Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC50 = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.It is well established that L-proline has several roles in the biology of trypanosomatids. In  Trypanosoma cruzi is the causative agent of Chagas' disease, which affects approximately 12–14 million people in endemic areas throughout Mexico, Central, and South America The protozoan parasite T. cruzi has a complex life cycle characterized by several forms in vertebrate and invertebrate hosts T. cruzi life cycle The successful survival of the parasite in different environments throughout its life cycle  depends on its ability to maintain intracellular homeostasis of ions and nutrients and the ability to catabolize different substrates to obtain energy. Thus, the activity of transporters allowing the uptake of solutes across the parasite cytoplasmic membrane and the metabolism of certain amino acids are crucial processes at various points in the T. cruzi genome database T. cruzi with respect to mammalian host cells, we hypothesize that inhibition of proline metabolism in T. cruzi could have more severe effects on the parasite biology that on host cells.Previous work showed that L-proline was taken up from the extracellular environment through two active transporters T. cruzi. We also investigated the synergism or antagonism of T4C with stress conditions faced by T. cruzi during its natural life cycle: temperature, pH and oxidative and nutritional stress.Various L-proline analogues such as T4C have been used as inhibitors of proline metabolism in prokaryote and eukaryote organisms T. cruziL-proline, a relevant energy source in trypanosomatids, is metabolized and converted into five intermediates of the tricarboxylic acid cycle  through its oxidation to glutamate in T. cruzi, 2.5×106 epimastigotes per ml were cultured in the presence of different concentrations of T4C, ranging from 0.1 to 1 mM. The growth of epimastigotes on LIT medium with or without 200 µM rotenone and 0.5 µM antymicin were also followed as controls. The epimastigotes reached the stationary phase on the tenth day of growth, with 94×106 cells/ml in LIT. T4C showed a dose-dependent inhibition of epimastigote growth at 28°C and pH 7.5, the standard temperature and pH for cultivating these cells. The observed growth differences between the treated cells and the LIT control were significant (p<0.01), and the IC50 was determined to be 0.89±0.02 mM . On the other hand, T4C had no influence on the incorporation of proline into proteins in plants. In addition, these authors found that concentrations between 0.2 and 1 mM T4C inhibited proline oxidation in mitochondria isolated from etiolated barley, and this inhibition was related to the activity of proline dehydrogenase and not the transport of proline into the mitochondrial matrix. All informations suggest that the inhibitory activity of T4C on T. cruzi growth could be due to any of the mentioned processes in which proline participates.It has been suggested that T4C inhibits the growth of T. cruzi, as well as other parasitic organisms that cycle among different conditions and environments, must deal with radical physical, chemical and physicochemical variations such as temperature, availability of nutrients, oxidative stress and pH, all constituting stress factors. The interaction of T4C with these stress factors could identify a possible role for proline metabolism in T. cruzi biology, so we analyzed this interaction. As proline metabolism is involved in resistance to several stress conditions in a variety of organisms ranging from yeast to plants T. cruzi. We initially evaluated the effects of T4C together with high temperature (33°C and 37°C), acidic pH (5.5 and 6.5), stress from nutrient deficiency  and oxidative stress due to different reactive oxygen species (incubation in the presence of H2O2).T. cruzi goes through temperature changes throughout its life cycle because the insect vector does not regulate its body temperature and because it alternates between an insect vector and a mammalian host. Therefore, this parasite faces environmental temperature variations and the high temperature of the mammalian body (up to 37°C). Epimastigotes grow optimally at a temperature of 28°C, a condition in which a cell density of 94×106 cells/ml was reached at the stationary phase. Our data showed, however, that cell growth reached a higher density at 33°C (120×106 cells/ml), while cell growth at 37°C was even less than that obtained at 28°C (70×106 cells/ml). These results are in opposition to those obtained by 50 values were calculated, being 0.83±0.07 mM and 0.54±0.01 mM for 33°C and 37°C, respectively (p<0.05).As already mentioned, ectively , showingT. cruzi also faces pH variations throughout its life cycle. In the intestinal tract of the insect vector, the pH varies between 5.9 and 8.9, depending on the starvation and blood meal periods 6 cells/ml in LIT medium, while parasites grown at pH 6.5 achieved similar cell densities (102×106 cells/ml at the stationary phase) to those seen at pH 7.5 (94×106 cells/ml) . This result suggests that proline metabolism is not involved in pH stress resistance mechanisms.ells/ml) . When thells/ml) . The IC5T. cruzi life cycle, particularly in the gut terminal portion, where metacyclogenesis occurs T. cruzi epimastigotes can metabolize not only proline, but also glucose, glutamate and aspartate. These energy sources are able to support the parasite survival to some extend after PBS starvation 6 cells/mL in the exponential growth phase were starved for 72 h in PBS (PBS) or in 3 mM L-proline in PBS (PRO). Both cultures were treated or not (controls) with T4C, then cell viabilities were determined using the MTT assay. As expected, the presence of 3 mM proline in the PBS increased parasite viability when compared with parasites starved in PBS. Both cultures treated with T4C showed significant differences (p<0.01) compared to their respective controls (not treated with T4C), with a greater difference in the parasites maintained with L-proline (93%) compared to PBS (38%) (p<0.01). Between these two essential nutrients incorporated in PBS, the most significant effect was obtained with glucose because it served as a carbon and energetic source. Other carbon and energy sources such as aspartate and glutamate could also have the same effect, since they seem to be as relevant as glucose or proline for the parasite survival in nutritional stress conditions Nutritional stress also occurs regularly during the BS (38%) . This coBS (38%) , treatme2O2. Cells (5×106 parasites/mL) were resuspended in PBS and challenged with different hydrogen peroxide concentrations (between 0 and 200 µM) at 28°C for 90 min. After removal of the H2O2, cells were re-inoculated in fresh-LIT medium, and their growth was evaluated, as described in 2O2 inhibiting 50% of parasite growth (IC50) was determined to be 160 µM under our experimental conditions. To determine the effects of H2O2 on T4C treatment, cells were then incubated under oxidative stress conditions (IC50) or in PBS in the presence or absence of 0.5 mM T4C, and parasite growth was followed (2O2 or T4C) and its respective control were significant (p<0.01). The number of cells previously challenged with T4C was reduced by 55% with respect to the control, and cells incubated simultaneously in T4C and H2O2 showed a 75% reduction in growth. The protective effect of L-proline against oxidative stress was previously reported by some authors, such as T. cruzi could generate different reactive oxygen species (ROS) by its own aerobic metabolism and the host immune response. These species lead to membrane disruption, inactivation of essential enzymes, mutagenesis and damage to the DNA repair machinery. The importance of these effects is primarily seen when the parasite invades the mammalian cell or when it is exposed to drugs used to treat Chagas' disease 2O2. In spite of the ROS detoxifying mechanisms in T. cruziIn order to study the possible interaction between T4C and oxidative stress, we first evaluated the dose-response of the epimastigotes to different concentrations of Hfollowed . The dif2O2 was due to the fact that this reagent affected a proline-dependent oxidative stress resistance mechanism. Alternatively, it could be argued that the enhancement of the effects of T4C seen under oxidative stress conditions was due to the fact that T4C made the cells more fragile by partially arresting proline metabolism, which is relevant for energy generation 6 cells/mL) were incubated for 48 h in PBS, supplemented or not with 3 mM proline. As done previously, cultures maintained in PBS supplemented with 3 mM glucose were also evaluated. All groups were maintained in the presence or absence of 0.5 mM T4C (T4C), and the cultures were then challenged with H2O2, as previously described, or mock challenged with H2O. The cell viability in each case was evaluated by a MTT assay. Before the 3 h challenge (T0), the viability of parasites incubated with carbon sources (proline or glucose) differed significantly (p<0.01) from the viability of those incubated with PBS  in all of the treatments. When the T4C-treated parasites were challenged with H2O2, a significant reduction in viability was observed (p<0.01) compared to parasites that were mock challenged .From the results presented above, it could be proposed that the enhanced effect of T4C in the presence of Hwith PBS . As expeallenged . Interes0), the intracellular free proline concentration was twice as high in the PRO group compared to the GLC group  (0), the concentration of intracellular L-proline in the presence of T4C was maintained (PBS = 0.88 µM/106 cells) or reduced  with respect to incubation without the analogue (2O2 (or H2O as mock stress) for 3 h. There were significant differences (p<0.01) in the intracellular L-proline concentration between the cells kept under oxidative stress and in the presence of T4C. These two results confirm the synergistic effects of T4C and oxidative stress. Higher sensitivity to H2O2 of the cells maintained in PRO/T4C with respect to those not treated with T4C could be attributed to lower intracellular free proline levels rather than to inhibition of its metabolism.The enhanced effect of T4C on the PRO group with respect to the GLC group was expected and could be explained by the fact that T4C interfered with proline rather than glucose metabolism. In order to confirm this, we measured the intracellular free-proline levels in parasites starved for 48 h with PBS or 3 mM proline, in the presence or absence of T4C. Parasites starved with 3 mM glucose were used as controls, as previously described. After 48 h of incubation (Tctively) , indicatctively) . After 4analogue . The effT. cruzi takes up proline through two active transport systems, one of them being a high affinity, low capacity transporter called system A, and the other a low affinity high capacity transporter called system B 50 (0.5 mM) concentration for both assays. As can be observed, both transporters resulted partially inhibited (68% inhibition for system A and 91% inhibition for system B) (ystem B) . In turn50 = 11.4±0.36 mM) .In order to evaluate the effectiveness of treating the infected host-cells, the toxicity of T4C on mammalian CHO-K1 cells was determined. Interestingly, the inhibitory T4C concentrations