The L1 cell adhesion molecule promotes neurite outgrowth and neuronal survival in homophilic and heterophilic interactions and enhances neurite outgrowth and neuronal survival homophilically, i.e. by self binding. We investigated whether exploitation of homophilic and possibly also heterophilic mechanisms of neural stem cells overexpressing the full-length transmembrane L1 and a secreted trimer engineered to express its extracellular domain would be more beneficial for functional recovery of the compression injured spinal cord of adult mice than stem cells overexpressing only full-length L1 or the parental, non-engineered cells. Here we report that stem cells expressing trimeric and full-length L1 are indeed more efficient in promoting locomotor recovery when compared to stem cells overexpressing only full-length L1 or the parental stem cells. The trimer expressing stem cells were also more efficient in reducing glial scar volume and expression of chondroitin sulfates and the chondroitin sulfate proteoglycan NG2. They were also more efficient in enhancing regrowth/sprouting and/or preservation of serotonergic axons, and remyelination and/or myelin sparing. Moreover, degeneration/dying back of corticospinal cord axons was prevented more by the trimer expressing stem cells. These results encourage the view that stem cells engineered to drive the beneficial functions of L1 via homophilic and heterophilic interactions are functionally optimized and may thus be of therapeutic value. In vitro and in vivo studies support the view that homophilic interactions of L1 not only promote neurite outgrowth and neuronal migration, but also neuronal survival in vitro and in vivo as well as in learning and memory The adhesion molecule L1 has been shown to favour conducive processes in a generally inhibitory environment In previous studies, it has been shown that soluble, recombinantly expressed dimeric L1 containing the extracellular domain of the molecule in fusion with the Fc portion of human immunoglobulin (L1-Fc) promotes locomotor recovery in adult rats after contusion spinal cord injury Given the potential of L1 to be of therapeutic value in nervous system diseases and that its homophilic interactions are conducive, it deemed important to improve L1's efficacy in injuries of the central nervous system. We hypothesized that secretion of a trimer presenting its extracellular domain by stem cells constitutively overexpressing full-length L1 at the cell surface under the control of a universal promoter would enhance regeneration after spinal cord injury even more so than the L1 overexpressing cells. A trimer of the extracellular domain of L1 was constructed for expression with the aim to enhance L1's capacity to cluster cell surface expressed full-length L1 via homophilic interactions, thus inducing signal transduction events conducive to neurite outgrowth and neuronal survival and conditioning the cellular environment of the injured host spinal cord to enrich the hostile host tissue with a regeneration-conducive adhesion molecule. Here we report that the thus engineered stem cells are more potent in recovery of locomotor functions than the parental cells not expressing L1 or overexpressing full-length L1.The construct p901-L1-ccCMP-His6 was generated using pECE L1-ccCMP-His6 as a template which was kindly provided by Dr. Heike Hall . In this construct, the mouse L1 extracellular domain  is connected at the carboxy-terminus with a sequence encoding the cartilage matrix protein's (CMP's) coiled-coil domain (CMPcc) to generate L1 homotrimers by covalently assembling the CMPcc domains To verify secretion of the trimer in full-length L1 overexpressing ESCs two stably transfected ESC lines were maintained for eight days in culture and the supernatants and cell lysates were collected, dialyzed against 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl, and concentrated ∼10-fold in an Amicon Ultra centrifugation filter device . Samples were subjected to 7.5% reducing and non-reducing SDS-PAGE and probed for by Western blot analysis using a polyclonal antibody against full-length mouse L1, kindly provided by Dr. Martin Grumet (Rutgers University). Protein was determined by BCA protein concentration assay kit .5 cells in phosphate-buffered saline, pH 7.3 (PBS)) was injected 0.5 mm both rostral and caudal to the lesion site using a glass micropipette (tip diameter 100 µm) 1 mm deep into the spinal cord Female C57BL/6J mice, 3 months old, were deeply anaesthetized by intraperitoneal injections of ketamine and xylazine (100 mg of Ketanest and 5 mg of Rompun per kg body weight). Surgery was performed as described 7+n−X7)/(X0−X7)]×100, where X0, X7 and X7+n are experimental values prior to injury, 7 days after injury, and a time-point n days after injury, respectively. Values for the left and right extremities were averaged. Only animals that showed a foot-stepping angle of 160° or more 7 days after injury were considered severely lesioned. All other animals were excluded from further analysis. One animal each was excluded from the tL1 NSC group and PBS vehicle only group, respectively.Ground locomotion was evaluated every week from 1 week to 6 weeks after injury using the Basso Mouse Score (BMS) rating scale Animals were deeply anesthetized by intraperitoneal injection of ketamine and xylazine (100 mg of Ketanest and 5 mg of Rompun per kg body weight) and perfused transcardially with cold phosphate buffer, pH 7.4 (0.1 M) followed by 4% paraformaldehyde in 0.1 M phosphate buffer. A 3 cm long tissue piece of the spinal cord centered at the lesion site was dissected out and processed for the different types of analysis as follows.For cryotomy, the spinal cords were post-fixed in the perfusion solution plus 10% sucrose overnight at 4°C, and then cryoprotected in 20% sucrose in PBS for 48 h at 4°C. Then, the spinal cord centered at the lesion site was dissected and embedded in mounting media  on dry ice. Cryostat sections (30 µm thick) were cut and stored at −70°C.For immunohistochemistry, sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min at room temperature. Non-specific adsorption was minimized by incubating the sections in 2% (v/v) normal goat serum in PBS for 20 min at room temperature. Sections were incubated in the blocking solution with primary antibody at 4°C overnight. For control, non-immune mouse IgM and IgG as well as rabbit IgG were used as primary antibodies. Slides were washed three times with PBS and incubated with Alexa 555-conjugated goat anti-mouse IgG or Alexa 555-conjugated goat anti-rabbit IgG  for 2 h at room temperature. Sections were then washed with PBS, and some sections were incubated with 4′-6-diamidino-2-phenylindole (DAPI) for 10 minutes, rewashed with PBS, mounted with Aqua Poly/Mount medium , and imaged with an Axiovert200 Fluorescence Live Cell Imaging Workstation (Zeiss). Control sections did not yield detectable signals.Lesion volume was estimated in parasagittal sections spaced 200 µm apart and stained with polyclonal antibodies against GFAP  and fibronectin  to estimate the scar volume using the Cavalieri principle.Parasagittal sections were double-immunostained for serotonin (5-HT)  and fibronectin  to determine 5-HT+ fibers which had sprouted/regrown into lesion site and caudal beyond the fibronectin-immunostained lesion site 6 weeks after the injury. 5-HT+ fibers were imaged with the Axiovert 200 Fluorescence Live Cell Imaging Workstation (Zeiss) and quantified by AxioVision software (Zeiss). Background intensity from an area with no 5-HT immunoreactivity was subtracted from the intensity value. The values are presented as the area of 5-HT+ fibers from 2 mm rostral to the lesion site and the % ratio of fluorescence measured 1 mm caudal to the lesion over the background immunofluorescence measured rostral (1 mm) to the lesion site, respectively.For the assessment of axons in the corticospinal tract (CST) by PKC-γ immunostaining, free-floating cross-sections (50 µm thick) were blocked and then incubated with anti-PKC-γ antibody  overnight at 4°C. After washes in PBS, the sections were incubated with fluorescent Alexa 555 goat-anti mouse secondary antibody . Images were taken of the dorsal spinal cord columns at 10×magnification using Axiovert 200 Fluorescence Live Cell Imaging Workstation (Zeiss) at 3, 6, 9, and 11 mm rostral to the injury center. Using ImageJ Software (NIH), the relative intensity of PKC-γ immunoreactivity was measured in a traced area within the dorsal columns. Background intensity from an area with no PKC-γ immunoreactivity was subtracted from the intensity value. Automatic thresholding for each image using ImageJ software was performed to determine the threshold for specific immunoreactivity. After setting the threshold, the immunostaining intensities above the threshold were quantified.M NaCl, 3 mM KCl, 1 mM NaHPO4, 26 mM NaHCO3, 1.5 mM MgSO4, 1.5 mM CaCl2, and 10 mM glucose pH 7.0), and cleaned from meninges and nerve roots. A 1 cm long tissue piece of the cord centered at the lesion site was dissected and homogenized . For slot blotting, 100 µg of total protein/well was transferred to polyvinylidene difluoride membranes using a Bio-Dot slot blot apparatus . The membranes were then incubated with rat L1 monoclonal antibody (555) , mouse monoclonal chondroitin sulfate antibody (CS56) , mouse monoclonal NG2 antibody , rabbit polyclonal glial fibrillary acidic protein (GFAP) antibodies , rat monoclonal myelin basic protein antibody , mouse monoclonal neurofilament H (NF200) antibody , mouse monoclonal beta-III tubulin antibody , rabbit polyclonal numb antibody  and mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody . Secondary mouse, rat or rabbit antibodies conjugated to horseradish peroxidase with ECL illuminescence intensification  were used for detection. The grey value of each band was measured and normalized to the grey value of the corresponding GAPDH band using Gel-Pro Analyzer software .Spinal cords were dissected, placed in ice-cold artificial CSF , followed by Tukey's post-hoc test. The p-values<0.05 were considered statistically significant.All numerical data are presented as group mean values with standard error of mean (SEM). Statistical analyses for locomotor