Orthogonal projections to latent structures as a strategy for microarray data normalization

Background  

During generation of microarray data, various forms of systematic biases are frequently introduced which limits accuracy and precision of the results. In order to properly estimate biological effects, these biases must be identified and discarded.     

The old and new biochemistry of polyamines

Polyamines are ubiquitous positively charged amines found in all organisms. These molecules play a crucial role in many biological functions including cell growth, gene regulation and differentiation. The three major polyamines produced in all mammalian cells are putrescine, spermidine and spermine. The intracellular levels of these polyamines depend on the interplay of the biosynthetic and catabolic enzymes of the polyamine and methionine salvage pathway, as well as the involvement of polyamine transporters. Polyamine levels are observed to be high in cancer cells, which contributes to malignant transformation, cell proliferation and poor patient prognosis. Considering the critical roles of polyamines in cancer cell proliferation, numerous anti-polyaminergic compounds have been developed as anti-tumor agents, which seek to suppress polyamine levels by specifically inhibiting polyamine biosynthesis, activating polyamine catabolism, or blocking polyamine transporters. However, in terms of the development of effective anti-cancer therapeutics targeting the polyamine system, these efforts have unfortunately resulted in little success. Recently, several studies using the iron chelators, O-trensox and ICL670A (Deferasirox), have demonstrated a decline in both iron and polyamine levels. Since iron levels are also high in cancer cells, and like polyamines, are required for proliferation, these latter findings suggest a biochemically integrated link between iron and polyamine metabolism.     

Microfluidic perfusion for regulating diffusible signaling in stem cells

Background

Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands.

Methodology/Principal Findings

We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs.

Conclusions/Significance

Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.     

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A₂ receptor

In humans, thromboxane (TX) A₂ signals through the TPα and TPβ isoforms of its G-protein coupled TXA₂ receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA₂ mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.     

Effects of training at simulated altitude on performance and muscle metabolic capacity in competitive road cyclists

Differences between the effects of training at sea level and at simulated altitude on performance and muscle structural and biochemical properties were investigated in 8 competitive cyclists who trained for 3-4 weeks, 4-5 sessions/week, each session consisting of cycling for 60-90 min continuously and 45-60 min intermittently. Four subjects, the altitude group (AG), trained in a hypobaric chamber (574 torr = 2300 m above sea level), and the other four at sea level (SLG). Before and after training work capacity was tested both at simulated altitude (574 torr) and at sea level, by an incremental cycle ergometer test until exhaustion. Work capacity was expressed as total amount of work performed. Venous blood samples were taken during the tests. Leg muscle biopsies were taken at rest before and after the training period. AG exhibited an increase of 33% in both sea level and altitude performance, while SLG increased 22% at sea level and 14% at altitude. Blood lactate concentration at a given submaximal load at altitude was significantly more reduced by training in AG than SLG. Muscle phosphofructokinase (PFK) activity decreased with training in AG but increased in SLG. All AG subjects showed increases in capillary density. In conclusion, work capacity at altitude was increased more by training at altitude than at sea level. Work capacity at sea level was at least as much improved by altitude as by sea level training. The improved work capacity by training at altitude was paralleled by decreased exercise blood lactate concentration, increased capillarization and decreased glycolytic capacity in leg muscle.     

Control of Root-knot Nematodes on Tomato by Lectins

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.     

Co-culture of human monocytes and thyrocytes in bicameral chamber: monocyte-derived IL-1alpha impairs the thyroid epithelial barrier

Pro-inflammatory cytokines are important mediators in tissue responses to a wide range of endogenous (e.g. autoantigens) and exogenous (e.g. infections, wounds, biomaterials) stimuli. The complex interactions taking place between different cell types in such processes are difficult to examine in vivo. Here we studied the effect of human monocytes on thyroid epithelial cells co-cultured in bicameral chambers. Freshly isolated monocytes (1x10(6)/ml) added to the basal compartment reduced the transepithelial resistance (from 300-600 to <100 Omega.cm(2)) and caused a disruption of the tight junctions in apically grown thyrocyte monolayers after co-culture for 24 h. The barrier function was further attenuated by monocytes exposed to lipopolysaccharide (10 microg/ml) or polystyrene microspheres (size: 3 microm; 1x10(7)/ml). Loss of transepithelial resistance was accompanied by release of interleukin 1alpha (maximally 550 pg/ml) from the monocytes. Conversely, the resistance remained high when co-cultures were simultaneously incubated with neutralizing anti-human interleukin 1alpha antibodies. The results show that the integrity of cultured thyroid epithelium is impaired by monocytes without requirement of direct cell-to-cell contact. This action, mediated by interleukin-1alpha, suggests a mechanism by which hidden (lumenal) autoantigens might be exposed to interstitial antigen-presenting cells in autoimmune thyroid disease. In perspective, the model provides a tool in which humoral and cell-cell dependent processes generated by bioactive agents and particulate materials, for instance, during the healing and repair of tissue around biomaterials and hybrid implants, can be selectively examined.     

Identification of distinct physiochemical properties of toxic prefibrillar species formed by Aβ peptide variants

The formation of amyloid-β peptide (Aβ) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aβ. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aβ-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aβ aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aβ peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aβ peptide to form nontoxic versus toxic species.     

Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor: independent modulation of TPalpha signaling by prostacyclin and nitric oxide

In humans, thromboxane (TX) A(2) signals through the TPalpha and TPbeta isoforms of the TXA(2) receptor that exhibit common and distinct roles. For example, Gq/phospholipase (PL)Cbeta signaling by TPalpha is directly inhibited by the vasodilators prostacyclin and nitric oxide (NO) whereas that signaling by TPbeta is unaffected. Herein, we investigated whether TPalpha and/or TPbeta regulate G(12)/Rho activation and whether that signaling might be differentially regulated by prostacyclin and/or NO. Both TPalpha and TPbeta independently regulated RhoA activation and signaling in clonal cells over-expressing TPalpha or TPbeta and in primary human aortic smooth muscle cells (1 degrees AoSMCs). While RhoA-signaling by TPalpha was directly impaired by prostacyclin and NO through protein kinase (PK)A- and PKG-dependent phosphorylation, respectively, signaling by TPbeta was not directly affected by either agent. Collectively, while TPalpha and TPbeta contribute to RhoA activation, our findings support the hypothesis that TPalpha is involved in the dynamic regulation of haemostasis and vascular tone, such as in response to prostacyclin and NO. Conversely, the role of TPbeta in such processes remains unsolved. Data herein provide essential new insights into the physiologic roles of TPalpha and TPbeta and, through studies in AoSMCs, reveal an additional mode of regulation of VSM contractile responses by TXA(2).