Changes in secondary structure of BSA on interaction with EC and ECG have been identified by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic characterization reveals the binding process to be exothermic, spontaneous and entropy driven. Mixed binding forces  exist between ECG and BSA. Binding site for EC is primarily site-II in sub-domain IIIA of BSA and for ECG; it is site-I in sub-domain IIA. ECG with its high antioxidant activity accompanied by high affinity for BSA could be a model in drug designing.The health benefits stemming from green tea are well known, but the exact mechanism of its biological activity is not elucidated. Epicatechin (EC) and epicatechin gallate (ECG) are two dietary catechins ubiquitously present in green tea. Serum albumins functionally carry these catechins through the circulatory system and eliminate reactive oxygen species (ROS) induced injury. In the present study ECG is observed to have higher antioxidant activity; which is attributed to the presence of galloyl moiety. The binding affinity of these catechins to bovine serum albumin (BSA) will govern the efficacy of their biological activity. EC and ECG bind with BSA with binding constants 1.0×10 Diets rich in antioxidants contribute to lower incidence of several major chronic diseases. In particular, cancer development or growth is inhibited by antioxidants. Antioxidants delay or prevent the oxidation of a given substrate by free radicals. Epicatechin (EC) and epicatechin gallate (ECG) are ubiquitous antioxidants present in green tea. These catechins have been investigated for various applications stemming from their antioxidant properties, like radioprotective properties Interactions of flavonoids and proteins have gained lot of attention recently In the present study interactions of EC and ECG with BSA were explored by fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Binding affinities and the accompanied structural changes during the binding process were measured. The binding site was identified using competition method with warfarin and ibuprofen, whose binding site to BSA is known. Binding mode and thermodynamic characterization of the binding process was done by isothermal microcalorimetry (ITC).BSA, EC, ECG, warfarin, ibuprofen and DPPH  were obtained from Sigma-Aldrich, St. Louis, MO, USA. All the experiments were carried out in 50 mM phosphate buffer of pH 6.7.Scavenging ability of EC and ECG on DPPH radical was measured according to the method reported by Turkmen et al. AA (%) = (control absorbance – sample absorbance)/control absorbance × 10050) values were calculated from data obtained graphically, using mathematical method based on the principle of the right-angled triangle: IE50 = D−[(A−50% maximum response)X]/Y, in which A is the immediately higher response of 50% maximum response; B is the immediately lower response of 50% maximum response; D = log concentration corresponding to A response; C = log concentration corresponding to B response; X = D−C; and Y = A−B The inhibitory concentration 50 (IC−6 M) in 50 mM phosphate buffer of pH 6.7 was followed on excitation at 280 nm.All the fluorescence measurements were made on a Perkin Elmer fluorescence spectrophotometer (LS-55) equipped with 150 W Xenon flash lamp and using fluorescence-free quartz cell of 1 cm path length at room temperature −6 M BSA in 50 mM phosphate buffer of pH 6.7 was titrated with successive addition of catechins EC (1×10−5 M) and ECG (1×10−5 M) separately. The excitation wavelength was 280 nm and the emission spectra were recorded from 290 nm to 450 nm. The intensities of emission maxima were used for the calculation of binding constants using Eq. 1 (vide infra).Solution containing 2.5×10−6 M each) was mixed and kept for 1 hr and titrated with EC (1×10−5 M) and ECG (1×10−5 M) in separate experiments. Similar solution containing equimolar ibuprofen and BSA was prepared and titrated with EC and ECG separately to identify the binding sites of EC and EG with BSA. Titration was followed fluorimetrically using excitation wavelength of 280 nm.To identify the binding sites of EC and ECG in BSA, displacement of warfarin and ibuprofen known to bind at site I and site II, respectively, was followed fluorimetrically. Equimolar solution of BSA and warfarin  equipped with a peltier cuvette holder and temperature controller PFD425 L/15. The BSA concentration and path length of the cuvette used were 1 µM and 0.1 cm, respectively. The instrument parameters were set at scanning speed of 50 nm/min, bandwidth of 1.0 nm and sensitivity of 100 milli degree. CD spectra of BSA in presence of EC and ECG in various D/P ratios were recorded separately. Four scans were averaged and smoothed to improve signal to noise ratio. The molar ellipticity values are expressed in terms of mean residue molar ellipticity, in units of deg. cm−1 with a resolution of 4 cm−1. The background was corrected before scanning the samples and the buffer spectrum collected. FT-IR spectra of free BSA (1×10−4 M), BSA-EC complex (1∶1 and 1∶2) and BSA-ECG complex (1∶1 and 1∶2) were recorded to identify changes in secondary structure in BSA on interaction with EC and ECG. All the experiments were performed at room temperature (25°C).FT-IR measurements were carried out on Perkin Elmer, Spectrum GX equipment using ZnSe window The energetics of the binding of catechins to BSA was studied by isothermal titration calorimetry (ITC) using a VP ITC unit  with calorimeter cell volume of 1.423 ml. All solutions were degassed under vacuum  on the MicroCal’s Thermovac unit to eliminate air bubble formation inside the calorimeter cell. Briefly, the calorimeter syringe was filled with a concentrated solution of EC or ECG (500 µM each). Successive injections of 10 µl of this solution into 50 µM solution of BSA, in the same buffer contained in the calorimeter cell was effected from the rotating syringe that enabled constant stirring of the solution. The data were corrected for the heat of dilution of catechins, which was determined in a separate set of experiments under identical conditions. The titration and analysis were performed through Origin 7 software provided with the unit.50 values for the antioxidant activity of EC and ECG are 475 µM and 120 µM, respectively. From these values it is evident that ECG is more efficient in scavenging the DPPH free radicals than EC, and thus a better antioxidant. The antioxidant activity of ECG is attributed to the galloyl group containing three phenolic groups capable of transferring hydrogen easily to the oxidizing agent. Higher antioxidant activity combined with higher binding efficacy (discussed later) with BSA would be effective in efficient transportation and combating reactive oxygen species (ROS) induced stress.The ICThere are two tryptophans (Trp 134 and Trp 213) in BSA of which Trp 134 is located on the surface and Trp 213 resides in the hydrophobic pocket of the protein molecule −6 M) as shown in −5 M) shows progressive red shift in λmax, evidencing formation of complex between BSA and ECG, leading to change in micro environment of the fluorophore. Such peak shift was not observed for BSA-EC interaction suggesting weaker interactions (0 is the fluorescence intensity of free BSA and F is the consecutive fluorescence on addition of catechins. Plot of log (F0– F)/F against log [Q] was used to determine the values of K and n from the intercept and the slope respectively. The binding constants (K) for BSA-EC and BSA-ECG complexes are 1.0×106 M−1 and 6.6×107 M−1, respectively, and are summarized in Albumins have high affinity for fatty acids, hematin, bilirubin and a broad affinity for small negatively charged aromatic compounds. Serum albumins facilitate the disposition and transport of variety of ligands like quercetin and other flavonoids ractions . Binding−6 M) was quenched upon addition of EC (1×10−5 M) and ECG (1×10−5 M) in separate experiments. The fluorescence emission quenching data were analyzed by the Stern-Volmer equation:0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, KSV is the Stern-Volmer quenching constant and [Q] is the concentration of quencher (here EC or ECG). The Stern-Volmer quenching constant (KSV) values for EC and ECG are 3.7×105 M−1 (R = 0.9951) and 3.8×106 M−1 (R = 0.9973), respectively, (SV for BSA-ECG is good measure of the strong ground state binding compared to EC. The quenching rate constants (Kq) were evaluated using the equation:−1 s−1 and 3.8×1014 M−1 s−1, respectively. The higher KSV and Kq values for the ECG-BSA system signify that ECG quenches BSA through complexation more efficiently than EC.The fluorescence emission intensity of BSA  fluorescence emission intensity by ECG (1×10−5 M) was measured in solutions of varying ionic strength (0.1 M –0.5 M) using NaCl and KSV values were calculated. The KSV value is 3.8×106 M−1 in absence of NaCl and this value decreased with increase in ionic strength to 3.7×105, 3.5×105 and 2.2×105 M−1 for 0.1, 0.3 and 0.5 M NaCl, respectively  fluorescence emission intensity by ECG (1×10−5 M) was followed in presence of 1 M sucrose; known for formation of hydrogen bonds through its hydroxyl groups SV) was measured to be 1.9×105 M−1 which is more than an order less than that observed in absence of sucrose (Studies on catechin-BSA interactions in the presence of sucrose will provide information on the involvement hydrogen bonding process since sucrose has hydroxyl groups which can interfere in the hydrogen bond formation between the drug and protein. Quenching of BSA (2.5×10 sucrose . From th−6 M) in presence of warfarin and ibuprofen (2.5×10−6 M) are shown in max to 363 nm, is observed whereas a slight blue shift to 342 nm is observed for BSA-ibuprofen complex. On addition of ECG to the solution containing BSA and warfarin, no further change in spectral profile  is a sensitive technique to monitor the conformational changes leading to protein unfolding upon interaction with the ligand. The CD spectra for BSA-EC (0–8 µM of EC) and BSA-ECG (0–3 µM of ECG) system are represented in −1 and 1548–1560 cm−1 respectively −4 M) is shown in −1 in free BSA to 1655 cm−1 in case of BSA:EC complex (1∶1) and 1656 cm−1 for BSA:ECG complex (1∶1). Increasing the concentration of EC keeping BSA concentration same (1∶2) did not result in further peak shift. However, in infrared spectrum of BSA-ECG complex (1∶2) further peak shift of amide-I to 1657 cm−1 was observed. In the amide-II region also slight peak shift from 1544 cm−1 to 1543 cm−1 was observed for both (1∶1 and 1∶2) complexes. CD as well as FT-IR results unravel that ECG when bound to BSA brings about changes in secondary structure mainly in the α-helix which are more pronounced than those observed for EC-BSA complex.Fourier transform infrared spectroscopy has long been used as a powerful method for investigating the secondary structures of proteins and their dynamics 4 M−1 and 2.7×105 M−1, respectively, as determined by the following equation:G° is the free energy, R and T are the gas constant and temperature, respectively, K is the binding constant. The value of the stoichiometry of the binding is fixed to one based upon the goodness of the fit to ITC data and fluorescence observations vide supra. From S° and a negative value of ΔG°. The binding process is always spontaneous as evidenced by the negative value of ΔG°. For typical hydrophobic interaction both ΔS° and ΔH° are positive, which are not observed in the present findings. The positive entropy observed accounts for hydrophobic interactions and negative enthalpy may play a role in electrostatic interactions which is also inferred from the decrease in quenching constant with increase in ionic strength. Furthermore, TΔS° contributes significantly to ΔG° signifying that the binding of the catechins EC and ECG is entropy driven.In this work, we have used isothermal titration calorimetry in determining binding affinity, enthalpy, entropy and stoichiometry of binding of catechins with BSA. Representative isothermal titration calorimetric heat profiles for the binding of EC and ECG with BSA at pH 6.7 and 25°C are shown in This paper provides a comparative approach for studying the interaction of bovine serum albumin with dietary flavonoids EC and ECG using different biophysical techniques. It is evident from this work that the ECG which is the galloylated form of EC interacts strongly with BSA. The galloyl moiety has three phenolic and a carboxyl group which contribute substantially to the higher antioxidant activity and higher binding efficiency of ECG to BSA compared to EC. The binding forces are established to be hydrophobic and electrostatic in nature and stabilized with hydrogen bonding. ECG with antioxidant activity comparable to gallic acid, combined with high affinity for blood carriers can be used as a model drug. This report thus has a huge significance in the field of pharmacology and clinical medicine.


CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories.There have been conflicting reports in the literature on association of gene copy number with disease, including In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts  of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations.QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number. Characterization of genetic variants is fundamental in understanding human heterogeneity and susceptibility to disease. Regions where humans differ in diploid DNA dosage are known as copy number variations (CNV) and are an important component of genetic variation. CNVs are believed to encompass more nucleotide content than single nucleotide polymorphisms (SNPs) DEFB4 and the DEFB103 which encode human β-defensins 2 and 3 , respectively. Defensin genes encode small cationic peptides that have antimicrobial activity and have multi-functional activity: they function as chemo-attractants for T-lymphocytes, monocytes and dendritic cells One region showing extensive CNV is found in the 8p23.1 chromosome en bloc, an assay for any point within the defensin cluster can be assumed to measure copy number across the whole region The beta-defensin cluster varies in copy number between 2 and 12 copies per diploid genome with most people having 2–7 copies PRT addresses one of the major drawbacks of QPCR, namely the problem of bias due to different amplification kinetics of test and reference amplicons Unrelated DNA samples from normal healthy individuals from five different demographic population groups, Denmark (n = 174), Czech Republic (n = 21), Ghana (n = 100), Portugal (n = 91), and the United Kingdom (the European Collection of Cell Cultures ECACC (Cat. No.: HRC-1 and HRC-2); (n = 190) were used in the study.All samples were gathered with full ethical consent and appropriate documentation as stipulated by the ethical consent, which normally involved written informed consent. For Portuguese samples, the local ethics committee of Hospital Garcia de Orta, Almada, Portugal, gave its approval for a genetic study using blood samples from local patients and normal volunteers. For Czech samples, the study has been approved by the Ethical commitee of the General Faculty Hospital in Prague. For Ghanaian samples, approval of the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana was given. For Danish samples, they are a standard set of anonymised DNA samples provided for standardisation purposes and quality assurance, and were not required under Danish law to pass the Danish Ethics Approval system. Other samples were from immortalised lymphoblastoid cell lines, which had been derived from B-cell lymphocytes with full ethical consent by the suppliers (ECACC and Coriell Cell Repositories).Genomic DNA was isolated from samples of venous blood, anti-coagulated with EDTA and purified using the QIAamp DNA Blood Mini Kit  according to the manufacturer's protocol and eluted in water. The UK samples were purified using an in-house magnetic bead affinity method and was obtained from ECACC . Czech DNA samples were extracted using a routine “salting out” and ethanol precipitation procedure.DEFB103 gene were designed using the Primer Express software, version 2.0 . The sequences of the primers and probes were as follows: DEFB103 forward primer (5′ CAT AGG GAG CTC TGC CTT ACC A 3′); DEFB103 reverse primer (5′TGC AGA ACA CAC CCA CTC ACT C 3′) and DEFB103 probe (5′ FAM - TGG GTT CCT AAT TAA C – MGB 3′). The sequence for RNaseP is not known since it is a commercial available kit . The amplification efficiencies of the target genes and the reference genes were tested to be approximately equal not varying more than 5% from each other. Optimizing runs were performed to define limiting primer concentrations for the duplex assay. The PCR reactions (20 µl) were carried out in triplicate with 10–75 ng of template DNA, 1 x Brilliant II QPCR master mix , 400 nM of DEFB103 probe, 600 nM DEFB103 primers and 1 x RNaseP primer mix. Each plate included triplicate wells of “no template control” and 4 control samples. QPCR was performed using a Stratagene MX3000P machine (Stratagene) using the following conditions 95°C for 10 min followed by 40 cycles of 95°C for 15 sec, and 60°C for 1 min. In all runs samples from Coriell Institute for Medical Research  with known copy number were includedA duplex TaqMan real-time quantitative PCR-based assay was developed for the detection of beta-defensin genomic copy number using RNaseP as reference gene. Primers and probes for amplification of a region near the PRT was performed as described previously HSPD21 on chromosome 21) 3 kb distal to the DEFB4 gene with only two copies per genome were PCR amplified. The resulting PCR amplicons differed at 10 positions with an 8 bp (BLAST) difference in length. One of the positions where the amplicons differed was used to quantify the two chromosome regions against each other by pyrosequencing across it. Primers for the pyrosequencing assay were designed using the PSQ assay design software version 1.0.6 . The following sequence was analysed with the position that varied between chromosome 8 and 21 marked with red: KATGCYAT (5′-GAGGTCACTGTGATCAAAGAT-3′) and a reverse primer (5′-Biotin- AACCTTCAGCACAGCTACTC-3′) was used was used together with Q-solution (Qiagen), 10 mM dNTP and Tempase polymerase. PCR was performed on a thermocycler using the following conditions: 15 min at 95°C followed by 35 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 45 s and one extension step at 72°C for 10 min. The biotin-labeled PCR products were immobilized on streptavidin Sepharose  by mixing 40 µl of the PCR product with 3 µl streptavidin Sepharose suspension, 40 µl water, and 37 µl 1 x binding buffer (Qiagen). The suspension was shaken at room temperature for 10 minutes. To remove unbiotinylated DNA the samples were sequentially washed in 70% ethanol for 5 seconds, denaturated in 0.5 M NaOH for 5 seconds and additional washing in 1x washing buffer (Qiagen) for 5 seconds. This was done using the PyroMark Vacuum Prep Tool (Qiagen). After the last wash the ssDNA biotinylated DNA was transferred to 39 µl 1 x annealing buffer (Qiagen) and 1 µl sequencing primer (5′-AGGTCACTGTGATCAAAGAT-3′). The suspension was heated to 80°C for 2 minutes and was equilibrated to room temperature for 5 minutes in order to let the sequencing primer anneal. The sequencing reaction was performed using the Pyro Gold Reagent Kit (Qiagen) in the PSQ 96 MA Pyrosequencer (Qiagen) according to the manufacturer's instructions. The relative percentages of the two variants were calculated by the accompanying software and were used for the gene copy number determination. Positive controls with known copy number in each run were used to generate a correction curve by linear regression, and corrected copy number estimates calculated for each sample calculated using this run-specific regression equation. A “no template control” was included in each run.PRT was carried out essentially as described previously KATGCYAT . For theStatistical analysis was performed using GraphPad InStat 3  and Microsoft Excel.DEFB4 co-amplifies a “reference” region on chromosome 21. Pyrosequencing of the product allows quantification of a particular sequence variant that reports copy number by distinguishing test from reference amplicons.We have developed a version of the PRT method that uses pyrosequencing and quantification of different sequence variants to distinguish test and reference amplicons . A pyrosequencing approach to determining beta-defensin copy number has been described previously We determined beta-defensin gene copy number using PPRT and two other independent methods on DNA samples from 576 individuals from five populations. Comparison of copy number estimates for 14 samples measured using several different methods shows that triplex PRT and MLPA give exactly the same copy numbers as the previously published PRT method . PPRT peBoth PPRT and triplex PRT give a copy number distribution of between 2 and 6 copies, with a few samples showing a higher copy number. However, the QPCR assay gives a considerably broader distribution, with more samples showing apparently higher copy number, and a significant number of samples showing copy number higher than 10. This is reflected in the Bland-Altman plots comparing the raw copy number estimates between the different methods , and theWe took the raw copy number data and divided, according to cohort, by separating the Ghanaian and Portuguese cohorts from the UK, Danish and Czech cohorts. Plotting this raw data as a histogram shows that DNA cohort origin is clearly responsible for systematic copy number calling bias in QPCR between “poor quality” DNA from the Portuguese and Ghanaian cohorts and “good quality” DNA from the UK, Danish and Czech cohorts  but had We therefore reasoned that systematic difference in DNA quality between the DNA sample cohorts may be responsible for this effect. Cukier and colleagues recently showed that degraded DNA could give spurious CNV findings despite the presence of multiplexed internal samples in QPCR assays a priori determination of the applicability of each sample for copy number calling by QPCR. Furthermore, it is unclear whether internal controls constructs would remove this effect, given that it is a property of the genomic DNA itself rather than any effect of aberrant normalisation.This raises questions about the applicability of QPCR in measuring DNA copy number, particularly as we cannot, as yet, identify the factor that is responsible for this effect, ruling out Taking the triplex PRT data as representing the correct copy number of the samples typed, we investigated whether there was any significant difference between copy number distributions in the five different populations. There was no significant difference between the means , reflecting essentially no difference between populations within Europe or between European and Ghanaian populations . This isDifferences in beta-defensin copy number may have important clinical consequences in the susceptibility to, and progression of, a variety of diseases with an inflammatory or infectious etiology. Accurate and precise methods for measuring copy number are essential when investigating subtle changes in copy number distribution between patients and healthy controls, which may reflect an effect of beta-defensin copy number on susceptibility to the disease under study. In summary, we show that PPRT is a practical high-throughput approach, although we would recommend multiplicate PPRTs per sample are required for sufficient accuracy for case-control analyses, and careful quality-control performed for every experiment. PPRT is cost-effective, with a single test priced around €0.75, compared with around €1.2 for a triplex PRT. We hope that with an increasing focus on reliable methods to type beta-defensin copy number variation we will be in a position to investigate the role of this complex locus in disease.


