Protein aggregation in silico

Protein aggregation is a challenge to the successful manufacture of protein therapeutics; it can impose severe limitations on purification yields and compromise formulation stability. Advances in computer power, and the wealth of computational studies pertaining to protein folding, have facilitated the development of molecular simulation as a tool to investigate protein misfolding and aggregation. Here, we highlight the successes of protein aggregation studies carried out in silico, with a particular emphasis on studies related to biotechnology. To conclude, we discuss future prospects for the field, and identify several biotechnology-related problems that would benefit from molecular simulation.     

The rational strategies for detecting developmental dysplasia of the hip at the age of 4-6 months old infants: a prospective study

Using ultrasound in evaluation of infant's hip development can reduce surgical procedures, hospitalization and late presentation of developmental dysplasia of the hip (DDH). The increasing incidence of DDH after ultrasound examination is observed and published by many authors. In a prospective study, radiograph of every single ultrasonographic positive hip in infants older than three months, was taken and analyzed in order to see whether it affects infants splintage rate in treating DDH. In a period of 30 months, clinical and simple static ultrasonographic examinations according to Graf were performed on 1430 consecutive infant hips in patients aged between 4 and 6 months. Sonographic positive hips were radiographed and acetabular index (AI) values on simple AP radiographs were analyzed. The sonographic DDH incidence was 51.75 per 1000 hips (51.75 per thousand). After X-ray examination of all 74 ultrasonographic positive hips, only 44 remained abnormal and required treatment indicating a true DDH incidence of 30.77 per 1000 hips (30.77 per thousand). The difference in incidence per ultrasonographic and X-ray positive hips is statistically significant p < 0.01 (t = 5,536). The rational approach in detection of DDH in a child more than 3 months old is to do radiographic assessment of every sonographic positive hip.     

Transition from nanodomains to microdomains induced by exposure of lipid monolayers to air

The morphology of monolayers prepared from ternary lipid mixtures that have coexisting fluid phases has been examined by atomic force microscopy for samples transferred to mica before and after exposure to air. Mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol with either egg sphingomyelin or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied at several surface pressures. Both lipid mixtures have a combination of small islands and large microdomains at low surface pressure (5-10 mN/m) for monolayers deposited in either air or nitrogen. By contrast, monolayers have small interconnected nanodomains when deposited under nitrogen at 30 mN/m but mixtures of large microdomains and small nanodomains when transferred after exposure to air. These results are consistent with an earlier report that concluded that the formation of large domains at high surface pressures (>30 mN/m) for monolayers exposed to air is caused by lipid oxidation. However, the higher spatial resolution available with atomic force microscopy indicates that exposure of the monolayers to air leads to an increase in the size of preexisting nanodomains, rather than a change in the miscibility pressure. Examination of changes in surface morphology as a function of surface pressure demonstrate a gradual evolution in size and surface coverage for both nano- and microdomains, before formation of a network of interconnected nanodomains. Similar studies for binary mixtures in the absence of cholesterol indicate that lipid oxidation results in analogous changes in domain size for monolayers with coexisting gel and fluid phases. These results illustrate the importance of using techniques capable of probing the nanoscale organization of membranes.     

Effect of maturation on the relationship between physical performance and body size

The purpose of the study was to explore the effect of maturation on the body size-physical performance relationship. Based on our previous study that evaluated only muscle strength, we hypothesized that the physical performance tested on pubescent subjects would be related to body size at a higher rate than predicted by standard scaling theory applied on pre- and postpubescent subjects. Six age groups of highly selected and trained junior soccer players (N = 478; age 12-17 years) presumably including prepubescent, pubescent, and postpubescent subjects were evaluated. They were tested using various standard tests of maximum physical performance including muscle strength of 3 leg muscle groups, 2 jumps, sit-ups, hand and foot tapping, and agility. The results revealed a steeper increase of the tested performance with an increase in body size for the pubescent age than for the pre- and postpubescent age. The observed phenomenon was interpreted by different level of maturation of the subjects tested within the same pubescent age group. We conclude that maturation alters the effect of body size on various physical performances and, therefore, this phenomenon needs to be taken into account when interpreting the data from the physical performance tests applied on pubescent subjects.     

