Study protocol: fish oil supplement in prevention of oxaliplatin-induced peripheral neuropathy in adjuvant colorectal cancer patients - a randomized controlled trial. (OxaNeuro).

BACKGROUND: Oxaliplatin-induced peripheral neuropathy (OIPN) in general and painful OIPN in particular is a debilitating late effect that severely affects cancer survivors' quality of life and causes premature cessation of potentially lifesaving treatment. No preventive treatments and no effective treatment for chronic OIPN exist despite many attempts. One of several suggested mechanisms includes neuroinflammation as a contributing factor to OIPN. Fish oil containing long-chain n-3 polyunsaturated fatty acids (n-3 LCPUFAs) are precursors to specialized proresolving mediators that mediate the resolution of inflammation. Our primary hypothesis is that a high supplementation of n-3 LCPUFAs will lower the prevalence and severity of OIPN. METHODS: The OxaNeuro project is an investigator-initiated, multicenter, double-blinded, randomized, placebo-controlled clinical study. We will include 120 patients eligible to receive adjuvant oxaliplatin after colorectal cancer surgery. Patients will receive fish oil capsules containing n-3 LCPUFAs or corn oil daily for 8 months. The primary endpoint is the prevalence of OIPN at 8 months defined as relevant symptoms, including one of the following: abnormal nerve conduction screening, abnormal vibration threshold test, abnormal skin biopsy, or abnormal pinprick test. Additional endpoints include the intensity and severity of OIPN-related neuropathic pain, patient-reported OIPN symptoms, quality of life, mental health symptoms, body composition, and cognitive evaluation. Furthermore, we will evaluate inflammatory biomarkers in blood samples and skin biopsies, including the potential OIPN biomarker neurofilament light protein (NfL) which will be measured before each cycle of chemotherapy. DISCUSSION: If readily available fish oil supplementation alleviates OIPN prevalence and severity, it will significantly improve the lives of both cancer survivors and palliative cancer patients receiving oxaliplatin; it will improve their quality of life, optimize chemotherapeutic treatment plans by lowering the need for dose reduction or premature cessation, and potentially increase survival. TRIAL REGISTRATION: ClinicalTrial.gov identifier: NCT05404230 Protocol version: 1.2, April 25th. 2023.

Facile Method for High-throughput Identification of Stabilizing Mutations.

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. However, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 °C increase in thermal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.

A randomized phase II study of full dose gemcitabine versus reduced dose gemcitabine and nab-paclitaxel in vulnerable patients with non-resectable pancreatic cancer (DPCG-01).

BACKGROUND: According to current evidence, the best treatment for fit patients with non-resectable pancreatic cancer (PC) is combination chemotherapy, whereas frail patients are recommended gemcitabine (Gem) monotherapy. Randomized controlled trials in colorectal cancer and a post-hoc analysis of gemcitabine and nab-paclitaxel (GemNab) in PC suggest, however, that reduced dose of combination chemotherapy may be feasible and more efficient compared to monotherapy in frail patients. The aim of this study is to investigate whether reduced dose GemNab is superior to full dose Gem in patients with resectable PC, who are not candidates for full dose combination chemotherapy in first line. METHODS: The Danish Pancreas Cancer Group (DPCG)-01 trial is a national multicenter prospective randomized phase II trial. A total of 100 patients in ECOG performance status 0-2 with non-resectable PC, not candidate for full dose combination chemotherapy in first line, but eligible for full dose Gem, will be included. Patients are randomized 1:1 to either full dose Gem or GemNab in 80% of recommended dose. The primary endpoint is progression-free survival. Secondary endpoints are overall survival, overall response rate, quality of life, toxicity and rate of hospitalizations during treatment. The correlation between blood inflammatory markers, including YKL-40 and IL-6, circulating tumor DNA, and tissue biomarkers of resistance to chemotherapy and outcome will be explored. Finally, the study will include measures of frailty (G8, modified G8, and chair-stand-test) to assess whether scoring would enable a personalized allocation to different treatments or indicates a possibility for interventions. DISCUSSION: Single-drug treatment with Gem has for frail patients with non-resectable PC been the main treatment option for more than thirty years, but the impact on outcome is modest. If improved results and sustained tolerability with reduced dose combination chemotherapy can be shown, this could change the future practice for this increasing group of patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05841420. Secondary Identifying No: N-20210068. EudraCT No: 2021-005067-52. PROTOCOL VERSION: 1.5, 16-MAY-2023.

From expert to novice and back: a qualitative study of interprofessional collaboration and the experiences of frontline healthcare professionals during the first wave of COVID-19.

