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THE OPTIMIZATION OF A TIME-RESOLVED, VESICLE-BASED FLUORESCENCE ASSAY FOR THE ACTIVITY OF THE LIPID KINASE PI4K-IIIBETA AND THE EFFECT OF LIPID TRANSFER PROTEINS ON ENZYME ACTIVITY
Get PDFHuman PI4K-IIIβ is an 89 kDa phosphatidylinositol (PI) kinase that phosphorylates its substrate headgroup at position C-4, thus producing PI(4)P. This phosphoinositide is the most abundant in the trans-Golgi network where it is essential for secretory vesicle formation, as well as the precursor for other phosphoinositides that are crucial for intracellular signalling. Among others, phosphoinositide homeostasis in eukaryotic membranes rely on PI kinases and PI transfer proteins (PITPs). In yeast, the PITP Sec14p is known to exchange PI and phosphatidylcholine between lipid bilayers in vitro and proposed to present PI to be phosphorylated by the PI4-kinase Pik1 in a heterotypic ligand exchange fashion. However, the precise mechanism by which this interaction occurs has yet to be elucidated. To explore how and if PITPs and PI4K-IIIβ work as hypothesized, we expressed and purified recombinant human PI4K-IIIβ in Escherichia coli and assayed lipid kinase activity using an optimized real-time, vesicle-based fluorescence assay. After comparing different affinity tags, deletion mutants and expressing cell lines, GST-tagged wild-type PI4K-IIIβ was chosen and expressed in Rosetta 2(DE3) cells with a 2.5-fold increase in the native protein yield when compared to other methods. Proteins were further purified by an addition heat shock protein removal wash. The resulting PI4K-IIIβ displayed activity comparable to the commercially available, insect cell expressed counterpart. Optimization of the activity assay afforded a robust assay that displayed protein concentration dependent response while using unilamellar liposomes as the substrate. Agreeing with previous reports, the activity of PI4K-IIIβ was greatly reduced by wortmannin and increased by Triton X-100. The activity of PI4K-IIIβ was tested in the presence of active human PITPα and PITPβ, as well as yeast Frequenin and Sec14p, but none of them elicited a reproducible enhancement on PI(4)P production by PI4K-IIIβ. A similar pattern was observed with the human PI3-kinase, PIK3C3. Our results demonstrate that a PI presentation model based on heterotypic exchange may not occur in vitro, suggesting either that PITPs’ role in phosphoinositide production could rely uniquely on maintaining sufficient PI pools in the Golgi membrane or that additional protein partners may be required for the regulation of PI4K-IIIβ by PITPs
Adverse Childhood Experience-conditioned serum from young adults promotes a dysregulated endothelial cell gene expression profile
Get PDFINTRODUCTION AND OBJECTIVES: Adverse Childhood Experiences (ACEs) are some of the most intensive and frequently occurring sources of stress that children may suffer in early life. There events are known to cause health problems across the life course leading to earlier mortality. In particular, ACEs have been associated with an increased risk of developing cardiovascular disease (CVD) in adulthood, although the mechanisms leading to these outcomes remain poorly understood. Endothelial dysfunction is one of the earliest signs of cardiac pathology and can establish long-term changes in inflammatory state, blood composition, and vasomotor activity. The objective of this study was to investigate gene expression changes in primary endothelial cells when exposed to serum of those who have experienced ACEs (0 vs. ≥4). METHODS: Serum samples came from the Niagara Longitudinal Heart Study (NLHS). Samples collected from young male participants were divided into groups based on their ACE exposure (0 ACEs and ≥4 ACEs), with 20 samples investigated for each group. The blood serum was then added to near-confluent male human coronary artery endothelial cells (HCAEC) for various lengths of time (0, 1, 4, 24 hours). The mRNA from these cells were collected and an amplification efficiency-optimized qPCR design was used to determine expression changes for a panel of genes linked to endothelial dysfunction. RESULTS: When exposed to serum from the high ACEs group, the endothelial cells had significantly higher expression of Intercellular Adhesion Molecule 1 (ICAM1) (p<0.001), Chemokine Ligand 2 (CCL2) (p=0.02), Vascular Cell Adhesion Molecule 1 (VCAM1) (p<0.001), and Endothelin-1 (EDN1) (p<0.01) and lower expression of Nitric Oxide Synthase 3 (NOS3) (p=0.048) than the cells that were exposed to serum from the zero ACEs group. CONCLUSION: Overall, higher expression of inflammatory mediators (ICAM1, CCL2, VCAM1), as well as decreased expression of the vasodilator NOS3 and increased expression of the vasoconstrictor EDN1 points to an endothelial transcriptional profile in which high ACEs promote circulating blood conditions in young adulthood that indicate endothelial dysfunction. This suggests that dysfunction of the endothelium may be an early mechanism through which ACEs can lead to increased risk for poor CVD trajectories across the life course
Relational Approach to the L-Fuzzy Concept Analysis
Get PDFModern industrial production systems benefit from the classification and processing of objects and their attributes. In general, the object classification procedure can coincide with vagueness. Vagueness is a common problem in object analysis that exists at various stages of classification, including ambiguity in input data, overlapping boundaries between classes or regions, and uncertainty in defining or extracting the properties and relationships of objects.
