Extensive Interindividual Variation Research Articles

Fungal-Bacterial Interactions in the Human Gut of Healthy Individuals.

Most studies of the microbiota in the human gut focus on the bacterial part, but increasing information shows that intestinal fungi are also important for maintaining health. This can be either by directly influencing the host or by indirectly influencing the gut bacteria that link to host health. Studies of fungal communities in large cohorts are scarce; therefore, this study aims at obtaining more insight into the mycobiome of healthy individuals and how this mycobiome interacts with the bacterial component of the microbiome. For this purpose, ITS2 and 16S rRNA gene amplicon sequencing was performed on fecal samples from 163 individuals which were available from two separate studies to analyze the fungal and bacterial microbiome, respectively, as well as the cross-kingdom interactions. The results showed a much lower fungal, as compared to bacterial, diversity. Ascomycota and Basidiomycota were the dominant fungal phyla across all the samples, but levels varied enormously between individuals. The ten most abundant fungal genera were Saccharomyces, Candida, Dipodascus, Aureobasidium, Penicillium, Hanseniaspora, Agaricus, Debaryomyces, Aspergillus, and Pichia, and here also extensive inter-individual variation was observed. Correlations were made between bacteria and fungi, and only positive correlations were observed. One of the correlations was between Malassezia restricta and the genus Bacteroides, which have both been previously described as alleviated in IBD. Most of the other correlations found were with fungi that are not known as gut colonizers but originate from food and the environment. To further investigate the importance of the observed correlations found, more research is needed to discriminate between gut colonizers and transient species.

Chronic Kidney Disease and the Exposome of Ageing.

The gap between improvements in lifespan and age-related health is widening. Globally, the demographic of ageing is increasing and there has emerged a 'diseasome of ageing', typified by a range of non-communicable diseases which share a common underlying component of a dysregulated ageing process. Within this, chronic kidney disease is an emerging global epidemic.The extensive inter-individual variation displayed in how people age and how their diseasome manifests and progresses, has required a renewed focus on their life course exposures and the interplay between the environment and the (epi)genome. Termed the exposome, life course abiotic and biotic factors have a significant impact on renal health.We explore how the exposome of renal ageing can predispose and affect CKD progression. We discuss how the kidney can be used as a model to understand the impact of the exposome in health and chronic kidney disease and how this might be manipulated to improve health span.Notably, we discuss the manipulation of the foodome to mitigate acceleration of ageing processes by phosphate and to explore use of emerging senotherapies. A range of senotherapies, for removing senescent cells, diminishing inflammatory burden and either directly targeting Nrf2, or manipulating it indirectly via modification of the microbiome are discussed.

Establishing a Novel Pipeline That Combines in-Silico Prediction with in-Vitro and Ex-Vivo Validation to Discover Secondary Drug Combinations Against Relapsed and/or Refractory Multiple Myeloma

Multiple myeloma (MM) is the second-most common hematological malignancy in the US. MM is an incurable, age-dependent plasma cell neoplasm with a 5-year survival rate of less than 50%. Extensive inter-individual variation in response to standard-of-care drugs like proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), drug resistance, and dose-limiting toxicities are critical problems for the treatment of MM. Clinical success in anti-myeloma treatment, therefore, warrants continuous development of novel combination therapy strategies with the explicit goal to improve the therapeutic efficacy by concomitantly targeting multiple signaling pathways.Previously, we have reported the development of an in-house computational pipeline called secDrug that applies greedy algorithm-based set-covering computational optimization method followed by a regularization technique to predict secondary drugs that can be repurposed as novel synergistic partners of standard-of-care drugs for the management of refractory/ resistant MM. Top among these secondary drugs (secDrugs) were the HSP90 inhibitor 17-AAG.In this study, we used 17-AAG as a proof of principle to establish a pipeline that integrates our in silico predictions with in vitro and ex vivo validation as well as multi-omics technologies to identify, validate, and characterize therapeutic agents that could be used either alone or in combination with standard-of-care drugs for the treatment of R/R MM patients (Figure 1).To screen and validate our in silico prediction results, we performed in vitro cytotoxicity assays using 17-AAG on a panel of human myeloma cell lines (HMCLs; in vitro model systems) that captures a wide range of biological and genetic heterogeneity representing the complexities encountered in clinical settings. These cell lines include HMCLs representing innate sensitive/resistance, >10 pairs of parental and clonally-derived PI- and IMiD-resistant pairs (P vs VR or LenR; representing acquired/emerging resistance/relapse), NRAS mutants which leads to the constitutive activation of oncogenic Ras signaling, and CRISPR-edited HSP90 knockdown cell line. Our results showed that 17-AAG has high synergistic activity in combination with PI in inducing apoptosis even in innate and acquired PI-resistant HMCLs and significantly reduces the effective dose of PI required to achieve IC 50 (Chou-Talalay's Dose Reduction Index or DRI 7±1.4). Moreover, 17-AAG+IMiD showed synergistic cell killing activity in clonally-derived IMiD resistant HMCL. Further, 17-AAG induced cell death was comparable with Hsp90 knockdown as evident from the cytotoxicity assay using PI and 17-AAG in combination in RPMI8226-wild type and RPMI-HSP90AA1 knocked down cell line. Notably, 17-AAG was strikingly effective against the NRAS-mutant cell line indicating an additional niche (NRas mutant myeloma) where 17-AAG could be most effective.Next, we performed RNA sequencing to elucidate the molecular mechanisms behind 17-AAG drug action, drug synergy, 17-AAG-induced cell death. Our gene expression profiling (GEP) followed by Ingenuity Pathway Analysis (IPA) analysis revealed protein ubiquitination, aryl hydrocarbon receptor signalling pathway as the top canonical pathways. 17-AAG induced apoptosis via mitochondrial mediated pathway in myeloma. 17-AAG exerts its cytotoxic effect by activating intrinsic pathway of apoptosis which we further confirmed through the increase in reactive oxygen species generation and decrease in mitochondrial membrane potential. 17-AAG was also effective in reducing the expression of hallmarks of MM such as p65/NF-kB, IRF4, c-Myc. Finally, we performed mass cytometry (CyTOF; Cytometry by time of flight) on primary bone-marrow cells (PMCs) from myeloma patients for further validation of proteomic signatures at the single-cell level. CyTOF analysis confirmed 17-AAG-induced cell death and key changes in MM-specific proteomic markers. 17-AAG treated PMCs showed elevated cleaved caspase levels and down-regulation of IRF4 and phospho-STAT3. GEP and CyTOF results were confirmed using immunoblotting assays.Together, our study demonstrates a unique pipeline for drug repositioning that has the potential to revolutionize clinical decision-making by minimizing the number of drugs required for discovering successful combination chemotherapy regimens against drug-resistant myeloma. [Display omitted] DisclosuresKumar: BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche-Genentech: Consultancy, Research Funding; Bluebird Bio: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Gene Expression Profiling of Kinetic Response to Proteasome Inhibitors in Innate and Acquired Resistant Multiple Myeloma

