The role of pollinators in generating and maintaining floral polymorphism : phylogeographic and behavioural aspects

Thesis (PhD)--Stellenbosch University, 2013.Pollinators play a fundamental role in floral evolution. They can exert selection on the flowers they visit in a plethora of different ways, ranging from innate floral preferences to differences in body size and shape and behavioural elements such as flower constancy and learning capacity. Since different pollinators exhibit differences in these characters, shifts between pollinating species are often considered the most likely drivers of floral diversification. While many lines of evidence support this claim, numerous angiosperms pollinated by a single species also exhibit floral variation. Throughout my thesis, I explore and investigate floral diversification in such species in the absence of pollinator shifts. In Chapter 2, I investigate variation in the preference of conspecific male and female pollinators for the floral traits of a sexually deceptive daisy that comprises distinct floral forms. I show that its pollinator exhibits gender-specific variation in floral preferences, and that some floral forms have specialized on the male pollinator. This chapter thus illustrates the importance of intraspecific variation in pollinator preference for floral diversification, an underappreciated mechanism in this field of research. The innate preferences of pollinators are likely to have a genetic basis, especially innate preferences that govern mate choice. Genetic structure within the pollinators of sexually deceptive plants, which mimic female insects to achieve pollination, may thus provide an important source of selection on the plants they pollinate. This depends on an association between genetic divergence and divergent mate preferences, and I explore this intriguing idea in Chapter 3. While pollinators associated with sexually deceptive floral forms did exhibit significant genetic structuring, male pollinators from different phylogeographic clades all exhibited preference for the same sexually deceptive floral form, thus rejecting this hypothesis. Another behavioural attribute of pollinators that may affect floral evolution, particularly in deceptive plant species, is learning ability. Studies on sexually deceptive orchids often report that male pollinators tend to avoid sexually deceptive flowers with experience. In Chapter 4, I systematically investigate learning abilities within male pollinators and the costs they suffer on sexually deceptive floral forms that vary in deceptiveness. Results reveal a positive relationship between the level of floral deceptiveness and the 4 associated mating costs that deceived males suffer. Pollinator learning, however, appears to occur only on the most deceptive floral forms, suggesting a link between the costs suffered to the occurrence of learning. In Chapter 4, I systematically investigate learning abilities within male pollinators and the costs they suffer on sexually deceptive floral forms that vary in deceptiveness. Results reveal a positive relationship between the level of floral deceptiveness and the associated mating costs that deceived males suffer. Pollinator learning, however, appears to occur only on the most deceptive floral forms, suggesting a link between the costs suffered to the occurrence of learning. In Chapter 5, I explore the importance of florivory damage in a polymorphic daisy. Studies on floral evolution often overlook the significance of florivorous visits and focus only on pollinator-mediated selection. I show that floral polymorphism is maintained by antagonistic selection exerted by pollinators and florivores on the same floral traits. Lastly, I focus on evolutionary history to explore similarity in the patterns of South African angiosperm evolution and the pollinator species used throughout my thesis. Molecular dating shows this pollinator exhibits broadly congruent evolutionary patterns to these angiosperms, indicative of a shared biogeography. Taken together, my thesis demonstrates the vast impact of floral visitors, in particular pollinating insects, on the evolution of floral form.My research was funded by the National Research Foundation of South Africa (NRF) and personal funding was provided by a NRF Innovation scholarship and merit bursaries from the Botany and Zoology department at Stellenbosch University. A WhiteSci Travel Grant and financial support from Prof. Erik Svensson at Lund University also allowed me to present parts of my research at international conference

Metformin in severe exacerbations of chronic obstructive pulmonary disease: a randomised controlled trial

Background Severe exacerbations of COPD are commonly associated with hyperglycaemia, which predicts adverse outcomes. Metformin is a well-established anti-hyperglycaemic agent in diabetes mellitus, possibly augmented with anti-inflammatory effects, but its effects in COPD are unknown. We investigated accelerated metformin therapy in severe COPD exacerbations, primarily to confirm or refute an anti-hyperglycaemic effect, and secondarily to explore its effects on inflammation and clinical outcome. Methods This was a multicentre, randomised, double-blind, placebo-controlled trial testing accelerated metformin therapy in non-diabetic patients, aged ≥35 years, hospitalised for COPD exacerbations. Participants were assigned in a 2:1 ratio to 1 month of metformin therapy, escalated rapidly to 2 g/day, or matched placebo. The primary end point was mean in-hospital blood glucose concentration. Secondary end points included the concentrations of fructosamine and C reactive protein (CRP), and scores on the COPD Assessment Test and Exacerbations of Chronic Pulmonary Disease Tool. Results 52 participants (mean (±SD) age 67±9 years) were randomised (34 to metformin, 18 to placebo). All were included in the primary end point analysis. The mean blood glucose concentrations in the metformin and placebo groups were 7.1±0.9 and 8.0±3.3 mmol/L, respectively (difference −0.9 mmol/L, 95% CI −2.1 to +0.3; p=0.273). No significant between-group differences were observed on any of the secondary end points. Adverse reactions, particularly gastrointestinal effects, were more common in metformin-treated participants. Conclusion Metformin did not ameliorate elevations in blood glucose concentration among non-diabetic patients admitted to hospital for COPD exacerbations, and had no detectable effect on CRP or clinical outcomes. Trial registration number ISRCTN66148745 and NCT01247870

Genome-Wide Association Study and Gene Expression Analysis Identifies CD84 as a Predictor of Response to Etanercept Therapy in Rheumatoid Arthritis

