A Transcriptomic Analysis of Higher-Order Ecological Interactions in a Eukaryotic Model Microbial Ecosystem.

Nonlinear ecological interactions within microbial ecosystems and their contribution to ecosystem functioning remain largely unexplored. Higher-order interactions, or interactions in systems comprised of more than two members that cannot be explained by cumulative pairwise interactions, are particularly understudied, especially in eukaryotic microorganisms. The wine fermentation ecosystem presents an ideal model to study yeast ecosystem establishment and functioning. Some pairwise ecological interactions between wine yeast species have been characterized, but very little is known about how more complex, multispecies systems function. Here, we evaluated nonlinear ecosystem properties by determining the transcriptomic response of Saccharomyces cerevisiae to pairwise versus tri-species culture. The transcriptome revealed that genes expressed during pairwise coculture were enriched in the tri-species data set but also that just under half of the data set comprised unique genes attributed to a higher-order response. Through interactive protein-association network visualizations, a holistic cell-wide view of the gene expression data was generated, which highlighted known stress response and metabolic adaptation mechanisms which were specifically activated during tri-species growth. Further, extracellular metabolite data corroborated that the observed differences were a result of a biotic stress response. This provides exciting new evidence showing the presence of higher-order interactions within a model microbial ecosystem. IMPORTANCE Higher-order interactions are one of the major blind spots in our understanding of microbial ecosystems. These systems remain largely unpredictable and are characterized by nonlinear dynamics, in particular when the system is comprised of more than two entities. By evaluating the transcriptomic response of S. cerevisiae to an increase in culture complexity from a single species to two- and three-species systems, we were able to confirm the presence of a unique response in the more complex setting that could not be explained by the responses observed at the pairwise level. This is the first data set that provides molecular targets for further analysis to explain unpredictable ecosystem dynamics in yeast.

The ecology of wine fermentation: a model for the study of complex microbial ecosystems.

The general interest in microbial ecology has skyrocketed over the past decade, driven by technical advances and by the rapidly increasing appreciation of the fundamental services that these ecosystems provide. In biotechnology, ecosystems have many more functionalities than single species, and, if properly understood and harnessed, will be able to deliver better outcomes for almost all imaginable applications. However, the complexity of microbial ecosystems and of the interactions between species has limited their applicability. In research, next generation sequencing allows accurate mapping of the microbiomes that characterise ecosystems of biotechnological and/or medical relevance. But the gap between mapping and understanding, to be filled by "functional microbiomics", requires the collection and integration of many different layers of complex data sets, from molecular multi-omics to spatial imaging technologies to online ecosystem monitoring tools. Holistically, studying the complexity of most microbial ecosystems, consisting of hundreds of species in specific spatial arrangements, is beyond our current technical capabilities, and simpler model systems with fewer species and reduced spatial complexity are required to establish the fundamental rules of ecosystem functioning. One such ecosystem, the ecosystem responsible for natural alcoholic fermentation, can provide an excellent tool to study evolutionarily relevant interactions between multiple species within a relatively easily controlled environment. This review will critically evaluate the approaches that are currently implemented to dissect the cellular and molecular networks that govern this ecosystem. KEY POINTS: • Evolutionarily isolated fermentation ecosystem can be used as an ecological model. • Experimental toolbox is gearing towards mechanistic understanding of this ecosystem. • Integration of multidisciplinary datasets is key to predictive understanding.

Combinatorial analysis of population dynamics, metabolite levels and malolactic fermentation in Saccharomyces cerevisiae/ Lachancea thermotolerans mixed fermentations.

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.

Evolution of mutualistic behaviour between Chlorella sorokiniana and Saccharomyces cerevisiae within a synthetic environment.

Yeast and microalgae are microorganisms with widely diverging physiological and biotechnological properties. Accordingly, their fields of applications diverge: yeasts are primarily applied in processes related to fermentation, while microalgae are used for the production of high-value metabolites and green technologies such as carbon capture. Heterotrophic-autotrophic systems and synthetic ecology approaches have been proposed as tools to achieve stable combinations of such evolutionarily unrelated species. We describe an entirely novel synthetic ecology-based approach to evolve co-operative behaviour between winery wastewater isolates of the yeast Saccharomyces cerevisiae and microalga Chlorella sorokiniana. The data show that biomass production and mutualistic growth improved when co-evolved yeast and microalgae strains were paired together. Combinations of co-evolved strains displayed a range of phenotypes, including differences in amino acid profiles. Taken together, the results demonstrate that biotic selection pressures can lead to improved mutualistic growth phenotypes over relatively short time periods.

