Vitality and nature in psychiatric spaces: Challenges and prospects for 'healing architecture' in the design of inpatient mental health environments.

Historically, nature has been considered central to healing and recovery in institutional mental health settings, with inpatient spaces designed to mirror the restorative forces nature may afford. Within contemporary healthcare architecture, the discourse surrounding nature's role has once again become prominent, especially in the concept of 'healing architecture'. While the literature on 'healing architecture' primarily considers how to connect recovery to nature through interventions in the built environment, less interest has been directed towards how nature is configured in design processes and what implications that has for the everyday experiences of patients and staff. In this paper we consider the design and implementation of one particular psychiatric hospital in Denmark to show that the 'nature' brought into this healthcare space can be experienced as anything but 'natural' and may reduce rather than enhance a felt sense of 'vitality' amongst patients. Based on our analysis, we end the paper by suggesting four principles for future healthcare design.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely-used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning based segmentation, "what you put is what you get" (WYPIWYG) - i.e., pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother-machine-based high-throughput imaging and analysis methods in their research.

Solvent-Producing Clostridia Revisited.

The review provides an overview of the current status of the solvent-producing clostridia. The origin and development of industrial clostridial species, as well as the history of the industrial Acetone Butanol Ethanol fermentation process, is reexamined, and the recent resurgence of interest in the production of biobutanol is reviewed. Over 300 fully sequenced genomes for solvent-producing and closely related clostridial species are currently available in public databases. These include 270 genomes sourced from the David Jones culture collection. These genomes were allocated arbitrary DJ codes, and a conversion table to identify the species and strains has now been provided. The expanded genomic database facilitated new comparative genomic and phylogenetic analysis. A synopsis of the common features, molecular taxonomy, and phylogeny of solvent-producing clostridia and the application of comparative phylogenomics are evaluated. A survey and analysis of resident prophages in solvent-producing clostridia are discussed, and the discovery, occurrence, and role of novel R-type tailocins are reported. Prophage genomes with R-type tailocin-like features were detected in all 12 species investigated. The widespread occurrence of tailocins in Gram-negative species is well documented; this survey has indicated that they may also be widespread in clostridia.

Intestinal transgene delivery with native E. coli chassis allows persistent physiological changes.

Live bacterial therapeutics (LBTs) could reverse diseases by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally raised (CR) hosts have been unsuccessful because engineered microbial organisms (i.e., chassis) have difficulty in colonizing the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli bacteria isolated from the stool cultures of CR mice were modified to express functional genes. The reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect physiology of and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts and enables LBT with curative intent.

Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon.

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (thlA), CoA-transferase (ctfAB), and acetoacetate decarboxylase (adc) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.

Diet and feeding pattern modulate diurnal dynamics of the ileal microbiome and transcriptome.

Compositional oscillations of the gut microbiome are essential for normal peripheral circadian rhythms, both of which are disrupted in diet-induced obesity (DIO). Although time-restricted feeding (TRF) maintains circadian synchrony and protects against DIO, its impact on the dynamics of the cecal gut microbiome is modest. Thus, other regions of the gut, particularly the ileum, the nexus for incretin and bile acid signaling, may play an important role in entraining peripheral circadian rhythms. We demonstrate the effect of diet and feeding rhythms on the ileal microbiome composition and transcriptome in mice. The dynamic rhythms of ileal microbiome composition and transcriptome are dampened in DIO. TRF partially restores diurnal rhythms of the ileal microbiome and transcriptome, increases GLP-1 release, and alters the ileal bile acid pool and farnesoid X receptor (FXR) signaling, which could explain how TRF exerts its metabolic benefits. Finally, we provide a web resource for exploration of ileal microbiome and transcriptome circadian data.

Cell-free prototyping enables implementation of optimized reverse ß-oxidation pathways in heterotrophic and autotrophic bacteria.

