Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in critically ill surgical patients. We hypothesized that the extent of pGSN reduction might correlate with different outcome of burn patients. The study was performed to evaluate the prognostic implications of pGSN levels on the development of multiple organ dysfunction syndrome (MODS) and fatal outcome in a group of severely burn patients.95 patients were included, and they were divided into three groups with different burn area: group I (n = 33), group II (n = 32) and group III (n = 30). According to whether there was development of MODS or not, patients were divided into MODS group (n = 28) and none-MODS group (n = 67); then the patients with MODS were further divided into non-survivor group (n = 17) and survivor group (n = 11). The peripheral blood samples were collected on postburn days (PBD) 1, 3, 7, 14, and 21. The levels of pGSN were determined and T cells were procured from the blood. The contents of cytokines  released by T cells were also measured. The related factors of prognosis were analyzed by using multivariate logistic regression analysis. The results showed that pGSN concentrations, as well as the levels of IL-2 and IFN-γ, decreased markedly on PBD 1–21, whereas, the levels of IL-4 increased markedly in all burn groups as compared with normal controls (P<0.05 or P<0.01), and there were obviously differences between group I and group III (P<0.05 or P<0.01). The similar results were found in MODS patients and the non-survivor group as compared with those without MODS and the survival group on days 3–21 postburn (P<0.05 or P<0.01). Moreover, as the pGSN levels decreased, the incidence of septic complication as well as MODS remarkably increased.pGSN levels appear to be an early prognostic marker in patients suffering from major burns. Burn injury is one of the most common and devastating forms of trauma. Patients with severe burns require immediate specialized care in order to minimize morbidity and mortality. It has been demonstrated that a large thermal injury induces a state of immunosuppression that predisposes the patient to infectious complications Gelsolin, a protein of 82 to 84 kDa, is a member of gelsolin protein superfamily, which exists in a cytoplasmic as well as an excreted plasma isoform, and contains six homologous repeats termed gelsolin-like (G) domains Numerous studies show that an increased burn size leads to higher mortality in burn patients 95 patients who were consecutively admitted to our burn departments with total body surface area (TBSA) larger than 30% were included in the present study over a time period of 18 months. Patients were successfully resuscitated using colloid and lactated Ringer's solution. Within 96 hours of admission most patients underwent burn wound excision for full-thickness burn, and the excision wounds were seeded with granulated autologous skin, and totally covered with allograft. Five to ten days after healing of the donor area, the remaining wounds, if there was any, were totally covered with autograft skin.g for 5 minutes, plasma was harvested and frozen at −80°C until analysis. The study was reviewed and approved by the Institutional Review Board of the Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital, Beijing, China. Prior to the study, an informed consent was signed by the patient or next of kin. All patients and volunteers were of Chinese Han origin.The thermally injured patients were stratified into three groups according to burn size: 30%–49% TBSA burns , 50%–69% TBSA burns , and >70% TBSA burns . Demographic, laboratory, and clinical data were collected, and the Sequential Organ Failure Assessment (SOFA) score pGSN levels were measured using an enzyme-linked immunosorbent assay (ELISA), in accordance with the manufacturer's instructions . The microtiter plate provided in this kit has been pre-coated with an antibody specific to GSN. Standards or samples were then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for GSN. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each microplate well and incubated. Then a TMB substrate solution was added to each well. Only those wells that contain GSN, biotin-conjugated antibody and enzyme-conjugated avidin would exhibit a change in color. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of GSN in the samples was then determined by comparing the O.D. of the samples to the standard curve. No significant cross-reactivity or interference was observed.6 cells/ml. Cells were treated with 5 µg/ml Con A for 48 hours at 37°C in 5% CO2/100% humidified air, and the contents of cytokines  released into supernatants were determined.Heparinised blood was diluted in Hanks' balanced salt solution, and Ficoll-Hypaque  was used for isolation and preparation of peripheral blood lymphocytes. The isolated lymphocytes were washed twice with phosphate buffered saline (PBS), centrifuged , resuspended in basal medium (unstimulated culture) RPMI-1640  containing 10% fetal calf serum , 1% penicillin/streptomycin , and 1% glutamine . Then cell populations were counted under a light microscope. Based on the different experimental demand, the peripheral blood mononuclear cells (PBMC) suspension was diluted with RPMI-1640 to 2×10IL-2, IL-4 and IFN-γ levels were determined by ELISA, strictly following the protocols provided by the manufacturer . The color reaction was also terminated by adding 100 µl of ortho-phosphoric acid. Plates were read in a microplate reader .P value of 0.05 or less was considered to indicate statistical significance.Data were expressed as mean ± standard deviation (SD) and analyzed with ANOVA  by means of SPSS 15.0 . Tukey Test was used to evaluate significant differences between groups. To assess the effect of pGSN levels and other independent variables on burn-derived complications and mortality, Logistic regressions were used as odds ratios (ORs) with 95% CI adjusting for the pGSN level, burn size, age, gender and inhalation injury. A 95 patients with burn injury were included in the present study. The patients' demography was illustrated in To investigate the changes in pGSN levels, the blood samples were detected by ELISA kits for human at different time points and in different groups after burns. As shown in The pGSN levels also gradually decreased with an increase in the mean SOFA score in MODS patients, as shown in As shown in The related factors of prognosis were analyzed by using multivariate logistic regression analysis. The results showed that the pGSN level, burn size, age and inhalation injury were independent predictors for the development of both MODS and fatal outcome of burn patients P<0. 05, . Gender Severe burn injury may induce a temporal shift in immune reactivity that can lead to septic syndrome or even death. The immune system responds to injury by a rapid production of early and late proinflammatory cytokines, and also suppression of T cell functions raison d'être for actin-binding proteins in plasma, which under normal circumstances does not contain detectable actin. A straightforward hypothesis emerged suggesting that pGSN scavenges actin which leaked into the circulation or interstitial space from injured tissue in order to abort subsequent damage instigated by extracellular actin filaments The actin cytoskeleton is an essential scaffold for integrating membrane and intracellular functions. It is very dynamic and is remodeled in response to a variety of signals. Gelsolin was first identified as a cytoplasmic actin-regulatory protein essential for cell locomotion and phagocytosis. When a circulating isoform was subsequently discovered, its physiological significance was far from clear. For over two decades, researchers have made concerted effort to define the de novo synthesis. As we know, pGSN is synthesized primarily in skeletal muscle Accordingly, the degree of pGSN depletion might reflect the extent of tissue injury leading to significant exposure of actin to the extracellular space after major burns. In the current study, a decreased level of pGSN from burned patients was also observed on PBD 1–21 compared with normal controls, and there were obvious differences among patients with different burn sizes. This finding was consistent with the result of a previous report in an animal model, showing a significant reduction in circulating gelsolin levels within the first 2 hours of injury, with the decrease persisting for at least 6 days postburn in burned rats Based on our findings, it was indicated that a burn injury involving larger than 50% TBSA caused remarkably prolonged length of stay in hospital, significant increase in number of operations, and incidence of infection and MODS. Indeed, several investigators also found that low pGSN levels were associated with poor outcome in trauma +CD25+ Tregs per se can trigger both excessive inflammation and suppressed adaptive immunity. Actin exposed at the site of an overwhelming injury may act as a sink to exhaust the supply of pGSN, in turn allowing redundant inflammatory cascades unfettered opportunity to wreak havoc upon multiple organ systems. After a phase of excessive immune activation, a marked apoptosis-induced depletion of lymphocytes and a nonspecific anergy of immune function subsequent to severe insults might be responsible for the increased susceptibility of the host to subsequent septic complications. Similarly, our results in this study showed that the levels of IL-4 produced by T cells increased markedly after burn injury, whereas levels of IL-2 as well as IFN-γ decreased markedly, indicating that Th cells might have shifted to Th2 cells.Severe thermal injury induces detrimental changes in immune function, often rendering the host highly susceptible to development of life-threatening opportunistic infections. Multiple mechanisms have been proposed to explain infection-induced immunosuppression, including an imbalance in the cellular helper T cell (Th1/Th2) or cytokine profile, induction of anergy, depletion of effector cells and most recently the activation of CD4These observations of 95 patients with burn injuries of different extent confirmed the strong correlation between pGSN levels and such important clinical complications as the development of MODS and fatal outcome. Still unsettled issues concern the particular or incremental utility of gelsolin levels as timely prognostic indicators, the generalization of these findings to diverse clinical situations In conclusion, pGSN may be a valuable marker for severe complications in patients suffering from major burns. Extensive burn injury could result in significantly decreased pGSN levels, which appears to be associated with the development of MODS and fatal outcome secondary to acute insults. It also suggested that the potential therapeutic benefits of infusions of recombinant human pGSN might be important to the Th1/Th2 cytokine balance in patients after extensive burns. The prognostic role of pGSN in a critical illness requires further investigation in a large cohort.


