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Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Objectives The aim of this study was to explore how university students’ participation in a two-staged health screening at the beginning of university studies associates with student health care utilisation in a 6-year follow-up.Design Nationwide, observational, register-based cohort study with a 6-year follow-up.Setting Student health care in Finland. Finnish Student Health Service (FSHS) provides statutory student health services to university students in Finland. The two-staged health screening of FSHS includes the electronic health questionnaire (eHQ) provided annually to university entrants and a subsequent health check, when necessary, based on students eHQ response.Participants A national cohort of university entrants from the 2011–2012 academic year (N=15 723) was assessed. After exclusions the study population consisted of 12 972 students, n (female)=7368, n (male)=5604.Outcome measures The primary outcome measures were students’ health service utilisation pattern obtained by clustering analyses method and the students’ participation in different stages of the health examination process.Results Four distinguishable health care utilisation patterns were identified: (1) constant low use, (2) constant high use, (3) increasing use and (4) decreasing use. The students’ OR for belonging to the constant high use group was significantly higher among females (OR 4.0, 95% CI 3.5 to 4.6) and students who attended the health check (OR 4.7, 95% CI 3.9 to 5.6).Conclusions Participating in the two-staged health screening was associated with increase in health care utilisation. The process detects students with health problems.

The SIRPα-CD47 axis plays an important role in T cell recruitment to sites of immune reaction and inflammation but its role in T cell antigen priming is incompletely understood. Employing OTII TCR transgenic mice bred to Cd47-/- (Cd47KO) or SKI mice, a knock-in transgenic animal expressing non-signaling cytoplasmic-truncated SIRPα, we investigated how the SIRPα-CD47 axis contributes to antigen priming. Here we show that adoptive transfer of Cd47KO or SKI Ova-specific CD4+ T cells (OTII) into Cd47KO and SKI recipients, followed by Ova immunization, elicited reduced T cell division and proliferation indices, increased apoptosis, and reduced expansion compared to transfer into WT mice. We confirmed prior reports that splenic T cell zone, CD4+ conventional dendritic cells (cDCs) and CD4+ T cell numbers were reduced in Cd47KO and SKI mice. We report that in vitro derived DCs from Cd47KO and SKI mice exhibited impaired migration in vivo and exhibited reduced CD11c+ DC proximity to OTII T cells in T cell zones after Ag immunization, which correlates with reduced TCR activation in transferred OTII T cells. These findings suggest that reduced numbers of CD4+ cDCs and their impaired migration contributes to reduced T cell-DC proximity in splenic T cell zone and reduced T cell TCR activation, cell division and proliferation, and indirectly increased T cell apoptosis.

Objective To evaluate the association between health and study-related factors measured by an Electronic Health Questionnaire (eHQ), participation in a health examination process and graduation in a university student population.Design Nationwide, retrospective, register-based cohort study with a 6-year follow-up.Setting Student health care in Finland. Finnish Student Health Service (FSHS) provides statutory student health services to university students in Finland. The health examination process of FSHS includes the eHQ provided annually to university entrants and a subsequent health check when necessary based on students’ eHQ response.Participants A national cohort of university entrants from the 2011–2012 academic year (n=14 329, n (female)=8075, n (male)=6254).Outcome measures The primary outcome measure was graduation, measured based on whether a student had completed a bachelor’s, licentiate or master’s degree during the 6-year follow-up.Results Some 72% of the women and 60% of the men had graduated during the follow-up. The predictors in the eHQ associated with non-graduation differed by sex. Among the women’s low enthusiasm about studies (OR 2.6, 95% CI 1.9 to 3.6), low engagement with studies (OR 2.5, 95% CI 1.8 to 3.4) and daily smoking (OR 1.9, 95% CI 1.4 to 2.6) were the strongest predictors to non-graduation. Among the men, low engagement with studies (OR 3.7, 95% CI 2.5 to 5.5) and obesity (body mass index≥35) (OR 4.0, 95% CI 1.9 to 8.8) were the strongest predictors to non-graduation. Not attending the health check when referred was associated with non-graduation in both sexes: the OR for not graduating was 1.6 (95% CI 1.3 to 1.9) in women and 1.3 (95% CI 1.0 to 1.6) in men.Conclusions Engagement and enthusiasm about studying in the first year are important predictors of graduation and therefore a potential intervention target. Health promotion initiatives conducted early in the studies may have a positive effect on students’ academic achievement.

