Drawing in the Domain: An investigation into the dialogue between analogue and digital tools through the design of a Paraparaumu sports clubrooms

<p>Tourism in New Zealand is often about nature. On the Kapiti Coast, tourism is preoccupied with the island, but this research looks at possibilities of cultural tourism in Paraparaumu township: away from risks of coastal erosion, sea level rise, and potential tsunamis. Sport is important to local culture and the objective is to explore the architectural potential for the Paraparaumu Domain. The existing Paraparaumu Rugby Club, Old Boys Cricket Club, Athletics Club, and Memorial Hall are all in separate buildings, used at separate times, during different parts of the year. What kind of architecture could help celebrate local sports culture? What kind of processes might be cultivated through such a project and how could they influence the design? Through the design of a shared sports clubrooms in the domain I searched for design processes and typological hybrids that could both connect to the local cultures, but also to discover more complex technical and spatial possibilities. Beginning with a typical cement block I explored an interior quality of the existing clubrooms; one that is unpretentious and modest. I experimented with analogue and digital processes to draw out a relationship between qualities, thinking how blunt instruments can come together with more sophisticated ones.</p>

Drawing in the Domain: An investigation into the dialogue between analogue and digital tools through the design of a Paraparaumu sports clubrooms

<p>Tourism in New Zealand is often about nature. On the Kapiti Coast, tourism is preoccupied with the island, but this research looks at possibilities of cultural tourism in Paraparaumu township: away from risks of coastal erosion, sea level rise, and potential tsunamis. Sport is important to local culture and the objective is to explore the architectural potential for the Paraparaumu Domain. The existing Paraparaumu Rugby Club, Old Boys Cricket Club, Athletics Club, and Memorial Hall are all in separate buildings, used at separate times, during different parts of the year. What kind of architecture could help celebrate local sports culture? What kind of processes might be cultivated through such a project and how could they influence the design? Through the design of a shared sports clubrooms in the domain I searched for design processes and typological hybrids that could both connect to the local cultures, but also to discover more complex technical and spatial possibilities. Beginning with a typical cement block I explored an interior quality of the existing clubrooms; one that is unpretentious and modest. I experimented with analogue and digital processes to draw out a relationship between qualities, thinking how blunt instruments can come together with more sophisticated ones.</p>

PRDM12 in Health and Diseases

PRDM12 is a member of the PRDI-BF1 (positive regulatory domain I-binding factor 1) homologous domain (PRDM)-containing protein family, a subfamily of Kruppel-like zinc finger proteins, controlling key processes in the development of cancer. PRDM12 is expressed in a spatio-temporal manner in neuronal systems where it exerts multiple functions. PRDM12 is essential for the neurogenesis initiation and activation of a cascade of downstream pro-neuronal transcription factors in the nociceptive lineage. PRDM12 inactivation, indeed, results in a complete absence of the nociceptive lineage, which is essential for pain perception. Additionally, PRDM12 contributes to the early establishment of anorexigenic neuron identity and the maintenance of high expression levels of pro-opiomelanocortin, which impacts on the program bodyweight homeostasis. PRDMs are commonly involved in cancer, where they act as oncogenes/tumor suppressors in a “Yin and Yang” manner. PRDM12 is not usually expressed in adult normal tissues but its expression is re-activated in several cancer types. However, little information is currently available on PRDM12 expression in cancers and its mechanism of action has not been thoroughly described. In this review, we summarize the recent findings regarding PRDM12 by focusing on four main biological processes: neurogenesis, pain perception, oncogenesis and cell metabolism. Moreover, we wish to highlight the importance of future studies focusing on the PRDM12 signaling pathway(s) and its role in cancer onset and progression.

