title: A modular plasmid system for expression of genes in Neisseria gonorrhoeae

authors: Flemming, Chloe Dale

subjects: Plasmid

abstract: Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Antimicrobial resistance in N. gonorrhoeae is of increasing concern and there is no current vaccine. Over the last 80 years, N. gonorrhoeae has become highly resistant to many frontline antibiotics, and as such, there are now limited treatment options. Research and development is critical for the discovery of new diagnostic assays, antimicrobials, and vaccines that combat N. gonorrhoeae. 
To date, there are a variety of genetic tools available for generating mutations, gene deletions, and gene complementation in N. gonorrhoeae. Plasmids are available for the introduction of new genes of interest and complementation, however replicating plasmids are often unstable, time consuming to manipulate, and suffer from poor transformation efficiencies.

Here, this project aimed to resolve the current issues with N. gonorrhoeae plasmid systems by using a Golden Gate assembly technique to design a modular cloning system specifically to enable custom gene expression in N. gonorrhoeae. Golden Gate cloning uses Type IIS restriction endonucleases to allow the assembly of complex multipart plasmids from reusable DNA parts. These include the gonococcal-specific cryptic plasmid maintenance region from pEG2, the Opa promoter, antibiotic resistance genes encoding kanamycin and erythromycin resistance, fluorescent proteins, and a ColE1 origin of replication for propagation in Escherichia coli. Our system was designed to be more efficient by enabling the rapid generation of very small, customised plasmids capable of replication in both E. coli and N. gonorrhoeae.
Currently, there are vast gaps in our knowledge of N. gonorrhoeae biology. This thesis offers a new system that is an efficient tool for custom gene expression in N. gonorrhoeae to improve our understanding of this pathogen. Improving research approaches, such as mutagenesis and complementation, will contribute to drug and vaccine discovery efforts searching for reliable treatments for this devastating pathogen.

text: A modular plasmid system for expression of genes in Neisseria gonorrhoeae Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Antimicrobial resistance in N. gonorrhoeae is of increasing concern and there is no current vaccine. Over the last 80 years, N. gonorrhoeae has become highly resistant to many frontline antibiotics, and as such, there are now limited treatment options. Research and development is critical for the discovery of new diagnostic assays, antimicrobials, and vaccines that combat N. gonorrhoeae. 
To date, there are a variety of genetic tools available for generating mutations, gene deletions, and gene complementation in N. gonorrhoeae. Plasmids are available for the introduction of new genes of interest and complementation, however replicating plasmids are often unstable, time consuming to manipulate, and suffer from poor transformation efficiencies.

Here, this project aimed to resolve the current issues with N. gonorrhoeae plasmid systems by using a Golden Gate assembly technique to design a modular cloning system specifically to enable custom gene expression in N. gonorrhoeae. Golden Gate cloning uses Type IIS restriction endonucleases to allow the assembly of complex multipart plasmids from reusable DNA parts. These include the gonococcal-specific cryptic plasmid maintenance region from pEG2, the Opa promoter, antibiotic resistance genes encoding kanamycin and erythromycin resistance, fluorescent proteins, and a ColE1 origin of replication for propagation in Escherichia coli. Our system was designed to be more efficient by enabling the rapid generation of very small, customised plasmids capable of replication in both E. coli and N. gonorrhoeae.
Currently, there are vast gaps in our knowledge of N. gonorrhoeae biology. This thesis offers a new system that is an efficient tool for custom gene expression in N. gonorrhoeae to improve our understanding of this pathogen. Improving research approaches, such as mutagenesis and complementation, will contribute to drug and vaccine discovery efforts searching for reliable treatments for this devastating pathogen.

year: 2023

title: Standardizing definitions and reporting guidelines for the infertility core outcome set: an international consensus development study†  ‡.

authors: Duffy, J.M.N.