Abstract A brief burst-suppressing isoflurane anesthesia has been shown to rapidly alleviate symptoms of depression in a subset of patients, but the neurobiological basis of these observations remains obscure. We show that a single isoflurane anesthesia produces antidepressant-like behavioural effects in the learned helplessness paradigm and regulates molecular events implicated in the mechanism of action of rapid-acting antidepressant ketamine: activation of brain-derived neurotrophic factor (BDNF) receptor TrkB, facilitation of mammalian target of rapamycin (mTOR) signaling pathway and inhibition of glycogen synthase kinase 3β (GSK3β). Moreover, isoflurane affected neuronal plasticity by facilitating long-term potentiation in the hippocampus. We also found that isoflurane increased activity of the parvalbumin interneurons, and facilitated GABAergic transmission in wild type mice but not in transgenic mice with reduced TrkB expression in parvalbumin interneurons. Our findings strengthen the role of TrkB signaling in the antidepressant responses and encourage further evaluation of isoflurane as a rapid-acting antidepressant devoid of the psychotomimetic effects and abuse potential of ketamine.

Mycoplasma (M.) bovis is an important pathogen of cattle implicated in a broad range of clinical manifestations that adversely impacts livestock production worldwide. In the absence of a safe, effective, commercial vaccine in Europe, reduced susceptibility to reported antimicrobials for this organism has contributed to difficulties in controlling infection. Despite global presence, some countries have only recently experienced outbreaks of this pathogen. In the present study, M. bovis isolates collected in Denmark between 1981 and 2016 were characterized to determine (i) genetic diversity and phylogenetic relationships using whole genome sequencing and various sequence-based typing methods and (ii) patterns of antimicrobial resistance compared to other European isolates. The M. bovis population in Denmark was found to be highly homogeneous genomically and with respect to the antimicrobial resistance profile. Previously dominated by an old genotype shared by many other countries (ST17 in the PubMLST legacy scheme), a new predominant type represented by ST94-adh1 has emerged. The same clone is also found in Sweden and Finland, where M. bovis introduction is more recent. Although retrieved from the Netherlands, it appears absent from France, two countries with a long history of M. bovis infection where the M. bovis population is more diverse.

Mycoplasma bovis is an important bovine pathogen. Artificial insemination (AI) using contaminated semen can introduce the agent into a naïve herd. Antibiotics, most often gentamycin, tylosin, lincomycin, spectinomycin (GTLS) combination are added to semen extender to prevent transmission of pathogenic bacteria and mycoplasmas. In a commercial AI straw production system with industrial scale procedures, we analyzed the mycoplasmacidal efficacy of GTLS and ofloxacin on M. bovis ATCC and wild type strain isolated from commercial AI straws. The strains were spiked at two concentrations (106 and 103 CFU/mL) into semen. Viable M. bovis in frozen semen straws was detected by enrichment culture and real-time PCR. We also compared different protocols to extract M. bovis DNA from spiked semen. None of the antibiotic protocols had any effect on the viability of either of the M. bovis strains at high spiking concentration. At low concentration, the wild type was inhibited by all other protocols, except low GTLS, whereas the ATCC strain was inhibited only by high GTLS. The InstaGene™ matrix was the most effective method to extract M. bovis DNA from semen. When there is a low M. bovis contamination level in semen, GTLS used at high concentrations, in accordance with Certified Semen Services requirements, is more efficient than GTLS used at concentrations stated in the OIE Terrestrial Code.

