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The peripheral innate immune response to West Nile virus (WNV) is crucial for control of virus spread to the central nervous system. Therefore, transcriptomes encoding the innate immune response proteins against WNV were investigated in peripheral blood mononuclear cells (PBMCs) of New Zealand White rabbits, a recently established novel rabbit model for WNV pathogenesis studies. PBMCs were challenged with an Australian WNV strain, WNVNSW2011, in vitro and mRNA expression of selected immune response genes were quantified at 2h, 6h, 12h and 24h post infection (pi) using qRT-PCR. Compared to mock-inoculated PBMCs, WNV-stimulated PBMCs expressed high levels of interferon (IFN) alpha (IFNA), gamma (IFNG), IL6, IL12, IL22, CXCL10 and pentraxin 3 (PTX3) mRNA. Likewise, TLR1, 2, 3, 4, 6 and 10 mRNA became up-regulated with highest expression seen for TLR3, 4 and 6. TLRs-signalling downstream genes (MyD88, STAT1, TRAF3, IRF7 and IRF9) subsequently became up-regulated. The high expression of IFNs, TLR3, TLR4, TRAF3, STAT1, IRF7 and IRF9 are in accordance with antiviral activities, while expression of TNFA, HO1, iNOS, caspase 3 and 9 transcripts suggests the involvement of oxidative stress and apoptosis in WNV stimulated rabbit PBMCs, respectively. The level of WNVNSW2011 RNA increased at 24h pi in PBMCs challenged with virus in vitro compared to input virus. The expression dynamics of selected genes were validated in PBMCs from rabbits experimentally infected with WNV in vivo. Higher expression of IFNA, IFN beta (IFNB), IFNG, TNFA, IL6, IL22, PTX3, TLR3 & TLR4, IRF7, IRF9, STST1, TRAF3, caspase 3 and caspase 9 were seen in PBMCs from WNV infected rabbits on day 3 post intradermal virus inoculation compared to PBMCs from uninfected control rabbits. This study highlights the array of cytokines and TLRs involved in the host innate immune response to WNV in the rabbit leukocytes, and suggests that these cells may be a useful in vitro model for WNV infection study.

Recent findings revealed that the commonly used 15N2 tracer assay for the determination of dinitrogen (N2) fixation can underestimate the activity of aquatic N2-fixing organisms. Therefore, a modification to the method using pre-prepared 15-15N2-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of 15-15N2-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add 15-15N2 gas to the water in amounts exceeding the standard N2 solubility, followed by vigorous agitation (vortex mixing ≥5 min). Optionally, water can be degassed at low-pressure (≥950 mbar) for ten minutes prior to the 15-15N2 gas addition to indirectly facilitate the 15-15N2 dissolution. This preparation of 15-15N2-enriched water can be done within one hour using standard laboratory equipment. The final 15N-atom% excess was 5% after replacing 2–5% of the incubation volume with 15-15N2-enriched water. Notably, the addition of 15-15N2-enriched water can alter levels of trace elements in the incubation water due to the contact of 15-15N2-enriched water with glass, plastic and rubber ware during its preparation. In our tests, levels of trace elements (Fe, P, Mn, Mo, Cu, Zn) increased by up to 0.1 nmol L-1 in the final incubation volume, which may bias rate measurements in regions where N2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with 15-15N2. The 15-15N2 was injected as a bubble directly to the incubation water, followed by gentle shaking. Immediately thereafter, the bubble was replaced with water to stop the 15-15N2 equilibration. This method achieved a 15N-atom excess of 6.6±1.7% when adding 2 mL 15-15N2 per liter of incubation water. The herein presented methodological tests offer guidelines for the 15N2 tracer assay and thus, are crucial to circumvent methodological draw-backs for future N2 fixation assessments.

Antibiotic resistance is a growing concern in human, as well as in veterinary medicine. Part of the problem concerns how to respond to the risk presented by animal reservoirs of resistant bacteria with the potential of spreading to humans. One example is livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA). In countries where LA-MRSA is endemic in the pig population, people in contact with pigs have a higher risk of being colonised with LA-MRSA, and persons from this group are subjected to precautionary measures when visiting health care facilities. In the present study, it is assumed that, if LA-MRSA was introduced to the Swedish pig population, the prevalence in the risk groups would be the same as in Denmark or the Netherlands (two countries with low human prevalence that have implemented measures to detect, trace and isolate human LA-MRSA cases and, therefore, have comprehensive data with good coverage regarding prevalence of LA-MRSA), and that similar interventions would be taken in Swedish health care facilities. It is also assumed that the Swedish pig population is free of MRSA or that the prevalence is very low. We analyse if it would be efficient for Sweden to prevent its introduction by testing imported live breeding pigs. Given that quarantining and testing at import will prevent introduction to the pig population, the study shows that the preventive measures may indeed generate a societal net benefit. Benefits are estimated to be between € 870 720 and € 1 233 511, and costs to € 211 129. Still, due to gaps in knowledge, the results should be confirmed when more information become available.

