Expression and purification of NEIL3, a human DNA glycosylase homolog

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.     

Integrated study of copy number states and genotype calls using high-density SNP arrays

We propose a statistical framework, named genoCN, to simultaneously dissect copy number states and genotypes using high-density SNP (single nucleotide polymorphism) arrays. There are at least two types of genomic DNA copy number differences: copy number variations (CNVs) and copy number aberrations (CNAs). While CNVs are naturally occurring and inheritable, CNAs are acquired somatic alterations most often observed in tumor tissues only. CNVs tend to be short and more sparsely located in the genome compared with CNAs. GenoCN consists of two components, genoCNV and genoCNA, designed for CNV and CNA studies, respectively. In contrast to most existing methods, genoCN is more flexible in that the model parameters are estimated from the data instead of being decided a priori. GenoCNA also incorporates two important strategies for CNA studies. First, the effects of tissue contamination are explicitly modeled. Second, if SNP arrays are performed for both tumor and normal tissues of one individual, the genotype calls from normal tissue are used to study CNAs in tumor tissue. We evaluated genoCN by applications to 162 HapMap individuals and a brain tumor (glioblastoma) dataset and showed that our method can successfully identify both types of copy number differences and produce high-quality genotype calls.     

Limbic Structures Show Altered Glial–Neuronal Metabolism in the Chronic Phase of Kainate Induced Epilepsy

A better understanding is needed of how glutamate metabolism is affected in mesial temporal lobe epilepsy (MTLE). Here we investigated glial–neuronal metabolism in the chronic phase of the kainate (KA) model of MTLE. Thirteen weeks following systemic KA, rats were injected i.p. with [1-¹³C]glucose. Brain extracts from hippocampal formation, entorhinal cortex, and neocortex, were analyzed by ¹³C and ¹H magnetic resonance spectroscopy to quantify ¹³C labeling and concentrations of metabolites, respectively. The amount and ¹³C labeling of glutamate were reduced in the hippocampal formation and entorhinal cortex of epileptic rats. Together with the decreased concentration of NAA, these results indicate neuronal loss. Additionally, mitochondrial dysfunction was detected in surviving glutamatergic neurons in the hippocampal formation. In entorhinal cortex glutamine labeling and concentration were unchanged despite the reduced glutamate content and label, possibly due to decreased oxidative metabolism and conserved flux of glutamate through glutamine synthetase in astrocytes. This mechanism was not operative in the hippocampal formation, where glutamine labeling was decreased. In neocortex labeling and concentration of GABA were increased in epileptic rats, possibly representing a compensatory mechanism. The changes in the hippocampus might be of pathophysiological importance and merit further studies aiming at resolving metabolic causes and consequences of MTLE. Special issue article in honor of Dr. Frode Fonnum.     

Evaluation of prospective, routine application of Ki-67 immunoquantitation in early CIN for assessment of short-term progression risk

OBJECTIVE: To prospectively validate, in early cervical intraepithelial neoplasia (CIN), routine assessment of a previously developed prognostic Ki-67 immunoquantitative progression-risk model. STUDY DESIGN: Two hundred sixty-six consecutive cervical biopsies taken for an abnormal cytologic smear were routinely diagnosed by experienced pathologists as CIN. Ki-67 immunoquantitation was performed routinely by 3 technicians blinded to clinical and pathologic information. Progression of CIN 1-2 to CIN 3 in histologic follow-up biopsies was used as the intermediate end point. RESULTS: In 58 (22%) biopsies, technical shortcomings prevented Ki-67 immunoquantitation, and in 22 biopsies no follow-up was available. The routine diagnosis in the 186 remaining biopsies was CIN 1 = 24, CIN 2 = 56 and CIN 3 = 106. In 52 marker biopsies with expert review diagnosis of CIN 1-2 and adequate follow-up, histologic biopsies revealed CIN 3 in 9 (17%) cases: 9 of 34 (26%) of Ki-67 high-risk and 0 of 18 (0%) of Ki-67 low-risk lesions (log rank = 5.0, P = .03). Routine CIN grade (1 or 2) was not prognostic (P = .65). Eleven (55%) of 20 CIN 1 and 7 of 32 (22%) CIN 2 cases were Ki-67 low risk and none progressed, contrasting with 4 of 9 (44%) progressions of Ki-67 high risk CIN 1s and 5 of 25 (20%) high risk CIN 2s. Expert CIN grades were stronger prognostically than routine CIN grade, but Ki-67 was still stronger. CONCLUSION: Routine Ki-67 immunoquantitative progression prediction in CIN 1-2 is more predictive of CIN 3 in follow-up than are routine and review CIN grades.     

HPV mRNA Is More Specific than HPV DNA in Triage of Women with Minor Cervical Lesions

Background

In Norway, repeat cytology and HPV testing comprise delayed triage of women with minor cytological lesions. The objective of this study was to evaluate HPV DNA and HPV mRNA testing in triage of women with an ASC-US/LSIL diagnosis.

Materials and Methods

We used repeat cytology, HPV DNA testing (Cobas 4800) and HPV mRNA testing (PreTect HPV-Proofer) to follow up 311 women aged 25–69 years with ASC-US/LSIL index cytology.