Extensive inter-individual variation in response to chemotherapy is a serious cause of concern in the treatment of multiple myeloma (MM). Drug resistance may be categorized into innate resistance already present in drug-naive refractory patients or emerging (acquired) resistance where a patient's tumor ultimately undergoes relapse in course of treatment despite initial response.Using a panel of human myeloma cell lines (HMCLs), we have earlier presented a gene expression signature that could not only distinguish good and poor Proteasome inhibitor (PI)-response in the HMCL panel, but was also successful in stratifying exceptional response to PI-based chemotherapy in MM clinical datasets.In this study, we used six (6) most PI-sensitive and six (6) most PI-resistant HMCLs representing innate PI response and clonally-derived PI-resistant (R) cell lines and PI-sensitive (P) HMCLs representing acquired PI-response to identify kinetic changes in gene expression patterns following test dosing with the 2nd generation PI drug Ixazomib. High quality RNA was extracted from untreated/baseline/0Hr and treated/24hrs post-treatment myeloma cell lines and RNA sequencing was performed on llumina's HiSeq 2000 using 50bp paired-end protocol with depth of >20million reads-per-sample. Transcriptome profiles were then compared to characterize differences in kinetic response to PIs.Our analysis revealed, at FDR<0.05, 74 genes changed significantly post-treatment in Ix-sensitive lines, with a |fold-change|> 1.5 while no gene showed any significant kinetic change in Ix-resistant cell lines. When our kinetic innate Ix-response profile was compared to Shaughnessy's 80-gene kinetic signature derived from Bz test-dosing, 16 genes were found similar, including PSMD4 that was identified earlier as novel high-risk feature in MM patients on Bz-based clinical trial. On the other hand, kinetic expression analysis of the clonally-related PI-sensitive parental and PI-resistant cell line pairs representing acquired PI-resistance revealed 651 genes were significantly differentially expressed in the sensitive/parental cell lines compared to 21 in clonally-derived resistant cell lines. Further, we observed that the gene signatures of kinetic innate PI-response were different from the signature of acquired PI-response. We also observed strikingly lower number of genes that significantly changed post-treatment in both innate and acquired PI-resistant cell lines when compared to the PI-sensitive cell lines. Ingenuity Pathway Analysis (IPA) identified the proteasome ubiquitination pathway, NRF2-mediated oxidative stress response pathway, oxidative stress response and Glutathione depletion/ Glutathione redox reactions as top canonical pathways associated with the most differentially regulated kinetic response genes. Several of these genes, including CCND1 (cyclin D1) and E2F, have been earlier shown involved in proliferative activity as well as susceptibility to chemotherapy.Our results thus provide key insights into the phenomenon of kinetic PI response in MM and highlights the notable differences between innate and acquired drug response. We are currently validating our in vitro findings using ex vivo kinetic transcriptome analysis in primary myeloma cells derived from drug-naive newly diagnosed MM patients representing variation in PI-response. DisclosuresKumar:Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Skyline: Honoraria.

Identification of a foetal epigenetic compartment in adult human kidney

ABSTRACT The mammalian kidney has extensive repair capacity; however, identifying adult renal stem cells has proven elusive. We applied an epigenetic marker of foetal cell origin (FCO) in diverse human tissues as a probe for developmental cell persistence, finding a 5.4-fold greater FCO proportion in kidney. Normal kidney FCO proportions averaged 49% with extensive interindividual variation. FCO proportions were significantly negatively correlated with immune-related gene expression and positively correlated with genes expressed in the renal medulla, including those involved in renal organogenesis (e.g., FGF2, PAX8, and HOXB7). FCO associated genes also mapped to medullary nephron segments in mouse and rat, suggesting evolutionary conservation of this cellular compartment. Renal cancer patients whose tumours contained non-zero FCO scores survived longer. The kidney appears unique in possessing substantial foetal epigenetic features. Further study of FCO-related gene methylation may elucidate regenerative regulatory programmes in tissues without apparent discrete stem cell compartments.

In vitro and ex vivo gene expression profiling reveals differential kinetic response of HSPs and UPR genes is associated with PI resistance in multiple myeloma