The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans and horses with particularly virulent strains causing recent outbreaks in Eastern Europe, the Middle East and North America. In Australia, a strain of WNV, Kunjin (WNVKUN), is endemic in the north and infection with this virus is generally asymptomatic. However, in early 2011, following extensive flooding, an unprecedented outbreak of WNVKUN encephalitis in horses occurred in south-eastern Australia, resulting in more than 1,000 cases and a mortality of 10-15%. Despite widespread evidence of equine infections, there was only a single mild human case reported during this outbreak. To understand why clinical disease was seen in horses without similar observations in the human population, a serosurvey was conducted using blood donor samples from areas where equine cases were reported to assess level of flavivirus exposure. The seroprevalence to WNVKUN in humans was low before the outbreak (0.7%), and no significant increase was demonstrated after the outbreak period (0.6%). Due to unusual epidemiological features during this outbreak, a serosurvey was also conducted in rabbits, a potential reservoir host. Out of 675 animals, sampled across Australia between April 2011 and November 2012, 86 (12.7%) were seropositive for WNVKUN, with the highest prevalence during February of 2012 (28/145; 19.3%). As this is the first serological survey for WNVKUN in Australian feral rabbits, it remains to be determined whether wild rabbits are able to develop a high enough viraemia to actively participate in WNV transmission in Australia. However, they may constitute a sentinel species for arbovirus activity, and this is the focus of ongoing studies. Collectively this study provides little evidence of human exposure to WNVKUN during the 2011 outbreak and indicates that the Australian population remains susceptible to the emergence of virulent strains of WNV.

Research demonstrates that cognitive biases toward drug-related stimuli are correlated with substance use. This study aimed to investigate differences in cognitive biases (including approach bias, attentional bias and memory associations) between smoking and non-smoking adolescents in the US and the Netherlands. Within the group of smokers, we examined the relative predictive value of the cognitive biases and impulsivity related constructs (i.e.,including inhibition skills, working memory and risk taking) on daily smoking and nicotine dependence.Method: A total of 125 American and Dutch adolescent smokers (n = 67) and non-smokers (n = 58) between 13-18 years old participated. Participants completed the smoking Approach-Avoidance Task (S-AAT), the classical and emotional Stroop task, brief Implicit Associations Task (bIAT), Balloon Analogue Risk Taking (BART), the Self-Ordering Pointing Task (SOPT) and a questionnaire assessing level of nicotine dependence and smoking behavior. Results: The analytical sample consisted of 56 Dutch adolescents (27 smokers and 29 non-smokers) and 37 American adolescents (19 smokers and 18 non-smokers). No differences in cognitive biases between smokers and non-smokers were found. Generally, Dutch adolescents demonstrated an avoidance bias towards both smoking and neutral stimuli whereas the American adolescents did not demonstrate a bias. Within the group of smokers, regression analyses showed that stronger attentional bias and weaker inhibition skills predicted greater nicotine dependence while weak working memory predicted more daily cigarette use. Conclusion: Attentional bias, inhibition skills and working memory might be important factors explaining smoking in adolescence. Cultural differences in approach-avoidance bias should be considered in future research.

The study aimed at detecting apoptotic endothelial cells (ECs) and smooth muscle cells (SMCs) together with determining expression of NF-kappaB (p105/p50) and Bax in varicose vein walls. Women (n=35) undergoing the excision of varicose veins were divided into 3 groups: younger than 35 years (I), 36–50 years (II), and older than 50 years (III). Apoptosis was determined by the TUNEL method, NF-kappaB and Bax expression by immunohistochemistry. The percentage of apoptotic ECs and SMCs in the layers of varicose vein wall increased in groups II and III. NF-kappaB expression had the lowest level in Group II with particularly low level in the media. Contrariwise, Bax expression levels in Group II were increased. The study revealed that in varicose veins ECs and SMCs apoptosis increased with advancing age. If increase in apoptosis during earlier stages of varicosities is probably regulated by intrinsic pathway, then in older patients other signaling pathways may be involved.

The object of study in this article is the analysis of what relations can be established by the colon in written French. The article is the outcome of our cand.ling.merc. thesis from June 1997. Our aim was then to examine how these relations can be expressed in written Danish and whether the colon can be preserved or not. In cases where these relations are not usually signalled by the Danish colon, we will examine what operations are necessary in order to render the meaning in idiomatic Danish. In this article we would like to give a brief account of our findings.

The concentrations of mercury (Hg), cadmium (Cd), lead (Pb) and selenium (Se) were determined in liver, kidney and muscle samples from 20Baltic ringed seals (Phoca hispida botnica) (3-32 years), and from 17 ringed seals (Phoca hispida) (0-20 years) from Svalbard, in the Arctic. The concentrations of Hg and Se were considerably higher in the Baltic ringed seals, but the Cd concentrations lower than in the Svalbard ringed seals. There was no big geographical difference with respect to Pb concentrations. Se and Hg concentrations showed a significant positive correlation in both regions. By comparison with earlier studies on Baltic seals, the metal concentrations have remained at thesame level since the 1980s. Of the metals we studied, only the levelof Hg in Baltic ringed seals can be considered high (mean 53 mg/kg, range 6.5-124 mg/kg wet wt. for liver), but probably not high enough to cause metal intoxication. No pathological changes associated with metal intoxication were observed in the seals. [ABSTRACT FROM AUTHOR]

