Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and l... more Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.
The cellular prion protein PrP C is highly expressed in neurons, but also present in non-neuronal... more The cellular prion protein PrP C is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrP C . Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrP C have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrP C (PRNP Ter/Ter ) compared with cells from normal (PRNP +/+ ) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu 2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO 4 and H 2 O 2 . Surprisingly, PrP C -negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H 2 O 2 and methyl methanesulfonate (MMS), revealed no effect of PrP C on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrP C . RNA sequencing of PBMCs (n = 8 of PRNP +/+ and PRNP Ter/Ter ) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrP C . Data presented here questions the in vitro cytoprotective roles previously attributed to PrP C , although not excluding such functions in other cell types or tissues during inflammatory stress.
Access to full text and tables of contents, including tentative ones for forthcoming issues: www.... more Access to full text and tables of contents, including tentative ones for forthcoming issues: www.karger.com/neo_issues No. 2 Reviews 77 Development of Neonatal Respiratory and Intensive Care: Chinese Perspectives Sun, B. (Shanghai); Ma, L. (Shijiazhuang); Liu, X. (Shenzhen); Gao, X. (Changsha); Ni, L. (Xi'an) 83 Pulmonary Effects of Prolonged Oligohydramnios following Mid-Trimester Rupture of the Membranes -Antenatal and Postnatal Management
Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in l... more Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining....
We have previously described the isolation of a replication competent (RC) complex from calf thym... more We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase a, DNA polymerase d and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G 2 /M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by speci®c cyclin/Cdk complexes.
Background: Infants with extremely low birth weight uniformly develop anemia of prematurity and f... more Background: Infants with extremely low birth weight uniformly develop anemia of prematurity and frequently require red blood cell transfusions (RBCTs). Although RBCT is widely practiced, the indications remain controversial in the absence of conclusive data on the long-term effects of RBCT. Objectives: To summarize the current equipoise and to outline the study protocol of the ‘Effects of Transfusion Thresholds on Neurocognitive Outcome of extremely low birth-weight infants (ETTNO)’ study. Methods: Review of the literature and design of a large pragmatic randomized controlled trial of restrictive versus liberal RBCT guidelines enrolling 920 infants with birth weights of 400–999 g with long-term neurodevelopmental follow-up. Results and Conclusions: The results of ETTNO will provide definite data about the efficacy and safety of restrictive versus liberal RBCT guidelines in very preterm infants.
Vacuum filtration of clean coal slurries and flocculated slurries with cationic, anionic and noni... more Vacuum filtration of clean coal slurries and flocculated slurries with cationic, anionic and nonionic surfactants has been investigated. Significant reductions in filter cake moisture content were obtained for all types of surfactant. The most effective surfactant was CTAB. The final moisture content of filter cakes was reduced from 21 to 11.7 wt% by using a moderate dosage of flocculant followed
Collection of Czechoslovak Chemical Communications, 2009
1R*,4S*,6S*)-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.2]octane-2,2-dimethanol ( ) and (1R*,4R*,5S*)-... more 1R*,4S*,6S*)-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.2]octane-2,2-dimethanol ( ) and (1R*,4R*,5S*)-5-(6-chloro-9H-purin-9-yl)bicyclo[2.2.2]octane-2,2-dimethanol (17) were prepared from (1R*,4R*)-bicyclo[2.2.2]oct-5-ene-2,2-dimethanediyl dibenzoate (7) using two approaches. The first procedure consists in hydroboration of 7, separation of obtained 6-exo-hydroxy derivative 8 and 5-exo-hydroxy derivative 9, conversion of 8 and 9 to endo-hydroxy derivatives 12 and 13, respectively, and the Mitsunobu reaction with 6-chloropurine. Only 5-(6-chloropurinyl) analogue 16 was obtained in an acceptable yield. The target analog 17 was prepared by reductive debenzoylation of 16. The further reactions were hydroboration of 7, treatment with hydroxylamine-O-sulfonic acid and debenzoylation. Chloropurine analogues 17 and 22 were built on the obtained 6-exo-amino-and 5-exo-aminobicyclo[2.2.2]octane-2,2-dimethanols 18 and 19, respectively. Compounds 17 and 22 were converted to adenine (23, 24) and 6-(cyclopropylamino)purine analogues (25, 26).
A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles wa... more A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low-and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits.