Extensive inter-individual variation in response to chemotherapy (sensitive vs resistant tumors) is a serious cause of concern in the treatment of multiple myeloma (MM). In this study, we used human myeloma cell lines (HMCLs), and patient-derived CD138+ cells to compare kinetic changes in gene expression patterns between innate proteasome inhibitor (PI)-sensitive and PI-resistant HMCLs following test dosing with the second-generation PI Ixazomib. We found 1553 genes that changed significantly post treatment in PI-sensitive HMCLs compared with only seven in PI-resistant HMCLs (p < 0.05). Genes that were uniquely regulated in PI-resistant lines were RICTOR (activated), HNF4A, miR-16-5p (activated), MYCN (inhibited), and MYC (inhibited). Ingenuity pathway analysis (IPA) using top kinetic response genes identified the proteasome ubiquitination pathway (PUP), and nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response as top canonical pathways in Ix-sensitive cell lines and patient-derived cells, whereas EIF2 signaling and mTOR signaling pathways were unique to PI resistance. Further, 10 genes were common between our in vitro and ex vivo post-treatment kinetic PI response profiles and Shaughnessy’s GEP80-postBz gene expression signature, including the high-risk PUP gene PSMD4. Notably, we found that heat shock proteins and PUP pathway genes showed significant higher upregulation in Ix-sensitive lines compared with the fold-change in Ix-resistant myelomas.

Chromosome polymorphisms track trans-Atlantic divergence and secondary contact in Atlantic salmon.

Pleistocene glaciations drove repeated range contractions and expansions shaping contemporary intraspecific diversity. Atlantic salmon (Salmo salar) in the western and eastern Atlantic diverged >600,000years before present, with the two lineages isolated in different southern refugia during glacial maxima, driving trans-Atlantic genomic and karyotypic divergence. Here, we investigate the genomic consequences of glacial isolation and trans-Atlantic secondary contact using 108,870 single nucleotide polymorphisms genotyped in 80 North American and European populations. Throughout North America, we identified extensive interindividual variation and discrete linkage blocks within and between chromosomes with known trans-Atlantic differences in rearrangements: Ssa01/Ssa23 translocation and Ssa08/Ssa29 fusion. Spatial genetic analyses suggest independence of rearrangements, with Ssa01/Ssa23 showing high European introgression (>50%) in northern populations indicative of post-glacial trans-Atlantic secondary contact, contrasting with low European ancestry genome-wide (3%). Ssa08/Ssa29 showed greater intrapopulation diversity, suggesting a derived chromosome fusion polymorphism that evolved within North America. Evidence of potential selection on both genomic regions suggests that the adaptive role of rearrangements warrants further investigation in Atlantic salmon. Our study highlights how Pleistocene glaciations can influence large-scale intraspecific variation in genomic architecture of northern species.

Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Variation in proviral content among human genomes mediated by LTR recombination

BackgroundHuman endogenous retroviruses (HERVs) occupy a substantial fraction of the genome and impact cellular function with both beneficial and deleterious consequences. The vast majority of HERV sequences descend from ancient retroviral families no longer capable of infection or genomic propagation. In fact, most are no longer represented by full-length proviruses but by solitary long terminal repeats (solo LTRs) that arose via non-allelic recombination events between the two LTRs of a proviral insertion. Because LTR-LTR recombination events may occur long after proviral insertion but are challenging to detect in resequencing data, we hypothesize that this mechanism is a source of genomic variation in the human population that remains vastly underestimated.ResultsWe developed a computational pipeline specifically designed to capture dimorphic proviral/solo HERV allelic variants from short-read genome sequencing data. When applied to 279 individuals sequenced as part of the Simons Genome Diversity Project, the pipeline retrieves most of the dimorphic loci previously reported for the HERV-K(HML2) subfamily as well as dozens of additional candidates, including members of the HERV-H and HERV-W families previously involved in human development and disease. We experimentally validate several of these newly discovered dimorphisms, including the first reported instance of an unfixed HERV-W provirus and an HERV-H locus driving a transcript (ESRG) implicated in the maintenance of embryonic stem cell pluripotency.ConclusionsOur findings indicate that human proviral content exhibit more extensive interindividual variation than previously recognized, which has important bearings for deciphering the contribution of HERVs to human physiology and disease. Because LTR retroelements and LTR recombination are ubiquitous in eukaryotes, our computational pipeline should facilitate the mapping of this type of genomic variation for a wide range of organisms.