G protein-coupled receptors (GPCRs) are a superfamily of integral membrane proteins vital for signaling and are important targets for pharmaceutical intervention in humans. Previously, we identified a group of ten amino acid positions , within the seven transmembrane domain (7TM) interhelical region, which had high mutual information with each other and many other positions in the 7TM. Here, we estimated the evolutionary selection pressure at those key positions. We found that the key positions of receptors for small molecule natural ligands were under strong negative selection. Receptors naturally activated by lipids had weaker negative selection in general when compared to small molecule-activated receptors. Selection pressure varied widely in peptide-activated receptors. We used this observation to predict that a subgroup of orphan GPCRs not under strong selection may not possess a natural small-molecule ligand. In the subgroup of MRGX1-type GPCRs, we identified a key position, along with two non-key positions, under statistically significant positive selection. G protein-coupled receptors (GPCRs) constitute a diverse superfamily of integral membrane proteins involved in intercellular signal transduction. Their genes are expressed in almost all eukaryotes New protein functions in paralogous protein superfamilies arise by the modulation of older existing ones dS  is not subject to selective pressure on the expressed protein. Nonsynonymous mutations alter the amino acid sequence and their substitution rate dN  is a function of selective pressure on the protein. The ratio dN/dS,, referred to as ω, gives a measure of the selection pressure at that site Here, we examine the nucleotide sequences corresponding to these GPCRs to probe the evolutionary selection pressure at these key positions. Synonymous nucleotide substitutions (‘silent’ mutations) do not change the translated amino acid sequence so their substitution rate All subgroups of human GPCRs were classified into three categories in terms of their natural ligands: 1) small molecules , 2) lipids, and 3) peptides. GPCR subgroups whose natural ligands could not be exclusively classified as any of the above were categorized as divergent. A number of human GPCRs are orphans with no known natural ligands. The list of GPCR subgroups and the chemical class of associated natural ligands is in key, is shown in key and its average, <ωkey>, of subgroups associated with small molecules differed from that of subgroups associated with lipids and peptides. The Kruskal-Wallis rank sum test showed that <ωkey> for small molecule-activated receptors had significantly lower values compared to subgroups of lipid-activated receptors, peptide-activated receptors and divergent receptors (p<0.003). The ωkey values from all ten subgroups activated by small molecules showed strong negative selection (ω<0.05).The ω values were determined for subgroups with at least three paralogs. Selection pressure at the key positions, ωWe confirmed that human MRGX1-type receptors are under positive selection key> to random sets of 7TM positions <ωrandom7TM> to see if there was stronger selection pressure at the key positions. The values are shown in key> was less than <ωrandom7TM> although within two standard deviations of <ωrandom7TM>. The selection pressure for subgroup 42 was atypical in that <ωkey> was larger than <ωrandom7TM> by two standard deviations. For six of nine subgroups associated with lipid-activated receptors, <ωkey> was nearly equal to <ωrandom7TM>. In subgroups activated by peptides, <ωkey> was less than or nearly equal to <ωrandom7TM>. Subgroup 38, which exhibits strong positive selection, was the only other case where <ωkey> exceeded <ωrandom7TM> by two standard deviations. Linear regression of <ωkey> vs. <ωrandom7TM> for the subgroups excluding subgroup 38 and 44, showed a linear dependence   and <ωrandom7TM> (p<4.9×10−3). The dependence remained significant even after including subgroup 38. The Yang and Swanson's “fixed sites” model key> was significantly lower than <ωrandom7TM> in two of the ten small molecule subgroups (subgroups 3 and 10). Subgroup 11, which consists of lipid-activated receptors, showed statistically significant differences between key and random positions. In 5 of the 19 subgroups of the peptide receptors, key positions have significantly higher selection pressure then random positions. Only subgroup 22 of the peptide-activated receptors was significantly lower. The results are summarized in We next compared <ω−16) See . Howeverkey values in subgroups was due to the dissimilarity among amino acid (AA) residues at a given MSA position since it is expected that stronger selection pressure should result in lower variability. However, the strength of the correlation between ωkey and variability was not known. We examined this with three different measures. First, we computed the Shannon entropy (H) for the key positions of each subgroup, which has a theoretical range of 0 bits≤H≤4.32 bits. H for every key position across all subgroups. key> for subgroups with average pair-wise max(dN)<1 . However, the regression of <Hkey> against log10 <ωkey> had much lower correlation when <ωkey> was restricted to <ωkey> <0.1 . However, this decrease in correlation could be due to the decrease in statistical power because the sample size is reduced. Similar results were found using the BLOSUM80 substitution matrix key to estimate the dissimilarity among residues within subgroups at key positions. key> and the correlation is weaker for subgroups under strong negative selection.We also tested if the diversity of ωN)<1 see . This fiWe have found that class A GPCR subgroups that are naturally activated by small molecules possessed strong negative selection in the key positions. Additionally, the selection pressure at the key positions is more likely to be stronger than the rest of the TM positions in small molecule receptors. The existence of strong negative selection supports coevolution over evolutionary drift as an explanation for the high mutual information between the key positions. We suggest that collective substitutions of key residues under strong selection pressure may have altered function in GPCRs. It has been shown previously that evolutionary characteristics such as phylogeny and sequence similarity of AA residues are a strong predictor of determinants of ligand specificity key≈0.05) and max(ωrandom7TM≈0.1)). We thus predict that receptor subgroups with ω>0.1 at the key positions do not possess a natural small molecule ligand. This would include orphan receptors MAS1L, MRGPRF of group 41, MRGPRX3, MRGPRX4 of 38 and 44, and TAAR5, TAAR6, TARR8, and TAAR9 of 42. The inclusion of subgroup 42 may be considered to be surprising because TAAR1 of the group binds β-phenylethylamine and p-tryamine, which is a small molecule trace amine. Although this subgroup exhibits negative selection in conformation of recent studies involving TAAR orthologs Under the rules of formal logic, the observation that small molecule receptors are always under strong negative selection at key positions allows for the prediction that GPCRs not under strong negative selection pressure are not naturally activated by small molecules. Based on our results from Positive selection can lead to adaptation of a previous function We examined entropy and measures of sequence similarity to test the hypothesis that strong selection pressure is related to low variability. Our results showed that even under strong negative selection pressure, sequence diversity remained. The wide diversity in selection pressure for receptors associated with the different classes of natural ligands was not attributable to the size of the subgroup. Diversity of ω values is well documented key and ωrandom7TM changed with subgroup size. Subgroup 13b contained a pseudogene GPR42. Studies of class A GPCR orthologs have been previously investigated using opsins, MAS-related receptors, P2Y receptors and melanocortin receptors The inclusion of orthologs would improve the accuracy of our analysis. We used three overlapping subgroups: 13b (overlapping with 13), 43 (overlapping with 37) and 44 (overlapping with 38 and 41) to probe how ωAn alignment of human non-olfactory class A 7TMs was obtained from Nucleotide sequence fragments that encoded the GPCR 7TMs were obtained from NCBI's nucleotide database dN and dS selection criteria described below. A consensus phylogenetic tree was obtained using the CONSENSE program of PHYLIP. A list of GPCRs for all subgroups is shown in We used AA sequence fragments for the 7TMs of class A GPCRs to reconstruct a nearest neighbor phylogenetic tree. Program PROTDIST of PHYLIP dN and maximum dS estimated from all pairs of receptors within subgroups unlike in a traditional analysis where subgroups may be clearly identified as distinct clades from a familial phylogenetic tree. We used the counting scheme of Nei-Gojobori to estimate the average dN and dS from pairs of sequences dN of all pair-wise comparisons within the subgroup did not exceed 1. If the condition of max(dN)<1 was not met, then the out group taxa was removed, and the subgroup reduced. There was no a priori scheme to identify subgroups to achieve the max(dN) and max(dS) conditions. To study the measurement uncertainties due to sample size, we analyzed subgroups having progressively larger numbers of closely related receptors. The subgroups in which it exceeded 1 were indicated by “N” in dN)<1 selection resulted in max(dS)<3 for forty of forty-five subgroups. Subgroups listed in dS)<3. The dN and dS obtained after maximum likelihood computation was more conservative compared to that obtained via the Nei-Gojobori counting method (results not shown).We analyzed forty-five subgroups, of which forty-two were non-overlapping and distinct. The number of constituent GPCRs in respective subgroups ranged from three to ten. Because GPCRs are highly divergent, we restricted the average maximum 0 (ω0<1), a free parameter determined from data, that was common for most 7TM positions. In addition, to represent neutral evolution, a portion of the remaining 7TM positions were constrained to ω1 = 1. Lastly, with another free parameter, the same model also accommodated representation of positive selection for the remaining fraction of positions (ω2>1). In contrast, model M1a was a special case of M2a, in which it excluded positive selection. Because ω for an AA position under near-neutral evolution was also constrained to unity, this was the most conservative of the three strategies. Test 1 compares M1a vs. M2a.PAML version 4.2b p and q). Model M8 represented the spectrum of ω across all MSA positions with ten discrete ωi categories to represent the beta function . An additional eleventh category ω10 accounted for a small fraction of positions under positive selection. In model M7, there was no provision for such positive selection . Test 2 compares M7 vs. M8.In the second strategy the spectrum of ω values from MSA positions was represented by a beta function .In all the three strategies, which we refer to as Tests 1–3 in In a fourth strategy, which we called Test 4, model M3 was compared to model M0 for all subgroups. The