tested on epimastigotes (between 0.1 and 1.0 mM) were not toxic to mammalian cells (IC0.36 mM) . It is wbursting . The higcells/ml . There wT. cruzi biology, involved in several processes that are essential for the parasite life cycle Proline is a relevant metabolite in 3H] proline, 100 Ci/mmol was purchased from NEN Life Science Products . RPMI culture medium and Fetal Calf Serum were from Cultilab . All other reagents were from Amresco.L-proline, L-thyazolidine-4-carboxylic acid, rotenone, antimycin and MTT -2,5-diphenyltetrazolium bromide) were from Sigma . L-[2, 3, 4, 5-3, 100 units mL−1 penicillin and 100 µg mL−1 streptomycin at 37°C in a humid atmosphere containing 5% CO2. Epimastigotes of T. cruzi, CL strain, clone 14 The Chinese Hamster Ovary cell line CHO-K1 was routinely cultivated in RPMI medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 0.15% (w/v) NaHCO6 cells/mL) were washed three times by centrifugation and resuspended in phosphate buffered saline (PBS), then cultured in fresh-LIT medium supplemented or not (controls) with different concentrations of T4C ranging from 0.1 to 1.0 mM at 28°C. The assays were carried out in 96 well plates by inoculating 2.5×106 cells/mL in 200 µL of medium. Cell growth was estimated by absorbance readings at 620 nm every day for ten days. The absorbance was transformed into a cell density value (cells/mL) using a linear calibration equation previously obtained under the same conditions . The concentration of T4C that inhibited 50% of parasite growth (IC50) was determined in the exponential growth phase (sixth day) by adjusting the effect  as a function of T4C concentration to a classical sigmoidal equation. As a cell growth inhibition control, growth curves in which 200 µM rotenone and 0.5 µM antimycin were added to the culture medium were run in parallel for all experiments.Epimastigotes in the exponential growth phase  or PBS supplemented with 3 mM glucose, 3 mM proline, or nothing (as control) for 72 h in Eppendorf tubes. Subsequently, cell viability was estimated by the MTT assay. To evaluate the combined effect of T4C and oxidative stress, 5×106 parasites/mL in the stationary phase were maintained for 1 h and 30 min at 28°C in PBS or with 160 µM H2O2 in the presence or absence of 0.5 mM T4C. The cells were then collected by centrifugation and resuspended in LIT medium, and after 5 days, the number of cells/mL was determined as described previously 6 epimastigotes/mL were washed twice, resuspended in PBS and pre-incubated for 48 h in PBS in the presence or absence of 3 mM L-proline and 3 mM glucose, with or without 0.5 mM T4C. After pre-incubation, the cells were washed twice, resuspended in PBS with or without 160 µM H2O2 and maintained under those conditions at 28°C for 3 h. Cell viability was then estimated using the MTT assay To analyze the combined effect of T4C at different pH values and at different temperatures, the growth curves were generated as described above, with adjustment of the LIT pH to the desired values or by incubating the cultures at 28°C (no stress), 33°C and 37°C. To evaluate the effect of T4C combined with nutritional stress conditions, 20×106 cells/ml and distributed in aliquots of 100 µl (containing 20×106 cells each). Transport assays were initiated by the addition to the assay tubes of 100 µl of radioactively traced proline (0.5 µCi) to a final concentration of 0.31 mM or 1.36 mM, supplemented or not with 0.5 mM T4C. V0 was measured at 28°C for 30 s, and proline transport was stopped by addition of 800 ml of cold 50 mM proline in PBS, and rapid washing by centrifugation at 10,000 g for 60 s. The T. cruzi proline dehydrogenase activity was assayed as previously described T. cruzi extracts by performing a coupled reaction, using D-proline as the enzyme substrate and L-proline dehydrogenase as the revelation system. Both assays were performed in the presence or absence of 0.5 mM T4C.The proline competition assays were performed as previously described 5 cells ml−1) were inoculated in 24-well plates in FCS supplemented RPMI medium, as previously described, in the presence of increasing concentrations of T4C or no T4C (controls). The viability of cells was determined by the MTT assay, and the IC50 was obtained by fitting the data to a typical dose-response sigmoidal curve.CHO-K1 cells  was performed by incubation the parasites in the presence of 0.5 µM antimycin and 200 µM rotenone.Growth curve of epimastigotes of (0.48 MB TIF)Click here for additional data file.Figure S2Trypanosoma cruzi treated with T4C at 37°C and 7.5 pH: ▪ 0 mM, ▵ 0.1 mM, ○ 0.25 mM, ⧫ 0.5 mM, □ 0.75 mM, ▴ 1.0 mM. Inhibition control (─) was performed by incubation the parasites in the presence of 0.5 µM antimycin and 200 µM rotenone.Growth curve of epimastigotes of (0.45 MB TIF)Click here for additional data file.Figure S3Trypanosoma cruzi. Percentage of growth inhibition of T. cruzi epimastigotes at different temperatures and T4C. The curves represent the sigmoidal equation at 28°C (—), 33°C (---), and 37°C (••••••••••••••••). It also shows the maximum cell growth obtained in the control at each condition of temperature (inset).Sigmoidal dose-response curve of epimastigotes of (0.53 MB TIF)Click here for additional data file.Figure S4Trypanosoma cruzi treated with T4C at 28°C and 5.5 pH: ▪ 0 mM, ▵ 0.1 mM, ○ 0.25 mM, ⧫ 0.5 mM, □ 0.75 mM, ▴1.0 mM. Inhibition control (•) was performed by incubation the parasites in the presence of 0.5 µM antimycin and 200 µM rotenone.Growth curve of epimastigotes of (0.55 MB TIF)Click here for additional data file.Figure S5Trypanosoma cruzi treated with T4C at 28°C and 6.5 pH: ▪ 0 mM, ▵ 0.1 mM, ○ 0.25 mM, ⧫ 0.5 mM, □ 0.75 mM, ▴1.0 mM. Inhibition control (─) was performed by incubation the parasites in the presence of 0.5 µM antimycin and 200 µM rotenone.Growth curve of epimastigotes of (0.45 MB TIF)Click here for additional data file.Figure S6Trypanosoma cruzi. Percentage of growth inhibition of T. cruzi epimastigotes at different temperatures and T4C. The curves represent the sigmoidal equation at pH 7.5 (─), 6.5 (---), and 5.5 (••••). It also shows the maximum cell growth obtained in the control at each condition of pH (inset).Sigmoidal dose-response curve of epimastigotes of (0.53 MB TIF)Click here for additional data file.


P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 Å. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 Å resolution electron density map. At 5–7 Å resolution, α helices and β sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive  Most proteins in eukaryotic cells exist as components of large protein, RNA-protein, or DNA-protein complexes, which carry out important biological functions in an integrated manner . The strWe have recently solved the structure, at 5.5 Å resolution, of the functional core of human U1 snRNP, which consists of U1 snRNA, seven Sm proteins, U1-70K, and U1-C . In thisP1 space group with unit cell parameters a = 127 Å, b = 128 Å, c = 156 Å, α = 96°, β = 107°, and γ = 101° and diffract to ∼6 Å resolution. Self-rotation and self-Patterson analyses suggested four U1 snRNPs in the asymmetric unit (ASU) (data not shown).The reconstitution and crystallization of U1 snRNP have been described . The fun6Br12) derivative , lower Cullis R factor (0.71 versus 0.76), and better overall figures of merit (0.413 versus 0.404) than a single point Gaussian model. A multiwavelength anomalous dispersion data set was collected from a tantalum bromide cluster (Tarivative  at the Trivative A and thein SHARP . Inspect6Br12 phases. The map had 123 peaks greater than 3.0 SD (σ) above background (data not shown).To improve phasing and to aid location of the Sm proteins, they were all labeled with SeMet then incorporated into U1 snRNP crystals. Se K-edge (λ = 0.9797 Å) data were collected and an anomalous difference map calculated using solvent-flattened TaTo further improve phasing and to locate the Zn-finger protein U1-C, an anomalous map was calculated from native data collected at the zinc K edge (λ = 1.2827 Å). The map showed four peaks greater than 7.8 σ with the next strongest being 4.4 σ. The four strongest peaks correspond to zinc atoms in U1-C protein, in agreement with there being four U1 snRNP complexes in the ASU.6Br12 clusters were refined together against their respective data sets in SHARP  and four of the protein structures have been determined as heterodimers of SmD1D2 and SmBD3 . One to The lowest energy NMR structure of the Zn-finger domain of U1-C , form a The RNA binding domain (RBD) of U1-70K was known to bind to SL1  and a laThe location and the structure of the N-terminal 62 residues of U1-70K were unknown, so we made several single methionine mutants in this region to locate these residues from the position of anomalous peaks when U1 snRNP was reconstituted with their SeMet derivative. The amino acids chosen for substitution with methionine had long and/or hydrophobic side chains, in order to minimize structural changes. Basic side chains were avoided because their mutation could disrupt RNA binding. Substitutions were made at approximately ten residue intervals to facilitate chain tracing. Data from crystals grown with SeMet derivatives of these mutants gave selenium anomalous difference peaks that revealed an extended polypeptide from residue 9 to 63 B. The po6Br12 derivative and zinc edge data, the original experimental map was calculated. The unit cell of this crystal had a c axis 3.7 Å shorter than the mean of the other crystals' c  and two in the α-helical region (Met-67 and Met-88). The Se atoms of the mutant sites all gave significant anomalous peaks, except for the E49M site in particle C . We also+) and (I−), measured in SCALA  protein  at 100 K. All these steps were done at 4°C. Freezing in a cryostream consistently reduced the mosaicity of the crystals, as opposed to plunging the crystal directly into liquid nitrogen.Diffraction data were collected on a Mar225 CCD detector at Swiss Light Source X06SA and X10SA beam lines with the X-rays focused on the detector. In order to minimize the effect of radiation damage inverse beam mode was used; i.e., after every 1° oscillation the crystal was rotated 180° such that Friedel pairs were measured nearly simultaneously. Data were integrated with MOSFLM  and scalFigures were prepared using PyMol  except f


