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Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2'-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms. Author summary: Herpes simplex virus type 1 (HSV-1) is a common virus carried approximately by half of the global population. Though it is mostly known by causing cold sores, it also causes herpes keratitis, which is the leading cause of infectious blindness in the world. The treatment for herpes keratitis and other severe disease forms of herpes infection is insufficient, as resistant variants arise upon long-term prophylactic treatments. We have earlier developed an anti-HSV siRNA swarm, which has proven safe and effective in many cell types, in animal models, and against variants resistant to current first-in-line treatment. Most recently, we added modifications to the anti-HSV siRNA swarm, which increased its efficacy and stability. In this study, we show the efficacy and safety of the modified anti-HSV siRNA swarm in a cell line representing the treatment target tissue in herpes keratitis. Our results show that our modified anti-HSV siRNA swarm is a possibility for future therapy for herpes keratitis. The results encourage further research in an animal model of herpes keratitis in order to uncover the potential of our modified anti-HSV siRNA swarm. [ABSTRACT FROM AUTHOR]

Background: Rheumatic heart disease (RHD) complicating acute rheumatic fever (ARF) remains an important health problem in developing countries. No definitive diagnostic test for ARF exists and the role of Doppler echocardiography (DEC) for long-term prognostic evaluation following ARF is not well established. Objective: To investigate the prognostic value of DEC in patients with ARF as a predictor of chronic valve dysfunction. Methods: Prospectively enrolled patients with clinical ARF had a DEC performed soon after diagnosis and repeated at 1, 3, 6 and 12 months and thereafter at every 1–2 years. We defined chronic valve dysfunction by ≥ 3 of the following: increased valve thickening, commissure fusion, subvalvular thickening, reduced leaflet mobility, non-trivial mitral and/or aortic regurgitation. We performed univariate analysis and developed multivariate logistic regression models to identify variables that may influence evolution to RHD. p <0.05 was considered significant. Results: We evaluated 70(57% men) patients, 10.8±5.6 years-old during the ARF episode and followed for 95±26 months. Chronic valve dysfunction was identified in 36(51.4%) which fulfilled criteria for RHD and 10(27.8%) of them died or underwent valve surgery. Univariate analysis showed that mitral valve thickening and presence of mitral regurgitation at baseline DEC, were associated with RHD(p<0.01). Multivariate logistic regression showed that only mitral valve thickness either as a continuous (Odds-Ratio:5.8;95%CI:1.7–19.7) or as a categorical variable (Odds-Ratio:4.04;95%CI:1.06–15.3) was an independent predictor of chronic valve dysfunction. Conclusions: Mitral leaflets thickening documented at the time of diagnosis of ARF is a consistent prognostic marker for the subsequent evolution to RHD. [ABSTRACT FROM AUTHOR]

Specific cleavage of proteins by proteases is essential for several cellular, physiological, and viral processes. Chymotrypsin-related proteases that form the PA clan in the MEROPS classification of proteases is one of the largest and most diverse group of proteases. The PA clan comprises serine proteases from bacteria, eukaryotes, archaea, and viruses and chymotrypsin-related cysteine proteases from positive-strand RNA viruses. Despite low amino acid sequence identity, all PA clan proteases share a conserved double β-barrel structure. Using an automated structure-based hierarchical clustering method, we identified a common structural core of 72 amino acid residues for 143 PA clan proteases that represent 12 protein families and 11 subfamilies. The identified core is located around the catalytic site between the two β-barrels and resembles the structures of the smallest PA clan proteases. We constructed a structure-based distance tree derived from the properties of the identified common core. Our structure-based analyses support the current classification of these proteases at the subfamily level and largely at the family level. Structural alignment and structure-based distance trees could thus be used for directing objective classification of PA clan proteases and to strengthen their higher order classification. Our results also indicate that the PA clan proteases of positive-strand RNA viruses are related to cellular heat-shock proteases, which suggests that the exchange of protease genes between viruses and cells might have occurred more than once. [ABSTRACT FROM AUTHOR]

During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly. [ABSTRACT FROM AUTHOR]

Rationale: Neonatal respiratory distress syndrome is a restrictive lung disease characterized by surfactant deficiency. Decreased vascular endothelial growth factor (VEGF), which demonstrates important roles in angiogenesis and vasculogenesis, has been implicated in the pathogenesis of restrictive lung diseases. Current animal models investigating VEGF in the etiology and outcomes of RDS require premature delivery, hypoxia, anatomically or temporally limited inhibition, or other supplemental interventions. Consequently, little is known about the isolated effects of chronic VEGF inhibition, started at birth, on subsequent developing lung structure and function. Objectives: To determine whether inducible, mesenchyme-specific VEGF inhibition in the neonatal mouse lung results in long-term modulation of AECII and whole lung function. Methods: Triple transgenic mice expressing the soluble VEGF receptor sFlt-1 specifically in the mesenchyme (Dermo-1/rtTA/sFlt-1) were generated and compared to littermate controls at 3 months to determine the impact of neonatal downregulation of mesenchymal VEGF expression on lung structure, cell composition and function. Reduced tissue VEGF bioavailability has previously been demonstrated with this model. Measurements and Main Results: Triple transgenic mice demonstrated restrictive lung pathology. No differences in gross vascular development or protein levels of vascular endothelial markers was noted, but there was a significant decrease in perivascular smooth muscle and type I collagen. Mutants had decreased expression levels of surfactant protein C and hypoxia inducible factor 1-alpha without a difference in number of type II pneumocytes. Conclusions: These data show that mesenchyme-specific inhibition of VEGF in neonatal mice results in late restrictive disease, making this transgenic mouse a novel model for future investigations on the consequences of neonatal RDS and potential interventions. [ABSTRACT FROM AUTHOR]

Inflammation is an important feature of carcinogenesis. Tumor-associated macrophages (TAMs) can be associated with either poor or improved prognosis, depending on their properties and polarization. Current knowledge of the prognostic significance of TAMs in bladder cancer is limited and was investigated in this study. We analyzed 184 urothelial bladder cancer patients undergoing transurethral resection of a bladder tumor or radical cystectomy. CD68 (pan-macrophage marker), MAC387 (polarized towards type 1 macrophages), and CLEVER-1/Stabilin-1 (type 2 macrophages and lymphatic/blood vessels) were detected immunohistochemically. The median follow-up time was 6.0 years. High macrophage counts associated with a higher pT category and grade. Among patients undergoing transurethral resection, all studied markers apart from CLEVER-1/Stabilin-1 were associated with increased risk of progression and poorer disease-specific and overall survival in univariate analyses. High levels of two macrophage markers (CD68/MAC387+/+ or CD68/CLEVER-1+/+ groups) had an independent prognostic role after transurethral resection in multivariate analyses. In the cystectomy cohort, MAC387, alone and in combination with CD68, was associated with poorer survival in univariate analyses, but none of the markers were independent predictors of outcome in multivariate analyses. In conclusion, this study demonstrates that macrophage phenotypes provide significant independent prognostic information, particularly in bladder cancers undergoing transurethral resection. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers. [ABSTRACT FROM AUTHOR]

