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.


P < 0.05). in all groups and had higher stimulatory effect in lymphocytes from day 10 than day 0 of estrous cycle. Progesterone inhibited lymphocyte proliferation, and inhibition was directly related to the dose, in all groups of either sex. Testosterone did not inhibit proliferation at any dose level and did not show any consistent and lucid effects on lymphocyte proliferation. Present study revealed that buffalo lymphocytes produce appreciable amounts of NO in culture system after treatment with estradiol. Significantly high levels of NO in culture supernatant were found in prepubertal buffalo calves and least in post pubertal buffaloes, which had an inverse relation with lymphocyte proliferation in presence of estradiol. NO in culture supernatant was high at the lowest dose of progesterone which was proportional to the lymphocyte proliferation when treated with progesterone. No significant difference in NO culture supernatant was observed between different concentrations of testosterone treatment.Present study was carried out on forty four apparently healthy Murrah buffaloes of different age groups of both sexes to investigate the effects of sex steroids on cell mediated immunity in vitro. Estrogen inhibited proliferation in mitogen-stimulated lymphocytes from prepubertal but not post pubertal buffaloes of either sex. Estrogen at 100 pg/mL concentration stimulating the proliferation significantly ( The sex steroids are in use for the last more than a millennium to correct reproductive disorders and augment reproductive efficiency in animals. However, it has been increasingly apparent that the effects of sex hormones extend far beyond their predominant role in sexual differentiation and reproduction and are now being realised as integral signalling modulators of mammalian immune system. Sexual dimorphism in disease incidence and host response in wide range of animal species provides an indirect evidence of role of sexual hormones in immune modulation –3. ImmunForty-four apparently healthy Murrah buffaloes of three age groups of both sexes  were selected from NDRI herd for conducting present experiments. Eighteen male buffaloes were divided into 3 groups of 6 buffaloes on the basis of their age. Group-I included buffaloes with age ranging between 11 and 13 months; group-II consisted of buffaloes with age ranging between 22 and 24 months. Group-III had postpubertal buffaloes 34 and 36 months of age. Similarly 18 female buffaloes were grouped exactly the same way as in males except that female post pubertal group had cyclic heifers at day 8–10 of estrous cycle. Eight cyclic heifers were selected and studied on day 0 and day 10 of estrous cycle. The estrum (day 0) was confirmed through rectal palpation by an expert and concerned veterinarian at the farm. All these Murrah buffaloes were maintained under routine management and nutritional practices as followed in the herd at the institute. The animal experiments were acceptable to the ethical standards of the National Dairy Research Institute, Karnal, India vide IAEC no. 23/09-21/11.The lymphocytes from peripheral blood were isolated as per the method followed by Young et al.  with som6 cells per culture well. The culture medium used in present study was RPMI-1640 from Sigma Chemical Co., St. Louis, MI, USA, supplemented with L-glutamine, sodium bicarbonate, sodium pyruvate, and antibiotics , besides 10% fetal calf serum. 20 μL of phytohemagglutinin (PHA-P) was added per well to yield a concentration of 5 μg/mL of culture medium. 20 μL of steroid hormone (working concentration) was added to each well. The final volume in each well after adding mitogen and or sex steroid hormone was made to 200 μL with culture medium. Blank wells contained only 200 μL of culture medium with no cells, whereas S–M and S + M wells contained no mitogen and mitogen, respectively, besides lymphocytes. Every observation was made in triplicate wells. The cells were cultured for 36 hours at 37°C in a humidified CO2 incubator (5% CO2). After 36 hours of incubation, 100 μL of culture supernatant was harvested in sterile 1.5 mL eppendorf tubes and stored at −20°C for estimation of nitric oxide. Equal volume of culture media with supplements, preincubated at 37°C, was added into the wells. 20 μL of the MTT solution (5 mg/mL dissolved in DPBS and filtered through 0.22 μm Millex-GV filter unit) was added to each well. The plates were again incubated for 4 hr at 37°C in humidified CO2 incubator. Thereafter, the supernatant was pipetted out completely without disturbing formazan crystal layer and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. After incubating the plate at room temperature for 15 minutes, it was shaken on a microplate shaker and the optical density was read using ELISA reader (Microscan MS-5608A) in dual wavelength measuring system, at a wavelength of 570 nm and a reference wavelength of 630 nm. Lymphocyte blastogenic response was expressed as Proliferation Index (PI) and was calculated as follows:Trypan blue dye exclusion method was used to determine the proportion of viable cells in the separated lymphocytes. When the lymphocyte viability was above 95%, the lymphocytes were processed further for culture. Culture of lymphocytes was carried out in 96-well, tissue culture, flat bottom, sterile microplates with lid from Greiner Bio-One, Germany. The cells were adjusted to 1 × 10μg/mL. 10 μL of it was used for every 90 μL culture medium in each culture well to yield concentration of PHA 5 μg/mL. Stock solution of estradiol, progesterone, and testosterone was prepared in ethanol which was diluted with culture medium to working concentrations of 10 ng/mL, 5 ng/mL, 1 ng/mL, and 100 pg/mL for estrogen, 10 μg, 5 μg, 1 μg, and 100 ng for progesterone, and 1000 ng, 100 ng, 10 ng, and 1 ng/mL for testosterone. 10 μL of working steroid solutions was added to every 90 μL culture medium in culture well yielding final concentration of steroids as 1 ng/mL, 500 pg/mL, 100 pg/mL, and 10 pg/mL for estrogen, 1000 ng/mL, 500 ng/mL, 100 ng/mL and 10 ng/mL for progesterone, and 100 ng/mL, 10 ng/mL, 1 ng/mL, and 0.1 ng/mL for testosterone. All the steroid hormones, β-Estradiol, Progesterone (Δ4-pregnen-3-20-dione) and Testosterone (Δ4-androsten-17β-ol-3 one), Histopaque 1077, Phytohemagglutinin (PHA-P), Trypan blue, and Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Sigma Chemical Co., St. Louis, MI, USA. Dulbecco's Phosphate Buffered Saline (DPBS) was purchased from Himedia Laboratories Pvt. Ltd. India. Phytohemagglutinin used as a mitogen for proliferation of T-lymphocytes was made in culture medium at 50 x) using modified Griess reaction as described by Miranda et al. [μL of test culture supernatant and standards  prepared in culture medium was pipetted out into a 96-well microtitre plate and blank wells contained culture media only. The tests, blanks, and standards were run in triplicates. 50 μL Griess I (1% sulphanilamide) was added to each well and held at room temperature for 5 minutes. 50 μL Griess II was then added to each well and plates were incubated at 37°C for 30 min. After incubation, the absorbance was read at 540 nm wavelength in ELISA plate reader (Microscan MS-5608A). The concentration was calculated from the standard curve using linear regression equation. Detection limit of NO was 1.56 μM. Inter-assay and intra assay difference was 3.4% and 4.62%, respectively.Plasma nitric oxide was estimated as total nitrite . Analysis of variance of the data was performed using three-way ANOVA with variables age, sex, and concentration included in the model as fixed effects and Tukey's honestly significant difference test was employed. Values are presented as mean ± S.E. Graphs and charts were prepared in Microsoft Excel 2007.) on lymphocyte proliferation response that was calculated as proliferation index (PI), in buffalo lymphocytes cultured in vitro, is presented in P < 0.001) variation between age groups and treatment levels was observed in the present study. Estrogen was found inhibiting proliferation in group-I and group-II buffaloes by 5% as compared to group-III post pubertal buffaloes where it stimulated the proliferation to the level of 4%. Among all the groups, post pubertal female buffaloes registered the highest (P < 0.05) PI (1.09 ± 0.03) with estrogen treatment. Among the four concentrations of estrogen, 100 pg/mL registered significant (P < 0.001) stimulatory effect on PI (1.05 ± 0.02) to the level of 5% as compared to 1000 pg/mL (0.93 ± 0.02) and 10 pg/mL (0.95 ± 0.01) that instead inhibited lymphocyte proliferation by 7% and 5% respectively.The effect of four different levels of estradiol  higher stimulatory effect on PI to as high as 10% (1.10 ± 0.03) as compared to 3% (1.03 ± 0.01) on day 0 of estrous cycle. Among the estrogen concentrations, 1000, 500, and 100 pg/mL stimulated the lymphocyte proliferation  significantly (P < 0.05) to the level of 12%, 10%, and 7%, respectively, whereas low level (10 pg/mL) inhibited proliferation (0.99 ± 0.03) up to 1% in cyclic buffaloes. Thus, in cycling females only low level of estrogen was found to be inhibitory to lymphocyte proliferation while all other concentrations were stimulatory.Estrogen treatment of the lymphocytes collected from cyclic buffaloes on day 10 registered significantly  in both males and females, the inhibitory effect was severe in males (0.90 ± 0.01) to the level of 10% as compared to females (0.97 ± 0.01) only 3%. There was no significant difference in the effects of progesterone between different age groups of buffaloes. Progesterone inhibitory effects were significantly (P < 0.001) dose-dependent and PI was inversely proportional to the progesterone concentration. The highest concentration of progesterone (1000 ng/mL) inhibited proliferation (0.81 ± 0.02) by 19% whereas the lowest (10 ng/mL) registered stimulatory effect (1.058 ± 0.02) of 6%. Among all groups, group-III post pubertal female buffaloes registered highest (P < 0.01) proliferation (1.065 ± 0.02) and group-III post pubertal males the lowest (0.86 ± 0.03) when treated with progesterone.Though progesterone inhibited lymphocyte proliferation significantly (P < 0.01) with highest concentration 1000 ng/mL progesterone inhibiting proliferation (0.976 ± 0.02) by 2% and 10 ng/mL stimulated proliferation (1.103 ± 0.02) by 10%. The trend in lymphocyte proliferation with