Human endogenous retroviruses (HERV) are relics of ancient retroviral infections in our genome. Most of them have lost their coding capacity, but proviral RNA or protein have been observed in several disease states (e.g. in inflammatory and autoimmune diseases and malignancies). However, their clinical significance as well as their mechanisms of action have still remained elusive. As human aging is associated with several biological characteristics of these diseases, we now analyzed the aging-associated expression of the individual proviruses of two HERV families, HERV-K (91 proviruses) and HERV-W (213 proviruses) using genome-wide RNA-sequencing (RNA-seq). RNA was purified from blood cells derived from healthy young individuals (n = 7) and from nonagenarians (n = 7). The data indicated that in the case of HERV-K (HML-2) 33 proviruses had a detectable expression but in only 3 of those the expression levels were significantly different between the young and old individuals. In the HERV-W family expression was observed in 45 loci and only in one case the young/old difference was significant. However, applying hierarchical clustering on the HERV expression data resulted in the formation of two distinct clusters, one containing the young individuals and another the nonagenarians. This suggests, that even though the aging-associated differences in the expression levels of individual proviruses are minor, there seems to be some underlying aging-related pattern. These data indicate that aging does not have a strong effect on the expression of individual HERV proviruses, but instead several proviruses are affected moderately, leading to age-dependent expression profiles.

Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) is a zoonotic pathogen for persons in contact with horses. In horses, S. zooepidemicus is an opportunistic pathogen, but human infections associated with S. zooepidemicus are often severe. Within 6 months in 2011, 3 unrelated cases of severe, disseminated S. zooepidemicus infection occurred in men working with horses in eastern Finland. To clarify the pathogen’s epidemiology, we describe the clinical features of the infection in 3 patients and compare the S. zooepidemicus isolates from the human cases with S. zooepidemicus isolates from horses. The isolates were analyzed by using pulsed-field gel electrophoresis, multilocus sequence typing, and sequencing of the szP gene. Molecular typing methods showed that human and equine isolates were identical or closely related. These results emphasize that S. zooepidemicus transmitted from horses can lead to severe infections in humans. As leisure and professional equine sports continue to grow, this infection should be recognized as an emerging zoonosis.

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.

Fatty liver is a major cause of obesity-related morbidity and mortality. The aim of this study was to identify early metabolic alterations associated with liver fat accumulation in 50- to 55-year-old men (n = 49) and women (n = 52) with and without NAFLD.Hepatic fat content was measured using proton magnetic resonance spectroscopy (1H MRS). Serum samples were analyzed using a nuclear magnetic resonance (NMR) metabolomics platform. Global gene expression profiles of adipose tissues and skeletal muscle were analyzed using Affymetrix microarrays and quantitative PCR. Muscle protein expression was analyzed by Western blot.Increased branched-chain amino acid (BCAA), aromatic amino acid (AAA) and orosomucoid were associated with liver fat accumulation already in its early stage, independent of sex, obesity or insulin resistance (p

The potential impact of nanoparticles on the environment and on human health has attracted considerable interest worldwide. The amount of transcriptomics data, in which tissues and cell lines are exposed to nanoparticles, increases year by year. In addition to the importance of the original findings, this data can have value in broader context when combined with other previously acquired and published results. In order to facilitate the efficient usage of the data, we have developed the NanoMiner web resource (http://nanominer.cs.tut.fi/), which contains 404 human transcriptome samples exposed to various types of nanoparticles. All the samples in NanoMiner have been annotated, preprocessed and normalized using standard methods that ensure the quality of the data analyses and enable the users to utilize the database systematically across the different experimental setups and platforms. With NanoMiner it is possible to 1) search and plot the expression profiles of one or several genes of interest, 2) cluster the samples within the datasets, 3) find differentially expressed genes in various nanoparticle studies, 4) detect the nanoparticles causing differential expression of selected genes, 5) analyze enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms for the detected genes and 6) search the expression values and differential expressions of the genes belonging to a specific KEGG pathway or Gene Ontology. In sum, NanoMiner database is a valuable collection of microarray data which can be also used as a data repository for future analyses.