To manage the ambiguity mentioned in the classification of objects, using a framework for L-fuzzy relations, and displaying such uncertainties by it can be a solution. Obtaining the least unreliable and uncertain output associated with the original data is the main concern of this thesis.
Therefore, my general approach to this research can be categorized as follows:
We developed an L-Fuzzy Concept Analysis as a generalization of a regular Concept Analysis. We start our work by providing the input data. Data is stored in a table (database). The next step is the creation of the contexts and concepts from the given original data using some structures. In the next stage, rules, or patterns (Attribute Implications) from the data will be generated. This includes all rules and a minimal base of rules. All of them are using L-fuzziness due to uncertainty. This requires L-fuzzy relations that will be implemented as L -valued matrices. In the end, everything is nicely packed in a convenient application and implemented in Java programming language. Generally, our approach is done in an algebraic framework that covers both regular and L -Fuzzy FCA, simultaneously.
The tables we started with are already L-valued (not crisp) in our implementation. In other words, we work with the L-Fuzzy data directly. This is the idea here. We start with vague data.
In simple terms, the data is shown using L -valued tables (vague data) trying to relate objects with their attributes at the start of the implementation. Generating attribute implications from many-valued contexts by a relational theory is the purpose of this thesis, i.e, a range of degrees is used to indicate the relationship between objects and their properties. The smallest degree corresponds to the classical no and the greatest degree corresponds to the classical yes in the table
Cycling and Running Yield a Similar Myokine and Osteokine Response in Young Adult Females
Get PDFThis study examined potential exercise-induced changes in the myokine (irisin), osteokines
(sclerostin, osteoprotegerin [OPG]), bone turnover markers (osteocalcin [OC]), and the bonemodulating
parathyroid hormone (PTH) in young women following two modes of high-intensity
interval exercise of high and low impact. Healthy, recreationally active, females (n=18; 22.5±2.7
years) performed two exercise trials in random order; high-intensity interval running on a
treadmill and high-intensity interval cycling on a cycle ergometer. Trials consisted of eight 1-
minute running or cycling intervals at ≥ 90% of maximal heart rate, separated by 1-minute passive
recovery intervals. Blood samples were collected at rest (pre-exercise), and 5 min, 1h, and 24h
following each exercise trial. Irisin, osteocalcin, sclerostin, osteoprotegerin (OPG), receptor
activator nuclear factor kappa-β ligand (RANKL), and parathyroid hormone (PTH) were analyzed
in serum with post-exercise concentrations being corrected for exercise-induced changes in
plasma volume. Irisin was higher than its resting values 24h post-exercise irrespective of impact
(time effect, p<0.05), and it was consistently higher during the cycling trial compared to running
at all time points (exercise mode effect, p<0.05) with no interaction. A significant time effect was
found for sclerostin, PTH, and OC, all of which increased from pre to 5 minutes post-exercise and
returned to baseline levels by 1 hour post-exercise, with no difference between exercise modes
and no exercise mode-by-time interaction. OPG also showed a significant main effect for time,
reflecting an overall increase at 5 minutes and 1 hour following high-intensity interval exercise
irrespective of exercise mode, which was not significant after the Bonferroni adjustment. In
conclusion, these results provide evidence that in young women, a single bout of high-intensity
exercise induces an immediate increase in upstream and downstream regulators of bone
remodeling, and a later response in irisin concentrations. These findings support the concept of
bone-muscle crosstalk during exercise independent of the impact of gravity loading
Post-activation potentiation and potentiated motor unit firing patterns in boys and men
Get PDFBackground
Post-activation potentiation (PAP) describes the enhancement of twitch torque following a conditioning contraction (CC) in skeletal muscle. In adults, PAP may be related to muscle fibre composition and is accompanied by a decrease in motor unit (MU) firing rates (MUFRs). Muscle fibre composition and/or activation is different between children and adults. This study examined PAP and MU firing patterns of the potentiated knee extensors in boys and men.