decreased dose of progesterone was linear.The progesterone effects were not significantly different between the lymphocytes from the day 0 and day 10 of reproductive cycle. However, dose dependent effect of progesterone on proliferation was significant (P < 0.01) inhibited proliferation by 4% in group-II males but stimulated by 13% in group-I males, irrespective of testosterone concentration. Contrary to this, testosterone significantly (P < 0.01) inhibited proliferation (0.958 ± 0.01) by 5% in group-I females but stimulated (1.101 ± 0.03) by 10% in group-III post pubertal females.The effects of testosterone treatment on in vitro proliferation of buffalo lymphocytes of different sex and age are presented in  There was no significant effect of testosterone on lymphocyte proliferation index in cycling buffaloes. However, proliferation was comparatively low in lymphocytes from day 0 (1.049 ± 0.03) as compared to day 10 (1.104 ± 0.03) of estrous cycle when treated with testosterone. The testosterone in all its concentrations stimulated proliferation to the level of 10% (1.104 ± 0.04) at 10 ng/mL and 5% (1.049 ± 0.04) at 0.01 ng/mL concentration in cyclic buffaloes.P < 0.001) higher (8.52 ± 0.39 μM/L) NO in culture than females (6.13 ± 0.39 μM/L). The difference was also significant (P < 0.05) among the three groups with lowest in group-III (6.23 ± 0.48 μM/L), followed by group-II (8.07 ± 0.48 μM/L) and group-I (7.68 ± 0.48 μM/L). Among all groups, group-II male buffaloes registered the highest (9.89 ± 1.18 μM/L) and group-III female buffaloes the lowest (4.87 ± 0.42 μM/L) levels of nitric oxide in culture supernatant. In cyclic buffaloes the nitric oxide concentration in culture supernatant did not differ significantly with the stage of estrous cycle and was 5.20 ± 0.29 and 4.88 ± 0.41 μM/L on day 0 (estrus) and day 10 (diestrus), respectively  higher levels of nitric oxide were recorded in culture supernatant of males (9.73 ± 0.30 μM/L) than females (6.75 ± 0.30 μM/L) after 36 hours of culture supplemented with estradiol. The NO levels in culture supernatants supplemented with estradiol were significantly (P < 0.001) different between age groups with group-I buffalo lymphocytes registering NO as high as 11.36 ± 0.37 μM/L followed by group-II and group-III (μM/L) in culture supernatant (P < 0.05) effect of different concentrations of estrogen on nitric oxide levels in culture supernatant; however, NO levels were maximum (8.77 ± 0.74 μM/L) at 100 pg/mL concentration of estrogen and minimum at 10 pg/mL level ( Significantly (roup-III . Among ternatant . There wmL level .μM/L) significantly (P < 0.001) higher than (3.47 ± 0.38 μM/L) day 0 (estrous) lymphocytes. Similarly different concentrations of estrogen in culture system had significant (P < 0.001) effect on NO in culture supernatant in cyclic buffaloes. Significantly higher levels of NO (5.82 ± 0.39 μM/L and 5.51 ± 0.39 μM/L) were recorded at 100 pg/mL and 500 pg/mL estradiol, respectively, as compared to (3.43 ± 0.93 and 2.79 ± 0.39 μM/L) at 1000 pg/mL and 10 pg/mL estradiol, respectively, in cyclic buffaloes  lymphocytes was  difference in levels of nitric oxide in culture supernatant supplemented with different concentrations of progesterone was registered between the sexes  and between the age groups . Among the groups, group-II males registered highest (P < 0.05) levels of NO in culture supernatant where as group-I females the lowest (P < 0.01) between various concentrations of progesterone treatment ( Significant (e lowest . Similarreatment .P < 0.01) higher (5.68 ± 0.40 μM/L) NO in culture supernatant as compared to day 0 (3.98 ± 0.40 μM/L) (P < 0.01) decline in NO production with increasing concentrations of progesterone supplementation with highest NO at 10 ng/mL progesterone followed by 100, 500, and 1000 ng/mL progesterone respectively in cyclic buffaloes  . There wuffaloes .μM/L NO, respectively. Further, NO in culture supernatant was significantly (P < 0.05) high (7.98 ± 0.29 μM/L) in group-II buffaloes as compared to group-III (7.08 ± 0.29) and group-I (6.96 ± 0.29 μM/L). Among the all groups, group-II male buffaloes recorded the highest (P < 0.01) NO whereas group-I males the lowest (μM/L) with 10 ng/mL supplementation.Analysis of variance revealed significant influence of sex on nitric oxide production in culture supernatant supplemented with testosterone and male and female buffaloes on an average, which produced 7.13 ± 0.23 and 7.54 ± 0.23 e lowest . The leve lowest . HoweverμM/L in day 10 and day 0 lymphocyte culture, respectively  as compared to females (3%). This sex-dependent variability in progesterone effects may probably be due to the adaptation of lymphocytes to progesterone because of being exposed to it in females. There was an inverse relationship between the dose and proliferation index. The highest concentration of progesterone (1000 ng/mL) inhibited proliferation by 19% whereas the lowest (10 ng/mL) registered stimulatory effect of 6%. These findings are in partial agreement with those reported by Marco et al.  reportedIn current investigation, testosterone treatment stimulated lymphocyte proliferation in both males and females to the tune of 2 and 5%, respectively, with