The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR.th century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples.We identified a mass grave in Douai, France dating from the early XVIIIB. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XXth century.In the Douai remains, we identified one case of th century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America.Louse-borne outbreaks were raging during the XVIII Yersinia pestis Orientalis Bartonella quintana in very ancient samples Bartonella quintana and Rickettsia prowazekiiAs mentioned by Zinsser, infectious illness has killed more soldiers during war than weapons Recently, a mass grave dating back to the 18th century was explored in Douai, France. The city of Douai was besieged from 1710 to 1712, being successively occupied by the French and then the Dutch and then retaken by French in 1712 during the war of Spanish succession . These eDouai is a village located in northern France. It was disputed between France and the Great Alliance of La Haye between 1702 and 1712 Bacillus anthracis (anthrax), Borrelia recurrentis (louse-borne relapsing fever), Bartonella quintana (trench fever), Rickettsia prowazekii (epidemic typhus), Salmonella enterica Typhi (typhoid fever), Poxvirus  and Yersinia pestis (plague)  . Real-tiRickettsia prowazekii DNA invertase Pin-like protein  by combining external primers RpDet-F1 5′-GTTGGATATATAAGGGTTTC-3′ and RpDet-R2 5′-CCGAGTCTATCTAATTTCCA-3′ and internal primers RpDet-F3 5′-ATGATCGTCAAGTGTTCGAT-3′ and RpDet-R4 5′-TAGACAGTCGCCATCTTGTA-3′; the final expected PCR product was 152 bp , 2 µL Master Mix , 1 µL of a 10 µM solution of RpDet-F1, 1 µL of a 10 µM solution of RpDet-R2, 8.9 µL sterile water and 5 µL DNA  or 5 µL sterile water (negative control tube). The first round of amplification was done using a LightCycler™ apparatus (Roche Diagnostics) and the following conditions: 10-min activation at 95°C followed by 45 cycles of 15-sec denaturation at 95°C, 20-sec hybridisation at 55°C and 25-sec elongation at 72°C. PCR products were recovered by centrifugation of capillaries at 380 g for 2 minutes. The nested PCR was performed in the capillary containing 1.6 µL MgCl2, 0.5 µL BSA, 2 µL Master Mix, 1 µL of 10 µM solution of RpDet-F3; 1 µL of 10 µM solution of RpDet-R4; 8.9 µL sterile water and 5 µL first round PCR product. The second amplification in the LightCycler™ apparatus used the following conditions: activation at 95°C for 10 minutes and 45 cycles of denaturation at 95°C for 15 seconds, hybridisation at 57°C for 20 seconds, elongation at 72°C for 25 seconds. Nested PCR products were detected on a 2% agarose gel  in the presence of molecular weight marker VI .The Douai specimens were further examined with a suicide nested PCR protocol after conventional phenol chloroform DNA extract s 152 bp . This reR. prowazekii glutamine amidotransferase-like protein  by combining external primers Rpro-F1 5′-ACTGTTATTACCGATCTTGCCA-3′ and Rpro-R1 5′-TGGTTGATGCTAGGTTATTTGG-3′ and internal primers Rpro-F11 5′-GTATTAAGAATTTGATGCCACCA-3′and Rpro-R11 5′-GTTATTAGTCCAAATGACGTGAA-3′; the final expected PCR product was 130 bp  or 5 µL sterile water (negative control tube). The first round of amplification was done in an ABI GeneAmp™ 2700 thermocycler  under the following conditions: 10-min activation at 95°C and 45 cycles of 30-sec denaturation at 95°C, 45-sec hybridisation at 58°C and 90-sec elongation at 72°C. The PCR products were purified using the Millipore plate protocol and suspended into 40 µL water. The second round of PCR was performed using 0.8 µL MgCl2, 0.2 µL HotStart Taq, 2.5 µL 10X PCR buffer, 2.5 µL dNTP, 0.5 µL BSA, 0.5 µL Rpro-F11, 0.5 µL Rpro-R11, 12.5 µL sterile water and 5 µL first round PCR product. The second amplification was performed using the following conditions: 10-min activation at 95°C and 45 cycles of 30-sec denaturation at 95°C, 45-sec hybridisation at 62°C and 90-sec elongation at 72°C. Nested PCR products were detected on a 2% agarose gel  in the presence molecular weight marker VI .A second suicide nested-PCR targeted a 187-base pair fragment of the s 130 bp . This reNested PCR products were purified using the Millipore plate protocol and suspended in 50 µL water. The sequencing reaction was carried out in a tube containing 3 µL Big Dye Terminator, 0.5 µL forward or reverse primer, 3.5 µL purified PCR product and 3 µL sterile water using the following conditions: activation at 95°C for 5 minutes followed by 25 cycles consisting of 30-sec denaturation at 96°C, 20-sec hybridisation at 55°C, 4-min elongation at 60°C and 7-min extension at 15°C. The sequencing products were purified on Séphadex® G50 5% gel and analysed with the ABI PRISM 3100 Genetic Analyser (HITACHI). The sequences were read and corrected by using the software ChromasPro version 1.34 and then aligned with BLAST to compare the sequences available in GenBank.5′-CCGGAAATGTAGTAAATCAATC-3′ and rpmE-R2 5′-CTGAGAATTTAAAGATTTATCTG-3′) and a 115-bp sequence  on the rpmE-tRNAfMet intergenic spacer (gi|56967982|gb|AY695449.1|)   or 5 µL sterile water (negative control tube) in a Stratagene plate. The first round of amplification was performed in a Stratagene™ thermocycler (Agilent Technologies Company) using the following conditions: activation at 95°C for 10 minutes, 45 cycles of denaturation at 95°C for 30 seconds, hybridisation at 59°C for 30 seconds, elongation at 72° C for 1 minute. Nested PCR was realised in a Stratagene plate containing 1.6 µL MgCl2, 0.5 µL BSA, 2 µL Master Mix, 1 µL of 10 µM solution of rpmE-F3, 1 µL of 10 µM solution of rpmE-R4, 8.9 µL sterile water and 5 µL first round PCR product. The second amplification in the Stratagene™ thermocycler was performed using the following conditions: activation at 95°C for 10 minutes followed by 45 cycles consisting of denaturation at 95°C for 30 seconds, hybridisation at 62°C for 30 seconds and elongation at 72°C for 1 minute. The nested PCR products were detected on a 2% agarose gel (Invitrogen™) in the presence of molecular weight marker VI (Boehringer Mannheim). The PCR products were sequenced as described above.The software PerlPrimer version 1.1.6 was used to design two pairs of primers targeting a 210-bp sequence  . GenotypR. prowazekii had never been previously amplified. Each step was conducted in a separate room under a hood with air-capture. All instruments were sterilised and used only once. No positive control was included, and one negative control consisting of sterile water was used for each three or five samples.all manipulations of ancient materials were done in two successive laboratories where B. quintana DNA in 1/40 dental pulp specimens, and no other pathogen was detected in these 40 specimens. We then attempted to detect R. prowazekii using a more sensitive technique.A total of 1.192 dental pulp specimens collected from several burial sites in France were analysed by high throughput detection, including 40 specimens collected in Douai tested blindly. Whereas the negative controls remained negative, high throughput real-time PCR detected R. prowazekii DNA in 2/38 (5.3%) ancient teeth collected from 2/19 (10.5%) different individuals (coded as A6-4 and A10-19). Sequence alignment yielded 100% sequence similarity with the reference R. prowazekii strain Madrid E (Genbank accession emb|AJ235273.1|RPXX04). As for the second suicide PCR, it detected R. prowazekii DNA in 1/17 (5.9%) dental pulp specimens collected from 1/9 (11%) different individuals (coded as 126/220-39)  .R. prowazekii rpmE/tRNAfMet intergenic spacer sequence in 3/15 (20%) ancient teeth collected from 3/7 (43%) different individuals . All PCR products yielded an identical sequence exhibiting 100% sequence similarity to R. prowazekii type B, which is characterised by a T to C substitution at position 111 (R. prowazekii DNA in 6/55 (11%) teeth collected from 6/21 (28.6%) individuals.In the genotyping, the negative controls remained negative while we amplified the tion 111 . MergingR. prowazekii had never been worked on, nor had R. prowazekii DNA been extracted. All operations were carried out under a hood with air-capture using sterilised instruments that were used only once. All the negative controls remained negative. We obtained an original sequence that was consistently found in several teeth collected in several individuals, its uniqueness thus giving much confidence. After blindly detecting B. quintana in one tooth from Douai, we decided to employ more sensitive techniques to recover R. prowazekii from other teeth from this site.The results presented herein were interpreted as authentic. Indeed, we selected teeth that had a closed apex and that were free of dental caries and traumatic lesions to minimise any risk of external contamination of the dental pulp. For the first time, we used a high throughput paleomicrobiological approach to screen 1192 teeth for seven infectious agents . This technique is less sensitive than suicide PCR but is well adapted to testing numerous samples. Our work was carried out in a laboratory where Yersinia pestis DNA R. prowazekii sequences were detected in 6/55 (11%) teeth collected in 6/21 (28.6%) individuals; this prevalence is significantly higher  than that previously reported in the Napoleon's Grand Army study, which detected R. prowazekii in 4/72 (5.6%) teeth collected from 3/35 (8.6%) soldiers B. quintana, we suspect the circulation of louse-borne diseases in this place, but we did not recover Borrelia recurrentis DNA.In this study, we observed that not all dental pulp specimens collected from a single individual yielded a positive PCR product. Such variability in the positivity of PCR-based detection has been previously observed in detecting R. prowazekii DNA. Dental pulp is a specialised conjunctive tissue occupying the central position in the tooth, where it is protected from the external environment by the dentine. We previously demonstrated that blood-borne bacteria could be detected in dental pulp both by culture and by molecular detection of specific DNA sequences R. prowazekii genotype B. It was later shown that real-time PCR is a rapid, specific and sensitive method for detecting R. prowazekii in blood fMet spacer in 15 modern R. prowazekii DNA samples found three genotypes: A, B and C. Genotype B had been sequenced in the avirulent R. prowazekii Madrid E strain (derived from a Spanish isolate R. prowazekii Evir in two blood isolates from Russia and Algeria, and in four lice collected in Rwanda and Burundi th-century Europe.In the present study, we also took advantage of the dental pulp to detect and genotype 300-year-old R. prowazekii issued from ancient materials have been provided. One individual diagnosed with fatal epidemic typhus in Maryland in 1901 was demonstrated by using immunohistological detection ninety years later to have died of Rocky Mountain spotted fever caused by Rickettsia rickettsiiR. prowazekii and B. quintana in an estimated one-third of the individuals Despite the fact that typhus epidemics has been depicted in historical sources for two millennia or so B. quintana epidemic persisting in Europe for an extremely long time. The oldest trace of infection of humans by this bacterium goes back 4.000 years th century in Spain. The first descriptions go back to Fracastor, and early in the 16th century a number of epidemics compatible with the diagnosis of typhus were reported. However, at the beginning of the 18th century, the clinical individualisation of epidemic typhus, like that of trench fever of the trenches, was not yet carried out. The first definition of typhus was provided by Boissier de Sauvages in 1772 Taken together, the results reported herein show clearly that an epidemic disease transmitted by lice prevailed during the long siege of Douai. Diseases transmitted by body lice were probably extremely frequent in the past. When sanitary arrangements are degraded, lice are likely to quickly expand and the population of lice can increase by 10% per day In conclusion, the molecular diagnosis of past epidemic infections related to the teeth made it possible to identify the first outbreak of epidemic typhus in the 18th century in the context of a pan-European Great War. Working together, molecular biologists, dentists and anthropologists have explored burials from catastrophes to identify the prevailing epidemics in past centuries.