subjects: female infertility

abstract: STUDY QUESTION: Can consensus definitions for the core outcome set for infertility be identified in order to recommend a standardized approach to reporting? 
SUMMARY ANSWER: Consensus definitions for individual core outcomes, contextual statements and a standardized reporting table have been developed. 
WHAT IS KNOWN ALREADY: Different definitions exist for individual core outcomes for infertility. This variation increases the opportunities for researchers to engage with selective outcome reporting, which undermines secondary research and compromises clinical practice guideline development. 
STUDY DESIGN, SIZE, DURATION: Potential definitions were identified by a systematic review of definition development initiatives and clinical practice guidelines and by reviewing Cochrane Gynaecology and Fertility Group guidelines. These definitions were discussed in a face-to-face consensus development meeting, which agreed consensus definitions. A standardized approach to reporting was also developed as part of the process. 
PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, researchers and people with fertility problems were brought together in an open and transparent process using formal consensus development methods. 
MAIN RESULTS AND THE ROLE OF CHANCE: Forty-four potential definitions were inventoried across four definition development initiatives, including the Harbin Consensus Conference Workshop Group and International Committee for Monitoring Assisted Reproductive Technologies, 12 clinical practice guidelines and Cochrane Gynaecology and Fertility Group guidelines. Twenty-seven participants, from 11 countries, contributed to the consensus development meeting. Consensus definitions were successfully developed for all core outcomes. Specific recommendations were made to improve reporting. 
LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations. There was limited representation from low- and middle-income countries. 
WIDER IMPLICATIONS OF THE FINDINGS: A minimum data set should assist researchers in populating protocols, case report forms and other data collection tools. The generic reporting table should provide clear guidance to researchers and improve the reporting of their results within journal publications and conference presentations. Research funding bodies, the Standard Protocol Items: Recommendations for Interventional Trials statement, and over 80 specialty journals have committed to implementing this core outcome set. 
STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Catalyst Fund, Royal Society of New Zealand, Auckland Medical Research Fund and Maurice and Phyllis Paykel Trust. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and an editor of the Cochrane Gynaecology and Fertility Group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. R.S.L. reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. C.N. reports being the Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and a financial interest in NexHand. E.H.Y.N. reports research sponsorship from Merck. A.S. reports consultancy fees from Guerbet. J.W. reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. A.V. reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from Human Fertilisation and Embryology Authority for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the work presented. All authors have completed the disclosure form. 
TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials Initiative: 1023.

text: Standardizing definitions and reporting guidelines for the infertility core outcome set: an international consensus development study†  ‡. STUDY QUESTION: Can consensus definitions for the core outcome set for infertility be identified in order to recommend a standardized approach to reporting? 
SUMMARY ANSWER: Consensus definitions for individual core outcomes, contextual statements and a standardized reporting table have been developed. 
WHAT IS KNOWN ALREADY: Different definitions exist for individual core outcomes for infertility. This variation increases the opportunities for researchers to engage with selective outcome reporting, which undermines secondary research and compromises clinical practice guideline development. 
STUDY DESIGN, SIZE, DURATION: Potential definitions were identified by a systematic review of definition development initiatives and clinical practice guidelines and by reviewing Cochrane Gynaecology and Fertility Group guidelines. These definitions were discussed in a face-to-face consensus development meeting, which agreed consensus definitions. A standardized approach to reporting was also developed as part of the process. 
PARTICIPANTS/MATERIALS, SETTING, METHODS: Healthcare professionals, researchers and people with fertility problems were brought together in an open and transparent process using formal consensus development methods. 
MAIN RESULTS AND THE ROLE OF CHANCE: Forty-four potential definitions were inventoried across four definition development initiatives, including the Harbin Consensus Conference Workshop Group and International Committee for Monitoring Assisted Reproductive Technologies, 12 clinical practice guidelines and Cochrane Gynaecology and Fertility Group guidelines. Twenty-seven participants, from 11 countries, contributed to the consensus development meeting. Consensus definitions were successfully developed for all core outcomes. Specific recommendations were made to improve reporting. 
LIMITATIONS, REASONS FOR CAUTION: We used consensus development methods, which have inherent limitations. There was limited representation from low- and middle-income countries. 
WIDER IMPLICATIONS OF THE FINDINGS: A minimum data set should assist researchers in populating protocols, case report forms and other data collection tools. The generic reporting table should provide clear guidance to researchers and improve the reporting of their results within journal publications and conference presentations. Research funding bodies, the Standard Protocol Items: Recommendations for Interventional Trials statement, and over 80 specialty journals have committed to implementing this core outcome set. 
STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Catalyst Fund, Royal Society of New Zealand, Auckland Medical Research Fund and Maurice and Phyllis Paykel Trust. Siladitya Bhattacharya reports being the Editor-in-Chief of Human Reproduction Open and an editor of the Cochrane Gynaecology and Fertility Group. J.L.H.E. reports being the Editor Emeritus of Human Reproduction. R.S.L. reports consultancy fees from Abbvie, Bayer, Ferring, Fractyl, Insud Pharma and Kindex and research sponsorship from Guerbet and Hass Avocado Board. B.W.M. reports consultancy fees from Guerbet, iGenomix, Merck, Merck KGaA and ObsEva. C.N. reports being the Editor-in-Chief of Fertility and Sterility and Section Editor of the Journal of Urology, research sponsorship from Ferring, and a financial interest in NexHand. E.H.Y.N. reports research sponsorship from Merck. A.S. reports consultancy fees from Guerbet. J.W. reports being a statistical editor for the Cochrane Gynaecology and Fertility Group. A.V. reports that he is a Statistical Editor of the Cochrane Gynaecology & Fertility Review Group and of the journal Reproduction. His employing institution has received payment from Human Fertilisation and Embryology Authority for his advice on review of research evidence to inform their 'traffic light' system for infertility treatment 'add-ons'. N.L.V. reports consultancy and conference fees from Ferring, Merck and Merck Sharp and Dohme. The remaining authors declare no competing interests in relation to the work presented. All authors have completed the disclosure form. 
TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials Initiative: 1023.