no significant dose-dependent effect. Testosterone treatment in lymphocyte culture did not yield any dependable and lucid effects either at any particular dose or in any age group. In group-I males it stimulated proliferation at all concentrations of testosterone with maximum at 0.1 ng/mL but in case of group-I female it inhibited proliferation at all concentrations of testosterone. In group-II males contrary to group-I males, testosterone inhibited proliferation except at 1 ng/mL concentration but at the same time in group-II females it stimulated proliferation at all concentrations of testosterone with maximum at 1 ng/mL. Whereas in group-III post pubertal males picture was same as that of group-II males but in group-III females, testosterone stimulated proliferation at all concentrations with maximum at 100 ng/mL. With these varied effects of testosterone, it is hard to arrive at any definitive conclusion. It may be possible that the lymphocytes possess the capability to convert testosterone into estrogen, which may govern the proliferation in adult buffaloes. This option may be working since we got similar observations with testosterone and estrogen in post pubertal female lymphocytes. Benten et al.  have dem Sex and age bias in NO production by lymphocytes in culture was evident in present study. Significantly high levels of NO were found in culture supernatant of males as compared to females treated with estrogen. Among the groups, group-I buffaloes had more NO in culture as compared to group-II and III. However, production rate was not affected by increasing dose of estrogen in culture. The proliferation was higher in group-III where NO was lowest suggesting inverse relationship between PI and nitric oxide production in culture and probably estrogen affected proliferation through NO levels in lymphocyte culture. However, the NO in culture supernatant of day 10 lymphocytes was significantly higher than day 0 while proliferation was also significantly high at diestrous than estrous phase. NO has been reported to be secreted by lymphocytes , 35 and  Nitric oxide in lymphocyte culture treated with different concentrations of progesterone was significantly high in males as compared to females. Among the groups, significantly high levels of NO in culture supernatant were found in group-III and least in group-I buffaloes. In cyclic buffaloes, diestrous phase lymphocytes produced more NO than estrous phase, treated with progesterone. Similarly the nitric oxide levels in supernatant differed between the concentrations of progesterone with lowest NO at highest progesterone concentration. The same trend was also observed in lymphocyte proliferation. This suggested that progesterone probably has no direct effect on NO production by lymphocytes and as such more proliferation is accompanied with more NO.μM/L NO, respectively, in culture. This indicated that the lymphocytes obtained from female animals were equally responsive to testosterone as males. However, levels tended to be significantly (P < 0.05) high in group-II buffaloes as compared to group-I and III. In general, low levels of proliferation were associated with less production of NO in culture in different age groups of buffaloes. Differences were not significant during different stages of estrous cycle which suggested that variations in the immune responses during estrous cycle were not related to testosterone as was evident through PI also. NO in lymphocyte culture supernatant at any level of testosterone did not show any significant difference between sexes with male and female buffaloes on an average, producing 7.13 ± 0.23 and 7.54 ± 0.23 Since it happens to be the first report to register NO in culture supernatant of lymphocytes from buffaloes of different sex and age with sex steroid treatment, it needs further investigation before arriving at any final conclusion on relationship of sex steroids, NO, and lymphocyte proliferation.Effect of female sex steroids on lymphocyte proliferation in vitro was found to be existing. Estradiol effects were dose dependent too, and high as well as too low levels of estradiol were inhibitory to lymphocyte proliferation. Progesterone was found to be inhibiting lymphocyte proliferation and there was inverse relationship between progesterone dose and lymphocyte proliferation. Testosterone did not show any consistent and lucid effects on lymphocyte proliferation, nor did it inhibit proliferation at any dose level. The proliferation was higher in group-III where NO was lowest suggesting inverse relationship between PI and nitric oxide production in culture and probably estrogen affected proliferation through NO levels in lymphocyte culture. NO was not the candidate mediator of effects of all sex steroids in lymphocyte proliferation. NO levels in supernatant differed between the concentrations of progesterone with lowest NO at highest progesterone concentration and lowest proliferation. This suggested that progesterone probably has no direct effect on NO production by lymphocytes and as such more proliferation is accompanied with more NO. No significant difference in NO culture supernatant was observed at different concentrations of estradiol or testosterone.