BACKGROUND: The global coronavirus disease 2019 pandemic put extreme pressure on healthcare systems worldwide, forcing a heavy workload on healthcare professionals. Frontline treatment and care for patients with coronavirus disease 2019 compelled healthcare professionals to rapidly adapt to new working conditions. This study explores the experiences of frontline healthcare professionals to learn more about how frontline work affects their learning and skills development but also interprofessional collaboration during a pandemic. METHODS: In-depth, one-to-one semi-structured interviews were conducted with 22 healthcare professionals. A broad interdisciplinary group, the participants were employed in public hospitals in four of Denmark's five regions. Using a reflexive methodology for the data analysis allowed reflexive interpretation when interpreting subjects and interpreting the interpretation. RESULTS: The study identified two empirical themes: into the unknown and in the same boat, which we critically interpreted using learning theory and theory on interprofessionalism. The study found that the healthcare professionals moved from being experts in their own fields to being novices in the frontline of the pandemic, and then back to being experts based on interprofessional collaboration that included shared reflection. Working in the frontline was imbued with a unique atmosphere in which workers were equals and functioned interdependently, the barriers normally obstructing interprofessional collaboration set aside to focus on combating the pandemic. CONCLUSIONS: This study reveals new insights regarding knowledge on frontline healthcare professionals in terms of learning and developing new skills, as well as the importance of interprofessional collaboration. The insights contributed to the understanding of the importance of shared reflection and how the development of expertise was a socially embedded process where discussions were possible without fear of being ridiculed and healthcare professionals were willing to share their knowledge.

The ReCoN intervention: a co-created comprehensive intervention for primary mental health care aiming to prevent involuntary admissions.

BACKGROUND: Reducing involuntary psychiatric admissions is a global concern. In Norway, the rate of involuntary admissions was 199 per 100,000 people 16 years and older in 2020. Individuals' paths towards involuntary psychiatric admissions usually unfold when they live in the community and referrals to such admissions are often initiated by primary health care professionals. Interventions at the primary health care level can therefore have the potential to prevent such admissions. Interventions developed specifically for this care level are, however, lacking. To enhance the quality and development of services in a way that meets stakeholders' needs and facilitates implementation to practice, involving both persons with lived experience and service providers in developing such interventions is requested. AIM: To develop a comprehensive intervention for primary mental health care aiming to prevent involuntary admissions of adults. METHODS: This study had an action research approach with a participatory research design. Dialogue conferences with multiple stakeholders in five Norwegian municipalities, inductive thematic analysis of data material from the conferences, and a series of feedback meetings were conducted. RESULTS: The co-creation process resulted in the development of the ReCoN (Reducing Coercion in Norway) intervention. This is a comprehensive intervention that includes six strategy areas: [1] Management, [2] Involving Persons with Lived Experience and Family Carers, [3] Competence Development, [4] Collaboration across Primary and Specialist Care Levels, [5] Collaboration within the Primary Care Level, and [6] Tailoring Individual Services. Each strategy area has two to four action areas with specified measures that constitute the practical actions or tasks that are believed to collectively impact the need for involuntary admissions. CONCLUSIONS: The ReCoN intervention has the potential for application to both national and international mental health services. The co-creation process with the full range of stakeholders ensures face validity, acceptability, and relevance. The effectiveness of the ReCoN intervention is currently being tested in a cluster randomised controlled trial. Given positive effects, the ReCoN intervention may impact individuals with a severe mental illness at risk of involuntary admissions, as more people may experience empowerment and autonomy instead of coercion in their recovery process.

DnaK response to expression of protein mutants is dependent on translation rate and stability.

Chaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the λ-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

The marked body - a qualitative study on survivors embodied experiences of a COVID-19 illness trajectory.