γ-Aminobutyric A Receptor (GABAAR) Regulates Aquaporin 4 Expression in the Subependymal Zone: RELEVANCE TO NEURAL PRECURSORS AND WATER EXCHANGE*

Activation of γ-aminobutyric A receptors (GABA_ARs) in the subependymal zone (SEZ) induces hyperpolarization and osmotic swelling in precursors, thereby promoting surface expression of the epidermal growth factor receptor (EGFR) and cell cycle entry. However, the mechanisms underlying the GABAergic modulation of cell swelling are unclear. Here, we show that GABA_ARs colocalize with the water channel aquaporin (AQP) 4 in prominin-1 immunopositive (P⁺) precursors in the postnatal SEZ, which include neural stem cells. GABA_AR signaling promotes AQP4 expression by decreasing serine phosphorylation associated with the water channel. The modulation of AQP4 expression by GABA_AR signaling is key to its effect on cell swelling and EGFR expression. In addition, GABA_AR function also affects the ability of neural precursors to swell in response to an osmotic challenge in vitro and in vivo. Thus, the regulation of AQP4 by GABA_ARs is involved in controlling activation of neural stem cells and water exchange dynamics in the SEZ.     

Revisiting tree-migration rates: Abies alba (Mill.), a case study

At northern temperate latitudes trees have adjusted their ranges substantially in response to changing climates during the Holocene. Results from dispersal model simulations suggest that postglacial migration rates may have been over-estimated from fossil pollen data. As a contribution to this debate, we infer the migration rates of Abies alba (Mill.), silver fir, as a case-study species, by using a spatially explicit approach based on fossil pollen but taking into account its modern genetic diversity pattern. Maximum estimates of migration rates from fossil pollen data alone are higher than 700 m yr^?1 during the Holocene. Considering the potential refugia as suggested from all the fossil data but restricting the area over which silver fir expanded from each glacial refugium using data on the current haplotype distribution, the estimated maximum migration rates of silver fir are less than 250 m yr^?1. Genetic information may allow for (1) the exclusion of those refugial areas where the species may have survived during the last glacial period but from which it did not spread or spread only very locally and (2) the delineation of the areas over which the species spread from each glacial refugium. The estimated rates in the present study are generally consistent with rates suggested from modelling approaches. This study shows that integrating fossil pollen records can improve simulations of dispersal processes and, thus, allow for better predictions of future changes in tree species’ ranges.     

Ischemia-Induced Inhibition of Mitochondrial Complex I in Rat Brain: Effect of Permeabilization Method and Electron Acceptor

In this study we have examined the effect of global brain ischemia/reperfusion on biochemical properties of the mitochondrial respiratory complex I (CI) in rat hippocampus and cortex. Since the inner mitochondrial membrane forms the permeability barrier for NADH, the methodology of enzymatic activity determinations employs membrane permeabilization methods. This action affects the basic character of electrostatic and hydrophobic interactions inside the membrane and might influence functional properties of membrane embedded proteins. Therefore we have performed the comparative analysis of two permeabilization methods (sonication, detergent) and their impact on CI enzymatic activities under global brain ischemic-reperfusion conditions. We have observed that ischemia led to significant decrease of CI activities using both permeabilization methods in both brain areas. However, significant differencies in enzymatic activities were registered during reperfusion intervals according to used permeabilization method. We have also tested the effect of electron acceptors (decylubiquinone, potassium ferricyanide, nitrotetrazolium blue) on CI activities during I/R. Based on our results we assume that the critical site where ischemia affects CI activities is electron transfer to electron acceptor. Further, the observed mitochondrial dysfunction was analyzed by means of one and 2-dimensional BN PAGE/SDS PAGE with the focus on 3-nitrotyrosine immunodetection as a marker of oxidative damage to proteins. Add to this, initialization of p53 mitochondrial apoptosis through p53, Bax, Bcl-X_L proteins and a possible involvement of GRIM-19, the CI structural subunit, in apoptotic processes were also studied.     

PPARα and PPARγ regulate the nucleoside transporter hENT1

Human equilibrative nucleoside transporter 1 (hENT1) is an important determinant for nucleoside analog based chemotherapy success. Preliminary data suggest hENT1 regulation by PPARs. Using A2780 cells, we investigated the role of PPARs on hENT1 expression and activity. PPARα and PPARγ agonists, Wy14,643 and RGZ, increased hENT1 expression, but only PPARα activation or overexpression resulted in higher hENT1 transport activity. On the other hand, promoter analysis showed two putative PPRE in hENT1 promoter and luciferase-coupled promoter constructs were generated and analyzed. Our results suggest that PPARα-but not PPARγ-mediated expression regulation of hENT1 is PPRE-dependent. In conclusion, PPARα and PPARγ activation modulate hENT1 expression.