Background: Previous military studies have shown an energy deficit during a strenuous field training course (TC). This study aimed to determine the effects of energy bar supplementation on energy balance, physical activity (PA), physical performance and well-being and to evaluate ad libitum fluid intake during wintertime 8-day strenuous TC. Methods: Twenty-six men (age 20 ± 1 yr.) were randomly divided into two groups: The control group (n = 12) had traditional field rations and the experimental (Ebar) group (n = 14) field rations plus energy bars of 4.1 MJ⋅Nday-1 . Energy (EI) and water intake was recorded. Fat-free mass and water loss were measured with deuterium dilution and elimination, respectively. The energy expenditure was calculated using the intake/balance method and energy availability as (EI/estimated basal metabolic rate). PA was monitored using an accelerometer. Physical performance was measured and questionnaires of upper respiratory tract infections (URTI), hunger and mood state were recorded before, during and after TC. Results: Ebar had a higher EI and energy availability than the controls. However, decreases in body mass and fat mass were similar in both groups representing an energy deficit. No differences were observed between the groups in PA, water balance, URTI symptoms and changes in physical performance and fat-free mass. Ebar felt less hunger after TC than the controls and they had improved positive mood state during the latter part of TC while controls did not. Water deficit associated to higher PA. Furthermore, URTI symptoms and negative mood state associated negatively with energy availability and PA. Conclusion: An easy-to-use protein-rich energy bars did not prevent energy deficit nor influence PA during an 8-day TC. The high content of protein in the bars might have induced satiation decreasing energy intake from field rations. PA and energy intake seems to be primarily affected by other factors than energy supplementation such as mood state. [ABSTRACT FROM AUTHOR]

A high-affinity divalent cation-binding site located proximal to the catalytic center has been identified in several RNA-dependent RNA polymerases (RdRps), but the characteristics of such a site have not been systematically studied. Here, all available polymerase structures that follow the hand-like structural motif were screened for the presence of a divalent cation close to the catalytic site but distinct from catalytic metal ions. Such non-catalytic ions were found in all RNA virus families for which there were high-resolution RdRp structures available. Bound ions were always located in structurally similar locations at an approximate 6-Ådistance from the catalytic site. Furthermore, the second aspartate residue in the highly conserved GDD sequence was found to be involved in the coordination of the bound ion in all viral RdRps studied. These results suggest that a non-catalytic ion-binding site is conserved across positive-sense, single-stranded, and doublestranded RNA viruses. Interestingly, a non-catalytic ion was also observed in a similar position in the reverse transcriptase of the human immunodeficiency virus. Moreover, two members of the DNA-dependent DNA polymerase B family displayed an ion at a comparable distance from the catalytic site, but the position was clearly distinct from the non-catalytic ion-binding sites of RdRps. [ABSTRACT FROM AUTHOR]

The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNAbinding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways. [ABSTRACT FROM AUTHOR]