8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair ... more 8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3 to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.
Background: Some viruses cause outbreaks which require immediate attention. Neutralizing antibodi... more Background: Some viruses cause outbreaks which require immediate attention. Neutralizing antibodies could be developed for viral outbreak management. However, development of monoclonal antibodies is often long, laborious, and unprofitable. Here we report a Norwegian platform for development of chicken polyclonal neutralizing antibodies with powerful therapeutic potential. Methods: Layers were immunized twice with 14-day interval using purified RBD of SARS-CoV-2. Eggs were harvested 14 days after the second immunization. Polyclonal IgY antibodies were extracted. Binding of anti-RBD IgY to RBD was measured by indirect ELISA. Neutralization capacity of anti-RBD IgYs was measured in Vero-E6 cells infected with SARS-CoV-2-mCherry strain using fluorescence and cell viability assay. In addition, the effect of IgYs on the expression of SARS-CoV-2 and host cytokine genes in lungs of Syrian Golden hamsters was examined using qRT-PCR. Results: Anti-RBD IgYs efficiently bind RBD of S protein of SARS-CoV-2 in situ, neutralize the virus in vitro, and lower viral RNA amplification without significant alteration of virus-mediated immune gene expression in vivo. Conclusions: Altogether, our results indicated that chicken polyclonal IgYs can be attractive targets for pre-clinical and clinical development for rapid management of outbreaks of emerging and re-emerging viruses.
Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-speci... more Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broadspectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of the general population from emerging and re-emerging viral diseases, reinforcing the arsenal of available antiviral options. Here, we review discovery and development of BSAAs and summarize the information on 120 safe-in-man agents in a freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:
Cytosine (C) in DNA is often modified to 5-methylcytosine (m<sup>5</sup>C) to execute... more Cytosine (C) in DNA is often modified to 5-methylcytosine (m<sup>5</sup>C) to execute important cellular functions. Despite the significance of m<sup>5</sup>C for epigenetic regulation in mammals, damage to m<sup>5</sup>C has received little attention. For instance, almost no studies exist on erroneous methylation of m<sup>5</sup>C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m<sup>5</sup>C into <i>N</i><sup>4</sup>,5-dimethylcytosine (m<i><sup>N</sup></i><sup>4,5</sup>C) in DNA, m<i><sup>N</sup></i><sup>4,5</sup>C is probably present <i>in vivo</i>. We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the <i>Escherichia coli</i> Nei and Fpg proteins at m<i><sup>N</sup></i><sup>4,5</sup>C residues <i>in vitro</i>. The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m<sup>5</sup>C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m<i><sup>N</sup></i><sup>4,5</sup>C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in <i>E. coli</i> to initiate its repair.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
<strong><em>Background:</em></strong> Perinatal asphyxia and ensuing reox... more <strong><em>Background:</em></strong> Perinatal asphyxia and ensuing reoxygenation change the antioxidant capacity of cells and organs. <b><i>Objectives:</i></b> To analyze the neuroprotective effect of the antioxidant N-acetylcysteine amide (NACA) after perinatal hypoxia-reoxygenation with an emphasis on proinflammatory cytokines and the transcription factor NF-κB in the prefrontal cortex of neonatal pigs. <b><i>Methods:</i></b> Twenty-nine newborn pigs, aged 12-36 h, were subjected to global hypoxia and hypercapnia. One sham-operated group (n = 5) and 2 experimental groups (n = 12) were exposed to 8% oxygen, until the base excess was -20 mmol/l or the mean arterial blood pressure fell to &lt;20 mm Hg (asphyxia with NACA or saline). The pigs were observed for 9.5 h after hypoxia. Samples of prefrontal cortex and plasma were analyzed. <b><i>Results:</i></b> Cortex: there was no significant difference in mRNA expression between the intervention groups regarding IL-1β, IL6, TNFα, MMP2, MMP9 or IL18. Pigs exposed to hypoxia-reoxygenation and treatment with NACA (NACA-pigs) had a significantly lower protein concentration of IL-1β than pigs treated with saline (placebo controls), i.e. 8.8 ± 3.9 versus 16.8 ± 10.5 pg/mg protein (p = 0.02). The activation of the transcription factor NF-κB (measured as the fold-change of phosphorylated p65<sup>Ser 536</sup>), was reduced in the NACA-pigs when compared to the placebo controls (5.2 ± 4.3 vs. 16.0 ± 13.5; p = 0.02). No difference between the intervention groups regarding brain histopathology or in the levels of 8-oxoguanine measured in the prefrontal cortex were observed. Plasma: the NACA-pigs had a stronger reduction of TNFα in the first 30 min following asphyxia compared with the placebo controls, i.e. 36 (30-44) versus 24 (14-32)% (p = 0.01). <b><i>Conclusion:</i></b> The reduced levels of the pivotal inflammatory markers IL-1β and TNFα and the transcription factor NF-κB may indicate that NACA has possible neuroprotective effects after perinatal asphyxia.
