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NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+ Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+ This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Background: In this research article, we review the infrequently considered long-term impact of the pandemic on inpatient mental health, by reviewing the clinical parameters of all psychiatric admissions to Mount Carmel Hospital, our region’s main psychiatric healthcare facility, from 2019–2021. Methods: 4292 patients were admitted during the research period of this retrospective longitudinal analysis. Taking 2019 as the pre-COVID reference year, we compared mean monthly admissions from 2020 and 2021, looking at patient demographics, status under the Mental Health Act, diagnosis, and self-injurious behaviour. Results: While the pandemic was reflected in a moderate increase in mean monthly presentations with suicidal ideation and suicidal self-injury, presentations in 2020 otherwise remained largely stable. This contrasted with a surge in presentations in 2021 with mood disorders, schizophrenia, anxiety, personality disorders, and autism spectrum disorders. Furthermore, presentations involving self-injurious behaviour continued to grow. Involuntary admissions also increased significantly in 2021. Conclusions: This paper highlights the pernicious long-term impact of the pandemic on mental health presentations, demonstrated by an increase in hospital admissions and more serious presentations. These findings should be considered in the guidance for responses to any future pandemic, giving attention to the evidence of the impact of restrictive measures on mental health.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense. The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors., The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense. The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

NADH (NAD(+)) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD(+) also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD(+) biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD(+) synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE(NH3) and octameric glutamine-dependent NadE(Gln), and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE(Gln) in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE(Gln) enzymes may constitute evolutionary intermediates between dimeric NadE(NH3) and octameric NadE(Gln). The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium.

Background: Calorie restriction (CR) retards aging and increases longevity in many animal models. However, it is unclear whether CR can be implemented in humans without adverse effects on body composition.Objective: We evaluated the effect of a 2-y CR regimen on body composition including the influence of sex and body mass index (BMI; in kg/m2) among participants enrolled in CALERIE-2 (Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy), a multicenter, randomized controlled trial.Design: Participants were 218 nonobese (BMI: 21.9-28.0) adults aged 21-51 y who were randomly assigned to 25% CR (CR, n = 143) or ad libitum control (AL, n = 75) in a 2:1 ratio. Measures at baseline and 12 and 24 mo included body weight, waist circumference, fat mass (FM), fat-free mass (FFM), and appendicular mass by dual-energy X-ray absorptiometry; activity-related energy expenditure (AREE) by doubly labeled water; and dietary protein intake by self-report. Values are expressed as means ± SDs.Results: The CR group achieved 11.9% ± 0.7% CR over 2-y and had significant decreases in weight (-7.6 ± 0.3 compared with 0.4 ± 0.5 kg), waist circumference (-6.2 ± 0.4 compared with 0.9 ± 0.5 cm), FM (-5.4 ± 0.3 compared with 0.5 ± 0.4 kg), and FFM (-2.0 ± 0.2 compared with -0.0 ± 0.2 kg) at 24 mo relative to the AL group (all between-group P < 0.001). Moreover, FFM as a percentage of body weight at 24 mo was higher, and percentage of FM was lower in the CR group than in the AL. AREE, but not protein intake, predicted preservation of FFM during CR (P < 0.01). Men in the CR group lost significantly more trunk fat (P = 0.03) and FFM expressed as a percentage of weight loss (P < 0.001) than women in the CR group.Conclusions: Two years of CR had broadly favorable effects on both whole-body and regional adiposity that could facilitate health span in humans. The decrements in FFM were commensurate with the reduced body mass; although men in the CR group lost more FFM than the women did, the percentage of FFM in the men in the CR group was higher than at baseline. CALERIE was registered at clinicaltrials.gov as NCT00427193.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

NADH (NAD

Aims In this work, an ammonium-excreting strain (HM053) of A. brasilense was further characterized genetically and biochemically, and its abilities to colonize and promote wheat growth were determined.