The utilization of artificial intelligence (AI) is expanding significantly within medical research and, to some extent, in clinical practice. Deep learning (DL) applications, which use large convolutional neural networks (CNN), hold considerable potential, especially in optimizing radiological evaluations. However, training DL algorithms to clinical standards requires extensive datasets, and their processing is labor-intensive. In this study, we developed an annotation tool named DLLabelsCT that utilizes CNN models to accelerate the image analysis process. To validate DLLabelsCT, we trained a CNN model with a ResNet34 encoder and a UNet decoder to segment the pancreas on an open-access dataset and used the DL model to assist in annotating a local dataset, which was further used to refine the model. DLLabelsCT was also tested on two external testing datasets. The tool accelerates annotation by 3.4 times compared to a completely manual annotation method. Out of 3,715 CT scan slices in the testing datasets, 50% did not require editing when reviewing the segmentations made by the ResNet34-UNet model, and the mean and standard deviation of the Dice similarity coefficient was 0.82±0.24. DLLabelsCT is highly accurate and significantly saves time and resources. Furthermore, it can be easily modified to support other deep learning models for other organs, making it an efficient tool for future research involving larger datasets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mustonen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Endophytic bacterium Serratia plymuthica A30 was identified as a superior biocontrol agent due to its effective colonization of potato tuber, tolerance to cold conditions, and strong inhibitory action against various soft rot pathogens, including Dickeya solani. We characterized transcriptome changes in potato tubers inoculated with S. plymuthica A30, D. solani, or both at the early and the late phases of interaction. At the early phase and in the absence of the pathogen, A30 influenced the microbial recognition system to initiate plant priming. In the presence of the pathogen alongside biocontrol strain, defense signaling was highly stimulated, characterized by the induction of genes involved in the detoxification system, reinforcement of cell wall structure, and production of antimicrobial metabolites, highlighting A30's role in enhancing the host resistance against pathogen attack. This A30-induced resistance relied on the early activation of jasmonic acid signaling and its production in tubers, while defense signaling mediated by salicylic acid was suppressed. In the late phase, A30 actively interferes with plant immunity by inhibiting stress- and defense-related genes expression. Simultaneously, the genes involved in cell wall remodeling and indole-3-acetic acid signaling were activated, thereby enhancing cell wall remodeling to establish symbiotic relationship with the host. The endophytic colonization of A30 coincided with the induction of genes involved in the biosynthesis and signaling of ethylene and abscisic acid, while downregulating those related to gibberellic acid and cytokinin. This combination suggested fitness benefits for potato tubers by preserving dormancy, and delaying sprouting, which affects durability of tubers during storage. This study contributes valuable insights into the tripartite interaction among S. plymuthica A30, D. solani, and potato tubers, facilitating the development of biocontrol system for soft rot pathogens under storage conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hadizadeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The present study examined whether people higher in psychopathy experienced less self-reported and psychophysiological nociceptive pressure than people lower in psychopathy. We also examined whether psychopathy affects empathy for others' pain via self-reported and psychophysiological measures. Three hundred and sixty-nine students (18-78 years; M = 26, SD = 9.34) were screened for psychopathic traits using the Youth Psychopathy Inventory (YPI). Stratified sampling was used to recruit 49 adults residing in the highest (n = 23) and lowest (n = 26) 20% of the psychopathy spectrum. Using skin conductance response (SCR) and self-report responses, participants responded to individually adjusted intensities of pneumatic pressure and others' pain images and completed self-reported psychopathy and empathy measures (Triarchic Psychopathy Measure, TriPm; Interpersonal Reactivity Index, IRI). People higher in psychopathy self-reported feeling less nociceptive pressure compared to people lower in psychopathy, yet we did not find any differences in SCR to nociceptive pressure. However, when viewing other people in pain, the high psychopathy group displayed lower SCR and lower self-reported empathy compared to those lower in psychopathy. Our results suggest psychopathic traits relate to problems empathising with others' pain, as well as the perception of nociceptive pressure. We also show support for the theory of dual harm which has been receiving increasing attention. Consequently, psychopathy interventions should focus both on recognising and empathising with the pain of others., Competing Interests: The authors have declared no competing interests exist., (Copyright: © 2024 Alshukri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Out-of-hospital cardiac arrest (OHCA) carries a relatively poor prognosis and requires multimodal prognostication to guide clinical decisions. Identification of previously unrecognized metabolic routes associated with patient outcome may contribute to future biomarker discovery. In OHCA, inhaled xenon elicits neuro- and cardioprotection. However, the metabolic effects remain unknown., Materials and Methods: In this post-hoc study of the randomised, 2-group, single-blind, phase 2 Xe-Hypotheca trial, 110 OHCA survivors were randomised 1:1 to receive targeted temperature management (TTM) at 33°C with or without inhaled xenon during 24 h. Blood samples for nuclear magnetic resonance spectroscopy metabolic profiling were drawn upon admission, at 24 and 72 h., Results: At 24 h, increased lactate, adjusted hazard-ratio 2.25, 95% CI [1.53; 3.30], p<0.001, and decreased branched-chain amino acids (BCAA) leucine 0.64 [0.5; 0.82], p = 0.007, and valine 0.37 [0.22; 0.63], p = 0.003, associated with 6-month mortality. At 72 h, increased lactate 2.77 [1.76; 4.36], p<0.001, and alanine 2.43 [1.56; 3.78], p = 0.001, and decreased small HDL cholesterol ester content (S-HDL-CE) 0.36 [0.19; 0.68], p = 0.021, associated with mortality. No difference was observed between xenon and control groups., Conclusions: In OHCA patients receiving TTM with or without xenon, high lactate and alanine and decreased BCAAs and S-HDL-CE associated with increased mortality. It remains to be established whether current observations on BCAAs, and possibly alanine and lactate, could reflect neural damage via their roles in the metabolism of the neurotransmitter glutamate. Xenon did not significantly alter the measured metabolic profile, a potentially beneficial attribute in the context of compromised ICU patients., Trial Registration: Trial Registry number: ClinicalTrials.gov Identifier: NCT00879892., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nummela et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Noise exposure during pregnancy may affect a child's auditory system, which may disturb fetal learning and language development. We examined the impact of occupational noise exposure during pregnancy on children's language acquisition at the age of one., Methods: A cohort study was conducted among women working in the food industry, as kindergarten teachers, musicians, dental nurses, or pharmacists who had a child aged <1 year. The analyses covered 408 mother-child pairs. Language acquisition was measured using the Infant-Toddler Checklist. An occupational hygienist assessed noise exposure individually as no (N = 180), low (70-78 dB; N = 108) or moderate/high exposure (>79 dB; N = 120)., Results: Among the boys, the adjusted mean differences in language acquisition scores were -0.4 (95% CI -2.5, 1.8) for low, and -0.7 (95% CI -2.9, 1.4) for moderate/high exposure compared to no exposure. Among the girls the respective scores were +0.1 (95% CI -2.2, 2.5) and -0.1 (95% CI -2.3, 2.2). Among the children of kindergarten teachers, who were mainly exposed to human noise, low or moderate exposure was associated with lower language acquisition scores. The adjusted mean differences were -3.8 (95% CI -7.2, -0.4) for low and -4.9 (95% CI -8.6, -1.2) for moderate exposure., Conclusions: In general, we did not detect an association between maternal noise exposure and children's language acquisition among one-year-old children. However, the children of kindergarten teachers exposed to human noise had lower language acquisition scores than the children of the non-exposed participants. These suggestive findings merit further investigation by level and type of exposure., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jungewelter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Medicine shortages prevail as a worldwide problem causing life-threatening situations for adults and children. Epinephrine auto-injectors are used for serious allergic reactions called anaphylaxis, and alternative auto-injectors are not always available in pharmacies. Healthcare professionals in Finland use the dedicated internet source, Physician's Database (PD), when seeking medical information in practice, while Health Library (HL) provides health information for citizens (S1 Data). The objectives were to assess whether (1) professionals' searches for epinephrine auto-injectors and (2) citizens' anaphylaxis article openings relate to epinephrine shortages in Finland., Methods: Monthly log data on epinephrine auto-injectors (EpiPen®, Jext®, Emerade®) from PD and on openings of anaphylaxis articles from HL were collected during 2016-2022. Professionals' searches of seven auto-injectors and citizens' openings of four anaphylaxis articles were compared to information on epinephrine shortages reported by Finnish Medicines Agency. Professionals' auto-injector prescriptions provided by Social Insurance Institution were also assessed., Results: Total searches in EpiPen® (N = 111,740), Jext® (N = 25,631), and Emerade® (N = 18,329) could be analyzed during 2016-2022. EpiPen® only could visually show seasonal patterns during summertime, peaking vigorously in the summer of 2018 when the major EpiPen® shortage appeared worldwide. Anaphylaxis articles equaled 2,030,855 openings altogether. Openings of one anaphylaxis article ("Bites and Stings") peaked during summertime, while another article ("Anaphylactic Reaction") peaked only once (three-fold increase) at the end of 2020 when COVID-19 vaccinations started, and auto-injector prescriptions were lowest. Fifty EpiPen®, one Jext®, and twelve Emerade® shortages were reported. Almost a two-fold increase in peaks of auto-injector prescriptions was found during summertime., Conclusion: This study shows that (1) epinephrine shortages related to professionals' searching for auto-injectors, and (2) citizens' information seeking on anaphylaxis related to summertime and shortages with lesser prescriptions. Therefore, the dedicated internet databases aimed at professionals and citizens could be used as additional information sources to detect anaphylactic reactions and auto-injector shortages., Competing Interests: The authors have read the journal’s policy and have the following competing interests: MK has held various trustee positions in the Finnish Medical Society Duodecim since the late 1990s. OH has held various trustee positions in the Finnish Medical Society Duodecim and Duodecim Publishing Company Ltd since 2009 and is a partner at iHealth Finland Ltd. The other authors have no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Mukka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Interventions that alter aspects of the physical environments in which unhealthy behaviours occur have the potential to change behaviour at scale, i.e., across populations, and thereby decrease the risk of several diseases. One set of such interventions involves reducing serving sizes, which could reduce alcohol consumption. The effect of modifying the available range of serving sizes of wine in a real-world setting is unknown. We aimed to assess the impact on the volume of wine sold of removing the largest serving size by the glass from the options available in licensed premises., Methods and Findings: The study was conducted between September 2021 and May 2022 in 21 licensed premises in England that sold wine by the glass in serving sizes greater than 125 ml (i.e., 175 ml or 250 ml) and used an electronic point of sale till system. It used an A-B-A reversal design, set over 3 four-weekly periods. "A" represented the nonintervention periods during which standard serving sizes were served and "B" the intervention period when the largest serving size for a glass of wine was removed from the existing range in each establishment: 250 ml (18 premises) or 175 ml (3 premises). The primary outcome was the daily volume of wine sold, extracted from sales data. Twenty-one premises completed the study, 20 of which did so per protocol and were included in the primary analysis. After adjusting for prespecified covariates, the intervention resulted in -420·8 millilitres (ml) (95% confidence intervals (CIs) -681·4 to -160·2 p = 0·002) or -7·6% (95% CI -12·3%, -2·9%) less wine being sold per day. There was no evidence that sales of beer and cider or total daily revenues changed but the study was not powered to detect differences in these outcomes. The main study limitation is that we were unable to assess the sales of other alcoholic drinks apart from wine, beer, and cider, estimated to comprise approximately 30% of alcoholic drinks sold in participating premises., Conclusions: Removing the largest serving size of wine by the glass from those available reduced the volume of wine sold. This promising intervention for decreasing alcohol consumption across populations merits consideration as part of alcohol licensing regulations., Trial Registration: ISRCTN https://doi.org/10.1186/ISRCTN33169631; OSF https://osf.io/xkgdb., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mantzari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In China, few molecular epidemiological data on hepatitis C virus (HCV) are available and all previous studies were limited by small sample sizes or specific population characteristics. Here, we report characterization of the epidemic history and transmission dynamics of HCV strains in China. We included HCV sequences of individuals belonging to three HCV surveillance programs: 1) patients diagnosed with HIV infection at the Beijing HIV laboratory network, most of whom were people who inject drugs and former paid blood donors, 2) men who have sex with men, and 3) the general population. We also used publicly available HCV sequences sampled in China in our study. In total, we obtained 1,603 Ns5b and 865 C/E2 sequences from 1,811 individuals. The most common HCV strains were subtypes 1b (29.1%), 3b (25.5%) and 3a (15.1%). In transmission network analysis, factors independently associated with clustering included the region (OR: 0.37, 95% CI: 0.19-0.71), infection subtype (OR: 0.23, 95% CI: 0.1-0.52), and sampling period (OR: 0.43, 95% CI: 0.27-0.68). The history of the major HCV subtypes was complex, which coincided with some important sociomedical events in China. Of note, five of eight HCV subtype (1a, 1b, 2a, 3a, and 3b), which constituted 81.8% HCV strains genotyped in our study, showed a tendency towards decline in the effective population size during the past decade until present, which is a good omen for the goal of eliminating HCV by 2030 in China., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: A recent meta-analysis suggested that using physical activity calorie equivalent (PACE) labels results in people selecting and consuming less energy. However, the meta-analysis included only 1 study in a naturalistic setting, conducted in 4 convenience stores. We therefore aimed to estimate the effect of PACE labels on energy purchased in worksite cafeterias in the context of a randomised study design., Methods and Findings: A stepped-wedge randomised controlled trial (RCT) was conducted to investigate the effect of PACE labels (which include kcal content and minutes of walking required to expend the energy content of the labelled food) on energy purchased. The setting was 10 worksite cafeterias in England, which were randomised to the order in which they introduced PACE labels on selected food and drinks following a baseline period. There were approximately 19,000 workers employed at the sites, 72% male, with an average age of 40. The study ran for 12 weeks (06 April 2021 to 28 June 2021) with over 250,000 transactions recorded on electronic tills. The primary outcome was total energy (kcal) purchased from intervention items per day. The secondary outcomes were: energy purchased from non-intervention items per day, total energy purchased per day, and revenue. Regression models showed no evidence of an overall effect on energy purchased from intervention items, -1,934 kcals per site per day (95% CI -5,131 to 1,262), p = 0.236, during the intervention relative to baseline, equivalent to -5 kcals per transaction (95% CI -14 to 4). There was also no evidence for an effect on energy purchased from non-intervention items, -5 kcals per site per day (95% CI -513 to 504), p = 0.986, equivalent to 0 kcals per transaction (95% CI -1 to 1), and no clear evidence for total energy purchased -2,899 kcals per site (95% CI -5,810 to 11), p = 0.051, equivalent to -8 kcals per transaction (95% CI -16 to 0). Study limitations include using energy purchased and not energy consumed as the primary outcome and access only to transaction-level sales, rather than individual-level data., Conclusion: Overall, the evidence was consistent with PACE labels not changing energy purchased in worksite cafeterias. There was considerable variation in effects between cafeterias, suggesting important unmeasured moderators., Trial Registration: The study was prospectively registered on ISRCTN (date: 30.03.21; ISRCTN31315776)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Reynolds et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dickeya solani is a soft rot bacterium with high virulence. In potato, D. solani, like the other potato-infecting soft rot bacteria, causes rotting and wilting of the stems and rotting of tubers in the field and in storage. Latent, asymptomatic infections of potato tubers are common in harvested tubers, and if the storage conditions are not optimal, the latent infection turns into active rotting. We characterized potato gene expression in artificially inoculated tubers in nonsymptomatic, early infections 1 and 24 hours post-inoculation (hpi) and compared the results to the response in symptomatic tuber tissue 1 week (168 hpi) later with RNA-Seq. In the beginning of the infection, potato tubers expressed genes involved in the detection of the bacterium through pathogen-associated molecular patterns (PAMPs), which induced genes involved in PAMPs-triggered immunity, resistance, production of pathogenesis-related proteins, ROS, secondary metabolites and salicylic acid (SA) and jasmonic acid (JA) biosynthesis and signaling genes. In the symptomatic tuber tissue one week later, the PAMPs-triggered gene expression was downregulated, whereas primary metabolism was affected, most likely leading to free sugars fueling plant defense but possibly also aiding the growth of the pathogen. In the symptomatic tubers, pectic enzymes and cell wall-based defenses were activated. Measurement of hormone production revealed increased SA concentration and almost no JA in the asymptomatic tubers at the beginning of the infection and high level of JA and reduced SA in the symptomatic tubers one week later. These findings suggest that potato tubers rely on different defense strategies in the different phases of D. solani infection even when the infection takes place in fully susceptible plants incubated in conditions leading to rotting. These results support the idea that D. solani is a biotroph rather than a true necrotroph., Competing Interests: The authors have declared that no competing interests exist.

