Labisia pumila, locally known as Kacip Fatimah, is a forest-floor plant that has tremendous potential in the herbal industry. It is one of the five herbal plants identified by the government as one of the national key economic areas to be developed for commercial purposes. There are three varieties of L. pumila namely, L. pumila var. pumila, L. pumila var. alata and L. pumila var. lanceolata and each has its own use.L. pumila Benth. were extracted using microwave assisted extraction (MAE). Antifungal activity of all plant extracts were characterized against Fusarium sp., Candida sp. and Mucor using the agar diffusion disc. Anti-inflammatory assays were performed using NO production by macrophage RAW 264.7 cell lines induced by LPS/IFN-g and cytotoxic activity was determined using several cancer cell lines and one normal cell line.The leaves and roots of the three varieties of L. pumila exhibited moderate to appreciable antifungal activity against Fusarium sp., Candida sp. and Mucor compared to streptomycin used as positive control. Leaf and root extracts of all varieties significantly decreased NO release. However, the root extracts showed higher activity compared to the leaf extracts. Cytotoxic activity against MCF-7, MDA-MB-231 and Chang cell lines were observed with all extracts.The overall result demonstrated that leaf and root extracts of all three varieties of L. pumila Benth. as a natural medicine and indicated the possible application of this medicinal plant such anti inflammatory activity and cytotoxic agents.These findings suggest the potential use of  Labisia pumila Benth. (Myrsinaceae) popularly known as Kacip Fatimah, is a herbaceous plant with creeping stems used by Malay women to induce and facilitate childbirth as well as a post-partum medicine. Recently, it was found that the bioactive compounds of L. pumila consisted of resorcinols, flavonoids and phenolic acids. These compounds have been identified as natural bioactive compounds with high biological activity [L. pumila (Blume) var. alata, L. pumila (Blume) var. pumila and L. pumila var. lanceolata. Each of these varieties has different applications. Uses of this herb include treatment for dysentery, dysmenorrheal, flatulence, and gonorrhea [L. pumila Benth. consisted of bioactive compounds including phenolics, flavonoids and saponins which might be responsible for the enhanced antioxidant activity observed in this plant [Labisia pumila Benth.Currently, there is growing interest in the application of plants as a medicinal agent since synthetic drugs have shown several side effects on the human body. Experimental investigations demonstrated that many naturally occurring agents in plant extracts have shown antioxidant, antimicrobial and anticancer potential in a variety of bioassay systems and animal models, having relevance to human disease . Medicinet al. indicatencer MCF-, gastriccer MKN-2 and coloLabisia pumila varieties were collected from places of origin at Hulu Langat (Selangor), Sungkai (Perak) and Kota Tinggi (Johore), respectively, and raised under glasshouse conditions for 18 months before use in the study. Voucher specimens were identified by the Herbarium unit, Institute of Bioscience, University Putra Malaysia ), pumila ((Stone 7233 (KLU)), and lanceolata ((Stone 8385 (KLU)). Healthy and uniform seedlings in terms of leaf numbers were selected from the three varieties. The leaves and roots of selected seedlings were cleaned, separated, freeze dried and stored until further analysis.Seedlings of the three et al.[L. pumila was weighed in a clean aluminum container, and then transferred into the vessel of the Ethos E Microwave Extraction System and extracted with 30 ml of methanol for 2 min (p = 750 w). The extraction temperature was 60°C. After extraction, the vessels were allowed to cool to room temperature, and the extracts were filtered and stored in a refrigerator.MAE was performed using a microwave apparatus with a closed vessel system under pressure  based on the method described by Xiao et al. with som5 spore/mL) and dispensed (100 μL) uniformly on the surface of the agar plate. Small wells were cut in the agar plate using a cork borer (6 mm). A fixed volume of the different extracts or amphotericin B  as positive control (at 450 μg/well), were loaded into the wells. The plates were incubated at 29°C for 72 h. The diameter of the inhibition zone around each well was then recorded in four different directions.The antifungal assay was carried out by the agar well diffusion method  with sli2 and were split twice a week. Approximately 1 × 106 cells/ml of RAW 264.7 cells were seeded in 96-well tissue culture plates and incubated for 24 h at 37°C with 5% CO2. The cells were then incubated in prepared DMEM medium containing 100 μl of test extract in DMSO and serially diluted, to give a final concentration of 100 μg/ml in 0.1% DMSO. Cells were then stimulated with 200 μ/ml of IFN-γ and 10 μg/ml LPS for 17 h. The presence of nitrite in the cell culture media was determined using Griess reagent and absorbance was read at 550 nm using the microplate reader . Nitrite concentration in the supernatants was determined by comparison with a sodium nitrite standard curve. The cell viability was detected by MTT cytotoxicity assay. L-NAME was used as iNOS inhibitor (control) at a concentration 250 μM [The murine monocytic macrophage cell line RAW 264.7 was cultured in Dulbecco’s Modified Eagle Media (DMEM)  with 10% foetal bovine serum (FBS). The cells were cultured at 37°C with 5% COn 250 μM .2 and 95% air atmosphere in Dulbecco’s Modified Eagle Media (DMEM) . Monolayers of cells (5 × 103/100 μl) were grown in 96-well microlitre plates and exposed to two fold serial dilutions of the extracts from 200 μg to 3.1 μg/100 μl. After 3 days incubation at 37°C, the cytotoxicity of extracts was determined using the MTT assay according to Ahmad et al.[Human cancer cell lines  and Human hepatocytes (Chang liver cells) cell lines obtained from the American Type Culture Collection (ATCC) were used in this study. Cells were grown at 37°C in humidified 5% COad et al.. TamoxifData were analyzed using the analysis of variance procedure in the Statistical Analysis System (SAS) Version 9 . Significant differences between means from triplicate analyses (p < 0.05) were determined by Duncan’s Multiple Range Test. GraphPad Prism 5 software  was also used in data analyses.Labisia pumila Benth against three fungi species are summarized in the Table pumila against Candida sp. (inhibition zone = 0.82 cm) and leaf extract of variety alata against Mucor sp. (inhibition zone = 0.70 cm). The lowest inhibitory activity was obtained with the root extract of variety lanceolata against Mucor sp. (inhibition zone = 0.33 cm). Antifungal activity of all the extracts is attributed to presence of bioactive compounds such as flavonoids, phenolics, saponins and various volatile compounds [in vitro[et al.[The results of antifungal activity of leaf and root extracts from the three varieties of ompounds . Various[in vitro. Vaqueroro[et al. revealedL. pumila was evaluated based on the colorimetric nitric oxide assay. In this study, all extracts were examined for ability to inhibit NO release in RAW 264.7 cells stimulated with bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-γ). All extracts were assayed at the two-fold dilution range (12.5-100 μg/ml) dissolved in 0.2% DMSO and compared with N-Nitro-L-Arginine Methyl Ester (L-NAME). The viability of RAW 264.7 cells after treatment was assessed by the MTT -2,5-diphenyl tetrazolium bromide) method. The activity profiles of the extracts in terms of percentage of NO inhibition were categorized according to Kim et al.[L. pumila . L-NAME inhibited NO production by 78.43%. The NO inhibition values of leaf extract of L. pumila var. pumila, alata and lanceolata were 45.66, 46.64 and 55.17%, respectively, at a concentration of 100 μg/ml, while the root extract of var. pumila, alata and lanceolata resulted in 66.45, 75.68 and 64.81%, inhibition, respectively  is an important radical molecule that is produced by a family of enzymes known as nitric oxide synthase (NOS) and participates in the physiology and pathophysiology of many systems. Producing large amount of NO is associated with numerous pathological conditions. NO released from cells can be detected and quantified photometrically as its stable product, nitrite in the cell culture supernatant . Anti-inim et al.. Inhibitmi et al. showed tl.[et al. suggesterophages . Meanwhiim et al. reportedLabisia pumila Benth are presented in Figures 50 values of extracts used in this study are presented in Table in vitro cytotoxic activity if the IC50 value (concentration that causes reduction in cell viability to 50%) was less than 30 μg/ml [Labisia pumila var. pumila exhibited more cytotoxic activity compared to var. alata followed by var. lanceolata. The IC50 concentrations for leaf extracts of L. pumila var. pumila, alata and lanceolata, with the MCF7 cell line were found to be 48.2, 54 and 59.9 μg/ml and for MDA-MB-231 the values were 55.80, 62.26 and 66.39 μg/ml, respectively. In terms of toxicity to the Chang liver cells, leaf and root extracts were considered as not toxic as the IC50 values were greater than 200 μg/ml. Tamoxifen was used as the positive control in this study , octadecanoic acid (16.24%) and 1,3-dioxolane,2,4,5-trimethyl (18.69%) were the main compounds in the leaf extracts of Labisia pumila var. alata, pumila and lanceolata. These compounds have been reported to have antibacterial and antifungal [et al.[et al.[Cancer is a group of diseases characterized by cells that grow out of control. In most cases, they form masses of cells, or tumors, that infiltrate, crowd out, and destroy normal tissue. Consumption of antioxidant rich fruits and vegetables in our daily diets significantly reduces the risk of many cancer diseases suggesting that antioxidants could be effective agents for the inhibition of cancer spread . The res30 μg/ml . Leaf exle et al. demonstrtifungal , antioxitifungal . It is atifungal . Oskoueil [et al. indicatel.[et al. showed tLabisia pumila plants and in particular the leaves for use in the development of anti fungal, anti-inflammatory and anti cancer drugs using microwave extraction. These activities might be due to the presence of various phenolic and flavonoid compounds in the leaves compare to root in all three varieties of L. pumila Benth [in vitro screening tests. Therefore isolation of bioactive compounds responsible for the observed activities is necessary and in vivo studies are needed for further confirmation.Medicinal plants are now increasingly getting more attention than ever before. The medicinal value of these plants lies in the bioactive phytochemical constituents such as phenolics, flavonoids and saponins that result in various biological activities . The resla Benth ,13. It sThe authors declare that they have no conflicts of interest concerning this article.EK conducted antifungal properties, cytotoxicity assay, analyze and interpretation of data, and drafted the manuscript. HZEJ was responsible for conception and design, drafted the manuscript and revised it critically for important intellectual content. SA conducted the anti-inflammatory assay, and assisted in the analysis and interpretation of data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/20/prepub


