Building a model of navigational strategies for queer undergraduate students in STEM.

Introduction: There is a critical need to foster inclusive educational spaces for Queer identifying students and to resist oppressive structures that seek to marginalize and inflict trauma on students because of their gender or sexual identity. Methods: Drawing on thematic analysis and Queer theory, we interviewed 11 Queer identifying STEM students to understand the navigational strategies they leveraged within higher education environments related to their Queer identity. Results: We developed a cyclical model of navigational strategies employed by Queer STEM students that involved evaluating the environments, performing psychological identity calculations, and engaging in behavioral actions. Students evaluated the environment by attending to the diversity of gender representation, presence of other Queer individuals, and contextual factors conveyed based on disciplinary expectations. Students engaged in psychological identity calculations whereby they assessed beliefs about the relevance, importance, and fears related to their Queer identity, with few perceiving any benefits. Behavioral actions resulted in students building a chosen community, disclosing or shelving their queer identity, and advocating for representation. Discussion: In order to support Queer students to thrive in educational contexts, researchers and practitioners should examine ways to increase representation, use inclusive pedagogical strategies, and understand the relevance of Queerness within disciplinary fields. Questioning the relevance or presence of Queerness in higher education environments only further serves to oppress, inflict trauma, and marginalize Queer students.

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Nitrification and beyond: metabolic versatility of ammonia oxidising archaea.

Ammonia oxidising archaea are among the most abundant living organisms on Earth and key microbial players in the global nitrogen cycle. They carry out oxidation of ammonia to nitrite, and their activity is relevant for both food security and climate change. Since their discovery nearly 20 years ago, major insights have been gained into their nitrogen and carbon metabolism, growth preferences and their mechanisms of adaptation to the environment, as well as their diversity, abundance and activity in the environment. Despite significant strides forward through the cultivation of novel organisms and omics-based approaches, there are still many knowledge gaps on their metabolism and the mechanisms which enable them to adapt to the environment. Ammonia oxidising microorganisms are typically considered metabolically streamlined and highly specialised. Here we review the physiology of ammonia oxidising archaea, with focus on aspects of metabolic versatility and regulation, and discuss these traits in the context of nitrifier ecology.

The effect of methane and methanol on the terrestrial ammonia-oxidizing archaeon 'Candidatus Nitrosocosmicus franklandus C13'.

The ammonia monooxygenase (AMO) is a key enzyme in ammonia-oxidizing archaea, which are abundant and ubiquitous in soil environments. The AMO belongs to the copper-containing membrane monooxygenase (CuMMO) enzyme superfamily, which also contains particulate methane monooxygenase (pMMO). Enzymes in the CuMMO superfamily are promiscuous, which results in co-oxidation of alternative substrates. The phylogenetic and structural similarity between the pMMO and the archaeal AMO is well-established, but there is surprisingly little information on the influence of methane and methanol on the archaeal AMO and terrestrial nitrification. The aim of this study was to examine the effects of methane and methanol on the soil ammonia-oxidizing archaeon 'Candidatus Nitrosocosmicus franklandus C13'. We demonstrate that both methane and methanol are competitive inhibitors of the archaeal AMO. The inhibition constants (Ki ) for methane and methanol were 2.2 and 20 µM, respectively, concentrations which are environmentally relevant and orders of magnitude lower than those previously reported for ammonia-oxidizing bacteria. Furthermore, we demonstrate that a specific suite of proteins is upregulated and downregulated in 'Ca. Nitrosocosmicus franklandus C13' in the presence of methane or methanol, which provides a foundation for future studies into metabolism of one-carbon (C1) compounds in ammonia-oxidizing archaea.