Real-time monitoring of population dynamics and physical interactions in a synthetic yeast ecosystem by use of multicolour flow cytometry.

Ecological interactions between different species of yeasts have been observed and described extensively, but the mechanisms of interaction remain poorly understood. A hindrance to the characterization of multispecies yeast ecosystems is the lack of accurate methods for rapid real-time analysis of population dynamics in synthetic multispecies consortia. Here, we sought to accelerate and improve the sensitivity of ecological modelling and characterization of a synthetic yeast ecosystem by developing a flow cytometry-based method that tracks and sorts fluorescently tagged individual yeast species in real time during growth in model multispecies consortia. A protocol for integrative genetic modification of non-conventional yeasts was developed. The application of the method was demonstrated in a model four-species synthetic wine-yeast ecosystem that consisted of species commonly isolated from natural wine fermentations. The data show that this method allows for rapid generation of meaningful ecological data that contributes to our understanding of multispecies synthetic yeast ecosystems. Furthermore, interspecies interactions have been shown to impact the evolution of yeasts in natural ecosystems, and this platform will provide an ideal tool to better evaluate the impact of biotic selection pressures.Key Points• Fluorescent labelling of yeast species in a consortium for multicolour flow cytometry• Method developed to track population dynamics of multispecies yeast consortia• Enables real-time visualization, manipulation and response analyses of population dynamics• Produces accurate, reproducible data with powerful visual analyses potential at a rapid rate.

Peer pressure: evolutionary responses to biotic pressures in wine yeasts.

In the macroscopic world, ecological interactions between multiple species of fauna and flora are recognised as major role-players in the evolution of any particular species. By comparison, research on ecological interactions as a driver of evolutionary adaptation in microbial ecosystems has been neglected. The evolutionary history of the budding yeast Saccharomyces cerevisiae has been extensively researched, providing an unmatched foundation for exploring adaptive evolution of microorganisms. However, in most studies, the habitat is only defined by physical and chemical parameters, and little attention is paid to the impact of cohabiting species. Such ecological interactions arguably provide a more relevant evolutionary framework. Within the genomic phylogenetic tree of S. cerevisiae strains, wine associated isolates form a distinct clade, also matched by phenotypic evidence. This domestication signature in genomes and phenomes suggests that the wine fermentation environment is of significant evolutionary relevance. Data also show that the microbiological composition of wine fermentation ecosystems is dominated by the same species globally, suggesting that these species have co-evolved within this ecosystem. This system therefore presents an excellent model for investigating the origins and mechanisms of interspecific yeast interactions. This review explores the role of biotic stress in the adaptive evolution of wine yeast.

Adjustment of trehalose metabolism in wine Saccharomyces cerevisiae strains to modify ethanol yields.

The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines.

Multipolar plasmonic resonances in silver nanowire antennas imaged with a subnanometer electron probe.

We detect short-range surface plasmon-polariton (SR-SPP) resonances setup in individual silver nanoantenna structures at high-spatial resolution with a scanning, subnanometer electron probe. Both even and odd multipolar resonant modes are resolved up to sixth order, and we measure their spatial distribution in relation to nanoantenna structures at energies down to 0.55 eV. Fabry-Perot type SR-SPP reflection phase shifts are calculated from direct measurements of antinode spacings in high-resolution plasmonic field maps. We observe resonant SR-SPP antinode bunching at nanoantenna terminals in high-order resonant modes, and antinode shifts in nonhomogeneous local environments. Finally, we achieve good agreement of our experimental SR-SPP maps with numerical calculations of photon excited near fields, using a novel integrated photon excitation geometry.

Radiosynthesis and in vitro stability evaluation of various radioiodine-labelled beta-iodoalkylether prosthetic groups linked to model compounds.

A systematic comparative investigation into the in vitro radiochemical stabilities of model compounds containing radioiodinated beta-iodoethoxyl units and derivatives thereof, as well as those of similar compounds lacking a beta-oxygen to serve as control references, was undertaken. The radioiodinations were carried out in fair to modest yields by means of substitution of a tosyl group by iodide. Stability evaluations were carried out by incubating the labeled compounds in human blood serum at 37 degrees C and measuring free radioiodide by means of radio-HPLC and radio-TLC. The compounds containing beta-iodoethoxyl units displayed much superior stabilities than those without, while the presence of small alkyl or aryl groups in such a unit rendered an additional degree of stability to the carbon-iodine bond, especially over a long period.

Posterior dislocation of the hip while playing basketball.