Objectives. Periodontitis is an inflammatory disease characterized by connective tissue loss and alveolar bone destruction. Interleukin-8 (IL8) is important in the regulation of the immune response. The aim of this study was to analyze four polymorphisms in the IL8 gene in relation to chronic (CP) and aggressive (AgP) periodontitis. Methods. A total of 492 unrelated subjects were included in this case-control association study. Genomic DNA of 278 patients with CP, 58 patients with AgP, and 156 controls were genotyped, using the 5′ nuclease TaqMan assay, for IL8  gene polymorphisms. Subgingival bacterial colonization was investigated by the DNA-microarray detection kit in a subgroup of subjects (N = 247). Results. Allele and genotype frequencies of all investigated IL8 polymorphisms were not significantly different between the subjects with CP and/or AgP and controls (P > 0.05). Nevertheless, the A(−251)/T(+396)/T(+781) and T(−251)/G(+396)/C(+781) haplotypes were significantly less frequent in patients with CP  than in controls. Conclusions. Although none of the investigated SNPs in the IL8 gene was individually associated with periodontitis, some haplotypes can be protective against CP in the Czech population. Porphyromonas gingivalis, Campylobacter rectus, Tannerella forsythia, Peptostreptococcus micro, and Treponema species [Treponema denticola, P. gingivalis, and T. forsythia, characterized as the “red complex,” were strongly associated with the clinical progression of chronic periodontitis [Aggregatibacter actinomycetemcomitans, but there were many individuals with AgP who did not harbor this microorganism [Periodontitis is an inflammatory disease which is initiated and maintained by the gram-negative bacteria of the subgingival biofilm . Specifi species , 4. Trepdontitis –5. In coorganism .IL8 expression rapidly in response to A. actinomycetemcomitans challenge, facilitating thus the recruitment of neutrophils as a host defense mechanism [IL8 expression in GEC is induced by P. gingivalis [T. forsythia [P. gingivalis and P. intermedia [In addition to the microbial challenge, other factors, such as genetics, environment, and host factors, play a role in the pathogenesis of these diseases –9. Varioechanism , 14. IL8ngivalis  and T. forsythia ; IL-8 prtermedia . The IL-termedia –20, and termedia .IL8 gene located on chromosome 4q13-21 (GenBank accession number M28130.1), consisting of four exons, three introns, and the proximal promoter region [IL8 gene [IL8 gene such as −845T/C (rs2227532), −738T/C, −251A/T , +396T/G (rs2227307), and +781C/T (rs2227306) have been studied in patients with AgP or CP in the Brazilian population [IL8 −251T allele, which was associated with higher production of IL-8, increased the risk of developing acute suppurative form of apical periodontitis (AP), whereas IL8 −251A “low-producing” allele was associated with chronic nonsuppurative form of AP in the Colombian population [IL8 −251A allele has been associated with decreased susceptibility to CP [IL8 gene but did not specify whether it was for patients with CP or AgP [IL8 gene polymorphisms in patients with periodontitis has been performed in Caucasians.IL-8 is encoded by the r region . SeveralIL8 gene –26 and s5T/C rs22532, −738pulation . In the ty to CP . The croIL8 gene  and their haplotypes to CP and AgP and subgingival bacterial colonization in the Czech population.The aim of this study was to associate four SNPs in the The study was performed with the approval of the Committees for Ethics of the Medical Faculty, Masaryk University Brno and St. Anne's Faculty Hospital. Written informed consent was obtained from all participants before inclusion in the study, in line with the Helsinki Declaration.All patients were recruited from the patient pool of the Periodontology Department, Clinic of Stomatology, St. Anne's Faculty Hospital, Brno, from 2005 to 2011. They had at least 20 remaining teeth and were in good general health. Exclusion criteria included history of cardiovascular disorders (such as coronary artery diseases or hypertension), diabetes mellitus, malignant diseases, immunodeficiency, current pregnancy, or lactation. Controls were selected from subjects referring to the Clinic of Stomatology for reasons other than periodontal disease  during the same period as patients and matched for age, gender, and smoking status. Similarly as patients, all controls had at least 20 remaining teeth and were in good general health. Exclusion criteria were the same as those applied for patients with periodontitis.N = 278): all patients with chronic periodontitis (CP) fulfilled the diagnostic criteria defined according to CAL levels by the International Workshop for a Classification of Periodontal Diseases and Conditions for Chronic Periodontitis [Generalized CP group (dontitis . InclusiN = 58): patients with aggressive periodontitis with age at disease onset <35 years, attachment loss of 4 mm or more in at least 30% of the teeth (at least three of the affected teeth were not first molars and incisors), and the severity of attachment loss being inconsistent with the amount of dental plaque were included in this study.Generalized AgP group (N = 156): controls were screened using a WHO probe and the CPITN  was assessed [Control group   and attachment loss (CAL) were collected with a UNC-15 periodontal probe from six sites on every tooth present. The loss of the alveolar bone was determined radiographically. We used the index of Mühlemann to evaluate decreases in alveolar bone levels.In order to adjust for the effect of smoking history on periodontal disease, the subjects (patients and controls) were classified into the following groups: subjects who never smoked (referred to as nonsmokers) and subjects who were former smokers for ≥5 pack years or current smokers (referred to as smokers). The pack years were calculated by multiplying the number of years of smoking by the average number of cigarette packs smoked per day.μL−1 at 4°C) as well as the genotyping of samples were conducted in the laboratory of the Department of Pathophysiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.DNA for genetic analysis was extracted from the peripheral blood leukocytes using standard phenol/chloroform procedures with proteinase K according to Sambrook et al. . IsolatiIL8 gene were genotyped using the 5′ nuclease TaqMan assay for allelic discrimination. Individual fluorogenic TaqMan probes, consisting of an oligonucleotide labelled with both a fluorescent reporter dye, FAM, and a quencher dye, VIC, were obtained from Life Technologies . Each reaction mixture was prepared using TaqMan Genotyping Master Mix (12.5 μL), TaqMan SNP Genotyping Assay (1.25 μL), and 50 ng of genomic DNA in 17.5 μL of dH2O to make a 25.0 μL reaction volume. Genotyping was carried out simultaneously with 88 samples on 96-well plate (+8 negative controls). Details on SNPs detection are summarized in Four SNPs  in the Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, Peptostreptococcus micros, and Fusobacterium nucleatum) in subgingival pockets was investigated by the DNA microarray based on a periodontal pathogen detection kit  in a subgroup of randomly selected subjects  before subgingival scaling. Microbial samples were collected from the deepest pocket in periodontitis patients  of each quadrant by inserting a sterile paper point into a base of the pocket for 20 seconds. Bacterial plaque samples from each individual were pooled in one tube. This test determined the individual pathogens semiquantitatively as follows: (−) undetected, which corresponds to the number of bacteria less than 103, (+) slightly positive corresponding to the number of bacteria 103 to 104, (++) positive corresponding to the number of bacteria 104 to 105, and (+++) strongly positive, with the number of bacteria higher than 105.Subgingival bacterial colonization . Variations in the quantity of subgingival bacteria corresponding to the particular genotypes/alleles were tested by χ2 and Fisher's exact tests. Differences were considered significant at P < 0.05.To examine the linkage disequilibrium (LD) between all SNPs, pairwise LD coefficients (D′) and haplotype frequencies were calculated using the SNP Analyzer 2 program  as end-point statistical measures. ORs with corresponding 95% confidence intervals (CI) were estimated using logistic regression models, adopting age, sex, and smoking as adjusting covariates. All calculations were performed using Statistica ver. 10.0  and SPSS software .P < 0.05). Nearly, 27% of the periodontitis patients (28.0% of CP and 26.0% of AgP) and 28.0% of healthy subjects were smokers. There were no significant differences between the subjects with periodontitis and controls regarding the mean percentage of smokers and ratio of males/females .The mean ages for AgP patients  and healthy subjects (41.0 ± 12.3) did not differ between the two groups. However, subjects with CP were significantly older (47.9 ± 8.7) than the patients with AgP  allowed a statistically significant detection of OR out of the range of 0.55–1.80 . In case of the AgP group , the statistically significantly detectable ORs estimates were out of the range of 0.39–2.59. IL8 polymorphisms were not significantly different between the subjects with CP and/or AgP and controls .All studied polymorphisms were in the Hardy-Weinberg equilibrium in the control group. Allele and genotype frequencies of all investigatedIL8 gene . All variants in the IL8 gene were in tight linkage disequilibrium with each other to various degrees . The complex analysis revealed differences in IL8 haplotype frequencies. Specifically, the A(−251)/T(+396)/T(+781) and T(−251)/G(+396)/C(+781) haplotypes were significantly less frequent in patients with CP  . Moreover, an uncommon ATT/ATT haplotype (1.15% of the studied population) was found more, but nonsignificantly, in non-periodontitis controls . There was also a nonsignificant trend in the ATC/TTC haplotype association with CP  . The decF. nucleatum occurred less frequently in nonperiodontitis subjects (N = 75) positive for T allele of IL8 +396G/T variant  or TT genotype . In contrast, IL8 −251T allele carriers had an increased OR for individual presence of A. actinomycetemcomitans in AgP (N = 21) patients  and also TT genotype was more often found in A. actinomycetemcomitans presence . Patients with CP (N = 151) carrying CC genotype of IL8 +781T/C variant had less frequent presence of T. forsythia in their subgingival microflora than subjects without this genotype . However, the relationship between periodontal bacteria and IL8 gene polymorphisms must be assessed very carefully regarding small numbers of subjects in the respective subgroups.IL8 and their genes, are important potential modifiers of individual susceptibility to AgP or CP. Although none of the investigated SNPs in the IL8 gene was individually associated with aggressive or chronic periodontitis, the patients with CP showed lower A(−251)/T(+396)/T(+781) and T(−251)/G(+396)/C(+781) haplotype frequencies than the controls. The association of TGC haplotype with CP was confirmed by the relationship between TGC/TTC haplotype (arranged as genotypes) and CP. These results confirm the hypothesis that haplotypes are more powerful for detecting susceptibility alleles than individual polymorphisms. Our results differ from those obtained by Scarel-Caminaga et al. [IL8 −845(T/C)/−738(T/A)/−353(A/T) variants showed significant association to, or protection against, CP [IL8 SNPs, only one polymorphism was the same as in our study  referred to as −353 in