behavior were performed by one-way repeated-measures ANOVA comparing groups followed by Tukey's Western blot analysis was performed to test L1 as full-length transmembrane molecule and as secreted trimer in cultured embryonic stem cells (ESCs). The expression and secretion of the trimeric L1 was observed in the cell lysate and supernatant of a stably transfected ESC line overexpressing trimeric L1 (tL1) with an apparent molecular mass of 380 kD in SDS-PAGE under non-reducing conditions (p<0.01) or wt NSCs at 3, 4, 5 and 6 weeks (p<0.01) at the different time points indicating superior locomotor recovery with the maximal mean score values being 5.1±0.16 at 5 weeks  score one week after injury Fig. 2A.p<0.01) or wt NSCs at 3 weeks (p<0.05) and at 6 weeks (p<0.01) (p<0.05) or wt NSCs at 3 weeks (p<0.05) and at 6 weeks (p<0.01) (p<0.05). No differences were observed between the mice transplanted with tL1 NSCs or L1 NSCs, although the former showed a tendency towards improved motor function than the latter three and six weeks after injury (p<0.01) or wt NSCs (p<0.01) at six weeks after transplantation  and showed a slightly, although not significantly reduced lesion volume in comparison with mice grafted with L1 NSCs  or wt NSCs (p<0.01) (p<0.01) or the mice grafted with wt NSCs (p<0.01)  Fig. 4F.p<0.05 for 21.5 kDa) and (p<0.01 for 18 kDa) or wt NSCs (p<0.01 for 21.5 kDa and 18 kDa)  and 9 mm (p<0.05) (p<0.01)  was analyzed. Axons caudal to the lesion site were observed to undergo Wallerian degeneration, whereas axons in the rostral segment were observed to undergo retrograde axonal degeneration (“dying back”). Integrity of the CST was assessed by immunostaining for PKC-γ p<0.01) (p<0.01).Western blot analysis under non-reducing conditions identified tL1 in homogenates from 1 cm long spinal cord segments containing the lesion site one week after transplantation with tL1 NSCs. The homogenates from these segments obtained 6 weeks after the injury and NSCs transplantation were probed by Western blot analysis using antibodies against chondroitin sulfate (ChS), GFAP and the chondroitin sulfate proteoglycan (CSPG) NG2. Reduction in the levels of CSPGs, GFAP and NG2 expression was observed in mice grafted with tL1 NSCs, when compared to mice grafted with wt NSCs (p<0.01) Fig. 7. The present study was undertaken to probe the need for improving the efficacy of the functions of L1 by engineering stem cells to overexpress full-length L1 and, in addition, a secreting the trimer of the extracellular domain of L1 in order to optimize not only for the beneficial homophilic and also heterophilic interactions benefitting the transplanted stem cells in a fashion that could be perceived to act via “autocrine” mechanisms (L1 interacts with itself in trans-interaction), but also to favourably condition the hostile tissue environment in a “paracrine” fashion to tip the balance towards regeneration-conducive cues. The hypothesis was that stem cells with this configuration of L1 expression would not only be stimulated in L1 signal transduction mechanisms leading to a combination of beneficial events in the full-length L1 overexpressing stem cells triggered by the L1 trimer, but also lead to diffusion of the trimeric L1 away from the engineered cells into the host tissue and thus condition it rostral and distal to the lesion site for tissue preservation and overcoming the inhibitory cellular environment of the host, respectively. An example of this conducive effect is that the transmembrane form of L1 is able to overcome its inhibitory signaling complex with neuropilin-1 in vitro by addition of L1-Fc Our study lends support to the view that we have optimized the beneficial functions of L1 in neural stem cells. It is noteworthy in this context that L1 not only interacts via homophilic mechanisms, but has many ways of interacting in heterophilic modes, one of which relates to its heterophilic cis-interactions resulting in neurite outgrowth and homophilic trans-interactions which results in neuronal survival, via distinct, but also more down-stream similar signal transduction cascades We have shown previously that full-length L1 overexpressing embryonic stem cells differentiated into the neural lineage are able, without trimer expression, to promote murine corticospinal tract axons regrowing into the lesion site of spinal cord and, in some cases, extending beyond it one month after transplantation. These results indicated that L1 expressed by stem cells may confer beneficial properties to the stem cells themselves by presenting a favorable substrate to the severed axons so that they would cross the lesion site and lend to them trophic support Based on these observations we sought to combine the regeneration-promoting effect of transmembrane-anchored L1 with diffusible L1 to promote regeneration after injury. We observed beneficial effects of this combination by assessment of locomotor recovery using the BMS score and quantitative evaluation of hind leg posture indices and, as observed previously The results of the present study encourage the view that neural stem cells can be used to optimize the beneficial functions of L1 and thus contribute to an improved amelioration of the behavioral deficits after injury in the acutely lesioned spinal cord. Based on our previous studies we hope that the optimized cells would also ameliorate the deficits in mouse models of neurodegenerative diseases, such as Huntington's


Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species.Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE.Intron-F's (inserted at L798 position) were found in 88% of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of  Phialophora verrucosa was described by Medlar in 1915  and [V]. Collectively, this tree displays that all introns of P. verrucosa generated by the core regions are members of subgroup IC1s.Alignment of the core regions consisting of highly conserved sequences of the elements of P, Q, R and S and the pairing segment P3 and the nucleotide sequences, in particular, the last two nucleotides GC of the Q element and the first and second GU nucleotides of the R element  Additio showed tP. verrucosa were predicted to be the same structures as the putative structure of intron-F derived from PV1 drawn in Figure The predicted secondary structure of the intron-F and G were constructed as follows. The conserved P, Q, R and S regions of intron-F (L798) from PV1 were initially aligned with the same regions from other taxa, and then regions of P1 through P10 were constructed and added on the basis of the secondary structure model as shown in Figure The structure of intron-G (L1921) from PV1 was drawn just as was done for intron-Fs Figure . A G-C pP. verrucosa belong to IC1 group 1 introns.In conclusion, we have identified that the ten intron-Fs and four intron-Gs of in vitro in a previous report , Fungi/Ascomycota; [Ac], Acanthamoebidae; [C], Chlorophyta; [H], Heterolobosea; [M], Mycetozoa and [R], Rhodophyta.For the comparison with highly conserved sequences of subgroup IC1 from 20 taxa, sequences of elements of P, Q, R and S and the pairing segment P3 were obtained from DDBJ database  regions of the P, Q, R and S were marked with dotted lines. Boxed nucleotides participate in the pairing segments of P1-P10 of the secondary structure model.Click here for fileAlignment of intron-G used for the phylogenetic analysis and the modeling of secondary structure. The gaps were marked with dashes. The highly conserved (ribozymatic core) regions of the P, Q, R and S were marked with dotted lines. Boxed nucleotides participate in the pairing segments of P1-P10 of the secondary structure model.Click here for file


A substantial number of “retrogenes” that are derived from the mRNA of various intron-containing genes have been reported. A class of mammalian retroposons, long interspersed element-1 , has been shown to be involved in the reverse transcription of retrogenes (or processed pseudogenes) and non-autonomous short interspersed elements (SINEs). The 3′-end sequences of various SINEs originated from a corresponding LINE. As the 3′-untranslated regions of several LINEs are essential for retroposition, these LINEs presumably require “stringent” recognition of the 3′-end sequence of the RNA template. However, the 3′-ends of mammalian L1s do not exhibit any similarity to SINEs, except for the presence of 3′-poly(A) repeats. Since the 3′-poly(A) repeats of L1 and Alu SINE are critical for their retroposition, L1 probably recognizes the poly(A) repeats, thereby mobilizing not only Alu SINE but also cytosolic mRNA. Many flowering plants only harbor L1-clade LINEs and a significant number of SINEs with poly(A) repeats, but no homology to the LINEs. Moreover, processed pseudogenes have also been found in flowering plants. I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized a specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution. Gene duplication is a fundamental process of gene evolution . There aEukaryotic genomes generally contain an extraordinary number of retroposons such as long terminal repeat (LTR) retrotransposons, LINEs or non-LTR retrotransposons, and SINEs , 14, 15.in vitro .