Injuries in basketball are usually to the ankles and knees. Dislocation of the hip is usually associated with severe trauma--for example, road traffic accidents. A case is reported here in which a 22 year old club basketball player slipped on landing from a jump shot, forcing him into a side splits position from which he sustained a posterior dislocation of the hip resulting in a sciatic nerve palsy.

A biomechanical evaluation of suture anchors in repair of the rotator cuff.

Repair of the rotator cuff requires secure reattachment, but large chronic defects cause osteoporosis of the greater tuberosity which may then have insufficient strength to allow proper fixation of the tendon. Recently, suture anchors have been introduced, but have not been fully evaluated. We have investigated the strength of suture-to-anchor attachment, and the use of suture anchors in repairs of the rotator cuff either to the greater tuberosity or the lateral cortex of the humerus. The second method gave a significant increase in the strength of the repair (p = 0.014). The repairs were loaded cyclically and failed at low loads by cutting into bone and tendon, casting doubt on the integrity of the repair in early mobilisation after surgery. Repairs with suture anchors did not perform better than those with conventional transosseous attachment.

The detection of blood contamination in human follicular fluid.

PURPOSE: The aim of this study was to compare the reliability of the methods conventionally used to identify low levels of blood contamination in human follicular fluid (hFF) as applicable in the clinical environment. METHODS: Follicular fluid (n = 339) and plasma samples (n = 20) were collected from patients (n = 138) attending the Centre for Fertility Studies, HF Verwoerd Hospital, University of Pretoria, South Africa. hFF blood contamination was assessed by means of (a) visual inspection, (b) hematocrit (Hct), (c) spectrophotometric analysis, (d) spectrophotometric hemoglobin kit, and (e) Combur-9-test urine sticks. RESULTS: (1) Neither hematocrit nor spectrophotometry provided reliable detection at low levels of blood contamination. (2) Visual inspection presented with a better discriminatory ability than either Hct or spectrophotometry. (3) Combur-9-test sticks identified up to 50% of blood-contaminated fluids. (4) Spectrophotometrically determined hemoglobin levels presented with weak discriminatory abilities for detecting blood-contaminated fluids. CONCLUSIONS: Visual inspection as performed in this study provides a fast and relatively reliable method for the determination of blood-contaminated hFFs. In a laboratory environment, however, it would be recommended that a combination of visual inspection, Hct, and spectrophotometric evaluation be employed for the selection of blood-free fluids.

Papulonecrotic tuberculid in children. A report of eight patients.

Papulonecrotic tuberculid (PNT), a form of cutaneous tuberculosis (TB), is uncommon in children. We identified eight children (six girls and two boys) with PNT. Their ages ranged from 19 to 139 months (median 47.5 months, mean:64.75 months). Skin lesions had been present for 2-24 weeks (median: 4 weeks) before diagnosis. All patients displayed scattered papulo- and/or pustulonecrotic lesions on the limbs, and the ears were involved in six patients. Lesions healed with varioliform scars. Associated pulmonary TB was present in seven patients. Additional clinical findings included fever (n = 4), hepatomegaly (n = 4), lymphadenopathy (n = 3), phlyctenular conjunctivitis (n = 3), and splenomegaly (n = 2). Histology of eight biopsies showed ulceration (n = 6), dermal necrosis (n = 6) (follicle-centered in two), granulomatous inflammation (n = 6) (palisading granuloma-like in three), superficial and deep infiltrate of lymphocytes (n = 7), erythrocyte extravasation (n = 7), and subepidermal edema (n = 3). Vasculitis was not a feature. A Ziehl-Neelsen stain was negative in all. Glycosaminoglycans were not increased. Immunohistochemistry found a predominance of T lymphocytes, macrophages, a few antigen-presenting cells, and no B lymphocytes, consistent with a type IV hypersensitivity reaction. Polymerase chain reaction (PCR) performed on deparaffinized tissue identified M. tuberculosis DNA in one biopsy. All patients received combination anti-TB treatment for 6 months. Six patients were compliant and were followed up for 6-30 months. Skin lesions and pulmonary TB healed in all. PNT in children resembles the adult form, but phlyctenular conjunctivitis and associated TB are more common, scrofuloderma and concomitant erythema induratum of Bazin are unusual, and vasculitis is not found. In cases where M. tuberculosis DNA can be confirmed with PCR, papulonecrotic TB is perhaps the more appropriate nomenclature.

Semiquantitative postembedding characterization of intermediate filaments in central nervous system lesions using immunoelectron microscopy.