Brazilian study). Polymorphism −845T/C (rs2227532) was also investigated in this study, but regarding a very low frequency of C allele, it was analyzed only in several individuals and therefore it was not included for any further haplotype assessment.Cytokines involved in the inflammatory process, such as a et al.  who assoa et al. . In the inst, CP . Of the IL8 SNPs  and CP or AgP in different populations (mostly in Brazilian but none in the Caucasian population) have been performed, with contradictory results [IL8 −251 variant and AgP; however, no association was found. Similarly, Kim et al. [IL8 −251 SNP. In contrast, another study discovered a significant association between IL8 −251 SNP and CP in nonsmokers. The IL8 −251TA heterozygote genotype was associated with increased levels of IL8 mRNA transcripts and A allele had an increased risk for developing periodontitis [IL8 −251 tended to be associated with higher IL-8 production in lipopolysaccharide- (LPS-) stimulated human whole blood [IL8 −251 variant was associated with decreased susceptibility to CP in Chinese population. Houshmand et al. [IL8 −251A/T and +396G/T SNPs between subjects with periodontitis and a control group in Hamedan, Iran, but did not specify whether it was for patients with CP or AgP. Conversely, Corbi et al. [IL8 gene was not associated with worse periodontal clinical parameters and increased IL-8 concentration. With the exception of Scarel-Caminaga et al. [To date, eight studies evaluating the association of  results , 34, 35. results  studied m et al.  failed tdontitis . This isi et al.  showed ta et al.  who assoIL8 −251T allele carriers had an increased OR for the individual presence of A. actinomycetemcomitans in AgP patients (P < 0.01) and an increased OR was also found for the presence of T. forsythia for T allele of IL8 +781 in CP patients (P < 0.05). These data are in agreement with the notion that individual genetic susceptibility may influence the host response to infection [IL6 SNPs and A. actinomycetemcomitans, which they confirmed by the haplotype analysis. Specifically, IL6 −174GG genotype was associated with high (above median) counts of A. actinomycetemcomitans  in Indians [IL1 gene cluster could be associated with subgingival colonization with A. actinomycetemcomitans and could thus be an independent risk indicator of AgP. In addition, Finoti et al. [IL8 ATC/TTC haplotype than in patients without this haplotype.Several studies have examined polymorphisms in interleukins in connection with a subgingival bacterial colonization in patients with periodontitis. In this study, SNPs in the gene encoding IL-8 were associated with the presence of pathogenic bacteria in subgingival dental plaque. The results showed that nfection . Nibali nfection  found as Indians . But Nib Indians  also sug Indians  found noi et al.  observedIL8) is the nature of periodontitis as a multifactorial disease in which interaction between multiple genes plays a role and each genetic polymorphism has generally only a small effect. In addition, the interaction of gene variants with environmental factors , socioeconomic factors, BMI, and others not analyzed in this study, potentially affect the observed phenotype. Second, the case-control approach used is generally quite vulnerable to the population stratification, for example, due to different ethnic origin. The present sample, however, is exclusively of the Czech Caucasian origin, restricted to the limited geographical area populated by quite homogeneous population with low admixture. Finally, we did not measure RNA expression or protein levels of IL-8, therefore, we do not know the functional consequences of these polymorphisms in our subjects.There are some limitations to this study that need to be considered. First, the major complicating factor in the study of isolated locus (such as IL8 gene were individually associated with periodontitis, some haplotypes can be protective against CP in the Czech population. Clinical significance of these findings is low due to a very low frequency of the “protective” haplotypes. The individual IL8 variants were associated with subgingival colonization with A. actinomycetemcomitans in AgP and with T. forsythia in CP in the Czech population. However, these relationships must be assessed very carefully regarding small numbers of subjects in the respective subgroups. Further studies are needed to clarify the association of these polymorphisms with periodontal diseases in other populations.In conclusion, although none of the investigated SNPs in the


From 5% to 10% of diabetic patients have type 1 diabetes. Here we describe three cases of adult-onset type 1 diabetes in pregnancy treated at our clinic between 2009 and 2012. Two patients came for specialist examination during pregnancy, the third after pregnancy. These women had no prior overt diabetes and shared certain characteristics, that is, no family diabetes history, age over 35, normal prepregnancy BMI, need for insulin therapy as of the early weeks of pregnancy, and high-titer anti-GAD antibody positivity. The patients had persistent diabetes after delivery, suggesting that they developed adult-onset type 1 diabetes during pregnancy. About 10% of GDM patients become pancreatic autoantibody positive and the risk of developing overt diabetes is higher when two or more autoantibodies are present (particularly GAD and ICA). GAD-Ab shows the highest sensitivity for type 1 diabetes prediction. We need to bear in mind that older patients might conceivably develop an adult-onset type 1 diabetes during or after pregnancy. So we suggest that women with GDM showing the described clinical features shall be preferably tested for autoimmunity. Pregnant patients at risk of type 1 diabetes should be identified to avoid the maternal and fetal complications and the acute onset of diabetes afterwards. Autoantibodies directed against insulin, islet cells, glutamic acid decarboxylase 65 (GAD), and tyrosine phosphatase IA-2 are markers of this immune destruction [From 5% to 10% of diabetic patients have type 1 diabetes, which results from an autoimmune destruction of pancreatic 2). Based on her glycemic profiles, insulin therapy was begun in her 17th g.w., in addition to the already-prescribed dietary restrictions. During the pregnancy, the patient's metabolic control was good (mean HbA1c 5.4%). The baby was delivered in the 39th g.w. (birth weight 3265 g). Postpartum OGTT revealed diabetes mellitus (2 h plasma glucose >200 mg/dL) with low blood insulin values (5–23.6 mU/mL), C-peptide 1.1 ng/mL, and anti-GAD Ab positivity (>2000 U/L), while HbA1c was 5.9%. In October 2010, the patient resumed insulin therapy and now has 4 injections a day, but her metabolic control remains poor (HbA1c 9.5%).The first concerns a 42-year-old woman who was referred for specialist examination in her 16th week of gestation (g.w.) to confirm a diagnosis of gestational diabetes. Her family history was negative for diabetes and her prepregnancy BMI was normal (22.5 kg/m2). GDM had been diagnosed in the third trimester of the first pregnancy, but within the first two weeks of the second. Insulin therapy was needed in both pregnancies (right from the first few weeks in the second). Diabetes mellitus was diagnosed on postpartum OGTT after the second pregnancy. The patient took metformin therapy for several months, with no benefit. In April 2011, she was hospitalized and laboratory tests revealed: glycemia 170 mg/dL; HbA1c 7.3%, C-peptide 1.2 ng/mL; and anti-GAD Ab positivity (2000 U/L). She is currently on dietary restrictions alone.The second patient was 39 years old when she came for her first specialist examination. Her family history was negative for diabetes. Between 2004 and 2007 she had successfully completed two pregnancies, both complicated by gestational diabetes. Her prepregnancy BMI was normal (20.4 kg/m2). Given her poor glycemic profiles, insulin therapy was begun immediately in addition to dietary restrictions. In the 29th g.w., blood tests revealed anti-GAD Ab positivity (268 U/L) and low fasting plasma C-peptide levels (1.8 ng/mL). During the pregnancy, the patient's metabolic control was good (mean HbA1c 6%). The baby was delivered in the 35th g.w. (birth weight 2520 g). The patient's metabolic control is currently good (HbA1c 5.6%) and she is on dietary restrictions alone.The third patient was 36 years old and came for specialist examination in her 28th g.w. in February 2012. Her family history was negative for diabetes and her prepregnancy BMI was normal . Before they were referred to us for examination, all three patients had fasting plasma glucose levels below 126 mg/dL [These pregnant women shared certain characteristics, that is, no family history of diabetes, age over 35 years, normal prepregnancy BMI, need for insulin therapy as of the early weeks of pregnancy, high-titer anti-GAD antibody positivity, and low fasting plasma C-peptide levels. The patients also had persistent diabetes after delivery, suggesting that they developed adult-onset type 1 diabetes during their pregnancy.The literature on autoimmunity and pregnancy is limited. In most cases, GDM is characterized by a higher insulin resistance and lower insulin secretion, both of which are defects typical of type 2 diabetes. Women with GDM are consequently believed to be at higher risk of developing type 2 diabetes after pregnancy. About 10% of GDM patients become pancreatic autoantibody positive, however, so—although type 1 diabetes generally develops in younger people (under 30 years old)—we need to bear in mind that older patients might conceivably develop an adult-onset type 1 diabetes during or after pregnancy . In our personal experience, these are the first cases of adult-onset type 1 diabetes occurring during pregnancy and our findings confirm the importance of identifying the risk of patients developing this type of diabetes. Most studies have demonstrated that the presence of diabetes-related autoimmunity in patients with GDM predicts the occurrence of type 1 diabetes; the risk of developing overt diabetes is higher when two or more autoantibodies are present (particularly GAD and ICA). Füchtenbusch et al. have reported that two years after delivery the risk was 17% with one antibody, 31% with two antibodies, and 84% with three antibodies positivity. In the study of Nilsson et al., 83% of the patients with GDM and positivity for at least one autoantibody developed type 1 diabetes 4 years after delivery. Moreover, GAD-Ab shows the highest sensitivity for type 1 diabetes prediction –7.In addition, it has been found out that women with GDM and autoantibodies more frequently need insulin therapy , 5; howeUp to now there are not guidelines about which patients should be checked for autoantibodies. According to our experience, we suggest that women with GDM showing clinical features as no family history of diabetes, normal pre-pregnancy BMI, need for insulin therapy in the early weeks of pregnancy, and age higher than 35 years, shall be preferably tested for autoimmunity.Pregnant patients at risk of type 1 diabetes should be identified to avoid the maternal and fetal complications of this form of diabetes developing during the pregnancy and, very importantly, the acute onset of diabetes afterwards. Women found anti-GAD positive should be considered at high risk of developing type 1 diabetes and should consequently be regarded as potential future candidates for immunomodulatory strategies .