Maple syrup urine disease (MSUD) is caused by a defect in branched chain a-ketoacid dehydrogenase... more Maple syrup urine disease (MSUD) is caused by a defect in branched chain a-ketoacid dehydrogenase complex (BCKD), an essential metabolon for the catabolism of the branched chain amino acids. Here, we report four novel mutations in the DBT gene, encoding the transacylase subunit (E2) of BCKD, resulting in intermittent MSUD in seven Norwegian patients. The patients had episodes with neurological symptoms including lethargy and/or ataxia during childhood infections. All seven patients were heterozygous for the annotated R301C mutation. The second allelic mutations were identified in five patients; one nonsense mutation (G62X), two missense mutations (W84C and R376C) and a mutation in the 3 0 untranslated region (UTR; c.*358A>C) in two patients. These four novel mutations result in near depletion of E2 protein, and the common R301C protein contributes predominantly to the residual (14%) cellular BCKD activity. Structural analyses of the mutations implied that the W84C and R376C mutations affect stability of intramolecular domains in E2, while the R301C mutation likely disturbs E2 trimer assembly as previously reported. The UTR mutated allele coincided with a strong reduction in mRNA levels, as did the non-R301C specific allele in two patients where the second mutation could not be identified. In summary, the pathogenic effect of the novel mutations is depletion of cellular protein, and the intermittent form of MSUD appears to be attributed to the residual R301C mutant protein in these patients.
Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and l... more Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.
The cellular prion protein PrP C is highly expressed in neurons, but also present in non-neuronal... more The cellular prion protein PrP C is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrP C . Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrP C have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrP C (PRNP Ter/Ter ) compared with cells from normal (PRNP +/+ ) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu 2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO 4 and H 2 O 2 . Surprisingly, PrP C -negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H 2 O 2 and methyl methanesulfonate (MMS), revealed no effect of PrP C on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrP C . RNA sequencing of PBMCs (n = 8 of PRNP +/+ and PRNP Ter/Ter ) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrP C . Data presented here questions the in vitro cytoprotective roles previously attributed to PrP C , although not excluding such functions in other cell types or tissues during inflammatory stress.
Access to full text and tables of contents, including tentative ones for forthcoming issues: www.... more Access to full text and tables of contents, including tentative ones for forthcoming issues: www.karger.com/neo_issues No. 2 Reviews 77 Development of Neonatal Respiratory and Intensive Care: Chinese Perspectives Sun, B. (Shanghai); Ma, L. (Shijiazhuang); Liu, X. (Shenzhen); Gao, X. (Changsha); Ni, L. (Xi'an) 83 Pulmonary Effects of Prolonged Oligohydramnios following Mid-Trimester Rupture of the Membranes -Antenatal and Postnatal Management
Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in l... more Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining....
We have previously described the isolation of a replication competent (RC) complex from calf thym... more We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase a, DNA polymerase d and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G 2 /M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by speci®c cyclin/Cdk complexes.
Background: Infants with extremely low birth weight uniformly develop anemia of prematurity and f... more Background: Infants with extremely low birth weight uniformly develop anemia of prematurity and frequently require red blood cell transfusions (RBCTs). Although RBCT is widely practiced, the indications remain controversial in the absence of conclusive data on the long-term effects of RBCT. Objectives: To summarize the current equipoise and to outline the study protocol of the ‘Effects of Transfusion Thresholds on Neurocognitive Outcome of extremely low birth-weight infants (ETTNO)’ study. Methods: Review of the literature and design of a large pragmatic randomized controlled trial of restrictive versus liberal RBCT guidelines enrolling 920 infants with birth weights of 400–999 g with long-term neurodevelopmental follow-up. Results and Conclusions: The results of ETTNO will provide definite data about the efficacy and safety of restrictive versus liberal RBCT guidelines in very preterm infants.