We report a case of congenital rubella syndrome in a child born to a vaccinated New Jersey woman who had not traveled internationally. Although rubella and congenital rubella syndrome have been eliminated from the United States, clinicians should remain vigilant and immediately notify public health authorities when either is suspected. Once a main cause of congenital abnormalities  and in 2003 received measles, mumps, and rubella (MMR) vaccine. Vaccination status of the father was unknown. Maternal rubella virus IgG titer at 4 months’ gestation was >400 IU/mL (immune >9).The infant was delivered by urgent cesarean section because of cardiac decelerations during labor. Apgar scores at 1 and 5 minutes were 9 . At birth, the child weighed 2.7 kg (10th percentile), and he was 48.25 cm (15th percentile) in length and had a head circumference of 31.75 cm (<3rd percentile). He had a petechial rash on the face, back, and upper extremities and a systolic heart murmur. No jaundice or hepatosplenomegaly was noted.9/L (reference 9–30) with atypical lymphocytes; a hemoglobin level of 202 g/L (reference 135−195); abnormal erythrocyte morphology, including macrocytosis, polychromasia, and poikilocytosis; and a platelet count of 98,000 × 109/L (reference 140–440). Computed tomographic scan of the head revealed 2 small calcifications in the left corona radiata. Results of neonatal hearing screening were normal. Urine samples were cultured for cytomegalovirus (CMV), and serum samples were tested for toxoplasma and parvovirus IgM; all results were negative. The infant was discharged from the hospital on day 4 with a suspected congenital infection.Initial tests showed a leukocyte count of 13.8 × 10At 16 days of age, the child was seen by an infectious disease physician. Examination revealed hepatosplenomegaly, and the systolic heart murmur was detected across the precordium, with radiation to the back. CMV quantitative PCR and testing for lymphocytic choriomeningitis IgM and IgG were requested, but not obtained. Congenital rubella was considered, but serologic tests were not ordered. A subsequent echocardiogram demonstrated supravalvular and peripheral pulmonic stenosis, a small patent ductus arteriosis, and a patent foramen ovale.The child was delayed in attaining developmental milestones. At 6.5 months of age, he was referred to a geneticist, who requested multiple tests, including tests for rubella, CMV, and lymphocytic choriomeningitis. Rubella virus serum IgM was 7.2 (positive >1.0) and IgG was 59 IU/mL (immune >9). The local health department received notification of the test results and immediately initiated an investigation in collaboration with the New Jersey Department of Health. During multiple interviews, the child’s family denied any exposure to international travelers or persons with a rash illness during the potential exposure period. No source contact or secondary transmission was identified. New Jersey Department of Health and the infectious disease physician requested that the child not attend day care and avoid exposure to unvaccinated infants until after his first birthday, when he would be presumed to be free of infection. MMR vaccination at 12 months of age was withheld until completion of additional diagnostic testing.>1.1).When the infant was 7.5 months of age, rubella virus RNA was detected from a nasal wash sample by using reverse transcription PCR (RT-PCR); results of a nasal wash culture and serum RT-PCR were negative. CDC culture and RT-PCR results for nasal samples collected when the infant was 10 and 12 months of age were also negative. However, ELISAs performed by CDC on a serum sample collected when the infant was 12 months of age confirmed the presence of rubella virus IgM antibodies  and IgG antibodies ; results were 2.43 and 5.9, respectively  were compatible with CRS and met 3 of the Council of State and Territorial Epidemiologists’ laboratory criteria for a confirmed case of CRS .,CRS occurs rarely in children of women with a history of vaccination or documented immunity by rubella virus titer. When CRS does occur, it is frequently a result of asymptomatic rubella reinfection in the mother  of adolescents 13–17 years of age had received >2 doses (We found no evidence of secondary rubella transmission. A model has suggested that to interrupt rubella transmission, Despite its elimination from the United States, rubella infection and CRS continue to occur rarely: 6 cases of CRS have been reported to CDC since 2004. Clinicians should remain vigilant to the possibility of rubella and CRS and immediately notify public health authorities when either is suspected.


Although whole human genome sequencing can be done with readily available technical and financial resources, the need for detailed analyses of genomes of certain populations still exists. Here we present, for the first time, sequencing and analysis of a Turkish human genome. We have performed 35x coverage using paired-end sequencing, where over 95% of sequencing reads are mapped to the reference genome covering more than 99% of the bases. The assembly of unmapped reads rendered 11,654 contigs, 2,168 of which did not reveal any homology to known sequences, resulting in ∼1 Mbp of unmapped sequence. Single nucleotide polymorphism (SNP) discovery resulted in 3,537,794 SNP calls with 29,184 SNPs identified in coding regions, where 106 were nonsense and 259 were categorized as having a high-impact effect. The homo/hetero zygosity  and transition/transversion ratios  were within expected limits. Of the identified SNPs, 480,396 were potentially novel with 2,925 in coding regions, including 48 nonsense and 95 high-impact SNPs. Functional analysis of novel high-impact SNPs revealed various interaction networks, notably involving hereditary and neurological disorders or diseases. Assembly results indicated 713,640 indels (1∶1.09 insertion/deletion ratio), ranging from −52 bp to 34 bp in length and causing about 180 codon insertion/deletions and 246 frame shifts. Using paired-end- and read-depth-based methods, we discovered 9,109 structural variants and compared our variant findings with other populations. Our results suggest that whole genome sequencing is a valuable tool for understanding variations in the human genome across different populations. Detailed analyses of genomes of diverse origins greatly benefits research in genetics and medicine and should be conducted on a larger scale. Following the publication of two draft sequences Efforts (other than WGS) that target discovery of human genome variations also exist, such as the HapMap project Despite the increasing use of human WGS for both research and clinical purposes, there remain two areas that require further attention: i) there are populations for which WGS or SNP discovery efforts have not been done; ii) very few of the human WGS performed so far provide high-coverage sequencing results with detailed analysis. Out of the 185 individuals for whom WGS has been performed in the 1000 Genomes Project's Pilot Phase, only six were subjected to high-coverage sequencing (∼42x) while the remaining individuals were subjected to low-coverage sequencing (2–6x). All of the individuals studied in Phase I of the project were analyzed using low-coverage sequencing (2–6x).There have been various efforts to perform high coverage WGS of different populations with detailed analysis Turkey, the most populous well-defined region inhabited by Turks, is an interesting geographical region as it lies at the crossroads between Europe and Asia. Historically, migration from Central Asia and ancestral contribution to regions surrounding Anatolia, such as the Balkans, Middle East, Caucasian and Caspian regions, has positioned the Turkish population as an interesting genetic resource that requires further detailed analysis. Certain important diseases, such as hemoglobinopathies (e.g. sickle-cell disease), thalassemias, and Behcet's disease, are highly prevalent in the Turkish population; and diseases exist where the Turkish population does not exhibit the variant believed to be the cause This study is approved by the Committee on Ethics in Research on Humans of Istanbul Bilgi University.The genomic DNA (gDNA) used in this study came from a healthy male individual, who was anonymous and was reported to come from Turkish ethnicity for at least three generations. Informed consent was obtained prior to the collection of the blood sample from which gDNA was isolated using a QIAamp DNA blood kit (Qiagen®). The individual gave written consent to the publication of his genome sequence. A quality control inspection and rough quantitation of the gDNA sample was performed by agarose gel electrophoresis and UV-induced ethidium bromide fluorescence . The quaThe genomic DNA sample was used to generate a paired-end library suitable for the HiSeq sequencing platform (Illumina®) prepared using the TrueSeq DNA Sample Preparation kit, following the manufacturer's instructions. Quality control analysis of the library using an Agilent 2100 Bioanalyzer indicated that the library was of acceptable quality, containing the expected fragment size and yield, for continued sample processing . The libSanger sequencing was used for validation of 20 small indels identified by computational analysis of the whole genome sequence data. DNA was isolated using a Dual DNA isolation kit . Twenty regions were amplified from 50 ng genomic DNA with 10 pmol of forward and reverse primer pairs. Polymerase chain reaction (PCR) was performed using the following cycling profile: initial denaturation at 95°C for 5 min. followed by 10 cycles of 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s; 25 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s; and a final extension step at 12°C for 5 min. Amplicons were purified using GenedireX PCR Clean-Up kit  and quantified. ABI 3100 DNA analyzer and ABI BigDye Terminator cycle sequencing was used for Sanger sequencing.bwasw algorithm to index the database and generated suffix array indices for two ends in a paired-end read separately. We then combined the two results with the sampe algorithm to produce the final sequence alignment/map (SAM) file. BWA analysis results were investigated with SAMStat v1.08 −10.The reference genome used in this study was NCBI human reference genome build 37.1 (GRCh37/hg19 assembly). We adopted two workflows for read mapping. First, we used the vendor supplied Eland and Casava (v1.8) pipeline using recommended settings with a variants covariance cut-off value of 3. Alternatively, we used the Burrows-Wheeler Alignment tool (BWA v0.6.2) for mapping reads to the reference genome SNP identification was done using the vendor-supplied Eland-Casava pipeline with the recommended settings and The Genome Analysis Toolkit (GATK v2.2) We used Ingenuity Software Knowledge Base (IKB),  for the functional interaction analysis of the genes affected by high-impact, novel SNPs. IKB uses interactions between genes and/or gene products based on manual curation of scientific literature providing a robust interaction database. Once a gene list of interest is identified, IKB uses known interactions between these genes to build an interaction network. The final network includes genes that are not in the input list but are highly connected to the genes in the input list. This feature enables the investigation of new modules of interactions that are not covered by existing canonical pathways. The results are also analyzed in terms of drugs, small metabolites, functions, and diseases that are overlaid on the resulting network. To this end, an annotation of the resulting interaction network is achieved where functional entities involved in the network are underlined.Prior to the sequencing step, we compared the microarray genotyping results of the individual used in this project, who was an anonymous, healthy male claiming to have come from Turkish ancestry for