I2D/IG ratios, and enhancements of G and 2D bands with suspended and supported graphene flakes were measured and analyzed. The peak shifts of G and 2D bands between the Raman and SERS signals demonstrate the doping effect induced by silver nanoparticles by n-doping. The I2D/IG ratio can provide a more sensitive method to carry out the doping effect on the graphene surface than the peak shifts of G and 2D bands. The enhancements of 2D band of suspended and supported graphenes reached 138, and those of G band reached at least 169. Their good enhancements are helpful to measure the optical properties of graphene. The different substrates that covered the graphene surface with doping effect are more sensitive to the enhancements of G band with respect to 2D band. It provides us a new method to distinguish the substrate and doping effect on graphene. The interactions between phonons and electrons induced by the dopants or the substrate of graphene in spectroscopic investigation reveal a rich source of interesting physics. Raman spectra and surface-enhanced Raman spectra of supported and suspended monolayer graphenes were measured and analyzed systemically with different approaches. The weak Raman signals are greatly enhanced by the ability of surface-enhanced Raman spectroscopy which has attracted considerable interests. The technique is regarded as wonderful and useful tool, but the dopants that are produced by depositing metallic nanoparticles may affect the electron scattering processes of graphene. Therefore, the doping and substrate influences on graphene are also important issues to be investigated. In this work, the peak positions of G peak and 2D peak, the 78.67.Wj ; 74.25.nd ; 63.22.Rc (phonons in graphene) With our analysis, details about the effects of charged impurities and substrate can be realized. The peak shift of G and 2D bands and the I2D/IG ratio are useful to demonstrate the dopants and substrate effects on the graphene. The well-enhanced G and 2D bands are obtained to enhance the weak Raman signals. Moreover, the enhancements of G band with respect to 2D band are found to be more sensitive to various substrate influences on the graphene surface. This paper provides a new approach to investigate the substrate and doping effect on graphene.Graphene, the thinnest operties -3. The woperties -9. The poperties -12. Ramaoperties -18, and operties -24. The operties ,26. Theroperties ,28. The 2 were first defined by photolithography on an oxidized silicon wafer with an oxide thickness of 300 nm. Reactive ion etching was then used to etch the squares to a depth of 150 nm. Highly ordered pyrolytic graphite was consequently cleaved with the protection of scotch tape to enable the suspended graphene flakes to be deposited over the indents. To study the SERS, silver nanoparticles were deposited on the graphene flake at a deposition rate of 0.5 nm/min by a thermal deposition system. A 5-nm-thick layer of silver nanoparticles on the graphene flake was thus formed. To measure the graphene flake, a micro-Raman microscope  was utilized to obtain the Raman and SERS signals of monolayer graphene. The monolayer graphene was identified through optical observation with various color contrast and by Raman spectroscopy with the different shape bandwidths and peak positions of 2D band under different graphene layers. During spectroscopic measurement, a 632-nm He-Ne laser was used as the excitation source; the power was monitored and controlled under 0.5 mW to avoid the heating of the graphene surface.Suspended graphene was fabricated by mechanical exfoliation of graphene flakes onto an oxidized silicon wafer. The optical image of suspended and supported graphenes and the illustration of their coverage by silver nanoparticles are shown in Figure To explore the SERS on graphene, the interactions between metallic nanoparticles and graphene surface has to be presumably understood. This is because the plasmonic resonances of nanoparticles with different shapes and sizes can affect the interactions between them, and then change the SERS signals ,29-33. TI2D/IG ratios of G and 2D bands were important indicators of doping effect on graphene [I2D/IG ratio is particularly more sensitive than the peak shifts to the doping effect. A lower I2D/IG ratio is related to more charged impurities in graphene. The Raman and SERS signals of the suspended and the supported graphenes are shown in Figure According to previous work, the peak positions and graphene -40, in wI2D/IG ratio of suspended graphene under Raman signals is 4.1 ± 0.1 and larger than supported graphene which is 3.6 ± 0.5, while the I2D/IG ratio of suspended graphene on the SERS signals is around 2.9 ± 0.1 and smaller than supported graphene which is 3.0 ± 0.2. The result disclosed the substrate effect on the supported graphene is stronger than the suspended graphene. To our understanding, this above effect can be reasonable because SiO2/Si substrate has a direct contact on the graphene surface so the silver nanoparticles deposition causes stronger doping effect on the suspended graphene than supported graphene.In order to minimize the random errors, each