Pimenta pseudocaryophyllus popularly referred to as craveiro is considered as a calming agent in different local preparations. The present study attempted to examine antidepressant-like effect of dichloromethane fraction (DF) and role of monoamine oxidase (MAO), tryptophan, and tyrosine hydroxylase. Based on the research focus, tail suspension (TS), forced swimming (FS), and open field (OF) tests were conducted after oral administration of DF . Ex vivo assay of MAO was also conducted to evaluate inhibitory effect of DF (250 mg/Kg). Administration of DF elicits antidepressant-like response in the TS and FS. However, DF 500 mg/Kg did not alter mice performance in these models. The data obtained in the OF showed a reduction in total crossing and rearing activity; these effects suggest motor interference in TS and FS performance. Mice pretreatment with p-chlorophenylalanine methyl ester (PCPA)  for 4 consecutive days or acute administration of α-methyl-p-tyrosine (AMPT)  blocked anti-immobility effect of DF in the FS. In ex vivo assay of MAO, DF did not inhibit catabolic activity of MAO. Our findings support antidepressant-like activity of DF and suggest an effect that depends on monoamine biosynthesis. Popular applications  of its leaf extract in different preparations have been reported especially in the county of Campos do Jordão, São Paulo, Brazil [u-cravo” , 2. The , Brazil .Pimenta pseudocaryophyllus leaf demonstrated behavioural alterations in the open field, elevated plus maze, light dark box, and barbiturate sleep induction tests without any form of motor incoordination [Pimenta pseudocaryophyllus is borne out of its anxiolytic property that has been associated with monoaminergic receptor [Previous neuropharmacological screening of essential oil, ethanolic leaf extract, and active fractions of the dination –9. Evalureceptor . Pimenta pseudocaryophyllus makes evaluation of its antidepressant effect imperative. The hypothesis of biogenic amine involvement in depression has produced several generations of antidepressant agents (monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), selective noradrenaline reuptake inhibitor (SNRI), and atypical antidepressants among others). The fact that clinical responses to drug effects take weeks of sustained treatment  and occuThus, we hypothesize that the efficacy of aforementioned antidepressive drugs or natural products to treat anxiety or vice versa is a function of the active principles in these compounds and underlining mechanism of their neuropharmacological action. In this manner, the aim of this study was to investigate antidepressant-like property of DF on mice and involvement of metabolic enzymes (tryptophan hydroxylase (TrOH), tyrosine hydroxylase (TOH), and monoamine oxidase (MAO)). The leaf collection, identification, voucher specimen (herbarium) deposit, extraction, and partitioning of ethanolic leaf extract to obtain DF as well as qualitative and quantitative analysis of this fraction by HPLC were achieved following the procedure described in our previous work .Male albino Swiss mice (30 ± 5 g) were provided by Central Animal House of Federal University of Goiás (UFG). They were housed under controlled environmental conditions  and allowed free access to standard food and water. All experimental procedures were conducted with strict adherence to the regulations of ethical principles in animal research as adopted by the Brazilian society of laboratory animal science. The experimental protocol was approved by research ethic council of the Federal University of Goiás (no. 104/08). α-methyl-p-tyrosine  used to deplete monoamine  were administered intraperitoneally to groups of mice; imipramine , diazepam , clorgyline 15 mg/kg (an irreversible and selective inhibitor of monoamine oxidase A), and tranylcypromine 15 mg/kg (a nonselective and irreversible inhibitor of monoamine oxidase) used as standard drug were dissolved in 0.9% saline, dichloromethane fraction  was dissolved in vehicle  while control animals received appropriate equivalent vehicle. Oleanolic acid  was used as a standard for HPLC analysis of DF. All solutions were freshly prepared on test days and administered (10 mL/kg of mice body weight).p-Chlorophenylalanine methyl ester  and According to the method described by Steru and his collaborators , duratioAssessment of antidepressant-like effect of DF was conducted in FS. Pretrial exposure  of mice to this apparatus was followed by a 6 min test period during which scoring of immobility time was realized. The FS was realized following the treatment procedure under Section  2.4.1 and placement of mice in a cylinder  filled with water (24°C) up to 30 cm. Experimental subject was considered to be immobile when it ceased struggling and making minimum movements necessary to keep afloat . In subsMice were individually placed at the centre of the open field apparatus in a sound proof experimental room to measure rearing and locomotor activity during 5 min after oral administration of vehicle, DF (500 mg/kg), or DZP (5 mg/kg). The floor of the OF is divided into 8 sectors of equal area. The number of crossing and rearing was registered for further statistical analysis. The apparatus was cleaned with 10% alcohol after mouse exposure.Mice were treated acutely with DF 250 mg/kg), clorgyline 15 mg/kg (a selective inhibitor of MAO-A), or tranylcypromine 15 mg/kg (a nonselective and irreversible inhibitor of monoamine oxidase) and sacrificed by decapitation after 60 min. Brain tissues homogenates were prepared according to  and stor0 mg/kg, t-test was used as described by Drummond and Tom [P < 0.05 were considered to be significant.In order to compare level of significance between two groups, unpaired Student's  and Tom , 27. To P < 0.05 and  **P < 0.01, resp.) and rearings  in the open-field test (OF) significantly when compared with the vehicle treated group. Reduction in these parameters is an indication of motor incoordination by DF 500 mg/kg or DZP 5 mg/kg treatment. Effect of biosynthetic enzymes inhibition that result in indoleamine (serotonin) depletion was shown in the FS with p-chlorophenylalanine methyl ester  pretreatment for four consecutive days followed by acute oral administration of vehicle, DF (250 mg/kg) or imipramine (30 mg/kg) as described under Section  2.4.2.#P < 0.001) with PCPA pretreatment. Administration of this tryptophan hydroxylase (TrOH) inhibitor alone did not elicit significant behavioural alteration in the FS. Similar observation was made with the α-methyl-p-tyrosine  pretreatment, an inhibitor of tyrosine hydroxylase (TOH), the rate-limiting enzyme for catecholamine biosynthesis, administered 4 h before the FS.  #P < 0.05). Effect of DF on brain MAO as investigated showed, unlike clorgyline and tranylcypromine that reduced MAO activities to 17.7 ± 6.4% (P < 0.001) and 47 ± 9.0% (P < 0.01), respectively, its ineffectiveness to inhibit MAO activity . With this result, it is obvious that DF do not have inhibitory effect on any of the MAO isoforms (A and B).The chromatograms in P. pseudocaryophyllus showed the presence of triterpenes, flavonoids, besides the (E)-methyl isoeugenol which constitute almost all of the essential oil (approximately 94%) [Previous phytochemical analysis of the fractions obtained through liquid-liquid partitioning of the ethanol leaf extract of ely 94%) . In the ely 94%) . Being oely 94%)  and unpuThe neuropharmacological activity of dichloromethane fraction (DF) had been demonstrated and was associated with the involvement of serotonergic pathway . As earl Acute treatment with the standard drug or DF seems not to satisfy the aspect of face validity in this test considering the notion that therapeutic actions of antidepressant drugs evolve gradually with chronic treatment –33. Howe The insinuations of acquirement of anxiolytic properties by antidepressants following chronic administration  may not  However, the intriguing nature of DF 500 mg/kg (highest dose) insignificance effect in antidepressive models (TS and FS) led us to its evaluation in the open field. In this animal model, parameters  that are susceptible to the effects of myorelaxant or sedative agent were evaluated to augment information obtained on antidepressant models. Interestingly, reduction in these parameters as a result of DF 500 mg/kg administration is an indication of motor incoordination. Moreover, CNS stimulatory effect is also one of the commonly found false positive effects of natural or synthetic product in these models. However, previous results , 9 did nα-methyl-p-tyrosine (AMPT) was used to deplete catecholamine storage [ In an attempt to investigate possible mechanism of action involved, biosynthetic enzymes was hypothesized to influence the synaptic level of monoamine. Metabolic activities of cytosolic enzymes like tryptophan hydroxylase (TrOH) and tyrosine hydroxylase (TOH) indirectly influence monoaminergic transmission. TrOH and TOH are rate limiting enzymes in serotonin and norepinephrine synthesis, respectively. Evidences in the literature showed that inhibition of norepinephrine and serotonin synthetic enzymes blocked antidepressant effect of desipramine or fluoxetine, respectively, and elicit a rapid return of symptoms in depressed patients , 43. Ser storage . Pretrea storage , 48. Reduction in immobility time in this research is a reflection of an increase in swimming and struggling. According to Millan , dual noHypericum perforatum that inhibits monoamine oxidase (A and B) [Moreover, activity of drugs on MAO has been employed in the treatment of depression. In the treatment of this neural disease, monoamine oxidase inhibitors especially MAO A inhibitors  have proved to be more effective compared to MAO B inhibitor-like selegiline , 51. MAOA and B) . Unlike A and B)  and tranA and B) ), we canIn conclusion this work reveals antidepressive-like property of dichloromethane fraction and integrates new findings of possible mechanisms underlining antidepressant action with a growing body of evidence on vital role of monoamine biosynthetic enzyme. Subsequent preclinical study will be focused on active principles that are responsible, toxicological study, and dose extrapolation for possible clinical trial.