at least three generations, with those obtained from the HapMap project DNA sequencing generated 1,238,722,496 paired-end reads corresponding to ∼125,111 M bases of data yielding ∼35x coverage. Quality and length-based trimming and filtering dropped 4.44% of the reads eliminating a total 5.03% of total base pairs. The remaining ∼1.18 billion reads  proved to be of high quality , with sufficient length (mode is ∼100 bp), and included no Ns . Of the Casava and GATK workflows identified 3,642,449 and 4,301,769 SNPs, respectively. In order to increase the reliability of our findings, all downstream analysis was performed with SNPs identified by both methods, which resulted in 3,537,794 variants. Of these concordant SNP calls, 97.8% were in agreement with the SNPs called by the genotyping performed on the array, showing high reproducibility. The transitions  transversions , Ts/Tv, ratio was 2.06, and the homozygosity  and heterozygosity  proportions were 40% and 60%, respectively, with both ratios and percentages resembling expected figures in similar studies www.genecards.org) SNPs, where (i) the variant hits a splice acceptor/donor site, (ii) a start codon is changed into a nonstart codon, or (iii) a stop codon is gained or lost due to the variant are categorized as having a “high-impact” effect. There were a total of 259 SNPs with a high-impact effect and 167 of these were found to be on gene sequences. Out of the ∼3.5 million SNPs identified, there were 480,396 potentially novel SNPs that did not exist in dbSNP. Of these potentially novel SNPs, 49% and 41% were in intergenic and intronic regions, respectively. Over 8% were upstream or downstream of a gene, and 2,925 (or 0.5%) of the SNPs were found to be in coding regions. There were 48 nonsense SNPs and a total of 95 SNPs with a high impact. These high-impact SNPs affected 47 genes (45 well characterized). In We identified 713,640 indels, which consisted of 341,382 insertions and 372,258 deletions. Of these indels, 159,593 (or 22%) were found to be novel. The length distribution ranged from -52 bp to 34 bp  and detailed analysis of the whole genome sequence of a Turkish individual. Although whole genome human sequencing is almost routinely done, very few of these efforts provide high coverage and analysis; and various populations are not included in large consortium efforts. Therefore, we believe the current study provides a reference data set in understanding human genome variation on a large scale and a population-dependent context and is an initial step in exploring Turkish genomic features. Despite its importance as the first Turkish whole genome analysis, we acknowledge that further studies are required to validate and generalize our findings to the population scale. Nevertheless, we believe the data presented here will provide a cornerstone for such studies and enrich the analysis of human genomic variation across diverse populations.Our genotyping results from chip and sequence data, and the comparative analysis of these with HapMap and other chip genotyping results suggest that the sequenced individual represents a typical member of the Turkish population. The sequencing analysis proves that the data generated is of high quality as only 5% of the total sequence is filtered out, and the remaining reads almost completely cover the reference human genome. High N50 values indicate successful assembly of unmapped reads, which also resulted in ∼1 Mbp of unmapped human genome sequence that did not reveal any homology in the RefSeq database. SNP identification rendered high reproducibility with ∼98% consistency between the sequencing and microarray genotyping results and validated about 86% of identified SNPs in the dbSNP database. We believe the remaining 480,396 potentially novel SNPs contribute to understanding the human genomic variation and the relationship between genotypes and phenotypes.The length distribution of the 9,109 identified CNV/SV events showed peaks at the 300 bp and 6,000 bp marks potentially representing short and long interspersed elements, respectively. A similar trend is seen in the 1,629 high confidence calls . The relIn order to position our results in a better population genetics context, we compared the SNPs identified in the sequenced Turkish individual to the SNPs found in Utah, USA inhabitants with ancestry from Europe (CEU) and to the SNPs found in Han Chinese in Beijing, China (CHB). Both CEU and CHB populations were included in the HapMap project, which constitute the source of the identified SNPs in these populations used in our analysis. From a historical perspective, precursors of the Turks originated in Central Asia and Turks are known to have been inhabitants of regions that are parts of modern day China. Turks' migration toward the West ended up mostly in Anatolia with minor settlements in Europe. Turks' presence in Europe was later expanded throughout the Ottoman era. We, therefore, performed our comparative analysis with CEU and CHB, which are the two most closely related populations to the Turkish population with available large-scale genetic data. We found 665,032 SNPs commonly shared by the three populations, which corresponds to 47% of all of the CEU SNPs and 50% of all the CHB SNPs . SNPs exNovel SNPs predicted by our results have the potential to explain genetic features specific to the Turkish population. The 23 well-characterized genes that were affected by the novel nonsense SNPs identified in this study were found to affect 25 different phenotypes listed in OMIM, potentially leading to additional genetic disease mechanisms see . We usedIKB analysis revealed three interaction networks that included 15, 13, and 3 of the 47 genes. In The whole genome sequencing reads have been deposited in the NCBI Short Read Archive (SRA) database under the accession number SRA056442. SNPs and indels have been deposited in the NCBI dbSNP database with the handle ID “BILGI_BIOE”. CNV/SV calls have been deposited in the NCBI dbVar database with project ID PRJNA171612.Figure S1QC gel image (0.8% agarose) of gDNA sample (1) compared to molecular weight marker (M). Sample quality was considered to be acceptable if the gDNA supplied a single visible band while lacking any significant degradation products .(TIF)Click here for additional data file.Figure S2Library quality. The electropherogram for the generated library displaying expected yield and size.(TIF)Click here for additional data file.Figure S3Quality statistics for the forward reads only  following trimming and filtering. a) Average base quality with respect to the position of the base in the read; b) Histogram of the sequence lengths; c) histogram of the average quality scores of the reads; d) Ns seen in the reads with respect to the position of the base in the read.(TIF)Click here for additional data file.Figure S4Length distribution of the identified 713, 640 indels.(TIF)Click here for additional data file.Figure S5Distribution of 9,109 identified CNV/SV calls across chromosomes. A) Number of CNV/SV events; B) Length normalized (per million base pairs) CNV/SV events.(TIF)Click here for additional data file.Figure S6Length distribution of 9,109 identified, 3870 novel, and 1629 high confidence CNV/SV calls. Note that the bin size for SVs less than 10 Kbp is 200 bp while the bin size for SVs more than 10 Kbp is 2 Kbp. The last bar in the graphs on the right-hand column represents SVs more than 250 Kbp.(TIF)Click here for additional data file.Figure S7Overlap of SNPs identified in the Turkish individual used in the manuscript (TUR); Utah, USA inhabitants with ancestry from Europe (CEU); and Han Chinese in Beijing, China (CHB).(TIF)Click here for additional data file.Figure S8IKB Network analysis of 45 genes affected by a high impact novel SNP. Genes indicated by red are affected by a nonsense SNP and genes indicated by green are affected by a SNP targeting a splice site donor/acceptor region. Hereditary and Neurological Disorders/Diseases are indicated where applicable.(TIF)Click here for additional data file.Table S145 well characterized genes that were affected by a high-impact SNP. Effect Types: 1: Stop gained; 2: Splice site acceptor; 3: Splice site donor; 4: Stop lost; 5: Start lost.(PDF)Click here for additional data file.Table S220 predicted indels used for validation by Sanger Sequencing .(PDF)Click here for additional data file.Table S3Forward and reverse primers used in Sanger Sequencing.(PDF)Click here for additional data file.Table S4Biological Function categories known to involve 45 well characterized genes that were affected by a high-impact SNP.(PDF)Click here for additional data file.


Lamotrigine trial in SPMS was a randomised control trial to assess whether partial blockade of sodium channels has a neuroprotective effect. The current study was an additional study to investigate the value of neurofilament (NfH) and other biomarkers in predicting prognosis and/or response to treatment.SPMS patients who attended the NHNN or the Royal Free Hospital, UK, eligible for inclusion were invited to participate in the biomarker study. Primary outcome was whether lamotrigine would significantly reduce detectable serum NfH at 0-12, 12–24 and 0–24 months compared to placebo. Other serum/plasma and CSF biomarkers were also explored.Treatment effect by comparing absolute changes in NfH between the lamotrigine and placebo group showed no difference, however based on serum lamotrigine adherence there was significant decline in NfH . Serum NfH correlated with disability: walking times, 9-HPT (non-dominant hand), PASAT, z-score, MSIS-29  and EDSS and MRI cerebral atrophy and MTR. Other biomarkers explored in this study were not found to be significantly associated, aside from that of plasma osteopontin.The relations between NfH and clinical scores of disability and MRI measures of atrophy and disease burden support NfH being a potential surrogate endpoint complementing MRI in neuroprotective trials and sample sizes for such trials are presented here. We did not observe a reduction in NfH levels between the Lamotrigine and placebo arms, however, the reduction in serum NfH levels based on lamotrigine adherence points to a possible neuroprotective effect of lamotrigine on axonal degeneration. Sodium channel blockade is postulated to protect axons from anoxia-induced injury secondary to neuroinflammation. However, the Phase II lamotrigine trial in secondary progressive MS encountered an unexpected result with lamotrigine causing early cerebral volume loss that partially corrected after discontinuation of treatment. This effect may have been due in part to reversible fluid shifts or a reduction in inflammation due to the treatment.Neurofilament proteins are the scaffolding proteins of neurons and are markers of neuro-axonal injury N-acetylaspartate (NAA), cytokines CXCL12, BLC, CCL19, CCL21, TNFα, TNFβ, IFNγ, interleukins 2, 4, 5, 6, 8, 10, 12p70, 1b, oxysterols 24OHC/27OHC, lanosterol and cholesterol, DJ-1, osteopontin, were measured based on their evidence as biomarkers of disease activity in MS.The biomarker study was to test whether lamotrigine could significantly reduce detectable NfH in sera at 12 and 24 months relative to baseline, compared to placebo. Other hypotheses explored included whether NfH predicts cerebral and spinal cord atrophy as measured using MRI and disability scales. A further exploratory panel of nitrite/nitrate, GFAP, BDNF and NGF NfL, ferritin, MBP, growth factors NCAM, GAP-43, + or Ca2+ channel blockers, or had received immunosuppressive agents recently were excluded. This study was approved by the University College London Hospitals Committee and National Research Ethics Committee; written consent was obtained as outlined by the ethics committees.120 subjects with SPMS were recruited into the lamotrigine trial lasting two years (NCT00257855) vs 40% on lamotrigine). The sample size was based on a power of 