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Hydrazines as Substrates and Inhibitors of the Archaeal Ammonia Oxidation Pathway.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. Hydroxylamine is an intermediate in both AOB and AOA, but homologues of hydroxylamine dehydrogenase (HAO), catalyzing bacterial hydroxylamine oxidation, are absent in AOA. Hydrazine is a substrate for bacterial HAO, while phenylhydrazine is a suicide inhibitor of HAO. Here, we examine the effect of hydrazines in AOA to gain insights into the archaeal ammonia oxidation pathway. We show that hydrazine is both a substrate and an inhibitor for AOA and that phenylhydrazine irreversibly inhibits archaeal hydroxylamine oxidation. Both hydrazine and phenylhydrazine interfered with ammonia and hydroxylamine oxidation in AOA. Furthermore, the AOA "Candidatus Nitrosocosmicus franklandus" C13 oxidized hydrazine into dinitrogen (N2), coupling this reaction to ATP production and O2 uptake. This study expands the known substrates of AOA and suggests that despite differences in enzymology, the ammonia oxidation pathways of AOB and AOA are functionally surprisingly similar. These results demonstrate that hydrazines are valuable tools for studying the archaeal ammonia oxidation pathway. IMPORTANCE Ammonia-oxidizing archaea (AOA) are among the most numerous living organisms on Earth, and they play a pivotal role in the global biogeochemical nitrogen cycle. Despite this, little is known about the physiology and metabolism of AOA. We demonstrate in this study that hydrazines are inhibitors of AOA. Furthermore, we demonstrate that the model soil AOA "Ca. Nitrosocosmicus franklandus" C13 oxidizes hydrazine to dinitrogen gas, and this reaction yields ATP. This provides an important advance in our understanding of the metabolism of AOA and expands the short list of energy-yielding compounds that AOA can use. This study also provides evidence that hydrazines can be useful tools for studying the metabolism of AOA, as they have been for the bacterial ammonia oxidizers.

Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that-like for AOB-ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes.

Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, "Candidatus Nitrosocosmicus franklandus" C13 and "Candidatus Nitrosotalea sinensis" Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from "Ca Nitrosocosmicus franklandus" and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits "Ca Nitrosocosmicus franklandus" and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from "Ca Nitrosocosmicus franklandus," and it does not compete with NH3 for binding at the active site.IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

Current margin practice and effect on re-excision rates following the publication of the SSO-ASTRO consensus and ABS consensus guidelines: a national prospective study of 2858 women undergoing breast-conserving therapy in the UK and Ireland.

INTRODUCTION: There is variation in margin policy for breast conserving therapy (BCT) in the UK and Ireland. In response to the Society of Surgical Oncology and American Society for Radiation Oncology (SSO-ASTRO) margin consensus ('no ink on tumour' for invasive and 2 mm for ductal carcinoma in situ [DCIS]) and the Association of Breast Surgery (ABS) consensus (1 mm for invasive and DCIS), we report on current margin practice and unit infrastructure in the UK and Ireland and describe how these factors impact on re-excision rates. METHODS: A trainee collaborative-led multicentre prospective study was conducted in the UK and Ireland between 1st February and 31st May 2016. Data were collected on consecutive BCT patients and on local infrastructure and policies. RESULTS: A total of 79 sites participated in the data collection (75% screening units; average 372 cancers annually, range 70-900). For DCIS, 53.2% of units accept 1 mm and 38% accept 2-mm margins. For invasive disease 77.2% accept 1 mm and 13.9% accept 'no ink on tumour'. A total of 2858 patients underwent BCT with a mean re-excision rate of 17.2% across units (range 0-41%). The re-excision rate would be reduced to 15% if all units applied SSO-ASTRO guidelines and to 14.8% if all units followed ABS guidelines. Of those who required re-operation, 65% had disease present at margin. CONCLUSION: There continues to be large variation in margin policy and re-excision rates across units. Altering margin policies to follow either SSO-ASTRO or ABS guidelines would result in a modest reduction in the national re-excision rate. Most re-excisions are for involved margins rather than close margins.

Eye fixation scanpaths of younger and older drivers in a hazard perception task.

Our previous research has shown that observing patterns of eye fixations is a successful method of establishing differences in underlying cognitive processes between groups of drivers. Eye movements recorded from drivers in a laboratory while they watch film clips recorded from a driver's perspective can be used to identify scanpaths and search patterns that reveal ability differences. In the present study 12 older subjects (60-75 years) and 12 younger subjects (30-45 years) watched clips for potential hazards such as other road users appearing on an intersecting trajectory. Acuity and visual field differences between the two groups were eliminated through screening, so that only age-related differences would emerge. Eye fixations were analysed on a frame-by-frame basis to generate sequences of codes representing the location and object of the viewer's interest, before and during the appearance of a hazard. These codes were analysed for the existence of two fixation scanpaths using Markov Matrices. Unique scanpaths were identified for each group of drivers before and during the hazard. Evidence from the inspection of different objects and from the spread of the search indicated that both groups of driver were sensitive to attentional capture by the appearance of the hazard. Detection of the hazards - both speed and accuracy - was similar for older and younger drivers, although the older drivers perceived the films as being more hazardous in general. There is little evidence in this study of an age-related decline in the search of the scene when detecting hazards.