The biological clock: the bodyguard of temporal homeostasis

In order for any organism to function properly, it is crucial that it be table to control the timing of its biological functions. An internal biological clock, located, in mammals, in the suprachiasmatic nucleus of the hypothalamus (SCN), therefore carefully guards this temporal homeostasis by delivering its message of time throughout the body. In view of the large variety of body functions (behavioral, physiological, and endocrine) as well as the large variety in their preferred time of main activity along the light:dark cycle, it seems logical to envision different means of time distribution by the SCN. In the present review, we propose that even though it presents a unimodal circadian rhythm of general electrical and metabolic activity, the SCN seems to use several sorts of output connections that are active at different times along the light: dark cycle to control the rhythmic expression of different body functions. Although the SCN is suggested to use diffusion of synchronizing factors in the rhythmic control of behavioral functions, it also needs neuronal connections for the control of endocrine functions. The distribution of the time-of-day message to neuroendocrine systems is either directly onto endocrine neurons or via intermediate neurons located in specific SCN targets. In addition, the SCN uses its connections with the autonomic nervous system for spreading its time-of-day message, either by setting the sensitivity of endocrine glands (i.e., thyroid, adrenal, ovary) or by directly controlling an endocrine output (i.e., melatonin synthesis). Moreover, the SCN seems to use different neurotransmitters released at different times along the light: dark cycle for each of the different connection types presented. Clearly, the temporal homeostasis of endocrine functions results from a diverse set of biological clock outputs.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 μm)³. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 micron)3. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Transient phases of OXPHOS inhibitor resistance reveal underlying metabolic heterogeneity in single cells

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

Influence of molecular noise on the growth of single cells and bacterial populations

During the last decades experimental studies have revealed that single cells of a growing bacterial population are significantly exposed to molecular noise. Important sources for noise are low levels of metabolites and enzymes that cause significant statistical variations in the outcome of biochemical reactions. In this way molecular noise affects biological processes such as nutrient uptake, chemotactic tumbling behavior, or gene expression of genetically identical cells. These processes give rise to significant cell-to-cell variations of many directly observable quantities such as protein levels, cell sizes or individual doubling times. In this study we theoretically explore if there are evolutionary benefits of noise for a growing population of bacteria. We analyze different situations where noise is either suppressed or where it affects single cell behavior. We consider two specific examples that have been experimentally observed in wild-type Escherichia coli cells: (i) the precision of division site placement (at which molecular noise is highly suppressed) and (ii) the occurrence of noise-induced phenotypic variations in fluctuating environments. Surprisingly, our analysis reveals that in these specific situations both regulatory schemes [i.e. suppression of noise in example (i) and allowance of noise in example (ii)] do not lead to an increased growth rate of the population. Assuming that the observed regulatory schemes are indeed caused by the presence of noise our findings indicate that the evolutionary benefits of noise are more subtle than a simple growth advantage for a bacterial population in nutrient rich conditions.     

The relative biological effectiveness in V79 Chinese hamster cells of the neutron capture reactions in boron and nitrogen

V79 Chinese hamster cells were irradiated in the presence of different amounts of boric acid with thermal neutrons at the Medical Research Reactor at Brookhaven National Laboratory. From the linear dose-survival curves observed, a D0 value of 66 rad for the 10B(n, alpha) 7Li neutron capture reaction was obtained. No dependence of this value on the concentration of boric acid was found. Comparing this value to the D0 value of 150 rad obtained with 250 kVp X rays between 10 and 0.01% survival, an extrapolated RBE value of 2.3 was calculated. By irradiation of the same line of cells with cold neutrons at the Institut Laue - Langevin , a D0 value for the 14N(n,p)14C reaction of 77 rad was obtained, with a corresponding RBE value of 1.9. Comparison is made with previously published RBE values for the 10B(n, alpha) 7Li reaction.     

Chromatographic capture of cells to achieve single stage clarification in recombinant protein purification

Recent advancements in cell culture engineering have allowed drug manufacturers to achieve higher productivity by driving higher product titers through cell line engineering and high-cell densities. However, these advancements have shifted the burden to clarification and downstream processing where the difficulties now revolve around removing higher levels of process- and product-related impurities. As a result, a lot of research efforts have turned to developing new approaches and technologies or process optimization to still deliver high quality biological products while controlling cost of goods. Here, we explored the impact of a novel single use technology employing chromatographic principle-based clarification for a process-intensified cell line technology. In this study, a 16% economic benefit ($/g) was observed using a single-use chromatographic clarification compared to traditional single-use clarification technology by improving the overall product cost through decreased operational complexity, higher loading capacity, increased product recovery, and higher impurity clearance. In the end, the described novel chromatographic approach significantly simplified and enhanced the cell culture fluid harvest unit operation by combining the reduction of insoluble and key soluble contaminants of the harvest fluid into a single stage.     

Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na⁺]_in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H⁺) or sodium ions (Na⁺), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H⁺- and Na⁺-driven components that functions as a sodium motor. Changing external sodium concentration ([Na⁺]_ex) in the range 1–85 mM resulted in changes in [Na⁺]_in between 5–14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na⁺]_in and [Na⁺]_ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2–3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.     

Measurement of green fluorescent protein concentration in single cells by image analysis

The gene encoding the green fluorescent protein (GFP) has been widely used in studies of gene expression. The GFP can be detected nondestructively in living cells or tissues by the green fluorescence of the protein under blue light. Solutions of enhanced GFP (EGFP) of known concentration were filled in glass capillaries and used to calibrate a method for quantitative determination of EGFP or GFP-S65T in plant cells. Images captured by a digital camera were analyzed to determine the linear range for measurement of EGFP expression. The value of the method was illustrated by analysis of the relative levels of GFP expression under control of different promoters in aleurone cells of barley.     

The secret lives of single cells

Looking back fondly on the first 15 years of Microbial Biotechnology, a trend is emerging that biotechnology is moving from studies that focus on whole-cell populations, where heterogeneity exists even during robust growth, to those with an emphasis on single cells. This instils optimism that insights will be made into myriad aspects of bacterial growth in communities.