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Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from Mycobacterium chubuense strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in Mycobacterium smegmatis mc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2 to C4), and their activity was much lower on liquid alkenes (C5 to C8). Despite intensive efforts to express the complete EtnABCD enzyme in Escherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 µmol cytochrome c reduced/min/mg protein). Cloning the EtnABCD gene cluster into Pseudomonas putida KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of cpn60 chaperonins from either Mycobacterium spp. or E. coli Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels.IMPORTANCE Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.
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Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica , Mycobacterium smegmatis/genética , Mycobacterium/enzimologia , Oxigenases/genética , Pseudomonas putida/genética , Alcenos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocromos c , Escherichia coli/metabolismo , Etilenos/metabolismo , Cinética , Mycobacterium/genética , Mycobacterium smegmatis/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Pseudomonas putida/metabolismoRESUMO
Background: Camouflaging is frequently reported in autistic people and entails the disguising of autistic traits in social situations. Camouflaging is associated with poor mental health in autistic people. This study examined the manifestation of camouflaging in a nonautistic sample, examining the relationship between autistic traits, self-reported camouflaging, gender, and mental health. Method: In total 110 nonautistic adults completed standardized self-report questionnaires that measured autistic traits, mental health symptoms, and camouflaging behaviors. Hierarchical multiple linear regression models were used to analyze data and examine the unique contributions of autistic traits and camouflaging to mental health. Results: Self-reported autistic traits were associated with increased symptoms of poor mental health. However, autistic traits were not associated with mental health symptoms when controlling for self-reported camouflaging, and self-reported camouflaging predicted increased mental health symptoms over and above the effects of autistic traits. Women had poorer mental health than men in our sample, and in women there was a stronger relationship between camouflaging and mental health than in men. Conclusions: Camouflaging may contribute to poor mental health outcomes in the general population, just as it does for autistic people, to the extent that camouflaging more clearly relates to mental health profile than self-reported autism traits. This suggests camouflaging is an important construct for understanding mental health in general, and for exploring the complex relationship between autism and autistic traits.
Why was this study done?: Some autistic individuals report trying to hide their autistic traits to blend in with others, referring to this as "camouflaging" or "masking." Many autistic people who camouflage describe it as being a very difficult and distressing process, which can be harmful to their mental health. In nonautistic people, a higher level of autistic traits is related to poor mental health. Nonautistic people have also reported camouflaging autistic traits, but we know less about whether this might also impact their mental health. What was the purpose of this study?: We did this study to find out more about the relationship between mental health and camouflaging of autistic traits in nonautistic people. What did the researchers do?: We asked 110 nonautistic adults to complete a series of questionnaires. These questionnaires asked them about their autistic traits, their camouflaging behaviors, and their mental health. We then used statistical tests to look at the relationships between autistic traits, camouflaging, gender, and mental health. What were the results of the study?: We found that people who camouflaged more had poorer mental health, including symptoms of anxiety, depression, and stress. People with more autistic traits also had poorer mental health. When we looked at both of these relationships at the same time, only camouflaging was related to poor mental health, and not autistic traits. When we also looked at gender, we found that women in our study had poorer mental health than men, and the relationship between camouflaging and mental health was stronger for women. What do these findings add to what was already known?: These findings help us understand the relationship between autistic traits and mental health by demonstrating that it may be the camouflaging of autistic traits that is related to poorer mental health. These findings also help us better understand camouflaging by showing that the negative consequences of camouflaging for mental health may apply to both autistic and nonautistic people. What are the potential weaknesses in the study?: Our study only looked at the relationship between autistic traits, camouflaging, gender, and mental health. It is possible that there are other factors our study did not investigate, such as ethnicity or sexual identity, which also play an important role. Our study also cannot tell us whether or not camouflaging is the cause of poor mental health, only that there is a relationship between camouflaging and mental health. How will these findings help autistic adults now or in the future?: By studying camouflaging in different groups and seeing what is shared and what is unique between autistic and nonautistic people, we can develop a better understanding of camouflaging. Knowledge of the mental health consequences of camouflaging can help mental health services to reduce the negative impact of camouflaging for autistic and nonautistic people. Our findings also show an important similarity between autistic and nonautistic people, showing that both groups of people may camouflage, and this negatively impacts mental health. Acknowledgment of this shared experience may help to reduce the discrimination and stigma that autistic people face.
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LAY ABSTRACT: Research has suggested that autistic people enjoy spending time with other autistic people and find them easier to talk to. We wanted to find out what autistic people say about spending time with other autistic people and whether this makes their life better. We found 52 papers which described this and reviewed what they found. We found that many autistic people had positive experiences of spending time with other autistic people and these experiences had positive impact on their lives in a range of different ways. The papers did not tell us whether this also happens for autistic people with a learning disability. More research is needed to find out more about why spending time with other autistic people helps some autistic people.
