Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions

A transient mechanistic two-fluid model for Direct Contact Condensation

Intensified heat treatment, using Direct Contact Condensation (DCC), is applied in the production of dairy products to ensure a high level of food safety. The key challenge with DCC is the protein reactions and fouling that limit operational efficiency and sustainability. The use of validated, transient models to predict temperature, phase fraction and velocity gradients is essential and can improve the operation of DCC. Pilot plant scale experiments were performed for a wide range of steam mass fluxes, inlet water temperatures, water Reynolds number and channel pressures to validate a transient 1D two-fluid model. The model was tested against steady-state experimental data and showed good agreement. In addition the model was tested against transient data in which either the water flow rate was step wise decreased or the pressure was adjusted throughout the experiment. In general the model followed the experimental trend well provided that the flow inside the channel is homogeneous.</p

Cloning and sequencing of the tuf genes of Streptomyces coelicolor A3(2)

Microbial Biotechnolog

Spectral analysis and resolving spatial ambiguities in human sound localization

This dissertation provides an overview of my research over the last five years into the spectral analysis involved in human sound localization. The work involved conducting psychophysical tests of human auditory localization performance and then applying analytical techniques to analyze and explain the data. It is a fundamental thesis of this work that human auditory localization response directions are primarily driven by the auditory localization cues associated with the acoustic filtering properties of the external auditory periphery, i.e., the head, torso, shoulder, neck, and external ears. This work can be considered as composed of three parts. In the first part of this work, I compared the auditory localization performance of a human subject and a time-delay neural network model under three sound conditions: broadband, high-pass, and low-pass. A “black-box” modeling paradigm was applied. The modeling results indicated that training the network to localize sounds of varying center-frequency and bandwidth could degrade localization performance results in a manner demonstrating some similarity to human auditory localization performance. As the data collected during the network modeling showed that humans demonstrate striking localization errors when tested using bandlimited sound stimuli, the second part of this work focused on human sound localization of bandpass filtered noise stimuli. Localization data was collected from 5 subjects and for 7 sound conditions: 300 Hz to 5 kHz, 300 Hz to 7 kHz, 300 Hz to 10 kHz, 300 Hz to 14 kHz, 3 to 8 kHz, 4 to 9 kHz, and 7 to 14 kHz. The localization results were analyzed using the method of cue similarity indices developed by Middlebrooks (1992). The data indicated that the energy level in relatively wide frequency bands could be driving the localization response directions, just as in Butler’s covert peak area model (see Butler and Musicant, 1993). The question was then raised as to whether the energy levels in the various frequency bands, as described above, are most likely analyzed by the human auditory localization system on a monaural or an interaural basis. In the third part of this work, an experiment was conducted using virtual auditory space sound stimuli in which the monaural spectral cues for auditory localization were disrupted, but the interaural spectral difference cue was preserved. The results from this work showed that the human auditory localization system relies primarily on a monaural analysis of spectral shape information for its discrimination of directions on the cone of confusion. The work described in the three parts lead to the suggestion that a spectral contrast model based on overlapping frequency bands of varying bandwidth and perhaps multiple frequency scales can provide a reasonable algorithm for explaining much of the current psychophysical and neurophysiological data related to human auditory localization

The (co-)occurrence of problematic video gaming, substance use, and psychosocial problems in adolescents

Aims. The current study explored the nature of problematic (addictive) video gaming and the association with game type, psychosocial health, and substance use. Methods. Data were collected using a paper and pencil survey in the classroom setting. Three samples were aggregated to achieve a total sample of 8478 unique adolescents. Scales included measures of game use, game type, the Video game Addiction Test (VAT), depressive mood, negative self-esteem, loneliness, social anxiety, education performance, and use of cannabis, alcohol and nicotine (smoking). Results. Findings confirmed problematic gaming is most common amongst adolescent gamers who play multiplayer online games. Boys (60%) were more likely to play online games than girls (14%) and problematic gamers were more likely to be boys (5%) than girls (1%). High problematic gamers showed higher scores on depressive mood, loneliness, social anxiety, negative self-esteem, and self-reported lower school performance. Nicotine, alcohol, and cannabis using boys were almost twice more likely to report high PVG than non-users. Conclusions. It appears that online gaming in general is not necessarily associated with problems. However, problematic gamers do seem to play online games more often, and a small subgroup of gamers – specifically boys – showed lower psychosocial functioning and lower grades. Moreover, associations with alcohol, nicotine, and cannabis use are found. It would appear that problematic gaming is an undesirable problem for a small subgroup of gamers. The findings encourage further exploration of the role of psychoactive substance use in problematic gaming

Treatment seeking for alcohol and drug use disorders by immigrants to the Netherlands: Retrospective, population-based, cohort study

Background We compared risks of first contact with services for an alcohol use disorder (AUD) or drug use disorder (DUD) between the largest immigrant groups to the Netherlands and Dutch nationals. We tested the hypothesis that the ethnic pattern for DUD is similar to the previously demonstrated pattern for schizophrenia. Methods Retrospective, population-based cohort study of First Admissions to Dutch psychiatric hospitals during the period 1990-1996 (national data) and First Contacts with inpatient or outpatient centres in Rotterdam for treatment of AUD or DUD during the period 1992-2001 (Rotterdam data). Results In both datasets the risk of service contact for AUD was significantly lower in immigrants from Surinam, Turkey and Morocco than in Dutch nationals. The risk was lower or moderately higher in immigrants from western countries. Analysis of the national data showed that, compared with Dutch males, the risk of first hospital admission for DUD was higher for male immigrants from the Dutch Antilles (RR = 4.6; 95% CI: 4.0-5.3), Surinam (RR = 4.3; 3.94.7) and Morocco (RR = 23; 2.0-2.6), but not for male immigrants from Turkey (RR = 0.9; 0.7-1.1). A similar pattern was found with the Rotterdam data. Female immigrants from Surinam and the Dutch Antilles had a higher risk for DUD according to the national data, but a lower risk according to the Rotterdam data. Female immigrants from Turkey and Morocco had a lower risk (both datasets). Immigrants from western countries had a higher risk for DUD, but many had developed the disorder before emigrating. Conclusion Those immigrant groups in the Netherlands that are at increased risk of schizophrenia appear also at increased risk of developing DUD, but not AUD

Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~ 45.9 kDa, ~ 37 kDa, and ~ 83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50 > 100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24 h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24 h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24 h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24 h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted

The Targeting of Plasmalemmal Ceramide to Mitochondria during Apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1—a member of a family of Ca2+ and membrane-binding proteins—to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this “kiss-of-death” increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis