Development of macroporous poly(ethylene glycol) hydrogel arrays within microfluidic channels

The mass transport of solutes through hydrogels is an important design consideration in materials used for tissue engineering, drug delivery, and protein arrays used to quantify protein concentration and activity. We investigated the use of poly(ethylene glycol) (PEG) as a porogen to enhance diffusion of macromolecules into the interior of polyacrylamide and PEG hydrogel posts photopatterned within microfluidic channels. The diffusion of GST-GFP and dextran-FITC into hydrogels was monitored and effective diffusion coefficients were determined by fitting to the Fickian diffusion equations. PEG-diacrylate (M(r) 700) with porogen formed a macroporous structure and permitted significant penetration of 250 kDa dextran. Proteins copolymerized in these macroporous hydrogels retained activity and were more accessible to antibody binding than proteins copolymerized in nonporous gels. These results suggest that hydrogel macroporosity can be tuned to regulate macromolecular transport in applications such as tissue engineering and protein arrays.     

Polarized light based scheme to monitor column performance in a continuous foam fractionation column

Background  

A polarized light scattering technique was used to monitor the performance of a continuously operated foam fractionation process. The S ₁₁ and S ₁₂ parameters, elements of the light scattering matrix, combined together (S ₁₁ +S ₁₂) have been correlated with the bubble size and liquid content for the case of a freely draining foam. The performance of a foam fractionation column is known to have a strong dependence on the bubble size distribution and liquid hold up in foam. In this study the enrichment is used as a metric, representative of foam properties and column performance, and correlated to the S ₁₁ +S ₁₂ parameter.     

Selective ion tracing and MSn analysis of peptide digests from FSBA-treated kinases for the analysis of protein ATP-binding sites

Kinases play a key role in many cellular processes by catalyzing the transfer of phosphoryl groups from ATP to a broad number of substrates, including amino acids on target proteins. The reagent 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA) has been widely used to identify ATP-binding sites in kinases since it reacts with nucleophilic amino acids occurring within these motifs, determining a mass increase of 433 Da. In this study, we present a versatile MS approach that has been developed to recognize labeled peptides generated after enzymatic digestion of FSBA-treated kinases. Using selective ion tracing and MS(2)/MS(3) experiments, we were able to easily identify peptides occurring at protein ATP-binding sites, also affording a complete characterization of the modified amino acids. This methodology may be used in the development of future parent ion scanning-based applications directed to large scale analysis of kinases within complex protein mixtures.     

Neonatal inhalatory anesthetic exposure: reproductive changes in male rats

We investigated the effects of an inhalatory anesthetic (ethyl ether) during the neonatal period of brain sexual differentiation on the later fertility and sexual behavior of male rats. Animals were exposed to ethyl ether immediately after birth. At adulthood, body weight, testes wet weight, and plasma testosterone levels were not affected; however, neonatal exposure to ether showed alterations on male fertility: a decrease in the number of spermatids and spermatozoa, an increase in the transit time of cauda epididymal spermatozoa and a decrease in daily sperm production. An alteration of sexual behavior was also observed: decreased male sexual behavior and appearance of homosexual behavior when the male rats were castrated and pretreated with exogenous estrogen. Probably, the ether delayed or reduced the testosterone peak of the sexual differentiation period, altering the processes of masculinization and defeminization of the hypothalamus. Our results indicate that perinatal exposure to ethyl ether during the critical period of male brain sexual differentiation, acting as endocrine disruptors, has a long-term effect on the fertility and sexual behavior of male rats, suggesting endocrine disruption through incomplete masculinization and defeminization of the central nervous system.     

Activation of human T lymphocytes under conditions similar to those that occur during exposure to microgravity: a proteomics study

A number of experiments, conducted under microgravity conditions, i.e. in space shuttle biolaboratories or in ground based systems simulating the conditions occurring in microgravity, show that in hypogravity, in vitro human lymphocyte activation is severely impaired. However, very early stimulation steps of T lymphocytes are not compromised, since CD69 receptor, the earliest membrane activation marker, is expressed by T cells at a level comparable to that observed on 1 g activated lymphocytes. Since CD69 engagement, together with submitogenic doses of phorbol esters, transduces an activation signal to T lymphocytes, we undertook a comparative study on the stimulation mediated through this receptor on human CD3+ cells cultured under conditions similar to those which occur during exposure to microgravity, i.e. in clinorotation, or at 1 g. During the early hours of activation, increased levels of intracellular calcium and increased mitochondrial membrane potential were detectable in clinorotating as well as in 1 g cells. However, after 48 hours clinorotation, interleukin 2 production by T lymphocytes was significantly reduced and cell proliferation was greatly decreased. By means of a differential proteomics approach on T cells activated in clinorotation or at 1 g for 48 hours, we were able to detect statistically significant quantitative protein alterations. Seven proteins with modified expression values were identified; they are involved in nucleic acids processing, proteasome regulation and cytoskeleton structure.     

Flavopiridol inhibits the growth of GL261 gliomas in vivo: implications for malignant glioma therapy

The mechanism of action of many chemotherapeutic agents targets the cell cycle. Recently, we demonstrated cytotoxic and other anti-tumor effects of flavopiridol, the first synthetic cyclin dependent kinase (CDK) inhibitor to enter clinical trials, on the murine GL261 glioma cell line in vitro (Newcomb et al., Cell Cycle 2003; 2:243). Given that flavopiridol has demonstrated anti-tumor activity in several human xenograft models, we wanted to evaluate it for anti-glioma activity in vivo in our established subcutaneous and intracranial GL261 experimental tumor models. In particular, the intracranial animal model recapitulates many of the histopathological and biological features of human high-grade glioma including both necrosis with pseudopalisading and invasion of the brain adjacent to tumor. Here we tested the activity of flavopiridol against tumors formed by GL261 cells, first as subcutaneous implants, and then in the intracranial model. We demonstrate efficacy of flavopiridol as a single modality treatment in delaying tumor growth in both animal models. We hypothesize that flavopiridol treatment induced tumor growth delay by two possible mechanisms involving growth arrest combined with recruitment of tumor cells to S-phase. Based on our findings, flavopiridol should be considered as a treatment approach for patients with high-grade glioma.     

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.Unconjugated bilirubin (UCB) is the metabolic product of heme degradation in mammals. The enzymes heme oxygenase 1 and 2 (HO-1, HO-2) catalyze the degradation of heme into biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase. In the liver, UCB is conjugated to glucuronic acid by UDP-glucuronosil transferase (UGT1a1), rendering it water soluble and excretable in the bile. In neonates, the late induction of Ugt1a1 gene expression may result in a limited capacity to conjugate bilirubin. The imbalance between bilirubin production and its elimination result in neonatal hyperbilirubinemia. Moderate jaundice is present in a high proportion of babies and is considered beneficial because of the antioxidant and cytoprotective properties of UCB.¹ However, excessive hyperbilirubinemia in newborns may produce acute bilirubin encephalopathy (BE), bilirubin-induced neurological disorders (BINDs) and eventually death by kernicterus.² BIND is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, because of UCB accumulation in the cerebellum, hippocampus and basal ganglia. Infants affected by null mutations in the Ugt1 gene develop Crigler–Najjar syndrome type I (CNSI).³ CNSI patients present high UCB plasma levels and are particularly exposed to BE and BIND if untreated. Current therapy initially consists in intensive phototherapy (PT) but liver transplantation is required later in life because of a reduction in PT efficiency.^{4, 5}Despite extensive investigations in animal models and in vitro tissue culture cells, the basic mechanisms of hyperbilirubinemia neurotoxicity have not been fully clarified yet.^{6, 7} UCB affects a large number of cellular functions and neurological damage appears to be the result of their concerted disruption rather than misregulation of a single pathway. The reported observations range from energy metabolism,⁸ cell proliferation,⁹ DNA and protein synthesis,¹⁰ receptor functionality,¹¹ to neurotransmitter uptake and release.¹² Our group and others reported that UCB is associated with an increased oxidative stress condition in cell culture.^{13, 14} In vitro studies showed that exposure to UCB decreases neuronal and glia viability.^{15, 16} In primary cultures, it has been observed that UCB permeabilizes mitochondrial membranes, resulting in mitochondrial swelling, release of cytochrome c into the cytosol, caspase-3 activation, Bax translocation and cell death by apoptosis.^{14, 17} Bilirubin also decreases NGF signaling to AKT and ERKs, interfering with prosurvival signaling pathways.¹⁸ However, most of the studies leading to potential mechanisms of bilirubin neurotoxicity were performed on monotypic cultures with artificial dosing of bilirubin, lacking the complexity of the interactions between different cell types, as it occurs in vivo.The aim of this study was to get a deeper unbiased insight into the molecular processes underlying bilirubin-induced neurodegeneration in vivo by using a mouse model of neonatal hyperbilirubinemia that shows early lethality because of bilirubin-induced neurological damage.¹⁹ Previous experiments conducted by our lab showed that the cerebellum of the Ugt1a^−/− mouse is the most vulnerable region of the brain to bilirubin toxicity.^{19, 20} Cerebellar susceptibility to bilirubin resulted in important alterations of its architecture, being the external germinal layer (EGL) and the Purkinje cell layer (PCL) the most affected regions, associated with an increase of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells.^{19, 20}In this study, we performed a differential proteomic analysis of the cerebellum from Ugt1 mutant and wild-type (WT) mice followed by validation of candidate genes both at protein and RNA levels. Our data point to an impairment of enzymatic antioxidant processes as important intracellular mechanisms involved in the onset of bilirubin-induced neurotoxicity in vivo.