Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.Peer reviewe

Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules

<p>Abstract</p> <p>Objective</p> <p>Here we report the improved results of a new siRNA design program and analysis tool called <b><it>siRNA_profile </it></b>that reveals an additional criterion for bioinformatic search of highly functional siRNA sequences.</p> <p>Methods</p> <p>We retrospectively analysed over 2400 siRNA sequences from 34 genes and with known efficacies to categorize factors that differentiate highly, moderately and non-functional siRNA sequences in more detail. We tested the biological relevance of <b><it>siRNA_profile </it></b>in CHO cells stably expressing human TRACP.</p> <p>Results</p> <p>The highly functional siRNA molecules exhibited lower overall stabilities than non-functional siRNAs after taking into consideration all the nucleotides from 5'-terminus to the 3'-terminus along the siRNA molecule, in addition to the 5'-section of the antisense strand and the region between 9–14 nucleotides as previously has been acknowledged. Comparison of the <b><it>siRNA_profile </it></b>program to five other programs resulted in a wide range of selected siRNA sequences with diverse gene silencing capacities, even when the target was only 197 nucleotides long. Six siRNA design programs selected 24 different siRNA sequences, and only 6 of them were selected by two or more programs. The other 18 sequences were individually selected by these six programs.</p> <p>Conclusion</p> <p>Low general stability of dsRNA plays a significant role in the RNAi pathway and is a recommended criterion to consider, in addition to 5'-instability, internal instability, nucleotide preferences and target mRNA position, when designing highly efficient siRNAs.</p

Spin coated chitin films for biosensors and its analysis are dependent on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.Peer reviewe

Total and Carboxylated Osteocalcin Associate with Insulin Levels in Young Adults Born with Normal or Very Low Birth Weight

Peer reviewe

Towards an ecology of participation: Process philosophy and co-creation of higher education curricula

This article brings together the authors' previous work on co-created curricula (Bovill et al., 2011; Bovill, 2013a; Bovill, 2014) and on partnership and ethics (Taylor and Robinson, 2014; Taylor, 2015), to develop the concept of co-created curricula as an ecology of participation. In doing so, it deploys Alfred North Whitehead’s process philosophy to formulate a new way of considering co-creation in the curriculum and co-creation of the curriculum in higher education. Two empirical examples are used to illuminate what such an approach offers. From this, we outline three dimensions of an ecology of participation: a process of becoming which recasts subjectivity; acting well in relation which enacts concern; and an orientation to harmony in which difference in equality is valued. The contribution of the article is twofold: first, the concept of an ecology of participation takes forward current thinking on higher education curricula and partnership ethics; second, its use of process philosophy provides a new lens to consider co-creation in the curriculum and co-creation of the curriculum

Learning spaces: built, natural and digital considerations for learning and learners

The goal of this chapter is to assess research that can inform understandings of places and spaces of learning. The chapter assesses evidence across three types of learning spaces: built spaces, digital spaces, and natural spaces. It looks at the role of these different kinds of spaces for learning, attainment, interpersonal relationships, skills development, wellbeing and behaviours ‒ across four pillars of learning to know, to be, to do and to live together. The chapter also explores how learning spaces can be actively shaped, felt and understood through practices and policies that occur within and around them

Reimagining Education: The International Science and Evidence based Education Assessment.

The goal of this chapter is to assess research that can inform understandings of places and spaces of learning. The chapter assesses evidence across three types of learning spaces: built spaces, digital spaces, and natural spaces. It looks at the role of these different kinds of spaces for learning, attainment, interpersonal relationships, skills development, wellbeing and behaviours ‒ across four pillars of learning to know, to be, to do and to live together. The chapter also explores how learning spaces can be actively shaped, felt and understood through practices and policies that occur within and around them.Additional co-authors: Jo Hickman Dunne, Sarah Howard, John Lupinacci, Jamie Mcphie, Manulani Aluli Meyer, Selena Nemorin, Fikile Nxumalo, Rosie Parnell, Jessica Pykett, Pauliina Rautio, Julian Sefton Green, Neil Selwyn, David Stovall, Erica Southgate, Robert Talbert, Cheryl Teelucksingh, Richard Tucker, Ola Uduku, Alex Wilson, Adam Woo

Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

Background: Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages 6150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl2,0.31), while the other two types of channels are slightl

Alendronate decreases orthotopic PC-3 prostate tumor growth and metastasis to prostate-draining lymph nodes in nude mice