alternative model demonstrated the heterogeneity of ω values across the 7TMs and the null model was representative of their common ω value. Test 4 is not specific for inferring positive selection and all results are shown in Subgroups were classified into three categories in terms of their natural ligands: 1) small molecules , 2) lipids and 3) peptides. If subgroups did not exclusively bind the same chemical class of natural ligand or if they had more than two orphan receptors, then we categorized them as divergent. If subgroups exclusively contained orphan receptors then they were categorized as orphan.key> with randomly selected 7TM positions, two hundred cohorts of AA positions were simulated. The average ω from each of the cohort of ten randomly selected 7TM positions was computed – this was denoted as <ωrandom7TM>. The average of the two hundred independent cohorts was computed from the distribution of <ωrandom7TM>.To compare <ωX, with AA residues x, was defined asr of the MSA, p(x) was the probability of having residue x at position X, and the summation is over all AA residues.Shannon entropy was first used to estimate the diversity in AA composition at key positions across all subgroups. The Shannon entropy at MSA position S to be the number of concatenated 7TM fragments in a subgroup. The AA similarity (and dissimilarity) among MSA positions of 7TM fragments due to substitutions among the S different paralogs of the subgroup was determined. We used BLOSUM80 substitution matrix to evaluate sequence similarity among the residues at key positions of the MSA. For a given key position, the average score of the key AA residues substituting with each other within the subgroup, we used the definition of Karlin and Brocchieri rc(x) is the AA at MSA position (or column) X in the sth fragment, and rsM scores for substitution between AA x and AA y. This similarity score rsM, for the defined  pairs of AAs in the rth and sth sequence fragment, is defined asrsm is the BLOSUM80 x in the rth row to AA y in the sth row of the alignment (or vice versa). We defined the BLOSUM similarity score for a given key position X as key = CKarlinBLO_80(X), and the average similarity score of all key positions <keyBLO_80> was averaged over the ten key positions.A variety of strategies exist to quantify sequence similarity key. The quantity −log10(Dkey) was computed to compare the attribute with previously computed measures of sequence similarity.In another approach, another estimate for dissimilarity was obtained using residues from MSA columns at key positions. To represent a distance measure, the average percentage of accepted mutation using program PROTDIST from PHYLIP software Figure S1key>) contrasted with average ω from randomly selected 7TM positions (<ωrandom7TM>).The average ω from key positions (<ωkey> while open triangle represents the average from random cohorts (from <ωrandom7TM> distribution). The error bar represents two standard deviations (2σrandom7TM) or the limits of 95% confidence interval from ωrandom7TM distribution.(TIFF)Click here for additional data file.Figure S2key> vs. <ωrandom7TM>.Graph of <ω Trend from <ωkey> vs. <ωrandom7TM> is shown using a logarithmic scale. Graph excludes subgroups labeled as “N” in (TIFF)Click here for additional data file.Figure S3Shannon entropy (H) for key positions across GPCR subgroups.(TIFF)Click here for additional data file.Figure S4Average Shannon entropy vs. average selection pressure for key positions across subgroups. Average scores from dN)<1) are represented here.(TIFF)Click here for additional data file.Figure S5Similarity scores for key positions across GPCR subgroups. Similarity scores (<BLO_80key>) in subgroup MSA defined by Karlin and Brocchieri, as in Reference 67, (described in (TIFF)Click here for additional data file.Figure S6key> vs. average selection pressure for key positions across subgroups.Average similarity score <BLO_80 Average scores from dN)<1) are represented here.(TIFF)Click here for additional data file.Figure S710Dkey>) for key positions across GPCR subgroups.Inverse protdist distance measure (<−log Plot showing the logarithm of inverse protdist distance (D) at key positions from GPCR subgroups.(TIFF)Click here for additional data file.Figure S8Average inverse protdist distance vs. average selection pressure for key positions across subgroups. The Y-axis represents <−log10Dkey> from dN)<1) are represented here.(TIFF)Click here for additional data file.Table S1Tenable PAML models representing molecular evolution of 7TMs of class A non-olfactory human GPCR subgroups. PAML's tenable models that represent molecular evolution of their 7TMs are illustrated across GPCR subgroups. (DOC)Click here for additional data file.Table S2Tenable PAML models representing molecular evolution of 7TMs of class A non-olfactory human GPCR subgroups. PAML's tenable models that represent molecular evolution of their 7TMs are illustrated across GPCR subgroups. (DOC)Click here for additional data file.


Recent advances reveal mRNA 3′end processing as a highly regulated process that fine-tunes posttranscriptional gene expression. This process can affect the site and/or the efficiency of 3′end processing, controlling the quality and the quantity of substrate mRNAs. The regulation of 3′end processing plays a central role in fundamental physiology such as blood coagulation and innate immunity. In addition, errors in mRNA 3′end processing have been associated with a broad spectrum of human diseases, including cancer. We summarize and discuss the paradigmatic shift in the understanding of 3′end processing as a mechanism of posttranscriptional gene regulation that has reached clinical medicine. Cleavage and polyadenylation of cellular pre-mRNAs at the 3′end is accomplished by a fine-tuned mechanism involving a number of RNA-binding proteins (RBPs) and regulatory ents Fig . In addicis-acting sequence elements determine the efficiency of a poly(A) (polyadenylation) site.In an mRNA's 3′UTR, the arrangement of cis-acting elements is a hexameric sequence motif that was first described by Proudfoot and Brownlee , which is located 30 to 45 nt 3′ of the hexameric sequence motif  resulted in a transcriptome-wide shift towards the use of proximal PASs, revealing a role of CFIm in APA . This is exemplified by hnRNPI/PTB1, which is a well-known global splicing repressor when bound to polypyrimidine tracts. hnRNPI/PTB1 can act as a regulator of 3′end processing efficiency upon binding to cis-acting RNA sequence elements playing a role in polyadenylation, such as the upstream sequence element (USE) and the DSE   genes via stimulated 3′end processing, the transcriptome-wide modulation of polyadenylation efficiency has been reported in response to various stress conditions, including thermal stress and DNA damage  class shift is triggered by APA  in the central nervous system (CNS). GFAP forms the intermediate cytoskeleton in mature astrocytes. APA results in the dynamic expression of two GFAP protein isoforms (GFAPɛ and GFAPκ)  upon antigen exposure. The stimulation of the T-cell antigen receptor triggers a switch from long protein isoforms in naïve T cells to a short isoform in mature T cells. Whereas in naïve T cells, the “weak” proximal PAS is not efficiently used, mature T cells show proximal poly(A) site usage and the short NF-ATc isoform is produced. The short NF-ATc isoform stimulates the expression of T-cell-specific genes, including lymphokine and cytokine genes  site usage in a set of genes under cold shock conditions, leading to elongated 3′UTRs  signal (AATAAA → AATAAG) of the α 2-globin gene causing α-thalassaemia  in the FOXP3 (forkhead box P3) gene causing the IPEX syndrome, which is characterized by immune dysfunction, polyendocrinopathy and enteropathy  transporter gene (SERT or 5-HTT)  that causes autosomal-dominant oculopharyngeal muscle dystrophy (OPMD). trePABPN1 interacts with WT PABPN1 in a dominant negative fashion and interferes with its function as a suppressor of proximal poly(A) site usage. Mouse models of OPMD as well as human cells expressing trePABPN1 show a global shift towards proximal, usually “weaker” PAS  site usage in otherwise normal transcripts is associated with malignancies. A number of diseases have been linked to either APA of single transcripts or to global changes in polyadenylation. The symptoms of such diseases include growth retardation, cancer predisposition syndromes and neurodegeneration.et al, et al, Lynch syndrome is a hereditary predisposition to several types of cancer, including colorectal and endometrial cancer, which is caused by a loss of function of one of the four DNA mismatch repair genes MSH2, MLH1, MSH6, PMS2. Some patients with this syndrome carry germline mutations in the coding region of one of those genes. Recently, a 20 nucleotide duplication in close vicinity to the poly(A) signal of MSH6 mRNA has been detected in two patients, associated with decreased MSH6 mRNA levels  of a CGG element within the promoter region of the fragile X mental retardation 1 (FMR1) gene, with the premutation (55–200 CGG repeats) already showing a clinically relevant phenotype  exhibited different polyadenylation patterns than control cells or tissues. MCL is related to the APA of cyclin D1. In proliferating MCL tumours, truncated cyclin D1 mRNA isoforms originating from processing at a proximal PAS are over-represented , a compound that inhibits polyadenylation by arresting the cleavage complex (Kondrashov et al, trans-acting proteins with their cognate RNA binding motifs such as those that have been reported between the USE and stimulatory and inhibitory proteins of 3′end processing (Danckwardt et al, Apart from using 3′end processing profiles as diagnostic tools, the polyadenylation step might serve as a target of future medical therapies. Since mRNA 3′end processing is regulated via et al, cis-acting elements playing a role in the cleavage and polyadenylation reaction might help to treat associated diseases in the future.In addition, directly modulating the function of RNA molecules in mammalian cells via the introduction of antisense oligonucleotides has gained much attention as a potential therapeutic strategy (Bennett & Swayze, The regulation of mRNA 3′end processing has gained much attention in recent years as a mechanism that can fine-tune gene expression in a quantitative and qualitative manner. Its role in pathophysiology and the underlying molecular mechanisms are intensively investigated at present. Certainly, the number of physiological and pathological cellular pathways controlled via regulated mRNA polyadenylation and cleavage will progressively increase. Prospectively, regulated mRNA 3′end processing could instruct new diagnostic approaches and serve as a therapeutic target, aiding in treating diseases, including cancer.The authors declare that they have no conflict of interest.Develop strategies to therapeutically target misregulated mRNA 3′end processing.Unravel the molecular mechanisms underlying misregulated mRNA 3′end processing to increase the level of understanding of its impact on pathological and physiological pathways.Explore the differences in mRNA 3′end processing as a diagnostic tool.