Background: Congenital heart defects (CHD) are structural defects of the heart affecting approximately 1% of newborns. They exhibit low penetrance and non-Mendelian patterns of inheritance as varied and complex traits. While genetic factors are known to play an important role in the development of CHD, the specific genetics remain unknown for the majority of patients. To elucidate the underlying genetic risk, we performed a genome wide association study (GWAS) of CHDs in general and specific CHD subgroups using the FinnGen Release 10 (R10) (N > 393,000), followed by functional fine-mapping through eQTL and co-localization analyses using the GTEx database., Results: We discovered three genome-wide significant loci associated with general CHD. Two of them were located in chromosome 17: 17q21.32 (rs2316327, intronic: LRRC37A2, Odds ratio (OR) [95% Confidence Interval (CI)] = 1.17[1.12-1.23], p = 1.5 × 10 -9 ) and 17q25.3 (rs1293973611, nearest: BAHCC1, OR[95%CI] = 4.48[2.80-7.17], p = 7.0 × 10 -10 ), respectively, and in addition to general CHD, the rs1293973611 locus was associated with the septal defect subtype. The third locus was in band 1p21.2 (rs35046143, nearest: PALMD, OR[95%CI] = 1.15[1.09-1.21], p = 7.1 × 10 -9 ), and it was associated with general CHD and left-sided lesions. In the subgroup analysis, two additional loci were associated with septal defects (rs75230966 and rs6824295), and one with left-sided lesions (rs1305393195). In the eQTL analysis the variants rs2316327 (general CHD), and rs75230966 (septal defects) both located in 17q21.32 (with a LD r2 of 0.41) were both predicted to significantly associate with the expression of WNT9B in the atrial appendage tissue category. This effect was further confirmed by co-localization analysis, which also implicated WNT3 expression in the atrial appendage. A meta-analysis of general CHD together with the UK Biobank (combined N = 881,678) provided a different genome-wide significant locus in LRRC37A2; rs16941382 (OR[95%CI] = 1.15[1.11-1.20], p = 1.5 × 10 -9 ) which is in significant LD with rs2316327., Conclusions: Our results of general CHD and different CHD subcategories identified a complex risk locus on chromosome 17 near BAHCC1 and LRRC37A2, interacting with the genes WNT9B, WNT3 and MYL4, may constitute potential novel CHD risk associated loci, warranting future experimental tests to determine their role., (© 2024. The Author(s).)

Importance: Maternal diabetes and overweight or obesity are known to be associated with increased risk of congenital heart defects (CHDs) in offspring, but there are no large studies analyzing outcomes associated with these factors in 1 model., Objective: To investigate the association of maternal diabetes and overweight or obesity with CHDs among offspring in 1 model., Design, Setting, and Participants: This nationwide, population-based register study was conducted in a birth cohort from Finland consisting of all children born between 2006 and 2016 (620 751 individuals) and their mothers. Data were analyzed from January 2022 until November 2023., Exposures: Maternal prepregnancy body mass index (BMI; calculated as weight in kilograms divided by height in meters squared), categorized as underweight (<18.5), normal (18.5-24.9), overweight (25.0-29.9), and obesity (≥30), was assessed. Maternal diabetes status, classified as no diabetes, type 1 diabetes (T1D), type 2 or other diabetes, and gestational diabetes, was assessed., Main Outcomes and Measures: Odds ratios (ORs) of isolated CHDs in children were found. In addition, 9 anatomical CHD subgroups were studied., Results: Of 620 751 children (316 802 males [51.0%]; 573 259 mothers aged 20-40 years [92.3%]) born in Finland during the study period, 10 254 children (1.7%) had an isolated CHD. Maternal T1D was associated with increased odds of having a child with any CHD (OR, 3.77 [95% CI, 3.26-4.36]) and 6 of 9 CHD subgroups (OR range, 3.28 [95% CI, 1.55-6.95] for other septal defects to 7.39 [95% CI, 3.00-18.21] for transposition of great arteries) compared with no maternal diabetes. Maternal overweight was associated with left ventricular outflow tract obstruction (OR, 1.28 [95% CI, 1.10-1.49]) and ventricular septal defects (OR, 0.92 [95% CI, 0.86-0.98]), and obesity was associated with complex defects (OR, 2.70 [95% CI, 1.14-6.43]) and right outflow tract obstruction (OR, 1.31 [95% CI, 1.09-1.58]) compared with normal maternal BMI., Conclusions and Relevance: This study found that maternal T1D was associated with increased risk for most types of CHD in offspring, while obesity and overweight were associated with increased risk for complex defects and outflow tract obstruction and decreased risk for ventricular septal defects. These different risk profiles of T1D and overweight and obesity may suggest distinct underlying teratogenic mechanisms.

Arrhythmogenic cardiomyopathy is an inherited cardiomyopathy that can involve both ventricles. Several genes have been identified as pathogenic in arrhythmogenic cardiomyopathy, including TMEM43 . However, there are limited data on cardiac MRI findings in patients with TMEM43 variants to date. In this case series, cardiac MRI findings and clinical outcomes are described in 14 patients with TMEM43 variants, including eight (57%) with the pathogenic p.Ser358Leu variant (six female patients; mean age, 33 years ± 15 [SD]) and six (43%) with a TMEM43 variant of unknown significance (three female patients; mean age, 38 years ± 11). MRI findings demonstrated left ventricular systolic dysfunction in eight (57%) patients and right ventricular dysfunction in four (29%) patients. Among the nine patients with late gadolinium enhancement imaging, left ventricular late gadolinium enhancement was present in seven (78%; all subepicardial) patients. In summary, TMEM43 variants are associated with high prevalence of subepicardial late gadolinium enhancement and left ventricular dysfunction. Keywords: Arrhythmogenic Cardiomyopathy, Arrhythmogenic Right Ventricular Cardiomyopathy, TMEM43 , Cardiac MRI, Genetic Variants Supplemental material is available for this article ., (© RSNA, 2023.)