Vacuum filtration of clean coal slurries and flocculated slurries with cationic, anionic and noni... more Vacuum filtration of clean coal slurries and flocculated slurries with cationic, anionic and nonionic surfactants has been investigated. Significant reductions in filter cake moisture content were obtained for all types of surfactant. The most effective surfactant was CTAB. The final moisture content of filter cakes was reduced from 21 to 11.7 wt% by using a moderate dosage of flocculant followed
Collection of Czechoslovak Chemical Communications, 2009
1R*,4S*,6S*)-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.2]octane-2,2-dimethanol ( ) and (1R*,4R*,5S*)-... more 1R*,4S*,6S*)-6-(6-Chloro-9H-purin-9-yl)bicyclo[2.2.2]octane-2,2-dimethanol ( ) and (1R*,4R*,5S*)-5-(6-chloro-9H-purin-9-yl)bicyclo[2.2.2]octane-2,2-dimethanol (17) were prepared from (1R*,4R*)-bicyclo[2.2.2]oct-5-ene-2,2-dimethanediyl dibenzoate (7) using two approaches. The first procedure consists in hydroboration of 7, separation of obtained 6-exo-hydroxy derivative 8 and 5-exo-hydroxy derivative 9, conversion of 8 and 9 to endo-hydroxy derivatives 12 and 13, respectively, and the Mitsunobu reaction with 6-chloropurine. Only 5-(6-chloropurinyl) analogue 16 was obtained in an acceptable yield. The target analog 17 was prepared by reductive debenzoylation of 16. The further reactions were hydroboration of 7, treatment with hydroxylamine-O-sulfonic acid and debenzoylation. Chloropurine analogues 17 and 22 were built on the obtained 6-exo-amino-and 5-exo-aminobicyclo[2.2.2]octane-2,2-dimethanols 18 and 19, respectively. Compounds 17 and 22 were converted to adenine (23, 24) and 6-(cyclopropylamino)purine analogues (25, 26).
A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles wa... more A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low-and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits.
8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair ... more 8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3 to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.
Background: Some viruses cause outbreaks which require immediate attention. Neutralizing antibodi... more Background: Some viruses cause outbreaks which require immediate attention. Neutralizing antibodies could be developed for viral outbreak management. However, development of monoclonal antibodies is often long, laborious, and unprofitable. Here we report a Norwegian platform for development of chicken polyclonal neutralizing antibodies with powerful therapeutic potential. Methods: Layers were immunized twice with 14-day interval using purified RBD of SARS-CoV-2. Eggs were harvested 14 days after the second immunization. Polyclonal IgY antibodies were extracted. Binding of anti-RBD IgY to RBD was measured by indirect ELISA. Neutralization capacity of anti-RBD IgYs was measured in Vero-E6 cells infected with SARS-CoV-2-mCherry strain using fluorescence and cell viability assay. In addition, the effect of IgYs on the expression of SARS-CoV-2 and host cytokine genes in lungs of Syrian Golden hamsters was examined using qRT-PCR. Results: Anti-RBD IgYs efficiently bind RBD of S protein of SARS-CoV-2 in situ, neutralize the virus in vitro, and lower viral RNA amplification without significant alteration of virus-mediated immune gene expression in vivo. Conclusions: Altogether, our results indicated that chicken polyclonal IgYs can be attractive targets for pre-clinical and clinical development for rapid management of outbreaks of emerging and re-emerging viruses.
Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-speci... more Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broadspectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of the general population from emerging and re-emerging viral diseases, reinforcing the arsenal of available antiviral options. Here, we review discovery and development of BSAAs and summarize the information on 120 safe-in-man agents in a freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections.
