Single cell biology beyond the era of antibodies: relevance,challenges, and promises in biomedical research

Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.     

Multiple time-series modelling: Another look at the canadian money and income data

The practice of modelling the components of a vector time series to arrive at a joint model for the vector is considered. It is shown that in some cases this is not unreasonable. A vector ARMA model is used to model the Canadian money and income data. We also use these data to discuss the issue of differencing a multiple time series. Finally, models based on first and second differences are compared using forecasts.     

Magnification of light from many distant quasars by gravitational lenses

Exceptionally bright quasars with redshifts up to z = 6.28 have recently been discovered. Quasars are thought to be powered by the accretion of gas onto supermassive black holes at the centres of galaxies. Their maximum (Eddington) luminosity depends on the mass of the black hole, and the brighter quasars are inferred to have black holes with masses of more than a few billion solar masses. The existence of such massive black holes poses a challenge to models for the formation of structures in the early Universe, as it requires their formation within one billion years of the Big Bang. Here we show that up to one-third of known quasars with z approximately equal to 6 will have had their observed flux magnified by a factor of ten or more, as a consequence of gravitational lensing by galaxies along the line of sight. The inferred abundance of quasar host galaxies, as well as the luminosity density provided by the quasars, has therefore been substantially overestimated.     

Spectral signature of cosmological infall of gas around the first quasars

Recent observations have shown that, only a billion years after the Big Bang, the Universe was already lit up by bright quasars fuelled by the infall of gas onto supermassive black holes. The masses of these early black holes are inferred from their luminosities to be >10(9) solar masses (M(O)), which is a difficult theoretical challenge to explain. Like nearby quasars, the early objects could have formed in the central cores of massive host galaxies. The formation of these hosts could be explained if, like local large galaxies, they were assembled gravitationally inside massive (> 10(12) M(O)) haloes of dark matter. There has hitherto been no observational evidence for the presence of these massive hosts or their surrounding haloes. Here we show that the cosmic gas surrounding each halo must respond to its strong gravitational pull, where absorption by the infalling hydrogen produces a distinct spectral signature. That signature can be seen in recent data.     

The formation of the first low-mass stars from gas with low carbon and oxygen abundances

The first stars in the Universe are predicted to have been much more massive than the Sun. Gravitational condensation, accompanied by cooling of the primordial gas via molecular hydrogen, yields a minimum fragmentation scale of a few hundred solar masses. Numerical simulations indicate that once a gas clump acquires this mass it undergoes a slow, quasi-hydrostatic contraction without further fragmentation; lower-mass stars cannot form. Here we show that as soon as the primordial gas--left over from the Big Bang--is enriched by elements ejected from supernovae to a carbon or oxygen abundance as small as approximately 0.01-0.1 per cent of that found in the Sun, cooling by singly ionized carbon or neutral oxygen can lead to the formation of low-mass stars by allowing cloud fragmentation to smaller clumps. This mechanism naturally accommodates the recent discovery of solar-mass stars with unusually low iron abundances (10(-5.3) solar) but with relatively high (10(-1.3) solar) carbon abundance. The critical abundances that we derive can be used to identify those metal-poor stars in our Galaxy with elemental patterns imprinted by the first supernovae. We also find that the minimum stellar mass at early epochs is partially regulated by the temperature of the cosmic microwave background.     

Importance of stirring in the development of an iron-fertilized phytoplankton bloom

The growth of populations is known to be influenced by dispersal, which has often been described as purely diffusive. In the open ocean, however, the tendrils and filaments of phytoplankton populations provide evidence for dispersal by stirring. Despite the apparent importance of horizontal stirring for plankton ecology, this process remains poorly characterized. Here we investigate the development of a discrete phytoplankton bloom, which was initiated by the iron fertilization of a patch of water (7 km in diameter) in the Southern Ocean. Satellite images show a striking, 150-km-long bloom near the experimental site, six weeks after the initial fertilization. We argue that the ribbon-like bloom was produced from the fertilized patch through stirring, growth and diffusion, and we derive an estimate of the stirring rate. In this case, stirring acts as an important control on bloom development, mixing phytoplankton and iron out of the patch, but also entraining silicate. This may have prevented the onset of silicate limitation, and so allowed the bloom to continue for as long as there was sufficient iron. Stirring in the ocean is likely to be variable, so blooms that are initially similar may develop very differently.     

Light stimulated 'shunt-metabolism' succinate-alpha-ketoglutarate-isocitrate cycle and accumulation of citric acid in Aspergillus niger.

Studies with light of the visible range had shown that light plays significant role in the biosynthesis and accumulation of citric acid in Aspergillus niger. Accumulation of 14C-labelled carbon atoms in alpha-ketoglutaric, isocitric, succinic and glycolic acids in the cultures grown under illumination suggest a probable 'shunt-metabolism' leading to the succinate-alpha-ketoglutarate-isocitrate (SKI) cycle. This shunt metabolism minimizes the accumulation of citric acid in cultures due to depletion of intermediates.     

Antibodies to paired helical filaments in Alzheimer's disease do not recognize normal brain proteins

During ageing of the human brain, and particularly in senile dementia of the Alzheimer type (AD), many neurones progressively accumulate abnormal cytoplasmic fibres, called paired helical filaments (PHF). Each such fibre consists of a pair of intermediate (10 nm) filaments twisted into a double helix with a periodicity of 160 nm. PHF accumulate in large perikaryal masses, called neurofibrillary tangles, and are also found in degenerating cortical neurites that form neurite plaques. The density of PHF-containing neurites and cell bodies correlates with the degree of dementia and the extent of loss of cholinergic neurotransmitter function in AD. Recently, we demonstrated that PHF from human cerebral cortex are large, rigid polymers with unusual molecular properties, including insolubility in SDS, urea and other denaturing solvents and apparent resistance to protease digestion. These properties have so far prevented complete purification and analysis of the constituents of PHF. Based on their insolubility, we have developed a new method of preparing highly enriched PHF fractions and have raised an antiserum that is highly specific for PHF. We report here that this antiserum specifically labels PHF, free of any associated normal fibrous proteins and, unexpectedly, it reacts with neither neurofilaments nor any other normal cytoskeletal protein in brain sections or on immunoblotted gels. These anti-PHF antibodies have been used for the specific detection of Alzheimer-type PHF and in the search for cross-reacting antigens in various tissues, and are suitable for immunoaffinity purification of PHF. Our results indicate that PHF contain determinants that are not shared with normal neuronal fibrous proteins.     

Application of intervention analysis to a road fatality series in ontario

Effect of an intervention on a road fatality time series is studied using (i) the usual intervention analysis of Box and Tiao (1975), and (ii) CUSUM charts for the one-step-ahead forecast errors. It is shown that the seat belt and speed limit legislations of the Ontario Government had some impact in bringing down the road toll level.     

Genomic alterations in cultured human embryonic stem cells

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.