ESGO-ESMO-ESP consensus conference recommendations on ovarian cancer: pathology and molecular biology and early, advanced and recurrent disease.

The European Society of Gynaecological Oncology, the European Society for Medical Oncology (ESMO) and the European Society of Pathology held a consensus conference (CC) on ovarian cancer on 15-16 June 2022 in Valencia, Spain. The CC panel included 44 experts in the management of ovarian cancer and pathology, an ESMO scientific advisor and a methodologist. The aim was to discuss new or contentious topics and develop recommendations to improve and harmonise the management of patients with ovarian cancer. Eighteen questions were identified for discussion under four main topics: (i) pathology and molecular biology, (ii) early-stage disease and pelvic mass in pregnancy, (iii) advanced stage (including older/frail patients) and (iv) recurrent disease. The panel was divided into four working groups (WGs) to each address questions relating to one of the four topics outlined above, based on their expertise. Relevant scientific literature was reviewed in advance. Recommendations were developed by the WGs and then presented to the entire panel for further discussion and amendment before voting. This manuscript focuses on the recommendation statements that reached a consensus, their voting results and a summary of evidence supporting each recommendation.

Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase.

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.

Structural insights into CodY activation and DNA recognition.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

Mortality and complications after percutaneous endoscopic gastrostomy: a retrospective multicentre study.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) is the method of choice for patients in need of long-term nutritional support or gastric decompression. Although it is considered safe, complications and relatively high mortality rates have been reported. We aimed to identify risk factors for complications and mortality after PEG in routine healthcare. METHODS: This retrospective study included all adult patients who received a PEG between 2013 and 2019 in Region Norrbotten, Sweden. RESULTS: 389 patients were included. The median age was 72 years, 176 (45%) were women and 281 (72%) patients received their PEG due to neurological disease. All-cause mortality was 15% at 30 days and 28% at 90 days. Malignancy as the indication for PEG was associated with increased mortality at 90 days (OR 4.41, 95% CI 2.20-8.88). Other factors significantly associated with increased mortality were older age, female sex, diabetes mellitus, heart failure, lower body mass index and higher C-reactive protein levels. Minor and major complications within 30 days occurred in 11% and 15% of the patients, respectively. Diabetes increased the risk of minor complications (OR 2.61, 95% CI 1.04-6.55), while those aged 75 + years were at an increased risk of major complications, compared to those younger than 65 years (OR 2.23, 95% CI 1.02-4.85). CONCLUSIONS: The increased risk of death among women and patients with malignancy indicate that these patients could benefit from earlier referral for PEG. Additionally, we found that age, diabetes, heart failure, C-reactive protein and body mass index all impact the risk of adverse outcomes.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Global DNA methylation changes in treated and untreated MS patients measured over time.

Multiple sclerosis (MS) is an autoimmune, neurological disease. We investigated genome-wide DNA methylation profiles of CD4+ and CD8+ T cells from MS patients and healthy controls at baseline and a follow-up visit. Patients were all treatment-naïve at baseline, and either on treatment or remained untreated at the follow-up visit. MS patients show more changes in their T cell DNA methylation profiles as compared to healthy controls over time, with the most pronounced differences observed in the untreated MS patients. These findings underline the potential of DNA methylation as biomarkers in MS.

Structural basis for transthyretin amyloid formation in vitreous body of the eye.

Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form.

The PERFORM-P (Principles of Evidence-based Reporting in FORensic Medicine-Pathology version).

INTRODUCTION: Most findings of forensic pathology examinations are presented as written reports. There are currently no internationally accepted recommendations for writing forensic pathology reports. Existing recommendations are also varied and reflect the differences in the scope and role of forensic medical services and local settings in which they are to be implemented. The legal fact-finder thus faces wide variation in the quality of forensic pathology reports, which poses a threat to the reliability of legal decision-making. To address this issue, the development of the "PERFORM-P (Principles of Evidence-based Reporting in FORensic Medicine-Pathology version)" was undertaken. The goal of the PERFORM-P is to provide common practice recommendations adaptable to local requirements to promote evidence-based practice (EBP) in forensic pathology. METHODS: An international consensus study was conducted in three phases by (1) developing a long-list of items to be considered in the reporting recommendations, (2) conducting a Delphi process (an iterative survey method to transform individual opinions into group consensus) with international forensic pathologists, and (3) designing the PERFORM-P prototype and its accompanying manual. RESULTS: With assistance from 106 forensic pathologists/forensic medical practitioners from 41 countries, the PERFORM-P was developed. The PERFORM-P consists of a list of 61 items to be included in a forensic pathology report, which is accompanied by its Explanation and Elaboration (E&E) document. DISCUSSION: To prepare forensic pathology (postmortem) reports that incorporate principles of evidence-based practice, internationally accepted recommendations might be helpful. The PERFORM-P identifies recommendations for necessary elements to include in a forensic pathology report. PERFORM-P can be applied to a wide range of matters requiring forensic pathological analysis, acceptable to forensic pathologists from a representative selection of jurisdictions and medico-legal systems.