Soluble CD40 ligand (sCD40L) and matrix metalloproteinase 9 (MMP-9) are inflammation markers and have been poorly described in infectious disease. In this prospective study, we describe the sera kinetics of these two molecules in the course of treatment follow up in human visceral leishmaniasis (VL).p-value < 0.05).Sera from VL patients were collected before and during follow up of regular Antimony treatment. sCD40L and MMP-9 were measured by Luminex assay. Paired analysis by Wilcoxon signed test was used for comparison of values of the same subjects before and after initiation of treatment. Correlations between clinical data and parasite load with the serum levels of sCD40L and MMP-9 were performed by Spearman test. Tests were considered statistically significant if the probability of a type I error was less than 5% . Additionally, negative correlations were found between spleen sizes and MMP-9 before treatment and sCD40L at day 15 of treatment. Negative correlations were also found between parasite load with both sCD40L and MMP-9.While sCD40L and MMP-9 were not observed in sera from non endemic controls which are at low risk of Serum sCD40L and MMP-9 are identified as new and simple biomarkers in two situations: (i) monitoring the success of therapy and (ii) predicting favorable clinical outcome of human VL. Leishmania, transmitted to mammalian hosts by phlebotomine sandflies. Human VL is characterized by fever, hepatosplenomegaly, anemia, leukopenia, and severe weight loss and is fatal if not treated. VL is associated with a marked impairment of leishmania-specific Th1 response, as evaluated by in vitro leishmania antigen stimulation of peripheral blood mononuclear cells (PBMC) [Visceral leishmaniasis (VL) is a disease caused by systemic infection with protozoa of the genus s (PBMC) -4. IL-12s (PBMC) . These cs (PBMC) . High ams (PBMC) -9 and dis (PBMC) . The pros (PBMC) .L. major amastigotes deviate the cellular CD40 signaling pathway by inducing ERK 1/2 and IL-10 production, which inhibits the p38MAPK/IL-12 pathway [CD40 was identified on B cells, monocytes, dendritic cells, endothelial and epithelial cells . Cellula pathway .T. gondii[-/-) mice are more susceptible to L. major infection [L. major or T. cruzi infection [CD40-CD40L signaling is also important for antibody isotype switching, and germinal center formation, and patients with congenital X-linked hyper IgM syndrome (X-HIM), caused by the lack of functional CD40L, present an increased incidence of infections with opportunistic pathogens (reviewed in ). PeriphT. gondii. CD40L knfection , while mnfection ,18. The nfection ,20.CD40L may be expressed as an heteromultimeric complex . After cT. cruzi, by favoring the infiltration of immune cells [MMPs are produced by different cell types, including macrophages, lymphocytes, endothelial cells, and are associated with remodeling and modulation of inflammation . In tubene cells .sCD40L and MMP-9 have been described in cardiovascular disease as mediators of inflammation in arterial coronary disease, and are considered markers of poor prognosis ,28. In iThis was a prospective study performed in Hospital Universitário – Universidade Federal de Sergipe – Brazil, approved by the Ethics Committee of University Hospital from Universidade Federal de Sergipe. All individuals, or their legal guardians, signed an informed consent form. Pregnant women, patients receiving immunosuppressive treatments and patients with comorbidities  were excluded.Leishmania on bone marrow aspirates by direct exam and culture in NNN media , and rK39 serological test . Patients were examined using a standard protocol containing the following information: identification, clinical complaints, physical exam, and the results of laboratory tests performed (distinct from data used in this study). After diagnosis, patients received regular Antimony treatment (20 mg Sbv/Kg for 20 days). Liver and spleen sizes were evaluated by two observers by palpation and reported as the distance to the rib border in cm at diagnosis and every five day during the treatment until the end of therapy, and later, every 3 months until one year post treatment.Initially, 45 patients  with confirmed VL were selected for this study. The diagnostic criteria used for inclusion were identification of Several groups were included to serve as controls. A group of individuals (either household contacts or relatives) living in the same area as the patients but with no signs of clinical disease were recruited (endemic controls) (n = 37). Healthy subjects, United States Citzens, thus not exposed to the infection (non endemic controls) (n = 24) were also included. Sera of chronic Chagas disease patients (n = 29) were obtained from a reference laboratory for diagnosis of public health diseases. Clinical data were not available regarding serum from patients with Chagas disease.Blood samples without anticoagulant were collected for all study participants, for VL patients, blood was collected before specific leishmania therapy, day 0 (D0) (n = 45) and at several times after the initiation of treatment . Sera were collected and stored at -70°C until sCD40L, and MMP-9 concentrations were determined by a Luminex assay, performed according to the manufacturer’s instructions . The levels of sCD40L were measured in all sera samples collected from VL patients, while MMP-9 concentrations were determined in D0, n = 37; D15, n = 30; D30, n = 36; D45, n = 29; D60, n = 29; and D180, n = 6. The measurement of transaminases (ALT and AST) by a chemistry system  was adopted as a hepatic damage marker before treatment in VL patients and was also tested in endemic controls . The few missing values for laboratorial data were due to samples limitations.Evaluation of parasite load was carried out in blood samples of VL patients before treatment D0 (n = 15), at D30 (n = 13) and D60 (n = 13) of treatment. The blood samples were collected in tubes containing Paxgene , according to manufacture’s instructions. Nucleic acid extraction was performed by using a QIAamp DNA, Blood Mini Kit . The Real-time PCR based on TaqMan Probe was applied for accurate quantification of Leishmania in blood samples of VL patients, described previously. Specifip-value < 0.05).Kolmogorov-Smirnov normality test was applied. Data were analyzed for different groups  by a nonparametric Kruskall-Wallis with post Dunn’s multiple comparison test. Student T test for parametric or Mann–Whitney for the non-parametric samples was used for two groups comparisons. Paired analysis by Wilcoxon signed test was used for comparison of values of the same subjects before and after initiation of treatment. Correlations between clinical data and parasite load with the serum levels of sCD40L and MMP-9 were performed by Spearman test. All tests were carried out using Graph Pad Prism, version 4.0, 2005. Tests were considered statistically significant if the probability of a type I error was less than 5%   Figure . Thus, bp < 0.001). Interestingly, levels of these analytes in endemic controls were also higher than those in VL patients (p < 0.005)    and MMP-9  levels higher than non endemic controls . In order to evaluate if MMP-9 is involved in hepatocyte damage, the sera levels of transaminases (ALT and AST) were correlated with the sera levels of sCD40 and MMP-9 from VL patients before treatment and from endemic controls. Although VL patients showed levels of ALT (79.20 ± 103.4 U/ml) and AST (119.6 ± 188.4 U/ml), higher than those of the endemic controls  . Similarly, sCD40L levels and spleen size correlated at D15 of treatment   and sCD40L  were also observed  and of free radicals, which require IL-12 and IFN-γ production. Mice infected with T. cruzi and treated with CD40L exhibit a decrease in parasitemia and in mortality that are accompanied by prevention of the immunosuppression that typically follows T. cruzi infection [Previous animal studies have indicated the importance of the CD40-CD40L signaling pathway for protection against a variety of parasites (reviewed in ). CD40 snfection .L. donovani infection [L. major amastigotes modulate the signaling pathway downstream of membrane CD40 engagement by inducing ERK 1/2 and IL-10 production, which inhibits the p38MAPK/IL12 pathway [In leishmania infection, the role of CD40L is clearly demonstrated. Knockout mice have an increased susceptibility to nfection . CD40-CDnfection . CD40L infection . It is anfection ). Responnfection ,38. Morenfection ,30,41,42nfection . Increasnfection . The levnfection . Our dat pathway . As sCD4 pathway , it mighDuring the course of VL infection there is extensive parasite multiplication that results in a high parasite burden in the spleen and liver. The enlargement of the spleen and liver is a cardinal feature of human VL and the return of these organs to their normal impalpable state has long been used to evaluate cure. The reductions of spleen or liver after the initiation of treatment (data not shown) are observed at D15 of treatment, and at this time point, the reduction of spleen size correlates with serum level of sCD40L.Trypanosoma cruzi infection also produces an intense inflammatory response in diverse tissues including the heart [Our data also indicate elevated sCD40L in Chagas disease patients and it is possible that sCD40L levels may be related to the control of parasitemia seen in the chronic stage of disease. Since he heart , the sCDThe CD40-CD40L signaling pathway also is involved in matrix metalloproteinases (MMPs) expression, including MMP-9 ,20. Our Alternatively, sCD40L is exposed to cleavage by the action of MMPs, liberating soluble CD40L . Thus, iConfirming their role as a marker of favorable clinical evolution in VL patients, inverse correlations between the serum sCD40L levels and MMP-9 with parasite load were found. Serum levels of IL-10 are also a good marker of disease severity and directly correlated with parasite load, considering its immunosuppressive role of macrophage microbicidal mechanisms. Although further studies are required to understand both the mechanisms by which the sCD40L and MMP-9 influence disease outcome in human VL, and the timing of the release of these molecules in sera after the infection, sCD40L and MMP-9 represent useful biomarkers with which to predict favorable response to VL therapy. In addition, as there is no marker of protection of clinical evolution from leishmania infection to clinical disease, and in some endemic areas it is difficult to assess clinical data of households contacts, sCD40L and MMP-9 represent important predictive clinical outcome biomarkers.MMP-9 in macrophage-hepatocyte co-culture supernatants is associated with liver damage, and the release of transaminases AST and ALT . LeishmaThis study identifies serum sCD40L and MMP-9 as new and simple biomarkers in two situations: (i) monitoring the success of therapy and (ii) predicting favorable clinical outcome of human VL.The authors declare that they have no competing interests.FAO helped to collect the data, built the database, performed the statistical analysis and wrote the paper. CVOS, NPD and ROP conduct the treatment follow up of the VL patients, and collected medical data and the serum samples. MSD, JAG and AB performed the immunoassays and helped to draft the manuscript. TRM helped to collect patients samples and draft the manuscript. SGR participated in study design and financed the immunological tests. RPA participated in study design, supervision of clinical study, and conduct the treatment and follow up of the VL patients. ARJ participated in the supervision of clinical study, provide major help to the first author to perform the statistical analysis and in the manuscript preparation. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/331/prepub


