between homocysteine and CA-IMT in patients with clinical hypothyroidism. Determination of homocysteine in clinical hypothyroidic patients may provide information on the cardiovascular burden. Also, therapy to reduce homocysteine levels may be an important approach in these patients to decrease the cardiovascular burden.