Scenario tools are widely used to support policymaking and strategic planning. Loss of biodiversity, climate change, and increase in biomass demand ways to project future forest resources considering e.g. various protection schemes, alterations to forest management, and potential threats like pests, wind, and drought. The European Forestry Dynamics Model (EFDM) is an area-based matrix model that can combine all these aspects in a scenario, simulating large-scale impacts. The inputs to the EFDM are the initial forest state and models for management activities such as thinning, felling or other silvicultural treatments. The results can be converted into user-defined outputs like wood volumes, the extent of old forests, dead wood, carbon, or harvest income. We present here a new implementation of the EFDM as an open-source R package. This new implementation enables the development of more complex scenarios than before, including transitions from even-aged forestry to continuous cover forestry, and changes in land use or tree species. Combined with a faster execution speed, the EFDM can now be used as a building block in optimization systems. The new user interface makes the EFDM more approachable and usable, and it can be combined with other models to study the impact of climate change, for example., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Health care professionals working in primary and specialized care typically search for medical information from Internet sources. In Finland, Physician's Databases are online portals aimed at professionals seeking medical information. As dosage errors may occur when prescribing medication to children, professionals' need for reliable medical information has increased in public health care centers and hospitals. Influenza continues to be a public health threat, with young children at risk of developing severe illness and easily transmitting the virus. Oseltamivir is used to treat children with influenza. The objective of this study was to compare searches for children's oseltamivir and influenza diagnoses in primary and specialized care, and to determine if the searches could aid detection of influenza outbreaks., Methods: We compared searches in Physician's Databases for children's oral suspension of oseltamivir (6 mg/mL) for influenza diagnoses of children under 7 years and laboratory findings of influenza A and B from the National Infectious Disease Register. Searches and diagnoses were assessed in primary and specialized care across Finland by season from 2012-2016. The Moving Epidemic Method (MEM) calculated seasonal starts and ends, and paired differences in the mean compared two indicators. Correlation was tested to compare seasons., Results: We found that searches and diagnoses in primary and specialized care showed visually similar patterns annually. The MEM-calculated starting weeks in searches appeared mainly in the same week. Oseltamivir searches in primary care preceded diagnoses by -1.0 weeks (95% CI: -3.0, -0.3; p = 0.132) with very high correlation (τ = 0.913). Specialized care oseltamivir searches and diagnoses correlated moderately (τ = 0.667)., Conclusion: Health care professionals' searches for children's oseltamivir in online databases linked with the registers of children's influenza diagnoses in primary and specialized care. Therefore, database searches should be considered as supplementary information in disease surveillance when detecting influenza epidemics., Competing Interests: MK has held various trustee positions in the Finnish Medical Society Duodecim since the late 1990s. OH has held various trustee positions in the Finnish Medical Society Duodecim and Duodecim Medical Publications Ltd since 2009 and is a partner at iHealth Finland Ltd. The other authors have no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication., Competing Interests: The authors have declared that no competing interests exist.

Purpose of the Study: School absenteeism and school dropout jeopardize the future health and wellbeing of students. Reports on the participation of school health care in absenteeism reduction are infrequent, although physical and mental health problems are the most common causes of school absenteeism. Our aim was to explore what reasons different professionals working in schools recognize for absenteeism and which factors either promote or inhibit the inclusion of school health care in absenteeism reduction., Materials and Methods: Data for this qualitative study was gathered from ten focus groups conducted in two municipalities in southern Finland. The groups included (vice) principals, special education/resource/subject teachers, guidance counselors, school social workers, school psychologists, school nurses, school doctors, and social workers working in child protective services. Data analysis was predominantly inductive but the categorization of our results was based on existing literature., Results: Study participants identified student-, family-, and school-related reasons for absenteeism but societal reasons went unmentioned. A number of reasons promoting the inclusion of school health care in absenteeism reduction arose, such as expertise in health-related issues and the confidentiality associated with health care. Inclusion of school health care was hindered by differences in work culture and differing perceptions regarding the aims of school health care., Conclusion: Professionals working in schools were knowledgeable about the different causes of school absenteeism. Clarifying both the aims of school health care and the work culture of different professionals could facilitate the inclusion of school health care in absenteeism reduction., Competing Interests: The authors have declared that no competing interests exist.