–22.20–22Eickbush's group conducted comprehensive phylogenetic analysis of LINEs using extended sequence alignment of their RT domains . All ideThe 3′-end sequences of various SINEs originated from a corresponding LINE  31]; fo; fo31]; Since the R2 LINE protein specifically recognizes the sequence near the 3′-end of the RNA transcript for the initiation of first-strand synthesis , 17, theρ = 0.25), the number of SINEs with a LINE tail is positively correlated with the number of LINEs belonging to each clade (R2 = 0.83); that is, more LINEs tend to lead to more SINE/LINE pairs. Therefore, although a few LINE clades are the predominant source of SINE/LINE pairs, it is plausible that this simply reflects the large number of LINEs in these clades. However, L1-clade LINEs are the only prominent exception to this. Although over 800 L1-clade LINEs appeared in the database, only 3 SINEs with L1 tails were found , on, on34], In accordance with this hypothesis, almost all L1-clade LINEs in flowering plants end in poly(A) repeats, while all RTE-clade LINEs end in (TTG)n or (TTGATG)n  34]. As. As34]. Comprehensive phylogenetic analysis of L1-clade LINEs revealed three important points . First, One monocot L1 lineage (monocot 1a in ME1) consisted of a large number of L1-clade LINEs that were identified mainly in the recently released maize and sorghum genomes. Moreover, one group of LINEs in this lineage retained a conserved 3′-end sequence . The aveFurthermore, the putative transcript from this region forms a putative hairpin structure  34]. Co. Co34]. Chlamydomonas SINEX-3_CR [The last example of a SINE/LINE pair in the L1-clade was found in a green alga. The 3′-end sequence (~80 nucleotides) of NEX-3_CR  was veryNEX-3_CR . Since lNEX-3_CR , the folIt is possible that the ancestral L1-clade LINE in the genome of the common ancestor of green plants possessed stringent, nonmammalian-type RNA recognition properties. During the course of plant evolution, an L1 lineage then lost the ability to recognize specifically the RNA template for reverse transcription, thereby introducing relaxed 3′-end recognition in land (flowering) plants as well as in mammals. This model assumes that rigid sequence specificity was an ancestral state, although the timing of its loss might be subject to debate. Since horizontal transfer of LINEs between eukaryotes is rare , 85–87, The ancestral L1-clade LINE might have required the 3′-end sequence and the terminal poly(A) repeats. A few L1 lineages might then have lost their specific interaction with the 3′-UTR of the template RNA, retaining some role for the 3′-repeats. As shown in Alternatively, the ancestral L1-clade LINE may have possessed relaxed, mammalian-type RNA recognition properties. During the course of plant evolution, the L1 lineages of land plants (ME1) and green algae might then have gained specific stringent-type recognition of the RNA template. However, it is difficult to imagine that the molecular machinery for rigid sequence specificity, such as the particular conformation of the RNA-binding domain, has arisen independently under reduced constraints.In vivo retroposition assays have been developed for several LINEs [al LINEs , 39, 48.L1 LINEs have contributed significantly to the architecture and evolution of mammalian genomes, whereas LTR retrotransposons are overwhelmingly found in certain flowering plants. Understanding the independent origins of flexible 3′-end recognition may help us to determine what distinguishes the fate of a retroposon in the eukaryotic genome and why it has succeeded so well in certain genomes –93.


We consider a model for the evolution of surname distribution under a gender-equality measure currently being discussed by the Spanish Parliament . We quantify how this would bias the alphabetical distribution of surnames, and analyze its effect on the present distribution of surnames in Spain. In Spain, as in many other countries, children usually inherit their father's surname. As a consequence, the mother's surname is lost in the child's generation . Nowadays, in Spain, parents can agree upon whether it is the mother's or father's surname that is given to their children, but if parents do not reach an agreement, it will automatically be the father's surname that is inherited by the children. Due to gender-equality issues, a new law is under review whereby, if either, parents do not reach an agreement, or if no wish is expressed, the surname inherited by the children will be selected according to the alphabetical order of the parents' two surnames.People have immediately realized that this implies a bias on the surnames favoring those beginning with the first letters in the alphabet  and could mean that surnames beginning with the last letters  disappear completely. In this short paper, we quantify the effect of this bias on the present distribution of surnames in Spain.Evolution of surnames distribution  has been studied previously. The pioneering work of Galton and Watson used a branching process to study the probability of extinction of surnames in English aristocracyAs a first order model aimed at capturing the essence of the process of surname inheritance we propose the following:We measure time The population evolves according to a bisexual Galton and Watson branching processThe model introduced above is a minimal model and does not consider some realistic issues such as geographical distribution of surnames, etc. but those are expected to be second order effects with little impact in the overall trend. The effect of immigration and population growth is analyzed later in the text.Let us define The distribution of surnames at time Eq. (4) shows that the distribution of surnames approaches a Kronecker-delta at www.ine.es . Similar data is available for other countries. Our simulation results only consider those We have applied the above results to the current distribution of Spanish surnames. Besides the analytical result of Eq.(5), we have performed a numerical simulation of the model by which The evolution in the frequency of a surname does not have to be monotonous, as it can first increase and then decrease in time. Let us take, for instance, the most common surname in Spain: “Garca”. According to the INE data, there are 1,481,923 people bearing this surname and the cumulative distribution is In the previous analysis we assumed that the population remains constant (there is an average of two children per couple) and that the only changes in the distribution of surnames correspond to the application of the alphabetical order rule. We now consider the effect that, both, population growth and new surnames brought in by immigration have in the distribution of surnames. This implies modifying condition Let The first term corresponds to mating of two people  both with surname in alphabetical position From this equation, and after some algebra, one can obtain the evolution of the number of people We see from Eq.(8) that the intrinsic growth, An analysis of this expression, shows that a critical value www.ine.es. Similar data is available for other countries. We have also performed numerical simulations of an agent-based model, which agree with the analytical result.We have developed a mathematical model for the evolution of the surnames distribution when an alphabetical-order rule on the progenitor's surnames is applied. The premises of the model lead to a differential equation governing the evolution of the probability distribution. As initial condition we have considered the data for the present distribution of the In our minimal model for surname transmission, we prove that the adoption of the alphabetical rule leads to an exponential decrease in the surnames that begin with letters that are towards the end of the alphabet, with a characteristic decay time of We believe that this study offers an example in which statistical methods and mathematical modeling can be used to quantitatively calculate the consequences of a political measure and, consequently, it can serve as a guide to institutions and policy makers.


Ca3.1 K+ channel-dependent cell processes contribute to IPF pathophysiology. Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that KCa3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of KCa3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]). KCa3.1 channel mRNA and protein. KCa3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. KCa3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. KCa3.1 was expressed strongly in IPF tissue. KCa3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility invitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca2+.Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed KCa3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking KCa3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic. K Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial pneumonia of unknown etiology . The terThe pathogenic mechanisms involved in IPF initiation and progression are poorly understood . Myofibr2+ activated K+ channel KCa3.1 plays an important role in Ca2+ signalling through its ability to maintain a negative membrane potential during cell activation -1H-pyrazole (TRAM-34) (200 nM)i were assessed with the use of a flurometric Ca2+ probe Fura-2 (Sigma-Aldrich). Myofibroblasts were grown on 35 mm fluorodish cell culture dishes  and when 40% confluent loaded with Fura-2 for 45 min at 37°C in normal physiological saline solution  containing 5 µmol/L fura-2-acetoxymethyl ester and 2.5 mmol/L probenecid. The cells were then washed and [Ca2+]i recorded using the single cell recording system previously described i occur following stimulation with TGFβ1. TGFβ1 (10 ng/ml) elicited an immediate, rapid rise in [Ca2+]i in 100% of IPF cells and 53% of NFC cells. There was a significant increase in [Ca2+]i for both IPF  and NFC 11a. In 01). These observations are in keeping with bFGF- and TGFβ1-dependent KCa3.1 upregulation in several cell types i concentrations which was blocked by TRAM-34. This demonstrates how both membrane hyperpolarization and Ca2+ influx is critical for myofibroblast function in keeping with observations in other cells types [Ca3.1 in lung myofibroblast responses. Upon TGFβ1 stimulation human lung myofibroblast exhibited a rise in [Cals types ,11,26, aCa3.1 channels were more highly expressed in IPF myofibroblasts than in NFC cells, the functional responses of the cells to growth factors were largely similar irrespective of their source. This is perhaps not surprising because membrane hyperpolarization can be achieved with as few as 12 channels per cell [2+ dependent cell processes can proceed with very low channel expression. Similar shifts in membrane potential in NFC and IPF myofibroblasts are evident in Ca3.1 blockers are attractive targets as they block all channels and will potentially attenuate pro-fibrotic responses as shown in this study.Although Kper cell , meaningCa3.1 was expressed in both NFC and IPF lung tissue, supporting the relevance of our in vitro findings. In IPF tissue, KCa3.1 was not only expressed in areas of αSMA positivity, but also highly expressed in alveolar epithelial cells, vessels and inflammatory cells. In addition to alveolar epithelial cells, both mast cells and fibrocytes are implicated in IPF progression and express KCa3.1 [Ca3.1 in IPF may therefore have additional benefits over and above those achieved through the inhibition of myofibroblast function.Ks KCa3.1 ,26. TargCa3.1 channel may play a key role in the development of lung fibrosis, in both IPF and other lung disorders. KCa3.1 is an attractive pharmacological target as it appears to play a minor role in healthy physiology, but contributes significantly to tissue remodeling and fibrosis [Ca3.1 opening contributes to pulmonary vascular relaxation in rats[Ca3.1 knockout mice are viable, of normal appearance, produce normal litter sizes, and exhibit rather mild phenotypes [Ca3.1 blocker, ICA-17043, has been administered to humans in phase 2 and 3 trials of sickle cell disease with minor side effects [Ca3.1 blockade in clinical trials of IPF and other fibrotic lung diseases.Our data indicate that the Kfibrosis . It has n in rats, but thin in rats. Importaenotypes . High doenotypes  and the  effects . There i


