Combining multiple fluorescence imaging techniques in biology: when one microscope is not enough

While fluorescence microscopy has proven to be an exceedingly useful tool in bioscience, it is difficult to offer simultaneous high resolution, fast speed, large volume, and good biocompatibility in a single imaging technique. Thus, when determining the image data required to quantitatively test a complex biological hypothesis, it often becomes evident that multiple imaging techniques are necessary. Recent years have seen an explosion in development of novel fluorescence microscopy techniques, each of which features a unique suite of capabilities. In this Technical Perspective, we highlight recent studies to illustrate the benefits, and often the necessity, of combining multiple fluorescence microscopy modalities. We provide guidance in choosing optimal technique combinations to effectively address a biological question. Ultimately, we aim to promote a more well-rounded approach in designing fluorescence microscopy experiments, leading to more robust quantitative insight.     

Dealing with DNA lesions: When one cell cycle is not enough

Subversion of genome integrity fuels cellular adaptation and is a prerequisite for organismal evolution, yet genomic lesions are also the harmful driving force of cancer and other age-related human diseases. Genome integrity maintenance is inherently linked to genome organization and nuclear architecture, which are substantially remodeled during the cell cycle. Here we discuss recent findings on how actively dividing cells cope with endogenous genomic lesions that occur frequently at repetitive, heterochromatic, and late replicating regions as byproducts of genome duplication. We discuss how such lesions, rather than being resolved immediately when they occur, are dealt with in subsequent cell cycle phases, and even after mitotic cell division, and how this in turn affects genome organization, stability, and function.     

When counting cattle is not enough: multiple perspectives in agricultural and veterinary research

A traditional approach in agricultural and veterinary research is focussing on the biological perspective where large cattle-databases are used to analyse the dairy herd. This approach has yielded valuable insights. However, recent research indicates that this knowledge-base can be further increased by examining agricultural and veterinary challenges from other perspectives. In this paper we suggest three perspectives that may supplement the biological perspective in agricultural and veterinary research; the economic-, the managerial-, and the social perspective. We review recent studies applying or combining these perspectives and discuss how multiple perspectives may improve our understanding and ability to handle cattle-health challenges.     

Multi-locus species delimitation in closely related animals and fungi: one marker is not enough

Despite taxonomy’s 250‐year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence‐rich and time‐intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population‐level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (～70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi‐locus integrative approach to species delimitation and taxonomy.     

When sex is not enough: ecological correlates of resprouting capacity in congeneric tropical forest shrubs

In moist tropical forests resprouting may be an important component of life history, contributing to asexual reproduction through the clonal spread of individuals derived from shoot fragments. However, in contrast to other ecosystems where resprouting is common, the ecological correlates of resprouting capacity in tropical forests remain largely unexplored. In this study we characterized shade tolerance, resprouting capacity and sexual reproductive success of eight co-occurring Piper species from lowland forests of Panama. In field experiments we found that shade-tolerant Piper species had a higher capacity to regenerate from excised or pinned stem fragments than light-demanding species in both gap and understory light conditions. In contrast, shade-tolerant species had lower recruitment probabilities from seeds, as a consequence of lower initial seed viability, and lower seedling emergence rates. All Piper species needed gap conditions for successful seedling establishment. Of 8,000 seeds sown in the understory only 0.2% emerged. In gaps, seed germination of light-demanding species was between 10 and 50%, whereas for shade-tolerant species it was 0.5–9.8%. We propose that the capacity to reproduce asexually from resprouts could be adaptive for shade-tolerant species that are constantly exposed to damage from falling litter in the understory. Resprouting may allow Piper populations to persist and spread despite the high rate of pre-dispersal seed predation and low seed emergence rates. Across Piper species, we detected a trade-off between resprouting capacity and the annual viable seed production per plant but not with annual seed mass produced per plant. This suggests that species differences in sexual reproductive success may not necessarily result from differential resource allocation. Instead we suggest that low sexual reproductive success in the understory may in part reflect reduced genetic diversity in populations undergoing clonal growth, resulting in self-fertilization and in-breeding depression.     

When simplicity is not parsimonious: a priori and a posteriori methods in historical biogeography

Despite using the same null hypothesis, a priori and a posteriori approaches in historical biogeography differ fundamentally. Methods such as Component Analysis (CA) and Reconciled Tree Analysis (RTA) may eliminate or modify input data in order to maximize fit to the null hypothesis, by invoking assumptions 1 and 2. Methods such as Brooks Parsimony Analysis (BPA) modify the null hypothesis, if necessary, to maintain the integrity of the input data, as required by assumption 0. Two exemplars illustrate critical empirical differences between CA/RTA and BPA: (1) CA rather than BPA may select the incorrect general area cladogram for a set of data (2) BPA, not RTA, provides the most parsimonious interpretation of all available data and (3) secondary BPA, proposed in 1990, applied to data sets for which dispersal producing areas with reticulate histories is most parsimonious, provides biologically realistic interpretations of area cladograms. These observations lead to the conclusion that BPA and CA/RTA are designed to implement different research programmes based on different conceptual frameworks. BPA is designed to assess the biogeographic context of speciation events, whereas CA/RTA are designed to find the best fitting pattern of relationships among areas based on the taxa that inhabit them. Unique distributional elements and reticulate (hybrid) histories of areas are essential for explaining complex histories of speciation. The conceptual framework for BPA, thus, assumes biogeographical complexity, relying on parsimony as an explanatory tool to summarize complex results, whereas CA/RTA assumes biogeographical simplicity, assuming conceptual parsimony a priori .     