80% at 5% significance level, with a combined loss to follow-up and/or non-compliance of 10%. Further modelling of serum biomarker data included all available samples  tubes, centrifuged at 2,500 rpm for 10 minutes before aliquoting, CSF was centrifuged at 1,500 rpm for 5 minutes, cell-free CSF was aliquoted and stored at −80°C. The first aliquot of CSF and paired blood sample were processed for microbiology, protein and CSF/serum oligoclonal bands.et alet alNfH , BDNF  and NGF  were measured using ELISA kits. NOx was measured using vanadium assay by Rejdak et alet alet alCSF 24OHC, 27OHC, cholesterol and plasma 24OHC and lanosterol  were measured by gas chromatography isotope-dilution mass spectrometry as in Leoni Changes in serum biomarkers NfH, NOx, GFAP, NGF and BDNF between 0–12 m, 12–24 m and 0–24 m were calculated for each patient. Differences in the distribution of changes between lamotrigine and placebo and lamotrigine adherent and non-adherent groups were determined by Kruskal-Wallis test. A comparison of subjects getting better  were determined by Chi-square test. To assess the relationships between the serum biomarkers and the clinical and MRI measures the levels were categorised. For NfH and GFAP three categories were defined: absent (below assay detection), and above and below the median of the detectable concentrations. For NOx, BDNF and NGF four categories were defined bounded approximately by the quartiles of each biomarker's distribution . GeneralScreening for relationships between the 31 biomarkers in CSF and associated blood samples and clinical and MRI parameters were carried out by tabulating the rank correlations between every pair of variables. If a rank correlation suggested a statistically significant relationship (p<0.05), it was examined in further detail. Most serum immune markers were present in only few samples and we therefore constructed single flag variable indicating the presence of any serum immune biomarker.A summary table of serum biomarker values is in Reduction in NfH levels by measuring differences 0–12, 12–24 and 0–24 m between lamotrigine adherent and non-adherent groups revealed significant differences between the two groups at NfH 12–24 and NfH 0–24 m, p = 0.043 and p = 0.023, respectively. This was further supported by measuring rate of change, where lamotrigine adherent group showed a significant change between 0–24 m in NfH levels (p = 0.041), with NfH levels at 12–24 m having a p-value of 0.055. Grouping by improvement in biomarker values (i.e. reduction in NfH) was significant in NfH 0–24 m (p = 0.042). NOx, GFAP, BDNF and NGF values did not differ between the two groups over time (p>0.05). Analysis based on tablet returns did not reveal a significant difference in the serum biomarker levels, except based on grouping by improvement in biomarker levels for NfH 0–24 m (p = 0.012). However, seven subjects who had been considered tablet compliant had neither detectable serum lamotrigine levels or only at 6 m.NfH was found to be related to components of the MSFC. It was a strong predictor of walking times, with patients having any detectable NfH taking nearly twice as long to complete the test as those with none p<0.001; . The 9-HEDSS was higher in patients with detectable NfH, and there was some indication of an association with NOx levels . MSIS-29Higher NfH was associated with lower MTR means in grey matter, white matter and whole brain . BDNF inMRICCV was highly correlated with all five serum biomarkers . It was T1 and T2 lesion volumes were higher in patients with higher levels of BDNF and NGF  m, n.CSF biomarker analysis are summarised in et al. We report on the prognostic value of serum neurofilament measurement in neuroprotective clinical trials. The primary hypothesis that lamotrigine will significantly reduce the proportion of cases with detectable NfH in sera at 12 and 24 months relative to baseline, compared to placebo was not evident in the intention to treat group, although the true effect of Lamotrigine may have been diluted by the low levels of compliance (40–50% non-adherence based on return of tablets and serum lamotrigine concentrations). Per protocol analysis based on serum and tablet adherence was therefore also performed, which also tested the pure pharmaceutical effect of the treatment. There was significant reduction in serum NfH of subjects with detectable serum lamotrigine between 12–24 m and 0–24 m, suggesting a possible neuroprotective effect on axons by lamotrigine. The delay in the reduction of NfH by 12 months implies a lead-lag effect between taking the drug and neuroprotection. Lim et al. found that MSIS-29 increased with EDSS deterioration NfH was a strong predictor of ambulation and disability based on MSFC and EDSS, supporting the use of NfH as a surrogate measure of disability. This is confirmed by the time-to-event modelling for completion of the timed 25-foot walk. Interestingly, performance in the 9-HPT and its relationship with NfH levels was found predominantly in the non-dominant hand which may be related to the loss in manual dexterity that may be more obvious with the non-dominant hand as the test is normally performed faster using the dominant hand NfH correlated well with all MRI-derived measures except for conventional T1 and T2 volumes. MRICCV and MTR were both lowered in patients with higher NfH, supporting earlier associations of NfH with clinical measures and connecting progressive brain atrophy in SPMS to NfH release and a reduction in cerebral volumes (MRICCV) and global disease burden (MTR). Our observed lack of correlation between spinal cord area and NfH levels may have been partly confounded by the lack of sensitivity of spinal cord MR imaging to axonal disease Based on the parameters obtained for NfH we have estimated the sample sizes that future studies would need to detect changes in the proportion of patients with detectable NfH. In this study 80% of samples had detectable NfH and we calculated the sample sizes required to detect differences between this and 50, 55, 60, 65 and 70% in a comparator group .et al. We also analysed serum NOx, GFAP, BDNF and NGF, but found significant association was between walking times or 9-HPT times. However, MRI central cerebral volumes highly correlated with NOx and GFAP as expected since inflammation and oedema may increase brain volume. Cheriyan J Of the 31 paired CSF/serum/plasma biomarkers studied, no clear trends emerged from the analyses aside from plasma osteopontin . Higher In conclusion, the results of this study failed to show any benefit of lamotrigine in the prevention of axonal breakdown by lowering NfH levels compared to the placebo arm. A further subgroup analysis based on lamotrigine adherence, however, suggests that it may have a beneficial effect on axonal protection and it would be premature to abandon this hypothesis in entirety. We have demonstrated that NfH is very good surrogate marker of neurodegeneration and correlates well with clinical and MRI biomarkers of progression. The sample size calculations provided in this paper should pave the way for neuroprotection trials using serum NfH.Figure S1Plasma osteopontin and its relationship with the z-score, MRICCV, MTR grey matter and whole brain are presented.(TIF)Click here for additional data file.Table S1Biomarker values in serum, plasma and CSF samples from the lamotrigine trial. They are grouped approximately by the quartiles of each biomarker's distribution, and in analysis where only a few were detectable as absolute values.(TIF)Click here for additional data file.Table S2Rank correlation coefficients between biomarkers and test scores (a) and MRI measures. Statistically significant correlations  are shaded, red for positive correlations and green for negative.(TIF)Click here for additional data file.


In immunocompromised patients, influenza infection may progress to prolonged viral shedding from the respiratory tract despite antiviral therapy. We describe chronic influenza A virus infection in an immunocompromised child who had prolonged shedding of culturable influenza virus in stool. Human influenza A virus infections are typically localized to the respiratory tract, and viral presence in the gastrointestinal (GI) tract is rarely observed. Isolation of influenza virus from the stool is most commonly documented in human infections of avian influenza subtype H5N1. In these infections, virus in the stool may be related to a disseminated infection with exceptionally high viral titers, atypical of seasonal, circulating influenza infections  testing of a nasopharyngeal swab specimen showed influenza A infection. Over the next several months, he had additional influenza A–positive DFAs and was given oral and aerosolized ribavirin for chronic infection. Despite this aggressive antiviral treatment, >24 respiratory specimens were positive for influenza A by DFA or culture over the next year.Clostridium difficile–toxin B cytotoxicity assay, and stool examination for ova and parasites were all negative. Results of a urine culture were positive for enterococcus. Culture of stool samples was enterovirus positive and over the subsequent 2 weeks, enterovirus was isolated from 2 additional viral stool cultures. These cultures were not evaluated for the presence of influenza virus. Three weeks after the initial stool testing, results of viral stool culture and repeat stool studies were negative. However, the diarrhea and emesis persisted, and ≈8 weeks after the onset of symptoms, another stool specimen was sent for viral culture.When the patient was ≈3 months into an extended hospitalization at the Lucile Packard Children’s Hospital , he experienced multiple daily episodes of nonbilious, nonbloody emesis (≈2–3/day) and loose stools without blood or mucous (up to 12/day). The clinical team suspected a graft versus host flare but also conducted a work up of the patient to identify an infectious process. Results of blood cultures, stool culture, After 3 days, the culture demonstrated cytopathic effect on primary RhMK cells but not on human foreskin fibroblasts, MRC-5 fibroblasts, or A549 lung carcinoma cells, which is consistent with enterovirus infection. However, results of immunofluorescent staining of the RhMK cells  were negative. Staining results were also negative with serotype group–specific reagents, including the coxsackie virus B blend, echovirus blend, enterovirus 70 and 71, poliovirus blend, coxsackie virus A9, and coxsackie virus A24 . An astute technologist associated the pattern of cells showing a cytopathic effect with the patient’s concurrent influenza A–positive respiratory specimen and long history of influenza infection. She then set up a standard respiratory virus DFA panel (Millipore/Light Diagnostics), which included fluorescein-conjugated antibodies for the detection of influenza A and B; respiratory syncytial virus; parainfluenza 1, 2, and 3; and adenovirus. Strikingly, the specimen was strongly positive for influenza A virus and showed obvious hemadsorption with guinea pig erythrocytes. Two months later, influenza A was again isolated from the patient’s stool, which suggested persistent infection of the GI tract with influenza A virus. Subsequent nucleic acid testing revealed that this chronic influenza A infection was caused by the seasonal, circulating subtype H1N1 virus. Overall, the patient shed influenza A from respiratory secretions for >1.5 years and from stool for >2 months.Because viral stool cultures from patients with respiratory infections are infrequently ordered, the true occurrence of influenza virus in stool is unknown. The few studies to date have considered viral RNA in fecal specimens as a marker of GI influenza infection, which may not accurately reflect the shedding of intact virus or the capacity for transmission. One study of 4 children with respiratory symptoms and confirmed influenza infection showed that half had influenza RNA in stool  2009 virus suggested that it produced diarrhea, vomiting, or both, in ≈25% of case-patients, more often than the previous seasonal, circulating influenza viruses (