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse ß-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL-1), as well as hexanoic acid (3.06 ± 0.03 gL-1) and 1-hexanol (1.0 ± 0.1 gL-1) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.

Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale.

Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

Choreographies of sexual safety and liminality: Forensic mental health and the limits of recovery.

Medium secure forensic psychiatric units are unique environments within the broader "post asylum" landscape of mental health services. Length of stay is much greater and restrictions on behavior, including sexual behavior, are legally and institutionally legitimated, due to concerns regarding risk. As a result, sexuality is rarely explored experientially with service users and no official policies on sexual conduct and sexual safety have yet been developed.

Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii.

Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

Stepping on the Gas to a Circular Economy: Accelerating Development of Carbon-Negative Chemical Production from Gas Fermentation.

Owing to rising levels of greenhouse gases in our atmosphere and oceans, climate change poses significant environmental, economic, and social challenges globally. Technologies that enable carbon capture and conversion of greenhouse gases into useful products will help mitigate climate change by enabling a new circular carbon economy. Gas fermentation usingcarbon-fixing microorganisms offers an economically viable and scalable solution with unique feedstock and product flexibility that has been commercialized recently. We review the state of the art of gas fermentation and discuss opportunities to accelerate future development and rollout. We discuss the current commercial process for conversion of waste gases to ethanol, including the underlying biology, challenges in process scale-up, and progress on genetic tool development and metabolic engineering to expand the product spectrum. We emphasize key enabling technologies to accelerate strain development for acetogens and other nonmodel organisms.

Bacterial Differential Expression Analysis Methods.

RNA-Seq examines global gene expression to provide insights into cellular processes, and it can be particularly informative when comparing contrasting physiological states or strains. Although relatively routine in many laboratories, there are many steps involved in performing a transcriptomics experiment to ensure representative and high-quality results are generated for analysis. In this chapter, we present the application of widely used bioinformatic methodologies to assess, trim, and filter RNA-seq reads for quality using FastQC and Trim Galore, respectively. High-quality reads are mapped using Bowtie2 and differentially expressed genes across different groups were estimated using the DEseq2 R-Bioconductor package. In addition, we describe the various steps to perform the sample-wise data quality assessment by generating exploratory plots through the DESeq2 package. Simple steps to calculate the significant differentially expressed genes, up- and down-regulated genes, and exporting the data and images are also included. A Venn diagram is a useful method to compare the differentially expressed genes across various comparisons and steps to generate the Venn diagram from DESeq2 results are provided. Finally, the output from DESeq2 is compared to published results from EdgeR. The Clostridium autoethanogenum data are published and publicly available.

Gene targets for engineering osmotolerance in Caldicellulosiruptor bescii.

BACKGROUND: Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. RESULTS: Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions-introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. CONCLUSIONS: Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra).

Transcriptomic Profiles of Zymomonas mobilis 8b to Furfural Acute and Long-Term Stress in Both Glucose and Xylose Conditions.

Zymomonas mobilis 8b is an ethanologenic bacterium engineered to utilize both glucose and xylose. The impacts of lignocellulosic hydrolyzate inhibitors on the growth of Zymomonas mobilis 8b have been investigated. However, the molecular responses of these inhibitors have not been completely elucidated yet. In this study, molecular responses to furfural were investigated using transcriptomic approaches of both chip-based microarray and a directional mRNA-Seq. Furfural acute shock time-course experiment with 3 g/L furfural supplemented when cells reached exponential phase and stress response experiment in the presence of 2 g/L furfural from the beginning of fermentation were carried out to study the physiological and transcriptional profiles of short-term and long-term effects of furfural on 8b. Furfural negatively affected 8b growth in terms of final biomass and the fermentation time. Transcriptomic studies indicated that the response of 8b to furfural was dynamic and complex, and differences existed between short-term shock and long-term stress responses. However, the gene function categories were similar with most down-regulated genes related to translation and biosynthesis, while the furfural up-regulated genes were mostly related to general stress responses. Several gene candidates have been identified and genetic studies indicated that expression of ZMO0465 and cysteine synthase operon ZMO0003-0006 driven by its native promoter in a shuttle vector enhanced the furfural tolerance of 8b. In addition, the relationship between microarray and mRNA-Seq was compared with good correlations. The directional mRNA-Seq data not only provided the gene expression profiling, but also can be applied for transcriptional architecture improvement to identify and confirm operons, novel transcripts, hypothetical gene functions, transcriptional start sites, and promoters with different strength.