Background: Novel immunisation methods against respiratory syncytial virus (RSV) are emerging, but knowledge of risk factors for severe RSV disease is insufficient for optimal targeting of interventions against them. Our aims were to identify predictors for RSV hospital admission from registry-based data and to develop and validate a clinical prediction model to guide RSV immunoprophylaxis for infants younger than 1 year., Methods: In this model development and validation study, we studied all infants born in Finland between June 1, 1997, and May 31, 2020, and in Sweden between June 1, 2006, and May 31, 2020, along with the data for their parents and siblings. Infants were excluded if they died or were admitted to hospital for RSV within the first 7 days of life. The outcome was hospital admission due to RSV bronchiolitis during the first year of life. The Finnish study population was divided into a development dataset (born between June 1, 1997, and May 31, 2017) and a temporal hold-out validation dataset (born between June 1, 2017, and May 31, 2020). The development dataset was used for predictor discovery and selection in which we screened 1511 candidate predictors from the infants', parents', and siblings' data, and developed a logistic regression model with the 16 most important predictors. This model was then validated using the Finnish hold-out validation dataset and the Swedish dataset., Findings: In total, there were 1 124 561 infants in the Finnish development dataset, 130 352 infants in the Finnish hold-out validation dataset, and 1 459 472 infants in the Swedish dataset. In addition to known predictors such as severe congenital heart defects (adjusted odds ratio 2·89, 95% CI 2·28-3·65), we confirmed some less established predictors for RSV hospital admission, most notably oesophageal malformations (3·11, 1·86-5·19) and lower complexity congenital heart defects (1·43, 1·25-1·63). The prediction model's C-statistic was 0·766 (95% CI 0·742-0·789) in Finnish data and 0·737 (0·710-0·762) in Swedish validation data. The infants in the highest decile of predicted RSV hospital admission probability had 4·5 times higher observed risk compared with others. Calibration varied according to epidemic intensity. The model's performance was similar to a machine learning (XGboost) model using all 1511 candidate predictors (C-statistic in Finland 0·771, 95% CI 0·754-0·788). The prediction model showed clinical utility in decision curve analysis and in hypothetical number needed to treat calculations for immunisation, and its C-statistic was similar across different strata of parental income., Interpretation: The identified predictors and the prediction model can be used in guiding RSV immunoprophylaxis in infants, or as a basis for further immunoprophylaxis targeting tools., Funding: Sigrid Jusélius Foundation, European Research Council, Pediatric Research Foundation, and Academy of Finland., Competing Interests: Declaration of interests PV reports an expert fee from Pfizer. SH reports being the site principal investigator in the MK1654-007 study, and subinvestigator in several maternal RSV vaccine and other paediatric vaccine trials at the Meilahti Vaccine Research Center (Helsinki, Finland) and reports lecture fees from Merck Sharp & Dohme and Swedish Orphan Biovitrum. All other authors declare no competing interests. SJ was supported by the Academy of Finland (grant number 341747). SH was supported by the Academy of Finland (grant number 323499)., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: Disclosures None.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10 -5 ), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model ( Xenopus laevis ) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2 707C>T and CAPN2 1112C>T variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis., Competing Interests: M.J.B. and J.X.C. are the editor-in-chief and deputy editor, respectively, of Human Genetics and Genomics Advances and were recused from the editorial handling of this article. J.J.W. is an employee and shareholder of Invitae. M.J.B. is chair of the Scientific Advisory Board of GeneDx. S.M. is on the Hypertrophic Cardiomyopathy Advisory Board of Bristol-Myers Squibb., (© 2023 The Author(s).)

Objectives: Several studies have reported mortality risk factors associated with hypoplastic left heart syndrome (HLHS). However, these data are ambiguous and mainly focused on the independent effects of these factors. We examined both the independent and the cumulative effects of preoperative risk factors for poor outcome in patients undergoing the Norwood procedure. Moreover, we studied the risk factors associated with prolonged initial hospital stays in these patients., Methods: We performed a retrospective national 18-year observational study of preoperative risk factors for 1 year, as well as total follow-up mortality or need for transplant in patients with HLHS (N = 99) born in Finland between 1 January 2004 and 31 December 2021., Results: Overall, one-year survival was 85.6%. In a multivariable analysis, having a major extracardiac anomaly and being small for gestational age were significant predictors of one-year mortality or the need for a transplant. Aortic atresia was a predictor of total follow-up mortality. An analysis of the cumulative effect indicated that the presence of 2 risk factors was associated with higher mortality., Conclusions: HLHS remains the defect with the highest procedural risks for mortality in paediatric cardiac surgery. From a prognostic point of view, recognition of independent preoperative risk factors as well as the cumulative effect of risk factors for mortality is essential.The results of this study were presented orally at the 55th Annual Meeting of the Association for European Paediatric and Congenital Cardiology, Geneva, Switzerland, 28 May 2022., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.)

Competing Interests: Conflict of interest: None declared.

Background: Higher maternal body mass index (BMI) and abnormal glucose metabolism during early pregnancy are associated with congenital heart defects in the offspring, but the exact mechanisms are unknown., Methods: We evaluated the association between maternal first trimester metabolic profile and transposition of the great arteries (TGA) in the offspring in a matched case-control study with 100 TGA mothers and 200 controls born in Finland during 2004-2014. Cases and controls were matched by birth year, child sex, and maternal age and BMI. Serum samples collected between 10- and 14-weeks of gestation were analyzed for 73 metabolic measures. Conditional logistic regression was used to assess the risk for TGA in the offspring, and a subgroup analysis among mothers with high BMI was conducted., Results: Higher concentrations of four subtypes of extremely large very-low-density lipoprotein (VLDL) particles and one of large VLDL particles were observed in TGA mothers. This finding did not reach statistical significance after multiple testing correction. The pooled odds ratio (OR) of the all metabolic variables was slightly higher in TGA mothers in the subgroup with maternal BMI over 25 (OR 1.25) and significantly higher in the subgroup with maternal BMI over 30 (OR 1.95) compared to the original population (OR 1.18)., Conclusions: Our findings indicate that an abnormal maternal early pregnancy metabolic profile might be associated with TGA in the offspring, especially in obese mothers. A trend indicating altered VLDL subtype composition in TGA pregnancies warrants further research., (© 2022 The Authors. Birth Defects Research published by Wiley Periodicals LLC.)

