Effectiveness and energy requirements of pasteurisation for the treatment of unfiltered secondary effluent from a municipalwastewater treatment plant

Pasteurisation was investigated as a process to achieve high microbial quality standards in the recycling of water from unfiltered secondary effluents from a wastewater treatment plants in Melbourne, Australia. The relative heat sensitivity of key bacterial, viral, protozoan and helminth wastewater organisms (Escherichia coli, Enterococcus, FRNA bacteriophage, adenovirus, coxsackievirus, Cryptosporidium, and Ascaris) were determined by laboratory scale tests. The FRNA phage were found to be the most heat resistant, followed by enterococci and E. coli. Pilot scale challenge testing of a 2 ML/day pasteurisation pilot plant using unfiltered municipal wastewater and male specific coliphage (MS2) phage showed that temperatures between 69 °C and 75 °C achieved log reductions values between 0.9 ± 0.1 and 5.0 ± 0.5 respectively in the contact chamber. Fouling of the heat exchangers during operation using unfiltered secondary treated effluent was found to increase the energy consumption of the plant from 2.2 kWh/kL to 5.1 kWh/kL. The economic feasibility of pasteurisation for the current municipal application with high heat exchanger fouling potential can be expected to depend largely on the available waste heat from co-generation and on the efforts required to control fouling of the heat exchangers

Repurposing triphenylmethane dyes to bind to trimers derived from Aß

Accepted author manuscriptSoluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer’s disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ17–36. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N,N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe19, Phe20, and Ile31 were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe20 side chains and is clasped by the Ile31 side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.Ye

Detection of tumour lymphovascular space invasion using dual cytokeratin and CD31 immunohistochemistry

Background: Lymphovascular space invasion (LVSI) is an important step in the complex process of tumour metastasis. Various methods have been used in the past to improve the histological detection of LVSI. Aims: To develop a sensitive immunohistochemical method for the detection of LVSI. Methods: Paraffin wax blocks from 108 patients who had undergone hysterectomy for stage I endometrial cancer were retrieved. Dual immunostaining for pancytokeratin and the CD31 endothelial cell marker was carried out on 4 μm sections cut from these bocks and compared with conventional haematoxylin and eosin staining. Results: The detection rate for LVSI increased threefold compared with conventional haematoxylin and eosin staining in the test group. Conclusion: This finding suggests that LVSI is a much more common phenomenon than previously thought and questions current understanding of tumour metastasis

X‑ray Crystallographic Structure of a Compact Dodecamer from a Peptide Derived from Aβ_16–36

The assembly of the β-amyloid peptide, Aβ, into soluble oligomers is associated with neurodegeneration in Alzheimer’s disease. The Aβ oligomers are thought to be composed of β-hairpins. Here, the effect of shifting the residue pairing of the β-hairpins on the structures of the oligomers that form is explored through X-ray crystallography. Three residue pairings were investigated using constrained macrocyclic β-hairpins in which Aβ_30–36 is juxtaposed with Aβ_17–23, Aβ_16–22, and Aβ_15–21. The Aβ_16–22–Aβ_30–36 pairing forms a compact ball-shaped dodecamer composed of fused triangular trimers. This dodecamer may help explain the structures of the trimers and dodecamers formed by full-length Aβ

Correction to “Repurposing Triphenylmethane Dyes To Bind to Trimers Derived from Aβ”

There was an error in listing the PDB ID numbers in the captionof Figure 7. The corrected caption should read as follows: (Figure Presented)

Controlling the Oligomerization State of Aβ-Derived Peptides with Light

A key challenge in studying the biological and biophysical properties of amyloid-forming peptides is that they assemble to form heterogeneous mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting groups have emerged as tools to control the properties of biomolecules with light. Blocking intermolecular hydrogen bonds that stabilize amyloid oligomers provides a general strategy to control the biological and biophysical properties of amyloid-forming peptides. In this paper we describe the design, synthesis, and characterization of macrocyclic β-hairpin peptides that are derived from amyloidogenic peptides and contain the <i>N</i>-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aβ_16–36 or Aβ_17–36 constrained into β-hairpins. The <i>N</i>-2-nitrobenzyl group is appended to the amide backbone of Gly₃₃ to disrupt the oligomerization of the peptides by disrupting intermolecular hydrogen bonds. X-ray crystallography reveals that <i>N</i>-2-nitrobenzyl groups can either block assembly into discrete oligomers or permit formation of trimers, hexamers, and dodecamers. Photolysis of the <i>N</i>-2-nitrobenzyl groups with long-wave UV light unmasks the amide backbone and alters the assembly and the biological properties of the macrocyclic β-hairpin peptides. SDS–PAGE studies show that removing the <i>N</i>-2-nitrobenzyl groups alters the assembly of the peptides. MTT conversion and LDH release assays show that decaging the peptides induces cytotoxicity. Circular dichroism studies and dye leakage assays with liposomes reveal that decaging modulates interactions of the peptides with lipid bilayers. Collectively, these studies demonstrate that incorporating <i>N</i>-2-nitrobenzyl groups into macrocyclic β-hairpin peptides provides a new strategy to probe the structures and the biological properties of amyloid oligomers

X‑ray Crystallographic Structures of Oligomers of Peptides Derived from β₂‑Microglobulin

Amyloid diseases such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes share common features of toxic soluble protein oligomers. There are no structures at atomic resolution of oligomers formed by full-length amyloidogenic peptides and proteins, and only a few structures of oligomers formed by peptide fragments. The paucity of structural information provides a fundamental roadblock to understanding the pathology of amyloid diseases and developing preventions or therapies. Here, we present the X-ray crystallographic structures of three families of oligomers formed by macrocyclic peptides containing a heptapeptide sequence derived from the amyloidogenic E chain of β₂-microglobulin (β₂m). Each macrocyclic peptide contains the heptapeptide sequence β₂m_63–69 and a second heptapeptide sequence containing an <i>N</i>-methyl amino acid. These peptides form β-sheets that further associate into hexamers, octamers, and dodecamers: the hexamers are trimers of dimers; the octamers are tetramers of dimers; and the dodecamers contain two trimer subunits surrounded by three pairs of β-sheets. These structures illustrate a common theme in which dimer and trimer subunits further associate to form a hydrophobic core. The seven X-ray crystallographic structures not only illustrate a range of oligomers that a single amyloidogenic peptide sequence can form, but also how mutation can alter the size and topology of the oligomers. A cocrystallization experiment in which a dodecamer-forming peptide recruits a hexamer-forming peptide to form mixed dodecamers demonstrates that one species can dictate the oligomerization of another. These findings should also be relevant to the formation of oligomers of full-length peptides and proteins in amyloid diseases