Bacterial curli protein promotes the conversion of PAP248-286 into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences

Fragments of prostatic acid phosphatase (PAP248-286) in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection), however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg) acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus) infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide) and Aβ1−40) with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1−40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed

The design, synthesis, and evaluation of compounds that bind to Alzheimer's-related and HIV-1-related amyloids

Amyloids--misfolded, aggregated peptides--have been implicated in over thirty human diseases. This thesis focused on the study of two different amyloids--A[beta](1- 42) and SEVI (semen-derived enhancer of virus infection)-- associated with two distinct conditions--Alzheimer's Disease (AD) and Acquired Immune Deficiency Syndrome (AIDS), respectively. A[beta] aggregates are a hallmark of AD and may play a central, causative role in the pathogenesis of this disease. A[beta]-amyloid-targeting small molecules have, therefore, attracted wide interest as potential agents for the treatment or diagnosis of AD. This thesis describes the development of a general method to evaluate small molecule-A[beta]-amyloid binding interactions via a modified quantitative ELISA protocol. The implementation of an in vitro model to evaluate the blood-brain barrier permeability of [beta]-amyloid- targeting compounds is also discussed in this thesis. The design and evaluation of a new class of fluorescent probes that bind to A[beta] aggregates is described in this thesis. The advantage of these compounds is that their spectroscopic properties can be altered and fine-tuned via simple synthetic methods. The second portion of this thesis discusses the study of small molecules that bind to SEVI, a naturally abundant amyloid found in semen. SEVI can potentially increase the infectivity of HIV-1 in cells by up to 400,000-fold. Although the mechanism of SEVI- mediated transmission of HIV-1 remains poorly understood, evidence suggests that SEVI binds to both HIV-1 virions and cell membranes, thereby facilitating viral infection. We hypothesized that BTA-EG₆, a derivative of the well- known amyloid-binding compound Thioflavin T, could coat SEVI fibrils, thereby inhibit HIV-1 interactions with SEVI fibrils, and thus, reduce SEVI-mediated enhancement of HIV -1 infectivity. The results of these investigations are presented in this thesis. The final project described in this thesis is the design, synthesis, and evaluation of multivalent analogs of BTA-EG6. The goals of this project were 2-fold : 1) Create compounds that bind with high affinity to both A[beta] fibrils and SEVI fibrils based on the multivalent design strategy and 2) evaluate whether oligomers of the BTA moiety exhibit improved ability over the BTA monomer to inhibit SEVI-mediated enhancement of HIV-1 infectivity. The results of this project are presented in this thesi

Noncovalent, Electrostatic Interactions Induce Positively Cooperative Binding of Small Molecules to Alzheimer's and Parkinson's Disease-Related Amyloids.

Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases

Experiences of young unwed mothers

This study focused on the experiences of 30 young unwed mothers, aged 15-21 years old. This was done by looking into the changes brought about by the pregnancy, feelings toward their situation and coping behaviors. The method used was descriptive exploratory. The respondents were obtained through chain-referral, they were either referred by the respondents or the friends of the researchers. Copies of a survey questionnaire were answered by the subjects, enabling the researchers to choose 9 young unwed mothers for interviews, it was found that most of these individuals regret. Although they claimed to regret because of the changes in their career and physical lifestyles, results showed that these aspects were interrelated with regrets brought about by the changes in their relationships

Performance and governance in selected business-centered microfinance institutions: The Metro Manila setting

The paper examines the relationship between governance factors and performance measures of selected microfinance institutions (MFIs). Using panel data estimations, the effects of governance factors, divided in two: internal and external, are going to be studied on the MFIs\u27 performance measures, mainly for financial performance and outreach to clients. The study will find out if the results of the governance-performance relationship are significant in the Metro Manila (Philippine) setting. The results will determine if the approach of measuring performance is significant through its governance

Enzyme-Linked Immunosorbent Assay-Based Method to Quantify the Association of Small Molecules with Aggregated Amyloid Peptides

This paper describes a simple enzyme linked immunosorbent assay (ELISA) protocol for quantifying the binding of small molecules to aggregated β-amyloid (Aβ) peptides. Amyloid-targeting small molecules have attracted wide interest as potential agents for the treatment or diagnosis of neurodegenerative disorders such as Alzheimer’s disease. The lack of general methods to evaluate small molecule–amyloid binding interactions, however, has significantly limited the number of amyloid-targeting molecules that have been studied to date. Here, we demonstrate a general method to quantify small molecule–amyloid binding interactions via a modified quantitative ELISA protocol. A key feature of this protocol is the treatment of commercial ELISA plates with an air plasma to help maintain the desired β-sheet content of the aggregated Aβ upon immobilization of these peptides on to the polystyrene surface. We developed an ELISA-based competition assay on these air plasma-treated plates and evaluated the binding of five previously known amyloid-binding small molecules to aggregated Aβ. We show that this general ELISA-based competition assay can be used to quantify small molecule–amyloid binding interactions in the low nanomolar to low micromolar range, which is the typical range of affinities for many amyloid-targeting diagnostic agents under current development. This simple protocol for quantifying the interaction of small molecules with aggregated Aβ peptides overcomes many limitations of previously reported spectroscopic or radioactivity assays and may, therefore, facilitate the screening and evaluation of a more structurally diverse set of amyloid-targeting agents than had previously been possible

Noncovalent, Electrostatic Interactions Induce Positively Cooperative Binding of Small Molecules to Alzheimer's and Parkinson's Disease-Related Amyloids.

Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases

Sources of cost and differentiation drivers of filstar Distributor Corporation and Fiesta Greetings Incorporated

The researchers employed a comparative case study to determine if there are significant differences in cost and differentiation drivers used for two firms. Exploratory research was used to find out the firms\u27 financial information and its value chain activities. In-depth interviews were conducted with key personnel of both firms to determine the firms\u27 perceived sources of cost advantage and focus group discussions were held with the products\u27 target market to determine the sources of differentiation advantage on the firm perceived by the consumer. Based from the in-depth interviews and focus group discussions, the researchers found that there are significant differences in the perceived sources of cost and differentiation advantage. It was found in the study that savings in costs may not automatically result to higher profit. The study stresses the importance of balancing a firm\u27s cost and differentiation advantages to maximize firm performance

Medicines management in the Philippine public sector during the response to Haiyan

Introduction: Health service delivery in the Philippines is constantly challenged by disasters and emergencies. This descriptive study documented existing policies for medicines management in the Philippines and then assessed these in the public sector response post-Haiyan. Method: We used desk a review of existing laws, regulations and related issuances and a series of interviews of key informants from various national and local health agencies. Results: We found that while numerous national policies covered critical aspects of medicines management, implementation post-Haiyan was problematic at all levels of the decentralized health-care system. We identified issues of quantification, warehousing, distribution, utilization monitoring and disposal. Donated medicines also added additional burden for storage and disposal, especially for expired and unwanted medicines. Discussion: While the process of managing medicines during disasters did not differ greatly from non-emergency situations, the Haiyan experience highlighted the system’s weaknesses. With the current gaps in implementation, as well as the logistical obstacles brought about by disasters, there is a need to have integrated mechanisms for medicines management in the Philippines. This assessment provided an important opportunity to review the medicines management policies at national and local levels