A 9-year-old boy developed progressive anthracycline-induced cardiomyopathy three months after completion of chemotherapy for osteosarcoma. Five months after completion of chemotherapy, at the age of 10 years, heart transplantation was performed. At 29 months since transplantation, the patient remains free of rejection and recurrence of osteosarcoma. ( Level of Difficulty: Intermediate. )., Competing Interests: Open access was funded by the Helsinki University Library. This work was financially supported by the Foundation for Pediatric Research, Helsinki, Finland, and by the Academy of Finland, Helsinki, Finland. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2021 The Authors.)

Cell-cell interactions are crucial for organ development and function. In the heart, endothelial cells engage in bidirectional communication with cardiomyocytes regulating cardiac development and growth. We aimed to elucidate the organotypic development of cardiac endothelial cells and cardiomyocyte and endothelial cell crosstalk using human induced pluripotent stem cells (hiPSC). Single-cell RNA sequencing was performed with hiPSC-derived cardiomyocytes (hiPS-CMs) and endothelial cells (hiPS-ECs) in mono- and co-culture. The presence of hiPS-CMs led to increased expression of transcripts related to vascular development and maturation, cardiac development, as well as cardiac endothelial cell and endocardium-specific genes in hiPS-ECs. Interestingly, co-culture induced the expression of cardiomyocyte myofibrillar genes and MYL7 and MYL4 protein expression was detected in hiPS-ECs. Major regulators of BMP- and Notch-signaling pathways were induced in both cell types in co-culture. These results reflect the findings from animal studies and extend them to human endothelial cells, demonstrating the importance of EC-CM interactions during development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Helle, Ampuja, Dainis, Antola, Temmes, Tolvanen, Mervaala and Kivelä.)

Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and environmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections have enabled genome-wide association studies (GWAS) in non-syndromic CHD. The ability to generate human to create human induced pluripotent stem cells (hiPSC) and further differentiate them to organotypic cells enables further exploration of genotype-phenotype correlations in patient-derived cells. Here we review how these technologies can be used in unraveling the genetics and molecular mechanisms of heart development.

During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.

Introduction: Newborn infants with transposition of the great arteries (d-TGA) need immediate care for an optimal outcome. This study comprised a nationwide 11-year population-based cohort of d-TGA infants, and assessed whether the implementation of a nationwide systematic fetal screening program, or other perinatal, or perioperative factors, are associated with mortality or an increased need for hospital care., Material and Methods: The national cohort consisted of all live-born infants with simple d-TGA (TGA ± small ventricular septal defect, n = 127) born in Finland during 2004-2014. Data were collected from six national registries. Prenatal diagnosis and perinatal and perioperative factors associated with mortality and length of hospitalization were evaluated., Results: Preoperative mortality was 7.9%, and the total mortality was 8.7%. The prenatal detection rate increased after introducing systematic fetal anomaly screening from 5.0% to 37.7% during the study period (P < .0001), but the total mortality rate remained unchanged. All prenatally diagnosed infants (n = 27) survived. Lower gestational age (odds ratio 0.68, P = .012) and higher maternal age at birth (odds ratio 1.16, P = .036) were associated with increased mortality in multivariable analysis. Older infant age at time of operation (P = .002), longer aortic clamp time (P < .001), and higher maternal body mass index (P = .027) were associated with longer initial hospital stay. An extended need for hospital care during the first year of life was multi-factorial., Conclusions: In our cohort, none of the prenatally diagnosed d-TGA infants died. As a result of the limited prenatal detection rates, however, the sample size was insufficient to reach statistical significance. The d-TGA infants born with lower gestational age and to older mothers had increased mortality., (© 2020 Nordic Federation of Societies of Obstetrics and Gynecology (NFOG). Published by John Wiley & Sons Ltd.)

Myocardial dysfunction is a known risk factor for morbidity and mortality in hypoplastic left heart syndrome (HLHS). Variants in some transcription factor and contractility genes, which are known to cause cardiomyopathy, have previously been associated with impaired right ventricular function in some HLHS patients. The care of HLHS patients is resource demanding. Identifying genetic variants associated with myocardial dysfunction would be helpful in tailoring the follow-up and therapeutic strategies. We tested whether a commercial cardiomyopathy gene panel could serve as a diagnostic tool in a Finnish cohort of HLHS patients with impaired right ventricular function to identify potentially pathogenic variants associated with poor prognosis. None of the patients had pathogenic or likely pathogenic variants in the studied cardiomyopathy-associated genes. Thus, our approach of performing a cardiomyopathy gene panel to identify pathogenic variants as directly causal or as modifiers for worse outcomes in hypoplastic left heart syndrome is not useful in clinical practice at the moment., (Copyright © 2020 Helle, Pihkala, Turunen, Ruotsalainen, Tuupanen, Koskenvuo and Ojala.)

The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development., (Copyright © 2020 Helle, Ampuja, Antola and Kivelä.)