Methods
Twenty-three boys (10.5 ± 1.3 years) and 20 men (23.1 ± 3.3 years) completed familiarization and experimental sessions. Maximal isometric evoked-twitch torque and MU firing patterns during submaximal contractions (20% and 70% maximal voluntary isometric contraction, MVIC) were recorded before and after a CC (5 s MVIC). PAP was calculated as the percent-increase in evoked-twitch torque after the CC. MU firing patterns were examined during submaximal contractions before and after the CC using Trigno Galileo surface electrodes (Delsys Inc) and decomposition algorithms (NeuroMap, Delsys Inc). MU action potential amplitudes (MUAPamp) and MUFRs were calculated for each MU and exponential MUFR-MUAPamp relationships were calculated for each participant and trial.
Results
PAP was higher in men than in boys (98.3 ± 37.1% vs. 68.8 ± 18.3%, respectively; p = 0.002). Following potentiation, the rate of decay of the MUFR-MUAPamps relationship decreased in both contractions, with a greater decrease among boys during the high-intensity contractions.
Conclusion
Lower PAP in the boys did not coincide with smaller changes in potentiated MU firing patterns, as boys had greater reductions in MUFRs with potentiation compared with men in high-intensity contractions.Natural Science and Engineering Research Council of Canada
(B. Falk). S. Woods and A. McKiel hold Ontario Graduate Scholarships
Letter by Geo. Hatchey, September 22, 1812
Get PDFA letter by Geo. Hatchey, Camp at Williamsport, Pennsylvania, September 22, 1812, to Haynie Hatchey, Lunenburg County, Virginia. The letter is two pages and begins “Dear Brother”. Hatchey was a soldier from a troop of Virginia Cavalry in the War of 1812. He writes that they arrived last evening and will only stay for two days. They have been very hospitably treated in what is called the Dutch settlement along the Susquehanna. Colonel Coles was visited by a gentleman and lady to know if the Colonel would permit them to treat the soldiers. They later served cakes and coffee and it was a great treat to the soldiers, but it was his turn to mount Guard which meant he was unable to attend their home and drink a little wine with them. They expect to reach Niagara in the course of 20 days and will be in active service until Christmas
Mobility-based Multi-layered Caching and Data Distribution in Vehicular Fog Computing
Get PDFIntelligent transportation systems provide valuable services to drivers and passengers, whereas vehicular networks enable fundamental underlying support through communication and data sharing. While edge and fog computing offers the benefits of providing support to access data closer to devices and applications, they come at the cost of requiring ongoing communication and computation. %Vehicular edge and fog computing became essential to access data almost instantaneously and perform computation closer to devices and applications; however, there is a high demand for continuous communication and computation. Therefore, caching data is a practical approach as it improves performance and enhances data availability.
Moreover, the significant latency of the communication makes it prohibitive to fetch data from other sources. In the context discussed, caching data is a feasible option as it helps to increase performance and make data more readily accessible. Also, finding the desired data in the cache enables services besides improving performance. This study proposes a multi-layer caching mechanism that magnifies data availability while enhancing communication between the layers. The cache in the layers is distributive and updated on time based on the demand and criteria of the requests. We also distribute data using vehicular mobility by placing limited but significant data into the cache of the vehicle. These communication and data exchange types are standardized through policies in the proposed methodology. This cache management design is extensively analyzed using established frameworks and vehicular networks through simulated environments and visual constructions. Our simulation results indicate that the proposed method improves the performance of data availability and latency in vehicular fog computing. This approach can be applied to the diverse vehicle-to-everything use cases of the intelligent transportation system
Sherman Zavitz fonds, 1991-2021, n.d.