Background: Prolapsed nictitating membrane gland (PNMG) is the most common disorder of the third eyelid in dogs. However, the epidemiology of PNMG in the wider dog population remains understudied., Methods: Using de-identified clinical data from the VetCompass Programme, this cohort study aimed to report the prevalence, demographic and breed-related risk factors of PNMG in dogs attending UK primary care veterinary practices in 2016., Results: There were 1,802 PNMG cases identified from 905,543 dogs, yielding an annual prevalence of 0.20% (95% confidence interval (CI) 0.19-0.21). The median age at first diagnosis was 0.63 years (IQR 0.33-1.98, range 0.11-18.00). Dogs aged under 1 year had 10.82 times the odds (95% CI 9.17-12.76) compared with dogs aged from 2 to under 4 years. Neutered animals had higher odds than entire animals within both sexes. Breeds with the highest odds of PNMG compared with crossbred dogs included Neapolitan Mastiff (odds ratio (OR) 34.26, 95%CI 15.92-73.75), English Bulldog (OR 24.08, 95% CI 20.62-28.13), Cane Corso (OR 14.66, 95% CI 8.18-26.28), Lhasa Apso (OR 12.37, 95% CI 10.26-14.92) and American Cocker Spaniel (OR 11.57, 95% CI 5.59-23.96). Purebred dogs had 1.43 times the odds (95% CI 1.26-1.63) of PNMG compared with crossbreed dogs. Breeds with brachycephalic skull conformation had 6.71 times the odds (95%CI 5.89-7.64) compared with breeds with mesocephalic skull conformation. Insured dogs had 1.89 times the odds (95% CI 1.65-2.16) compared with uninsured dogs., Conclusions: This study reports the largest cohort of primary-care PNMG cases assembled to date. The results showing young age at diagnosis along with the breed, purebred and brachycephalic skull conformation predispositions suggest a hereditary involvement in PNMG development. These results may help to guide breeding strategies to reduce the prevalence of PNMG and improve welfare in predisposed individuals., Competing Interests: The authors have declared that no competing interests exist.

Background: Overconsumption of energy from food is a major contributor to the high rates of overweight and obesity in many populations. There is growing evidence that interventions that target the food environment may be effective at reducing energy intake. The current study aimed to estimate the effect of decreasing the proportion of higher energy (kcal) foods, with and without reducing portion size, on energy purchased in worksite cafeterias., Methods and Findings: This stepped-wedge randomised controlled trial (RCT) evaluated 2 interventions: (i) availability: replacing higher energy products with lower energy products; and (ii) size: reducing the portion size of higher energy products. A total of 19 cafeterias were randomised to the order in which they introduced the 2 interventions. Availability was implemented first and maintained. Size was added to the availability intervention. Intervention categories included main meals, sides, cold drinks, snacks, and desserts. The study setting was worksite cafeterias located in distribution centres for a major United Kingdom supermarket and lasted for 25 weeks (May to November 2019). These cafeterias were used by 20,327 employees, mainly (96%) in manual occupations. The primary outcome was total energy (kcal) purchased from intervention categories per day. The secondary outcomes were energy (kcal) purchased from nonintervention categories per day, total energy purchased per day, and revenue. Regression models showed an overall reduction in energy purchased from intervention categories of -4.8% (95% CI -7.0% to -2.7%), p < 0.001 during the availability intervention period and a reduction of -11.5% (95% CI -13.7% to -9.3%), p < 0.001 during the availability plus size intervention period, relative to the baseline. There was a reduction in energy purchased of -6.6% (95% CI -7.9% to -5.4%), p < 0.001 during the availability plus size period, relative to availability alone. Study limitations include using energy purchased as the primary outcome (and not energy consumed) and the availability only of transaction-level sales data per site (and not individual-level data)., Conclusions: Decreasing the proportion of higher energy foods in cafeterias reduced the energy purchased. Decreasing portion sizes reduced this further. These interventions, particularly in combination, may be effective as part of broader strategies to reduce overconsumption of energy from food in out-of-home settings., Trial Registration: ISRCTN registry ISRCTN87225572., Competing Interests: The authors have declared that no competing interests exist.

Acute exacerbation of ILD (AE-ILD) is a common reason for hospitalization; it is also associated with significant mortality. Less is known about the prognostic significance of other events causing acute, non-elective hospitalizations in ILD patients. ILD patients hospitalized due to acute respiratory worsening were collected from medical records. Reasons for respiratory deterioration were classified into AE-ILDs and other causes. Clinical features and survival data of idiopathic pulmonary fibrosis (IPF) and other types of ILDs were evaluated and compared. In all, 237 patients (138 with IPF and 99 with other ILD) fulfilled the inclusion criteria. Of the non-IPF ILD types, the most prevalent subgroups were connective tissue disease-associated ILD (n = 33) and asbestosis (n = 22). The most common cause for hospitalization was AE-ILD explaining 41% of hospitalizations. Lower respiratory tract infection (22%), subacute progression of ILD (12%) and cardiovascular causes (7.2%) were other common reasons for hospital treatment. Patients with a lower respiratory tract infection had a more favorable prognosis compared with patients with AE-ILD. AE-ILDs were less fatal than cardiovascular or concurrent non-ILD-related causes for hospitalizations in non-IPF patients. High Gender-Age-Physiology (GAP) index was a marker for shortened survival and earlier AE-ILDs in all patients. IPF patients had a significantly shorter overall and post-hospitalization survival time compared with other ILDs. Most respiratory hospitalizations in ILD patients were related to causes other than AE-ILD, which highlights the importance of accurate differential diagnosis in order to target the appropriate treatment for each ILD patient., Competing Interests: JS reports congress fees and travel costs from Boehringer-Ingelheim, GlaxoSmithKline, Novartis, Orion Pharma and Roche, and lecture fees from Boehringer-Ingelheim, Chiesi, GlaxoSmithKline, Mundipharma, Orion Pharma and Roche outside the submitted work. HV has nothing to disclose. MP reports a lecture fee from Boehringer-Ingelheim Finland Ltd and a congress fee and travel costs from Roche, outside the submitted work. RK reports consultant fees from Boehringer-Ingelheim, lecture fees from Roche and Boehringer-Ingelheim, and a congress travel subsidy from Orion pharma outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In this article, we compared the content validity of two instruments used in measuring pediatric pain knowledge and attitudes. This was considered necessary due to the universal differences in culture, semantics and healthcare resources in different parts of the globe. Thirteen (13) pediatric experts in Ghana assessed the content validity of two instruments: the 42-item Pediatric Nurses' Knowledge and Attitudes Survey Regarding Pain (PNKAS) and the 41-item Pediatric Healthcare Providers' Knowledge and Attitudes Survey Regarding Pain (PHPKASRP). The relevance and clarity of each item on these instruments were rated on a four-point likert scaled options from 1 (not relevant/ not clear) to 4 (very relevant/ very clear). The item-level content validity index (I-CVI) was calculated by dividing the number of experts who rated an item with 3 or 4 by the total number of experts. The average scale-level content validity index (S-CVI/Ave) was also estimated by summing up the I-CVIs of all items and dividing them by the total number of items. The I-CVIs on the PNKAS ranged from 0.62 to 1.00 for the relevance component and 0.69 to 1.00 for the clarity component. The I-CVIs on the PHPKASRP ranged from 0.62 to 1.00 for both the relevance and clarity components. The S-CVI/Ave were 0.87 and 0.89 for the relevance and clarity aspects on the PNKAS respectively. The S-CVI/Ave for the PHPKASRP instrument were 0.86 and 0.89 for the relevance and clarity aspects correspondingly. At the end of the validation process, 5 items were revised on both instruments whilst 37 and 36 items were maintained on the PNKAS and PHPKASRP instruments respectively. The PNKAS and PHPKASRP have an acceptable level of content validity in the Ghanaian context and recommended for educational and research purposes. Other forms of validity and reliability of these instruments should also be examined in future studies., Competing Interests: The authors have declared that no competing interests exist.