RBP levels were within normal ranges but declined with age. The two indicators of iron levels suggested that although levels of stored iron were within normal levels , levels of functional iron (sTfR levels) were reduced in 28.6% of the population. Schistosome infection alone was not associated with levels of any of the micronutrients, but altered the relationship between parasite-specific IL-4 and IL-5 and levels of ferritin and sTfR.Micronutrients play an important role in the development of effective immune responses. This study characterised a populations exposed to schistosome infections in terms of the relationship between micronutrients and immune responses. Levels of retinol binding protein , vitamin D, ferritin and soluble transferrin receptor (sTfR), and C reactive protein (CRP) were related to levels of schistosome specific cytokines (IFN- Schistosoma trematode species [Schistosoma haematobium is the causative agent of urogenital schistosomiasis and is widely distributed in Africa [S. haematobium affected growth and nutritional status of children [Micronutrients are known to play an important role in health and the development of an effective immune system. In tropical and subtropical regions there is an overlapping distribution of helminth infections and micronutrient deficiencies. –3. Schis species , 4, 5. Sn Africa . Infectin Africa . Simultachildren . It is cREG) through direct contact stimulation and IL-10 production [H2 which occurs during helminth infection is an effective antiparasitic response, it is unclear whether superimposition of regulatory responses primarily benefits the worms or the host. Downregulation of the inflammatory response would reduce host mediated immunopathology but also reduce protection [Acquired immunity to schistosomiasis develops slowly and only provides partial protection . Schistooduction , 13. Whiotection , 14. TheH2, TH17 and TREG responses through the activation of retinoid receptors. Retinoic acid activates the FoxP3 transcription factor, which stimulates the development of naïve T cells into TREG[H2 antibodies IgE, IgG1, and IgA [Traditionally vitamin A has been known for its role in vision, with deficiency resulting in xerophthalmia, which is the leading cause of preventable childhood blindness. However, it has a wide range of physiological functions and is essential for haematopoiesis and prevention of anaemia, as well as immune function. It is acquired from foods such as liver, milk, cheese, eggs, green leaves, carrots and ripe mangos. Infants acquire vitamin A through breast feeding . Vitamininto TREG–17. Vitainto TREG. Hypovit and IgA .2 vitamin D3 in the kidney [H1 cytokines IFN-γ and IL-2, and upregulates IL-4 to create a TH2 polarisation. Vitamin D can stimulate TREG through production of TGFβ-1 and CD25 expression by CD4+ T cells [REG induction [Vitamin D is historically known for its role in calcium and bone homeostasis. It is produced in the skin when 7-dehydrocholesterol reacts with UVB radiation to form vitamin D3, which modified in the liver to form 25(OH) vitamin D3, and converted to its active metabolite 1,25(OH)e kidney . Vitamine kidney . A role e kidney . The imm T cells –24. It anduction , 23.S. japonicum and iron deficiency anaemia has been established [REG responses whereas iron supplementation has been linked to TH1 responses and decreased IL-10 [Anaemia affects 1.62 billion people worldwide , and aroablished . It is lablished , 28. Itsablished , 29. Expablished . Iron deed IL-10 , 31. Theed IL-10  since feA few studies have shown a recent review of data collected in Zimbabwe between 1980 and 2006 showed that a significant proportion of preschool children, school children, and adult women (lactating or pregnant) experienced malnutrition with significant proportions of these groups suffering from vitamin A and iron deficiencies .S. haematobium infection.The aim of this study was to determine the relationship between the micronutrients vitamin A, D, and iron as well as a measure of inflammatory responses C-reactive protein (CRP) and schistosome-specific cytokine levels in Zimbabweans exposed to The study received ethical and institutional approval from the Medical Research Council of Zimbabwe and the University of Zimbabwe, respectively. Permission to conduct the work in this province was obtained from the Provincial Medical Director. Informed consent/assent was obtained from all participants or their parents/guardians prior to enrolment into the study. Project aims and procedures were explained to the community, school children, and their teachers prior the study, and survey was conducted amongst all compliant participants. After sample collection, all participants were offered treatment with the standard dose of 40 mg/Kg body weight of the antihelminthic drug Praziquantel.S. haematobium is endemic. Participants were part of a larger immunoepidemiology study which was carried out between 2002 and 2005, and the study area is described in detail elsewhere [The study was conducted in two rural villages in the Mashonaland East Province of Zimbabwe  where lsewhere . The maiS. mansoni as well as negative for HIV and Plasmodium falciparum, (5) should have provided a blood sample to obtain sera. Furthermore, only sera samples were used for these analyses, which have not been used previously and therefore were defrosted for the first time. Following these criteria samples from 40 people aged 7–54 years  were included in this study. Data were subsequently separated into 3 age groups: 7–10 years (N = 6), 11–20 years (N = 23), 21+ years (N = 11), which represent a typical age-infection profile for S. haematobium as shown in All samples used in this study were obtained at baseline in 2002 were selected using following criteria: (1) participants should be life-long residents in this area (assessed by questionnaire), (2) should not have received antihelminthic treatment prior this study, (3) should have provided at least two urine and 2 stool samples on consecutive days to allow parasitological diagnosis, (4) should have been test negative for soil transmitted helminth and S. mansoni eggs and other intestinal helminths, while the urine filtration method [S. haematobium eggs in urine samples. Serum samples obtained from 20 mL of venous blood from each participant were frozen and stored in duplicate at −20°C in the field and transferred to a −80°C freezer in the laboratory. One complete set of the samples was subsequently transported frozen from Zimbabwe to the UK, stored at −80°C and defrosted for the first time for use in this study. Small aliquots of blood were used to prepare thick and thin smears for the microscopic detection of Plasmodium parasites.Parasitology samples (at least 2 urine and 2 stool samples collected on 3 three consecutive days) and 20 mL of venous blood were collected from each participant. Stool samples were processed following the Kato-Katz procedure  to detecn method  was usedγ, (marker for TH1 responses) IL-4, IL-5 (markers of TH2 responses), and IL-10 (marker for regulatory responses) were measured by enzyme linked immunosorbent assays (ELISA) in supernatants obtained after stimulation of whole blood samples using cercarial, egg, and adult schistosome antigens following published methods [The parasite-specific cytokines IFN- methods . SpontanMicronutrients and C reactive Protein (CRP) were measured using enzyme linked immunosorbent assay (ELISA) kits according to manufacturers' instructions. Serum transferrin receptor (sTFR) is a marker of iron deficiency and is required for lymphocyte activation and proliferation. It was assayed using an ELISA kits from R&D Systems (Cat. #DTFR1). Ferritin is a marker of iron status, but rises with inflammation , 38 and μg/L, male normal range 15.0–200.0 μg/L) [Statistical analyses were performed using the software PASW 17 (formerly SPSS). Vitamin D status was described using previously published ranges  . The Wor.0 μg/L) . R&D Sys.0 μg/L) . There i.0 μg/L) , 44. TheP values ≤0.05 were taken as significant.For the statistical analyses, host infection intensity was recorded into infection status, that is, infected and uninfected, cytokine absorbencies were square root transformed, and levels of all micronutrients were log transformed to satisfy the assumptions of parametric tests. In order to determine if the relationship between micronutrients and immune responses differed between schistosome infected versus uninfected people, a multivariate analysis of variance (MANOVA) was conducted. The dependent variables were the transformed micronutrient data and the independent variables were cytokine levels, infection status (infected/uninfected) age ), sex . The effects of interactions between infection status and micronutrients were also included in the MANOVA model. Sequential sums of squares were used to calculate the test statistics so that the potentially confounding effects of all other variables could be allowed for testing for the effects of infection status which was entered last in the single effects list. n = 12) of the population being classified as vitamin D replete (≥50.00 nmol/L); 17.9% (n = 7) were mildly deficient (25–49.90 nmol/L), 10.3% (n = 4) were moderately deficient (12.50–24.90 nmol/L), and 38.5% (n = 15) were severely deficient (≤12.49 nmol/L). Levels of CRP were within the normal range while 28.6% (n = 10) of the participants had elevated sTfR based on the 95 percentile data provided with the assay as detailed in the methods section.Schistosome infection prevalence in the study population was 60% (95% CI: 43–75%) and the mean infection intensity was 39.3 eggs/10 mL urine (SEM = 13.5) with a range of 0–362 eggs/10 mL urine. Infection intensity