Does proliferation capacity of lymphocytes depend on human blood types?

In vitro human lymphocyte culture methodology is well established yet certain confounding factors such as age, medical history as well as individual's blood type may potentially modulate in vitro proliferation response. These factors have to be carefully evaluated to release reliable test report in routine cytogenetic evaluation for various genetic conditions, radiation biodosimetry, etc. With this objective, the current study was focused on analyzing the proliferation response of lymphocytes drawn from 90 individuals (21-29 years) with different blood types. The proliferation response was assessed in the cultured lymphocytes by cell cycle, mitotic index (MI), and nuclear division index (NDI) after stimulation with phytohaemagglutinin (PHA). To investigate the toxic effect on proliferation, MI was calculated in representative samples of each blood type were X-irradiated. The results showed that there was no significant difference among the cell cycle phases of lymphocytes in different blood types (P > 0.05). Similarly, both MI and NDI of lymphocytes derived from different blood types also did not show significant difference ( P > 0.05). The extensive interindividual variation within and among the blood types is likely responsible for the lack of significant difference in lymphocyte proliferation. Although spontaneous proliferation efficiency of lymphocytes of different blood types after PHA stimulation was grossly similar, the MI observed after radiation exposure showed a significant difference ( P < 0.05) indicating a differential proliferation response among the blood types. Our results suggest that the blood types did not have any impact on PHA-induced proliferation; however, a specific differential lymphocyte proliferation observed after radiation exposure needs to be considered.

Variation in CYP2A6 Activity and Personalized Medicine.

The cytochrome P450 2A6 (CYP2A6) enzyme metabolizes several clinically relevant substrates, including nicotine—the primary psychoactive component in cigarette smoke. The gene that encodes the CYP2A6 enzyme is highly polymorphic, resulting in extensive interindividual variation in CYP2A6 enzyme activity and the rate of metabolism of nicotine and other CYP2A6 substrates including cotinine, tegafur, letrozole, efavirenz, valproic acid, pilocarpine, artemisinin, artesunate, SM-12502, caffeine, and tyrosol. CYP2A6 expression and activity are also impacted by non-genetic factors, including induction or inhibition by pharmacological, endogenous, and dietary substances, as well as age-related changes, or interactions with other hepatic enzymes, co-enzymes, and co-factors. As variation in CYP2A6 activity is associated with smoking behavior, smoking cessation, tobacco-related lung cancer risk, and with altered metabolism and resulting clinical responses for several therapeutics, CYP2A6 expression and enzyme activity is an important clinical consideration. This review will discuss sources of variation in CYP2A6 enzyme activity, with a focus on the impact of CYP2A6 genetic variation on metabolism of the CYP2A6 substrates.

A gene expression signature distinguishes innate response and resistance to proteasome inhibitors in multiple myeloma

Extensive interindividual variation in response to chemotherapy is a major stumbling block in achieving desirable efficacy in the treatment of cancers, including multiple myeloma (MM). In this study, our goal was to develop a gene expression signature that predicts response specific to proteasome inhibitor (PI) treatment in MM. Using a well-characterized panel of human myeloma cell lines (HMCLs) representing the biological and genetic heterogeneity of MM, we created an in vitro chemosensitivity profile in response to treatment with the four PIs bortezomib, carfilzomib, ixazomib and oprozomib as single agents. Gene expression profiling was performed using next-generation high-throughput RNA-sequencing. Applying machine learning-based computational approaches including the supervised ensemble learning methods Random forest and Random survival forest, we identified a 42-gene expression signature that could not only distinguish good and poor PI response in the HMCL panel, but could also be successfully applied to four different clinical data sets on MM patients undergoing PI-based chemotherapy to distinguish between extraordinary (good and poor) outcomes. Our results demonstrate the use of in vitro modeling and machine learning-based approaches to establish predictive biomarkers of response and resistance to drugs that may serve to better direct myeloma patient treatment options.