The purpose of this study was to design a valid questionnaire suitable to the Iranian culture to measure the stress mounted on dementia caregivers.In order to design a valid and reliable tool, the stages of content validation were performed as follows: 1- Development: search of relevant electronic databanks and use of experts and caregivers’ opinions to prepare appropriate content, review and correction of the content through consecutive focus group discussions with experts. 2- Judgment Quantification: determination of interrater agreement (IRA), relevancy and clarity of each of the items and the tool as a whole. Reliability was measured with Cronbach’s alpha, and repeatability was measured with intracluster correlation through repeated test-piloting at 2-3 week intervals.Using a conservative approach, the IRA for the overall relevancy and clarity of the tool was 87.87% and 81.81%, respectively. Through overall agreement  the overall relevancy of the tool obtained was 98.62%. The overall clarity of the tool was calculated through the mean clarity of the questions and was 99.3%, and eventually its comprehensiveness was 100%. The overall Cronbach’s alpha was 94% and the intracluster correlation that was obtained through comparing the overall score of the questionnaire in the pretest and test phase was 97%.The new tool has good reliability and validity suitable to Iranian dementia patients and their caregivers’ culture. Researchers can use this tool to monitor the pressure mounted on dementia caregivers and to assess interventions in this group. Population aging could be considered as an important consequence of demographic transition, a decline in death rate results in an increase in the proportion of the elderly. On the other hand, a rise in the prevalence of geriatric diseases is a consequence of elderly population growth in all countries which is more remarkable in developing countries like Iran. Dementia is one of the diseases that an increase in its prevalence among the elderly is predictable. Approximately 31 million Iranians are in their middle age nowTo design the questionnaire, appropriate content was prepared through literature review and interviews with content experts and lay experts . Then, the reliability of the instrument was evaluated.To find out the pre-existing instruments, we started this stage by searching different databases . According to the experts’ opinion, the ‘Zarit Burden Interview’ (ZBI) was considered as the main instrument and one of the most commonly used and comprehensive instruments for measuring caregivers’ burden of care for patients with dementia. This questionnaire, which contains 22 questions, has been used in different studies and articles throughout the world.The selected questions were translated to Persian two times by two different translators and after approving the translations, the final Persian version was prepared by the research team. The Persian version was back-translated to English by a person who was proficient in English language and this version was compared to the original questionnaire by a person who knew English as much as a native speaker. Cases of any discrepancies between the concepts of the translated and original version questionnaire were recognized and the necessary changes were made to the Persian translation.The initial Persian version of the questionnaire was sent to three neurologists with enough experience of dementia and two psychiatrists to compare it with the original questionnaire and to gain reassurance about its representativeness. To equalize the experts’ conception of content validity indices , the definitions of these indices were sent along with the questionnaire. The ability of selected questions to reflect the content was defined as relevancy while the questions lucidity concerning their wording and concept was labeled as clarity, and finally, the instrument’s ability to include all content domains was considered as instrument comprehensiveness. Then, the experts were asked to compare the Persian version with the original one and after reviewing every item, rate its clarity and relevancy from 1 to 4 . Moreover, we asked them to:Edit the questions in case it was necessary to improve the item’s clarityDelete the question(s) which did not have appropriate relevancy from their point of view.“Suggest the question(s), adaptable to the Iranian patients and their caregivers’ culture.”The questions of the questionnaire were sent to five alert caregivers as well. To make the definitions of relevancy, clarity, and instrument comprehensiveness more understandable to caregivers, the definitions sent to the content experts were modified a bit. They were asked to share their opinion on the indices and suggest the questions which they believed were appropriate to the Iranian culture. At this stage, the face validity of the questionnaire was investigated too. The experts’ answers were collected in a 1 to 3-week period. Their opinions about the items wordings, in addition to their suggested questions that were not present in ‘Zarit’, ‘Caring for The Caregivers’, and ‘Caregiver Burden Scale’ were evaluated in several brain-storming sessions in the presence of experts and the team of investigators. In all, twelve brain-storming sessions were held to prepare and approve the questions.In the summer of 2009, to determine the reliability of the questionnaire and to find out the executive problems, a pretest was carried out on the caregivers referred to ‘Iran Alzheimer Association’. After a 2-3 week interval, the respondents filled in the questionnaire again to assess the repeatability. Caregivers were included in the study only if they had the inclusion criteria of the study which was having a close familial relationship  and agreement to participate in the study. The questionnaires were filled in via surveying. The participants were asked to mark the unclear items, if they found any, and explain the ambiguity (unclearness) of each one. Reliability was evaluated in two aspects, repeatability and internal consistency.A conservative approach was chosen to determine the interrater agreement (IRA) for the instrument relevancy and clarity. In this approach, the number of the questions which all (100%) of the experts  chose “quite appropriate” and “appropriate” or “somewhat appropriate𠇜 and “inappropriate” for relevancy  was divided by the total number of items. IRA for the instrument clarity was calculated exactly by the same method.171820To calculate the clarity of each question, the total number of experts who chose “appropriate” or “quite appropriate” for clarity of each item, was divided by the total number of the experts. Calculation of the relevancy of each item was performed exactly the same way.1620 To calculate the relevancy of the total instrument, Scale-Content Validity Index/Universe Agreement approach (S-CVI/UA) was used. In this approach the total number of the questions with appropriate relevancy was divided by the total number of the items.1620 In different articles, the minimum acceptable relevancy for a new instrument has been suggested as 80%.s by the total number of questions (mean of the questions clarity or mean of I-CVIs). The instrument comprehensiveness was achieved via dividing the number of experts who judged the comprehensiveness of instrument appropriate by the total number of experts. The questionnaire’s total score, which is the indicator of the burden on the caregiver, was calcu-lated by adding the item scores.The clarity and relevancy of the total instrument are achievable through different methods. In this study, after combining choices “inappropriate” and “somewhat appropriate” and also “appropriate” and “quite appropriate” together, binary choices for each question were computed: appropriate and inappropriate.Intra-class correlation (ICC) and alpha-Cronbach were used to estimate the reliability and internal consistency, respectively. ICC and alpha-Cronbach’s higher than 0.7 were considered acceptable. The total ICC was obtained by comparing the total scores of the questionnaire in two stages at 2 to 3 weeks interval. The questions, which had reliability problems in either repeatability or internal consistency, were evaluated and proper decisions were made about this group of items by researchers and experts. SPSS (version 15) was used for statistical analysis.All experts’  feedbacks on relevancy, clarity, and comprehensiveness of the 23 questions extracted from the literature review were collected in 1 to 3 weeks (response rate=100%). Besides the 23 items selected from ZBI, Caring for The Caregiver, and Caregiver Burden Scale, the psychiatrists and neurologists suggested 10 more questions which were fitted to characteristics of Iranian Alzheimer patients and their caregivers. These 10 questions were finalized by the team of investigators in brainstorming sessions. After asking the experts for their opinion about the relevancy and clarity of the ten suggested questions, the draft of the questionnaire was prepared which included 33 items. According to the lay and content experts’ questions rating, the IRA used in conservative approach for the clarity and relevancy of these 33 questions were 81.81% and 87.87%, respectively. The relevancy, clarity, and