Congenital heart disease (CHD) is the most common anatomical malformation occurring live-born infants and an increasing cause of morbidity and mortality across the lifespan and throughout the world. Population-based observations have long described associations between maternal cardiometabolic disorders and the risk of CHD in the offspring. Here we review the epidemiological evidence and clinical observations relating maternal obesity and diabetes mellitus to the risk of CHD offspring with particular attention to mechanistic models of maternal-fetal risk transmission and first trimester disturbances of fetal cardiac development. A deeper understanding of maternal risk factors holds the potential to improve both prenatal detection of CHD by identifying at-risk pregnancies, along with primary prevention of disease by improving preconception and prenatal treatment of at-risk mothers.

Loss of function variants in NOTCH1 cause left ventricular outflow tract obstructive defects (LVOTO). However, the risk conferred by rare and noncoding variants in NOTCH1 for LVOTO remains largely uncharacterized. In a cohort of 49 families affected by hypoplastic left heart syndrome, a severe form of LVOTO, we discovered predicted loss of function NOTCH1 variants in 6% of individuals. Rare or low-frequency missense variants were found in 16% of families. To make a quantitative estimate of the genetic risk posed by variants in NOTCH1 for LVOTO, we studied associations of 400 coding and noncoding variants in NOTCH1 in 1,085 cases and 332,788 controls from the UK Biobank. Two rare intronic variants in strong linkage disequilibrium displayed significant association with risk for LVOTO amongst European-ancestry individuals. This result was replicated in an independent analysis of 210 cases and 68,762 controls of non-European and mixed ancestry. In conclusion, carrying rare predicted loss of function variants in NOTCH1 confer significant risk for LVOTO. In addition, the two intronic variants seem to be associated with an increased risk for these defects. Our approach demonstrates the utility of population-based data sets in quantifying the specific risk of individual variants for disease-related phenotypes., (© 2018 Wiley Periodicals, Inc.)

Background: Early term birth is associated with increased need for hospital care during the early postnatal period. The objective of this study was to assess the morbidity and health care-related costs during the first 3 years of life in children born early term., Methods: Data come from a population-based birth cohort study in the municipalities of Helsinki, Espoo, and Vantaa, Finland using data from the national medical birth register and outpatient, inpatient, and primary care registers. All surviving infants born in 2006-08 (n = 29 970) were included. The main outcome measures were morbidities, based on ICD-10 codes recorded during inpatient and outpatient hospital visits, and health care costs, based on all care received, including well child visits (specialised care, primary care, private care, and medications)., Results: 7.0% of children born full term had at least one of the studied morbidities by 3 years of age. This percentage was significantly higher in children born early term: 8.6% (adjusted odds ratio 1.2, 95% confidence interval (CI) 1.1, 1.4). The increased morbidity of children born early term was attributed to obstructive airway diseases and ophthalmological and motor problems. Health care-related costs during the first 3 years of life were 4813€ (95% CI 4385, 5241) per child in the early term group, higher than for full term children 4047€ (95% CI 3884, 4210)., Conclusions: Infants born early term have increased morbidity and higher health care-related costs during early childhood than full term infants. Early term birth seems to be associated with a health disadvantage., (© 2016 John Wiley & Sons Ltd.)

Objective: To evaluate the amount of antenatal care days in level III hospitals caused by regionalization of very preterm deliveries., Study Design: We included all 171,997 pregnancies registered in Finland between January 1, 2004 and December 31, 2006. Data on deliveries from the Medical Birth Register were linked to the Hospital Discharge Register. Maternal zip codes were used to define whether a mother lived inside or outside a level III hospital region. Regionalization was defined as care in level III hospitals between gestational weeks 22 and 32 among mothers living outside level III hospital regions. Pregnancies were divided into 3 groups based on the gestational age at delivery: very preterm (<32 weeks), late preterm (32-36 weeks), and term (≥37 weeks)., Results: There were 12,354 antenatal care days in level III hospitals caused by regionalization, which amounts to a need for 12 antenatal maternal beds annually. In the very preterm pregnancies, the antenatal length of stay was comparable for mothers living inside or outside level III hospital regions (median 4 days, P = .81) but significantly longer for mothers living outside level III hospital regions in the late preterm (median 9 vs 7 days, P = .001) and term groups (median 3 vs 2 days, P < .0001)., Conclusions: The costs of regionalization of very preterm deliveries were low, as measured by antenatal care days. Regionalization did not increase the antenatal length of stay in very preterm deliveries., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although ringed seals are important components in oceanic and fresh water ecosystems at high latitudes, little is known about how they exploit these harsh environments. Seasonal activity and diving behaviour of 19 adult Baltic ringed seals were studied by satellite telemetry. We elaborated an activity budget for ten months of the year, extending over the period from moult to the breeding season. Seals from three main regions showed explicit site fidelity and the distributions of animals tagged from different areas did not overlap, suggesting separate stocks. Both the mean duration and the mean depth of dives peaked in June and July. Seals spent 70% (females) to 85% (males) of their time diving in June and July which decreased to 50% in late autumn. Less than one percent of dives exceeded 10 min in females, while 10% of male dives lasted longer than 10 min in June to September. Less than one percent of dives lasted for more than 25 min. Both females and males were most active during day time and hauled out predominantly during the night. Activity patterns during the summer are suggested to be correlated to energy accumulation and prey availability. The information on seasonal activity budget is crucial for developing population energetic models where interactions between ringed seals and other trophic levels can be evaluated.