On June 11, 2009 the World Health Organization (WHO) declared a new H1N1 influenza pandemic. This pandemic strain is as transmissible as seasonal H1N1 and H3N2 influenza A viruses. Major concerns facing this pandemic are whether the new virus will replace, co-circulate and/or reassort with seasonal H1N1 and/or H3N2 human strains. Using the ferret model, we investigated which of these three possibilities were most likely favored. Our studies showed that the current pandemic virus is more transmissible than, and has a biological advantage over, prototypical seasonal H1 or H3 strains. The swine-origin H1N1 pandemic virus was initially linked to human outbreaks in North America in April 2009 and has since spread to over 140 countries in the last five months (http://www.who.int/csr/disease/influenza/en/).  The WHO’s decision to proceed to Phase 6, pandemic influenza, was mainly due to the transmissibility, not the morbidity, of the virus.  This virus has continued to be isolated during the summer months in North America and spread to the regions of the southern hemisphere where the influenza season is at its peak.  Numbers from South American countries indicate this pandemic strain predominates over the seasonal H1 and H3 strains, although all 3 strains appear to circulate. Studies to determine the possibility of reassortment following co-infection of an avian and human virus has been reported in vivo, we performed co-infection studies in ferrets.  The ferret is a well-established model for human influenza infection, pathogenesis and transmission studies     To establish whether the current pandemic H1N1 virus has a biological advantage over the seasonal H1N1 and/or H3N2 strain(s) and whether reassortment between the pandemic and seasonal strains is rapidly favored 6 TCID50 of the corresponding virus combination. Direct contact ferrets and respiratory droplet contact ferrets were introduced at 24 h.p.i. Black and white bars represent individual ferrets. Respiratory droplet contacts shown: (A) A/California/04/09 [Ca/04] H1N1 virus, (B) A/Brisbane/59/07 [BR/59] H1N1, (C) co-infected with BR/59 and Ca/04, (D) A/Brisbane/10/07 [BR/10] H3N2, (E) or co-infected with BR/10 and Ca/04 (E). In (E), day 10 p.c., the respiratory contact from the group represented by the black bars died, as noted by a star in the bar graph. Post-mortem examination indicated intestinal hemorrhage and virus was isolated from respiratory tissues. Titers are expressed as log10 values of TCID50/ml with the limit of detection at 0.699 log10TCID50/ml. Viruses were grown and titrated in MDCK cells as previously described [6]Ferrets were inoculated with 106 TCID50 of either Ca/04, BR/59, BR/10 or nasal washes from the last day of respiratory droplet contact shedding in the co-infected groups (passed once in MDCKs to achieve a titer of 106 TCID50). Tissues were collected 4 dpi and samples were taken for histopathology and to determine titers and tropism. Nasal turbinate, trachea, lung, intestine, liver, spleen, heart and olfactory bulb were homogenized and titrated in MDCKs. 10% (w/v) of the homogenates were used to determine viral titers in MDCK cells. Abbreviations on figure include: NT, nasal turbinate. Asterisk indicates when only one out of two ferrets was positive for virus. For histopathology studies tissues were paraffin-embedded and stained for hematoxylin and eosin. PBS , Ca/04 , BR/59 , BR/10 , BR/59:Ca/04 [H1/H1] , BR/10:Ca/04 [H3/H1] . Lung  and trachea  samples are shown.(A) Ferrets were infected with 10     To determine the possibility of co-infection of pandemic and seasonal influenza viruses, ferrets were either co-infected with Ca/04 H1N1 and the seasonal BR/59 H1N1 virus [BR/59:Ca/04] or co-infected with Ca/04 and the seasonal BR/10 H3N2 virus [BR/10:Ca/04].  Successful co-infection was confirmed through PCRs performed directly on nasal washes collected 1-day post-infection (p.i.) with virus-specific primers , as well as through seroconversion against both viruses in inoculated ferrets (Table 1).  Transmission to direct and respiratory droplet contact ferrets was observed in both co-infections .  In each case, co-infection led to an intermediate transmission phenotype where virus transmitted to direct and respiratory droplet contacts earlier than the seasonal strain and within a day of the pandemic strain.  In addition, ferrets shed viral titers higher than the seasonal strain but comparable to the pandemic strain.  Clinical signs including onset of sneezing and body temperatures were similar in the co-infected groups; however, ferrets from the BR/59:Ca/04 [H3/H1] group presented with diarrhea and weight loss and one respiratory droplet contact had to be euthanized while still shedding virus .  Tissues collected from the euthanized ferret indicated that virus was present in the nasal turbinate, trachea, lung and olfactory bulb (data not shown). Nasal washes from co-infected ferrets  were collected days 1-11 p.i.  Nasal washes from respiratory droplet contacts first and last day of shedding were analyzed by PCR using primers specific for BR/59, BR/10, and Ca/04.  Data for the last day of shedding from each respiratory droplet contact is shown for the BR/59:Ca/04 (H1:H1) pair day 8 post-contact  and the BR10:Ca/04 (H3:H1) pair days 7 and 10 post-contact .  Nasal wash from ferrets directly inoculated with BR/59:Ca/04 (H1:H1) and BR10:Ca/04 (H3:H1) virus combinations were analyzed 1 dpi to confirm efficient replication and shedding of both viruses .  adenotes non-specific binding confirmed by sequencing the PCR product to eliminate suspicion of reassortment.    To establish the virus population(s) selected for respiratory droplet transmission following co-infection in both BR/59:Ca/04 [H1/H1] and BR/10:Ca/04 [H3/H1] groups, viral RNA was purified and RTPCRs were performed directly from the nasal washes collected from the respiratory droplet contacts.  We selected the first and last days of viral shedding as the most fit virus population to a) transmit to respiratory droplets, b) to survive transmission, and c) to be passed on in respiratory droplet form.  We performed RTPCRs on purified viral RNA from nasal washes collected from respiratory droplet contacts and/or stock virus used in our single Ca/04, BR/59, and BR/10 infection studies, as controls for virus-specific primers. In addition, virus in nasal washes from co-infected and single infected ferrets in the respiratory droplet contact groups were amplified once in cell culture (P1 MDCK) to determine which type of virus population was prevalent. Viruses isolated from the first and last days of shedding from respiratory droplet contacts co-infected with either BR/59:Ca/04 (H1/H1) or BR/10:Ca/04 (H3/H1) show a single virus population, a full Ca/04 virus. The data was consistent regardless of whether we analyzed RNA obtained directly from nasal washes or P1 MDCK passage virus   In contrast, directly inoculated ferrets with either the BR/59:Ca/04 (H1/H1) or BR/10:Ca/04 (H3/H1) virus combinations showed both virus populations at 1 dpi, from either nasal washes or P1 MDCK grown virus .  HI analysis of the BR/59:Ca/04 (H1/H1) ferret sera indicated that only inoculated ferrets had an antibody response to the seasonal BR/59 while all ferrets had strong responses to Ca/04, suggesting that the Ca/04 out-competes the seasonal H1N1 virus, most likely at direct transmission (Table 1). Interestingly, although a majority of the ferret sera from the BR/10:Ca/04 (H3/H1) group responded to both BR/10 and Ca/04 (Table 1), only Ca/04 was isolated in all nasal washes and organs tested . To further determine the nature of the predominant virus population in respiratory droplet contacts in the co-infected groups, the nasal washes from the respiratory droplet contact ferrets, collected on the last day of shedding (P1 MDCKs), were used to infect a new set of ferrets and determine which virus population(s) prevailed . Nasal washes and respiratory tissues collected from this new set of ferrets revealed the presence of high virus titers comparable to Ca/04 single infection . Interestingly, PCR analysis of tissue homogenates  from this set of ferrets revealed the presence of Ca/04 and no evidence of BR/59 or BR/10 (not shown).  In addition, histopathology of lungs and trachea from both co-infected groups show similar bronchopneumonia and inflammatory infiltration like Ca/04 single infection .    These studies emphasize the notion that the pandemic strain has growth and transmissibility advantages over the seasonal strains.  More importantly, no evidence of reassortment was detected in PCRs performed on direct nasal washes or nasal washes passed once in MDCK cells in either of the respiratory droplet contacts from either co-infection group . It should be noted that the pre-infection HI titers for both the H1 and H3 seasonal strains were minimal, although detectable, in all ferrets (Table 1). Thus, we cannot exclude the possibility that the relative transmissible advantage of the Ca/04 H1N1 is merely a consequence of prior immunity in ferrets against the seasonal influenza strains.  Our data, however, clearly showed that both the BR/59 (H1N1) and BR/10 (H3N2) replicated and transmitted well in single virus infections.  It remains to be determined whether the minimal antibody levels in ferrets against the seasonal flu strains contributed to the Ca/04’s ability to out-compete them. However, we must note that our studies reflect the most likely scenario in the human population as it is expected that most people carry antibodies against H1 and H3 seasonal strains. This is particularly true in those within the 15-40 age group that appear to have shown more exacerbated disease with the pandemic strain.  Nevertheless, these observations are consistent with the notion that the emergence of novel pandemic strains in humans may be favored, not only because of lack of immunity against the new virus but also because of the presence of immunity against extant seasonal strains.Recent reports have speculated on the possibility that the new pandemic H1N1 strain may become more virulent and/or more transmissible if it were to reassort with seasonal influenza strains 50) in MDCKs.  For A/Brisbane/59/07 (H1N1) and A/Brisbane/10/07 (H3N2) the viruses were received as egg passage 1 (EP1) was grown and titrated in MDCKs (EP1/MDCKP1).  For A/California/04/09 (H1N1) virus was received as MDCK P1 and then grown twice in MDCKs for MDCK P3 and titrated.  All experiments with live virus were performed in a biosafety level-3+ containment facility.     Viruses were grown in Madin-Darby Canine kidney (MDCK) cells and titrated with tissue culture infectious dose 50 , Triple F Farms, Sayre, PA were used for studies.  Animal studies were conducted under guidelines approved by the Animal Care and Use Committees of the University of Maryland and the Centers for Disease Control and Prevention. All animal studies were conducted in a biosafety level-36 TCID50 of virus in PBS, 250 µl per nostril.  Twenty-four hours later, two naïve ferrets were introduced into the isolator.  A direct contact was introduced into the same cage as the infected ferret while the respiratory droplet contact was placed into a cage separated from the infected and direct contact by a wire mesh wall.  The wire mesh prevented physical contact between the respiratory droplet contact and the infected/direct contact ferrets therefore, only air was shared among the group.  Individual weight and body temperatures were measured daily; fevers were defined as 3 standard deviations above an individual’s baseline temperature.  To monitor viral shedding nasal washes were collected daily for up to two weeks.  Ferrets were anesthetized as described above and 1 ml of PBS was used to induce sneezing.  Nasal washes were immediately tested for virus using the FLU DETECTTM Antigen Capture Test  and additional aliquots were stored at -800C before performing TCID50 titrations in MDCK cells.  Two weeks after shedding blood was collected and seroconversion determined by HI Assay.     The basic transmission study scheme consisted of groups of three ferrets: one infected, one direct contact and one respiratory droplet contact, as described previously     Serum samples were collected 14-15 days p.i. and treated with receptor-destroying enzyme  to remove nonspecific receptors.  The anti-viral antibody titers were evaluated using the HI assay system outlined by the WHO Animal Influenza Training Manual (WHO/CDS/CSR/NCS/2002.5).  HI assays were performed using homologous and heterologous viruses.6 TCID50 of either: A/California/04/09 (H1N1), A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), nasal wash collected from BR/59:Ca/04 co-infected respiratory droplet contacts on day 8 post-contact (p.c) (passed once in MDCK), nasal wash collected from BR/10:Ca/04 co-infected respiratory droplet contacts on days 7 and 10 p.c. (passed once in MDCK), or mock-infected with PBS.  Ferrets were euthanized on day 4 p.i.  Olfactory bulb, nasal turbinate, trachea, lung, heart, spleen, liver and intestine were collected and stored at ─80 ºC for virus titration as previously described     Groups of two ferrets were inoculated with 10    To establish the virus population(s) during the co-infection transmission studies RNA was collected directly from nasal washes, nasal washes passed once in MDCKs and tissue homogenates .  Reverse transcriptase polymerase chain reaction was used to determine the origin of each gene in all samples.  Nasal washes analyzed included: day 1 post-infection (p.i.) from both infected ferrets in both co-infection groups (BR/59:Ca/04 [H1/H1] and BR/10:Ca/04 [H3/H1]), the last day of shedding for each respiratory droplet contact from both co-infected groups (8 days post-contact (pc) from both respiratory droplet contacts in BR/59:Ca/04 [H1/H1] and days 10 and 7 pc from respiratory droplet contacts in BR/10:Ca/04 [H3/H1]).  The aforementioned nasal wash samples from all 4 respiratory droplet contacts (both H1/H1 and H3/H1 groups) were also passed once in MDCK cells to determine viral population after passage in cell culture.  Tissue homogenates collected 4 dpi, described above, were also processed for RNA.    Primers were designed to specifically hybridize to Ca/04, BR/59 or BR/10 genes and PCR products were visualized on 1% agarose gels.  Primer sequences and reaction conditions are available upon request.  Any background bands were cloned and sequenced to confirm non-specific binding. This research was possible through funding by the CDC-HHS grant (1U01CI000355), NIAID-NIH grant, (R01AI052155), CSREES-USDA grant (2005-05523), and NIAID-NIH contract (HHSN266200700010C).  We are indebted to Ivan Gomez-Osorio for his excellent laboratory techniques and animal handling assistance.  We would like to thank Andrea Ferrero and Theresa Wolter for their laboratory managerial skills and the Centers for Disease Control and Prevention, Atlanta, GA, for supplying the wild type viruses used in this study. The opinions of this manuscript are those of the authors and do not necessarily represent the views of the granting agencies. The authors have declared that no competing interests exist.