followed the typical schistosome age-infection pattern, rising with age to a peak in childhood and declining thereafter . The ager = −0.315, P = 0.033) as shown in The statistical analyses showed that sex affected only levels of ferritin, which was significantly lower in females and did not have a significant effect on levels of any of the other micronutrients . Age sigP = 0.049, β = 0.314) as well as between ferritin and parasite-specific IL-4 . In some cases, the relationship between the cytokine levels and micronutrients varied with schistosome infection status as shown in γ showed a significant positive correlation with sTFR in egg negative people but a negative but nonsignificant association in egg positive people  as well as from home-grown vegetables. Vegetables are amongst the prominent cash crops for commercial and small-scale farmers . This maIn this study serum retinol levels declined with age which is contradictory to reports from primary aged school children in Zimbabwe and Kenya , 48 whicS. haematobium infections with serum retinol levels in Zimbabwe, similar to observations in this current study. Interestingly, Friis et al. found, a strong negative association between S. mansoni infection and serum retinol levels in both Zimbabwe and Kenya, which suggests that the intestinal niche of S. mansoni infection may interfere with vitamin A absorption [S. mansoni infections. However, all participants of our study were negative for S. mansoni and therefore it was excluded as confounding factor.Friis et al. found no association between sorption , 48. Howsorption , which mIt has also been shown that all trans retinoic acid (ATRA) binds retinoic acid receptors, which induce FoxP3 expression polarizing immune responses towards a regulatory phenotype . Our fin3 occurs in the skin under the influence of ultraviolet light. Most studies of Vitamin D levels have been in Caucasian populations with reference to osteoporosis. It is possible that our findings may be explained by ethnic differences in skin pigmentation and skin UV penetration [H2 phenotype [Vitamin D produced the most surprising data, with 38.5% of subjects being severely deficient. There is a paucity of Vitamin D surveys in Africa compared to those conducted in Western countries. Since no clinical examination were conducted in this study, it is impossible to say whether the deficiencies observed in this study results are associated with pathology or remained asymptomatic. Production of pre-vitamin Detration , 51. Givetration . In thishenotype .γ and IL-4 in egg positive people, while IL-5 levels showed a positive association with ferritin in the same people. Iron replete people use iron in mounting inflammatory immune responses [H1 responses [γ is seen in iron deficiency, where it has a role in preserving iron stores [γ and IL-4 may be adaptive to preserving iron stores during schistosome infection. However, in the absence of mechanistic studies, this remains speculative.Iron deficiency is one of the most prevalent micronutrient deficiencies in the world affecting at least half of all pregnant women and young children in developing countries. In a survey conducted by the Ministry of Health and Child Welfare in 1997, 9% of the surveyed population  had depleted iron stores that is, ferritin. At the time of the study, pregnant and postpartum women were not offered iron supplementation by local healthcare providers, thus pregnancy and childbirth-related iron and blood loss may explain why male participants have significantly higher levels of ferritin. In this study, while ferritin levels were within normal ranges, sTfR levels were elevated in 28.6% of the population. Ferritin is an indicator of stored iron reserves in the body while sTfR indicates the functional iron component of the body and becomes elevated soon after the onset of iron deficiency. Ferritin is often decreased in iron deficiency anaemia, but can be raised in inflammatory conditions , 39. Howesponses . Iron suesponses , but alsesponses . Howevern stores , 28. ThuIn conclusion the study showed that while levels of vitamin A and iron where within normal ranges, there was a deficiency of vitamin D in 67.2% of the study population as well as elevated levels of sTfR in 28.6% of the participants. Thus, the 2 indicators of iron levels suggested that although levels of stored iron were within normal levels , levels of functional iron (measure by sTfR) may have been reduced in some participants. Schistosome infection intensity or status was not associated with levels of any of the micronutrients, but altered the relationship between parasite-specific IL-4 and IL-5 and the measures of iron levels (ferritin and sTfR). Cohort studies following a larger group of people through a cycle of antihelminthic treatment will clarify the effects of helminth infection on micronutrient levels and their subsequent effect on immune responses.


Limnoperna fortunei by characterizing the genetic structure in native and introduced ranges in Asia and South America. We surveyed 24 populations: ten in Asia and 14 in South America using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as well as eight polymorphic microsatellite markers. We performed population genetics and phylogenetic analyses to investigate population genetic structure across native and introduced regions. Introduced populations in Asia exhibit higher genetic diversity (HE = 0.667–0.746) than those in South America (HE = 0.519–0.575), suggesting higher introduction effort for the former populations. We observed pronounced geographical structuring in introduced regions, as indicated by both mitochondrial and nuclear markers based on multiple genetic analyses including pairwise ФST, FST, Bayesian clustering method, and three-dimensional factorial correspondence analyses. Pairwise FST values within both Asia  and South America  were lower than those between continents . Fine-scale genetic structuring was also apparent among introduced populations in both Asia and South America, suggesting either multiple introductions of distinct propagules or strong post-introduction selection and demographic stochasticity. Higher genetic diversity in Asia as compared to South America is likely due to more frequent propagule transfers associated with higher shipping activities between source and donor regions within Asia. This study suggests that the intensity of human-mediated introduction vectors influences patterns of genetic diversity in non-indigenous species.We explored possible links between vector activity and genetic diversity in introduced populations of  Blackburn et al. The distribution and genetic structure of introduced populations can exhibit complex patterns Dreissena polymorpha in North America exhibit lower but not severely diminished haplotype diversity relative to putative source populations in the Black Sea Genetic variation in introduced populations also depends on the structure of the source population The nature of transmitting vector can define the extent of propagule pressure and the genetic composition of introduced populations received from the source region Limnoperna fortunei, a freshwater mytilid native to mainland China, Korea, Cambodia, Indonesia, Laos, and Vietnam Limnoperna thereafter expanded its distribution very rapidly into Uruguay, Paraguay, Brazil and Bolivia, traveling an average of 240 km per year In this study, we explore genetic consequences of global spread of the golden mussel L. fortunei via discharged ballast water L. fortunei was introduced to Japan via aquaculture as a ‘fellow traveler’ with stocked Asian clams Corbicula fluminea imported from China L. fortunei could be introduced in Asia via at least two possible vectors , whereas only a single vector  appears possible for invasions in South America. Consequently the likelihood of admixed introduced populations should, be lower in South America than in Asia. This pattern serves as the basis of our first hypothesis: introduced populations of L. fortunei in South America will be genetically impoverished relative to introduced ones in Asia. Introduced populations in Asia (Japan and Taiwan) are separated by geographical barriers (marine water) whereas introduced South American populations have spread upstream from the putative initial invasion site in the Río de la Plata estuary along the Paraná, Uruguay, and Paraguay rivers. This pattern serves as the basis of our second hypothesis: gene flow is more limited among introduced Asian populations than among South American ones, and, as a result, genetic differentiation is more pronounced among introduced Asian populations. To test these hypotheses we used the cytochrome c oxidase subunit I (COI) gene and microsatellite markers to study genetic structure of this species in both continents.Japan, Taiwan, Korea, and parts of China are considered at high risk for biological invasion on the basis of ship traffic volume, while the opposite is true for South America No specific permits were required for the described field studies in Asia and South America. The species collected is an invasive pest in South America, Japan, and Taiwan and is not protected throughout this range. Sampling points did not include protected or private lands.Limnoperna fortunei was sampled from 24 locations in Asia and South America, distributed across both native and introduced regions .Sequences were aligned using CodonCode Aligner 2.0  and then manually edited. The possibility of doubly uniparental inheritance (DUI) of mitochondrial genome observed in other Mytilidae species was tested and ultimately excluded according to the method described by Gillis et al. L. fortunei from Japan were retrieved from GenBank (Accession nos. AB520611– AB520627) and included in phylogenetic analyses.A parsimony haplotype network with 95% connection probability n), haplotype diversity (h), and nucleotide diversity (π), were estimated using DnaSP 5.0 ST with the Tamura-Nei substitution model implemented in ARLEQUIN version 3.1 a priori expectations according to their geographical origin into two groups: i) all South American populations versus all populations from Asia; and ii) based on their geographic region within Asia  and within South America (samples were divided based on river basins into six regions: I Upper Paraguay (CO), II Paraná River , III Uruguay River (UR), IV Río Tercero (RT), V Sao Gonçalo (SO), and VI Paraná Delta - Río de la Plata .The number of haplotypes (A), allelic frequency (F), allelic richness (Ar), observed heterozygosity (HO), and expected heterozygosity (HE) were measured using FSTAT v.2.9.3 Ar), which is an estimate of allelic diversity adjusted by the lowest sample size, was calculated using Fstat. Genetic differentiation among