When one tail isn't enough: abnormal caudal regeneration in lepidosaurs and its potential ecological impacts

Abnormal caudal regeneration, the production of additional tails through regeneration events, occurs in lepidosaurs as a result of incomplete autotomy or sufficient caudal wound. Despite being widely known to occur, documented events generally are limited to opportunistic single observations – hindering the understanding of the ecological importance of caudal regeneration. Here we compiled and reviewed a robust global database of both peer‐reviewed and non‐peer reviewed records of abnormal regeneration events in lepidosaurs published over the last 400 years. Using this database, we qualitatively and quantitatively assessed the occurrence and characteristics of abnormal tail regeneration among individuals, among species, and among populations. We identified 425 observations from 366 records pertaining to 175 species of lepidosaurs across 22 families from 63 different countries. At an individual level, regenerations ranged from bifurcations to hexafurcations; from normal regeneration from the original tail to multiple regenerations arising from a single point; and from growth from the distal third to the proximal third of the tail. Species showing abnormal regenerations included those with intra‐vertebral, inter‐vertebral or no autotomy planes, indicating that abnormal regenerations evidently occur across lepidosaurs regardless of whether the species demonstrates caudal autotomy or not. Within populations, abnormal regenerations were estimated at a mean ± SD of 2.75 ± 3.41% (range 0.1–16.7%). There is a significant lack of experimental studies to understand the potential ecological impacts of regeneration on the fitness and life history of individuals and populations. We hypothesised that abnormal regeneration may affect lepidosaurs via influencing kinematics of locomotion, restrictions in escape mechanisms, anti‐predation tactics, and intra‐ and inter‐specific signalling. Behaviourally testing these hypotheses would be an important future research direction.     

The markup is the model: reasoning about systems biology models in the Semantic Web era

Metabolic control analysis, co-invented by Reinhart Heinrich, is a formalism for the analysis of biochemical networks, and is a highly important intellectual forerunner of modern systems biology. Exchanging ideas and exchanging models are part of the international activities of science and scientists, and the Systems Biology Markup Language (SBML) allows one to perform the latter with great facility. Encoding such models in SBML allows their distributed analysis using loosely coupled workflows, and with the advent of the Internet the various software modules that one might use to analyze biochemical models can reside on entirely different computers and even on different continents. Optimization is at the core of many scientific and biotechnological activities, and Reinhart made many major contributions in this area, stimulating our own activities in the use of the methods of evolutionary computing for optimization.     

Effects of malaria double infection in birds: one plus one is not two

Avian malaria parasites are supposed to exert negative effects on host fitness because these intracellular parasites affect host metabolism. Recent advances in molecular genotyping and microscopy have revealed that coinfections with multiple parasites are frequent in bird-malaria parasite systems. However, studies of the fitness consequences of such double infections are scarce and inconclusive. We tested if the infection with two malaria parasite lineages has more negative effects than single infection using 6 years of data from a natural population of house martins. Survival was negatively affected by both types of infections. We found an additive cost from single to double infection in body condition, but not in reproductive parameters (double-infected had higher reproductive success). These results demonstrate that malaria infections decrease survival, but also have different consequences on the breeding performance of single- and double-infected wild birds.     

The value of applying molecular biology tools in environmental engineering: Academic and industry perspective in the USA

The integration of molecular biology tools in environmental engineering is a challenge. We discuss our views on the following four critical issues: (i) faculty career development, (ii) tool standardization, (iii) teaching, and (iv) the application of molecular biology tools in practice. For (i), we suggest that administrators and faculty need to understand the special challenges inherent to research and teaching within this highly interdisciplinary area. Furthermore, we suggest preparing two white papers aimed at educating administrators in universities and agencies. For (ii), we conclude that, because molecular biology tools are still in a state of rapid development, proposing standards at this time is premature. In the future, standards for widely applied tools should be in an on-line, peer-reviewed format. Concerning (iii), we believe that molecular biology should be taught only to the degree needed to achieve program goals. For example, environmental engineering practitioners only need to know the vocabulary and basic concepts of molecular biology tools, not be experts at doing them hands on. To help engineering students gain the right level and type of information, learning modules should be developed for them. Finally, although engineering successes applying molecular biology tools are available (iv), the biggest value will come when the tools are fully integrated with practice. Therefore, we encourage the creation of a demonstration project to document the value of applying molecular biology tools in environmental engineering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology

Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor–matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor–ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis.     