For most sample periods, daytime GEM flux was strongly correlated with solar radiation. The average nighttime GEM flux was slightly negative and was not well correlated with any of the measured meteorological variables. Preliminary, empirical models were developed to estimate GEM emissions from snow surfaces in northern New York. These models suggest that most, if not all, of the Hg deposited with and to snow is reemitted to the atmosphere.Snow surface-to-air exchange of gaseous elemental mercury (GEM) was measured using a modified Teflon fluorinated ethylene propylene (FEP) dynamic flux chamber (DFC) in a remote, open site in Potsdam, New York. Sampling was conducted during the winter months of 2011. The inlet and outlet of the DFC were coupled with a Tekran Model 2537A mercury (Hg) vapor analyzer using a Tekran Model 1110 two port synchronized sampler. The surface GEM flux ranged from −4.47 ng m Hg is a potent neurotoxin and regulated by the U.S. EPA −3 (GEM) remaining on the surface While the role of snow surfaces in Hg cycling has been widely studied in arctic regions −2 hr−1), with little contribution from deeper snow layers In the arctic, the snow surface-to-air flux of Hg is mainly the result of a diurnal pattern of GEM production in the interstitial air near the surface of the snowpack during the daytime  in Potsdam, NY  near the Clarkson University Observatory. This site remains largely undisturbed throughout the year and has served as a background site for the New York State particulate matter (PM) monitoring network. Sampling periods were determined based on access to the site and snow conditions. Special considerations were made to ensure that the chamber was never buried in snow and that all inlets and outlets remained above the snow during sampling. Measurements were conducted on a concrete slab, isolating the snowpack from the soil surface.−1 flow rate as the Tekran Model 2537A to maintain a constant turnover time (TOT) of 0.78 minutes and an optimized flushing flow rate (FFR) of 5 L min−1−1 FFR and 0.78 minute TOT are also similar to those used in a study by Eckley, et al. (2010).Concentrations of GEM were measured using a DFC with a method previously described in Choi & Holsen (2009). Briefly, the ambient sampling line (inlet) and chamber sampling line (outlet) of the DFC (described below) were coupled with a Tekran Model 2537A Hg vapor analyzer operated at room temperature in a field shed  using a Tekran Model 1110 two-port synchronized sampler. The Tekran 1110 unit allowed for alternating five minute sampling pairs to be made between the inlet and outlet sample lines every 20 minutes . During inlet sampling, outlet air is bypassed at the same 1 L min2 of the snow surface.Modified Teflon fluorinated ethylene propylene (FEP) chambers were used in the study. The modified Teflon chamber was constructed using a polycarbonate (PC) chamber frame and thin, 25 µm thick Teflon FEP film  to cover the top and side windows . In prev−1 (20°C) or approximately 0.26 ng into an operating chamber using a calibrated (ANSI/NCSL Z540-1-1994) Hamilton Digital Syringe  and a Tekran Model 2505 Hg vapor calibration unit . The recorded Hg concentrations after each manual spike test were roughly 9 ng m−3, on the same order as the average daytime Hg concentrations around 2 ng m−3. The recovery was 97.5±3.8%. Flow rates were calibrated using a Bios Definer 220 volumetric flow meter  at the beginning of each sampling period.Manual spike Hg recovery tests were conducted at the start of each sampling period by injecting 20 µL of Hg at roughly 13.23 pg µLPrior to all field measurements, the Tekran Model 2537A was calibrated with an internal permeation source to ensure acceptable response factors  and that the concentration difference between the inlet and outlet samples was less than 5%. In addition, all soda-lime traps and 0.2 µm polytetrafluoroethylene (PTFE) membrane filters were replaced at the start of each sampling period.−2) at a 10 minute time resolution.Meteorological data was collected using a weather station  located 1–2 m away from the chamber. The weather station measured ambient air temperature (°C), relative humidity (%), and solar radiation ; outletC and inletC are the concentrations of GEM (ng GEM m−3) at the outlet and inlet, respectively; Q is the FFR (m3 h−1) through the chamber; and A is the surface area (m2) of the snow exposed in the chamber. When fluxes were negative (-), Hg was being deposited on the snow surface, and when fluxes were positive (+) Hg was being emitted from the snow surface. All flux data was then smoothed using a Savitzky-Golay smoothing filter Where 4*F is the smoothed flux (ng GEM m−2 h−1), 1–7F are the range of measured abscissa flux values (ng GEM m−2 h−1), and 21 is the normalizing factor.Where Histograms of the GEM flux and the three meteorological predictor variables  showed that none of the variables were normally distributed. −2 h−1 . Fluxes also tended to follow a diurnal pattern   . The slo0<0.050) , similarIn the past, empirical models have been developed using meteorological data in order to estimate surface GEM flux from soils in temperate regions of eastern North America 2 = 0.481)Winter 2011 (Daytime): (R2 = 0.0616)Winter 2011 (Nighttime):  to 1.53±1.69 ng m−2 hr−1 (daytime) or ∼17.22 ng m−2 year−1. During the same time period Mercury Deposition Network (MDN) data from the same site yield a similar value with an average deposition flux of 0.48±0.41 ng m−2 hr−1 or 11.52±9.84 ng m−2 year−1. The reason for the slightly higher modeled flux compared to the measured flux is likely due to the fact that some of the measurements used to make the empirical model were made after fresh snowfall when GEM fluxes would be at their maximum values.Using the multiple linear regression models in conjunction with 5-year winter  EPA Clean Air Status and Trends Network (CASTNET) meteorological data from the National Atmospheric Deposition Program (NADP) site, NY20, it is estimated that the average snow surface emissions from the open Huntington Wildlife Forest (HWF) site range from −0.10±0.07 ng m−2 hr−1, zero change in surface snow Hg concentration after deposition events, and up to 54% GEM reemission during the first 24 hours after a snowfall.Overall, these models suggest that most if not all the Hg deposited to snow surfaces is promptly recycled. Similar reemission phenomena have been reported by other research groups −2 hr−1 occurred during separate sampling periods, one on 25 January and one on 05 February. Both of these event followed snowfalls ≥3 cm  with temperature and displayed patterns opposite to the diurnal patterns typically seen.Snow surface-to-air exchange of gaseous elemental Hg (GEM) was measured using a modified Teflon fluorinated ethylene propylene (FEP) dynamic flux chamber (DFC) in a remote, open site in Potsdam, New York during the winter months of 2011. The surface GEM flux ranged from −4.47 ng m


PSI+] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion “strains” or “variants” are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, Δ19, and E9, with limited transmissibility of [PSI+] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI+] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI+], all indistinguishably “weak” [PSI+], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI+]. Notably, a “strong [PSI+]” can have any of several different transmission efficiency patterns, showing that “strong” versus “weak” is insufficient to indicate prion variant uniformity.[ PSI+] prion (infectious protein) of yeast is a self-propagating amyloid (filamentous protein polymer) of the Sup35 protein, a subunit of the translation termination factor. A single protein can form many biologically distinct prions, called prion variants. Wild yeast strains have three groups of Sup35 sequences (polymorphs), which partially block transmission of the [PSI+] prion from cell to cell. We find that [PSI+] variants (including the rare [PSI+] from wild yeasts) show different transmission patterns from one Sup35 sequence to another. Moreover, we find segregation of different prion variants on mitotic growth and evidence for generation of new variants with growth under non-selective conditions. This data supports the “prion cloud” model, that prions are not uniform structures but have an array of related self-propagating amyloid structures.The [ PSI+] prion was first recognized as a non-chromosomal genetic element enhancing the read-thru of the premature termination codon in ade2-1Prions in yeast are a new form of gene, composed of proteins instead of nucleic acids Sup35p consists of C, an essential C-terminal domain (residues 254–685), responsible for the translation termination function S. cerevisiae also show considerable sequence variation in Sup35p sequence PSI+] Prions can often be transmitted between species, as was first recognized by infectivity of sheep scrapie brain extracts for goats A single prion protein can propagate any of a number of prion variants , with biological differences due to different self-propagating conformations of the amyloid S. cerevisiae, an ‘intraspecies barrier’. These intraspecies barriers are of particular interest since they must operate in nature, when S. cerevisiae strains mate among themselves. Interspecies matings are less efficient than intraspecies matings  deletion in the prion domain; E9 is representative of a group with N109S and several polymorphisms in the M domain −10). This indicates that intraspecies transmission barriers are variant-specific.Wild PSI+] (−10), unlike two [PSI+] isolates initially selected in cells expressing Δ19, which propagated poorly to E9 or reference −10 and .002). This again shows the prion variant specificity of transmission barriers. Note that these two E9G→Δ19 cytoductants differ in that [PSI+E9G]Δ19A was white (a strong [PSI+]) while [PSI+E9G]Δ19B was pink (a weak [PSI+]). This indicates that either the original E9G was a mixture of two prions or that new prion variants were selected by the difficulty of transmission into Δ19.Two of the few E9G→Δ19 cytoductants that were [PSI+]  were tesPSI+] . Each haPSI+] , p<10−10−10) and the original E9 sequence from which the prion originated (69%), but only poor transmission to the Δ19 sequence (PSI+ref]ref (originating and propagating in the ref sequence) which propagates poorly into E9  PSI+E9G] , transfer well to all polymorphs (−10). The [PSI+ref]E9 transfers well to both reference and E9 sequences . As expected, the prion originating in E9 and transmitted to E9, or that originating in the reference sequence and transmitted to the reference sequence, each maintain their original properties.The variant-dependence of transmissibility was again evident in cytoduction of  to each of the Sup35 polymorphs, we transferred them back to the original host (cured of [PSI+]) and re-examined their transmission properties to see if they had changed as a result of their experience . Moreover, the prion that passed through Δ19 could transmit 91% to another Δ19  without selection on ½ YPD plates for at least 75 generations. We subcloned single colonies of the 779-6A  from Y5 by growth on guanidine, and cytoducing cytoplasm from Y1, Y2 or Y5 into strain 4830 and then 8 cytoductants from each were cytoduced into a rho° derivative of the cured Y5 (2N+1)/(2N−1)(N+1)]n , where H0 is the starting fraction of mixed cells, N is the copy number of the plasmid, and n is the number of generations 0.The segregation of a mixed prion population could be considered analogous to the segregation of differently marked plasmids with the same replicon. The latter situation has been carefully examined by Novick and Hoppenstadt PSI+] strains for a number of generations comparable to the presumed copy number, so segregation of different prions is not surprising.The copy number in the case of yeast prions might be taken as the ‘seed number’ determined by the methods developed by Cox et al. However, we find that even when we have apparently purified a variant, further non-selective growth and subcloning leads to further appearance of the full range of variants among the