Warburgia ugandensis (Canellaceae). Ten individuals each were assessed from two locations east and west of the Great Rift Valley, Kenya, Africa, which differed in humidity and vegetation, closed forest versus open savannah. Despite organ- and partially site-specific variation of drimane sesquiterpenes, no clear effects on bacterial and fungal endophyte communities could be detected. The former were dominated by gram-negative Gammaproteobacteria, Pseudomonadaceae and Enterobacteriaceae, as well as gram-positive Firmicutes; the fungal endophyte communities were more diverse but no specific groups dominated. Despite initial expectations, the endophyte community of the pepper bark tree did not differ from other trees that much.Metabolite profiles (GC–−MS), drimane sesquiterpenes, sugars and sugar alcohols, were compared with bacterial and fungal endophyte communities  in leaves and roots of the pepper bark tree,  Warburgia ugandensis Sprague [=W. salutaris (Bertol.f.) Chiov], the pepper bark tree belongs to the Canellaceae, a small family of tropical trees, all of them aromatic and most with medicinal properties. W. ugandesis has a restricted distribution in evergreen forests and woodland ravines of northern KwaZulu-Natal, Swaziland, Mpumalanga, Uganda, and Kenya. This species is widely used in traditional medicine within local communities in Eastern Africa, known to cure several ailments such as stomach-ache, constipation, toothache, common cold, cough, fever, muscle pains, weak points, measles, and malaria  and one east (Rumuruti) of the Great Rift Valley, Kenya, Africa, were chosen. Kitale forest is a tropical area in western Kenya situated between Mount Elgon and the Cherengani Hills at an elevation of around 2000 m above sea level. The Rumuruti forest is a dry upland forest at an elevation of 1700–2000 m above sea level. Both differ in their humidity as documented by annual rainfall amounts. Based on AFLP comparisons, the two Warburgia populations were recently suggested to constitute two different species as a consequence of allopatric speciation, which also occurs for species of other genera that occur both west and east of the Great Rift Valley , the second one east of the Great Rift Valley in Kitale forest near the town Kitale , west of the Great Rift Valley. Kitale is located in a moist savannah in western Kenya situated between Mount Elgon and the Cherengani Hills at an elevation of around 2000 m. a. s. l. and has an average annual precipitation of 1269 mm; Rumuruti is located in a savannah with W. ugandensis mostly growing in or close to moist ravines embedded in semi-arid land at an elevation of 1700–2000 m. a. s. l. in the Laikipia district situated northwest of Mount Kenya with 739 mm average annual precipitation (http://www.climatedata.eu)  were extracted with 80 ml methanol for 24 h at ambient temperature. The extract was filtered  and concentrated under vacuum. Two hundred mg crude extract were fractioned over Amberlite XAD-1180 . Glass columns (15 mm diameter) were filled with 20 g resin and prepared according to the manufacturer's guidelines. Two 50 ml fractions were eluted, one with water, one with absolute ethanol. The evaporated eluates were dissolved in 10 ml methanol and stored at −20°C until further use. All used solvents were at least p.a. quality. This procedure was performed for all extracts to obtain fractions that could be analyzed in terms of drimane sesquiterpene composition. The crude extracts contained high quantities of sugar alcohols, specifically mannitol. Metabolite quantitation only was performed with the ethanol fraction, in which the drimanes were accumulated but which still contained notable amounts of sugars and sugar alcohols. A clean separation proved impossible. Consequently, the ethanol fraction represented the only fraction that provided a dataset allowing a relative comparison of drimane sesquiterpenes, fatty acids and sugar alcohols, assuming that lower sugar alcohol or sugar amounts present in the ethanol fraction represent a lower ethanol—drimane sesquiterpene ratio in the crude extract.For GC–MS measurements, 100 μg of the dried ethanolic eluate  for derivatisation into trimethylsilyl ethers. One μl of this solution was injected into an AutoSystem XL gas chromatograph  in the splitless mode, the injector temperature was 250°C. The column was a Zebron 5 ms column , the helium follow rate 0.8 ml/min. The temperature gradient started at 70°C and, after 3 min, rose to 300°C at a rate of 3°C/min. The gas chromatograph was linked to a TurboMass™ quadrupole mass analyzer ; the transfer line temperature was set to 280°C, the ion source to 200°C, the filament current to 70 eV. The mass spectrometer was run in the TIC mode from 40–620 amu. The obtained chromatograms were integrated with TurboMass 4.1.1  and the peak areas were expressed as relative amounts of the total peak area (100%). The majority of drimane structures were identified on basis of a tentative fragmentation pattern analysis of the silylated derivatives. Published structures, both from natural sources and from synthesis, served as templates for spectrum interpretation.2PO4 buffer, MT buffer was added and everything was transferred to the lysing matrix E tube followed by 30 s bead-beating with a bead beater .Prior to isolation of microbial community DNA, microbial cells were dislodged from plant tissue as previously described GGATTAGATACCC(GT)-3′)  according to the manufacturer's instructions. For a more detailed description of the cloning procedure see supplementary information. Clones have been sequenced with the primer M13f and/or M13r making use of the sequencing service of LGC Genomics . Retrieved sequences were visualized and vector sequences were removed with sequence alignment editor package of BioEdit . For identification sequences were subjected to the Basic Local Alignment Search Tool (BLAST) analysis with the National Center for Biotechnology Information (NCBI) database.Ribosomal DNA libraries were constructed from a pool of aliquots of all DNA samples as well as selected plant samples making use of the Strata Clone PCR Cloning Kit  and the StrataClone SoloPack Pseudomonadaceae-, Enterobacteriaceae- and Firmicutes-specific 16S rRNA genes within selected plant samples were analyzed in more detail by real-time PCR. Following primers were as used: Pseudomonadaceae; 8f: 5′-AGAGTTTGATCCTGGCTCAG-3′  employing Bray–Curtis similarity as resemblance measure; similarity boundaries were determined by group average clustering of the Bray–Curtis similarity matrix. SIMPER analyses were performed to identify variables that contribute to similarities and dissimilarities of defined sample groups Clarke, . Diversin = 3) were performed with Statgraphics Centurion XV .Parametric analyses of variance (ANOVA), provided normal distribution and variance homogeneity was given, or non-parametric Kruskal–Wallis analysis of variance, in case the above mentioned criteria failed, were carried out with respective multiple range tests . These and simple linear regression analyses , sugar alcohols (4), fatty acids (6) and glycerol, which also were detected. Only one triterpene was detected, β-sitosterol.GC–MS analyses identified 173 analytes in the extracts from 10 root, 10 leaf, and 5 fruit accessions, the latter originating only from the locality Rumuruti , of which 141 were assigned as terpenoids on basis of their fragmentation patterns, A multivariate analysis, MDS  of a Bray–Curtis resemblance matrix, revealed an organ-specific clustering Figure  that was1 to 18. Their MS spectra and the tentative interpretation of the fragmentation pattern are presented in datasheet S1: 1, drimendiol, was originally isolated from Drymis winteri, also a member of the Canellaceae  ; the single amounts varied considerably and this also contributed to dissimilarity . By contrast, Rumuruti leaves showed the dialdehyde mukaadial (7) and the acids 10 and 11. In fruits, which were only available from some individuals at Rumuruti, again other drimane sesquiterpenes contributed to similarity: triol 5 and the corresponding dialdehyde warburganal (6). By its presence, the sugar alcohol mannitol contributed to similarity in all analyzed organs; its detected concentration fluctuations, however, also added to the dissimilarity. In fruits, another sugar alcohol, was prominent, myo-inositol; in roots, it was the monosaccharide fructose. In aerial organs, palmitic acid was more prominent than in roots. These facts  revealed a low complexity for all accessions; only three peaks were prominent Table . One peaTable 3Relevant bacterial T-RFs (T-RF 153 bp was resolved further by qPCR).Gammaproteobacteria, with 52% of the clones being Pseudomonadaceae, predominantely the genus Pseudomonas, and the rest Enterobacteriaceae. The remaining clones belonged to the divisions of Actinobacteria (5%) and Firmicutes (5%).In order to identify the dominant genera in the bacterial assemblages we constructed a 16S rRNA gene clone library from a pool of aliquots of all DNA samples. Among 53 ribotypes five chimeric sequences, two chloroplast sequences and one mitochondrial sequence were found and excluded from further analysis. Two thirds of the clearly bacterial sequences showed at least 97% similarities to known 16S rRNA genes in the NCBI database, and 30% of the clones were distantly (92–96%) related to known species (datasheet S2). The majority (90%) of the sequences belonged to the Gammaproteobacteria and Bacillus group (data not shown). Altogether five families were identified, Pseudomonadaceae, Enterobacteriaceae, Bacillaceae, Paenibacillaceae, and Staphylococcaceae, with the latter being present only in Kitale leaf accession L1. The main difference between the individual clone libraries was the varying Pseudomonadaceae–Enterobacteriaceae abundance ratio. Quantitative PCR detection of 16S rDNA genes specific for the taxa Firmicutes, Enterobacteriaceae and Pseudomonadaceae confirmed the clone library data. Conversely, some accessions contained mainly Pseudomonadaceae and hardly any Enterobacteriaceae or Frimicutes and again other accessions contained no Pseudomonadaceae but were dominated by Enterobacteriaceae or Firmicutes, respectively  in ty Table  revealedty Table . Root T-ty Table .Table 4Relevant fungal T-RFs.W. ugandensis trees, we constructed an ITS region clone library from a pool of aliquots of all DNA samples. In total, 96 clones were analyzed and assigned by RFLP analysis to 47 ribotypes for which sequences were determined (datasheet S3). Three % were chimeric sequences and excluded from further analysis. The clearly non-chimeric sequences belonged to Ascomycota (81%) and Basidiomycota (16%). The two biggest classes of fungi were Dothidiomycetes (28%) and Sordariomycetes (27%), followed by Microbotryomycetes with 16%. The remaining sequences could be assigned to the classes of Saccharomycetes (12%), Leotiomycetes (12%), Pezizomycetes (2%), Eurotiomycets (2%), and Tremellomycetes (1%). The library comprised a total number of 20 fungal species; the basidiomycete Sporidobolus ruinae was the most abundant, representing 16% of the clones in the library. None of the identified clones unambiguously correlated with the peak at bp 72.In order to identify the dominant species in the fungal assemblages in Warburgia metabolites and predominance of specific microbial community member groups, which were explored further by simple regression analyses. The identification of correlations in the data was hampered by the fact that similarity within accession groups was rather low, in many cases less than 50%. This not only applied to drimane sesquiterpenes but also to bacterial and fungal endophyte communities. Thus we defined the following