Background: Antiangiogenic therapy, although part of standard treatment in ovarian cancer, has variable efficacy. Furthermore, little is known about the prognostic biomarkers and factors influencing angiogenesis in cancer tissue. We evaluated the expression of angiopoietin-2 and two endothelial tyrosine kinase receptors, Tie-1 and Tie-2, and assessed their value in the prediction of survival in patients with malignant epithelial ovarian cancer. We also compared the expression of these factors between primary high grade serous tumors and their distant metastasis., Materials and Methods: We evaluated 86 women with primary epithelial ovarian cancer. Matched distal omental metastasis were investigated in 18.6% cases (N = 16). The expression levels of angiogenic factors were evaluated by immunohistochemistry in 306 specimens and by qRT-PCR in 111 samples., Results: A high epithelial expression level of Tie-2 is a significant prognostic factor in primary high grade serous ovarian cancer. It predicted significantly shorter overall survival both in univariate (p<0.001) and multivariate survival analyses (p = 0.022). Low angiopoietin-2 expression levels in primary ovarian tumors were significantly associated with shorter overall survival (p = 0.015) in the univariate survival analysis. A low expression of angiopoietin-2 was also significantly related to high grade tumors, size of residual tumor after primary surgery and the recurrence of cancer (p = 0.008; p = 0.012; p = 0.018) in the whole study population. The expression of angiopoietin-2 and Tie-2 was stronger in distal omental metastasis than in primary high grade serous tumors in matched-pair analysis (p = 0.001; p = 0.002)., Conclusions: The angiogenic factor, angiopoietin-2, and its receptor Tie-2 seem to be significant prognostic factors in primary epithelial ovarian cancer. Their expression levels are also increased in metastatic lesions in comparison with primary tumors., Competing Interests: The authors have declared that no competing interests exist.

The objective of this study was to analyze the associations of various individual- and environmental-related factors with subgroups of daily, frequent, moderate and low moderate-to-vigorous physical activity (MVPA) among children and adolescents. Data were obtained from the Finnish School-age Physical Activity (FSPA) study 2016 from 4677 national representative 11-, 13-, and 15-year-old children and adolescents. MVPA and individual- and environmental-related factors were assessed by a questionnaire and analyzed by two-level logistic regression. Seventeen of the twenty-one variables were statistically significantly associated with MVPA. However, only three variables were statistically significant in all MVPA subgroups, whereby self-directed PA at least twice a week, fewer perceived barriers, and higher peer support increased the odds of participating in more MVPA. The results from this study showed essential differences among the MVPA subgroups, also supporting previous findings, whereby various individual- and environmental-based factors are associated with children and adolescents' levels of MVPA. Challenges to designing and implementing effective interventions are based on the need to account for individual differences within the population, as well as the varied connections between PA with different social and physical environments where children and adolescents' PA takes place. PA interventions with various actions at multiple levels are warranted., Competing Interests: The authors have declared that no competing interests exist.

Vitamin D is important for normal skeletal homeostasis, especially in growing children. There are no previous genome-wide association (GWA) studies exploring genetic factors that influence vitamin D metabolism in early childhood. We performed a GWA study on serum 25-hydroxyvitamin D (25(OH)D) and response to supplementation in 761 healthy term-born Finnish 24-month-old children, who participated in a randomized clinical trial comparing effects of 10 μg and 30 μg of daily vitamin D supplementation from age 2 weeks to 24 months. Using the Illumina Infinium Global Screening Array, which has been optimized for imputation, a total of 686085 markers were genotyped across the genome. Serum 25(OH)D was measured at the end of the intervention at 24 months of age. Skeletal parameters reflecting bone strength were determined at the distal tibia at 24 months using peripheral quantitative computed tomography (pQCT) (data available for 648 children). For 25(OH)D, two strong GWA signals were identified, localizing to GC (Vitamin D binding protein) and CYP2R1 (Vitamin D 25-hydroxylase) genes. The GWA locus comprising the GC gene also associated with response to supplementation. Further evidence for the importance of these two genes was obtained by comparing association signals to gene expression data from the Genotype-Tissue Expression project and performing colocalization analyses. Through the identification of haplotypes associated with low or high 25(OH)D concentrations we used a Mendelian randomization approach to show that haplotypes associating with low 25(OH)D were also associated with low pQCT parameters in the 24-month-old children. In this first GWA study on 25(OH)D in this age group we show that already at the age of 24 months genetic variation influences 25(OH)D concentrations and determines response to supplementation, with genome-wide significant associations with GC and CYP2R1. Also, the dual association between haplotypes, 25(OH)D and pQCT parameters gives support for vertical pleiotropy mediated by 25(OH)D., Competing Interests: OM declares consultancy for Kyowa Kirin, Alexion and Sandoz. All other authors declare no conflict of interest.

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types., Competing Interests: The authors have declared that no competing interests exist.

Objective: Ischaemia/reperfusion-injury degrades endothelial glycocalyx. Graft glycocalyx degradation was studied in human liver transplantation., Methods: To assess changes within the graft, blood was drawn from portal and hepatic veins in addition to systemic samples in 10 patients. Plasma syndecan-1, heparan sulfate and chondroitin sulfate, were measured with enzyme-linked immunosorbent assay., Results: During reperfusion, syndecan-1 levels were higher in graft caval effluent [3118 (934-6141) ng/ml, P = 0.005] than in portal venous blood [101 (75-121) ng/ml], indicating syndecan-1 release from the graft. Concomitantly, heparan sulfate levels were lower in graft caval effluent [96 (32-129) ng/ml, P = 0.037] than in portal venous blood [112 (98-128) ng/ml], indicating heparan sulfate uptake within the graft. Chondroitin sulfate levels were equal in portal and hepatic venous blood. After reperfusion arterial syndecan-1 levels increased 17-fold (P < 0.001) and heparan sulfate decreased to a third (P < 0.001) towards the end of surgery., Conclusion: Syndecan-1 washout from the liver indicates extensive glycocalyx degradation within the graft during reperfusion. Surprisingly, heparan sulfate was taken up by the graft during reperfusion. Corroborating previous experimental reports, this suggests that endogenous heparan sulfate might be utilized within the graft in the repair of damaged glycocalyx., Competing Interests: The authors have declared that no competing interests exist.

Two long-term potentially oil exposed Baltic Sea coastal sites near old oil refineries and harbours were compared to nearby less exposed sites in terms of bacterial, archaeal and fungal microbiomes and oil degradation potential. The bacterial, archaeal and fungal diversities were similar in oil exposed and less exposed sampling sites based on bacterial and archaeal 16S rRNA gene and fungal 5.8S rRNA gene amplicon sequencing from both DNA and RNA fractions. The number of genes participating in alkane degradation (alkB) or PAH-ring hydroxylation (PAH-RHDα) were detected by qPCR in all water and sediment samples. These numbers correlated with the number of bacterial 16S rRNA gene copies in sediment samples but not with the concentration of petroleum hydrocarbons or PAHs. This indicates that both the clean and the more polluted sites at the Baltic Sea coastal areas have a potential for petroleum hydrocarbon degradation. The active community (based on RNA) of the coastal Baltic Sea water differed largely from the total community (based on DNA). The most noticeable difference was seen in the bacterial community in the water samples were the active community was dominated by Cyanobacteria and Proteobacteria whereas in total bacterial community Actinobacteria was the most abundant phylum. The abundance, richness and diversity of Fungi present in water and sediment samples was in general lower than that of Bacteria and Archaea. Furthermore, the sampling location influenced the fungal community composition, whereas the bacterial and archaeal communities were not influenced. This may indicate that the fungal species that are adapted to the Baltic Sea environments are few and that Fungi are potentially more vulnerable to or affected by the Baltic Sea conditions than Bacteria and Archaea., Competing Interests: The authors have declared that no competing interests exist.

