CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries.

Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii.

Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.

Reducing violence in forensic care - how does it resemble the domains of a recovery-oriented care?

BACKGROUND: Forensic psychiatry is characterized by involuntary treatment and risk of violence. The concept of recovery is rarely in focus as the primary focus is on risk assessment, violence prevention and reducing coercion in care. AIM: To determine what resources forensic staff use to avoid or prevent violent situations, and to explore how these practices resemble the domains of recovery-oriented care. METHOD: Semi-structured interviews with staff who were identified by forensic patients as key workers in their recovery process. Interview texts were analyzed using interpretive content analysis. RESULTS: Staff prevent violent situations using tacit knowledge and experience, and through a shared collegial responsibility. Staff safeguard patients, encourage patient participation, and provide staff consistency. CONCLUSIONS: The results have implications for forensic care as well as psychiatry regarding the process of making recovery a reality for patients in the forensic care setting.

Reducing or increasing violence in forensic care: a qualitative study of inpatient experiences.

Semi-structured interviews with 13 forensic psychiatric inpatients that had decreased their assessed risk of violence were analyzed using interpretive description. The main contribution from this study is a detailed description of patients' own strategies to avoid violence. Participants described having an ongoing inner dialog in which they encouraged themselves, thereby increasing their self-esteem and trying to accept their current situation. An unsafe and overcrowded ward with uninterested and nonchalant staff increased the risk of aggressive behavior. In the process of decreasing violence, the patients and the forensic psychiatric nursing staff interacted to create and maintain a safe environment.

Reaching a turning point--how patients in forensic care describe trajectories of recovery.

In Sweden, the duration of treatment is increasing for patients admitted to forensic psychiatric care. To reduce the length of stay, it is important for the forensic rehabilitation and recovery process to be effective and safe. Not much is known about how the process of recovery and transition begins and how it is described by the forensic patients. The purpose of this study was to explore how forensic patients with a history of high risk for violence experienced the turn towards recovery. A qualitative content analysis was used to analyse interviews with 10 patients who had decreased their assessed risk for violence on the risk assessment instrument HCR-20 and who were successfully managed a lower level of security. Three themes were identified: (i) the high-risk phase: facing intense negative emotions and feelings (ii) the turning point phase: reflecting on and approaching oneself and life in a new way (iii) the recovery phase: recognising, accepting and maturing. In the high-risk phase, chaotic and overwhelming feelings were experienced. The turning point phase was experienced as a sensitive stage, and it was marked by being forced to find a new, constructive way of being. The recovery phase was characterised by recognising personal circumstances in life, including accepting the need for structure, a feeling of maturity and a sense of responsibility for their own life. In order to ensure a successful recovery, the forensic nursing staff needs to recognise and support processes related to treatment motivation and turning points. Recommendations for best nursing practice are given accordingly.

Decreased risk for violence in patients admitted to forensic care, measured with the HCR-20.

AIM: The aim of this study was to explore if patients admitted to forensic psychiatric care decreased their assessed risk for violence over time, to identify patients who decreased their assessed risk for violence exceptionally well (30% or more) on the clinical (C) and risk management (R) scales in the (HCR-20), and to compare them in terms of demographic data. METHODS: The HCR-20 risk assessment instrument was used to assess the risk for violence in 267 patients admitted to a Swedish forensic psychiatric clinic between 1997 and 2010. Their assessments at admission were compared with a second, and most recent, risk assessment. RESULTS: The risk for violence decreased over time. Demographic criteria had no impact on differences on decreased risk. Only two factors, namely gender and psychopathy showed a difference. Risk factors associated with stress and lack of personal support were the items that turned out to be the most difficult to reduce. CONCLUSION: The results show that risk prevention in forensic care does work and it is important to continue to work with risk management. The study highlights the importance of a careful analysis of the patient's risk for violence in order to work with the patient's specific risk factors to reduce the risk.

Membrane engineering of S. cerevisiae targeting sphingolipid metabolism.

The sustainable production of fuels and chemicals using microbial cell factories is now well established. However, many microbial production processes are still limited in scale due to inhibition from compounds that are present in the feedstock or are produced during fermentation. Some of these inhibitors interfere with cellular membranes and change the physicochemical properties of the membranes. Another group of molecules is dependent on their permeation rate through the membrane for their inhibition. We have investigated the use of membrane engineering to counteract the negative effects of inhibitors on the microorganism with focus on modulating the abundance of complex sphingolipids in the cell membrane of Saccharomyces cerevisiae. Overexpression of ELO3, involved in fatty acid elongation, and AUR1, which catalyses the formation of complex sphingolipids, had no effect on the membrane lipid profile or on cellular physiology. Deletion of the genes ORM1 and ORM2, encoding negative regulators of sphingolipid biosynthesis, decreased cell viability and considerably reduced phosphatidylinositol and complex sphingolipids. Additionally, combining ELO3 and AUR1 overexpression with orm1/2Δ improved cell viability and increased fatty acyl chain length compared with only orm1/2Δ. These findings can be used to further study the sphingolipid metabolism, as well as giving guidance in membrane engineering.

Alexithymia, memory of emotion, emotional awareness, and perfectionism.

The Toronto Alexithymia Scale (TAS-20; R. M. Bagby, J. D. A. Parker, & G. J. Taylor, 1994) is a self-assessment instrument designed to measure deficits in meta-emotional functioning (e.g., difficulties in identifying and describing emotions). Four studies were carried out to examine the association between the TAS-20 and (a) performance measures of meta-emotional functioning (memory of emotion and emotional awareness; Studies 1-2) and (b) measures of perfectionistic standards that may possibly be involved in the self-assessment of abilities-difficulties (Studies 3-4). The TAS-20 failed to correlate in the predicted direction with the performance measures but showed sizable correlations with measures of perfectionism. Moreover, perfectionism was found to predict TAS-20 scores independent of depression, anxiety, and somatic complaints. The results are discussed in terms of the TAS-20 measuring primarily certain aspects of meta-emotional self-efficacy.