progeny . This inPSI+E9]ref, in analogy with similar nomenclature for [URE3] PSI+E9]ref was transferred to Sup35 Δ19” to mean “[PSI+E9]ref was transferred to cells expressing Sup35 Δ19”.We refer to the standard laboratory yeast sequence ade2-1, with an ochre termination codon in the middle of the ADE2 gene. In addition to ade2-1, strains carry the SUQ5 weak suppressor mutation, which leaves cells Ade- unless the [PSI+] prion is also present Sup35p is a subunit of the translation termination complex, and the incorporation of a large proportion of Sup35p into the prion amyloid filaments makes it inactive, resulting in increased read-through of termination codons. This is measured by read-through of The strains used are listed in kar1-1 mutation URA3 SUP35C) was selected by growth on 5-fluoroorotic acid media and Ade- transformants were made rho° by growth on YPAD containing 1 mg/ml ethidium bromide. Donor and recipient strains at high density were mixed in water at a ratio of about 5∶1, and the mixture was spotted onto a YPAD plate. After 18 hours at room temperature, the mating mix was streaked for single colonies on media selective against growth of the donor strain. Clones are shown to be cytoductants by their growth on glycerol and failure to grow on media selective for diploids. As further tests of a sample confirm, Ade+ cytoductants are judged to have received and propagated [PSI+].Cytoplasm may be transferred from one strain to another utilizing the PSI+] Strain 779-6A 7 cells per colony, indicating a total of at least 75 generations of growth of clones Y1-Y12 before cytoduction. Additional subclones were handled in the same manner with only ten colonies selected from the initial ½ YPD plate. In experiments to rule out selection during stationary growth phase, subclones of Y1 and Y2 were grown in a 125 ml Erlenmyer flask containing 25 ml of liquid YPD medium. When A600 reached 0.3, the culture was diluted, transferring 1000 cells of each to a fresh flask. These subclones were propagated in exponential phase for 84 generations and were then streaked for single colonies on ½ YPD plates. After one day of growth on ½ YPD, 10 subclones were selected for each of Y1 and Y2, expanded and tested for transmission via cytoduction.[PSI+] MATα strains were cytoduced into strain 4830 carrying pRS316 (URA3) for selection. Lys2 mutants of MATa strains were selected on plates with DL-α-aminoadipic acid as a nitrogen source Strains reported to be [+PSI] or failure of its transmission. However, because of the large number of observations, the results should be approximately normally distributed. We want to calculate the probability that two sets of data are due to chance. Let p1 and p2 be the observed proportions of transmission in cytoductions 1 and 2, and ni the number of cytoductants tested in each experiment. Let p = (p1n1+p2n2)/(n1+n2) be the average of the proportion of transmission in the two experiments. The estimated standard error of the difference between the two proportions is1−p2)−0]/S = z = the number of standard deviations that the observed difference in proportions differs from the expected difference (0). The frequency of “z” being greater or equal to the observed value (assuming the null hypothesis) is obtained from a table of the normal distribution. The calculated “p values” are shown in the tables and at appropriate points in the text.The cytoduction data follows the binomial distribution, because each data point expresses two alternative results, transmission of [Cytoductants examined have been treated as independent since the chance that they represent sister cells is close to zero. This is because cytoductant mixtures were incubated at 20C where the cells divide slowly and because only about 30 cells were examined from several million in the zygote mixture on each plate.Figure S1PSI+] strains. Wild strains reported to carry [PSI+] kanMX, and examined microscopically as described in Methods. Strains UCD#521, 779 and 824 do not show obvious dots. Strains UCD#885, 978 and 2534 show dots which appear smaller than in the laboratory [PSI+] strain 779-6A. Dots in strain 5672 are comparable to those in the laboratory strain. Strains UCD#587 and 939 were indeterminate.Aggregation of Sup35-GFP in reported wild  prion using growth in the presence of guanidine, and of the mitochondrial DNA using ethidium bromide. [+PSI] was cytoduced from strains Y1, Y2 and Y5 to strain 4830 rho°, and ten cytoductants from each were then used as cytoduction donors to the [psi−] rho° derivative of Y5. One cytoductant from each of these cytoductions was picked and used as a donor into recipients expressing each of the three Sup35 polymorphs.Y1, Y2 and Y5 transmission phenotypes are not due to chromosomal mutations. Strain Y5 was cured of the [(XLS)Click here for additional data file.Table S4Y1 Subclones of Y1-3 diverge in transmission phenotype indicating inability to purify a specific variant of PSI transmission.(XLS)Click here for additional data file.Table S5Y1 transmission is unchanged after months in frozen stock.(XLS)Click here for additional data file.Table S6Cytoductions of individual clones summed in (XLS)Click here for additional data file.


In this study we quantified changes in [NAD(H)] and markers of inflammation and oxidative damage  in the cerebrospinal fluid (CSF) of healthy humans across a wide age range (24–91 years). CSF was collected from consenting patients who required a spinal tap for the administration of anesthetic. CSF of participants aged >45 years was found to contain increased levels of lipid peroxidation (F2-isoprostanes) (p = 0.04) and inflammation (IL-6) (p = 0.00) and decreased levels of both total antioxidant capacity (p = 0.00) and NAD(H) (p = 0.05), compared to their younger counterparts. A positive association was also observed between plasma [NAD(H)] and CSF NAD(H) levels (p = 0.03). Further analysis of the data identified a relationship between alcohol intake and CSF [NAD(H)] and markers of inflammation. The CSF of participants who consumed >1 standard drink of alcohol per day contained lower levels of NAD(H) compared to those who consumed no alcohol (p<0.05). An increase in CSF IL-6 was observed in participants who reported drinking >0–1 (p<0.05) and >1 (p<0.05) standard alcoholic drinks per day compared to those who did not drink alcohol. Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced [NAD(H)] in the brain which may be exacerbated by alcohol intake.An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the degenerative changes of systemic tissues in aging. However a comparatively limited amount of data is available to verify whether these processes also contribute to normal aging within the brain. High levels of oxidative damage results in key cellular changes including a reduction in available nicotinamide adenine dinucleotide (NAD In 1956 Harman proposed the oxidative stress theory of aging suggesting that the accumulation of unrepaired oxidative damage results in the typical aging phenotype The brain is particularly vulnerable to oxidative damage as a consequence of its high oxygen demand, high level of both polyunsaturated fatty acids and transition metals, and poor antioxidant defenses Both normal brain aging and neurodegenerative disease are characterized by increased inflammation associated with microglial over activation and a subsequent rise in pro-inflammatory cytokines +) has been observed in multiple organs of the rat + is a ubiquitous molecule that is required for a number of vital cellular processes. In addition to its role in cellular energy and metabolism there are several enzymes, including poly(ADP-ribose) polymerase 1 (PARP) and silent information regulators (e.g. SIRT1), that use NAD+ as their substrate + resulting in the formation of poly(ADP-ribose) subunits. Under conditions of mild-to-moderate DNA damage this process facilitates DNA repair + depletion + must be sustained.As inflammation and oxidative damage rise with age a decrease in available nicotinamide adenine dinucleotide  a gradual increase in oxidative damage burden in many of the body’s tissues. If experienced in the brain, neurodegenerative changes are likely. While age is the major risk factor for the development of most neurodegenerative disorders + availability) within the central nervous system. While evidence from cell culture, animal and limited postmortem brain tissue studies support this hypothesis, to date no study has investigated this in a healthy human cohort.Collectively these reports indicate that certain lifestyle choices may accelerate the development of an age associated oxidative-inflammatory state leading to increased tissue damage and reduced DNA repair capacity . We further correlated changes in CSF oxidative/inflammatory markers and [NAD(H)] with specific modifiable lifestyle factors.This study was conducted in accordance with the Helsinki declaration. Ethical approval was obtained from the Human Research Ethics Committee, Sydney Adventist Hospital (HREC# 2011-005). Written informed consent was obtained from all participants prior to commencement.Male (n = 20) and female (n = 50) participants, who required a spinal tap for the administration of anesthetic as part of routine care, were recruited at Sydney Adventist Hospital, Australia. The average age of participants was 53 years . Participants were excluded from the cohort if they were smokers or had a confirmed diagnosis of a neurological/neurodegenerative disorder or central nervous system infection. In total 70 CSF and 38 matched blood samples were collected from consenting participants considered in general good health.Fasting (≥10 h) blood and CSF samples were collected by an accredited anesthetist no longer than 30 minutes apart. CSF samples were collected, prior to injection of spinal anesthetics, via standard lumber puncture. Blood samples were collected into heparinized tubes from an intravenous cannula inserted into a superficial vein on an upper limb, prior to the administration of fluids or anesthetics. Samples were prepared by centrifuging at 1800 rpm for 10 minutes and stored within 1 hour of collection, at −194 degrees Celsius until analysis. Samples intended for F2-isoprostane analysis where stored in the presence of a glutathione/butylated hydroxytoluene preservative.Do you drink alcohol.’ If this was affirmative participants were asked to provide the number of standard drinks consumed per day.Alcohol consumption was assessed via questionnaire upon hospital admission. Specifically participants were asked ‘Total NAD(H) concentrations in plasma and CSF samples were measured spectrophotometrically using a thiazolyl blue microcycling assay established by Bernofsky and Swan (1973) CSF total antioxidant capacity was measured using a standardized commercial kit . This assay relies on the ability of antioxidants to inhibit oxidation of 2,2′-Azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS) by metmyoglobin. Briefly, 10 µL of metmyoglobin was added to 10 µL of diluted sample. 150 µL of chromagen, containing ABTS, was subsequently added and the reaction initiated by adding 40 µL of 441 µM hydrogen peroxide. The plate was incubated on a shaker for 5 minutes and the amount of oxidized ABTS was measured spectrophotometrically, at an absorbance of 750 nm, using a Model 680XR microplate reader .2SO4 was added to the wells to stop the enzyme-substrate reaction. The intensity of this colored product is directly proportional to IL-6 concentration. Absorbance was measured at 450 nm using a Model 680XR microplate reader .IL-6 was measured using a standardized commercial solid phase sandwich enzyme linked-immuno-sorbent assay (ELISA) . Briefly, 100 µL of CSF was added to a plate pre-coated with a monoclonal antibody specific for IL-6. 