Although note taking during trials is known to enhance jurors' recall of trial evidence, little is known about whether individual differences in note taking underpin this effect. Individual differences in handwriting speed, working memory, and attention may influence juror's note taking. This, in turn, may influence their recall. It may also be the case that if jurors note down and recall more incriminating than non-incriminating evidence (or vice versa), then this may predict their verdict. Three studies examined the associations between the aforementioned individual differences, the amount of critical evidence jurors noted down during a trial, the amount of critical evidence they recalled, and the verdicts they reached. Participants had their handwriting speed, short-term memory, working memory, and attention assessed. They then watched a trial video (some took notes), reached a verdict, and recalled as much trial information as possible. We found that jurors with faster handwriting speed (Study 1), higher short-term memory capacity (Study 2), and higher sustained attention capacity (Study 3) noted down, and later recalled, the most critical trial evidence. However, working memory storage capacity, information processing ability (Study 2) and divided attention (Study 3) were not associated with note taking or recall. Further, the type of critical evidence jurors predominantly recalled predicted their verdicts, such that jurors who recalled more incriminating evidence were more likely to reach a guilty verdict, and jurors who recalled more non-incriminating evidence were less likely to do so. The implications of these findings are discussed., Competing Interests: The authors have declared that no competing interests exist.

The bilingual executive advantage (BEA) hypothesis has attracted considerable research interest, but the findings are inconclusive. We addressed this issue in the domain of working memory (WM), as more complex WM tasks have been underrepresented in the previous literature. First, we compared early and late bilingual vs. monolingual WM performance. Second, we examined whether certain aspects of bilingual experience, such as language switching frequency, are related to bilinguals' WM scores. Our online sample included 485 participants. They filled in an extensive questionnaire including background factors such as bilingualism and second language (L2) use, and performed 10 isomorphic verbal and visuospatial WM tasks that yielded three WM composite scores (visuospatial WM, verbal WM, n-back). For verbal and visuospatial WM composites, the group comparisons did not support the BEA hypothesis. N-back analysis showed an advantage of late bilinguals over monolinguals and early bilinguals, while the latter two groups did not differ. This between-groups analysis was followed by a regression analysis relating features of bilingual experience to n-back performance, but the results were non-significant in both bilingual groups. In sum, group differences supporting the BEA hypothesis were limited only to the n-back composite, and this composite was not predicted by bilingualism-related features. Moreover, Bayesian analyses did not give consistent support for the BEA hypothesis. Possible reasons for the failure to find support for the BEA hypothesis are discussed., Competing Interests: The authors have declared that no competing interests exist.

Background: An association between maternal prenatal stress and increased rates of respiratory tract infections in the offspring has been described earlier. Data regarding the father's role is lacking. In this study our aim was to evaluate, whether mothers' and fathers' depressive symptoms and loneliness during pregnancy predict higher rates of respiratory tract infections in the offspring., Methods: In this longitudinal cohort study we gathered information on parental psychological risk during gestational week 20 using the BDI-II and UCLA loneliness scale questionnaires for the parents of 929 children. Loneliness was divided into social and emotional components, the former describing patterns of social isolation and the latter a perceived lack of intimate attachments. Episodes of acute otitis media, physician visits due to respiratory tract infections, and antibiotic consumption relating to respiratory tract infections were documented in the infants, excluding twins, from birth until 10 months of age using study diaries. Analyses were carried out by structural equation modeling, which provides dynamic estimates of covariances., Results: Maternal depressive symptoms during pregnancy predicted higher rates of acute otitis media in the infant and maternal emotional loneliness predicted higher rates of physician visits. Acute otitis media, physician visits and antibiotic consumption in the infant were slightly less frequent for families who reported social loneliness in the father or mother. Associations remained when taking into account confounders., Conclusions: Maternal prenatal depression and emotional loneliness predicted a higher burden of respiratory tract infections in the offspring. The protective influence of parental social loneliness on the burden of respiratory tract infections in infants was not in line with our study hypothesis, but could be explained by reduced use of healthcare services in these socially isolated families., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Osteoarthritis (OA) is the most common degenerative joint disease and one of the major causes of disability worldwide. It is a multifactorial disorder with a significant genetic component. The heritability of OA has been estimated to be 60% for hip OA and 39% for knee OA. Genetic factors behind OA are still largely unknown. Studying families with strong history of OA, facilitates examining the co-segregation of genetic variation and OA. The aim of this study was to identify new, rare genetic factors and novel candidate genes for OA., Methods: Eight patients from three Finnish families with hip and knee OA were studied using whole exome sequencing. We focused on rare exonic variants with predicted pathogenicity and variants located in active promoter or strong enhancer regions. Expression of identified candidate genes were studied in bone and cartilage tissues and the observed variants were investigated using bioinformatic analyses., Results: Two rare variants co-segregated with OA in two families. In Family 8 a missense variant (c.628C>G, p.Arg210Gly) was observed in the OLIG3 gene that encodes a transcription factor known to be associated with rheumatoid arthritis and inflammatory polyarthritis. The Arg210Gly variant was estimated to be pathogenic by Polyphen-2 and Mutation taster and the locus is conserved among mammals. In Family 12 the observed variant (c.-127G>T) was located in the transcription start site of the FIP1L1 gene. FIP1L1 participates in the regulation of polyadenylation. The c.-127G>T is located in the transcription start site and may alter the DNA-binding of transcription factors. Both, OLIG3 and FIP1L1 were observed in human bone and cartilage., Conclusion: The identified variants revealed novel candidate genes for OA. OLIG3 and FIP1L1 have specific roles in transcription and may effect expression of other genes. Identified variants in these genes may thus have a role in the regulatory events leading to OA., Competing Interests: Leena Ala-Kokko is employed by The Connective Tissue Gene Tests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Previous research on statement analysis has mainly concerned accounts by witnesses and plaintiffs. In our studies we examined true and false statements as told by offenders. It was hypothesized that SVA and MASAM techniques would enhance the ability to discriminate between true and false offenders' statements. Truthful and deceptive statements (confessions and denials) were collected from Swedish and Polish criminal case files. In Experiment 1, Swedish law students (N = 39) were asked to assess the veracity of statements either after training in and usage of MASAM or without any training and using their own judgements. In Experiment 2, Polish psychology students (N = 34) assessed veracity after training in and usage of either MASAM or SVA or without prior training using their own judgements. The veracity assessments of participants who used MASAM and SVA were significantly more correct than the assessments of participants that used their own judgements. Results show, that trained coders are much better at distinguishing between truths and lies than lay evaluators. There were significant difference between total scores of truthful and false statements for both total SVA and MASAM and it can be concluded that both veracity assessment techniques are useful in assessing veracity. It was also found, that the content criteria most strongly associated with correct assessments were: logical structure, contextual embedding, self-depreciation, volume of statement, contextual setting and descriptions of relations. The results are discussed in relation to statement analysis of offenders' accounts., Competing Interests: The authors have declared that no competing interests exist.