BACKGROUND: Research on COVID-19 has reported data on epidemiology and pathophysiology but less about what it means to be a person living through this illness. Research involving the patients' perspectives may help to improve healthcare professionals' understanding of ways to support patients. AIMS: To gain in-depth understanding of the meaning of a COVID-19 illness trajectory from the patients' perspective. METHODS: Fifteen participants who had undergone an illness trajectory due to confirmed COVID-19 infection participated in individual qualitative interviews. Data collection, analysis and interpretation were inspired by Ricoeur's philosophy and Merleau-Ponty's phenomenology of perception and embodiment has been applied as a theoretical frame. FINDINGS: Being infected with coronavirus is expressed as an experience in which the participants oscillate between relief, security, imprisonment and raw fear. A predominant focus on the physical dimensions of the diseased body was found in the encounters between patient and healthcare system, and distance may furthermore be a consequence of use of protective equipment. Stigma and fear of infection were also expressed. After COVID-19, an overwhelming feeling of a door opening to freedom is perceived. However, the body is marked, and bears witness to decay from this insidious and frightening virus. The responsibility for assessing their bodily symptoms is placed with the individual patients themselves, who feel lonely and fearful and this keeps them indoors. CONCLUSIONS: During a COVID-19 illness, trajectory concerns about the unknown course of this disease are highlighted. Isolation is confrontational; however, a companionship between patients might emerge. The study shed light on an unavoidable gap between the patients and healthcare professionals due to the use protective equipment. After COVID-19, the body is labelled as something others fear and become a symbol of awe and alienation for others.

The heart & mind trial: intervention with cognitive-behavioural therapy in patients with cardiac disease and anxiety: randomised controlled trial protocol.

INTRODUCTION: Patients with cardiac disease often experience anxiety (prevalence about 20%-25%) and have a doubled mortality risk when suffering from anxiety compared with patients without anxiety. This calls for interventions aiming to reduce anxiety. METHODS AND ANALYSIS: The Heart & Mind Trial consists of three parts: (1) screening of all hospitalised and outpatient cardiac patients with arrhythmia, heart failure or ischaemic heart disease at four university hospitals in Denmark using the Hospital Anxiety and Depression Scale-Anxiety subscale (HADS-A); Patients scoring ≥8 is invited to participate; (2) Assessment of the type of anxiety by Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders and (3) Randomised clinical superiority trial with blinded outcome assessment, with 1:1 randomisation to cognitive-behavioural therapy (CBT) performed by a CBT-trained cardiac nurse plus usual care or, usual care alone. The primary outcome is anxiety measured with HADS-A at 5 months. Secondary outcomes include anxiety symptoms measured with Becks Anxiety Inventory and heart rate variability. Exploratory outcomes measured at 12 months include blood cortisol (stress response), blood C reactive protein (stress response), health-related quality of life, readmission, mortality and attributable direct costs. A total of 336 patients will be included. The primary analyses are based on the intention-to-treat principle. For the primary outcome, we will use a linear regression model. For the long-term outcomes, mixed regression models will be used including repeated measurements. ETHICS AND DISSEMINATION: The trial is performed in accordance with the Declaration of Helsinki. All patients must give informed consent prior to participation and the trial is initiated after approval by the Danish Data Protection Agency (P-2020-894) and the National Committee on Health Research Ethics (H-20066739). Positive, neutral and negative results of the trial will be published. TRIAL REGISTRATION NUMBER: NCT04582734.

Like soldiers on the front - a qualitative study understanding the frontline healthcare professionals' experience of treating and caring for patients with COVID-19.

BACKGROUND: While people in the societies must stay home to reduce spread of the newly discovered coronavirus, healthcare professionals do the exact opposite. For them the coronavirus is an enemy that should be defeated as a part of one's job. They do, however, also have a daily life with family while doing their work obligations. The purpose of this study was to gain an in-depth understanding of the frontline healthcare professionals' experience of balancing work life and family life during the COVID-19 pandemic. METHODS: A sample of 22 frontline healthcare professionals caring for patients with COVID-19 was included and interviewed individually from May to August 2020. Ricoeur's phenomenological hermeneutical philosophy inspired the methodology in this study. RESULT: Frontline healthcare professionals treating and caring for patients with COVID-19 are, voluntarily or involuntarily, forced to be ready to change departments as well as being ready to face the unknown coronavirus. The frontline work leads to feelings of being abandoned among their families and friends due to the threat of bringing the infection home and spreading the virus. Although healthcare professionals are facing a working life filled with uncertainty and unpredictability impacting their family life, they express opposing feelings of being a part of something bigger. CONCLUSIONS: The work life balance for these healthcare professionals is threatened by changes in professional responsibilities, working hours and shifts. Fear of bringing the infection home challenges them ethically and creates a distance between healthcare professionals and their families, leading to a conflict within the individual if their work on the frontline is worth it - or if it is a too high price to pay. Despite facing a working life filled with uncertainty and unpredictability the healthcare professionals are being a part of something bigger that contributes to a fighting spirit and professional pride outweighing the negative consequences; like being soldiers on the front.

Testing for COVID-19 regulates behavior in the general population: A qualitative study of experiences of awaiting test result for COVID-19.