50 µL of biotinylated anti-IL6 was then added and the plate incubated for 3 hours. After incubation, the plate was washed before the addition of the enzyme, horseradish peroxidase. The plate was incubated for 30 minutes and then washed again to remove any unbound enzymes. The 3,3′,5,5′-Tetramethylbenzidine substrate was added and the plate was incubated in the dark for 12–15 minutes, after which H8-OHdG was measured in CSF samples using a standardized commercial competitive ELISA . Briefly 50 µL of sample or standard and 50 µL monoclonal antibody was adsorbed onto a 96-well plate precoated with 8 OHdG. Following an overnight incubation at 4 degrees Celsius the plate was washed and incubated with a secondary antibody for 1 hour. The plate was washed again before the addition of a chromatic solution for 15 minutes, after which the reaction was terminated and absorbance was measured at 450 nm using a Model 680XR microplate reader .Total F2-Isoprostanes were measured in CSF by gas chromatography–mass spectrometry (GC-MS) using electron capture negative ionization according to a modification in the method of Mori et al (1999) Statistical analyses were performed using SPSS version 16.0 and GraphPad Prism version 5 for Windows. Data is presented as means ± standard deviation unless otherwise stated. Multiple linear regression, controlling for age, was used to identify significant relationships between CSF [NAD(H)], plasma [NAD(H)], and markers of oxidative damage and inflammation. The Independent T Test or Mann-Whitney U Test was employed to analyze the effect of age on markers of oxidative damage, inflammation and metabolism. The Wilcoxon Signed Ranks Test was used to identify the association between mean plasma and CSF NAD(H) levels. Kruskal-Wallis with Dunn’s post-hoc test was used to assess the effect of alcohol consumption on markers of oxidative damage, inflammation and metabolism. Both the Kolmogorov-Smirnov, Shapiro-Wilk and histogram analysis was used to check normality of the variables. When required the Levene’s Test of Equality was used to check homogeneity of variances between groups. If the variances of the groups were found to be either not homogenous and/or normality tests for the variables were not significant then further investigation with graphical displays was performed to assess the distributions of the variables. Both adjusted and non-adjusted P-values are provided throughout with test significance set at P value ≤0.05.A number of studies have shown that lifestyle behaviors in midlife (i.e. around 45–50 years) are associated with reduced cognitive function in later life In this cohort older age was associated with an increase in a number of CSF oxidative and inflammatory markers . SpecifiAssessing these trends in each gender revealed that the CSF of females >45 years contained significantly higher levels of IL-6  and lower TAC levels  than their younger counterparts. Due to low number of male participants ≤45 years, valid comparisons were not possible.CSF NAD(H) levels were significantly lower in participants aged >45 years compared to those aged ≤45 years; 75.88±30.14 vs. 88.59±21.07 µg/mL respectively (p = 0.05) . AssessiAfter controlling for age, a significant inverse association was observed between CSF log TAC and CSF 8 OHdG  . A signiAfter controlling for age, a significant positive relationship was observed between plasma and CSF NAD(H) concentrations  . An incrThe data was further analyzed to identify possible relationships between alcohol intake and CSF [NAD(H)] and markers of inflammation and oxidative damage. For this analysis, participants were divided into three groups, those who consumed zero (n = 32), >0–1 (n = 14) and >1 (n = 8) standard alcoholic drinks per day. CSF NAD(H) levels were significantly different between the groups (p = 0.02). Specifically the CSF of participants who consumed >1 standard drink of alcohol per day contained significantly lower levels of NAD(H) compared to those who reported consuming zero drinks per day; 62.39±19.93 vs. 86.93±25.32 µg/mL respectively (p<0.05) . A signiIn this cohort, the CSF of male participants (n = 18) contained significantly higher levels of IL-6 compared to females (n = 42) (p = 0.00); 2.82±2.23 vs. 1.03±0.91 pg/mL respectively. However when the data was stratified into age groups, this observation did not remain statistically significant . No signConsiderable evidence now indicates that both inflammation and oxidative stress contribute to the development of various neuropathologies including Alzheimer’s and Parkinson’s disease + depletion It is well established, that oxidative DNA damage activates the NAD-dependent DNA repair enzyme, PARP, which is involved in base excision repair + as the sole substrate for their activities + it has been suggested that depletion of NAD+ results in reduced SIRT1 deacetylase activity + depletion resulting in neuronal death + depletion facilitates cell death is supported by observations in rodent models of brain ischemia and Alzheimer’s disease where significantly reduced levels of total cellular NAD+ occur prior to neuronal death + levels are therefore required to maintain cellular energy and robust SIRT1 activity. However further functional studies are required to determine the level of biochemical impact the relatively modest (∼14%) decrease in NAD(H) levels would have on cell metabolism.Adequate levels of NAD(H) are required to maintain normal cellular functions. In addition to its role in cellular energy metabolism there are several enzymes, in addition to PARP, that use the oxidized form, NAD+/NADH dependant mechanisms’ in both the central nervous system and periphery is apparent, it was not previously known whether peripheral stores of NAD(H) influence central NAD(H) levels. This study is the first to show a positive correlation between matched CSF and plasma NAD(H) levels in a healthy human cohort. This is consistent with a previous study by Rex and colleagues (2002) who observed that NAD(H) in both its oxidized and reduced forms is capable of crossing the blood brain barrier in rats +While the physiologic and pathologic importance of NADAlthough age is the major risk factor for the development of most neurodegenerative disorders The suggested pathways by which alcohol may damage the brain are numerous and include, but are not limited to, disruption of neural cell adhesion molecules Drosophila that NAD+ levels decrease by at least 20% in response to ethanol stress We also report for the first time an inverse relationship between alcohol consumption and CSF [NAD(H)]. Specifically the CSF of participants who consumed greater than one glass of alcohol per day had significantly lower levels of NAD(H) compared to those who did not drink alcohol. While research investigating the effect of alcohol on NAD(H) is scarce our results are consistent with a very early report by McElfresh and McDonald (1983) who also observed in While the observations reported in this study are statistically valid it is recognized that these associations have been obtained from a modest number of participants (38 plasma/CSF matched and 70 CSF). Though both genders were well represented in the older age groups, due to the difficulty in obtaining CSF samples from essentially healthy individuals greater numbers of females were represented in the younger age range. The disproportionately low number of younger male participants, as well as the small number of participants for which alcohol consumption data was available, prevented a comprehensive analysis on how gender may influence our findings. Self-reported alcohol consumption was also relied upon in this study and may have introduced some error into our analysis. However while such error may cause the levels of alcohol reported to slightly differ from the number of glasses actually consumed, it is unlikely to significantly affect the rank order of participants. Finally the restricted volume of sample collected as part of this study limited our analytical profile negating comparisons with other important molecular species such as the range of anti-inflammatory cytokines. Future studies overcoming these limitations are required to verify the consistency of our observations.An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the physiology of aging. However, comparatively limited data are available to verify whether these processes also contribute to normal aging within the brain. This study reports for the first time a potential link between aging, increased oxidative stress, inflammation and alcohol consumption and a decline in the essential pyridine nucleotide [NAD(H)], in the CSF of a healthy human population. We also provide evidence of a relationship between peripheral and central NAD(H) stores.Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced NAD(H) in the brain which may be exacerbated by certain lifestyle choices such as regular alcohol consumption.As reduced NAD(H) levels impact at least PARP and SIRT1 activity the observed decrease in NAD(H) availability within the aging brain may facilitate cell metabolic and genomic instability increasing an individuals’ susceptibility to degenerative disease. However further follow-up of the participants characterized in this study is required to confirm this hypothesis.


Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFNγ compared to NKT cells derived from male mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4− NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease.Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan  Entamoeba histolytica. This parasite primarily colonizes the human gut where it can reside and multiply for months or even years without inducing any clinical symptoms. However, in a small proportion of infected individuals the parasite invades the tissue and causes colitis or extraintestinal abscesses, most commonly in the liver. Interestingly, ALA is rare in children and women. The vast majority of more than 80% of all ALA cases occur in adult males Susceptibility to and outcome of infectious diseases may be greatly influenced by the gender. There are various examples, in particular for parasitic diseases, in which male individuals are more frequently infected with the respective pathogen, suffer from higher parasite burden or develop more severe clinical courses in comparison to females leishmaniasis or malaria, testosterone treatment decreases the resistance to parasite infections γ. The importance of IFNγ in the early control of E. histolytica invasion has been documented from various in vitro studies as well as from animal models for experimental ALA The influence of steroid hormones on the outcome of parasitic diseases, either by direct interactions with the parasite or by altering immune functions is well documented γ in early response to microbial infections are natural killer T (NKT) cells, which are immune cells, bridging the early innate and the adaptive immune response. NKT cells are activated by a range of microbial-derived lipids and glycolipids that are presented by the major histocompatibility complex (MHC) class I-like molecule CD1d on antigen presenting cells (APCs) γ by an E. histolytica lipopetidophosphoglycan (EhLPPG) present on the surface of ameba trophozoites One of the main producer of IFNγ by EhLPPG-activated NKT cells.In this communication, we report on the use of the C57BL/6 ALA mouse model to analyze the role of sexual hormones for ALA susceptibility and provide evidence that testosterone increases susceptibility for ALA by modulation the secretion of IFNhttp://ec.europa.eu/environment/chemicals/lab_animals/home_en.htm). All work was conducted with the approval of the Behörde für Gesundheit und Verbraucherschutz der Stadt Hamburg according to §8 TierSchG .Mice were maintained in a specific pathogen-free microenvironment in the animal facility of the Bernhard-Nocht-Institute and received care in compliance with the European Union guidelines for the handling of laboratory animals . Parasites from the log growth stage were used for intrahepatic infection of C57BL/6 mice.Trophozoites of the E. histolytica trophozoites as described previously g at 4°C for 10 min and subsequently the supernatant was recovered and the trophozoite membranes were enriched by ultracentrifugation at 150,000 g for 40 min. The obtained pellet was extracted with a mixture of chloroform/methanol/water 10∶10:3 (by volume) and the insoluble material was recovered by centrifugation, dried, resuspended in