Small bowel adenocarcinoma (SBA) is an aggressive disease with limited treatment options. Despite previous studies, its molecular genetic background has remained somewhat elusive. To comprehensively characterize the mutational landscape of this tumor type, and to identify possible targets of treatment, we conducted the first large exome sequencing study on a population-based set of SBA samples from all three small bowel segments. Archival tissue from 106 primary tumors with appropriate clinical information were available for exome sequencing from a patient series consisting of a majority of confirmed SBA cases diagnosed in Finland between the years 2003-2011. Paired-end exome sequencing was performed using Illumina HiSeq 4000, and OncodriveFML was used to identify driver genes from the exome data. We also defined frequently affected cancer signalling pathways and performed the first extensive allelic imbalance (AI) analysis in SBA. Exome data analysis revealed significantly mutated genes previously linked to SBA (TP53, KRAS, APC, SMAD4, and BRAF), recently reported potential driver genes (SOX9, ATM, and ARID2), as well as novel candidate driver genes, such as ACVR2A, ACVR1B, BRCA2, and SMARCA4. We also identified clear mutation hotspot patterns in ERBB2 and BRAF. No BRAF V600E mutations were observed. Additionally, we present a comprehensive mutation signature analysis of SBA, highlighting established signatures 1A, 6, and 17, as well as U2 which is a previously unvalidated signature. Finally, comparison of the three small bowel segments revealed differences in tumor characteristics. This comprehensive work unveils the mutational landscape and most frequently affected genes and pathways in SBA, providing potential therapeutic targets, and novel and more thorough insights into the genetic background of this tumor type.

Background: Studies have shown altered vitamin D metabolism in obesity. We assessed differences between obese and normal-weight subjects in total, free, and bioavailable 25-hydroxyvitamin D (25(OH)D, 25(OH)DFree, and 25(OH)DBio, respectively), vitamin D binding protein (DBP), parathyroid hormone (PTH) and bone traits., Methods: 595 37-47-year-old healthy Finnish men and women stratified by BMI were examined in this cross-sectional study. Background characteristic and intakes of vitamin D and calcium were collected. The concentrations of 25(OH)D, PTH, DBP, albumin and bone turnover markers were determined from blood. 25(OH)DFree and 25(OH)DBio were calculated. pQCT was performed at radius and tibia., Results: Mean±SE (ANCOVA) 25(OH)DFree (10.8±0.6 vs 12.9±0.4 nmol/L; P = 0.008) and 25(OH)DBio (4.1±0.3 vs 5.1±0.1 nmol/L; P = 0.003) were lower in obese than in normal-weight women. In men, 25(OH)D (48.0±2.4 vs 56.4±2.0 nmol/L, P = 0.003), 25(OH)DFree (10.3±0.7 vs 12.5±0.6 pmol/L; P = 0.044) and 25(OH)DBio (4.2±0.3 vs 5.1±0.2 nmol/L; P = 0.032) were lower in obese. Similarly in all subjects, 25(OH)D, 25(OH)DFree and 25(OH)DBio were lower in obese (P<0.001). DBP (399±12 vs 356±7mg/L, P = 0.008) and PTH (62.2±3.0 vs 53.3±1.9 ng/L; P = 0.045) were higher in obese than in normal-weight women. In all subjects, PTH and DBP were higher in obese (P = 0.047and P = 0.004, respectively). In obese women, 25(OH)D was negatively associated with distal radius trabecular density (R2 = 0.089, P = 0.009) and tibial shaft cortical strength index (CSI) (R2 = 0.146, P = 0.004). 25(OH)DFree was negatively associated with distal radius CSI (R2 = 0.070, P = 0.049), radial shaft cortical density (CorD) (R2 = 0.050, P = 0.045), and tibial shaft CSI (R2 = 0.113, P = 0.012). 25(OH)DBio was negatively associated with distal radius CSI (R2 = 0.072, P = 0.045), radial shaft CorD (R2 = 0.059, P = 0.032), and tibial shaft CSI (R2 = 0.093, P = 0.024)., Conclusions: The associations between BMI and 25(OH)D, 25(OH)DFree, and 25(OH)DBio, DBP, and PTH suggest that obese subjects may differ from normal-weight subjects in vitamin D metabolism. BMI associated positively with trabecular bone traits and CSI in our study, and slightly negatively with cortical bone traits. Surprisingly, there was a negative association of free and bioavailable 25(OH)D and some of the bone traits in obese women.

Repetition suppression (RS) is evident as a weakened response to repeated stimuli after the initial response. RS has been demonstrated in motor-evoked potentials (MEPs) induced with transcranial magnetic stimulation (TMS). Here, we investigated the effect of inter-train interval (ITI) on the induction of RS of MEPs with the attempt to optimize the investigative protocols. Trains of TMS pulses, targeted to the primary motor cortex by neuronavigation, were applied at a stimulation intensity of 120% of the resting motor threshold. The stimulus trains included either four or twenty pulses with an inter-stimulus interval (ISI) of 1 s. The ITI was here defined as the interval between the last pulse in a train and the first pulse in the next train; the ITIs used here were 1, 3, 4, 6, 7, 12, and 17 s. RS was observed with all ITIs except with the ITI of 1 s, in which the ITI was equal to ISI. RS was more pronounced with longer ITIs. Shorter ITIs may not allow sufficient time for a return to baseline. RS may reflect a startle-like response to the first pulse of a train followed by habituation. Longer ITIs may allow more recovery time and in turn demonstrate greater RS. Our results indicate that RS can be studied with confidence at relatively short ITIs of 6 s and above.

The ruminal microbiome, comprising large numbers of bacteria, ciliate protozoa, archaea and fungi, responds to diet and dietary additives in a complex way. The aim of this study was to investigate the benefits of increasing the depth of the community analysis in describing and explaining responses to dietary changes. Quantitative PCR, ssu rRNA amplicon based taxa composition, diversity and co-occurrence network analyses were applied to ruminal digesta samples obtained from four multiparous Nordic Red dairy cows fitted with rumen cannulae. The cows received diets with forage:concentrate ratio either 35:65 (diet H) or 65:35 (L), supplemented or not with sunflower oil (SO) (0 or 50 g/kg diet dry matter), supplied in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and four 35-day periods. Digesta samples were collected on days 22 and 24 and combined. QPCR provided a broad picture in which a large fall in the abundance of fungi was seen with SO in the H but not the L diet. Amplicon sequencing showed higher community diversity indices in L as compared to H diets and revealed diet specific taxa abundance changes, highlighting large differences in protozoal and fungal composition. Methanobrevibacter ruminantium and Mbb. gottschalkii dominated archaeal communities, and their abundance correlated negatively with each other. Co-occurrence network analysis provided evidence that no microbial domain played a more central role in network formation, that some minor-abundance taxa were at nodes of highest centrality, and that microbial interactions were diet specific. Networks added new dimensions to our understanding of the diet effect on rumen microbial community interactions.

Osteoarthritis (OA) is the most common degenerative joint disorder and genetic factors have been shown to have a significant role in its etiology. The first metatarsophalangeal joint (MTP I) is highly susceptible to development of OA due to repetitive mechanical stress during walking. We used whole exome sequencing to study genetic defect(s) predisposing to familial early-onset bilateral MTP I OA inherited in an autosomal dominant manner. A nonsynonymous single nucleotide variant rs41310883 (c.524C>T, p.Thr175Met) in TUFT1 gene was found to co-segregate perfectly with MTP I OA. The role of TUFT1 and the relevance of the identified variant in pathogenesis of MTP I OA were further assessed using functional in vitro analyses. The variant reduced TUFT1 mRNA and tuftelin protein expression in HEK293 cells. ATDC5 cells overexpressing wild type (wt) or mutant TUFT1 were cultured in calcifying conditions and chondrogenic differentiation was found to be inhibited in both cell populations, as indicated by decreased marker gene expression when compared with the empty vector control cells. Also, the formation of cartilage nodules was diminished in both TUFT1 overexpressing ATDC5 cell populations. At the end of the culturing period the calcium content of the extracellular matrix was significantly increased in cells overexpressing mutant TUFT1 compared to cells overexpressing wt TUFT1 and control cells, while the proteoglycan content was reduced. These data imply that overexpression of TUFT1 in ATDC5 inhibits chondrogenic differentiation, and the identified variant may contribute to the pathogenesis of OA by increasing calcification and reducing amount of proteoglycans in the articular cartilage extracellular matrix thus making cartilage susceptible for degeneration and osteophyte formation.