AIM: This study aimed to explore experiences of awaiting a test result for COVID-19 among individuals from the general population. METHODS: Fifteen participants were recruited from COVID-19 testing tents in the Capital Region of Denmark in March and April 2020. A phenomenological-hermeneutic approach inspired by Ricoeur's theory of interpretation was used. RESULTS: The analysis revealed five themes. (1) The participants' experiences of awaiting a COVID-19 test result illuminated concerns related to infecting others rather than their own health. Experiences of guilt for not taking all possible precautions to avoid the spread of COVID-19 were described and thoughts of potentially having exposed others bothered the participants. (2) The test result would guide their precautions and therefore regulate behaviour at home and in society. (3) Even though the participants did not take all possible precautions they made some changes in their everyday lives. (4) Leaving the individual with the responsibility for taking precautions based on their subjective experiences created feelings of insecurity and uncertainty. (5) Being met by health professionals was an experience that meant for the particpants that behaviour towards limiting the infection became very clear. The seriousness experienced around the test situation facilitated this attitude and behaviour in the participants. Conclusion: This study illuminated how testing for COVID-19 regulates behavior in the general population. The testing was both important for the individual's cautious behavior towards other people, work and in getting around in society but also a way to regulate behavior from a societal perspective to quicken suppression and avoid transmission of COVID-19.

It's Not Just a Virus! Lived Experiences of People Diagnosed With COVID-19 Infection in Denmark.

The purpose of this study was to explore the lived experiences of people infected with the coronavirus in Denmark during the first phase of the pandemic. An explorative qualitative design underpinned by a phenomenological hermeneutical approach was applied. Fifteen individuals with confirmed COVID-19 infection were included and interviewed individually by telephone. Analyses were inspired by Ricoeur's interpretation theory. The study illuminated how being diagnosed with COVID-19 was experienced not just a virus infecting the participants' biology; it was also a threat to their existence and bodily perception as well as an interference in ordinary social relationships. Beyond a supportive approach, the participants experienced being a special case where people around them acted with excitement and curiosity. Responsibility for existential and emotional care after COVID-19 has been placed with the individual ill person and within their ordinary social circle. We suggest follow-up and rehabilitation for people during and after COVID-19 to support recovery.

Physical activity and serious adverse events in patients with atrial fibrillation and/or atrial flutter treated with catheter ablation.

BACKGROUND: Atrial fibrillation (AF) and atrial flutter (AFL) are cardiac arrhythmias associated with cardiovascular morbidity. Physical activity (PA) can trigger AF and AFL recurrence, but can also improve physical functional capacity in this patient group. Guidelines do not include concrete recommendations regarding PA for this patient group. OBJECTIVE: To assess the impact of the level of PA on risk of serious adverse events (SAEs) in patients with AF and/or AFL treated with catheter ablation. METHODS: A prospective cohort study including 462 patients with AF and/or AFL treated with catheter ablation from the CopenHeart Survey. The International Physical Activity Questionnaire (IPAQ) was used to explore patients' self-reported level of PA. SAEs were identified in the Danish National Patient Register and the Danish Civil Registration System one year after study onset. Cox regression analysis was carried out to assess the risks of SAE. RESULTS: During the one-year follow-up period, 98 patients (21.8%) experienced at least one SAE. Patients with a moderate-high PA level had a 36% lower risk of experiencing SAEs during the follow-up period, compared to patients in the low PA group, after adjusting for confounders. CONCLUSION: A moderate-high vs. low level of PA was found to be associated with a lower incidence of SAEs in patients undergoing AF and/or AFL ablation.

A stoic and altruistic orientation towards their work: a qualitative study of healthcare professionals' experiences of awaiting a COVID-19 test result.

BACKGROUND: Extensive measures to reduce person-to-person transmission of COVID-19 are required to control the current outbreak. Special attention is directed at healthcare professionals as reducing the risk of infection in healthcare is essential. The purpose of this study was to explore healthcare professionals' experiences of awaiting a test result for a potential COVID-19 infection. METHODS: Qualitative interviews with 15 healthcare professionals were performed, underpinned by a phenomenological hermeneutical analytical framework. RESULTS: The participating healthcare professionals' experiences of awaiting a COVID-19 test result were found to be associated with a stoic and altruistic orientation towards their work. These healthcare professionals presented a strong professional identity overriding most concerns about their own health. The result of the coronavirus test was a decisive parameter for whether healthcare professionals could return to work. The healthcare professionals were aware that their family and friends were having a hard time knowing that the COVID-19 infection risk was part of their jobs. This concern did not, however, cause the healthcare professionals to falter in their belief that they were doing the right thing by focusing on their core area. The threat to own health ran through the minds of the healthcare professionals occasionally, which makes access to testing particularly important. CONCLUSION: The participating healthcare professionals had a strong professional identity. However, a discrepancy between an altruistic role as a healthcare professional and the expectations that come from the community was illuminated. A mental health coronavirus hotline for healthcare professionals is suggested.