To identify the disease locus for autosomal recessive congenital cataract in a consanguineous Pakistani family.All affected individuals underwent detailed ophthalmologic and medical examination. Blood samples were collected and DNA was extracted. A genome-wide scan was performed with polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and logarithm of odds (LOD) scores were calculated.The clinical records and ophthalmological examinations suggested that all affected individuals have nuclear cataracts. Maximum LOD scores of 5.01, 4.38, and 4.17 at θ=0 were obtained with markers D7630, D7S657, and D7S515, respectively. Fine mapping refined the critical interval and suggested that markers in a 27.78 cM (27.96 Mb) interval are flanked by markers D7S660 and D7S799, which co-segregate with the disease phenotype in family PKCC108.We have identified a new locus for autosomal recessive congenital cataract, localized to chromosome 7q21.11-q31.1 in a consanguineous Pakistani family. Congenital cataracts are one of the major causes of vision loss in children worldwide and are responsible for about one-third of cases of blindness in infants ,2. They GJA8, GCNT2, HSF4, LIM2, BFSP1, CRYAA, CRYβB1, and CRYβB3 have been identified [Approximately one-third of congenital cataract cases are familial . To dateentified ,11,13-17Here, we report a new locus for autosomal recessive nuclear cataract in a large consanguineous Pakistani family (PKCC108). Genome-wide linkage analyses localized the critical interval to chromosome 7q, whereas fine mapping refined the critical interval to a 27.78 cM (27.96 Mb) flanked by markers D7S660 and D7S799 that co-segregates with the disease phenotype in PKCC108.A total of 100 consanguineous Pakistani families with nonsyndromic cataract were recruited to participate in a collaborative study between the Center of Excellence in Molecular Biology, Lahore, Pakistan, and the National Eye Institute, Bethesda, MD, to identify new disease loci causing inherited visual diseases. Institutional Review Board (IRB) approval was obtained from the National Eye Institute and the National Centre of Excellence in Molecular Biology. The participating subjects gave informed consent consistent with the tenets of the Declaration of Helsinki. A detailed medical history was obtained by interviewing family members. Ophthalmic examinations were conducted with slit-lamp microscopy. Approximately 10 ml of blood samples were drawn from affected and unaffected members of the family and stored in 50 ml Sterilin® falcon tubes containing 400 μl of 0.5 M EDTA. Blood samples were kept at -20 °C for long-term storage. DNA was extracted by a nonorganic method, as described by Grimberg et al. with minor modifications . Briefly2, and 0.2 U Taq DNA polymerase (Applied Biosystems). Initial denaturation was performed for 5 min at 95 °C, followed by 10 cycles of 15 s at 94 °C, 15 s at 55 °C, and 30 s at 72 °C and then 20 cycles of 15 s at 89 °C, 15 s at 55 °C, and 30 s at 72 °C. The final extension was performed for 10 min at 72 °C. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems). The resulting PCR products were separated in an ABI 3100 DNA Analyzer (Applied Biosystems) and genotypes were assigned with GeneMapper software (Applied Biosystems).A genome-wide scan was performed with 382 highly polymorphic fluorescent markers from the ABI PRISM Linkage Mapping Set MD-10  having an average spacing of 10 cM. Multiplex polymerase chain reaction (PCR) was completed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). Briefly, each reaction was carried out in a 5 μl mixture containing 40 ng genomic DNA, various combinations of 10 mM dye-labeled primer pairs, 0.5 ml 10× GeneAmp PCR Buffer (Applied Biosystems), 1 mM dNTP mix, 2.5 mM MgClMarshfield database and the National Center for Biotechnology Information (NCBI) chromosome 7 sequence maps. For the initial genome scan, equal allele frequencies were assumed, while for fine mapping allele frequencies were estimated from 96 unrelated and unaffected individuals from the Punjab province of Pakistan.Two-point linkage analyses were performed using the FASTLINK version of MLINK from the LINKAGE Program Package  ,20. Maxiprimer3 program. The sequences, and annealing temperatures, and product size are shown in 2, and 0.2 U Taq DNA polymerase in the standard PCR buffer provided by the manufacturer (Applied Biosystems). PCR amplification consisted of a denaturation step at 96 °C for 5 min followed by 40 cycles, each consisting of 96 °C for 45 s followed by 57 °C for 45 s and 72 °C for 1 min. PCR products were analyzed on 2% agarose gel and purified by ethanol precipitation. The PCR primers for each exon were used for bidirectional sequencing using BigDye Terminator Ready reaction mix, according to manufacturer instructions. Sequencing products were precipitated and resuspended in 10 μl of formamide (Applied Biosystems) and denatured at 95 °C for 5 min. Sequencing was performed on an ABI PRISM 3100 Automated sequencer (Applied Biosystems). Sequencing results were assembled with ABI PRISM sequencing analysis software version 3.7 and analyzed with SeqScape software (Applied Biosystems).Primer pairs for individual exons were designed using the The family described in this study, PKCC108  is from During the genome-wide linkage scan, LOD scores greater than 1.0 were obtained for markers D1S142, D7S630, D7S657, and D7S515. Of these, D1S142 has closely flanking markers yielding large negative LOD scores and thus was excluded from further consideration. However D7S630, D7S657, and D7S515, adjacent markers in the MD-10 mapping set, yielded LOD scores of 5.01, 4.38 and 4.17 at θ=0, respectively. Fine mapping with closely spaced markers D7S660, D7S2540, D7S2482, D7S2459, D7S2456, D7S799, and D7S471 further confirmed linkage to markers on chromosome 7q. Maximum LOD scores of 5.08, 2.94, 4.76, and 4.32 at θ=0 were obtained with markers D7S2540, D7S2482, D7S2459, and D7S2456, respectively .Visual inspection of the haplotypes further supported the linkage results of the linkage analysis. A recombination event in individual 10 established the proximal boundary of the linked region at D7S660. Similarly, there is a distal recombination event in affected individual 11 and unaffected individual 14 at D7S799. Taken together, this places the disease locus in the 27.78 cM (27.96 Mb) interval of chromosome 7, flanked by markers D7S660 and D7S799 . All affGJE1 but did not identify any pathogenic mutations.The critical interval of 27.78 cM (27.96 Mb) is a gene-rich region and among these resides GJE1, a gap junction protein. As other gap junction proteins have been associated with autosomal recessive cataracts [Here we report identification of a new locus for autosomal recessive cataract mapped to chromosome 7q21.11-q31.1 in a consanguineous Pakistani family (PKCC108). A maximum LOD score of 5.08 was obtained with D7S2540 at θ=0, and the disease locus co-segregates with chromosome 7q markers in 27.78 cM (27.96 Mb) interval flanked by D7S660 and D7S799. The lack of significant LOD scores other than those in the chromosome 7q region during the genome-wide linkage scan, homozygous alleles for all the affected individuals for markers of 7q, and lack of homozygosity in all the unaffected individuals strongly suggest that the disease locus maps to a new region on chromosome 7q. No previously reported cataract loci or genes are located in the critical interval.CRYBB3 were identified [To date, 12 loci for recessive congenital cataract have been identified, illustrating the genetic heterogeneity of the disease. Previously, nuclear cataracts segregating in consanguineous families have been mapped to chromosome 2p12, 19q13.4, and 22q12.3, in the latter of which mutations in entified ,21. All GJE1, a gene that is a member of the gap junction protein family. As the lens is an avascular structure that lacks almost all cell organelles, cell–cell communication is brought about by an extensive system of gap junction proteins. Gap junction proteins GJA1 (connexin 43), GJA3 (connexin 46), and GJA8 (connexin 50) are expressed in human lens, and mutations in GJA3 have been reported to cause cataract with different morphology [GJA8 [GJE1 but found no pathogenic mutations.The critical interval harbors rphology ,23. Simigy [GJA8 ,25. We sTransparency and precise shape and optical density are distinctive features of the lens that are critical for proper light refraction. Elucidating the molecular mechanisms that maintain or disrupt lens transparency is a fundamental step toward preventing cataract. Identification of new genes involved in cataract will help us understand the molecular biology of the human lens and the structural and metabolic mechanisms involved in maintenance of the transparency of the lens.


TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates  and has an ability to hydrolyse xylan that is lacking in TfCel9A.A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is  Cellulose is a main structural component of plant biomass and the most abundant carbohydrate on Earth. It is composed of repeating d-anhydroglucose units linked by β1→4) glycoside bonds →4 glycos. IndividIn Nature, degradation of cellulose is carried out by the synergistic action of endo-acting and exo-acting enzymes that include glycosyl hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs) ,5. HowevThe temperature optima of cellulases in currently available commercial enzyme cocktails are typically around 50°C , i.e., nMetagenomics has proven to be a powerful tool for the discovery of thermostable enzymes from microbial sources. The crucial advantage of this approach is the ability to access extremophile genomes in a culture-independent manner . Metagenin silico mining of a metagenome from the Jan Mayen hydrothermal vent field. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the moderately thermophilic bacterium Thermobifida fusca [TfCel9A, which was then used as a reference enzyme in a comparative assessment of AMOR_GH9A functional properties. The results show that AMOR_GH9A has higher thermal stability and broader substrate specificity than its homolog from T. fusca.In recent years, we have been exploring the biodiversity of the Jan Mayen hydrothermal vent field at the Arctic Mid-Ocean Ridge, where temperatures can rise up to 260°C . In thisda fusca . We havePicea abies) that had been pretreated by sulfite-pulping using the BALI process [csbl.bmb.uga.edu/dbCAN) [A sample of unbleached Norway spruce  . The sigu/dbCAN) . The fulhttps://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify homologues of AMOR_GH9A. The sequence of AMOR_GH9A has been submitted to Genbank under accession number MK869727, and the DNA sequence of TfCel9A was obtained from GenBank (accession number L20093.1).The sequence-based mining led to the identification of a 2065 bp gene encoding a putative GH9 cellulase (AMOR_GH9A). The NCBI BLAST server . The genes were then amplified by PCR using Q5 high-fidelity DNA polymerase . The forward and reverse PCR primers incorporated plasmid-specific regions for ligation-independent cloning [E. coli competent cells  as recommended by the supplier. The transformed cells were incubated in SOC medium for 60 minutes at 37°C prior to plating on LB agar medium supplied with 50 μg/ml kanamycin and 5% (w/v) sucrose. The clones from overnight incubation at 37°C were screened for the target inserts by colony PCR using RedTaq polymerase  and pNIC-CH forward and reverse sequencing primers .The AMOR_GH9A and  cloning  to the pmers see . PositivTfCel9A expression strains were established through transformation of the expression plasmids to competent E. coli BL-21 StarTM (DE3) cells  according to the supplier’s protocol. The transformed cells were incubated in LB medium at 37°C for 1 hour prior to plating on LB agar medium with 50 μg/ml kanamycin, followed by overnight cultivation at the same temperature. The resulting clones were transferred to 500 ml of Terrific Broth (TB) medium with 50 μg/ml kanamycin and cultivated for 24 hours in a Harbinger system  at 23°C. The cultures were then induced by adding ITPG to a final concentration of 1 mM, and incubated for another 24h at 23 oC. The cells were harvested by centrifugation at 5000 x g for 15 minutes at 4°C, using a Beckman Coulter centrifuge  and resuspended in 50 ml 50 mM Tris-HCl buffer pH 8.0 containing 500 mM NaCl and 5 mM imidazole. The cell suspensions were subjected to sonication on ice using a Vibracell sonicator  with 5 seconds on/off pulses for 3 minutes at 30% amplitude. The debris was removed by centrifugation at 15,000 x g for 15 minutes at 4°C and the supernatant was filtered through a 0.22 μm syringe filter , yielding sterile cell-free extracts, which were stored at 4°C prior to enzyme purification.AMOR_GH9A and TfCel9A proteins were isolated from cell-free extracts using metal affinity chromatography on a Ni2+ affinity HisTrapTM HP 5 ml column . The enzymes were eluted with a linear gradient of imidazole (5–500 mM) in 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl. Chromatography fractions were analyzed by SDS-PAGE . Fractions containing purified enzymes were pooled and concentrated using 10,000 MWCO Vivaspin ultrafiltration tubes , with concomitant buffer exchange to 50 mM Tris-HCl, pH 8.0, containing 200 mM NaCl. The enzyme concentrations were determined by measuring optical absorbance at 280 nm with a Biophotometer UV-VIS spectrophotometer , using theoretical extinction coefficients (web.expasy.org/protparam). The protein stock solutions were stored at 4°C.AMOR_GH9A and TfCel9A with 1% (w/v) carboxymethyl cellulose (CMC) for 6 minutes at temperatures ranging from 20°C to 100°C in citrate-phosphate buffer pH 5.7 (AMOR_GH9A) or pH 6.2 (TfCel9A). The pH optima were determined by carrying out the same reactions in various citrate-phosphate (pH 3.0–7.6) and glycine-NaOH buffers (pH 9.3–10.8) at 98°C (AMOR_GH9A) or 65°C (TfCel9A). The pH of the buffer solutions was set at room temperature. The experiments were conducted in a thermomixer  at 600 rpm. The cellulase activity was determined by measuring the release of reducing sugars using the 3,5-dinitrosalicylic acid (DNS) reagent [The temperature optima of the enzymes were assessed by incubation of 1μM AMOR_GH9A or 2 μM  reagent  and glucThe effect of salt on the performance of AMOR_GH9A was determined by incubation of 1 μM enzyme and 1% (w/v) CMC in citrate-phosphate buffer pH 5.7 at 98°C with 0, 100, 500, 1000 or 2000 mM NaCl. Product formation was analyzed using the DNS assay, as described above.TfCel9A was assessed by measuring the residual activity of the enzymes on 1% (w/v) CMC after up to 24 hours of pre-incubation in citrate-phosphate buffer pH 5.7 (AMOR_GH9A) or pH 6.2 (TfCel9A). The pre-incubation was performed at 98, 90, 85, and 80°C (AMOR_GH9A) or at 65, 60, 55 and 50°C (TfCel9A). The reactions with CMC were carried out for 6 minutes at 98°C (AMOR_GH9A) or 65°C (TfCel9A) and product formation was quantified with the DNS assay as described above.The thermal stability of AMOR_GH9A and TfCel9A. The protein solutions were desalted using MiniTraptm G-25 gel filtration columns and citrate-phosphate running buffers with pH 5.7 (AMOR_GH9A) or pH 6.2 (TfCel9A). These running buffers were utilized as reference samples in the subsequent calorimetry experiments. The protein solutions  and the reference solutions were filtered through a 0.22 μm syringe filter  and degassed for 5 minutes using a ThermoVac system  prior to data collection. The calorimetry was carried out in a pressurized chamber (4 atm) at 20–130°C temperature range with 1°C/min scan rate. The data were processed using the NanoAnalyze software provided by TA Instruments . The buffer baselines were subtracted from the enzyme melting curves.Differential scanning calorimetry  was used to determine the apparent melting temperatures of AMOR_GH9A and TfCel9A) with 1 μM enzyme. The substrate concentrations were 1% (w/v) for Avicel and 0.5% (w/v) for the other substrates. Aliquots were taken at various time points and the reactions were stopped by addition of NaOH to 100 mM final concentration. The products were quantified using the DNS assay and glucose standards.Avicel PH-101  was selected as a model crystalline substrate in this study. Phosphoric-acid swollen cellulose (PASC) was prepared from Avicel as described in . BeechwoDegradation products from cellulose and xylan were analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The cellooligosaccharides were separated using a Dionex ICS3000 system  equipped with a CarboPac PA1 2 × 250 mm analytical column. A stepwise gradient with an increasing amount of eluent B  was applied starting right after sample injection, as follows: 0–10% B over 10 min, 10–30% B over 25 min, 30–100% B over 5 min, 100–0% B over 1 min, 0% B over 9 min. Data analysis was performed using Chromeleon 7.0 software. Cellooligosaccharide standards with a degree of polymerization of one to five (DP1—DP5) and xylo-oligosaccharide standards with a degree of polymerization of one to six (DP1 –DP6) were purchased from Megazyme  and used to identify the products.The products of xylan degradation were identified using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) UltrafleXtreme mass spectrometer  equipped with a Nitrogen 337-nm laser. 1 μl of reaction mixture was added to 2 μl of 9 mg/ml 2,5-dihydrooxybenzoic acid (DHB) solution on a MTP 384 ground steel target plate . After air-drying, spectral data was acquired and processed using Bruker flexControl and flexAnalysis software.After sequencing and assembly of metagenomic data, a 2065 bp gene encoding a putative GH9 cellulase was identified using the dbCAN annotation tool. The candidate enzyme was named AMOR_GH9A. According to the Pfam domain classification server , AMOR_GHArdenticatena maritima as having the highest degree of sequence similarity to AMOR_GH9A . Ardenticatena maritima was isolated from a hydrothermal field sediment and can grow at temperatures as high as 75° C [TfCel9A from the cellulolytic model actinomycete Thermobifida fusca . TfCel9A is a well-known thermostable GH9 cellulase with a complex domain architecture and an endo-processive mode of action [TfCel9A consists of a signal peptide, an N-terminal catalytic domain, two cellulose binding modules and a fibronectin type III domain  and belongs to subfamily CBM3c [Of note, both Pfam and dbCAN did not predict the domain boundaries of the CBM3 in AMOR_GH9A correctly, recognizing only the N-terminal half of this domain . SequencTfCel9A  and the ly CBM3c , 35.TfCel9A were codon optimized for expression in E. coli, synthesized and then cloned into pNIC-CH vectors using ligation-independent cloning. The enzymes were produced (E. coli BL-21 StarTM (DE3), without signal peptides and with a C-terminal affinity tag for purification by metal affinity chromatography. AMOR_GH9A and TfCel9A were produced in soluble form and the final yield was approximately 90 mg of purified protein per liter of E. coli culture for both enzymes.Genes encoding AMOR_GH9A and produced  in E. coTfCel9A were assessed using carboxymethyl cellulose (CMC) as a model substrate , which can heat up to approximately 70°C due to exothermic reactions [The pronounced difference between the temperature optima of AMOR_GH9A and eactions . In compeactions  with steCaldicellulosiruptor bescii [In a recent review, Escuder-Rodríguez et al.  summarizr bescii  with a roC showed that the activity of AMOR_GH9A was almost insensitive to salt. The highest activity was obtained in reactions without added NaCl. The increase of salt concentration gradually reduced activity but even at 2 M NaCl, the remaining activity was still approximately 85% of the base level (0 M NaCl) (CMC assays at pH 5.7 and 98  M NaCl) . These fTfCel9A was assessed and compared by measuring residual activity on CMC after pre-incubation of the enzymes in citrate-phosphate buffer at optimal pH and various temperatures . Both AMOR_GH9A and TfCel9A displayed irreversible unfolding. The melting curve for AMOR_GH9A showed a single peak at approximately 105°C while TfCel9A demonstrated a two-phase transition with peaks around 65°C and 78°C  and retains 64% of its activity after 4 hours of incubation at 95°C. Direct functional comparison with its closest characterized homolog (TfCel9A from the model thermophilic bacteria Thermobifida fusca) revealed that AMOR_GH9A possesses broader substrate specificity and higher activity on soluble and amorphous substrates . Thus, the novel GH9 cellulase demonstrates a set of industrially relevant properties and has the potential to become part of the enzymatic toolbox for biomass conversion.S1 TableVector complimentary sequences are underlined.(PDF)Click here for additional data file.S1 FigTfCel9A were produced and purified exactly in the same way.The predicted MW of AMOR_GH9A and TfCel9A is 73.2 kDa and 91.4 kDa respectively. The sample loading was 5μg for each protein. Note that there is some heterogeneity in the AMOR_GH9A band. We were not able to remove this heterogeneity by additional purification steps or extended boiling of the samples. Note that AMOR_GH9A and (TIF)Click here for additional data file.S2 FigThe graph shows product formation after a 6 min reaction with CMC at 98°C in citrate-phosphate buffer, pH 5.7, supplied with NaCl at different concentrations. Error bars indicate standard deviations between triplicates.(TIF)Click here for additional data file.