Raman spectra data point was obtained by five-time repetitions. As presented in Figure To understand the Raman and SERS signals, the enhancements of G and 2D bands with the suspended and supported graphenes are shown in Figure I2D/IG ratios were varied. The enhancement effect of suspended and supported graphenes was calculated and analyzed. The peak shifts of G and 2D bands and their Raman spectra and I2D/IG of SERS signals are found very useful in the investigation of the substrate and doping effect on the optical properties of graphene. The enhancements of G and 2D bands have been found to cause the great improvement of weak Raman signals. Otherwise, the more sensitive enhancements of G band with respect to 2D band are related to the doping effect with various substrates that covered the graphene surface. The optical emission spectra of suspended and supported graphenes have provided us a with new identification approach to understand the substrate and doping effect on graphene.In our work, Raman and SERS signals of supported and suspended monolayer graphenes were measured systematically. The peak positions of G and 2D bands and the The authors declare that they have no competing interests.C-H and B-JL carried on the experimental parts: the acquisition of data and analysis and interpretation of data. C-H also had been involved in drafting the manuscript. H-YL and C-HH analyzed and interpreted the data. They also had been involved in revising the manuscript. F-YS and W-HW  prepared the samples, suspended graphene using by micromechanical method, and captured the OM and AFM images. C-YL have made substantial contributions to the conception and design of the study and revising it critically for important intellectual content. H-CC, the corresponding author, had made substantial contributions to the conception and design of the study and had been involved in drafting the manuscript and revised it critically for important intellectual content. All authors read and approved the final manuscript.


Sarcoidosis is a multisystem granulomatous disease with an unknown etiology. It most commonly affects young and middle-aged females. It can affect any organ, but mostly lung, skin, and eyes. Up to half of patients are asymptomatic and the disease is often detected incidentally on abnormal chest radiography. We report the case of a 31-year-old male with bilateral recurrent dacryostenosis. The nasolacrimal obstruction was the initial manifestation of systemic sarcoidosis. Sarcoidosis is a multisystem granulomatous inflammation of unknown etiology . It typiThis is a 31-year-old male diagnosed with nasolacrimal sarcoidosis. He first presented complaining of bilateral epiphora worse on the left. There is no history of endonasal surgery nor facial trauma. He complained of a chronic nasal obstruction and had been treated for sinusitis in the past. He was otherwise asymptomatic and in good general health.Exam revealed complete dacryostenosis of the left and partial obstruction of the right ducts. He was initially presumed to have idiopathic obstruction and underwent external left dacryocystorhinostomy. On followup one year later, he presented with complete obstruction on the right and bilateral chronic maxillary sinusitis as seen on sinus CT . We perfHistologic evaluation of ethmoidal tissues revealed granulomatous inflammation compatible with sarcoidosis. Pulmonary CT demonstrated bilateral hilar adenopathy and parenchymatous infiltration consistent with stage 2 sarcoidosis . AngioteOn follow-up exam one year later, we noted right endonasal synechia . The patSarcoidosis affects primarily the respiratory tract. Although periorbital involvement is uncommon, any part of of the eye, orbit, and adnexal structures may be involved. The lacrimal gland seem to be the most affected, followed by the orbit, eyelids and the lacrimal sac . There aDemirci and Christianson  report aAlthough there is some nasolacrimal involvement cases reported in literature, most of them presented with known sarcoidosis. Fergie et al. reported 4 patients with nasolacrimal involvement, 3 of whom did not show evidence of systemic disease . VasquezMoreover, the utility of routine histopathologic examination of tissue obtained during DCR is controversial. While most nasolacrimal obstructions are idiopathic, some authors recommend histopathologic analysis in young patients, as we report here, and in the context of atypical clinical presentation. Heindl et al. performed biopsy of the lacrimal sac in 19 of 500 cases which were found to be atypical. 3 out of 19 biopsies performed were positive for sarcoidosis . Tucker et al. obtained 162 lacrimal sac biopsy specimens in 150 consecutive patients undergoing dacryocystorhinostomy for clinical primary-acquired nasolacrimal duct obstruction . BiopsieNasolacrimal obstruction as an initial presentation of sarcoidosis is rare. Although uncommon and unrelated to specific symptoms, this condition should be considered in the differential diagnosis of epiphora and sinus complaints. This case emphasizes the clinical purpose of routine histopathological analysis of the lacrimal sac when performing DCR.