Crystal Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.

Educational interventions to improve outcomes in patients with atrial fibrillation-a systematic review.

BACKGROUND: Atrial fibrillation (AF) is an emerging epidemic associated with poor mental health and quality of life, as well as morbidity and mortality. Whilst other cardiovascular conditions have demonstrated positive outcomes from educational programmes, this approach is not well integrated in clinical practice in patients with AF. Though evidence in this area is mounting, a thorough overview seems to be lacking. AIM: To assess benefits and harms of educational interventions compared with no intervention in adults with AF. METHOD: A systematic review and meta-analysis were performed including the outcomes: Serious adverse events (mortality and readmission), mental health (anxiety and depression), physical capacity, quality of life and self-reported incidence of symptoms of AF. PubMed, Embase, CINAHL, Cochrane Library and PsycINFO were searched between June and august 2018. Data extraction and quality assessment were performed independently by two reviewers. The Cochrane Risk of Bias tool was applied for the randomised controlled trials and the Amstar Checklist for the systematic reviews. RESULTS: Eight randomised controlled trials and one non-randomised interventional study were included, with a total of 2388 patients. Comparing with controls patient education was associated with a reduction in: Serious adverse events (Risk Ratio 0.78, CI 95% 0.63-0.97), anxiety with a mean difference of -0.62 (CI 95% -1.21, -0.04) and depression with a mean difference of -0.74 (CI 95% -1.34, -0.14). Health-related quality of life and physical capacity was found to increase after patient education, yet, only one study found statistically significant differences between groups. No differences were observed with regards to self-reported incidence of symptoms of AF. CONCLUSIONS: Educational interventions significantly decrease the number of serious adverse events in patients with AF and seem to have a positive impact on mental health and self-reported quality of life. However, the evidence is limited, and more studies are warranted.

Oxidoreductase disulfide bond proteins DsbA and DsbB form an active redox pair in Chlamydia trachomatis, a bacterium with disulfide dependent infection and development.

Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and-using recombinant purified proteins-demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein's thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB differentiation coincided with the oxidation of the chlamydial outer membrane complex (COMC). Together with our demonstration of an active redox pairing, our findings suggest a potential role for CtDsbA and CtDsbB in the critical disulfide bond formation step in the highly regulated development cycle.

Life inside and out: making and breaking protein disulfide bonds in Chlamydia.

Disulphide bonds are widely used among all domains of life to provide structural stability to proteins and to regulate enzyme activity. Chlamydia spp. are obligate intracellular bacteria that are especially dependent on the formation and degradation of protein disulphide bonds. Members of the genus Chlamydia have a unique biphasic developmental cycle alternating between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body. The proteins in the envelope of the EB are heavily cross-linked with disulphides and this is known to be critical for this infectious phase. In this review, we provide a comprehensive summary of what is known about the redox state of chlamydial envelope proteins throughout the developmental cycle. We focus especially on the factors responsible for degradation and formation of disulphide bonds in Chlamydia and how this system compares with redox regulation in other organisms. Focussing on the unique biology of Chlamydia enables us to provide important insights into how specialized suites of disulphide bond (Dsb) proteins cater for specific bacterial environments and lifecycles.

Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase.

The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins.

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template.

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations.

Fast, cross cultivar determination of total carotenoids in intact carrot tissue by Raman spectroscopy and Partial Least Squares calibration.

In order to speed up the breeding of orange carrots for high carotenoid content it is imperative to develop a fast and non-destructive technique. 332 roots from 86 carrot varieties grown in 2014 at the experimental farm in Høje Taastrup (DK) form the basis of this study. All roots were measured by Raman spectroscopy. The carotenoid content of the very same roots was estimated through a wet chemistry method coupled with UV-VIS at 447nm and 540nm. For the Raman spectroscopy, measurements were made on a cross section disk approximately 10cm from the root top at three different positions in the phloem. Since the top of the carrot is intact, it may still be used for growing. The final calibration model shows an uncertainty (RMSECV) of 20.5ppm, and a R(2)=0.86. It has thus proven to be well suited for prediction of carotenoids in orange carrots, and especially for ranking them according to the content.