Sexuality, risk, and organisational misbehaviour in a secure mental healthcare facility in England.

Sexuality in secure mental healthcare has been overlooked in both clinical praxis and academic research. In the UK, there exist no formal policies to inform staff approaches to managing inpatient sexuality. The limited research that has been undertaken in this field has found that often, prohibitive approaches are favoured, which may affect how inpatients conceptualise and experience their sexuality in the long-term. The aim of this study was to identify discursive constructions of inpatient sexuality, as articulated in semi-structured group interviews with inpatients and ward staff from a secure mental healthcare facility in England. The analysis identified constructions of inpatient sexuality within two overarching and conflicting discourses: one of the normalcy and legitimacy of sexual expression in human experience; and the other of risk, wherein sexuality needed to be regulated and obstructed. Inpatients' expressions of sexuality could often only be conceptualised in terms of 'organisational misbehaviour', acts that violated the implicit norms and codes of the institution. It is suggested that recoding inpatient sexuality as misbehaviour could have implications for inpatients' long-term recovery.

Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations.

Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems, but factors governing its production are poorly understood. Recent work has shown that the anaerobic microbial conversion of mercury (Hg) to MeHg requires the Hg-methylation genes hgcAB and that these genes can be used as biomarkers in PCR-based estimators of Hg-methylator abundance. In an effort to determine reliable methods for assessing hgcA abundance and diversity and linking them to MeHg concentrations, multiple approaches were compared including metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing and cloning/sequencing hgcAB gene products. Hg-methylator abundance was also determined by quantitative hgcA qPCR amplification and metaproteomics for comparison to the above measurements. Samples from eight sites were examined covering a range of total Hg (HgT; 0.03-14 mg kg-1 dry wt. soil) and MeHg (0.05-27 µg kg-1 dry wt. soil) concentrations. In the metagenome and amplicon sequencing of hgcAB diversity, the Deltaproteobacteria were the dominant Hg-methylators while Firmicutes and methanogenic Archaea were typically ∼50% less abundant. This was consistent with metaproteomics estimates where the Deltaproteobacteria were steadily higher. The 16S rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB+ species. Metagenomic and hgcAB results were similar for Hg-methylator diversity and clade-specific qPCR-based approaches for hgcA are only appropriate when comparing the abundance of a particular clade across various samples. Weak correlations between Hg-methylating bacteria and soil Hg concentrations were observed for similar environmental samples, but overall total Hg and MeHg concentrations poorly correlated with Hg-cycling genes.

Vital spaces and mental health.

The impact of social and material conditions on mental health is well established but lacking in a coherent approach. We offer the concept of 'vitality' as means of describing how environments facilitate 'feelings of being alive' that cut across existing diagnostic categories. Drawing on the work of Stern, Fuchs, Worms and Duff, we argue that vitality is not solely a quality of an individual body, but rather emerges from attunements and resonances between bodies and materials. We use vitality as a lens to explore how movements within and between assembled sets of relations can facilitate or disable feelings and expressions of being alive. Building on extended discussions of both inpatient and community-based mental healthcare, we sketch out a research agenda for analysing 'vital spaces'.

Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii.