comprehensiveness of the final instrument were 98.18%, 98.78%, and 80%, respectively. From the 21 participants who filled in the questionnaire at the test stage, only one was not available to refill it (response rate=95%). The participants’ characteristics are showed in The average time for completing the questionnaire (by interview) for each person was 14 minutes (range: 10 to 25 minutes). From the respondents’ point of view, there were no unclear question in the questionnaire. As the phrase “spouse and children” in question number 30 , “Do youAlthough the total ICC of the instrument was acceptable, it was not desirable (minimum 70%) for 6 questions. For this reason, and to improve the total ICC of the instrument, these items were discussed in brainstorming sessions by content experts and methodologists and finally the following decisions were made about them: Questions 3 (Do you feel stuck between caring for your patient and fulfilling your other family or work responsibilities?), 13 (Do you feel uncomfortable about inviting your friends to your house because of your patient’s presence?) and 25 (Have you ever felt frustrated over caring for your patient?) had unacceptable ICC, and the content experts and methodologists believed that deletion of these questions would not have any effect on the relevancy (representativeness) and total instrument comprehensiveness. Moreover, these deletions would lead to a shorter time and less expense for completion. So, we decided to delete them. In addition, the content experts believed that the concept of question 22 covered that of question 3 and that its omission would not have any impact on the relevancy and validity of the instrument.Although the ICC of questions 19 , 26  and 27  were lower than the acceptable level , the content experts decided to keep them in the questionnaire because of their high relevancy.Questions 20 (Do you feel that you should pay more attention to your patient?) and 21 (Do you feel that you could have taken better care of your patient) were replaced by “Do you feel that you could have taken care of your patient better and more than what you have done so far?“ which includes the concept of both.After making the abovementioned revisions, the relevancy, clarity, ICC, and Cronbach’s alpha of the total instrument changed to 0.986, 0.993, 0.967, and 0.943, respectively. The changes were implemented according to the content experts’ view and by considering the following two principles arrived at the brain-storming sessions:Improving the relevancy and clarity indices of the questions with ICC lower than appropriate level by editing their wordingReducing the number of the questions by deleting the items with inappropriate ICC in order to decrease the time (and cost) needed for questionnaire.As shown in “Using the content validation process we carried out this study to develop a questionnaire adaptable to Iranian culture for evaluation of “the patient with dementia burden on their family members.” Today, the more quantitative content validation process, using content and lay experts’ views, make this process as a proper tool to design a questionnaire with acceptable relevancy, clarity, and comprehensiveness.1617The unacceptable ICC in question number 25, which hints at the caregivers’ disappointment, can be attributed to an ambiguity in “frustration” and its uncommonness in Iranian culture. Due to these reasons we left this question out of the final instrument. The participants’ concern over answering question 13, which asked about the caregivers’ unwillingness in inviting their friends to their houses because of their patient’s presence, might be the reason behind its low ICC. Because the concept of question 22 covered the meaning of question 3, this question’s omission did not influence the relevancy of the instrument. Obtaining the ICC of the question which expresses the concept of questions 20 and 21 (Do you feel that you could have taken care of your patient better and more than what you have ever done?) could be one of the subjects of future studies. The noteworthy point about the questions suggested by the Iranian content and lay experts is the appropriateness of their validity index (relevancy and clarity); none of them was omitted and all of them were included in the final questionnaire. However, the ICC of questions 22 and 23 was not at the appropriate level. Three questions  were left out of the items selected from other questionnaires . After iIn July 2009, after gaining confidence about the questionnaire content validity and to determine its reliability on ICC and internal consistency  we entered the retest stage on the caregivers referred to the Iranian Alzheimer Association in a 2 to 3-week interval. After making the final changes, the total ICC of the instrument was calculated at 0.97; this showed the test repeatability. The ICC in the Brazilian and Chinese versions of the questionnaire was 0.88 and 0.99, respectively.77The results of this study confirmed the usefulness of the content validation process to develop valid and reliable content for questionnaires in medical research. Therefore, using the mentioned process, the developed questionnaire has a very appropriate validity and reliability adaptable to Iran’s culture and circumstances. The necessity of precise execution of different stages of content validation, performing required revisions of the generated questions, evaluating the instrument in each revision phase, and returning to the previous stage if it is necessary to determine the validity of a new instrument have been emphasized. Designing “Caregivers Burden of Dementia Patients” questionnaire could be considered as an essential step towards focusing on patients with dementia and their caregivers in Iran because this group of people deserves more consideration and this disease is worthy of more investment.


Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments.The dimorphic fungus  Paracoccidioides spp. is the causative agent of paracoccidioidomicosis (PCM), the most prevalent invasive fungal disease in South America The dimorphic fungus Paracoccidioides spp. have been evaluated PbFKS1) PbMLS) PbICL) PbSMT) Paracoccidioides lutzii Pb01.New therapeutic approaches for PCM have been performed ParacoccidioidesCandida albicans and Mycobacterium tuberculosis have been described in the literature PbICL has been reported to date. PbICL transcripts are highly abundant in P. lutzii Pb01 yeast cells PbICL by phosphorylation is reversible, indicating a new strategy for the rapid adaptation to changing environmental conditions PbICL inhibitors.Since expression of glyoxylate cycle genes malate synthase and isocitrate lyase are detected during specific stages of the interaction between host and pathogen in a variety of human-pathogenic bacteria and fungi PbICL, we focused on argentilactone, the major constituent of the essential oil of Hyptis ovalifolia, as this compound is known to suppress the proliferation of such microorganisms as Cryptococcus neoformans, C. albicans, and the dermatophytes Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton rubrumThere are abundant natural compounds from the Brazilian Savannah flora endowed with antifungal activity. Thus, in an effort to identify agents active against P. lutzii Pb01 yeast cells, during the differentiation from mycelium to yeast, and on recombinant and native PbICL enzymes in the presence of different carbon sources. In silico analyses were performed to corroborate the in vivo studies.Herein, we report the inhibitory action of argentilactone and its semi-synthetic derivatives reduced argentilactone, epoxy argentilactone, and diol argentilactone on m-chloroperoxybenzoic acid was visualized as a means of obtaining the epoxides 3 and 3a through electrophilic attack exclusively at the isolated double bond. The major epoxide 3 was stirred in HClO4 solution to furnish the diol 4.Compounds reduced argentilactone (2), epoxy argentilactone (3) and diol argentilactone (4) were synthesized efficiently using simple and well-established reactions . The hydH. ovalifolia and its derivatives against P. lutzii Pb01 yeast cells was verified in this study. Considering that pathogenic microorganisms utilize different carbon sources during pathogenesis PbICL is regulated by the carbon source, we evaluated the P. lutzii Pb01 yeast cells growth using glucose or acetate as the carbon source in the presence of argentilactone, reduced argentilactone, epoxy argentilactone, and diol argentilactone.The activity of argentilactone from P. lutzii Pb01 yeast cells growth when the fungus was cultured in the presence of glucose or acetate as the carbon source (1 and 2) (1 and 2)  P. lutzii Pb01 exposed to argentilactone (18 µg/mL), showed that after 6 h of incubation there was a decrease of approximately 15% viability ((18 µg/mL) was not toxic to MRC5 cells (data not show).The results showed a dose-dependent inhibition by argentilactone and reduced argentilactone on n source . In contn source . The min1 and 2)  than glu1 and 2) . Argentiiability . ArgentiP. lutzii Pb01 grows as a saprophytic mycelium, resulting in the formation of propagules that initiate the infection in humans when inhaled into the respiratory tract; the mycelial propagules subsequently develop into yeast cells in the lung. The transition is important for infection and disease in vitroet al. PbICL gene in this fungus increase during the mycelium to yeast transition. Considering that the carbon source used regulates PbICL expression, we evaluated whether argentilactone and its derivatives reduced argentilactone, epoxy argentilactone, and diol argentilactone interfered with the dimorphic transition of P. lutzii Pb01 from mycelium to yeast when cultured in the presence of glucose or acetate.In the soil, P. lutzii Pb01 when the fungus was cultured in the presence of glucose or acetate  than glucose (∼30%). The PbICL activity was higher on yeast phase, differentiation, and mycelium, respectively. Those results are in according with the higher level of PbICL transcript and protein during differentiation when compared to mycelium Most microorganisms that are capable of utilizing acetate or other fatty acids as the sole carbon source employ the glyoxylate cycle