Results

Of 311 women scheduled for secondary screening, 30 women (9.6%) had ASC-H/HSIL cytology at triage and 281 women (90.4%) had ASC-US/LSIL or normal cytology. The HPV DNA test was positive in 92 (32.7%) of 281 instances, and 37 (13.2%) were mRNA positive. Of the 132 women with repeated ASC-US/LSIL, we received biopsies from 97.0% (65/67) of the DNA-positive and 92.9% (26/28) of the mRNA-positive cases. The positive predictive values for CIN2+ were 21.5% (14/65) for DNA positive and 34.6% (9/26) for mRNA positive (ns). The odds ratio for being referred to colposcopy in DNA-positive cases were 2.8 times (95% CI: 1.8–4.6) higher that of mRNA-positive cases. Compared to the mRNA test, the DNA test detected four more cases of CIN2 and one case of CIN3.

Conclusions

The higher positivity rate of the DNA test in triage leads to higher referral rate for colposcopy and biopsy, and subsequent additional follow-up of negative biopsies. By following mRNA-negative women who had ASC-US/LSIL at triage with cytology, the additional cases of CIN2+ gained in DNA screening can be discovered. Our study indicates that in triage of repeated ASC-US/LSIL, HPV mRNA testing is more specific and is more relevant in clinical use than an HPV DNA test.     

Assimilation of inorganic nutrients from salmon (Salmo salar) farming by the macroalgae (Saccharina latissima) in an exposed coastal environment: implications for integrated multi-trophic aquaculture

This paper investigated the assimilation of dissolved inorganic nitrogen (DIN) in Saccharina latissima in proximity to salmon cages in coastal waters. The bioassays were performed on plants from three stations located in the vicinity of a salmon farm (Salmo salar) in exposed waters at Tristein (63° 52′ N, 9° 37′ E) in Central Norway. The growth, the C and N content, and the nitrogen isotope ratios (δ¹⁵N) of S. latissima were monitored over 1 year. The DIN concentrations in seawater were higher at the salmon farm stations than at the reference station during the winter, and the N/P ratio at the salmon farm stations was higher from September to January and in June. S. latissima at the salmon farm stations grew faster than at the reference station. The length of S. latissima increased by 50 % when integrated with the salmon farm compared to the reference station. The N content of S. latissima was positively correlated to the DIN concentration in seawater (p?<?0.05), but the increased N supply from salmon did not result in N accumulation in S. latissima at the salmon farm station because of the dilution by a higher growth rate. The δ¹⁵N in S. latissima was higher at the salmon farm station from April to June and changed in the direction of the δ¹⁵N signature in urine. This indicated that N in S. latissima at the salmon farm station partly originated from the salmon. One hectare of S. latissima may absorb 0.8～1.2 t N during one growth season. Large-scale cultivation of S. latissima should be considered to mitigate the environmental effects of DIN wastes from salmon farms.     

Effects of outplanting time on growth,shedding and quality of Saccharina latissima (Phaeophyceae) in its northern distribution range

Journal of Applied Phycology - To reach the goal of large-scale seaweed cultivation in Norway and the rest of Europe, new knowledge about the commercially important kelp species Saccharina...     

E-Rehabilitation an Internet and mobile phone based tailored intervention to enhance selfmanagement of Cardiovascular Disease: study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Cardiac rehabilitation is very important for the recovery and the secondary prevention of cardiovascular disease, and one of its main strategies is to increase the level of physical activity. Internet and mobile phone based interventions have been successfully used to help people to achieve this. One of the components that are related to the efficacy of these interventions is tailoring of content to the individual. This trial is studying the effect of a longitudinally tailored Internet- and mobile phone based intervention that is based on models of health behaviour, on the level of physical activity and the adherence to the intervention, as an extension of a face-to-face cardiac rehabilitation stay. METHODS: A parallel group, cluster randomized controlled trial. The study population is adult participants of a cardiac rehabilitation programme in Norway with home Internet access and mobile phone, who in monthly clusters are randomized to the control or the intervention condition. Participants have access to a website with information regarding cardiac rehabilitation, an online discussion forum and an online activity calendar. Those randomized to the intervention condition, receive in addition tailored content based on models of health behaviour, through the website and mobile text messages. The objective is to assess the effect of the intervention on maintenance of self-management behaviours after the rehabilitation stay. Main outcome is the level of physical activity one month, three months and one year after the end of the cardiac rehabilitation programme. The randomization of clusters is based on a true random number online service, and participants, investigators and outcome assessor are blinded to the condition of the clusters. DISCUSSION: The study suggests a theory-based intervention that combines models of health behaviour in an innovative way, in order to tailor the delivered content. The users have been actively involved in its design, and because of the use of Open-Source software, the intervention can easily and at low-cost be reproduced and expanded by others. Challenges are the recruitment in the elderly population and the possible underrepresentation of women in the study sample. Funding by Northern Norway Regional Health Authority. Trial registration Trial registry www.clinicaltrials.gov: NCT01223170.