title: Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent

authors: Steemson, John Durand

subjects: OB-fold

abstract: Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies.

text: Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies.

year: 2011

title: Prevalence and risk factors for musculoskeletal pain when running during pregnancy: A survey of 3102 women

authors: Wyatt, Hannah E.

abstract: Background: Musculoskeletal pain while running is a concern to women during pregnancy and can lead to running cessation. To support women who wish to run during pregnancy, it is essential to understand the sites, severities and personal risk factors associated with musculoskeletal pain. Objective: The aim was to investigate prevalence and risk factors for musculoskeletal pain when running during pregnancy. Methods: An online survey was completed by women who had a child in the past 5 years and ran prior to and during pregnancy. Pain frequency informed prevalence rates by body site, and logistic regression odds ratios (ORs) and 95% confidence intervals were calculated. Results: A total of 3102 women of 23 ethnicities from 25 countries completed the survey. Women were 22–52 years old when they gave birth and ran 2–129 km/week during the 0.5–35 years before the birth of their youngest child. Women ran significantly less distance and less often during pregnancy than before pregnancy. Most women (86%) experienced pain while running during pregnancy (59% pelvis/sacroiliac joint, 52% lower back, 51% abdomen, 44% breast, 40% hip). The highest prevalence of severe-to-worst pain was at the pelvis/sacroiliac joint (9%). Women at greatest risk of pain while running during pregnancy had a previous injury (OR = 3.44) or were older (OR = 1.04). Women with a previous child were less likely to experience breast pain (OR = 0.76) than those running during their first pregnancy. Conclusion: Healthcare practices to reduce pain should focus on regions of greatest musculoskeletal change during pregnancy, specifically the pelvis, lower back and abdomen. Efforts to support women to run for longer throughout pregnancy should focus on pain at the pelvis and breasts.

text: Prevalence and risk factors for musculoskeletal pain when running during pregnancy: A survey of 3102 women Background: Musculoskeletal pain while running is a concern to women during pregnancy and can lead to running cessation. To support women who wish to run during pregnancy, it is essential to understand the sites, severities and personal risk factors associated with musculoskeletal pain. Objective: The aim was to investigate prevalence and risk factors for musculoskeletal pain when running during pregnancy. Methods: An online survey was completed by women who had a child in the past 5 years and ran prior to and during pregnancy. Pain frequency informed prevalence rates by body site, and logistic regression odds ratios (ORs) and 95% confidence intervals were calculated. Results: A total of 3102 women of 23 ethnicities from 25 countries completed the survey. Women were 22–52 years old when they gave birth and ran 2–129 km/week during the 0.5–35 years before the birth of their youngest child. Women ran significantly less distance and less often during pregnancy than before pregnancy. Most women (86%) experienced pain while running during pregnancy (59% pelvis/sacroiliac joint, 52% lower back, 51% abdomen, 44% breast, 40% hip). The highest prevalence of severe-to-worst pain was at the pelvis/sacroiliac joint (9%). Women at greatest risk of pain while running during pregnancy had a previous injury (OR = 3.44) or were older (OR = 1.04). Women with a previous child were less likely to experience breast pain (OR = 0.76) than those running during their first pregnancy. Conclusion: Healthcare practices to reduce pain should focus on regions of greatest musculoskeletal change during pregnancy, specifically the pelvis, lower back and abdomen. Efforts to support women to run for longer throughout pregnancy should focus on pain at the pelvis and breasts.

year: 2024-02-06

title: Monitoring te mana o te wai: Intergrating an indigenous cultural health index with conventional biomonitoring tools for improved freshwater management

authors: Edgecombe, Shana Jade Te Iaa Tarere

subjects: Freshwater

abstract: Globally, there are universal efforts to quantify and address human impacts on ecosystems. In particular, changes in land use and river regulation have led to drastic declines in stream and river ecosystem health. These human impacts are driving the global freshwater biodiversity crisis and degrade ecosystem services provided by aquatic habitats. In Aotearoa New Zealand there is widespread concern about the state of our Nation’s freshwater ecosystems. Coupled with a renaissance in Māori culture, there has been a drive to develop indicators that help articulate cultural values, assess ecosystem health from a cultural perspective, and provide greater agency for Māori in environmental monitoring and management. In my thesis, I sought to integrate an established cultural monitoring framework with conventional biomonitoring tools for improved freshwater management. I hypothesized that using a suite of monitoring approaches would help detect the impacts of human land use on stream ecosystem health, and that cultural indicators would be consistent with conventional measures in diagnosing land use impacts. I also hypothesized that there could be discrepancies between cultural monitoring and conventional approaches, as each represent different perspectives and ways of knowing streams and rivers. 
To test my hypotheses, I selected ten stream sites across the catchment of the Kuratau River, a major tributary of Lake Taupō. Based on River Environment Classification land cover types, I used a balanced study design with five sites draining indigenous forest, and five sites draining pastoral land. I measured key stream physicochemical properties and collected water samples for determination of nutrient concentration and faecal coliform counts. I also characterised instream and riparian habitat. I collected benthic macroinvertebrate samples and sampled water for environmental DNA (eDNA) to assess different facets of biodiversity. I measured decomposition using the Cotton Strip Assay and quantified periphyton biomass using a portable fluorometer. In combination with these approaches, I also used a Cultural Health Index, an established cultural monitoring framework that assesses three components: customary significance, taonga species/mahinga kai (food gathering), and the Cultural Stream Health Measure (CSHM) to assess identified attributes for stream health.
I found that pastoral land uses lead to a decline in stream ecosystem health when compared to sites draining indigenous forest. Pastoral sites had higher specific conductivity and concentrations of total nitrogen and nitrate. Habitat variables changed, with increased sedimentation and more degraded riparian zones in pastoral streams. Decomposition rates as measured by the cotton strip assay increased in the pasture sites, which was likely a response to increased nutrient availability. There were shifts in macroinvertebrate composition, with key indicator taxa for Indigenous forest sites including pollution-sensitive mayflies and stoneflies. Total taxa, EPT (Ephemeroptera, Plecoptera, Trichoptera), and aquatic insect richness all significantly declined in pastoral sites. Increases in primary production were less pronounced, but did increase alongside abundances of grazing caddisflies and snails in the sites draining pastoral catchments. 
The Cultural Health Index indicated that sites in the Kuratau River catchment had customary and mahinga kai values. CSHM responded negatively to pastoral land uses, and the effect size it described was congruent with other responses. The CSHM was strongly correlated with an indicator of riparian habitat condition (the Riparian Condition Index) and had weak negative correlations with sedimentation and stream temperatures. The CSHM was not correlated with macroinvertebrate indices, which may have been because of the relatively few sites sampled, the narrow impact gradient in the Kuratau catchment, and the lack of temporal replication. However, cultural monitoring approaches should be seen to complement conventional environmental monitoring methods for assessing freshwater and not be expected to duplicate them. The strength of the Cultural Health Index approach comes from establishing Māori agency to assess the environment through a Te Ao Māori lens, and to support and foster inclusion for tangata whenua epistemologies in environmental monitoring and resource management. Future research should consider greater hapū involvement and further explore the mechanisms driving changes in stream health and biodiversity.