populations was examined by FST using ARLEQUIN. Similar to ФST, sequential Bonferroni corrections were used to adjust the significance level for multiple comparisons FST and the interpretation of population differences, we also calculated one of the corrected FST –like indices Jost’s D Genetic diversity indices including the number of alleles  was tested from one to 24  and ten independent runs for each K value were set at 106 Markov chain Monte Carlo (MCMC) iterations, with an initial burn-in of 105. We followed the method of Evanno et al. K. We assessed possible hierarchical genetic structure by conducting separate Bayesian analyses on populations from South America (K from 1 to 14), and Asia (K from 1 to 10), plus Taiwan and Japan (K from 1 to 5). A three-dimensional factorial correspondence analysis (3D-FCA) was performed using GENETIX v. 4.05 To further investigate population genetic structure of L. fortunei. Of the 16 Japanese haplotypes retrieved from GenBank, eight were identical to those detected in this study. The haplotype frequency map revealed a high level of geographic structure . All haplotypes were recovered after we used only female mussels (F-type haplotypes), suggesting that doubly uniparental inheritance (DUI) is not characteristic of tructure . Native tructure , Table 1tructure . Twenty-tructure .The Bayesian phylogenetic tree showed a shallow structure lacking apparent phylogeographic structure . We obsen = 9) in population CH3 sampled from mainland China, while only two haplotypes were recovered from population TW2 collected from Taiwan. Mean haplotype diversity (h) and nucleotide diversity (π) in Chinese populations were 0.611 and 0.007, respectively, higher than those observed in invaded areas. Comparable values for South America were 0.595 and 0.004, while those in introduced populations in Asia were 0.439 and 0.004, respectively. Populations from Taiwan (TW1 and TW2) exhibited the lowest haplotype and nucleotide diversity  ranged from 5 to 9.5, while mean expected heterozygosity (HE) and observed heterozygosity (HO) ranged from 0.518 to 0.858, and from 0.128 to 0.528, respectively (HE) and mean allelic richness (Ar) were higher in Asia as compared to South America . Many loci (150 of 192) deviated from Hardy-Weinberg equilibrium (HWE) and all exhibited heterozygosity deficiency  and South America (0.046) were lower than those between the continents (0.257). Almost all FST values were significant in Asia and between Asia and South America while most of the FST values were non-significant in South America. In Asia, pairwise FST values were lower inside each geographic region  as compared to the overall value (0.067) for the continent . We observed relatively high genetic differentiation between neighbouring populations in South America, for example, FST = 0.072 between EC and UR (separated by ∼ 50 km), while some geographically distant populations exhibited relatively low FST values, for example, 0.004 between CO and QU (separated by ∼ 2000 km). Similar to FST values, Jost’s D values were lower among populations in South America (D = 0.00–0.074) than in Asia (D = 0.024–0.404), and between Asia and South America (D = 0.351–0.726)  . GeneticP<0.001; .K = 2) were supported when Asia (K = 2). Two populations from Taiwan , and one Japanese population (JP1) were grouped together, while the other two Japanese populations (JP2 and JP3) were clustered into another group  and nuclear markers (microsatellites) to contrast the geographical distribution of genetic diversity of L. fortunei has been introduced more than once and/or in large inocula (i.e. high propagule pressure). This conclusion is consistent with Tominaga et al.’s (2009) L. fortunei was introduced to Japan via aquaculture as a ‘fellow traveler’ with stocked Asian clams, C. fluminea, imported from China L. fortuneiL. fortunei to neighboring countries Expected heterozygosity (0.667–0.746) for introduced populations in Japan and Taiwan was higher than that in introduced South American ones (0.519–0.575). Both groups exhibited lower expected heterozygosity as compared to native populations in mainland China and Korea (0.701–0.858). The high number of haplotypes at the COI locus recovered from Japan, coupled with relatively high heterozygosity, suggests that n = 3), but rather high level of allelic richness (Ar = 7.3) and heterozygosity (HE = 0.7052) at microsatellite loci (Ne) relative to nuclear DNA owing to its maternal inheritance and haploid nature. Therefore, the mitochondrial genome is expected to be more sensitive to bottleneck events than the nuclear genome Ciona intestinalisL. fortunei introduction from Japan through aquaculture appears to be low. L. fortunei has not yet been reported in USA or Canada. While aquaculture remains a possible vector for introduction to Taiwan, it is more likely active in Japan. The low haplotype diversity observed in Taiwan could be also result from inhospitable environmental conditions in primary introduction areas. It is also possible that the putative source population carries similar level of genetic diversity. Korea was also represented by a low number of haplotypes, and high nuclear allelic diversity, relative to native populations in mainland China (We observed a low number of mtDNA haplotypes in Taiwan (ite loci . MtDNA hnd China . HoweverD. rostriformis bugensis) and zebra mussels (D. polymorpha) L. fortunei can be transported both up- and downstream through recreational boating, natural inland currents, and seasonal flooding, temporal and/or spatial Wahlund effect could account for the heterozygosity deficit, although inbreeding and selection cannot be completely dismissed.Heterozygosity deficit was found in 77.6% of all analyzed microsatellite loci, including native populations in mainland China and Korea . This paL. fortunei in each geographic region by a saltwater dispersal barrier, with inter-region gene flow limited to human-mediated translocation of propagules. In addition, distinct sources of introduction can drive genetic differentiation among regions. We observed high genetic differentiation between South American and Asian populations (FST = 0.180 – 0.306). High genetic differentiation has been reported in other freshwater invasive mussels, including D. polymorpha (FST = 0.006 – 0.263) and D. rostriformis bugensis (FST = 0.008 – 0.267) L. fortunei, to upstream areas due to ship-mediated translocation appears responsible for the patchy post-establishment spread of the species in South America L. fortunei populations violated the HWE assumption for STRUCTURE analyses. However, similar results were observed using another method (i.e. 3D-FCA) without this assumption.In Asia, both mtDNA and microsatellite markers exhibited lower genetic differentiation within geographic regions as compared to that among regions. This finding is supported by a higher percentage of variance allocated to among groups as compared to among populations within groups AMOVA; . JapanesL. fortunei. It appears that a recent geographic expansion of L. fortunei throughout its native distribution can explain of its low phylogeographic structure. This finding suggests that even in the native region, populations may still be expanding to areas not previously populated. Vector activity such as ship traffic between local ports in native region (e.g. mainland China and Korea) may have contributed to recent expansion of L. fortunei across its native range The parsimony network analysis and Bayesian phylogenetic reconstruction revealed a close relationship among haplotypes of 4 ships from countries considered native for L. fortunei, whereas Argentina received only 26 ships. Second, Taiwan and especially Japan may benefit from an aquaculture transfers from adjacent Asian countries, notably mainland China. Third, given the comparatively short distance between Asian countries, hull fouling could effect local or regional spread. L. fortunei larger than 20 mm can tolerate anoxia for up to 18 days at 20°C L. fortunei in these regions.Our study suggests that introduced golden mussels carry less allelic diversity in South America than in Asia. A number of factors may contribute to this pattern. First, data from global ship traffic between 2005–2006 L. fortunei, D. polymorpha and D. rostriformis bugensis share similar ecological and biological characteristics including planktonic larvae and a sessile, benthic adult stage. These life history traits suggest similar vectors could effect their human-mediated spread . Also, golden mussel can be transported as a ‘fellow traveler’ in aquaculture D. polymorpha and D. rostriformis bugensis in North America and Eurasia L. fortunei provides a good basis for comparison of the genetic characteristics of the three mussel species (L. fortunei (40 haplotypes) as compared to 11 found for D. polymorphaD. rostriformis bugensisD. polymorpha, the Great Lakes appear to have served as a ‘hub’ for subsequent expansion across North America Although taxonomically distinct,  species . When us species . The higL. fortunei’s introduction in both Asia and South America likely involved multiple introductions and high propagule pressure, resulting in populations with high genetic diversity relative to sampled native populations in Asia. Introduced populations exhibiting lower genetic diversity (South America) likely received lower propagule pressure relative to those with higher diversity (Japan). Our genetic survey shows how human-mediated introduction of NIS can create genetic complexities across introduced locations. Our study evaluates possible links between vector activity and genetic composition of a nuisance NIS at a global scale, and highlights the utility of incorporating population genetics and vector activity data to understand species dispersal patterns Our findings suggest that Figure S1Values of ΔK calculated as in Evanno et al.for detecting the biologically relevant clusters of Limnoperna fortunei collected from all 24 locations (A) and Asia (B).(TIF)Click here for additional data file.Table S1Limnoperna fortunei, sampled from 24 locations across the global range in East Asia and South America. A, number of alleles; Ar, allele richness; HO, observed heterozygosity; HE, expected heterozygosity; PHW, exact P-value for Hardy-Weinberg equilibrium test. The significance after sequential Bonferroni correction was bolded.Genetic diversity at eight microsatellite loci for the golden mussel, (DOC)Click here for additional data file.Table S2Dreissena polymorpha, quagga mussel Dreissena rostriformis bugensis, and golden mussel Limnoperna fortunei.Comparison of microsatellite-based genetic features of the three highly invasive freshwater mussels, zebra mussel (DOC)Click here for additional data file.