Climate variation has long been studied in relation to human health. The aim of the present work was to evaluate the relationship between environmental humidity, and air temperature with the prevalence of diabetes, among elderly islanders.During 2005–2011, 1959 elderly (aged 65 to 100 years) individuals from 13 Mediterranean islands were enrolled. Socio-demographic, clinical and lifestyle factors were assessed using standard procedures. Diabetes was defined as fasting blood glucose levels > 125 mg/dl. Relative environmental humidity was measured as a percentage of air moisture and mean daily temperature in degrees Celsius.For the present analysis 713 men (74 ± 7 years) and 596 women (73 ± 7 years) with complete data were studied; 27% of both men and women had diabetes. The prevalence of diabetes was 42% in the elders living in high relative humidity areas  as compared with 24% among those living at low relative humidity residential areas (p < 0.001). After adjusting for age, sex and mean temperature, an increase in the area’s relative humidity by 1 degree, increased the likelihood of having diabetes by 12% . No significant association was observed between mean temperature and diabetes .A considerable proportion of elderly, especially those living in high relative humidity areas, had diabetes. Further research is needed to confirm this observation and to understand the underlying mechanisms. Worldwide 347 million of people have diabetes . There aClimate variation -15 and aDiabetes is a clinical condition, often asymptomatic and with high prevalence in older adults. Given the lack of data regarding the effect of environmental factors, and particularly climatological, on the prevalence of diabetes, the aim of the present work was to evaluate the association between relative humidity and mean air temperature, with the prevalence of diabetes, among elders (> 65 years old) that are lifelong inhabitants of Mediterranean islands.This work is under the context of the MEDIS Study . During nd WMA General Assembly, Edinburgh, Scotland, October 2000). The Institutional Ethics Board of Harokopio University approved the study design (16/19-12-2006). Participants were informed about the aims and procedures of the study and gave their consent prior to being interviewed.The study followed the ethical considerations provided by the World Medical Association  during the past 5 years before sampling, were recorded using data from the National Meteorological Service . Areas wAs already has been presented in previous reports from the MEDIS study ,23 the dContinuous variables were presented as mean ± standard deviation (SD), and categorical variables as frequencies. Comparisons of normally distributed continuous variables  between groups were performed using Student’s t-test. Spearman rho coefficient was applied to evaluate the correlation between mean relative humidity, mean temperature and physical activity, the MedDietScore of the participants. Normality was tested using P-P plots. Independence between categorical variables was tested using the chi-square criterion. Additive logistic regression models were used to evaluate the association between participants’ characteristics  and presence of diabetes. Results are expressed as odds ratios and the 95% confidence intervals. Deviance residuals and Hosmer-Lemeshow criterion evaluated models’ goodness-of-fit. P-value <0.05 was considered to be statistically significant. SPSS software (version 18) was used for all calculations .The prevalence of diabetes was 27% for the entire cohort. No significant variations were observed between islands. When comparing data from participants living in high humidity areas vs. participants living in low humidity ones, the prevalence of diabetes was 42% and 23%, respectively. Specifically, participants 65 to 80 years old living in high humidity areas, had almost double prevalence of diabetes compared with those living in low humidity areas . In the next age group  the percentage increased for participants living in high humidity areas while for those living in low humidity areas remained at the same levels . Demographic, behavioral and lifestyle characteristics of the sample, by relative humidity, are summarized in Table The clinical characteristics and the prevalence of diabetes, based on humidity conditions, are presented in Table Furthermore, an inverse correlation was observed between high humidity areas of living and the physical activity level . An inverse association was also observed between humidity and adherence to the Mediterranean dietary pattern . Both findings suggest that people living in high humidity areas tend to be less physically active and away from the Mediterranean dietary pattern.models 1, 2 and 3), even after adjusting for several confounders, such as physical activity, adherence to the Mediterranean diet, smoking habits, living conditions (model 2), hypertension, hypercholesterolemia and obesity (model 3).Age, sex and mean air temperature adjusted logistic regression analysis showed that people living in high humidity areas were 3.1-times more likely to have diabetes, compared to those