Baltic grey seals (Halichoerus grypus) alternate between land and ice breeding, depending on ice conditions. We show that the fitness of grey seal females in terms of pup mortality and quality is reduced when breeding on land as compared with ice. The mean preweaning mortality rate on land was 21.1% (range 0% to 31.6%), and correlated with birth density (range 0.5-5.2 pups 100 m(-2)). The mean mortality rate on ice was 1.5%, where the highest density was 0.2 pups 100 m(-2) in particularly dense breeding groups. Mean weights of pups born on ice were significantly greater (48.3 +/- 8.1 kg) at the onset of moult as compared with pups born on land (37.4 +/- 7.8 kg). Because indices of life-time net reproductive rate (pup survival) and pup quality (weaning weight and health) were more auspicious on ice as compared with land, diminishing ice fields will lower the fitness of Baltic grey seal females and substantially increase the risk for quasi-extinction.

We studied the accumulation of polychlorinated biphenyls (PCBs), 1,1,1.trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) and its metabolites, and vitamins A and E in the top levels of the Baltic Sea food web. The investigation focused on the transfer of contaminants and vitamins to the ringed seal (Phoca hispida baltica) and the grey seal (Halichoerus grypus) from their main prey species. The trophic level of the seals was investigated using stable isotopes of nitrogen and the results indicated that both species of Baltic seal feed at approximately the same level. PCBs accumulated to a greater extent in the grey seal than in the ringed seal. Biomagnification factors for DDT compounds were similar for both species of Baltic seal (85-140). Differences in observed DDT levels were due to different prey selection by the two species, while differences in PCB levels indicated a species-specific metabolic system. There was a clearly greater accumulation of DDT compounds than of PCBs in both species of seal. The supply of dietary vitamin A was normally above the recommended level in all the seal populations studied. Low levels of hepatic vitamin A in the Baltic seals could therefore indicate the toxic effects of a high level of persistent organic pollutants on vitamin A dynamics, at least in the ringed seal. In the grey seals, low hepatic vitamin A levels could also be explained by lower levels of dietary vitamin A, compared to the reference grey seals, as it is not known if seals can store unlimited amounts of vitamin A. The greater uptake of vitamin E by Baltic seals, compared to seals in the reference areas, demonstrated by elevated levels of vitamin E in the blubber, could be a response to oxidative stress caused by the high contaminant load. These results further support our previous hypothesis that the toxic effects of environmental contaminants could be causing the observed divergence in vitamin levels between the Baltic seals and the reference seal populations.

The Baltic Sea ecosystem has suffered from a heavy pollutant load for more than three decades. Persistent organic pollutants (POPs) and heavy metals have been of most concern due to their persistence and toxic properties. Ringed seals (Phoca hispida baltica) and grey seals (Halichoerus grypus) living in the Baltic Sea have been suffering from pathological impairments, including reproductive disturbances, which have resulted in a depressed reproductive capacity. We investigated several biochemical parameters as potential biomarkers for exposure to and effects of the contaminant load in the Baltic seals. Seals from less polluted areas were used as reference material in terms of the pollution load. In both Baltic seal populations, the levels of some biochemical parameters diverged from those in the reference seals, and some of these showed a clear correlation with the individual contaminant load. Of the potential bioindicators, we propose cytochrome P4501A activity and vitamin E levels, in blubber or plasma, as exposure biomarkers for polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) in both species. The arylhydrocarbon receptor-mediated chemical-activated luciferase gene expression (CALUX) response reflects the whole PCB and DDT burden in ringed seals. Retinyl palmitate (vitamin A) levels showed a negative correlation with the individual POP load, and is proposed as potential effect biomarkers for the depletion of the vitamin A stores. As the nutritional levels of both vitamin A and E have an impact on the vitamin levels in the seals, more information on the dietary vitamin levels is needed before any conclusions can be drawn. As the relationship between biochemical parameters and contaminants varied between the two species, species-specific characteristics has to be considered when monitoring the health status and possible toxic effects of the contaminant load in ringed and grey seals.

Because of the likelihood that Corynosoma magdaleni Montreuil, 1958. has been confused with C. strumosum (Rudolphi, 1802) in reports of parasites from seals and to clarify its distribution in the Baltic Sea, acanthocephalans from 26 young gray seals from the southwestern Finnish archipelago (western Baltic Sea) were examined. All harbored C. semerme (Forssell. 1904). In addition to C. semerme, 12 had both C. strumosum and C. magdaleni, 3 had only C. strumosum, and 9 had only C. magdaleni. Most anatomical structures of C. strumosum are similar to, but larger than, those of C. magdaleni. The most conspicuous differences are the longer and more robust trunk of C. strumosum, the routinely longer proboscis (mean, 653 vs. 476 microm) and larger proboscis hook (mean, 69 vs. 58 microm) of C. strumosum, and the greater extent of ventral trunk spines (mean, 61 vs. 47%) in C. magdaleni. In addition, C. strumosum consistently possesses 18 longitudinal rows of proboscis hooks, whereas C. magdaleni has 17-23, with 20 being the usual number by far. In seals aged 3.6 mo, on average, C. strumosum was more prevalent and abundant than was C. magdaleni, whereas in seals of age 2.0-3.0 mo the reverse was true, with C. strumosum being nearly absent. These differences might reveal the age-dependent food habits of the very young seals. No site segregation was found between C. strumosum and C. magdaleni in the small intestine, but they clearly segregated from C. semerme, which occurred mainly in the cecum, large intestine, and rectum. All species matured equally well in gray seals, with 62, 67, and 53% of the C. magdaleni, C. strumosum, and C. semerme, respectively, comprising gravid worms (possessing eggs with fully formed acanthors).