Standardized postembedding immunoelectron microscopy was performed to demonstrate glial fibrillary acidic protein (GFAP) and vimentin in individual intermediate filaments to determine the diagnostic value of demonstrating ultrastructural and immunophenotypic characteristics of intermediate filaments in routine brain biopsy specimens. Dual expression of GFAP and vimentin was observed in the astroblastoma and astrocytes of Alexander's disease. The antigen availability for vimentin, however, was too low to allow reliable assessment of the GFAP:vimentin ratio in individual intermediate filaments and/or filament bundles. In meningioma, only vimentin positive intermediate filaments were found. GFAP positive intermediate filaments were present in all other specimens except the oligodendroglial components of the mixed glioma, which were devoid of intermediate filaments. GFAP positivity in the filamentous periphery and electron-dense core of Rosenthal fibers was demonstrated. Technical and tissue processing factors had a significant effect on particle density values obtained for individual specimens. Although the number, distribution, and density of glial intermediate filaments varies in different astroglial entities, correlation of particle density values determined by immunoelectron microscopy with relative GFAP concentrations in different lesions requires utmost caution. Nevertheless, application of the postembedding approach to routinely fixed biopsy specimens indicated an association of different entities with the exclusive presence of GFAP and/or vimentin in individual intermediate filaments, thus emphasizing the diagnostic value of intermediate filament typing for pathological characterization.

Planar myocardial imaging in the baboon model with iodine-123-15-(iodophenyl)pentadecanoic acid (IPPA) and iodine-123-15-(P-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP), using time-activity curves for evaluation of metabolism.

The purpose of this study was to compare by planar myocardial scintigraphy the kinetics of iodine-123-15-(iodophenyl)pentadecanoic acid (123I-pPPA and 123I-oPPA), and of iodine-123-(p-iodophenyl)-3-R,S-methyl-pentadecanoic acid (BMIPP), firstly in normal baboons, and subsequently after blocking fatty acid oxidation by a carnitine palmitoyl transferase I(CPT1) inhibitor. The induced changes in myocardial metabolism were reflected in the dynamic behaviour of the three tracers. pPPA and oPPA to a large extent, provided information on beta-oxidation changes in the myocardium: beta-oxidation participation changed from 47% and 50%, respectively to 17% and 23% after inhibition. BMIPP provided better images and reflected largely on changed tracer incorporation into the neutral lipid pools. The beta-oxidation contributed only about 10% towards the metabolic pathway of BMIPP. The information obtained in this study could help determine the tracer of choice for SPECT, whereby myocardial viability could optimally be revealed.

Erythema induratum of Bazin. A clinicopathological study of 20 cases and detection of Mycobacterium tuberculosis DNA in skin lesions by polymerase chain reaction.

Erythema induratum of Bazin (EIB) is a chronic, recurring panniculitis that is found predominantly on the legs of women with tuberculin hypersensitivity. A causal relationship between EIB and Mycobacterium tuberculosis remains elusive because of the absence of demonstrable organisms in skin lesions. We reviewed the clinicopathological features of 20 patients (all women) with positive Mantoux tests (1:10,000 dilution) and characteristic skin lesions of EIB that cleared up with combined antituberculous treatment. Histological examination of skin lesions confirmed panniculitis with varying combinations of granulomatous inflammation, primary vasculitis, and necrosis. Sections from 20 formalin-fixed, paraffin-embedded skin biopsies obtained from the 20 patients were submitted for polymerase chain reaction (PCR) using oligonucleotide primers for the detection of a 123-bp DNA fragment specific for the M. tuberculosis complex. M. tuberculosis DNA was identified in five of the 20 biopsies. Meticulous care was taken to prevent contamination as a source of false-positive results. Mycobacterial DNA was absent in all negative controls and in normal skin biopsies from purified protein derivative-positive patients with and without EIB. These results provide direct evidence that mycobacterial components are present in EIB lesions and strongly suggest that M. tuberculosis is involved in the pathogenesis of EIB.

A comparative cerebral blood flow study in a baboon model with acetazolamide provocation: 99mTc-HMPAO vs 123I(IMP).

Pharmacological interactions are important when nuclear medical procedures are applied to patients under drug therapy, or drug provocation. This study compares in baboon models (regional) cerebral blood flow [(r)CBF] results from 99mTc-HMPAO and 123I-iodoamphetamine [123I(IMP)] each with and without acetazolamide, the latter a suggested drug for testing cerebrovascular reserve. Expected differences in cerebral uptake were observed between the two radio-tracers without acetazolamide. The increase in tracer uptake resulting from acetazolamide is significantly enhanced for 123I(IMP), which could have diagnostic implications.