Flow cytometry: illuminating microbiology

By means of flow cytometry, a technique whereby a hydrodynamically directed stream of cells is passed through a focus of exciting light, one can measure cell size and the macromolecular content of individual bacteria. The sensitivity and versatility of the flow cytometer make it a powerful tool in studies of the bacterial cell cycle, in identifying and characterizing bacterial infections, and in selecting bacteria of desired qualities. We review some of these applications of flow cytometry and conclude that this method holds great promise in many other areas of microbiology.     

Synaptic physiology: illuminating the road ahead

A number of genetically encoded reporters of neuronal activity are being developed to assay synaptic activity with single synapse resolution. A recently engineered probe allows imaging of glutamatergic transmission with high sensitivity, and similar probes may help pave the way for optical imaging of excitatory synaptic function in vivo.     

Visual neuroscience: illuminating the dark corners

Recent experiments suggest that our perception of lightness involves a sophisticated interpretation of illumination and shadow. This finding challenges common notions about hierarchical processing and the neural basis of perception.     

Two course illuminating scheme improves aminolevulinic acid photodynamic therapy in cell cultures.

Photodynamic therapy with the pro-drug 5-aminolaevulinic acid (ALA-PDT) is being used for the treatment of Barrett's oesophagus. We postulated that a first early course of ALA-PDT would increase protoporphyrin IX (PPIX) accumulation and thus the efficacy of a second course of ALA-PDT, by manipulating ferrochelatase (FC) and porphobilinogen deaminase (PBG-d) activity. Human EBV-transformed lymphoblastoid cells were used as a model of human tumour cells for the ability to form haem is present in all cells. After a single course of illumination (633 nm, 100 mW/cm2) the FC activity decreased significantly whereas the PBG-d activity did not change. During continued incubation with ALA following the first illumination, cells accumulated up to four times more PPIX than non-illuminated controls [220% +/- 30% versus (vs) 55% +/- 5%; p<0.001]. Two illuminations resulted in more cell death than one illumination (97% +/- 1% vs 80% +/- 2%; p<0.001). Since a second course of ALA-PDT within 3 hr after the first course resulted in a four fold increase in PPIX accumulation and significantly more cell death, we propose that a two course ALA-PDT scheme might improve the efficacy of this treatment for Barrett's oesophagus.     

Synaptic endocytosis: illuminating the role of clathrin assembly

Acute photo-inactivation of clathrin in Drosophila synapses sheds new light on a 35-year-old debate over mechanisms of synaptic-vesicle endocytosis: clathrin is essential for reformation of functional synaptic vesicles, but not for bulk membrane internalization.     

Genetic methods for illuminating the function of neural circuits

Guided by the notion that biology itself offers some of the most incisive tools for studying biological systems, neurophysiologists rely increasingly on cell biological mechanisms and materials encoded in DNA to visualize and control the activity of neurons in functional circuits. Optical reporter proteins can broadcast the operational states of genetically designated cells and synapses; remote-controlled effectors can suppress or induce electrical activity. Many challenges, however, remain. These include the development of novel gene expression systems that target reporters and effectors to functionally relevant neuronal ensembles, the capacity to monitor and manipulate multiple populations of neurons in parallel, the ability to observe and elicit precisely timed action potentials, and the power to communicate with genetically designated target neurons through electromagnetic signals other than light.     

Bimolecular fluorescence complementation: illuminating cellular protein interactions

Many cellular processes depend on the establishment of selective stable or transient interactions between proteins. Therefore, the ability to identify and characterize these contacts in physiologically relevant environments is crucial to understanding the networks of contacts that allow the transmission and integration of biological information in living cells. Protein-fragment complementation assays (PCA) have emerged as approaches that report on the proximity of two given proteins in the cell at a given location and time. In particular, bimolecular fluorescence complementation (BIFC) allows noninvasive imaging of protein binding in living cells at high spatial resolution and without the requirement for exogenous substrates. In the present review, we discuss PCA and BIFC fundamentals, the implementation of BIFC assays and selected applications of BIFC in drug discovery, developmental studies or neurological disorders.     

FRET-based biosensors for protein kinases: illuminating the kinome

Protein kinases are crucial components of intracellular signaling pathways which transmit signals by phosphorylation of downstream targets, altering their function. Efficient signal transduction requires precise kinase regulation within specific biological contexts, making tools that allow study of their dynamics in situ critical for understanding kinase function. Highlighted in this article is the design of genetically-encodable, FRET-based kinase biosensors with examples of their implementation to study kinase regulation in live biological contexts with high spatial and temporal resolution.     

Marker and reporter genes: illuminating tools for environmental microbiologists

Fluorescent and luminescent marker and reporter genes provide easily detectable phenotypes to microbial cells and are therefore valuable tools for the study of microorganisms in the environment. Although these tools are becoming widely adopted, there are still issues that remain to be solved, such as the dependence of the reporter output on the physiological status of the cell. Eventually it might be the use of marker and reporter genes themselves that will contribute towards better understanding of the physiological status of specific microbial populations in nature.     

Effect of increased gas density on pulmonary gas exchange in man.

Pulmonary gas exchange was measured in seven resting supine subjects breathing air or a dense gas mixture containing 21% O2 in sulfur hexafluoride (SF6). The mean value of the alveolar-arterial oxygen difference (AaDO2) decreased from 12.4 on air to 7.0 on SF6 (P less than 0.01), and increased again to 13.4 when air breathing resumed (P less than 0.01). No differences occurred between gas mixtures for O2 consumption, respiratory quotient, minute ventilation, breathing frequency, heart rate, or blood pressure, and the improved oxygen transfer could not be attributed to changes in cardiac output or mixed venous oxygen content in the one subject in which they were measured. These results are best explained by an altered distribution of ventilation during dense gas breathing, so that the ventilation-perfusion ratio (VA/Q) variance was reduced. Of several considered mechanisms, we favor one in which SF6 promotes cardiogenic gas mixing between peripheral parallel units having different alveolar gas concentrations. This mechanism allows for observed increases in arterial carbon dioxide tension and dead space-to-tidal volume ratio during dense gas breathing, and suggests that intraregional VA/Q variance accounts for at least one-half of the resting AaDO2 in healthy supine young men.     

The dynamics of adaptation: an illuminating example and a Hamilton-Jacobi approach

Our starting point is a selection-mutation equation describing the adaptive dynamics of a quantitative trait under the influence of an ecological feedback loop. Based on the assumption of small (but frequent) mutations we employ asymptotic analysis to derive a Hamilton-Jacobi equation. Well-established and powerful numerical tools for solving the Hamilton-Jacobi equations then allow us to easily compute the evolution of the trait in a monomorphic population when this evolution is continuous but also when the trait exhibits a jump. By adapting the numerical method we can, at the expense of a significantly increased computing time, also capture the branching event in which a monomorphic population turns dimorphic and subsequently follow the evolution of the two traits in the dimorphic population. From the beginning we concentrate on a caricatural yet interesting model for competition for two resources. This provides the perhaps simplest example of branching and has the great advantage that it can be analyzed and understood in detail.