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BACKGROUND: A rapid, accurate, non-invasive diagnostic screen is needed to identify people with SARS-CoV-2 infection. We investigated whether organic semi-conducting (OSC) sensors and trained dogs could distinguish between people infected with asymptomatic or mild symptoms, and uninfected individuals, and the impact of screening at ports-of-entry. METHODS: Odour samples were collected from adults, and SARS-CoV-2 infection status confirmed using RT-PCR. OSC sensors captured the volatile organic compound (VOC) profile of odour samples. Trained dogs were tested in a double-blind trial to determine their ability to detect differences in VOCs between infected and uninfected individuals, with sensitivity and specificity as the primary outcome. Mathematical modelling was used to investigate the impact of bio-detection dogs for screening. RESULTS: About, 3921 adults were enrolled in the study and odour samples collected from 1097 SARS-CoV-2 infected and 2031 uninfected individuals. OSC sensors were able to distinguish between SARS-CoV-2 infected individuals and uninfected, with sensitivity from 98% (95% CI 95-100) to 100% and specificity from 99% (95% CI 97-100) to 100%. Six dogs were able to distinguish between samples with sensitivity ranging from 82% (95% CI 76-87) to 94% (95% CI 89-98) and specificity ranging from 76% (95% CI 70-82) to 92% (95% CI 88-96). Mathematical modelling suggests that dog screening plus a confirmatory PCR test could detect up to 89% of SARS-CoV-2 infections, averting up to 2.2 times as much transmission compared to isolation of symptomatic individuals only. CONCLUSIONS: People infected with SARS-CoV-2, with asymptomatic or mild symptoms, have a distinct odour that can be identified by sensors and trained dogs with a high degree of accuracy. Odour-based diagnostics using sensors and/or dogs may prove a rapid and effective tool for screening large numbers of people.Trial Registration NCT04509713 (clinicaltrials.gov).
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COVID-19 , Cães , Animais , Infecções Assintomáticas , COVID-19/diagnóstico , Humanos , Programas de Rastreamento , SARS-CoV-2 , Sensibilidade e Especificidade , Compostos Orgânicos Voláteis/análiseRESUMO
Vitamin K is essential for blood coagulation and plays important roles in bone and cardiovascular health. Menaquinone-7 (MK-7) is one form of vitamin K that is especially useful due to its long half-life in the circulation. MK-7 is difficult to make via organic synthesis, and is thus commonly produced by fermentation. This study aimed to genetically modify Bacillus subtilis cultures to increase their MK-7 yield and reduce production costs. We constructed 12 different strains of B. subtilis 168 by overexpressing different combinations of the rate-limiting enzymes Dxs, Dxr, Idi, and MenA. We observed an 11-fold enhancement of production in the best-performing strain, resulting in 50 mg/L MK-7. Metabolite analysis revealed new bottlenecks in the pathway at IspG and IspH, which suggest avenues for further optimization. This work highlights the usefulness of Bacillus subtilis for industrial production of high value compounds.
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Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Receptor EphB6/metabolismo , Transdução de Sinais/fisiologia , Vitamina K 2/análogos & derivados , Meios de Cultura/metabolismo , Fermentação/fisiologia , Engenharia Metabólica/métodos , Vitamina K 2/metabolismoRESUMO
The continued use of platinum-based chemotherapeutic drugs in the clinic mandates the need for further investigation of the biological activity of structural analogues of the clinically approved complexes. Of interest are monofunctional platinum(II) complexes, which bear only one labile ligand, for which it is believed that each complex binds to DNA only once. Pyriplatin ([PtCl(NH3)2(py)]+) and enpyriplatin ([PtCl(en)(py)]+) are both monofunctional platinum(II) complexes that bear a pyridine ligand and a labile chlorido ligand, differing in their cisammine and ethane-1,2-diamine (en) ligands respectively. Despite their similar structure, the complexes exhibit dramatically different cytotoxicities. In this study, we synthesized and characterized both complexes in terms of their cytotoxicity, lipophilicity, DNA binding and cellular accumulation. There was no significant difference between the lipophilicities of the complexes and both complexes exhibited monofunctional type binding, but it was the temporal accumulation profiles of the two complexes which differed greatly. The complexes were further analyzed with size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS) to determine the platination state of the proteins. Consistent with the accumulation studies, pyriplatin bound to proteins in far greater amounts than enpyriplatin, and this study also revealed some different protein targets between the bifunctional cisplatin and monofunctional pyriplatin. This study highlights the need for more sophisticated techniques, such as SEC-ICP-MS, to determine not only how much of a platinum complex accumulates in cells, but also the speciation and metabolites of platinum anticancer drugs.