text: Monitoring te mana o te wai: Intergrating an indigenous cultural health index with conventional biomonitoring tools for improved freshwater management Globally, there are universal efforts to quantify and address human impacts on ecosystems. In particular, changes in land use and river regulation have led to drastic declines in stream and river ecosystem health. These human impacts are driving the global freshwater biodiversity crisis and degrade ecosystem services provided by aquatic habitats. In Aotearoa New Zealand there is widespread concern about the state of our Nation’s freshwater ecosystems. Coupled with a renaissance in Māori culture, there has been a drive to develop indicators that help articulate cultural values, assess ecosystem health from a cultural perspective, and provide greater agency for Māori in environmental monitoring and management. In my thesis, I sought to integrate an established cultural monitoring framework with conventional biomonitoring tools for improved freshwater management. I hypothesized that using a suite of monitoring approaches would help detect the impacts of human land use on stream ecosystem health, and that cultural indicators would be consistent with conventional measures in diagnosing land use impacts. I also hypothesized that there could be discrepancies between cultural monitoring and conventional approaches, as each represent different perspectives and ways of knowing streams and rivers. 
To test my hypotheses, I selected ten stream sites across the catchment of the Kuratau River, a major tributary of Lake Taupō. Based on River Environment Classification land cover types, I used a balanced study design with five sites draining indigenous forest, and five sites draining pastoral land. I measured key stream physicochemical properties and collected water samples for determination of nutrient concentration and faecal coliform counts. I also characterised instream and riparian habitat. I collected benthic macroinvertebrate samples and sampled water for environmental DNA (eDNA) to assess different facets of biodiversity. I measured decomposition using the Cotton Strip Assay and quantified periphyton biomass using a portable fluorometer. In combination with these approaches, I also used a Cultural Health Index, an established cultural monitoring framework that assesses three components: customary significance, taonga species/mahinga kai (food gathering), and the Cultural Stream Health Measure (CSHM) to assess identified attributes for stream health.
I found that pastoral land uses lead to a decline in stream ecosystem health when compared to sites draining indigenous forest. Pastoral sites had higher specific conductivity and concentrations of total nitrogen and nitrate. Habitat variables changed, with increased sedimentation and more degraded riparian zones in pastoral streams. Decomposition rates as measured by the cotton strip assay increased in the pasture sites, which was likely a response to increased nutrient availability. There were shifts in macroinvertebrate composition, with key indicator taxa for Indigenous forest sites including pollution-sensitive mayflies and stoneflies. Total taxa, EPT (Ephemeroptera, Plecoptera, Trichoptera), and aquatic insect richness all significantly declined in pastoral sites. Increases in primary production were less pronounced, but did increase alongside abundances of grazing caddisflies and snails in the sites draining pastoral catchments. 
The Cultural Health Index indicated that sites in the Kuratau River catchment had customary and mahinga kai values. CSHM responded negatively to pastoral land uses, and the effect size it described was congruent with other responses. The CSHM was strongly correlated with an indicator of riparian habitat condition (the Riparian Condition Index) and had weak negative correlations with sedimentation and stream temperatures. The CSHM was not correlated with macroinvertebrate indices, which may have been because of the relatively few sites sampled, the narrow impact gradient in the Kuratau catchment, and the lack of temporal replication. However, cultural monitoring approaches should be seen to complement conventional environmental monitoring methods for assessing freshwater and not be expected to duplicate them. The strength of the Cultural Health Index approach comes from establishing Māori agency to assess the environment through a Te Ao Māori lens, and to support and foster inclusion for tangata whenua epistemologies in environmental monitoring and resource management. Future research should consider greater hapū involvement and further explore the mechanisms driving changes in stream health and biodiversity.

year: 2024