for the anaplerosis of oxaloacetate, which is dispensable during growth on glucose. Therefore, the glyoxylate cycle and the tricarboxylic acid (TCA) cycle must operate simultaneously because some reactions from the glyoxylate cycle are shared with the TCA cycle st phase . The argParacoccidioides Pb01 yeast cells was obtained to investigate the action of argentilactone and its derivatives reduced argentilactone, epoxy argentilactone, and diol argentilactone on native PbICL activity. Argentilactone and reduced argentilactone inhibited PbICL activity, as shown in 50 = 50 µM) than glucose , possibly due to the high level of PbICL inactivity on glucose, which would bind to argentilactone and reduced argentilactone. No inhibition was observed for epoxi or diol argentilactone. The post-translational regulation of PbICL depending of carbon source could be one of the explanations to the high level of PbICL inactivity on glucose. According to Cruz et al.PbICL protein level in glucose-grown cells is slightly lower than in acetate-grown cells, the PbICL specific activity in yeast cells grown on glucose is much lower than would be expected based on the protein abundance due post-translational regulation of enzymatic activity. The action of argentilactone and derivatives reduced argentilactone, epoxy argentilactone, and diol argentilactone was also investigated using recombinant isocitrate lyase from Paracoccidioides Pb01 (PbICLr). PbICLr inhibition was higher in the presence of argentilactone (IC50 = 28.8 µM) than reduced argentilactone (IC50 = 30.2 µM). In contrast, epoxy argentilactone and diol argentilactone had no effect against PbICLr.A crude extract of PbICL thus far, the amino acid sequence was compared to sequences in Brookhaven Protein Data Bank (PDB) using the BlastP program Aspergillus nidulans ICL (AnICL) (PDB code: 1dqu) was found to be 85% identical to PbICL and thus was selected as the best template among the structures deposited in PDB.Because there is no experimentally resolved 3D structure for PbICL showed very little conformational change when compared to the template AnICL. PbICL had 0.49 Å of root-mean-square deviation (RMSD) when superimposed with respect to the non-hydrogen atoms, and significant conformational changes were only observed for the N-terminal and C-terminal regions  were considered. The conformation of the binding pocket was preserved after the molecular dynamic simulations. More pronounced deviations were observed in sections involving C-terminal residues from ASN500 and, in particular, between PRO269 and ALA300. However, those segments are far outside the region of the binding pocket and could not influence the interaction between a ligand and the residues involved in this region of the protein. Differences in the secondary structure content were rarely observed between the homology and molecular dynamic models, with only higher helix content from residue ALA452 being observed in the homology model.The selected structure of PbICL were limited to only a region surrounding the binding pocket of the protein, which was defined as involving the same residues of the template provided by PbICL binding site (ASP24-GLN55 and ILE227-THR236) were 100% identical to the template AnICL.Simulations involving the search for conformations with the substrates on the surface of PbICL and isocitrate was conducted to evaluate its energy profile in relation to the PbICL binding pocket. As expected for an original ligand, a very specific energy was found, Fscore =  −6.8 Kcal/mol for 100% of the simulations  also had Fscores of approximately −6.8 Kcal/mol, but the energies were less specific, ranging from conformations with an Fscore around or slightly above this value. PbICL. The ammonium group of LYS47 is involved in the interaction with all the compounds. ARG43 appears only to stabilize isocitrate; in the other compounds, this interaction is replaced by ASN53 and GLY235. The ammonium portion of TRP21 is also involved in the interactions with reduced argentilactone and diol argentilactone.PbICL) can be seen in Supplementary PbICL.Mutations involving the ASN53, GLY235 and LYS47 residues were performed to evaluate the importance of the stereochemistry of each one of these residues to provide stability to argentilactone. The multi-point mutations in the key amino acid residues were performed successfully using Swiss-PDB viewer. The point mutations were chosen strategically to dramatically change the size and nature of their side chains to affect the binding pocket. The effect of these mutations on stability of argentilactone compared to the original binding pocket . The ΔGsolv values for the compounds and the magnitude of the error in evaluating ΔGsolv are reported in The solvation free energies ΔGPbICL binding pocket more favorably contributes to the increase in the entropy of the medium. Derivative diol argentilactone also has a ΔGsolv very close to that observed for argentilactone. However, considering the amount of ASA in the PbICL binding pocket and the ΔGsolv proportional to this area, only 1 Kcal/mol more energy would be required to desolvate diol argentilactone compared to argentilactone.Almost four times more free energy is necessary to desolvate isocitrate  in comparison to argentilactone  , indicatPbICL binding pocket favors the formation of several hydrogen bonds between isocitrate-PbICL and isocitrate-solvent. Isocitrate is the most polar among the compounds, therefore a great difference in ΔGsolv can be observed in relation to the other compounds , and the nonpolar groups are very well protected in the PbICL binding pocket . This hypothesis could be evaluated to estimate the ligand dissociation path to calculate the smooth reaction path that links the bound and unbound states. In our next study, these estimates may be determined using umbrella sampling to calculate the potential of mean force and estimate the binding free energy of each compound.Our hypothesis is that torsion and the nonpolar content of argentilactone can provide sufficient flexibility to increase its specificity and facilitate accessibility to the in vivo, in vitro, and in silico results document the inhibitory activities of compound argentilactone and its derivatives reduced argentilactone, epoxy argentilactone, and diol argentilactone on P. lutzii Pb01. Argentilactone inhibited PbICL activity, P. lutzii Pb01 yeast cells growth, and differentiation from mycelium to yeast. The lowest inhibition was found for reduced argentilactone when compared to argentilactone; the lack of inhibition by epoxy argentilactone and diol argentilactone is most likely due to the change in the polarity of the molecules. In silico analyses allowed an assessment of the important aspects of the stability and kinetics of the compounds for accommodation in the binding pocket. The characteristics observed for argentilactone indicate its ability to be accommodated in the PbICL binding pocket, suggesting a higher stability in relation to the other compounds. Therefore, our results indicate that argentilactone is an excellent candidate as an antifungal model.Our 1H and at 75.457 MHz for 13C. CDCl3 was used as the solvent, with Me4Si (TMS) used as the internal standard. TLC was performed using precoated Kiesegel 60 F254 plate (Merck and M. Nagel). The spray reagent used for TLC was 5% anisaldehyde in ethanol NMR spectra were recorded with a Varian Mercury plus BB spectrometer operating at 300 MHz for H. ovalifolia was obtained from fresh leaves by hydrodistillation in a Clevenger-type apparatus for 5 h. The aqueous phase was extracted with diethyl ether, and the organic phase was dried over anhydrous sodium sulfate and concentrated under N2 to yield 0.05% of the essential oil based on the fresh leaf weight. The identification of the compounds was based on the comparison of their retention indices and mass spectra with data in the literature and through computerized matching of the acquired mass spectra with the GC/MS data system. Separation of argentilactone was performed by CC using silica gel (Merck) and hexane - ethyl ether (4∶1). Argentilactone (1): [α]D: −21°  ). IR (KBr) νmax/cm−1: 2928, 1723, 1380. EIMS m/z (rel. int.): 194 [M]+· (3), 97 (20), 68 (100); 1H NMR : Δ H 0.89 , 1.40–1.30 , 2.10 , 2.40 , 5.22 , 5.56 , 5.66 , 6.05 , 6.90 . 13C NMR  Δc 13.7 (C-11), 27.5 (C-8), 28.8 (C-9), 29.6 (C-4), 31.1 (C-10), 73.7 (C-5), 121.6 (C-2), 126.3 (C-6), 135.6 (C-7), 145.0 (C-3), 164.3 (C-1). These data are consistent with the literature The essential oil of 2 (1 atm) and the mixture was allowed to a stand at room temperature for 3 h. The solvent was evaporated. Chromatography of the crude product afforded the compound reduced argentilactone .The reduction of argentilactone was performed using a stirred solution of argentilactone  in ethanol (10 mL), 8 mg of Pd/C under Hm-cloroperbenzoic acid (2.3 g) was added to a stirred solution of argentilactone  in dry CH2Cl2 (15 mL), and the mixture was allowed to a stand at room temperature for 2 days. The CH2Cl2 solution was washed with 10% aqueous NaHCO3 (3×25 mL), dried (Na2SO4), and evaporated. Chromatography of the crude product afforded the mixture of epoxides (3)  and epoxy (3a) . Epoxide (3) (yield 62%). IR (KBr) νmax/cm−1: 2936, 2847, 1734, 1379, 1245, 1042, 808 cm−1. 1H NMR: CDCl3, 500 MHz ΔH 0.90 , 1.34–1.67 , 2.60 , 3.09 , 3.14 , 4.23 , 6.06 , 6.94 ; NMR 13C  ΔC 13.8 (C-12); 22.3 (C-11); 25.9 (C-9); 27.3 (C-7); 27.6 (C-8); 31.4 (C-10); 56.2 (C-6); 57.5 (C-7); 74.5 (C-5); 121.4 (C-2); 144.5 (C-3); 162.6 (C-1).The epoxidation of argentilactone was performed according Alves 3a) (yield: 28%). IR (KBr) νmax/cm−1: 2936, 2847, 1734 (C = O), 1379, 1239 (C = C), 1062 (C-O-C),808 cm−1. 1H NMR : ΔH 0.90 , 1.35–1.60 , 2.35 , 2.53 , 3.03 , 3.18 , 4.32 , 6.06 , 6.89 ; NMR 13C:  ΔC 13.8 (C-12); 22.4 (C-11); 25.9 (C-4); 26.5 (C-9); 28.1(C-8); 31.4 (C-10) 55.7(C-7); 56.8 (C-6); 77.4 (C-5); 121.6 (C-2); 143.6 (C-3); 162.8 (C-1).Epoxide . IR (KBr): 3361, 2924, 2854, 1708, 1385, 1259, 1075, 822 cm-1. Diol argentilactone: [α]D: −82° . 