Interaction Study between ESIPT Fluorescent Lipophile-Based Benzazoles and BSA

In this study, the interactions of ESIPT fluorescent lipophile-based benzazoles with bovine serum albumin (BSA) were studied and their binding affinity was evaluated. In phosphate-buffered saline (PBS) solution these compounds produce absorption maxima in the UV region and a main fluorescence emission with a large Stokes shift in the blue–green regions due to a proton transfer process in the excited state. The interactions of the benzazoles with BSA were studied using UV-Vis absorption and steady-state fluorescence spectroscopy. The observed spectral quenching of BSA indicates that these compounds could bind to BSA through a strong binding affinity afforded by a static quenching mechanism (Kq~1012 L·mol−1·s−1). The docking simulations indicate that compounds 13 and 16 bind closely to Trp134 in domain I, adopting similar binding poses and interactions. On the other hand, compounds 12, 14, 15, and 17 were bound between domains I and III and did not directly interact with Trp134.

Novel dynamic residue network analysis approaches to study homodimeric allosteric modulation in SARS-CoV-2 Mpro and in its evolutionary mutations

The rational search for allosteric modulators and the allosteric mechanisms of these modulators in the presence of evolutionary mutations, including resistant ones, is a relatively unexplored field. Here, we established novel in silico approaches and applied to SARS-CoV-2 main protease (Mpro). First, we identified six potential allosteric modulators (SANC00302, SANC00303, SANC00467, SANC00468, SANC00469, SANC00630) from the South African Natural Compounds Database (SANCDB) bound to the allosteric pocket of Mpro that we determined in our previous work. We also checked the stability of these compounds against Mpro of laboratory strain HCoV-OC43 and identified differences due to residue changes between the two proteins. Next, we focused on understanding the allosteric effects of these modulators on each protomer of the reference Mpro protein, while incorporating the symmetry problem in the functional homodimer. In general, asymmetric behavior of multimeric proteins is not commonly considered in computational analysis. We introduced a novel combinatorial approach and dynamic residue network (DRN) analysis algorithms to examine patterns of change and conservation of critical nodes, according to five independent criteria of network centrality (betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigencentrality (EC) and katz centrality (KC)). The relationships and effectiveness of each metric in characterizing allosteric behavior were also investigated. We observed highly conserved network hubs for each averaged DRN metric on the basis of their existence in both protomers in the absence and presence of all ligands, and we called them persistent hubs (residues 17, 111, 112 and 128 for averaged BC; 6, 7, 113, 114, 115, 124, 125, 126, 127 and 128 for averaged CC; 36, 91, 146, 150 and 206 for averaged DC; 7, 115 and 125 for EC; 36, 125 and 146 for KC). We also detected ligand specific signal changes some of which were in or around functional residues (i.e. chameleon switch PHE140). Using EC persistent hubs and ligand introduced hubs we identified a residue communication path between allosteric binding site and catalytic site. Finally, we examined the effects of the mutations on the behavior of the protein in the presence of selected potential allosteric modulators and investigated the ligand stability. The hit compounds showed various levels of stability in the presence of SARS-CoV-2 Mpro mutations, being most stable in A173V, N274D and R279C, and least stable in R60C, N151D V157I, C160S and A255V. SANC00468 was the most stable compound in the 43 mutant protein systems. We further used DRN metric analysis to define cold spots as being those regions that are least impacted, or not impacted, by mutations. One crucial outcome of this study was to show that EC centrality hubs form an allosteric communication path between the allosteric ligand binding site to the active site going through the interface residues of Domain I and II; and this path was either weakened or lost in the presence of some of the mutations. Overall, the results of this study revealed crucial aspects that need to be considered in drug discovery in COVID-19 specifically and in general for rational computational drug design purposes.

Cannabidiol Selectively Binds to the Voltage-Gated Sodium Channel Nav1.4 in Its Slow-Inactivated State and Inhibits Sodium Current

Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M−1 s−1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 μM vs. 51.48 μM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

The intervening domain is required for DNA-binding and functional identity of plant MADS transcription factors

The MADS transcription factors (TF) are an ancient eukaryotic protein family. In plants, the family is divided into two main lineages. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the MADS domain (M domain) called the Intervening domain (I domain) that was previously defined only in type II lineage MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a conserved fold with the I domain acting to stabilise the M domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters genome-wide DNA-binding specificity and dimerisation specificity. Introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant resulted in strong complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts as an integral part of the DNA-binding domain and significantly contributes to the functional identity of the MADS TF.