Little research has been conducted exploring the issues surrounding parenting with an acquired brain injury (ABI). This study aimed to explore the experiences and needs of parents who suffered an ABI in the last 2 years from their own perspectives. For individuals with an ABI who have dependent children their ABI has the potential to impact upon their parenting abilities, skills and relationships.Interpretive phenomenological analysis (IPA) was used to analyse the data. Using IPA allowed the research questions and inquiry to be positioned epistemologically and directed towards 'meaning' rather than 'difference' or 'causality'.Five participants (two female, three male) were interviewed using a semi-structured interview schedule.Four main themes were identified: (i) Multiple losses, (ii) A mix of resigned acceptance and uncertain future, (iii) Giving and receiving support is part of the healing process and (iv) Hopes and aspirations.The results indicated that the participants experienced an oscillation between experiencing the multiple losses of their parental role and attempting to adapt and adjust to these changes. These findings are discussed in relation to clinical and theoretical implications for parents who are inpatients with an ABI up to 2 years post-injury.

Orientadora : Prof. Dr. Luciano Fernandes Huergo Co-orientador : Profª Drª. Vivian Rotuno Moure e Prof. Dr. Marco A. S. de Oliveira Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências : Bioquímica. Defesa: Curitiba, 21/03/2016 Inclui referências : f. 77-83 Resumo: A enzima nicotinamida adenina dinucleotídeo sintetase (NadE) catalisa a amidação do ácido nicotínico adenina dinucleotídeo (NaAD) para formar NAD+. Essa reação é o último passo da biossíntese de NAD, e portanto é uma enzima chave no metabolismo. Enzimas NadE tipicamente usam glutamina ou amônia como doadores de nitrogênio, em uma reação dependente de ATP. Dada a função de NadE para o metabolismo de procariotos, NadE vêm atraindo interesse como alvo para antibióticos. Em vários organismos, podem ser encontradas várias cópias de genes que codificam para NAD sintetases. A bactéria associativa de plantas Herbaspirillum seropedicae possui dois genes que codificam para duas enzimas NadE1 e NadE2. O gene nadE1 é ligado por contexto genômico ao gene glnB que codifica para a proteína transdutora de sinal GlnB. Anteriormente a NadE1 foi caracterizada cineticamente, e ela mostrou ter preferência por glutamina. Este trabalho mostra a confirmação da preferência de glutamina por NadE1 por ensaio de LC/MS, assim como a caracterização da proteína NadE2 de H. seropedicae. Após a clonagem, expressão e purificação da proteína NadE2, a análise de cromatografia de gel filtração sugere que NadE2 é uma enzima homo-dimérica. A caracterização cinética de NadE2 indica que seu domínio glutaminase é funcional, porém há preferência por glutamina em relação a amônia é sutil. Essa é a primeira investigação cinética de uma enzima glutamina-dependente dimérica. Palavras chave: NAD+, glutaminase, enzima. Abstract: Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amidation of nicotinic acid adenine dinucleotide (NaAD) to form NAD+. This reaction is the last step of NAD biosynthesis, therefore is a key enzyme of metabolism. NadE enzymes typically use glutamine or ammonium as nitrogen donors in an ATP-dependent reaction. Given the NadE function to prokaryote metabolism, it has attracted interest as a potential target for novel antibiotics. In several organisms, it is possible to find several copies of genes that codify to NAD synthetases. The plant associative bacterium Herbaspirillum seropedicae has two genes that encode for two different NAD synthetases, NadE1 and NadE2. The nadE1 gene is genetically linked to glnB, encoding the signal transduction protein GlnB. Previously, NadE1 was characterized kinetically, and it shows preference for glutamine. In this work, the preference for glutamine is confirmed by LC/MS assay, as well as the characterization of H. seropedicae NadE2. After cloning, expression and purification of NadE2, gel filtration chromatography analysis suggests that NadE2 is a homodimer. Kinetic characterization of NadE2 indicates a functional glutaminase domain, although the preference for glutamine vs ammonium as nitrogen donor was only modest. This is the first biochemical characterization of a dimeric glutamine-dependent NAD synthetase. Keywords: NAD+, glutaminase, enzyme.

Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

This paper explores the examination procedures and protocols for the DBA degree through a survey of universities offering the degree in Australia. The findings indicate general consistency among institutions in understanding the differences between a DBA and PhD, and a relatively uniform acceptance of key criteria for assessment of the DBA. However, in some instances the information provided to examiners of DBA theses was either unavailable or limited, or did not articulate clearly the differences between a DBA and PhD and how these differences are to be addressed when examining a DBA thesis. A framework is provided for assisting potential examiners of DBA theses and graduate schools or business faculties in their drafting of DBA examination guidelines in order to obtain a level of consistency in DBA examination procedures and protocols.

If a rock is to retain a geologically meaningful magnetic record of its history, it is essential that it contains magnetic minerals which are capable of carrying stable magnetic remanence. Of the natural occurring magnetic minerals, magnetite is the most important because of its abundance and strong magnetic signature. The stability, i.e., the resistance to demagnetisation or reorientation, of magnetic remanence is related to grain size; in smaller grains the magnetic moments align to have single domain (SD) structures, in larger grains complex magnetic patterns are formed (multidomain (MD)). “Classical” domain theory predicts that SD remanence is stable, whilst MD remanence is not. However experimental evidence has shown that both SD and MD grains can have stable remanences. In this thesis the origin of stable MD remanence is examined. There are two opposing theories; one suggests that the stability is due to independent SD-like structures, the other postulates that the stability is due to metastable MD structure. A series of experiments were designed to examine the stability using a selection of characterised synthetic and natural samples. Low-stress hydrothermal recrystallised samples where grown for this study.For the first time, the stability of thermoremanence induced in hydrothermal crystals to cooling was examined. The results agree with previous observations for crushed and natural magnetites, and support kinematic models.The behaviour of SIRM and thermoremanences in MD magnetite to low-temperature cooling to below the crystallographic Verwey transition at 120-124 K (Tv) and the cubic magnetocrystalline anisotropy isotropic point (Tk) at 130 K was investigated. On cooling through Tv, SIRM was observed to decrease and demagnetise, however thermoremanence was found to display a large increase in the magnetisation at Tv, which was partially re- versible on warming. The size of the anomaly is shown to be dependent on the temperature at which the thermoremanence is acquired, internal stress and grain size. The anomaly is attributed to the large increase in the magnetocrystalline anisotropy which occurs on cooling through Tv . It is postulated that low-temperature cycling demagnetisation is due to kinematic processes which occur on cooling between room temperature and Tk. Characterisation of low-temperature treated remanence and partially alternating field demagnetised remanence, suggest that the stable remanence is multidomain.Low-temperature cooling of remanence in single sub-micron crystals was simulated using micromagnetic models. The models predict the observed anomaly for thermoremanence on cooling through Tv, and also the relative behaviour of SIRM and thermoremanence. The single domain threshold was calculated for the low-temperature phase of magnetite, and was found to be 0.14 microns, compared to 0.07 microns at room temperature.

Using conventional diagnostic methods to test for plant viruses requires knowledge of the pathogens likely to be associated with a host species. This knowledge can either be applied when using a targeted test method (e.g. ELISA or PCR) to identify required tests, or in bioassay, through knowing which viruses will transmit into which assay host. In the last decade High Throughput Sequencing (HTS) has revolutionised plant virology diagnostics, however, the knowledge and skills of the diagnostic virologist are needed to interpret the results of sequencing and to investigate the myriad of novel viruses reported using this technique. This thesis presents a body of published work and an accompanying linking document focussing on the development and application of these diagnostic technologies in a plant health/biosecurity setting. The thesis explores the use of diagnostic technologies in virus detection and discovery, but also in supporting research applications such as gathering the data necessary to support plant health risk assessment or carry out epidemiological studies on vector efficiency. The publications include a review of new virus records from the United Kingdom over a 35-year period, discussing the factors driving virus discovery such as changes in trade, research focus, and diagnostic technologies. Two case studies are presented which investigate diseases of unknown aetiology utilising contrasting approaches to infer the causal agent/s of disease, one utilising biological demonstration, the other experimental design and statistical analysis. Two publications discuss the evaluation and validation of diagnostic techniques. The final publication describes an investigation into the relative efficiency of transmission of potato virus Y and potato virus A by a range of aphid species. The accompanying linking document discusses each publication in the context of the current literature, as well as discussing alternative approaches to inferring causation where traditional biological approaches may not be possible.