1H NMR , ΔH: 0.90 , 1.31–1.61 , 2.57 , 3.63 , 3.89 , 4.49 , 6.09 , 6.96 ; 13C NMR , Δc: 13.9 (C-12), 22.6 (C-11), 25.3 (C-10), 25.5 (C-4), 31.7 (C-9), 33.6 (C-8), 69.2 (C-7),74.1 (C-6), 77.9 (C-5),120.9 (C-2), 146.1 (C-3), 164.1 (C-1).Epoxy argentilactone  was treated with 2 mL of HClOP. lutzii Pb01 (ATCC-MYA-826) was previously investigated in our laboratory P. lutzii Pb01 yeast cells were sub-cultured every 7 days in solid Fava-Netto’s medium at 36°C. For viability experiments, yeast cells were kept in liquid McVeigh Morton (MMcM) chemically defined medium The differentiation from mycelium to yeast was performed in Mc Veigh and Morton (MMcM) liquid minimal medium P. lutzii Pb01 was grown at 36°C, 200 rpm shaking for 10 days. For the sensitivity assay, 7-day-old P. lutzii Pb01 yeast cells were grown in liquid MMcM overnight at 36°C. Samples containing 105, 104, and 103 cells were spotted onto MMcM agar medium supplemented with argentilactone, reduced argentilactone, epoxy argentilactone, or diol argentilactone at concentrations of 9 µg/mL, 18 µg/mL, 36 µg/mL, or 72 µg/mL. Control plates were did not include inhibitor. The plates were incubated for 6 days at 36°C before being photographed.Inhibition assays were performed by broth macro dilution method according to NCCLS M27-A2 guidelines P. lutzii Pb01 yeast cells grown for 24 h in liquid minimal MMcM medium containing glucose or acetate as the carbon source were centrifuged for 5 min at 2.500×g. The proteins were extracted as previously described g, and the supernatant was centrifuged for 15 min at 4°C and 12,000×g. The cell-free extract was used for the ICL activity assays or stored at −20°C for further analyses. Quantification of the protein content was performed according to Bradford PbICL protein was obtained as described by Cruz Pbicl cDNA was inserted into the pET-32a (+) expression vector . The resulting plasmid was transformed into E. coli BL21 C43 (DE3) cells, and expression was induced at an A600 of 0.6 by the addition of 1 mm  isopropyl thio-β-D-galactoside (IPTG) (Sigma-Aldrich). After induction, the cells were incubated for another 2 h at 36°C with shaking at 200 rpm. The cells were harvested by centrifugation at 10,000×g for 5 min at 4°C and resuspended in 1 × NaCl/Pi buffer. After incubation for 30 min with 100 µg/mL lysozyme, the cells were lysed by extensive sonication. The sample was centrifuged at 4°C and 8,000×g for 15 min, and the supernatant, which contained the soluble protein fraction, was collected. His-tagged ICL was purified using the Ni-NTA Spin Kit (Qiagen), and the tags were subsequently removed by the addition of EKMax™ enterokinase (Invitrogen).Recombinant et al. 2, 10 mM phenylhydrazine HCl, 2 mM dithiothreitol, and 50 mM Tris HCl buffer (pH 7.0). The product glyoxylate-phenylhydrazone was followed at 324 nm using an extinction coefficient of 16.8 mM−1.cm−1. One unit of enzyme activity was defined as the formation of 1 µmol of glyoxylate-phenylhydrazone per minute using threo-D,L-isocitrate as the substrate. The specific activities were given as U.mg−1 protein.ICL activity was determined according to Cruz PbICL and 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM and 90 µM for native PbICL. 3-nitropropinate (50.7 µM) was used as positive control PbICL from mycelium, transition, and yeast, the cells were cultured for 10 days on 23°C and 36°C, and were used 18 µg/mL of argentilactone.The compound concentrations were 10 µM, 20 µM, 30 µM, and 40 µM for recombinant PbICL (537 aa) has not yet been resolved experimentally. Therefore, the amino acid sequences were compared against the PDB using the BlastP program The 3D structure of Molecular dynamic simulations of these structures were performed to improve the relaxation and orientation of the side chains. This procedure is usually crucial in molecular docking to reproduce the structural stability of the receptor in its native environment The particle mesh Ewald method PbICL with a constant temperature of 300 K, 1 atm pressure, time-step of 2 fs, and without any restriction of protein conformations/segments. All information concerning the trajectory of these times were collected every 5 ps. The equilibration of the trajectory was evaluated by monitoring the equilibration of quantities, such as the RMSD of the non-hydrogen atoms, with respect to the initial structure. The analysis of the total energy, potential energy, and kinetic energy were all obtained using GROMACS analysis tools. The RMSD values between the final and template structures also helped to identify the common segments corresponding to the structurally conserved region.The first step in the equilibration of the system was energy relaxation of the solvent for 100 ps; the system was subjected to molecular dynamic only after this step. The simulations were performed for 20 ns for gromos, as described in Daura PbICL. Aiming to prepare the structures for docking, all of the water molecules were removed from the selected structures. ASA was determined using the function of get_area pymol The program g_cluster (GROMACS tool) was used to determine the conformations that best represent the structures of the entire trajectory obtained during the simulation. The algorithm The 3D structures of the compounds were generated using GlycoBioChem PRODRG2SERVER exaustiveness = 8.AutoDock Vina g, given byNrot is the number of active rotatable bonds between heavy atoms in the ligand and w is the associated weight. Because of the exhaustive sampling performed for each ligand, in this work, we consider the Fscore given byAutoDock Vina The conformations of the protein-ligand complexes with the lowest Fscore values extracted from AutoDock Vina PbICL at a constant temperature of 300 K, 1 atm. pressure, time-step of 2 fs, and considering restriction of the protein conformations.The minimization of the complex followed the same protocol used for the receptor in section 4.11 (receptor preparation). A time of 100 ps was required for the equilibration; the system was subjected to molecular dynamic only after this step. The simulations were performed for 2 ns for in vacuum). The free energy differences were determined directly by the sample properly states involved in the transition between A and B by mapping states that are close enough to connect the states AB are functions of the coupling parameter λ, which can be determined according to the fluctuations occurring in Eq. 3, i.e., determining ∂E/∂λ over the entire range between adjacent values of λ. Simulations were performed at different values of λ to obtain a ∂E/∂λ curve from which ΔGAB was derived for each ligand.In this work, the ligand solvation free energy was determined when the ligand was taken from state A (solvated) to another state, B  method for calculating free energy differences PbICL activity experiments were performed in triplicate, and the results are presented as the means (±) standard deviation, Tukey's test was applied to compare the values from enzymatic assays with treated and untreated the compounds and viability assay. A one-way ANOVA multiple test was applied to compare the values obtained from minimal inhibitory concentration and transition. Statistical comparisons were performed using STATISTICA software version 8.0 p<0.05.The Figure S1Fscore of argentilactone obtained in the binding pocket of mutated PbICL. Vertical bars represent the total number of hits observed for each Fscore . Fscore values refer to modes of lower energies observed in the simulations with AutoDock Vina involving mutated and non-mutated binding pocket. Green bars correspond to non-mutated PbICL (native PbICL) Fscore. Bars in blue, red and cyan correspond to mutations involving ASN53, GLY235 and LYS47 residues.(TIFF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective medical therapies. Recent research has focused on identifying the biological processes essential to the development and progression of fibrosis, and on the mediators driving these processes. Lysophosphatidic acid (LPA), a biologically active lysophospholipid, is one such mediator. LPA has been found to be elevated in bronchoalveolar lavage (BAL) fluid of IPF patients, and through interaction with its cell surface receptors, it has been shown to drive multiple biological processes implicated in the development of IPF. Accordingly, the first clinical trial of an LPA receptor antagonist in IPF has recently been initiated. In addition to being a therapeutic target, LPA also has potential to be a biomarker for IPF. There is increasing interest in exhaled breath condensate (EBC) analysis as a non-invasive method for biomarker detection in lung diseases, but to what extent LPA is present in EBC is not known.In this study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess for the presence of LPA in the EBC and plasma from 11 IPF subjects and 11 controls.A total of 9 different LPA species were detectable in EBC. Of these, docosatetraenoyl (22:4) LPA was significantly elevated in the EBC of IPF subjects when compared to controls . A total of 13 different LPA species were detectable in the plasma, but in contrast to the EBC, there were no statistically significant differences in plasma LPA species between IPF subjects and controls.These results demonstrate that multiple LPA species are detectable in EBC, and that 22:4 LPA levels are elevated in the EBC of IPF patients. Further research is needed to determine the significance of this elevation of 22:4 LPA in IPF EBC, as well as its potential to serve as a biomarker for disease severity and/or progression. Idiopathic pulmonary fibrosis is a progressive and ultimately fatal disease in which normal lung is replaced by fibrous scar tissue. The cause of the disease is unknown; however, exposure to refluxed gastric acid, occupational exposures, and viral infections have been postulated as inciting insults -3. The aRecent advances have occurred in our understanding of the mechanisms involved in IPF pathogenesis. Specifically, aberrant wound healing responses to tissue injury, such as epithelial cell apoptosis, increased vascular permeability, extravascular coagulation, and fibroblast migration and activation, have all been implicated in the development of lung fibrosis ,7. Reseasn-1 or sn-2 position. There are numerous LPA species present in biological fluids, identified by the length and degree of saturation of the fatty acid moiety [1-6) [1 receptor, and genetic deficiency or pharmacologic