living in low humidity areas (95% CI 1.65 to 5.94). Specifically, when the relative humidity in an area increases by one degree, the likelihood of an elder having diabetes increases by 12% . However, residual confounding may exists, and therefore multiple logistic analyses were further applied. Results have been displayed in Table n = 341), and those above 80 years (n = 76), it was found that the positive association of relative humidity with diabetes was significant only in the group of very old people , while among those 65–80 years no significant association was found . Moreover, when the analysis was stratified by the level of adherence to the Mediterranean diet according to the MedDietScore cutoffs, it was observed that relative humidity was associated with higher likelihood of diabetes, only for the low adherence group . For the moderate/high adherence group, relative humidity was not associated with diabetes . Moreover, no association was observed between mean temperature and diabetes among those <80 or those >80 years, as well as those close or away to the Mediterranean dietary pattern.In addition, when data analysis was repeated separately for those between 65–80 years old  was similar to other recent studies conducted in European countries  and in GTo further explore the aforementioned finding, additional analysis of clinical and lifestyle components and the area’s relative humidity was performed. It was also observed higher prevalence of hypertension, central fat, along with lower physical activity in the elders living in high humidity areas, compared those living in low humidity ones. These factors could play a moderating role in the investigated research hypothesis. Despite the lack of previous studies on relative humidity and prevalence of diabetes and the other aforementioned clinical conditions, a number of studies have already investigated the effect of climate variations  on human health -20,22. SAccording to Köppen’s climate classification the Mediterranean climate is a particular variety of the subtropical climate found around the Mediterranean Sea. The climate in this area is characterized by relatively warm to hot, dry summers, mild to cool, rainy winters and extended sunshine almost all the year. The islands included in the MEDIS study have Mediterranean climate temperatures, with high air relative humidity and long, hot summers. In the mainland, the mean humidity is around 65% while in the islands is around 75% . High aiMoreover, several studies have proposed that higher rates of relative humidity are associated with lower mood scores ,34. DiscAdditional analysis showed a consistent association between relative humidity and the likelihood of an elder having diabetes, only in the group of 80 years old and above, and only in elders with low adherence to the Mediterranean diet. The first could be attribute to the fact that prolonged age accumulate several harmful factors, including the additive effect of lifelong living in an environment with high humidity. Regarding the second finding, recent studies have associated high adherence to the Mediterranean diet with better glucose homeostasis in the human body. The Mediterranean dietary pattern emphasizes on a consumption of high monounsaturated fats, mainly from olive oil, and encourages consumption of fruits, vegetables, tree nuts, legumes, whole grains, fish and poultry in low to moderate amounts, a relatively low consumption of red meat, as well as a moderate consumption of alcohol. The proposed mechanism was through regulations in plasma glucose and insulin levels . This efPhysical activity and adherence to the Mediterranean diet have been associated with insulin resistance and the development of diabetes in many previous studies ,37. The The present study has several strengths. It is one of the first to evaluate the effect of climate variations in the health status of a large sample of “healthy”, free-living elderly people in the Mediterranean islands. The limitation of the study is mainly caused by its cross-sectional design and therefore the lack of causal relationships. Also, measuring climate condition only by environmental humidity (and not indoor) can be considered as a study limitation.The prevalence of diabetes was high in the studied elderly and free of cardiovascular disease, insular population, and elders living in high humidity areas had almost double prevalence of diabetes, as compared with those living in low humidity residences. The latter attempted to be partially explained by the accumulation of cardiovascular disease factors and by the modifications in lifestyle habits, this population appeared to have made. However, further research is needed to confirm this observation and to understand the underlying mechanisms.The authors declare that there are not any financial or non-financial competing interests to declare in relation to this manuscript.ST, DP, CH, CL, EP conceived of the study, carried out its designing and implementation, drafted the manuscript, and performed the statistical analysis. MM, KK, NT, AZ, EG, GM, VB participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.