Anti-tumor necrosis factor alpha (anti-TNF) biologic therapy is a widely used treatment for rheumatoid arthritis (RA). It is unknown why some RA patients fail to respond adequately to anti-TNF therapy, which limits the development of clinical biomarkers to predict response or new drugs to target refractory cases. To understand the biological basis of response to anti-TNF therapy, we conducted a genome-wide association study (GWAS) meta-analysis of more than 2 million common variants in 2,706 RA patients from 13 different collections. Patients were treated with one of three anti-TNF medications: etanercept (n = 733), infliximab (n = 894), or adalimumab (n = 1,071). We identified a SNP (rs6427528) at the 1q23 locus that was associated with change in disease activity score (ΔDAS) in the etanercept subset of patients (P = 8×10-8), but not in the infliximab or adalimumab subsets (P>0.05). The SNP is predicted to disrupt transcription factor binding site motifs in the 3′ UTR of an immune-related gene, CD84, and the allele associated with better response to etanercept was associated with higher CD84 gene expression in peripheral blood mononuclear cells (P = 1×10-11 in 228 non-RA patients and P = 0.004 in 132 RA patients). Consistent with the genetic findings, higher CD84 gene expression correlated with lower cross-sectional DAS (P = 0.02, n = 210) and showed a non-significant trend for better ΔDAS in a subset of RA patients with gene expression data (n = 31, etanercept-treated). A small, multi-ethnic replication showed a non-significant trend towards an association among etanercept-treated RA patients of Portuguese ancestry (n = 139, P = 0.4), but no association among patients of Japanese ancestry (n = 151, P = 0.8). Our study demonstrates that an allele associated with response to etanercept therapy is also associated with CD84 gene expression, and further that CD84 expression correlates with disease activity. These findings support a model in which CD84 genotypes and/or expression may serve as a useful biomarker for response to etanercept treatment in RA patients of European ancestry. © 2013 Cui et al

Quantitative analysis of WC stars: Constraints on neon abundances from ISO/SWS spectroscopy

Neon abundances are derived in four Galactic WC stars -- gamma Vel (WR11, WC8+O7.5III), HD156385 (WR90, WC7), HD192103 (WR135, WC8), and WR146 (WC5+O8) - using mid-infrared fine structure lines obtained with ISO/SWS. Stellar parameters for each star are derived using a non-LTE model atmospheric code (Hillier & Miller 1998) together with ultraviolet (IUE), optical (INT, AAT) and infrared (UKIRT, ISO) spectroscopy. In the case of gamma Vel, we adopt results from De Marco et al. (2000), who followed an identical approach. ISO/SWS datasets reveal the [NeIII] 15.5um line in each of our targets, while [NeII] 12.8um, [SIV] 10.5um and [SIII] 18.7um are observed solely in gamma Vel. Using a method updated from Barlow et al. (1988) to account for clumped winds, we derive Ne/He=3-4x10^-3 by number, plus S/He=6x10^-5 for gamma Vel. Neon is highly enriched, such that Ne/S in gamma Vel is eight times higher than cosmic values. However, observed Ne/He ratios are a factor of two times lower than predictions of current evolutionary models of massive stars. An imprecise mass-loss and distance were responsible for the much greater discrepancy in neon content identified by Barlow et al. Our sample of WC5--8 stars span a narrow range in T* (=55--71kK), with no trend towards higher temperature at earlier spectral type, supporting earlier results for a larger sample by Koesterke & Hamann (1995). Stellar luminosities range from 100,000 to 500,000 Lo, while 10^-5.1 < Mdot/(Mo/yr) < 10^-4.5, adopting clumped winds, in which volume filling factors are 10%. In all cases, wind performance numbers are less than 10, significantly lower than recent estimates. Carbon abundances span 0.08 < C/He < 0.25 by number, while oxygen abundances remain poorly constrained.Comment: 16 pages,7 figures accepted for MNRA

Integration of Sequence Data from a Consanguineous Family with Genetic Data from an Outbred Population Identifies PLB1 as a Candidate Rheumatoid Arthritis Risk Gene

Integrating genetic data from families with highly penetrant forms of disease together with genetic data from outbred populations represents a promising strategy to uncover the complete frequency spectrum of risk alleles for complex traits such as rheumatoid arthritis (RA). Here, we demonstrate that rare, low-frequency and common alleles at one gene locus, phospholipase B1 (PLB1), might contribute to risk of RA in a 4-generation consanguineous pedigree (Middle Eastern ancestry) and also in unrelated individuals from the general population (European ancestry). Through identity-by-descent (IBD) mapping and whole-exome sequencing, we identified a non-synonymous c.2263G>C (p.G755R) mutation at the PLB1 gene on 2q23, which significantly co-segregated with RA in family members with a dominant mode of inheritance (P = 0.009). We further evaluated PLB1 variants and risk of RA using a GWAS meta-analysis of 8,875 RA cases and 29,367 controls of European ancestry. We identified significant contributions of two independent non-coding variants near PLB1 with risk of RA (rs116018341 [MAF = 0.042] and rs116541814 [MAF = 0.021], combined P = 3.2×10−6). Finally, we performed deep exon sequencing of PLB1 in 1,088 RA cases and 1,088 controls (European ancestry), and identified suggestive dispersion of rare protein-coding variant frequencies between cases and controls (P = 0.049 for C-alpha test and P = 0.055 for SKAT). Together, these data suggest that PLB1 is a candidate risk gene for RA. Future studies to characterize the full spectrum of genetic risk in the PLB1 genetic locus are warranted

Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings

Data_Sheet_2_Quantitative proteomics of small numbers of closely-related cells: Selection of the optimal method for a clinical setting.zip

Supplementary Table S10. Comparative analysis of -omics platforms Supplementary Table S10. Comparative analysis of -omics platforms Supplementary Table S7.xlsx Supplementary Table S8.xlsx Supplementary Table S9.xlsxMass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations.Peer reviewe

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries