Engineered Proteins in Materials Research

Peptides and proteins have attracted scientific and technological interest largely because of their intriguing properties as catalysts, receptors, signalling molecules, and therapeutic agents. In attempts to understand and exploit these properties, protein engineering has been used primarily to obtain precious proteins in increased quantities, or to explore systematic alterations in protein sequence through site-directed mutagenesis. Design of protein structures de novo ("from scratch") has attracted less attention, and has been directed in the main toward studies of protein folding (Kamtekar et al., 1993). Such studies represent a key element in the current vigorous investigation of the connections between amino acid sequence and the three-dimensional structures of isolated protein chains in aqueous solution. This chapter describes protein engineering of quite another sort, in which the proteinacious nature of the product is less important than its macromolecular character

Treatment of Branch Retinal Artery Occlusion With Transluminal Nd:YAG Laser Embolysis

The purpose of this paper was to report a successful treatment of transluminal Nd:YAG laser embolysis (NYE) for branch retinal artery occlusion (BRAO) with visible emboli. Two patients with acute, severe vision loss secondary to a branch retinal artery occlusion with visible emboli in one eye underwent NYE. A complete ocular examination was performed which included biomicroscopy of the posterior pole of the retina, intraocular pressure measurement, fundus color photographs, and fluorescein angiography (FA). After the NYE, the two patients showed dramatic improvements in best-corrected visual acuity, as well as, immediate and dramatic restorations in flow past the obstructed arteriole in FA. NYE is a treatment modality to be considered in patients with BRAO who present acutely with severe vision loss and a visible embolus

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors

Migration through physical constraints is enabled by MAPK-induced cell softening via actin cytoskeleton re-organization

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics. This article has an associated First Person interview with the first author of the paper

Migration through physical constraints is enabled by MAPK-induced cell softening via actin cytoskeleton re-organization

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics

Analysis of Serum Levels of Anti-Müllerian Hormone, Inhibin B, Insulin-Like Growth Factor-I, Insulin-Like Growth Factor Binding Protein-3, and Follicle-Stimulating Hormone with Respect to Age and Menopausal Status

This study was undertaken to investigate age-dependent and postmenopausal changes in the serum levels of anti-Müllerian hormone (AMH), inhibin B, insulin-like growth factor (IGF)-I, IGF-binding protein-3 (IGFBP-3), and follicle-stimulating hormone (FSH), and to determine which of these markers best reflects the aging process in women. A total of 144 women aged 20-59 yr were enrolled in this cross-sectional study. Blood samples were obtained on cycle day 3 of regularly menstruating women (n=111), or at random in postmenopausal women (n=33). Data were analyzed with respect to premenopausal women age groups and compared in pre- and postmenopausal women. Area under the receiver operating characteristic curve (ROCAUC) analyses were performed to assess the ability of each marker to discriminate between the pre- and postmenopausal status. Serum levels of AMH, IGF-I, and IGFBP-3 decreased and serum levels of FSH increased significantly with age in premenopausal women. Serum luteinizing hormone (LH) was higher and inhibin B was lower in women in their 20-30's than in 40's. Serum levels of AMH and IGF-I showed a consistent decrease with all age groups. ROCAUC analysis showed that the diagnostic accuracy of AMH for menopausal status was similar to those of FSH, LH, and inhibin B, and was better than that of IGF-I. In conclusion, the serum AMH level appears to be the best marker of the aging process in premenopausal women

A Genetic Animal Model of Alcoholism for Screening Medications to Treat Addiction

The purpose of this review is to present up-to-date pharmacological, genetic, and behavioral findings from the alcohol-preferring P rat and summarize similar past work. Behaviorally, the focus will be on how the P rat meets criteria put forth for a valid animal model of alcoholism with a highlight on its use as an animal model of polysubstance abuse, including alcohol, nicotine, and psychostimulants. Pharmacologically and genetically, the focus will be on the neurotransmitter and neuropeptide systems that have received the most attention: cholinergic, dopaminergic, GABAergic, glutamatergic, serotonergic, noradrenergic, corticotrophin releasing hormone, opioid, and neuropeptide Y. Herein, we sought to place the P rat's behavioral and neurochemical phenotypes, and to some extent its genotype, in the context of the clinical literature. After reviewing the findings thus far, this chapter discusses future directions for expanding the use of this genetic animal model of alcoholism to identify molecular targets for treating drug addiction in general

From whole gland to hemigland to ultra-focal high-dose-rate prostate brachytherapy: A dosimetric analysis

Purpose: To assess the magnitude of dosimetric reductions of a focal and ultra-focal high-dose-rate (HDR) prostate brachytherapy treatment strategy relative to standard whole gland (WG) treatment. Methods and Materials: HDR brachytherapy plans for five patients treated with WG HDR monotherapy were optimized to assess different treatment strategies. Plans were generated to treat the hemigland (HG), one-third gland (1/3G), and one-sixth gland (1/6G), as well as to assess treating the WG with a boost to one of those sub-volumes (WG+HG, WG+1/3G, WG+1/6G). Dosimetric parameters analyzed included Target D90%, V100%, V150%, Bladder (B), Rectal (R), Urethral (U) D0.1, 1 and 2cc, Urethral V75%, and the V50% to the contralateral HG. Two-tailed t tests were used for comparison of means, and p-values less than 0.05 were considered statistically significant. Results: Target objectives (D90>100% and V100>97%) were met in all cases. Significant organs at risk dose reductions were achieved for all approaches compared with WG plans. 1/6G vs WG plans resulted in the greatest reduction in dose with a mean bladder D2cc 24.7 vs 64.8%, rectal D2cc 32.8 vs 65.3%, urethral D1cc 52.1 vs 103.8%, and V75 14.5 vs 75% (p < 0.05 for all comparisons). Conclusion: Significant dose reductions to organs at risk can be achieved using HDR focal brachytherapy. The magnitude of the reductions achievable with treating progressively smaller sub-volumes suggests the potential to reduce morbidity, but the clinical impact on morbidity and tumor control remain to be investigated