with specic help available everywhere you see the i O symbol. The following versions of software ... more with specic help available everywhere you see the i O symbol. The following versions of software and data (see references i O) were used in the production of this report:
Cytosine (C) in DNA is often modified to 5-methylcytosine (m<sup>5</sup>C) to execute... more Cytosine (C) in DNA is often modified to 5-methylcytosine (m<sup>5</sup>C) to execute important cellular functions. Despite the significance of m<sup>5</sup>C for epigenetic regulation in mammals, damage to m<sup>5</sup>C has received little attention. For instance, almost no studies exist on erroneous methylation of m<sup>5</sup>C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m<sup>5</sup>C into <i>N</i><sup>4</sup>,5-dimethylcytosine (m<i><sup>N</sup></i><sup>4,5</sup>C) in DNA, m<i><sup>N</sup></i><sup>4,5</sup>C is probably present <i>in vivo</i>. We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the <i>Escherichia coli</i> Nei and Fpg proteins at m<i><sup>N</sup></i><sup>4,5</sup>C residues <i>in vitro</i>. The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m<sup>5</sup>C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m<i><sup>N</sup></i><sup>4,5</sup>C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in <i>E. coli</i> to initiate its repair.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
<strong><em>Background:</em></strong> Perinatal asphyxia and ensuing reox... more <strong><em>Background:</em></strong> Perinatal asphyxia and ensuing reoxygenation change the antioxidant capacity of cells and organs. <b><i>Objectives:</i></b> To analyze the neuroprotective effect of the antioxidant N-acetylcysteine amide (NACA) after perinatal hypoxia-reoxygenation with an emphasis on proinflammatory cytokines and the transcription factor NF-κB in the prefrontal cortex of neonatal pigs. <b><i>Methods:</i></b> Twenty-nine newborn pigs, aged 12-36 h, were subjected to global hypoxia and hypercapnia. One sham-operated group (n = 5) and 2 experimental groups (n = 12) were exposed to 8% oxygen, until the base excess was -20 mmol/l or the mean arterial blood pressure fell to &lt;20 mm Hg (asphyxia with NACA or saline). The pigs were observed for 9.5 h after hypoxia. Samples of prefrontal cortex and plasma were analyzed. <b><i>Results:</i></b> Cortex: there was no significant difference in mRNA expression between the intervention groups regarding IL-1β, IL6, TNFα, MMP2, MMP9 or IL18. Pigs exposed to hypoxia-reoxygenation and treatment with NACA (NACA-pigs) had a significantly lower protein concentration of IL-1β than pigs treated with saline (placebo controls), i.e. 8.8 ± 3.9 versus 16.8 ± 10.5 pg/mg protein (p = 0.02). The activation of the transcription factor NF-κB (measured as the fold-change of phosphorylated p65<sup>Ser 536</sup>), was reduced in the NACA-pigs when compared to the placebo controls (5.2 ± 4.3 vs. 16.0 ± 13.5; p = 0.02). No difference between the intervention groups regarding brain histopathology or in the levels of 8-oxoguanine measured in the prefrontal cortex were observed. Plasma: the NACA-pigs had a stronger reduction of TNFα in the first 30 min following asphyxia compared with the placebo controls, i.e. 36 (30-44) versus 24 (14-32)% (p = 0.01). <b><i>Conclusion:</i></b> The reduced levels of the pivotal inflammatory markers IL-1β and TNFα and the transcription factor NF-κB may indicate that NACA has possible neuroprotective effects after perinatal asphyxia.
Maple syrup urine disease (MSUD) is caused by a defect in branched chain a-ketoacid dehydrogenase... more Maple syrup urine disease (MSUD) is caused by a defect in branched chain a-ketoacid dehydrogenase complex (BCKD), an essential metabolon for the catabolism of the branched chain amino acids. Here, we report four novel mutations in the DBT gene, encoding the transacylase subunit (E2) of BCKD, resulting in intermittent MSUD in seven Norwegian patients. The patients had episodes with neurological symptoms including lethargy and/or ataxia during childhood infections. All seven patients were heterozygous for the annotated R301C mutation. The second allelic mutations were identified in five patients; one nonsense mutation (G62X), two missense mutations (W84C and R376C) and a mutation in the 3 0 untranslated region (UTR; c.*358A>C) in two patients. These four novel mutations result in near depletion of E2 protein, and the common R301C protein contributes predominantly to the residual (14%) cellular BCKD activity. Structural analyses of the mutations implied that the W84C and R376C mutations affect stability of intramolecular domains in E2, while the R301C mutation likely disturbs E2 trimer assembly as previously reported. The UTR mutated allele coincided with a strong reduction in mRNA levels, as did the non-R301C specific allele in two patients where the second mutation could not be identified. In summary, the pathogenic effect of the novel mutations is depletion of cellular protein, and the intermittent form of MSUD appears to be attributed to the residual R301C mutant protein in these patients.
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