inhibition of LPA1 confers protection against bleomycin-induced lung fibrosis in mice [1 pathway for the development of lung fibrosis, a Phase II clinical trial of an oral LPA1 antagonist for the treatment of IPF has recently been initiated . Recent evidence indicates that the LPA2 receptor can also mediate profibrotic effects of LPA, such as activation of latent transforming growth factor-β (TGF-β), and genetic deficiency of this receptor also results in protection against the development of lung fibrosis in mice [LPA is a biologically active lysophospholipid that has been shown to mediate numerous biological processes thought to contribute to tissue fibrosis . Structud moiety . The majd moiety ,10. LPA’ty [1-6) ,11,12. Tty [1-6) -17. In t in mice ,18,19. F in mice . Based o in mice ,20,21.Given its potentially important and central role in the development of pulmonary fibrosis, LPA is not only a therapeutic target but also a potential biomarker in IPF. While elevated LPA levels have been detected in the BAL from IPF patients , the extSubjects with IPF were identified from those being cared for in the Massachusetts General Hospital (MGH) outpatient pulmonary clinic or inpatient pulmonary consult service. For inclusion in this study, subjects had to meet criteria for a diagnosis of IPF based on the recent joint consensus statement of the American Thoracic Society (ATS), European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT) . ControlEBC was collected using the handheld RTube™ exhaled breath condensate collector , according to the manufacturer’s instructions, and following the ATS/ERS methodological recommendations for EBC collection . CollectBlood was obtained via venipuncture into tubes containing CTAD (citrate-theophylline-adenosine-dipyridamole) additive, in order to potently inhibit platelet activation, as activated platelets are known to release abundant amounts of LPA . Within EBC samples were subjected to lipid extraction using the modified Bligh and Dyer method as described ,32. Brie4COOH as solvent A and acetonitrile:chloroform:water:HCOOH, 80:20:0.5:0.5, v/v with 5 mM NH4COOH as solvent B. LPA molecular species were analyzed in negative ionization mode with declustering potential and collision energy optimized for each LPA molecular species. Individual saturated and unsaturated LPA molecular species  were used as reference compounds. 17:0-LPA was used as the internal standard, and LPA quantitation was performed by creating standard curves with variable amounts of each available LPA molecular species versus fixed amount of the internal standard (17:0-LPA). Total lipid extract from fetal bovine serum was used as a source of otherwise unavailable LPA molecular species to determine their chromatographic behavior and parameters of ionization and collision-induced decomposition, and the quantitation of these LPA molecular species was achieved via the use of the best possible approximation from the standard curves obtained with available individual LPA standards. The identification of LPA molecular species was achieved via monitoring for selected transitions from molecular to product (m/z 153) ions specific for each LPA molecular species, and by the analyte retention time identified by the available LPA standards and by comparing with LPA extracted from bovine serum.LPA levels were determined using electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) with an AB Sciex 5500 QTRAP hybrid triple quadrupole/ion trap mass spectrometer coupled with an Agilent 1200 liquid chromatography system. Lipids were separated on Ascentis Express C8  column using methanol:water:HCOOH, 60:40:0.5, v/v with 5 mM NHStatistical analysis was performed using Prism 6.0 . Differences in LPA levels between IPF patients and controls were analyzed for statistical significance using a two-tailed Student’s t-tests or Mann Whitney tests for parametric and nonparametric data, respectively. To adjust for multiple comparisons, we used the Bonferroni method to calculate the accepted α (Type I) error rate for each individual comparison performed, keeping the family-wise error rate at 0.05. Therefore, for EBC LPA levels, in which 9 different LPA species measured were measured, p values ≤ 0.0055 (0.05/9) were considered statistically significant. For plasma LPA levels, in which 13 different LPA species were measured, p values ≤ 0.0038 (0.05/13) were considered statistically significant.Relevant demographic and clinical data for IPF subjects n = 11) and controls without lung disease (n = 11) on whom EBC and plasma LPA measurements were performed are summarized in Table 1 and conNine different LPA species were detected in the EBC from IPF subjects and controls Table . Of thesThe average EBC 22:4 LPA level in IPF patients was 9.18 +/- 5.19 pM. There was no correlation between 22:4 LPA levels and disease severity, as determined by percent predicted FVC or DLCO (data not shown). One subject had an EBC 22:4 LPA level of 60 pM, which far exceeded the standard deviation of the mean. This subject was a 46 year-old man with biopsy-proven usual interstitial pneumonia (UIP) who required hospital admission for worsening respiratory status in the setting of a suspected IPF exacerbation. Spirometry performed during the patient’s hospitalization and within 10 days of EBC collection demonstrated a severe restrictive deficit, with a FVC of 33% predicted. He subsequently developed respiratory failure and eventually underwent lung transplantation within one month of sample collection. Pathologic review of the explanted lung revealed UIP in the accelerated phase. None of the other ten IPF patients in this study were in the midst of IPF exacerbations during sample collection.LPA has emerged as an important pro-fibrotic mediator in multiple organ systems, particularly the lungs, and the first clinical trial of an LPA receptor antagonist has recently been initiated in IPF patients . In this study, we analyzed the exhaled breath condensate (EBC) and plasma from eleven IPF patients and eleven controls without lung disease for the presence of lysophosphatidic acid (LPA), using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We demonstrated that at least nine LPA species are detectable in EBC, and that one of these species, docosatetraenoyl (22:4) LPA, is significantly elevated in the EBC of IPF patients compared to controls. Thirteen LPA species were detectable in plasma; however, none of these differed significantly between the two groups.Multiple species of LPA exist in biological fluids and are identified according to the composition of their fatty acid side chain. While all LPA species are thought to signal through LPA receptors, there are data indicating that the different species may have differing affinities for the various receptors . Very liIn addition to being a therapeutic target, LPA may also serve as a useful biomarker for IPF. Elevations in LPA have been detected in the bronchoalveolar lavage (BAL) fluid from mice after intratracheal bleomycin administration and from humans with known IPF . 22:4 LPIt is notable that the EBC 22:4 LPA level in one patient was far outside the standard deviation of the mean, and that this patient was in the midst of an IPF exacerbation at the time of sample collection. This observation raises the hypothesis that EBC 22:4 LPA levels may be a useful biomarker of disease activity and/or acute exacerbations in IPF. Analysis of our data failed to reveal an association between EBC 22:4 LPA levels and disease severity or outcomes , although this study was likely underpowered to detect any such associations. Further study of EBC LPA levels in IPF, specifically in patients with rapidly progressive disease and those suffering from acute exacerbations, may shed light on the potential role of EBC 22:4 LPA levels as a prognostic biomarker in this disease.While BAL has long been considered the optimal means of sampling the alveolar surfaces for analysis, it is invasive and not without risk, especially in subgroups of patients with advanced respiratory disease, such as those with pulmonary fibrosis. In comparison, EBC provides a method for non-invasive sampling of the lower respiratory tract. There are concerns regarding the accuracy with which EBC reflects the distal lung microenvironment, however, as there is risk of contamination with oral and gastrointestinal secretions, as well as an unknown  dilution factor due to condensed water vapor . RecommeAdditional limitations to this study exist. Most notably, the sample size of the study was small. Though we were able to detect a difference in the amount of 22:4 LPA in IPF subjects versus controls, we were likely underpowered to detect differences in the other LPA species should any exist. While the subjects in the study population of interest all met current consensus guidelines for diagnosis of IPF, they differed in disease severity. Whether or not significant differences in LPA species could be detected when stratified for disease severity is not known. However, despite our limitations, we were able to detect a significant elevation in the amount of 22:4 LPA in EBC from IPF subjects. This study builds on previous work showing that LPA is increased in BAL fluid in IPF patients , and advLPA is detectable in exhaled breath condensate (EBC), and 22:4 LPA levels are elevated in the EBC of IPF patients compared to controls.IPF: Idiopathic pulmonary fibrosis; LPA: Lysophosphatidic acid; EBC: Exhaled breath condensate; BAL: Bronchoalveolar lavage; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; FVC: Forced vital capacity; DLCO: Diffusion capacity of carbon monoxide.AMT has applied for the following patent: U.S. Patent Application, 12/450,051, filed September 9, 2009, “Lysophosphatidic Acid Receptor Targeting for the Treatment of Lung Disease”.SBM assisted in data collection, performed statistical analyses, and prepared the draft of the manuscript. SKM and LNB assisted in subject enrollment and performed clinical data collection and analyses. IG and EVB performed lipid extraction and measurement of LPA species by LC-MS/MS. BSS and AMT designed and oversaw all aspects of this study. All authors read and approved of the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/14/5/prepub