criteria for acceptable correlations: (1) the correlation had to be supported by at least three cases (n ≥ 3); (2) reproducibility was to be at least 90% (p ≤ 0.1), and (3) at least 50% of all cases should support the correlation (r2 ≥ 0.5). Table 4 with the fungal TRF 141, isodrimenol (2) and deacetylugandensiolide (16) with Firmicutes rDNA copy numbers. The same drimanes, however, 2 and 16, also were positively correlated, 2 with bacterial TRF 165, similarly as 12-hydroxy-epi-albrassitriol (12), and 16 with root-occurring Enterobacteriaceae rDNA copy numbers. The latter phenomenon was detected in both localities, Kitale and Rumuruti. Further positive correlations comprised the alcohol 5 and its oxidized derivative 8 with Pseudomonadaceae rRNA gene copy numbers. Moreover the metabolite diversity in W. ugandensis trees did not correlated.Explorative PLS regression (not shown) indicated only a few correlations between With bacterial and fungal endophyte diversity, at least on basis of GC–MS and T-RFLP results Table .7) and the corresponding acids 10 and 11; the Kitale leaves showed drimendiol (1) as major compound, which represents the most reduced derivative. The co-occurring hemiacetal 7 and the simple acid 3 somehow suggest a lower degree of oxidation in Kitale leaf tissues than in those of Rumuruti. Oxidation reactions on drimane sesquiterpene structural diversity will be discussed in detail later in this text. If the more humid climate of the Kitale site, which was suggested by more intensive colonization by epiphytic lichens and ferns, caused this phenomenon can only be clarified after more study sites have been studied with a focus on this aspect.Drimane sesquiterpenes diversity turned out to be higher than that of bacterial and fungal endophyte communities, both in the leaves and roots the two accessed sites. The T-RFLP patterns of all assessed accessions did not form any specific clusters in terms of collection site and plant organ. By contrast, metabolite patterns, especially if only drimane sesquiterpenes were considered, formed organ-specific clusters. Leaf profiles differed between Kitale and Rumuruti, but root patterns were similar Figures . The RumWarburgia ugandensis trees, and the abundant genera Pseudomonas, Pantoea, Bacillus/Paenibacillus represented both endophyte species frequently encountered in other plant species   Weber, , NigrospWarburgia colonizers.Contrary to initial expectations, the endophyte communities of the pepper bark tree do not differ from literature reports from other trees that much. The variation of drimane sesquiterpene profiles that was hinted in preliminary investigations was confirmed, but no substantial correlations were found with endophyte community composition. This suggests that any potential antimicrobial metabolites—drimane sesquiterpenes reportedly possess this activity  the endophyte community is not shaped by host drimane sesquiterpenes; (3) the diversity of the endophytic microflora in Warburgia ugendensis does not correlate with that of host drimane sesquiterpenes; and (4) other factors rather than endophytic microbes might be responsible for the high variations in the content and composition of drimane sesquiterpenes in the pepper bark tree.In conclusion, our study revealed that (1) the endophyte community of the tropical tree Further studies including also other tropical trees are required to explore if the conclusions from this paper can be confirmed on a broader scale. Ideally, such studies should consider the effect of climatic stress such as drought or high light exposure for the individual trees, if possible, to facilitate a more insightful assessment of the implications of variation in the analyzed microbial assemblages in geographically different host populations. Endophytes can affect the metabolism and health of their hosts in some cases, but they may fail to do so in other cases (Porras-Alfaro and Bayman, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Meles meles), have produced equivocal results. Culling-induced social perturbation of badger populations may lead to unexpected outcomes. We test predictions from the ‘perturbation hypothesis’, determining the impact of culling operations on badger populations, movement of surviving individuals and the influence on the epidemiology of bTB in badgers using data dervied from two study areas within the UK Government's Randomised Badger Culling Trial (RBCT). Culling operations did not remove all individuals from setts, with between 34–43% of badgers removed from targeted social groups. After culling, bTB prevalence increased in badger social groups neighbouring removals, particularly amongst cubs. Seventy individual adult badgers were fitted with radio-collars, yielding 8,311 locational fixes from both sites between November 2001 and December 2003. Home range areas of animals surviving within removed groups increased by 43.5% in response to culling. Overlap between summer ranges of individuals from Neighbouring social groups in the treatment population increased by 73.3% in response to culling. The movement rate of individuals between social groups was low, but increased after culling, in Removed and Neighbouring social groups. Increased bTB prevalence in Neighbouring groups was associated with badger movements both into and out of these groups, although none of the moving individuals themselves tested positive for bTB. Significant increases in both the frequency of individual badger movements between groups and the emergence of bTB were observed in response to culling. However, no direct evidence was found to link the two phenomena. We hypothesise that the social disruption caused by culling may not only increase direct contact and thus disease transmission between surviving badgers, but may also increase social stress within the surviving population, causing immunosuppression and enhancing the expression of disease.In the UK, attempts since the 1970s to control the incidence of bovine tuberculosis (bTB) in cattle by culling a wildlife host, the European badger ( Attempts to manage infectious diseases in wildlife populations have varying consequences for the ecology of the target populations, depending on the methods used Epidemiological patterns of directly transmitted infectious diseases are the product of contacts between individuals that permit disease transmission Mycobacterium bovis , can infect a wide range of mammals Meles meles) has been implicated as a reservoir for bTB in cattle Undisturbed, moderate to high density badger populations in lowland England are often organised into relatively stable, mixed-sex social groups Social disruption of remaining badger populations following culling has been shown to influence individual badger movements between social groups, with young females moving into depopulated setts Evidence that badger culling operations can cause social perturbation has been reported from badger populations in Gloucestershire, UK We report here on a field experiment carried out within the RBCT Triplet E , January 2003 , and August 2003 . The estimated population density in the treatment area fell to its lowest level of 1.2 badgers per km2 following the August 2003 cull, compared with approximately 6 badgers per km2 in the control site and approximately 5 badgers per km2 in the treatment site during the same season in the previous year. From our trapping records, the 77 badgers killed during culling operations amounted to between 34–43% of the badgers resident in the targeted social groups, based on the estimated number of animals within each group from our capture records.Over the four years of the study, a total of 663 badger captures with release was made in the culling (n = 481) and non-culling (n = 182) study areas, involving 423 individual animals. The overall rate of recapture was 29.7%, being approximately equal for males and females (28.3% and 30.1% respectively). Recapture rates were also consistent between study areas, being 30.1% in the culling area and 29.1% in the non-culling control area. Over the course of the study, the estimated overall density of badgers varied between 1.2 and 6.9 badgers per kmM. bovis (excreting) from one or more clinical samples. Of these animals with confirmed disease, eight were recaptured and none was found to excrete M. bovis intermittently .Of the 423 individuals captured during the study, 40 individuals were found to be positive for M. bovis between treatment and control sites , with estimates of 8.1% and 7.1% respectively. However prevalence increased in both sites post-culling, from 5.2% to 15% in the treatment site, and from 2.7% to 15.9% in the control site . The effect of culling on the prevalence of detected M. bovis in badgers differed among social group types in the treatment area  following culling , with the greatest increase being in Neighbouring groups: from 1.0% (sd = 4%) to 13.6% (sd = 12.3%)  .M. bovis among cubs in the treatment population following culling, with only one excreting individual identified out of 101 cubs captured before culling (0.9%), and eight out of 76 cubs (10.5%) testing positive for bTB after culling (Two-tailed Fisher's Exact Test: P = 0.01). Of these eight cubs, four tested positive for M. bovis from urine samples, three from sputum and one from a faecal sample. No M. bovis was detected among the 47 bager cubs tested in the control population during this study.There was a statistically significant increase in detected Radio-collars were fitted to 50  and 20  adult badgers in the treatment and control study areas, respectively. From the culture of clinical samples, six of the collared individuals were found to be infected with bTB: four males and one female in the treatment; and one female in the control population. In total, 8,311 fixes were collected from tracked individuals over 239 nights. Radio-collars remained attached to badgers for an average of 230 days (sd = 54 days).3,81 = 12.87 P<0.01). Within the treatment area the variation among cull phases was not consistent among group types , from an average of 21.4 Ha (sd = 22.8 Ha) pre-culling, to 9.4 Ha (sd = 12.7 Ha) post-culling. However, size of summer range areas of badgers from Removed groups increased between the pre- and inter-culling phases during which they were measured, from 28.3 Ha (sd = 11.3 Ha) to 40.6 Ha (sd = 27.6 Ha). In Neighbouring groups, home range areas decreased between the pre- and inter-culling phases from 22.0 Ha (sd = 26.2 Ha) to 10.0 (sd = 11.3 Ha), remaining low post-culling , with this trend approaching statistical significance . There was an upward trend in home range size for badgers from Other and Control groups .Home range size, as measured by 95% minimum convex polygons (MCP), varied seasonally, with maximum range (40.6 Ha) areas observed in summer in the treatment area and in autumn (23.4 Ha) in the control area . Core areas in the treatment population tended to expand in response to culling, with increased variability, from a mean of 2.2 Ha (sd = 2.1 Ha) to 2.5 Ha (sd = 6.3 Ha).In the control population, core areas were smaller than in the treatment and decreased through the culling phases, from 1.2 Ha (sd = 1.4 Ha) to 0.8 Ha (sd = 0.6 Ha). Within the treatment population, however, there was no statistically significant difference in badger core area size between social group types with respect to culling .The effect of culling phase on the size of badger home range core areas, based on 50% kernels, varied between study areas .Within the treatment area, the effect of culling phase on proportional overlap between badger summer home ranges varied significantly among group types . Neighbouring and Other group overlaps increased following culling, from an average of 0.8 to 2.7 groups with which individuals affiliated, compared with a decline in Removed groups from approximately 0.8 to 0.2 on average. The number of overlaps appeared to remain relatively high