Brain-derived neurotrophic factor (BDNF) importantly regulates learning and memory and supports the survival of injured neurons. Reduced BDNF levels have been detected in the brains of Alzheimer's disease (AD) patients but the exact role of BDNF in the pathophysiology of the disorder remains obscure. We have recently shown that reduced signaling of BDNF receptor TrkB aggravates memory impairment in APPswe/PS1dE9 (APdE9) mice, a model of AD. The present study examined the influence of Bdnf gene deficiency (heterozygous knockout) on spatial learning, spontaneous exploratory activity and motor coordination/balance in middle-aged male and female APdE9 mice. We also studied brain BDNF protein levels in APdE9 mice in different ages showing progressive amyloid pathology. Both APdE9 and Bdnf mutations impaired spatial learning in males and showed a similar trend in females. Importantly, the effect was additive, so that double mutant mice performed the worst. However, APdE9 and Bdnf mutations influenced spontaneous locomotion in contrasting ways, such that locomotor hyperactivity observed in APdE9 mice was normalized by Bdnf deficiency. Obesity associated with Bdnf deficiency did not account for the reduced hyperactivity in double mutant mice. Bdnf deficiency did not alter amyloid plaque formation in APdE9 mice. Before plaque formation (3 months), BDNF protein levels where either reduced (female) or unaltered (male) in the APdE9 mouse cortex. Unexpectedly, this was followed by an age-dependent increase in mature BDNF protein. Bdnf mRNA and phospho-TrkB levels remained unaltered in the cortical tissue samples of middle-aged APdE9 mice. Immunohistological studies revealed increased BDNF immunoreactivity around amyloid plaques indicating that the plaques may sequester BDNF protein and prevent it from activating TrkB. If similar BDNF accumulation happens in human AD brains, it would suggest that functional BDNF levels in the AD brains are even lower than reported, which could partially contribute to learning and memory problems of AD patients.

A comprehensive in vitro assessment of two commercial metal oxide nanoparticles, TiO2 and ZnO, was performed using human monocyte-derived macrophages (HMDM), monocyte-derived dendritic cells (MDDC), and Jurkat T cell leukemia-derived cell line. TiO2 nanoparticles were found to be non-toxic whereas ZnO nanoparticles caused dose-dependent cell death. Subsequently, global gene expression profiling was performed to identify transcriptional response underlying the cytotoxicity caused by ZnO nanoparticles. Analysis was done with doses 1 µg/ml and 10 µg/ml after 6 and 24 h of exposure. Interestingly, 2703 genes were significantly differentially expressed in HMDM upon exposure to 10 µg/ml ZnO nanoparticles, while in MDDCs only 12 genes were affected. In Jurkat cells, 980 genes were differentially expressed. It is noteworthy that only the gene expression of metallothioneins was upregulated in all the three cell types and a notable proportion of the genes were regulated in a cell type-specific manner. Gene ontology analysis revealed that the top biological processes disturbed in HMDM and Jurkat cells were regulating cell death and growth. In addition, genes controlling immune system development were affected. Using a panel of modified ZnO nanoparticles, we obtained an additional support that the cellular response to ZnO nanoparticles is largely dependent on particle dissolution and show that the ligand used to modify ZnO nanoparticles modulates Zn(2+) leaching. Overall, the study provides an extensive resource of transcriptional markers for mediating ZnO nanoparticle-induced toxicity for further mechanistic studies, and demonstrates the value of assessing nanoparticle responses through a combined transcriptomics and bioinformatics approach.

BACKGROUND: Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy). METHODOLOGY/PRINCIPAL FINDINGS: We obtained a mean RBC speed (0.9±0.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.9±0.6 sec) as compared to the age-matched non-exposed rats (1.2±0.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats. CONCLUSIONS/SIGNIFICANCE: The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method.