CD8+ T cell gene expression analysis identifies differentially expressed genes between multiple sclerosis patients and healthy controls.

BACKGROUND: Genetic and clinical observations have indicated T cells are involved in MS pathology. There is little insight in how T cells are involved and whether or not these can be used as markers for MS. OBJECTIVES: Analysis of the gene expression profiles of circulating CD8+ T cells of MS patients compared to healthy controls. METHODS: RNA from purified CD8+ T cells was sequenced and analyzed for differential gene expression. Pathway analyses of genes at several p-value cutoffs were performed to identify putative pathways involved. RESULTS: We identified 36 genes with significant differential gene expression in MS patients. Four genes reached at least 2-fold differences in expression. The majority of differentially expressed genes was higher expressed in MS patients. Genes associated to MS in GWAS showed enrichment amongst the differentially expressed genes. We did not identify enrichment of specific pathways amongst the differentially expressed genes in MS patients. CONCLUSIONS: CD8+ T cells of MS patients show differential gene expression, with predominantly higher activity of genes in MS patients. We do not identify specific biological pathways in our study. More detailed analysis of CD8+ T cells and subtypes of these may increase understanding of how T cells are involved in MS.

Structural Basis for GTP versus ATP Selectivity in the NMP Kinase AK3.

ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3, which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle, where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and nonproductively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and nonproductive binding by the incorrect substrate bear a strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together, we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from nonlinearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These nonspecific interactions likely act to lower the available intracellular GTP and ATP concentrations and may have driven evolution of the Michaelis constants of NMP kinases accordingly.

A Novel Growth-Based Selection Strategy Identifies New Constitutively Active Variants of the Major Virulence Regulator PrfA in Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections. Its major virulence regulator is the transcriptional activator PrfA, a member of the Crp/Fnr family of transcriptional regulators. To establish a successful L. monocytogenes infection, the PrfA protein needs to be in an active conformation, either by binding the cognate inducer glutathione (GSH) or by possessing amino acid substitutions rendering the protein constitutively active (PrfA*). By a yet unknown mechanism, phosphotransferase system (PTS) sugars repress the activity of PrfA. We therefore took a transposon-based approach to identify the mechanism by which PTS sugars repress PrfA activity. For this, we screened a transposon mutant bank to identify clones able to grow in the presence of glucose-6-phosphate as the sole carbon source. Surprisingly, most of the isolated transposon mutants also carried amino acid substitutions in PrfA. In transposon-free strains, the PrfA amino acid substitution mutants displayed growth, virulence factor expression, infectivity, and DNA binding, agreeing with previously identified PrfA* mutants. Hence, the initial growth phenotype observed in the isolated clone was due to the amino acid substitution in PrfA and unrelated to the loci inactivated by the transposon mutant. Finally, we provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.IMPORTANCE The Gram-positive bacterium Listeria monocytogenes is a human pathogen affecting mainly the elderly, immunocompromised people, and pregnant women. It can lead to meningoencephalitis, septicemia, and abortion. The major virulence regulator in L. monocytogenes is the PrfA protein, a transcriptional activator. Using a growth-based selection strategy, we identified mutations in the PrfA protein leading to constitutively active virulence factor expression. We provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.

Influence of collisions on ion dynamics in the inner comae of four comets.

