Multivariate curve resolution of time course microarray data

Background  

Modeling of gene expression data from time course experiments often involves the use of linear models such as those obtained from principal component analysis (PCA), independent component analysis (ICA), or other methods. Such methods do not generally yield factors with a clear biological interpretation. Moreover, implicit assumptions about the measurement errors often limit the application of these methods to log-transformed data, destroying linear structure in the untransformed expression data.     

Euler buckling-induced folding and rotation of red blood cells in an optical trap

We investigate the physics of an optically driven micromotor of biological origin. When a single, live red blood cell (RBC) is placed in an optical trap, the normal biconcave disc shape of the cell is observed to fold into a rod-like shape. If the trapping laser beam is circularly polarized, the folded RBC rotates. A model based on geometric considerations, using the concept of buckling instabilities, captures the folding phenomenon; the rotation of the cell is rationalized using the Poincaré sphere. Our model predicts that (i) at a critical power of the trapping laser beam the RBC shape undergoes large fluctuations, and (ii) the torque that is generated is proportional to the power of the laser beam. These predictions are verified experimentally. We suggest a possible mechanism for the emergence of birefringent properties in the RBC in the folded state.     

Exploitation of the endoplasmic reticulum by bacterial pathogens

The endoplasmic reticulum (ER) has unique properties that are exploited by microbial pathogens. Exotoxins secreted by bacteria take advantage of the host transport pathways that deliver proteins from the Golgi to the ER. Transport to the ER is necessary for the unfolding and translocation of these toxins into the cytosol where their host targets reside. Intracellular pathogens subvert host vesicle transport to create ER-like vacuoles that support their intracellular replication. Investigations on how bacterial pathogens can use the ER during host infection are providing important details on transport pathways involving this specialized organelle.     

Diet and temperature modify the relationship between energy use and ATP production to influence behavior in zebrafish (Danio rerio)

Food availability and temperature influence energetics of animals and can alter behavioral responses such as foraging and spontaneous activity. Food availability, however, is not necessarily a good indicator of energy (ATP) available for cellular processes. The efficiency of energy transduction from food‐derived substrate to ATP in mitochondria can change with environmental context. Our aim was to determine whether the interaction between food availability and temperature affects mitochondrial efficiency and behavior in zebrafish (Danio rerio). We conducted a fully factorial experiment to test the effects of feeding frequency, acclimation temperature (three weeks to 18 or 28°C), and acute test temperature (18 and 28°C) on whole‐animal oxygen consumption, mitochondrial bioenergetics and efficiency (ADP consumed per oxygen atom; P:O ratio), and behavior (boldness and exploration). We show that infrequently fed (once per day on four days per week) zebrafish have greater mitochondrial efficiency than frequently fed (three times per day on five days per week) animals, particularly when warm‐acclimated. The interaction between temperature and feeding frequency influenced exploration of a novel environment, but not boldness. Both resting rate of producing ATP and scope for increasing it were positively correlated with time spent exploring and distance moved in standardized trials. In contrast, behavior was not associated with whole‐animal aerobic (oxygen consumption) scope, but exploration was positively correlated with resting oxygen consumption rates. We highlight the importance of variation in both metabolic (oxygen consumption) rate and efficiency of producing ATP in determining animal performance and behavior. Oxygen consumption represents energy use, and P:O ratio is a variable that determines how much of that energy is allocated to ATP production. Our results emphasize the need to integrate whole‐animal responses with subcellular traits to evaluate the impact of environmental conditions on behavior and movement.     

On the origin and evolution of thermophily: reconstruction of functional precambrian enzymes from ancestors of Bacillus

Thermophily is thought to be a primitive trait, characteristic of early forms of life on Earth, that has been gradually lost over evolutionary time. The genus Bacillus provides an ideal model for studying the evolution of thermophily as it is an ancient taxon and its contemporary species inhabit a range of thermal environments. The thermostability of reconstructed ancestral proteins has been used as a proxy for ancient thermal adaptation. The reconstruction of ancestral "enzymes" has the added advantages of demonstrable activity, which acts as an internal control for accurate inference, and providing insights into the evolution of enzymatic catalysis. Here, we report the reconstruction of the structurally complex core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, E. C. 1.1.1.85) from the last common ancestor (LCA) of Bacillus using both maximum likelihood (ML) and Bayesian inference. ML LeuB from the LCA of Bacillus shares only 76% sequence identity with its closest contemporary homolog, yet it is fully functional, thermophilic, and exhibits high values for k(cat), k(cat)/K(M), and ΔG(?) for unfolding. The Bayesian version of this enzyme is also thermophilic but exhibits anomalous catalytic kinetics. We have determined the 3D structure of the ML enzyme and found that it is more closely aligned with LeuB from deeply branching bacteria, such as Thermotoga maritima, than contemporary Bacillus species. To investigate the evolution of thermophily, three descendents of LeuB from the LCA of Bacillus were also reconstructed. They reveal a fluctuating trend in thermal evolution, with a temporal adaptation toward mesophily followed by a more recent return to thermophily. Structural analysis suggests that the determinants of thermophily in LeuB from the LCA of Bacillus and the most recent ancestor are distinct and that thermophily has arisen in this genus at least twice via independent evolutionary paths. Our results add significant fluctuations to the broad trend in thermal adaptation previously proposed and demonstrate that thermophily is not exclusively a primitive trait, as it can be readily gained as well as lost. Our findings also demonstrate that reconstruction of complex functional Precambrian enzymes is possible and can provide empirical access to the evolution of ancient phenotypes and metabolisms.     

Identification of a redox-sensitive switch within the JAK2 catalytic domain

Four cysteine residues (Cys866, Cys917, Cys1094, and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(NΔ661)rJAK2), which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated by in vitro autokinase assays and by in situ autophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pretreatment with dithiothreitol or o-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.