Get PDFSherman Zavitz was originally from Welland but moved to Niagara Falls in 1960. He was a teacher for 35 years and taught at several elementary schools in the city during his career. After moving to Niagara Falls he became interested in the City’s history and learned about it by reading, talking to others, and exploring the city. In 1994 the Lundy’s Lane Historical Society nominated Zavitz for the position of Official Historian for the City of Niagara Falls, which was approved by city council. He remained in this role until he retired in 2019. He was also Official Historian of the Niagara Parks Commission.
Zavitz wrote many columns for the Niagara Falls Review on the history of Niagara Falls, including two regular series, “A Niagara Note” and a photo feature known as “Niagara Then and Now”. In addition to these articles, Zavitz wrote several books on Niagara’s history including Niagara Falls: Historical Notes, Then and Now: Niagara Falls, Ontario, and It Happened at Niagara. Some of his other activities included hosting local walking tours, appearances on local TV and radio, past president of the Lundy’s Lane Historical Society, and serving on the Board of Directors for the Canadian Canal Society, the Friends of Fort George, and Niagara Falls Museums.Fonds contains news clippings concerning Sherman Zavitz, official historian for Niagara Falls, Ontario, from 1994 to 2019. Most of the clippings are weekly columns written by Zavitz on Niagara’s history including “A Niagara Note” and a photo feature known as “Niagara Then and Now”. There are also some articles that feature Sherman Zavitz and a few on general Niagara history
Distributed Supervised Statistical Learning
Get PDFWe live in the era of big data, nowadays, many companies face data of massive size that, in most cases, cannot be stored and processed on a single computer. Often such data has to be distributed over multiple computers which then makes the storage, pre-processing, and data analysis possible in practice. In the age of big data, distributed learning has gained popularity as a method to manage enormous datasets. In this thesis, we focus on distributed supervised statistical learning where sparse linear regression analysis is performed in a distributed framework. These methods are frequently applied in a variety of disciplines tackling large scale datasets analysis, including engineering, economics, and finance. In distributed learning, one key question is, for example, how to efficiently aggregate multiple estimators that are obtained based on data subsets stored on multiple computers. We investigate recent studies on distributed statistical inferences. There have been many efforts to propose efficient ways of aggregating local estimates, most popular methods are discussed in this thesis. Recently, an important question about the number of machines to deploy is addressed for several estimation methods, notable answers to the question are reviewed in this literature. We have considered a specific class of Liu-type shrinkage estimation methods for distributed statistical inference. We also conduct a Monte Carlo simulation study to assess performance of the Liu-type shrinkage estimation methods in a distributed framework
What is the influence of music on performance in practice and competition among university competitive fencers?
Get PDFFencing as a sport and music as an expressive form are two topics that may seem very distant in comparison, but both have many aspects that are intertwined. The purpose of this study was to understand how music is used within practice and competition settings and how rhythm, tempo and timing in fencing might be influenced by music. This study used grounded theory and its three-phase thematic analysis and applied a social-constructivist lens. The research question was: What is the influence of music on performance in practice and competition among university competitive fencers? The participants were interviewed using semi-structured interviews and the researcher kept retrospective notes on observations as an insider to the fencing community. The main findings were split into two groups that included practice and competition. Practice music influence showed that music was used to increase motivation but could also cause distraction from the practice. It also showed how one learned to develop fencing rhythm using music, and how auditory cues from music and from saying sounds that correspond to physical movements help with development of timing. Other findings were that fencers have practice structured around the way they learn, moving from learning in parts to wholes or easy to complex. Also noted was that each weapon has its own style that is free to be discovered and developed. Competition music influence was discovered to be almost non-existent other than for the use of pre-competition preparation and was used sometimes between bouts for relaxation purposes. Other findings were that due to external stressors, fencers tend to not be aware of what their body is doing. In their minds, the action feels correct, but it might have been too big or small or too fast or slow. Also, partner rhythm within a competition is difficult to manipulate as both opponents are trying not to follow each other’s footwork. Music seems to have an influence on those who use it to their advantage, but is connected to the athletes, coaches, and their way of learning