BOLA-DRB3 gene polymorphisms influence bovine leukaemia virus infection levels in Holstein and Holstein×Jersey crossbreed dairy cattle.

Bovine leukemia virus (BLV) infections, causing persistent lymphocytosis and lethal lymphosarcoma in cattle, have reached high endemicity on dairy farms. We observed extensive inter-individual variation in the level of infection (LI) by assessing differences in proviral load in peripheral blood. This phenotypic variation appears to be determined by host genetics variants, especially those located in the BoLA-DRB3 MHCII molecule. We performed an association study using sequencing-based typed BOLA-DRB3 alleles from over 800 Holstein and Holstein×Jersey cows considering LI invivo and accounting for filial relationships. The DBR3*0902 allele was associated with a low level of infection (LLI) (<1% of circulating infected B-cells), whereas the DRB3*1001 and DRB3*1201 alleles were related to a high level of infection (HLI). We found evidence that 13 polymorphic positions located in the pockets of the peptide-binding cleft of the BOLA-DRB3 alleles were associated with LI. DRB3*0902 had unique haplotypes for each of the pockets: Ser13 -Glu70 -Arg71 -Glu74 (pocket 4), Ser11 -Ser30 (pocket 6), Glu28 -Trp61 -Arg71 (pocket 7) and Asn37 -Asp57 (pocket 9), and all of them were significantly associated with LLI. Conversely, Lys13 -Arg70 -Ala71 -Ala74 and Ser13 -Arg70 -Ala71 -Ala74 , corresponding to the DRB3*1001 and *1201 alleles respectively, were associated with HLI. We showed that the specific amino acid pattern in the DRB3*0902 peptide-binding cleft may be related to the set point of a very low proviral load level in adult cows. Moreover, we identified two BOLA-DRB3 alleles associated with a HLI, which is compatible with a highly contagious profile.

An Amperometric Cytochrome P450-2D6 Biosensor System for the Detection of the Selective Serotonin Reuptake Inhibitors (SSRIs) Paroxetine and Fluvoxamine

Paroxetine is the second most prescribed selective serotonin reuptake inhibitor (SSRI) antidepressant drug, characterized by extensive inter-individual variation in steady state plasma concentrations resulting in drug toxicity amongst patinets. A nanopolymeric biosensor for studying the biotransformation of paroxetine is presented. The bioelectrode system consists of cytochrome P450-2D6 enzyme encapsulated in nanotubular poly (8-anilino-1-napthalene sulphonic acid) electrochemically deposited on gold. The biosensing procedure involved the determination of the extent of modulation of fluvoxamine responses to the P450-2D6 enzyme electrode after incubation in paroxetine standard solutions. Paroxetine inhibited the activity of cytochrome P450-2D6 (CYP2D6) resulting in a decrease in the fluvoxamine signal of the biosensor. The biosensor gave a linear analytical response for the paroxetine in the interval 0.005 and 0.05 μM, with a detection limit of 0.002 μM and a response time of 30 s. Electrochemical Michaelis–Menten kinetics of the reversible competitive inhibition of the fluvoxamine responses of the biosensor by 0, 0.05 and 0.1 μM paroxetine gave apparent Michaelis–Menten constant (KMapp) values of 1.00 μM, 1.11 μM and 1.25 μM, respectively. The corresponding value for the maximum response, IMAX was 0.02 A. The dissociation constant, KI, value evaluated from Dixon analysis of the paroxetine modulation data was estimated to be-0.02 μM while Cornish-Bowden analysis confirmed the competitive inhibitory characteristics of the enzyme.

The composition of the zebrafish intestinal microbial community varies across development.

The assembly of resident microbial communities is an important event in animal development; however, the extent to which this process mirrors the developmental programs of host tissues is unknown. Here we surveyed the intestinal bacteria at key developmental time points in a sibling group of 135 individuals of a model vertebrate, the zebrafish (Danio rerio). Our survey revealed stage-specific signatures in the intestinal microbiota and extensive interindividual variation, even within the same developmental stage. Microbial community shifts were apparent during periods of constant diet and environmental conditions, as well as in concert with dietary and environmental change. Interindividual variation in the intestinal microbiota increased with age, as did the difference between the intestinal microbiota and microbes in the surrounding environment. Our results indicate that zebrafish intestinal microbiota assemble into distinct communities throughout development, and that these communities are increasingly different from the surrounding environment and from one another.

Convergence in generic pronouns: Language contact and Faroese mann

Despite state-driven language policy against Danish linguistic influence, the Faroese language has borrowed the Danish generic pronoun mann ‘one’. As in Danish, this pronoun varies with generically used tú ‘you’. An analysis of the variation in Faroese shows that Faroese tú is used more often than its Danish equivalent du (26% vs. 16.5%) and that although there is extensive inter-individual variation, different linguistic factors (inclusion of the addressee in the referent of the pronoun, use in a conditional construction and verb tense) and social factors (age, gender and peripherality of location; possibly also speech style) constrain this variation in Faroese and in Danish. This suggests that extensive bilingualism has not led to sociolinguistic convergence between Faroese and Danish. Inclusion of sociolinguistic analysis in language contact research can help further our understanding of contact processes and inform language policy.

Telomeres, atherosclerosis, and human longevity: a causal hypothesis.

Human telomere length, as expressed in leukocytes, is strongly fashioned by heritability.1–5 A number of genes that explain some of the inter-individual variation in leukocyte telomere length have already been identified.6–9 Leukocyte telomere length is longer in women than in men3,10,11 and in African Americans than in whites of European descent.12,13 In addition, offspring of older fathers display a longer leukocyte telomere length, a finding that may reflect longer telomeres in sperm of older men.14–17 In contrast to the male germline, telomeres in replicative somatic tissues, including the hematopoietic system, display age-dependent shortening. A shorter leukocyte telomere length is associated with aging-related diseases, principally atherosclerosis18,19 and reduced longevity.20,21 The prevailing interpretation is that a shorter leukocyte telomere length is the outcome of processes, mainly the age-dependent accruing burden of oxidative stress and inflammation22 that ultimately shorten the human lifespan. As such, leukocyte telomere length is considered to be a non-causal biomarker, ie, a telomeric “clock,” of human aging. We propose that telomere length is not only associated with atherosclerosis and longevity but is also a causal determinant of both. This hypothesis is based on a model of leukocyte telomere length dynamics (leukocyte telomere length and its attrition rate) that incorporates two key features. First, the model differentiates the findings of studies of telomere length dynamics in vitro from those in vivo. Second, the model distinguishes two phases of human leukocyte telomere length dynamics in vivo: growth and adulthood.

The developing xylem transcriptome and genome-wide analysis of alternative splicing in Populus trichocarpa(black cottonwood) populations

BackgroundAlternative splicing (AS) of genes is an efficient means of generating variation in protein structure and function. AS variation has been observed between tissues, cell types, and different treatments in non-woody plants such as Arabidopsis thaliana (Arabidopsis) and rice. However, little is known about AS patterns in wood-forming tissues and how much AS variation exists within plant populations.ResultsHere we used high-throughput RNA sequencing to analyze the Populus trichocarpa (P. trichocarpa) xylem transcriptome in 20 individuals from different populations across much of its range in western North America. Deep transcriptome sequencing and mapping of reads to the P. trichocarpa reference genome identified a suite of xylem-expressed genes common to all accessions. Our analysis suggests that at least 36% of the xylem-expressed genes in P. trichocarpa are alternatively spliced. Extensive AS was observed in cell-wall biosynthesis related genes such as glycosyl transferases and C2H2 transcription factors. 27902 AS events were documented and most of these events were not conserved across individuals. Differences in isoform-specific read densities indicated that 7% and 13% of AS events showed significant differences between individuals within geographically separated southern and northern populations, a level that is in general agreement with AS variation in human populations.ConclusionsThis genome-wide analysis of alternative splicing reveals high levels of AS in P. trichocarpa and extensive inter-individual AS variation. We provide the most comprehensive analysis of AS in P. trichocarpa to date, which will serve as a valuable resource for the plant community to study transcriptome complexity and AS regulation during wood formation.