in Other groups in autumn 2003, compared with 2002, when radio-tracking of six animals from five adjacent social groups revealed no home range overlaps. Numerical overlap between home ranges in Control groups did not change significantly in relation to culling .Similarly, the numbers of home ranges from different groups with which each badger overlapped in summer also varied among group types in the treatment population in response to culling  by radio-collared badgers twice out of 891 below-ground positions between 2001 and 2003. Both of these were in the treatment population and were made by males to groups immediately neighbouring their original groups. One of these moves was made during the culling operation in November 2002 and the other six months after culling in July 2003.21 = 7.20; P<0.01), with nine post-culling IGMs occurring in the treatment population all being made to Removed groups. Five of these nine were from Neighbouring social groups, and four from Removed groups.Out of a total of 663 trapping events (including 197 recaptures) in the treatment and control study areas, 12 instances of badgers moving between social groups (IGMs) were detected . Of these IGMs, nine (75%) occurred in the treatment population following the culls in Nov02/Jan03, and one occurred in the control study area . In tota1 = 3.64; P = 0.056), with more movements observed from Removed (n = 5) and Neighbouring groups (n = 5) compared with Other groups (n = 1). The size of the donor social group influenced IGM probability , with animals being more likely to move from larger groups , compared with an average group size of 3.4 animals (sd = 1.9) where no movement was observed). IGM probability was not influenced by an individual's sex , with IGMs being made by seven females and six males; or by age , although nine of the movers were adults and only four were cubs. The adult sex ratio of the donor group was also not a significant predictor of IGM .Within the treatment population, social group type influenced the probability of individuals moving from a group, with marginal statistical significance . However, M. bovis excretion was not detected amongst the individuals identified as having made IGMs.Overall there was a tendency for increases in the prevalence of detected M donors  and reciM donors , althougM. bovis infection from culture of clinical samples increased from 1% to 14% in Neighbouring groups following culling and this increase tended to be greater in groups involved in IGMs, whether as donors or receivers. The culture of clinical samples alone may prove insensitive, partly due to the potential for intermittent shedding of bacilli by badgers Information on the behavioural responses of badgers to culling is critical to furthering our understanding of the perturbation effect and the potential value of this management approach for the control of bTB infection in both badgers and cattle. In our study although inter-group movementss (IGMs) by badgers were rare, they increased significantly following culling. This usually involved the movement of animals into groups that had been subjected to culling either from other culled groups or their unculled immediate neighbours. The prevalence of detectable Within the treatment population, the relative sizes of badger core home range areas among group types were unchanged by culling. However, overall home range areas of individuals from Removed and Other groups, measured as MCP, increased significantly in response to culling, suggesting an increased range of movement by individuals from these groups. Individuals from Neighbouring groups increased the amount of home range overlap with individuals from surrounding groups, following culling, although the tendency was for their home range sizes to decrease through the culling phases. The increase in overlap is consistent with the observation from a separate study of the same population using bait-marking which showed that the group ranges of Removed and Neighbouring social groups also increased their overlap with adjacent social groups One simple prediction arising from the perturbation hypothesis is that the movement of infected animals from disturbed groups presents enhanced opportunities for disease transmission in the population. Our results were not consistent with this prediction, with instead there being a pervasive emergence of bTB across the badger population following culling Data on contact rates between individual badgers is scant, largely due to the practical difficulties of observing animals directly, particularly underground. In one undisturbed high density population, agonistic contacts between badgers were found to increase with density, as indicated by rates of bite wounding The evidence from radio-tracking individual movements also demonstrated a widespread effect of culling. In Other groups there was a behavioural response to culling, with increases in home range size and overlap between individuals from Neighbouring and Other groups. The IGM rate increased between Neighbouring and Removed groups following culling, with the majority of movements being into Removed groups. IGMs in the control area, and prior to culling in the treatment area, were rare. Animals were more likely to move from larger groups, with no effect of sex or age. This contrasts with the observations of Tuyttens et al. It is difficult to generalise about badger movement patterns 2. The comparative control population, which was not subject to culling by Defra, occupied an 18.9 km2 area within the survey only area of triplet E (51° 28′N 2° 03′W). Both areas included mixed farming, with arable and livestock production, the latter being principally dairy cattle. The agricultural landscapes of both areas created mosaics of woodland, pasture and arable fields.Study areas were located on the borders of Wiltshire, Somerset and South Gloucestershire in South-West England. The treatment badger population was positioned within the reactive culling area of the RBCT triplet E (51° 27′N 2° 25′W), occupying an area of 37.3 km−2 in both areas.Both treatment and control study areas were surveyed for badger setts initially in autumn 2000. Setts were categorised as either active or inactive based on the condition of entrance holes and the freshness of badger sign in the vicinity. In total, 71 and 38 active setts were identified in the treatment and control study areas respectively, giving a density of approximately two setts kmWithin the treatment study area, Defra performed three badger culling operations in November 2002 , January 2003  and August 2003 .Badgers were captured at active setts using wire mesh cage traps, with a 2.5 cm mesh size, placed at or near to setts and baited using peanuts. Traps were set over two consecutive nights, following at least one week of pre-baiting. Trapping was carried out between September 2002 and August 2003, with study areas trapped during winter (January), spring/early summer (May/June), summer (August) and autumn .Mycobacterium bovisCaptured badgers were sedated using a combination of ketamine hydrochloride , medetomidine hydrochloride  and butorphanol tartrate  by intramuscular injection at a ratio of 2∶1∶2 by volume respectively and a dose rate of approximately 0.2 mL/kg The home ranges of individual badgers were determined by radio-tracking, using radio-transmitters in the 173 MHz band, attached to a leather collar . Adult badgers were selected for radio-collaring based on existing collars within the same social group, spatial proximity of collared animals from adjacent social groups, and sex. The objective was to obtain a wide distribution of several clusters of collared animals of approximately equal sex ratio. Clusters were initiated by the opportunistic capture of two more adults of different sex from a social group. On subsequent trapping occasions, adults captured from these social groups, and their immediate neighbours, were collared preferentially, until two animals of each sex had been collared from each social group. This event only occurred at two social groups; one in the treatment area and one in the control area. Clustering collared animals over three or four neighbouring social groups provided the ability to examine overlap and interactions between individuals from different groups. The wide distribution of these clusters minimised bias due to the locations of  reactive culling operations which might have subsequently taken place within the treatment study area. Radio-collars were also deployed opportunistically as the need arose when practicalities thwarted adherence to the decision strategy, such as a lack of candidate individuals from a particular social group.Fixes were taken at a maximum interval of 15 minutes, decreasing to 5 minutes when badgers were moving. Observers radio-tracked individual focal animals at any one time preferentially, with fixes also established for other collared animals in the vicinity of the focal animal. Fixes were obtained using either Mariner  or R-1000  receivers with six-element Yagi-style directional antennae . Positional data were recorded on 1∶10,000 scale maps or with global positioning system (GPS) receivers (Garmin eTrax 12-channel). Badger positions were established either visually, with the aid of image intensifying equipment or by triangulation.Badger day-time sett locations were also established using radio-telemetery. Only badger presence could be determined, as failure to locate an animal at a particular sett did not necessarily demonstrate absence. Failure to locate a badger at either the sett at which it was captured or the sett to which it was last radio-tracked initiated a survey of all surrounding setts until either the badger had been located or no more setts remained to survey.Following the protocol of Tuyttens et al. www.esri.com) and Ranges home range analysis software (www.anatrack.com). Total home ranges of badgers were defined as 95% minimum convex polygons (MCP) and core areas within badger home ranges were identified using 50% kernels Individual ranges were estimated from radio-tracking data using geographical information system software , summer , autumn , and winter  . Radio-tracking data were collected throughout the study, from Nov 2001 to Dec 2003, except during culling operations, at Defra's request, to avoid compromising RBCT results. Comparisons of individual movements before and after culling were adjusted for season, with spring and summer comparisons being made relative to culling in Nov 02 and Jan 03; winter comparisons being made relative to Nov 02 culling and autumn comparisons being made relative to all culling events.Defining dispersal is difficult, and the approach used should depend on whether trapping or radio-tracking data are used. Trapping provides at most a ‘snap-shot’ of residency, with longer term data needed to distinguish short-term excursions from more permanent shifts in residency The SAS software was used for data analyses Home range MCP and core areas, were log-transformed (log10+1) to satisfy the condition of normality, as was numerical overlap between home ranges. Responses expressed as proportions were arcsine-square root transformed . We first fitted a model using the predictors study area (treatment or control), cull , season and all two-way interactions. Separate models were defined for the treatment population alone, comparing differences between social group types (RNO). No valid model could include both study area (treatment or control) and group type, as group type was entirely confounded with study area.The factors influencing the likelihood of an individual moving from one social group to another (IGM) were examined using logistic regression (SAS GENMOD procedure), with animal movement entered as a binary response. Donor social group type (RNO), sex ratio and estimated size were entered as covariates, along with the individual's sex and age (adult or cub).