Using phase resetting to predict 1:1 and 2:2 locking in two neuron networks in which firing order is not always preserved

Our goal is to understand how nearly synchronous modes arise in heterogenous networks of neurons. In heterogenous networks, instead of exact synchrony, nearly synchronous modes arise, which include both 1:1 and 2:2 phase-locked modes. Existence and stability criteria for 2:2 phase-locked modes in reciprocally coupled two neuron circuits were derived based on the open loop phase resetting curve (PRC) without the assumption of weak coupling. The PRC for each component neuron was generated using the change in synaptic conductance produced by a presynaptic action potential as the perturbation. Separate derivations were required for modes in which the firing order is preserved and for those in which it alternates. Networks composed of two model neurons coupled by reciprocal inhibition were examined to test the predictions. The parameter regimes in which both types of nearly synchronous modes are exhibited were accurately predicted both qualitatively and quantitatively provided that the synaptic time constant is short with respect to the period and that the effect of second order resetting is considered. In contrast, PRC methods based on weak coupling could not predict 2:2 modes and did not predict the 1:1 modes with the level of accuracy achieved by the strong coupling methods. The strong coupling prediction methods provide insight into what manipulations promote near-synchrony in a two neuron network and may also have predictive value for larger networks, which can also manifest changes in firing order. We also identify a novel route by which synchrony is lost in mildly heterogenous networks.     

When partitioning is not an option: Resource availability predicts intraguild interactions in two isolated Amazonian primate assemblages

Assemblage structure and acquisition of high-value resources will usually be affected by changes in resource availability and differential competitive abilities of assemblage members. In fragmented habitats where carrying capacity limits are exceeded due to high population densities and biomass, interspecific interactions can be expected to occur at a high frequency, potentially turning into an important cost for coexistence. We studied assemblage- and guild-level patterns of interspecific interactions in two highly diverse isolated primate assemblages in southern Amazonia. Specifically, we assessed the effects of temporal variation in fruit availability on the rates of interspecific interactions between gray woolly monkeys (Lagothrix lagotricha cana), one of the largest tree-dwelling mammals of the Amazon forests, and nine syntopic primate species. We found that fruit availability positively predicted rate of intraguild interactions in contrast to overall assemblage interaction rate. We did not find statistical evidence for the effect of fruit availability on the assemblage rates of type-dependent (i.e., agonistic or non-agonistic) and context-dependent (i.e., feeding or non-feeding) interactions. However, there was a clear trend toward increased feeding-related and agonistic-related interactions as fruit availability increased, with agonistic interactions mostly occurring at guild-level. These results provide support for a significant role of fruit availability in structuring spatial–temporal intraguild interactions at our study sites. Costly interspecific interactions and spatial habitat overlap can be expected to occur at a high frequency in highly diverse assemblages living in human-modified landscapes, which in turn, can have potentially negative impacts to the species involved.     

Chondrocyte apoptosis is not essential for cartilage calcification: evidence from an in vitro avian model

The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte apoptosis is not a pre-requisite for calcification in this culture system.     

Hypoxic preconditioning-induced mitochondrial protection is not disrupted in a cell model of mtDNA T8993G mutation-induced F1F0-ATP synthase defect: the role of mitochondrial permeability transition

Transient opening of the mitochondrial permeability transition pore plays a crucial role in hypoxic preconditioning-induced protection. Recently, the cyclophilin-D component of the mitochondrial permeability transition pore has been shown to interact with and regulate the F1F0-ATP synthase. However, the precise role of the F1F0-ATP synthase and the interaction between cyclophilin-D and F1F0-ATP synthase in the mitochondrial permeability transition pore and hypoxic preconditioning remain uncertain. Here we found that a 1-h hypoxic preconditioning delayed apoptosis and improved cell survival after stimulation with various apoptotic inducers including H₂O₂, ionomycin, and arachidonic acid in mitochondrial DNA T8993G mutation (NARP) osteosarcoma 143B cybrids, an F1F0-ATP synthase defect cell model. This hypoxic preconditioning protected NARP cybrid cells against focal laser irradiation-induced oxidative stress by suppressing reactive oxygen species formation and preventing the depletion of cardiolipin. Furthermore, the protective functions of transient opening of the mitochondrial permeability transition pore in both NARP cybrids and wild-type 143B cells can be augmented by hypoxic preconditioning. Disruption of the interaction between cyclophilin-D and F1F0-ATP synthase by cyclosporin A attenuated the mitochondrial protection induced by hypoxic preconditioning in both NARP cybrids and wild-type 143B cells. Our results demonstrate that the interaction between cyclophilin-D and F1F0-ATP synthase is important in the hypoxic preconditioning-induced cell protection. This finding improves our understanding of the mechanism of mitochondrial permeability transition pore opening in cells in response to hypoxic preconditioning, and will be helpful in further developing new pharmacological agents targeting hypoxia–reoxygenation injury and mitochondria-mediated cell death