Residue levels of polychlorinated biphenyls (PCBs) and 1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) were determined from liver samples of ringed seals (Phoca lispida) from the Baltic Sea and Svalbard, and of grey seals (Halichoerus grypus) from the Baltic Sea and Sable Island in Canada. Both Baltic seal populations showed clearly higher average sum PCB (SPCB) and sum DDT (SDDT) levels than the reference seal populations. Among the Baltic seals, SPCB levels were twice as high as SDDT levels, and both contaminants were higher in ringed seals than in grey seals. A difference in gender was observed only in the Sable Island grey seal population, in which males showed a higher level of contamination than females. A reduction of the SDDT levels, and to a lower extent of the SPCB levels can be observed in the Baltic seals since the peak contaminant levels during the 1970s. The decrease has been more rapid in the grey seals than in the ringed seals. The SPCB levels in the Baltic ringed seals are still high enough to cause a threat to their well being. Residue levels of the trace elements mercury, cadmium, lead and selenium were determined from liver, kidney and muscle samples of grey seals from the Baltic Sea and Sable Island. Only cadmium showed a geographic difference being higher in the seals from Sable Island than from the Baltic Sea. No clear reduction of the metal burden can be observed in the Baltic grey seal population since the 1970s.

The effect of gonadal steroids (GS) on proliferation of lymphocytes and distribution of lymphocyte subpopulations in cell culture was examined. The involvement of protein kinase C (PKC) and calcium ionophore in the proliferative response was tested. Estradiol benzoate (EB) or testosterone propionate (TP) had no significant influence on proliferation of peripheral blood lymphocytes (PBL) when cells were not stimulated by mitogen. At high concentration (10-6 M), EB and dihydrotestosterone (DHT) decreased lymphocyte proliferative response to concanavalin A (ConA) and lipopolysaccharide (LPS) at 24 and 72 h of incubation. However, at physiological doses (10(-12) to 10(-16) M), EB significantly enhanced the proliferative response at 24 h of incubation, whereas DHT had no effect. The inhibitory effect of the high dose of EB or DHT on proliferation of T and B lymphocytes was independent of time of hormone presentation to the cells or age and gender of cell donor. In all cultures, pre-incubation of lymphocytes with 10(-6) M of EB or DHT significantly reduced their proliferative responses to ConA, phytohemagglutinin (PHA), and LPS. The percentage of CD3+ cells was significantly reduced by EB, whereas DHT had no such effect. In contrast to inhibition of proliferation in response to mitogens, 10(-6) M EB dramatically enhanced the proliferation of lymphocytes in response to the PKC activator, phorbol 12-myristate 13-acetate, and calcium ionophore, A23187. Results suggest that high doses of EB do not damage the viability or proliferation capability of lymphocytes and, therefore, suppress the proliferative response to mitogens in a different manner, perhaps by reducing gene transcription for receptors that recognize the mitogens, or suppressing some postreceptor events. The enhancement of proliferation in response to mitogens by low doses of EB may support this assumption, because the biphasic effects of steroids on gene transcription are well documented.

Perfluorooctane sulfonate (PFOS) is a perfluorinated molecule that has recently been identified in the sera of nonindustrially exposed humans. In this study, 247 tissue samples from 15 species of marine mammals collected from Florida, California, and Alaskan coastal waters; and northern Baltic Sea; the Arctic (Spitsbergen); and Sable Island in Canada were analyzed for PFOS. PFOS was detected in liver and blood of marine mammals from most locations including those from Arctic waters. The greatest concentrations of PFOS found in liver and blood were 1520 ng/g wet wt in a bottlenose dolphin from Sarasota Bay, FL, and 475 ng/mL in a ringed seal from the northern Baltic Sea (Bothnian Sea), respectively. No age-dependent increase in PFOS concentrations in marine mammals was observed in the samples analyzed. The occurrence of PFOS in marine mammals from the Arctic waters suggests widespread global distribution of PFOS including remote locations.

Immunisation may induce myocardial complications. In this pilot study clinical, electrocardiographic, chemical and immunological findings have been studied during a six weeks' follow-up after routine immunisation (mumps, polio, tetanus, smallpox, diphtheria and type A meningococcal disease) among 234 Finnish conscripts at the beginning of their military service. Serial pattern of ECG changes suggestive of myocarditis was recorded in eight of the 234 conscripts one to two weeks after vaccination against smallpox and diphtheria. Changes were mainly minor ST segment elevations and T wave inversions and usually they disappeared in a few weeks. The ECG positives more often had a history of atopy, and their mean body temperatures and heart rates after the vaccinations were higher than among the other subjects (p less than 0.01). However, clinical myocarditis was never noted, nor were immunological or enzymological changes different among the ECG positives. Thus in 3% of the study population, evidence of postvaccinal myocarditis was noted, based on serial ECG patterns, but without any other evidence of cardiac disease.

Ten albino rats injected with hydrocortisone were infected with 36-66 cystacanths of Corynosoma semerme in order to study the course of infection in a warm-blooded animal. Surveys were carried out 2, 5, 8, 14, 18, 21 and 28 days after infection. Recovery decreased from 60% after 2-5 days to 22% after 28 days. The proportion of males dropped markedly before the 8th day of infection. The worms were concentrated in the latter half of the intestine, the males being in a more anterior location on average. The males matured between the 2nd and 5th day of infection. Disintegrated ovaries were observed in some of the 2-day-old females, but mature free ovaries were seen only in 5-day-old females. By the 14th day many females contained fully developed eggs, but it was only after the 18th day that the number of eggs increased considerably. Male worms were not encountered in the 4-week-old infection. The females were longer than the males. In the present study, experimental infections are compared with other acanthocephalan infections in both birds and mammals.