Tribolium castaneum. Within a four-patch landscape we modified resource level (constant and diminishing), patch connectivity (high and low) and patch configuration (static and dynamic) to conduct a 23 factorial experiment, consisting of 8 metapopulations, each with 3 replicates. For comparison, two control populations consisting of isolated and static subpopulations were provided with resources at constant or diminishing levels. Longitudinal data from 22 tri-weekly counts of beetle abundance were analyzed using Bayesian Poisson generalized linear mixed models to estimate additive and interactive effects of factors affecting abundance. Constant resource levels, low connectivity and dynamic patches yielded greater levels of adult beetle abundance. For a given resource level, frequency of colonization exceeded extinction in landscapes with dynamic patches when connectivity was low, thereby promoting greater patch occupancy. Negative density dependence of pupae on adults occurred and was stronger in landscapes with low connectivity and constant resources; these metapopulations also demonstrated greatest stability. Metapopulations in control landscapes went extinct quickly, denoting lower persistence than comparable landscapes with low connectivity. When landscape carrying capacity was constant, habitat destruction coupled with low connectivity created asynchronous local dynamics and refugia within which cannibalism of pupae was reduced. Increasing connectivity may be counter-productive and habitat destruction/recreation may be beneficial to species in some contexts.Interactive effects of multiple environmental factors on metapopulation dynamics have received scant attention. We designed a laboratory study to test hypotheses regarding interactive effects of factors affecting the metapopulation dynamics of red flour beetle,  Metapopulations are local populations distributed patchily in space and linked by dispersal Numerous prior studies have examined the role of each of these factors separately. Level of resource availability  has emerged predictably as an important determinant of metapopulation viability Drosophila hydei) Reduced connectivity of resource patches has lowered persistence for metapopulations of fruit flies ). We tested the predicted main effects summarized in the preceding paragraphs and examined all pairwise interactions of these effects on colonization, extinction, and abundance of beetles.We manipulated resource availability, patch connectivity, and dynamics of patch configuration in experimental metapopulations to investigate their additive and interactive effects. Specifically, we tested these effects by manipulating the amount of resources and the level of boundary permeability Tribolium castaneum was obtained from the U.S. Grain Marketing Research Laboratory in Manhattan, Kansas, in 2005. Beetles were cultured in 95% wheat flour and 5% yeast medium by mass. Beetles were maintained in an environmental chamber at 33±1°C and 70±5% relative humidity. The life cycle in T. castaneum (egg to adult) takes roughly one month, with an average 4 days for egg, 3 weeks for larval and 6 days for pupal development Red flour beetle is a stored grain pest that infests a variety of stored products worldwide. The stock population (Berlin) of We designed experimental landscapes with two habitat and two “marginal” habitat patches, arranged in an alternating sequence . Habitatwww.zinsser.com) containing fine-grained sand to facilitate beetle traction. To confine insects, the sides of the tray were treated with Fluon . A patch in a landscape consisted of a small (35 mm diameter×10 mm height) Petri dish affixed with glue inside the center of a larger (60×15 mm) Petri dish. Gluing was done all along the rim of the smaller Petri dish to seal the base and prevent adults or larvae from crawling under the smaller dish and thus getting trapped over the course of the experiment. To facilitate beetle movement between the inner and outer portions of the patch, an inverted-V paper ramp (24×4 mm) was attached to the rim of the inner dish. The lid of the inner small Petri dish was notched where the inverted-V paper ramp joined the inner Petri dish to allow the exit of beetles to the outer Petri dish. A circular hole of diameter 2 mm was made in the outer dish and oriented 180 degrees from the inverted paper ramp of the inner Petri dish to allow emigration of beetles from a patch into the surrounding landscape. Connectivity, specifically, patch boundary permeability Each constructed landscape consisted see  of a 17 −6, and the probability of 7–12 larvae of a given sex is 0.90.All experimental patches received 6 adults and 18 larvae released on 3 g of medium  inside the small dish of each patch . Both adults and larvae were added as a starter population to mimic more closely established local populations, avoid time lags, and buffer against crashes associated with density-dependent cannibalism Tribolium eggs in the medium For each treatment, observations were made every 3 weeks, a time interval chosen to correspond with the larval developmental period, allow sufficient time for beetles to respond to the treatments imposed, and minimize the disturbance associated with counting. Sifting the resources with #80 mesh sieve retains most of the We used a factorial design including 3 factors, each at 2 levels, for a total of eight treatments. Each treatment was replicated three times. The factors were landscape connectivity, resource level, and patch configuration. Connectivity was manipulated via high (exit holes at 2.5 mm) and low (holes at 4.0 mm) boundary permeability. Resource levels of landscapes either remained constant throughout the experiment, i.e., the medium in each patch was replenished every 3 weeks, or diminished to represent habitat degradation. For the latter treatment, at the end of every 3-week period, the medium in habitat and marginal-habitat patches was replaced with fresh medium, but in an amount reduced by 0.5 g from what had been present in the patch 3 weeks earlier. For instance, at the end of the first 3 weeks, a habitat patch with 3 g of resource was replenished with 2.5 g of fresh resource and similarly, a marginal-habitat patch was replenished with 2.5 g of dextrose. Reduction of patch resources by 0.5 g every 3 weeks was continued until the total resource available in a patch was reduced to 0.5 g, at which point a final reduction to 0.2 g was made. Below this resource level cannibalism is quite high 3 factorial experiments, we included as references two controls with no landscape connectivity, i.e., no dispersal of beetles from static patches. In one control, carrying capacity remained constant, whereas in the other carrying capacity diminished over time as described above. Only static patch configurations were used in control landscapes, because extinction would be inevitable in landscapes with dynamic patch configuration  and no dispersal. Each control was replicated three times.In addition to the 2Probabilities of colonization and extinction for each patch state  were calculated as the proportion of those colonization and extinction events occurring from time t-1 to time t during 22 tri-weekly surveys (except as noted below) divided by the total number of patches in a particular state and available for colonization  or extinction (occupied) respectively at time t-1. For analyses involving comparison across landscapes with diminishing resources or controls, only data from the first 18 tri-weekly surveys were used, because metapopulations in all three replicate landscapes suffered extinction beyond this time.Patch extinction was defined as absence of adult beetles at time t after being occupied by adults at time t-1. Conversely, patch colonization was defined as the presence of at least one adult in a patch following extinction. Data on colonization and extinction frequencies in both patch states of each replicate landscape were used to derive mean colonization and extinction probabilities at the landscape level for each treatment. Because count data from the experiment were overdispersed, a quasi-binomial generalized linear model was fitted We applied a generalized linear mixed effects Poisson model (GLMM) to determine how resource level, patch connectivity and patch configuration affected metapopulation attributes. The GLMM was implemented within a Bayesian framework ijC is the count observed in landscape j (j = 1 to 24) at time step i (i = 1 to 22). The intercept μ represents the grand mean effect, and βs are the coefficients associated with fixed effects. The parameters jα and iε account for random variation in beetle count data due to landscape and time effects, respectively. Uninformative normal priors with mean zero were used for μ, β, jα and iε. Standard deviations of 100 were specified for the fixed effects parameters, whereas the hyperparameters ασ and εσ reflect the random variation due to landscapes and time, respectively, and were drawn from a Uniform  distribution.Response variables in the GLMMs included total live adults, live adults inside patches, adults outside patches, larvae inside patches, and pupae inside patches. We fitted a repeated measures model with all main and two-way fixed effects and two random effects using the model:Our control landscapes lacked connectivity between patches. To model the effect of complete isolation on the number of live adults in patches, we modified the GLMM described above to include three levels of connectivity  and to exclude main and interaction effects associated with patch configuration. We also fitted a Poisson GLMM to assess the nature of density dependence on pupae and larvae at time t in patches of control and treatment populations. For this analysis, time and number of live adult beetles in patches at time t-1 were treated as fixed factors, along with random landscape and time effects.The GLMMs were fitted by calling WinBUGS 1.4 We estimated stability of metapopulations by measuring the mean amplitude of fluctuations in population size over time. Specifically, we computed a fluctuation index For colonization frequency, the QAIC-best model included a significant (P = 0.0003) interaction effect of patch connectivity and configuration. Increased connectivity dampened the positive effect of a dynamic patch configuration on colonization . SpecifiThe frequency of patch extinctions was 1.65 times greater for landscapes with diminishing resources (P = 0.01) and nearly three times greater in landscapes with dynamic patches (P≪0.001). No interactive effects on patch extinction were observed.At the landscape level, colonization probabilities were on average 2.6 times greater when resource was constant versus diminishing (P≪0.0001) and 4.9 times greater when patch configuration was dynamic versus static (P≪0.0001) . Ratios Abundance of one or more beetle life stages was influenced by the main effects of resource level, patch connectivity, and patch configuration. Not surprisingly, resource level was the most influential factor affecting abundance of all life stages, with standardized coefficients that were 1.7–33.8 times larger than the next most influential main effect , 3. AbunDynamics of adult beetles were affected by interactions of time with each of the experimental variables, and by interactions of resource×connectivity and resource×patch configuration . Adult aDynamics of subadult beetles were affected by interactions of time with each of the experimental variables, and by interactions of resource×patch configuration and connectivity×patch configuration . Larval For landscapes with static patch configuration, including control landscapes, significant interactions of time×resource and time×connectivity were evident . The avePoisson GLMM revealed that pupal abundance at time t declined with increasing adult abundance at time t-1 for both treatment and control landscapes . Negativβ = −0.12, P = 0.0008) or low connectivity  produced lower fluctuations in amplitude than those with diminishing resources or high connectivity, respectively. Moreover, the effect of low connectivity on fluctuations tended to be greater for landscapes with constant resources . Mean fluctuations of metapopulations in landscapes with static versus dynamic patch configurations did not differ . For all landscapes, habitat patches (0.41) always fluctuated less than marginal habitats (3.34) .Metapopulations with constant resources  for habitat patches experiencing static (0.34) versus dynamic (3.4) configuration, but did not differ for other main effects. Fluctuations of subpopulations in habitat patches were influenced by the interaction of resource and configuration  as well as connectivity and configuration . Effects of configuration dominated in both instances, with greatest fluctuations in habitat patches in dynamic landscapes. When resource levels were constant, the effect of dynamic patch configuration on fluctuations in habitats was 1.52 times greater than when resource levels were diminishing. Similarly, when connectivity of habitat patches was low, the effect of dynamic configuration on fluctuations was 1.34 times greater than when connectivity was high. Fluctuations in marginal habitats were lower in landscapes with low patch connectivity  and a static configuration of patches , but no interaction effects were significant.Patterns of fluctuations at the subpopulation level  differed dramatically from those observed for entire metapopulations. Fluctuation in amplitude was 10 times lower  In our study, reduced patch connectivity increased the difference in colonization rate between dynamic and static landscapes by 3–30-fold. Connectivity had no effect on extinction rate, but high connectivity resulted in more adults, and more adult mortality, in the matrix for landscapes with static patch configuration. Thus, the net effect of low connectivity was increased patch occupancy and abundance for metapopulations in dynamic landscapes. Despite fluctuations of habitat subpopulations that were 10 times greater for dynamic landscapes, metapopulations in dynamic landscapes displayed stability comparable to those in static landscapes. Asynchrony in the dynamics of subpopulations likely contributed to this effect, which was especially notable when connectivity was low. Following patch destruction, marginal habitats served as more effective refugia for mature larvae, pupae and callow when connectivity was low; a similar effect on recruitment was shown in a coral reef fish (Cardamine pensylvanica) and observed increased extinction due to increased connectivity when all subpopulations in a metapopulation fluctuated in synchrony and consequently experienced simultaneous decline. Conflicting results regarding the impact of connectivity on metapopulation dynamics suggest that predicting the effects of connectivity between habitat patches requires consideration of the dispersal ability of a species and how behaviors modified by landscape heterogeneity influence survival or reproduction Other investigators have noted potentially deleterious effects of enhanced connectivity. Hess Answering questions about optimal connectivity will likely require an understanding of the biology of a target species and the resource requirements for each of its life stages. Our findings suggest that questions of connectivity should simultaneously consider the temporal dynamics of preferred and marginal habitats. Clearly, additional empirical studies are needed to explore the impact of patch turn-over rates and habitat complexity on metapopulations residing in spatially and temporally varying landscapes.Appendix S1WinBUGS script for specifying Poisson mixed effect regression model for the response variable to estimate model parameters. The modelled data are C, which represent counts of adult beetles in landscape j at time i. Comments follow a #.(DOC)Click here for additional data file.