year: 2020

title: A core phyllosphere microbiome exists across distant populations of a tree species indigenous to New Zealand

authors: Noble, Anya S.

subjects: Science & Technology

abstract: The phyllosphere microbiome is increasingly recognised as an influential component of plant physiology, yet it remains unclear whether stable host-microbe associations generally exist in the phyllosphere. Leptospermum scoparium (mānuka) is a tea tree indigenous to New Zealand, and honey derived from mānuka is widely known to possess unique antimicrobial properties. However, the host physiological traits associated with these antimicrobial properties vary widely, and the specific cause of such variation has eluded scientists despite decades of research. Notably, the mānuka phyllosphere microbiome remains uncharacterised, and its potential role in mediating host physiology has not been considered. Working within the prevailing core microbiome conceptual framework, we hypothesise that the phyllosphere microbiome of mānuka exhibits specific host association patterns congruent with those of a microbial community under host selective pressure (null hypothesis: the mānuka phyllosphere microbiome is recruited stochastically from the surrounding environment). To examine our hypothesis, we characterised the phyllosphere and associated soil microbiomes of five distinct and geographically distant mānuka populations across the North Island of New Zealand. We identified a habitat-specific and relatively abundant core microbiome in the mānuka phyllosphere, which was persistent across all samples. In contrast, non-core phyllosphere microorganisms exhibited significant variation across individual host trees and populations that was strongly driven by environmental and spatial factors. Our results demonstrate the existence of a dominant and ubiquitous core microbiome in the phyllosphere of mānuka, supporting our hypothesis that phyllosphere microorganisms of mānuka exhibit specific host association and potentially mediate physiological traits of this nationally and culturally treasured indigenous plant. In addition, our results illustrate biogeographical patterns in mānuka phyllosphere microbiomes and offer insight into factors contributing to phyllosphere microbiome assembly.

text: A core phyllosphere microbiome exists across distant populations of a tree species indigenous to New Zealand The phyllosphere microbiome is increasingly recognised as an influential component of plant physiology, yet it remains unclear whether stable host-microbe associations generally exist in the phyllosphere. Leptospermum scoparium (mānuka) is a tea tree indigenous to New Zealand, and honey derived from mānuka is widely known to possess unique antimicrobial properties. However, the host physiological traits associated with these antimicrobial properties vary widely, and the specific cause of such variation has eluded scientists despite decades of research. Notably, the mānuka phyllosphere microbiome remains uncharacterised, and its potential role in mediating host physiology has not been considered. Working within the prevailing core microbiome conceptual framework, we hypothesise that the phyllosphere microbiome of mānuka exhibits specific host association patterns congruent with those of a microbial community under host selective pressure (null hypothesis: the mānuka phyllosphere microbiome is recruited stochastically from the surrounding environment). To examine our hypothesis, we characterised the phyllosphere and associated soil microbiomes of five distinct and geographically distant mānuka populations across the North Island of New Zealand. We identified a habitat-specific and relatively abundant core microbiome in the mānuka phyllosphere, which was persistent across all samples. In contrast, non-core phyllosphere microorganisms exhibited significant variation across individual host trees and populations that was strongly driven by environmental and spatial factors. Our results demonstrate the existence of a dominant and ubiquitous core microbiome in the phyllosphere of mānuka, supporting our hypothesis that phyllosphere microorganisms of mānuka exhibit specific host association and potentially mediate physiological traits of this nationally and culturally treasured indigenous plant. In addition, our results illustrate biogeographical patterns in mānuka phyllosphere microbiomes and offer insight into factors contributing to phyllosphere microbiome assembly.

year: 2020