distilled pyrogen free water and extracted three times with an equal volume of 90% phenol at 68°C for 30 min with constant stirring g for 30 min and dialysis against distilled water.EhLPPG was purified from the membranes of Female and male C57BL/6J mice at an age of 8 weeks were used for the experiments. The following groups of mice were assembled:Naive mice, ovariectomized (ovx) mice supplemented with testosterone, ovariectomized and orchiectomized mice treated with placebo, non-ovariectomized mice supplemented with testosterone and non-ovariectomized mice treated with placebo.Female mice were ovariectomized at an age of 8 weeks. The mice were prepared for surgery using an anesthetic protocol that enabled the abrogation of the narcosis immediately after finishing the surgical intervention. Inititially, a solution consisting of 6.3 µl Domitor , 27 µl Ketanest  and 66 µl phosphate buffered saline (PBS) was injected intraperitoneally. The tolerance time using this method was approximately 60 min. The intra muscular injection of 5 µl Antisedan (Pfizer)/45 µl phosphate buffered saline/animal abrogated the narcosis within the next 10 min.After skin desinfection, a dorsal midline skin incision was made caudal to the border of the ribs. Lateral to the incision, the muscle layer of the posterior abdominal wall and the peritoneum were separated to enter the abdominal cavity. The periovarian fat tissue was grasped of to lift and exteriorize the ovary. The cranial fixation of the ovary and the arteria ovarica, as well as the fallopian tube were carefully heat coagulated and the ovary was removed. The blunt end of the uterine horn was slowly relocated in the abdominal cavern. When no further bleedings were observed, the peritoneum and the muscle layers were sutured and the skin was closed using skin clamps. The process was then repeated to remove the second ovary.Surgical orchiectomy was performed via a midline scrotal incision allowing bilateral access to the hemiscrotal contents. After exposing each testicle, a 3-0 vicryl suture was used to ligate the spermatic cord and then remove the testicle.For hormone treatment, female mice were implanted subcutaneously with pellets releasing testosterone designed to yield blood levels of 6–9 ng/ml . Control mice were implanted with Placebo pellets for testosterone .5 axenically cultured trophozoites in a volume of 25 µl TY medium were injected in the visualized left liver lobe using a U-100 insulin syringe . Peritoneum and muscle layer were sutured and wound clips closed the skin. To maintain virulence, trophozoites were regularly passaged through the liver of male C57BL/6 mice. On day 7 post infection, the mice were sacrificed, the abscess size was calculated by measuring the size in mm and the introduction of score values  and the abscess material was maintained in TY medium at 37°C to provide optimal conditions for the re-isolation of amebic trophozoites.The mice were infected by direct hepatic inoculation of virulent trophozoites as previously reported Since no steroid ELISA kits are available for mouse samples, a human ELISA kit has been adopted. Prior to the measurement, steroids were extracted from the matrix by liquid/liquid-extraction to avoid mouse plasma matrix effects. The plasma was extracted three times in each case with a tenfold excess of tetra-butylmethylether (TBME). After evaporation of the combined organic extracts the dried residues can be stored at −20°C. For the subsequent ELISA, the material is reconstituted in steroid free serum. The serum testosterone concentration was quantified using a competitive ELISA according to the manufacturer’s protocols . The plates were read in a standard microplate reader at 450 nm . The concentrations were calculated upon the calibration from the standard curve and reported in ng/ml. The sensitivity of the testosterone assay is 0.083 ng/ml.g, cell pellets were resuspended in RPMI medium, underlayed with a 30% Nycodenz solution  and centrifuged at 900 g for 20 min. The liver lymphocytes were collected from the interface, treated with hypotonic ammonium chloride solution, washed and diluted in RPMI medium.Livers were perfused with ice cold PBS/20% FCS solution and subsequently filtered through a 40 µm mesh. Following centrifugation at 400 Gradient purified liver lymphocytes were further characterized by staining with APC-labelled anti-CD3, PE-labelled CD11b and FITC-labelled anti-GR1 in phosphate buffered saline containing 1% bovine serum albumine. The cells were subsequently subjected to flow cytometry on a FACS Calibur  and the data were analyzed using the CellQuestPro software (BD Bioscience).NKT-cells were stained for cell sorting using αGalCer  loaded to PE labelled recombinant CD1d-tetramer  and FITC- labelled anti-CD4 . Cell sorting was performed on a FACS Aria III (BD).Generation of APCs. Bone marrow was harvested from femurs of male and female, 6- to 10-weeks-old C57BL/6 mice and cultured as described by Lutz et al. In vitro stimulation of liver lymphocytes from testosterone treated female mice and orchiectomized male mice. In brief, gradient purified liver lymphocytes from hormone-modified mice were subjected to the PAN T cell isolation kit  according to the manufacturers instructions. Purified T cells (1×105) were added to gender matched APCs (5×104) previously pulsed with 4 µg/ml αGalCer and 8 µg/ml EhLPPG for 3 h. The cells were cultured in triplicates in 96-well round bottom plates with RPMI-20 medium supplemented with 200 mM L-glutamine, 50 µg gentamicin, 10% fetal calf serum, 1 mM sodium pyruvate and, 50 µM 2-mercaptoethanol. Cell supernatants were collected after 48 h of coincubation and assayed for IFNγ by enzyme-linked immunosorbent assay (ELISA) using antibody pairs purchased from R&D Systems .In vitro stimulation of purified murine NKT cells. Gradient purified liver lymphocytes from female and male C57BL/6 mice (10–12 weeks old) were stained with PE-labelled- αGalCer -CD1d-tetramer, sorted by fluorescence activated cell sorting (BD-FACS-Aria) and added in a cell density of 5×104 to 1×104 gender matched APCs prepulsed for 3 h with either αGalCer (4 µg/ml) or purified amebic EhLPPG (8 µg/ml). The cell supernatant was assayed for IFNγ as described above.In vitro stimulation assay using iNKT cell subpopulations. Gradient purified liver lymphocytes from female and male C57BL/6 mice (10–12 weeks old) were stained with PE-labelled- αGalCer -CD1d tetramer (Proimmune) and FITC-labelled anti-CD4. Tetramer positive, CD4+ or tetramer positive, CD4− NKT cells were sorted by fluorescence activated cell sorting (BD-FACS-Aria) and added at a cell density of 3×104 to 1×104 gender matched APCs previously pulsed with αGalCer or EhLPPG as described above. The amount of IFNγ was measured by ELISA in the cell supernatant after 48 h of co-cultivation.E. histolytica. The student`s t test was applied for the statistical analysis of the IFNγ values obtained by the NKT cell activation assay.The Mann Whitney U test was applied to compare the liver abscess sizes between male and female mice over the time period monitored and the Paired t-test was used to determine the difference between abscesses that were culture positive for E. histolytica trophozoites. Development of ALA was assessed on day 7 after infection (p<0.009) and parasite survival rates (p<0.02) in orchiectomized male mice compared to respective male controls. The sizes of ALA in orchiectomized mice were similar to those found in female mice. In contrast, ovariectomy did not influence sizes of ALA or parasite survival rates in female mice suggesting that testosterone rather than estrogens influences susceptibility to ALA.The C57BL/6 mouse model for ALA is characterized by the development of significant larger abscesses in male mice within 7 days following intraheptic ameba challenge, whereas female mice control the infection and develop only small lesions. To investigate whether sexual hormones might have an influence on ALA development, gonadectomy was performed in groups of 8 weeks old male and female mice. Seven weeks following castration, animals were challenged by intrahepatic application of cultured nfection . The resp<0.05), regardless whether mice were ovariectomized or not   . In contE. histolytica lipopeptidophosphoglycan (EhLPPG), similar to αGalCer, specifically activates liver or spleen murine NKT cells to secrete significant amounts of protective IFNγγ in female compared to male derived cells   . To furts gender . The resγ. However, whereas we found no gender specific differences in the number of CD4− NKT cells (data not shown), CD4− NKT cells from female mice produced significant higher amounts of IFNγ   (To determine whether gender specific differences in IFNsed APCs . Experimsed APCs  u. B as sed APCs  u. D. Aned<0.02)  u. C. Ined<0.05)  u. D.To further elucidate the mechanisms responsible for the higher susceptibility of males to develop ALA we have used a mouse model for the disease to investigate the role of sexual hormones and in particular the contribution of testosterone and of NKT cells. The mouse model used in our study is considered suitable for the analysis of ALA associated immune reactions and gender related functions as the histopathological cellular infiltrates of parasite infected livers and the strong gender bias towards males indicates clear similarities to findings in human ALA patients Because of the complex and strong host specificity of human parasites, rodent models that enable studies on the influence of hormones on infectious diseases are rare and often do not reflect the same gender bias that occurs in humans There are numerous examples of infectious diseases with predispositions in males. In general, this phenomenon is attributed to a reduced ability of male individuals to generate an effective humoral or cellular immune response γ are critically important for the control of ALA in a mouse model for the disease and that NKT cells can be specifically activated not only by αGalCer but similarly by EhLPPG to produce protective IFNγγ. Our results clearly indicate increased IFNγ production by αGalCer or EhLPPG stimulated NKT cells from female mice compared to male mice, which is in line with earlier studies, indicating higher amounts of IFNγ in the serum of female mice after administration of αGalCer γ production between male and female mice are obviously not the result of differences in the numbers of NKT cells, as in our in vitro experiments identical numbers of isolated NKT cells were used. Furthermore the quantification of liver-derived NKT cells from mice at various ages did not reveal significant differences between the genders.Previous studies have shown that NKT cells and IFNγ upon stimulation with αGalCer or EhLPPG compared to female mice, while orchiectomy increased NKT cell dependent IFNγ secretion in male mice. These results were independent whether cells were isolated from naïve or from ameba infected mice. Testosterone, or steroid hormones in general, have been shown to activate lymphocyte immune functions either directly or via the modulation of macrophages γ production in response to stimulation with EhLPPG in female mice is not due to an expansion of NKT cells but primarily due to IFNγ secretion by CD4− NKT cells, which are also present in male mice but without producing significant amounts of IFNγ. However, it remains to be determined whether NKT cells possess plasma membrane, intracellular or nuclear androgen receptors as conventional T lymphocytes do +, CD4−CD8+(DN) and CD8+ NKT cells. In contrast, mice lack CD8 as a marker for NKT cells. Thus, murine NKT cells are subdivided by the presence or absence of the CD4 marker only −CD8− NKT cell subset in humans exhibits a Th1-biased cytokine profile  +CD8+ NKT cell subset in human is supposed to induce a Th2-type immune response similar to the CD4+NKT cell subset in mice γ and MIP1-α in males but similar frequencies of cells in both genders producing IL-4 Interestingly, cytokine production of murine NKT cells can be modulated by testosterone. Liver lymphocytes isolated from testosterone substituted female mice produced significantly less IFN