<p>Abstract</p> <p>Background</p> <p>Metastatic prostate cancer is associated with a high morbidity and mortality but the spreading mechanisms are still poorly understood. The aminobisphosphonate alendronate, used to reduce bone loss, has also been shown to inhibit the invasion and migration of prostate cancer cells <it>in vitro</it>. We used a modified orthotopic PC-3 nude mouse tumor model of human prostate cancer to study whether alendronate affects prostate tumor growth and metastasis.</p> <p>Methods</p> <p>PC-3 cells (5 × 10⁵) were implanted in the prostates of nude mice and the mice were treated with alendronate (0.5 mg/kg/day in PBS, s.c.) or vehicle for 4 weeks. After sacrifice, the sizes of tumor-bearing prostates were measured and the tumors and prostate-draining regional iliac and sacral lymph nodes were excised for studies on markers of proliferation, apoptosis, angiogenesis and lymphangiogenesis, using histomorphometry and immunohistochemistry.</p> <p>Results</p> <p>Tumor occurrence in the prostate was 73% in the alendronate-treated group and 81% in the control group. Mean tumor size (218 mm³, range: 96–485 mm³, <it>n </it>= 11) in the alendronate-treated mice was 41% of that in the control mice (513 mm³, range: 209–1350 mm³, <it>n </it>= 13) (<it>p </it>< 0.05). In the iliac and sacral lymph nodes of alendronate-treated mice, the proportion of metastatic area was only about 10% of that in control mice (<it>p </it>< 0.001). Immunohistochemical staining of tumor sections showed that alendronate treatment caused a marked decrease in the number of CD34-positive endothelial cells in tumors (<it>p </it>< 0.001) and an increase in that of ISEL positive apoptotic cells in tumors as well as in lymph node metastases (<it>p </it>< 0.05) compared with those in the vehicle-treated mice. The density of m-LYVE-1-stained lymphatic capillaries was not changed.</p> <p>Conclusion</p> <p>Our results demonstrate that alendronate treatment opposes growth of orthotopic PC-3 tumors and decreases tumor metastasis to prostate-draining lymph nodes. This effect could be at least partly explained by decreased angiogenesis and increased apoptosis. The results suggest that bisphosphonates have anti-tumoral and anti-invasive effects on primary prostate cancer.</p

Automated Essay Scoring in Australian Schools: Key Issues and Recommendations

This summary outlines critical issues associated with the use of Automated Essay Scoring (AES) technology in the Australian education system. The key insights presented in this paper emerged from a collaborative, multi-stakeholder workshop held in July 2022 that explored an automated essay-scoring trial and generated future possibilities aligned with participant interests and expertise. Drawing on the workshop and our expert understanding of the wider landscape, we propose recommendations that can be adopted by various stakeholders, schools, and educational systems. There are compelling reasons for Australian schools and education departments to investigate the use of AES. AES could potentially alleviate aspects of teachers’ workload at a time when teacher attrition is historically high and teacher recruitment historically low. At the same time, AES also has the potential to de-professionalise and deskill of teachers. Educationalists are acutely aware that quality feedback can help students improve their learning across multiple subjects and domains, however parents and many are reluctant to hand that responsibility over to AES. In 2018, concerns among teachers, teachers’ unions, principles, and parents became apparent when the federal Department of Education, Skills and Employment attempted to implement a form of AES in The National Assessment Program – Literacy and Numeracy (NAPLAN). These concerns primarily registered around three issues: − de-professionalisation of teachers, − inequitable infrastructure in Australian schools, and − lack of transparency from examination authorities as to how marking decisions are made. The use of AES in NAPLAN ultimately proved to be politically unpopular, leading to its suspension. However, the growing implementation of AES in schools across the globe means that the use of this technology is likely to re-emerge as a controversial issue in Australia. Without political leadership in this area, it is ultimately up to educational institutions and agencies, policymakers, and school communities to assess the benefits and pitfalls of AES and navigate the way forward. Our recommendations will assist the emergence of good governance in this area. To begin, it is crucially important to identify whether AES will be used in high-stakes or low-stakes tests. High-stakes tests are defined as those with consequential outcomes for students or educators, such as the determination of progression of students or rankings of school institutions. If AES is to be used in Australian schools, the following issues must be considered: − the capacity of stakeholders, including principals, teachers, and parents, to understand how AES systems work − the infrastructure required to support the use of AES − the potential impacts of AES on assessment and workload practices which requires adequate professional development resources − competing interests and values between schools, departments, and institutions associated with using AES − how the use of AES relates to and integrates with broader policy frameworks. The investigation of these issues requires information sharing, dialogue, and negotiation among diverse stakeholders, including teachers, parents, students, leaders, and policymakers. In addition to this engagement, schools and other educational institutions must also discuss the implementation of AES tools with AES system developers and commercial vendors, so as to better understand the functions and limitations of the AES tool, as well as its implications for professional and assessment practices. Only then can decision-makers evaluate whether a specific AES system is worth the investment of funds and resources, including teacher workload, in both the medium and longer term. Although it appears as yet another drag on teacher time, the participatory and collaborative development of AES guidance, policy, and regulation is crucial. It ensures that pluralistic views and shared values are reflected in any innovations or reforms across the education sector. To ensure a collaborative foundation, the introduction of AES must be informed by stakeholder expertise across multiple locations and decision-making levels, including classrooms, schools, organisations, and state, territory, and national jurisdictions. For Australia, we recommend multi-scalar policy development informed by educators, policymakers, and representatives from educational technology companies engaging in cooperative learning and action