O-glucoside (Q3G), a glycosylated derivative of quercetin (Q), is a natural polyphenolic compound known to possess antioxidant activity. The hydrophilic/lipophilic nature of an antioxidant molecule is considered as an important factor governing the accessibility to the active sites of oxidative damages in vivo. Six long chain fatty acid esters of Q3G were evaluated with comparison to Q and Q3G, for their cytoprotective activity under H2O2-induced oxidative stress using cell culture model systems through cell viability, lipid peroxidation and fluorescence microscopy studies. Pre-incubation of α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) esters of Q3G exhibited significantly (p ≤ 0.05) greater cell viability in both human lung fibroblast (WI-38) and human primary hepatocytes upon exposure to H2O2 insult when compared to the control. Cytoprotection due to oleic acid and linoleic acid esters of Q3G was observed only in human primary hepatocytes. All the derivatives, Q3G and quercetin showed ability to significantly (p ≤ 0.05) lower production of lipid hydroperoxides under induced oxidative stress, compared to the control. However, ALA and DHA esters of Q3G resulted in significantly lower lipid hydroperoxidation than Q and Q3G. Based on fluorescence microscopy study, H2O2-induced apoptosis was attenuated by the fatty acid derivatives of Q3G. The fatty acid derivatives of Q3G possess better cytoprotective effect than Q3G against H2O2-induced cytotoxicity in vitro and the concentration should be selected to avoid cytotoxicity.Cellular oxidative stress causes detrimental effects to macromolecules, such as lipids, nucleic acids and proteins, leading to many pathological conditions. Quercetin-3- In biological environments, an excess production of partially reduced forms of molecular oxygen and insufficient antioxidant defense system disturbs the cellular redox balance resulting in oxidative stress  which isin vitro and in vivo  at 37 °C in a 5% CO® immobilized lipase from Candida antarctica L. (2 g) as the biocatalyst followed by an incubation at 45 °C for approximately 48 h. After the completion of the reaction, the product was isolated by silica gel column chromatography using acetone–toluene, 40:60 to 50:50 elution monitored by preparative thin layer chromatography (TLC).Six fatty acids, namely stearic acid, oleic acid, linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derivatives of Q3G were enzymatically synthesized according to the method described in . Briefly4 cells/100 µL per well and incubated at 37 °C for 24 h to allow for the attachment of cells. The cells were then treated with 100 µL test compounds  in DMSO  and incubated at 37 °C for 48 h. The cells in control group contained equal volume of DMSO without adding any test compounds and were not subjected to oxidative injury. The cells in model group contained equal volume of DMSO, no addition of test compounds, but were oxidatively injured by H2O2 addition. Exposure time and concentration of H2O2 were optimized . Then, media was removed and cells were rinsed with PBS to remove the oxidant and 100 µL of fresh serum free media was added into the wells. CellTitier 96® aqueous non-radioactive cell proliferation assay  was used to determine the cell viability. The absorbance was measured at 490 nm using a plate reader . Cells were cultured in 96-well plates at a density of 1 × 10ptimized . After i4 cells/100 µL per well and incubated at 37 °C for 24 h. Cells were treated with 100 µL of the test compounds  and incubated at 37 °C for 48 h. Then, the culture medium was removed and cells were washed with PBS twice. Cell viability was determined as described before.WI-38 cells were cultured in 96-well plates at a density of 1 × 104 cells/100 µL per well and incubated at 37 °C for 24 h. Then, cells were treated with 100 µL of the fatty acids separately: stearic acid, oleic acid, linoleic acid, ALA, EPA, DHA in 0.01, 0.1, 1, 10, 100, and 200 µM concentrations and incubated at 37 °C for 48 h. Cell viability was determined as described before.WI-38 cells were cultured in 96-well plates at a density of 1 × 103 cells/100 µL per well and incubated at 37 °C for 24 h. According to the result from the MTS assay, 0.1 and 1 µM concentrations were selected for the test compounds. After treating the cells with 100 µL test compounds, they were incubated at 37 °C for 48 h. The cells in the control group contained equal volume of DMSO  without adding any test compounds and were not oxidatively injured. The cells in the model group contained equal volume of DMSO, no addition of test compounds, but subjected to oxidative injury by H2O2 addition. Then the culture medium was removed and cells were washed with PBS twice. Cells were subjected to H2O2 insult of 800 µM for 3 h. After incubation, the plate was centrifuged at 1000 rpm for 2 min and 50 µL of the supernatant was added into a new 96-well plate. The released LDH was assayed using CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega Corporation) according to the manufacturer’s protocol. Absorbance was measured at 490 nm using a plate reader (FLUOstar OPTIMA). After subtracting the absorbance of blank and control wells, from all the model wells and test wells, cytoprotection was calculated as below: WI-38 cells were cultured in 96-well plates at a density of 1 × 105 cells/mL per well and incubated at 37 °C for 24 h. Then, the cells were treated with 1 µM of test compounds for 48 h at 37 °C. After incubation, the cells were washed with PBS to remove the test compounds and incubated with 800 µM H2O2 for 3 h at 37 °C. At the end of the oxidative injury period, supernatant (200 µL) of each well was taken into glass vials and lipid hydroperoxides were extracted by adding the equal volume of Extract R saturated methanol and 1 mL of cold chloroform followed by centrifuging the pre-vortexed mixture at 1500× g for 5 min at 0 °C. The bottom chloroform layer (500 µL) was collected to a glass vial and stored on ice. Methanol and chloroform was deoxygenated prior to use by bubbling nitrogen through the solvents for about 30 min. Deoxygenated chloroform–methanol (2:1) mixture (450 µL) was added into each extracted sample of 500 µL. Then freshly prepared standard chromogen (50 µL) was added into the assay vials and vortexed. The assay vials were closed tightly and kept at room temperature for 5 min. A volume of 300 µL from each vial was transferred into a glass 96-well plate and absorbance was read at 490 nm using a plate reader (FLUOstar OPTIMA). Lipid hydroperoxides in the samples were calculated using the equation obtained from the linear regression of the standard curve prepared using 0, 0.5, 1.0, 2.0, 3.0 and 4.0 nmol of lipid hydroperoxide standards.Lipid peroxidation was measured using a lipid hydroperoxide assay kit . WI-38 cells were cultured in 24-well plates at a density of 1 × 105 cells/ml per well and incubated at 37 °C for 24 h. Cells were treated with 1 µM of test compounds for 48 h at 37 °C. After incubation, the cells were washed with PBS to remove the test compounds and treated with 800 µM H2O2 for 3 h at 37 °C. The cells were carefully washed with PBS twice to remove the oxidants. The dual detection reagents; apoptosis detection reagent (Annexin V-EnzoGold) and necrosis detection reagent which is similar to the red emitting dye 7-AAD, supplied by GFP-certified™ apoptosis/necrosis detection kit  were prepared in binding buffer (1×). After careful removal of the supernatant, the prepared detection reagent was dispensed in a volume sufficient for covering the cell monolayer in each slide. The cells were incubated for 15 min at room temperature, protected from the light. Then, the staining solution was flicked onto a paper towel and few drops of binding buffer were added to prevent the cells from drying out. The cells were then observed under fluorescence microscope  with a filter sets for Cyanine-3 (Ex/Em: 550/570 nm) and 7-AAD (Ex/Em: 546/647 nm), coupled with digital camera . Number of affected cells were also counted for quantification of apoptosis and late apoptosis/necrosis.WI-38 cells were cultured in chamber slides  at a density of 1 × 105 cells/1 ml per well and incubated at 37 °C for 24 h. The cells were treated with 1 µM of test compounds for 48 h at 37 °C. After incubation, the cells were washed with PBS to remove the test compounds and treated with 800 µM H2O2 for 3 h at 37 °C. Morphology of the cells was examined under inverted microscope  using phase contrast optics under 10× magnification. The images were recorded using the Lumenara Infinity camera  including capture and analyzing software .WI-38 cells were cultured in 24-well plates at a density of 1 × 10p ≤ 0.05 was considered as statistically significance. For the statistical determination of the assays conducted for cytoprotection effects and cytotoxic effects using MTS assay, compounds were analyzed individually to understand their concentration effect. For the LDH assay, the series of test compounds was analyzed statistically under two concentration levels separately. The statistical determination for the lipid peroxidation assay was performed to determine the significance effect of the different test compounds at one concentration level.The experiments done with MTS assay were carried out in six replicates and all others in three replicates. The results were expressed as mean ± standard error mean (SEM). Statistical analysis was performed using one way ANOVA in Minitab 16 statistical software and multiple mean comparison was carried out by Tukey’s method. 2O2-induced cytotoxicity was attenuated by ALA, EPA and DHA acid esters of Q3G in human primary hepatocytes and lung fibroblasts as demonstrated by significantly higher % cell viability (p ≤ 0.05) under the experimental condition of H2O2-induced oxidative injury in cell culture model systems. Oleic acid and linoleic acid esters of Q3G exhibited significant (p ≤ 0.05) cytoprotection only in primary hepatocytes. ALA and DHA esters of Q3G exhibited significantly (p ≤ 0.05) lower production of lipid hydroperoxides under oxidative stress conditions. Low apoptotic cell death was demonstrated with the pre-incubation of cells with the novel ester compounds. Therefore, fatty acid esters of Q3G can be considered as potential cytoprotective agents under oxidative stress.In conclusion, H


Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.The phylum  Aquificae is a deeply branching bacterial phylum that contains chemolithoautotrophic thermophilic to hyperthermophilic bacteria. The phylum Aquificae consists of a single order, Aquificales, and three families, Aquificaceae, Hydrogenothermaceae, and Desulfurobacteriaceae, as well as Thermosulfidibacter takaii, a member of uncertain taxonomic affiliation within the phylum  and Thermocrinis albus DSM 14484T (WP_012991466) contain nifH genes encoding nitrogenase reductase, a key enzyme for biological nitrogen fixation thermophilic microbial communities  and small amounts of nitrogen compounds , as previously described  and streamers  were collected on March 29 and May 7, 2017 at [36°23′20″N 137°44′52″E]  and  (Stream Site), respectively, in Nakabusa hot springs, Japan. This hot spring water is slightly alkaline (pH 8.0 to 8.9) and contains sulfide 2SO4 with Na2S2O3 (1 g L−1). CO2 gas was used as the sole carbon source in the medium. A total of 0.75 mL of a modified trace mineral solution was used per L medium (2-EDTA·2H2O and 0.1 g Na2SeO4 (L−1), while nitrilotriacetic acid, H2SeO3, and KAl (SO4)2·12H2O were omitted. The pH of the medium was adjusted to 7.0 using HCl prior to autoclaving. Twenty milliliters of the medium was placed into a 120-mL glass vial. The vial was sealed with a butyl rubber stopper and aluminum cap, and autoclaved after the gas phase was replaced with N2:CO2 . After autoclaving, 15–30% vol. H2 gas and 1–10% vol. O2 gas were aseptically added to the gas phase (described in detail below).Nitrogen compound-free (N-free) modified TK-6 medium , 11 was L medium , and the2 gas. One milliliter of each culture was subcultured 4 times every 1–2 weeks into fresh N-free modified TK-6 medium for the enrichment of nitrogen-fixing bacteria under two conditions of 1 and 5% vol. O2 as well as 30% vol. of H2 gas in the headspace of the vials.Microbial mats and streamers were homogenized using an aseptic glass homogenizer. Two hundred microliters of the homogenized samples were inoculated into 20 mL of N-free modified TK-6 medium and incubated at 70°C under static conditions in the presence of 30% vol. of H2 concentrations  in the gas phase were applied for appropriate conditions to obtain visible colonies (Solid medium of N-free modified TK-6 medium containing 0.8% (w/v) gellan gum  was prepared in sealed vials as described above. A diluted enrichment culture was injected into the vials before cooling and then solidified. Different Ocolonies  for isol2:CO2:H2  with 5% vol. O2. One hundred microliters of the pre-culture solutions in the stationary phase of growth ([2.9 to 3.9]×107 cells mL−1) were inoculated into 20 mL of N-free modified TK-6 medium in 70-mL vials under a gas phase of either N2:CO2:H2  or Ar:CO2:H2  with 1, 5, or 10% vol. O2. Vials were incubated at 70°C and shaken by hand once a day for 5 s. When indicated, 2 mmol L−1 of NH4Cl was added to the medium. Bacterial growth was assessed by cell counting using a phase-contrast microscope  and a counting chamber . The experiment was conducted in triplicate.Bacterial isolates were pre-cultured in N-free modified TK-6 medium in 70-mL vials. The gas phase consisted of Net al.  as described previously , which is a 4Fe-4S iron sulfur cluster ligating cysteine, and were used to construct a phylogenetic tree as previously reported  and chloroform phenol extraction, as described by Noll et al. . The 16S2/1492R2 , 21 and olF/PolR  under theviously , 29. Purin MEGA7 . The dedreported . Phylogereported  and the reported , respectNitrogenase activity was measured using the acetylene reduction method , 39. The2:CO2  gas in the headspace of 20-mL sealed vials. H2 (~25% vol.) and O2 (1 to 10% vol.) were added to the headspace prior to the inoculation. After incubation at room temperature overnight to solidify the medium, vials were incubated at 70°C for 3 d with the addition of 15% vol. of 99.99% acetylene gas . This experiment was conducted in duplicate.The nitrogen-fixing activities of the isolates in solid media were tested as follows: 300 μL of culture solutions were injected into 8 mL of N-free modified TK-6 medium containing 0.8% gellan gum and N2:H2:CO2  and the addition of 5% vol. O2. Two milliliters of the cultures were placed into a 7-mL sealed vial. The headspace was replaced with Ar:H2:CO2 . NH4Cl (2 mmol L−1) was added when indicated.To assess acetylene reduction in liquid medium, bacteria were cultivated for 2 d in N-free modified TK-6 medium under NAfter 10-min pre-incubation at 70°C in a water bath, 15% vol. of 99.99% acetylene gas (Tatsuoka) was injected into the headspace. After 3-h incubation at 70°C, 200 μL of 37% formaldehyde was added to stop the reaction producing ethylene. Examination under each condition was conducted in triplicate.A negative control without bacteria was prepared to estimate any ethylene background generated from non-biological sources.2:H2:CO2  or N2:CO2  gas phase with the addition of 5% vol. O2. Two milliliters of each culture was placed into a 7-mL sealed vial. The headspace was replaced with Ar:H2:CO2  with O2 (5% vol.) or Ar:CO2 , respectively. A total of 20 mmol L−1 of sodium molybdate was added when indicated. Sample preparation for the acetylene reduction assay was the same as that described above.Bacteria were cultivated for 2 d in liquid N-free modified TK-6 medium under the NnifH gene sequences were deposited in DDBJ/EMBL/GenBank with the accession numbers LC375847 (strain 1–6) and LC375848 (strain 2–18) for the 16S rRNA gene and LC375850 (strain 1–6) and LC375849 (strain 2–18) for the nifH gene.16S rRNA and 2:CO2:H2:O2 atmosphere. After repetitive cultivation, bacterial growth was observed in all cultures containing both 1% and 5% vol. O2 in the headspace. The cultures were transferred into solid media under 1 to 10% vol. O2 and visible colonies were obtained in all cultures. Twenty-seven colonies were selected, purified, and used in the acetylene reduction assay tests: 14, 11, 1, and 1 isolates were obtained from the cultures with 1, 5, 8, and 10% vol. O2, respectively (2:CO2:H2:O2 atmosphere (data not shown), indicating that these seven isolates exhibited nitrogen-fixing abilities under the conditions tested. Based on 16S rRNA gene sequence analysis, the seven isolates were classified into two phylotypes; six sequences showed 100% nucleotide sequence identity to one another. The 16S rRNA gene sequences of these six strains were found in all samples; pale-tan microbial mats (four isolates), pale-tan streamers (one isolate), and gray streamers (two isolates). The representative isolate, strain 1–6, obtained from pale-tan mats with 5% vol. O2 in the vial headspace, was used for further analyses. Strain 2–18 was isolated from a gray streamer at 5% vol. O2.After homogenization, pale-tan microbial mats and two types of streamers  were cultivated under diazotrophic conditions in a Nectively . In seveHydrogenobacter and Thermocrinis . The two isolates shared 96.6% NifH sequence identity with each other.Putative Nakabusa , from hoNakabusa , 36. The2:CO2 or N2:H2:CO2 with 5% vol. of O2   .2H4×106 cells−1 h−1 in strain 1–6 and 16.6±7.51 pmol C2H4×106 cells−1 h−1 in strain 2–18 in nitrogen-free medium with 5% vol. of O2. Nitrogenase activity in the presence of 2 mmol L−1 NH4Cl in the acetylene reduction assay was observed in both isolates, but was lower than that in the absence of NH4Cl , showing 7.01±2.23 pmol C2H4×106 cells−1 h−1 in strain 1–6 and 3.37±2.10 pmol C2H4×106 cells−1 h−1 in strain 2–18.The nitrogenase activities of both strains were observed at 70°C, as shown in 2 concentrations for growth under nitrogen-fixing conditions, growth under different initial concentrations of O2 was compared in the presence of both thiosulfate and H2. As shown in 2 than at 1 and 5% vol. O2; however, initial growth for 1.5 d was similar. In contrast, in 2, but was better at 5% vol. O2 than at 1% vol. O2. In the presence of NH4Cl, growth at 10% O2 was better than under the other conditions for both strains    . On the s <0.05) .Aquificae were isolated from Nakabusa hot springs, Japan. The N2-fixing capability of two novel strains in the phylum Aquificae was demonstrated by positive acetylene reduction activities and diazotrophic growth. Genome analyses indicated that two species of bacteria in Aquificae, H. thermophilus TK-6T (WP_012963773) and T. albus DSM 14484T (WP_012991466), possessed putative nifH genes, whereas their active nitrogen-fixing abilities have never been demonstrated. Kawasumi et al. reported that H. thermophilus TK-6 did not grow using nitrogen gas as a sole nitrogen source (H. thermophilus TK-6 (kindly provided by Dr. H. Arai) did not exhibit nitrogen-fixing activity under the same conditions tested in the present study (data not shown). The nitrogen-fixing activities and diazotrophic growth of these novel isolates were observed at 70°C. Prior to the present study, the maximum temperature observed for active nitrogen fixation by bacteria was 63.4°C, as demonstrated in cyanobacteria from hot springs  for their nitrogenase activities, suggesting that nitrogenase activity is associated with anaerobic autotrophic sulfate reduction in microbial communities. In contrast, the isolates showed nitrogenase activity with thiosulfate, which was inhibited by molybdate only under anaerobic (thiosulfate disproportionating) conditions. Therefore, the isolates may not be responsible for the previously measured nitrogenase activity , and the findings indicated the global distribution of nitrogen-fixing Hydrogenobacter in thermal environments   . The dettrations .2 and thiosulfate at 5% vol. O2 . The presence of diazotrophic and autotrophic sulfate-reducing members in the thermophilic communities at Nakabusa hot springs cannot be excluded based on the data obtained, and further studies are needed to answer this question.As shown in our previous study , the anaHydrogenobacter strains appears to require semi-aerobic conditions with lower O2 concentrations than atmospheric O2 concentrations, in addition to reduced sulfur compounds and/or hydrogen as electron sources. Therefore, microbial mats and streamers appear to be appropriate environments for nitrogen fixation, i.e., the low O2 concentration achieved by O2 consumption through aerobic respiration, anaerobic sulfide production, sulfide supply from hot spring water, elemental sulfur production by abiotic or biotic processes, and hydrogen production by fermentative bacteria (Hydrogenobacter species have been shown to widely populate neutral to alkaline environments at a temperature range higher than 70°C, at which nitrogen compounds are limited, and some may be capable of fixing and supplying nitrogen in these communities (Nitrogen fixation in bacteria , 29, 44.munities , 29, 41.