Temperature-Dependent Reproductive Development ofLygus hesperus(Hemiptera: Miridae)

Recent studies to elucidate relationships between the western tarnished plant bug, Lygus hesperus Knight, and injury to cotton (Gossypium spp.) have identified important differences in feeding behaviors among adults of different gender and reproductive states. These findings suggest a need for improved knowledge of L. hesperus temperature-dependent reproductive development. We used nonlinear biophysical development rate models to describe the reproductive development of adult L. hesperus held under constant temperatures from 12.8 to 35.0°C. In addition, abdominal coloration was measured as a nondestructive indicator of reproductive maturity. The nonlinear models indicated most stages of ovary, seminal vesicle, and medial accessory gland development were subject to mild high-temperature inhibition, except for the stage of filled seminal vesicles. Development of filled seminal vesicles was subject to minor low-temperature inhibition and severe high-temperature inhibition. Estimated development times reflected extensive interindividual variation, especially at low temperatures. This variation suggests the opportunity to select for more rapid or more consistent reproductive development under the conditions of laboratory culture. Although presence of a dark aqua coloration of the ventral abdominal cuticle was statistically associated with reproductive maturity, especially in female L. hesperus, color-based predictions of reproductive maturity were often inaccurate for bugs reared at temperatures above or below 26.7°C. Therefore, use of abdominal coloration as an indicator of reproductive development in ecological studies seems inappropriate. The results provide mathematical descriptions of L. hesperus temperature-dependent reproductive development which should facilitate improved planning and interpretation of studies involving manipulation of adult reproductive status.

Comparative Analysis of the Humoral Immune Response to Moraxella catarrhalis and Streptococcus pneumoniae Surface Antigens in Children Suffering from Recurrent Acute Otitis Media and Chronic Otitis Media with Effusion

A prospective clinical cohort study was established to investigate the humoral immune response in middle ear fluids (MEF) and serum against bacterial surface proteins in children suffering from recurrent acute otitis media (rAOM) and chronic otitis media with effusion (COME), using Luminex xMAP technology. The association between the humoral immune response and the presence of Moraxella catarrhalis and Streptococcus pneumoniae in the nasopharynx and middle ear was also studied. The levels of antigen-specific IgG, IgA, and IgM showed extensive interindividual variation. No significant differences in anti-M. catarrhalis and anti-S. pneumoniae serum and MEF median fluorescence intensity (MFI) values (anti-M. catarrhalis and antipneumococcal IgG levels) were observed between the rAOM or COME groups for all antigens tested. No significant differences were observed for M. catarrhalis and S. pneumoniae colonization and serum IgG levels against the Moraxella and pneumococcal antigens. Similar to the antibody response in serum, no significant differences in IgG, IgA, and IgM levels in MEF were observed for all M. catarrhalis and S. pneumoniae antigens between OM M. catarrhalis- or S. pneumoniae-positive and OM M. catarrhalis- or S. pneumonia-negative children suffering from either rAOM or COME. Finally, results indicated a strong correlation between antigen-specific serum and MEF IgG levels. We observed no significant in vivo expressed anti-M. catarrhalis or anti-S. pneumoniae humoral immune responses using a range of putative vaccine candidate proteins. Other factors, such as Eustachian tube dysfunction, viral load, and genetic and environmental factors, may play a more important role in the pathogenesis of OM and in particular in the development of rAOM or COME.