Genes responsible for the anaerobic catabolism of benzoate in the thermophilic archaeon Ferroglobus placidus were expressed in the thermophilic lignocellulose-degrading bacterium Caldicellulosiruptor bescii, as a first step to engineering this bacterium to degrade this lignin metabolite. The benzoyl-CoA ligase gene was expressed individually, and in combination with benzoyl-CoA reductase and a putative benzoate transporter. This effort also assessed heterologous expression from a synthetically designed operon whereby each coding sequence was proceeded by a unique C. bescii ribosome binding site sequence. The F. placidicus benzoyl-CoA ligase gene was expressed in C. bescii to produce a full-length protein with catalytic activity. A synthetic 6-gene operon encoding three enzymes involved in benzoate degradation was also successfully expressed in C. bescii as determined by RNA analysis, though the protein products of only four of the genes were detected. The discord between the mRNA and protein measurements, especially considering the two genes lacking apparent protein abundance, suggests variable effectiveness of the ribosome binding site sequences utilized in this synthetic operon. The engineered strains did not degrade benzoate. Although the heterologously expressed gene encoding benzoyl-CoA ligase yielded a protein that was catalytically active in vitro, expression in C. bescii of six benzoate catabolism-related genes combined in a synthetic operon yielded mixed results. More effective expression and in vivo activity might be brought about by validating and using different ribosome binding sites and different promoters. Expressing additional pathway components may alleviate any pathway inhibition and enhance benzoyl-CoA reductase activity.

Curriculum requirements and subsequent civic engagement: is there a difference between 'forced' and 'free' community service?

Despite figures showing the growth of mandatory community service programmes, there is mixed empirical evidence of their effectiveness. This paper addresses the relationship of mandated community service to one of its purported aims: subsequent volunteerism. It compares current volunteerism among four university student cohorts: those doing no service in secondary school, those volunteering with no requirement, those volunteering both before and after the introduction of a requirement, and those introduced to service through a requirement. The analysis indicates that (1) students who were introduced to service through a mandated programme exhibit current levels of engagement no greater than non-volunteers; (2) this relationship stems largely from the different service experiences of our four cohorts and relates to the fact that service satisfaction and duration, as well as background variables account for current levels of civic engagement. The findings suggest that mandatory service programmes might well be failing the very population they seek to target, particularly in weaker, less structured programmes.

The effects of provider-prescribed obesogenic drugs on post-laparoscopic sleeve gastrectomy outcomes: a retrospective cohort study.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is one of the most commonly performed bariatric procedures and has proven effective in providing weight loss. However, considerable variance has been noted in the degree of weight loss. Physician prescription practices may be negatively affecting weight loss post-LSG and, thus, contributing to the broad range of weight loss outcomes. The aim of our study was to determine whether commonly prescribed obesogenic medications negatively affect weight loss outcomes post-LSG. SUBJECTS/METHODS: This single center retrospective cohort study performed at a University hospital included 323 patients (≥18 years) within the University California, San Diego Healthcare System who underwent LSG between 2007 and 2016. We identified a list of 32 commonly prescribed medications that have weight gain as a side effect. We compared the percent excess weight loss (%EWL) of patients divided into two groups based on post-LSG exposure to obesogenic medications. A linear regression model was used to analyze %EWL at 12 months post-LSG while controlling for age, initial body mass index (BMI), and use of leptogenic medications. RESULTS: A total of 150 patients (Meds group) were prescribed obesogenic medications within the one-year post-LSG follow up period, whereas 173 patients (Control group) were not prescribed obesogenic medications. The Meds group lost significantly less weight compared to the Control group (%EWL ± SEM at 12 months 53.8 ± 2.4 n = 78, 65.0 ± 2.6, n = 84 respectively, P = 0.002). This difference could not be attributed to differences in age, gender, initial BMI, co-morbidities, or prescription of leptogenic medications between the two groups. CONCLUSIONS: The use of provider-prescribed obesogenic medications was associated with worse weight loss outcomes post-LSG. Closer scrutiny of patient medications may be necessary to help improve outcomes of weight loss treatments.