CONTEXT: Collisions between cometary neutrals in the inner coma of a comet and cometary ions that have been picked up into the solar wind flow and return to the coma lead to the formation of a broad inner boundary known as a collisionopause. This boundary is produced by a combination of charge transfer and chemical reactions, both of which are important at the location of the collisionopause boundary. Four spacecraft measured ion densities and velocities in the inner region of comets, exploring the part of the coma where an ion-neutral collisionopause boundary is expected to form. AIMS: The aims are to determine the dominant physics behind the formation of the ion-neutral collisionopause and to evaluate where this boundary has been observed by spacecraft. METHODS: We evaluated observations from three spacecraft at four different comets to determine if a collisionopause boundary was observed based on the reported ion velocities. We compared the measured location of the ion-neutral collisionopause with measurements of the collision cross sections to evaluate whether chemistry or charge exchange are more important at the location where the collisionopause is observed. RESULTS: Based on measurements of the cross sections for charge transfer and for chemical reactions, the boundary observed by Rosetta appears to be the location where chemistry becomes the more probable result of a collision between H2O and H2O+ than charge exchange. Comparisons with ion observations made by Deep Space 1 at 19P/Borrelly and Giotto at 1P/Halley and 26P/Grigg-Skjellerup show that similar boundaries were observed at 19P/Borrelly and 1P/Halley. The ion composition measurements made by Giotto at Halley confirm that chemistry becomes more important inside of this boundary and that electron-ion dissociative recombination is a driver for the reported ion pileup boundary.

Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase ϵ.

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe-S cluster. Here, we present two new crystal structures of Pol2CORE with an Fe-S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe-S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT are inviable. In conclusion, Pol ϵ has a single Fe-S cluster bound at the base of the P-domain, and this Fe-S cluster is essential for cell viability and polymerase activity.

Control of Bacterial Virulence through the Peptide Signature of the Habitat.

To optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent-treated (PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione (GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Opp-imported oligopeptides promote PrfA induction intracellularly and PrfA repression outside the host.

The health promoting conversations intervention for families with a critically ill relative: A pilot study.

BACKGROUND: After intensive care unit treatment, patients often have prolonged impairments that affect their physical, cognitive and mental health. Family members can face overwhelming and emotionally challenging situations and their concerns and needs must be addressed. OBJECTIVE: We investigated the outcomes of pilot randomised control trial, a nurse-led family intervention, Health Promoting Conversations, which focused on family functioning and wellbeing in families with a critically ill member. STUDY DESIGN: This randomised controlled pilot study used a pre-test, post-test design with intervention and control groups to investigate the outcomes of the nurse-led intervention in 17 families. OUTCOME MEASURES: The Health Promoting Conversations intervention was evaluated using validated instruments that measure family functioning and family wellbeing: the General Functioning sub-scale from the McMaster Family Assessment Device; the Family Sense of Coherence, the Herth Hope Index, and the Medical Outcome Short-Form Health Survey. Descriptive and analytical statistical methods were used to analyse the data. RESULTS: After 12 months, the intervention group reported better family functioning than the control group. The intervention group also had better social functioning and mental health after 12 months. CONCLUSION: This intervention may improve family wellbeing by improving family function, reducing stress, and promoting better mental health.

Firearm deaths in Sweden.

BACKGROUND: Sweden's firearm legislation obligates physicians to report patients that are deemed unsuitable to possess a firearm. This study aimed to explore the involvement of firearm use in firearm fatalities and to evaluate physician reporting concerning cases of firearm deaths. METHODS: Fatal firearm suicides and homicides in Sweden were studied for the years 2012-2013, accidental deaths and undetermined manner of deaths for the period 1987-2013. Police reports and autopsy protocols were collected from the National Board of Forensic Medicine, health care data in 1 year before the fatality from the National Board of Health, and information about physician reports and firearm licences from the Swedish Police. RESULTS: A total of 291 firearm deaths (213 suicides, 52 accidental deaths, 23 solved homicides and 3 cases with undetermined manner of death) were identified. Firearm suicides were positively correlated with the number of licensed firearm owners. Legal firearm use predominated in firearm suicides and accidental deaths, illegal in homicides. No suicide victim or shooter in an accidental death was previously reported by a physician to the police according to the firearm law. The majority of the shooters in accidental deaths and suicides had no registered health care visits. Less than half (42%) of all suicide victims had a previous health care contact due to mental health problems. CONCLUSIONS: Not one single suicide victim nor any shooter in accidental deaths in the present study had been reported according to the firearm law, bringing the evidence of a suboptimal framework.

Interspecies Variation between Fish and Human Transthyretins in Their Binding of Thyroid-Disrupting Chemicals.

Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream ( Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (>500-fold lower affinity). Crystal structures of the seabream TTR-TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR. Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.