Isolation of a novel protein,P12—from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3′-5′- exonuclease activity

ABSTRACT

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10–13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3′-5′-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.     

Training in High-Throughput Sequencing: Common Guidelines to Enable Material Sharing,Dissemination, and Reusability

The advancement of high-throughput sequencing (HTS) technologies and the rapid development of numerous analysis algorithms and pipelines in this field has resulted in an unprecedentedly high demand for training scientists in HTS data analysis. Embarking on developing new training materials is challenging for many reasons. Trainers often do not have prior experience in preparing or delivering such materials and struggle to keep them up to date. A repository of curated HTS training materials would support trainers in materials preparation, reduce the duplication of effort by increasing the usage of existing materials, and allow for the sharing of teaching experience among the HTS trainers’ community. To achieve this, we have developed a strategy for materials’ curation and dissemination. Standards for describing training materials have been proposed and applied to the curation of existing materials. A Git repository has been set up for sharing annotated materials that can now be reused, modified, or incorporated into new courses. This repository uses Git; hence, it is decentralized and self-managed by the community and can be forked/built-upon by all users. The repository is accessible at http://bioinformatics.upsc.se/htmr.     

Community Resilience throughout the Lifespan – The Potential Contribution of Healthy Elders

An increase in the exposure and predisposition of civilian populations to disasters has been recorded in the last decades. In major disasters, as demonstrated recently in Nepal (2015) and previously in Haiti (2010), external aid is vital, yet in the first hours after a disaster, communities must usually cope alone with the challenge of providing emergent lifesaving care. Communities therefore need to be prepared to handle emergency situations. Mapping the needs of the populations within their purview is a trying task for decision makers and community leaders. In this context, the elderly are traditionally treated as a susceptible population with special needs. The current study aimed to explore variations in the level of community resilience along the lifespan. The study was conducted in nine small to mid-size towns in Israel between August and November 2011 (N = 885). The Conjoint Community Resiliency Assessment Measure (CCRAM), a validated instrument for community resilience assessment, was used to examine the association between age and community resilience score. Statistical analysis included spline and logistic regression models that explored community resiliency over the lifespan in a way that allowed flexible modeling of the curve without prior constraints. This innovative statistical approach facilitated identification of the ages at which trend changes occurred. The study found a significant rise in community resiliency scores in the age groups of 61–75 years as compared with younger age bands, suggesting that older people in good health may contribute positively to building community resiliency for crisis. Rather than focusing on the growing medical needs and years of dependency associated with increased life expectancy and the resulting climb in the proportion of elders in the population, this paper proposes that active "young at heart" older people can be a valuable resource for their community.     

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.     

Specific Detection of Two Divergent Simian Arteriviruses Using RNAscope In Situ Hybridization

Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers.     

Using an Inbred Horse Breed in a High Density Genome-Wide Scan for Genetic Risk Factors of Insect Bite Hypersensitivity (IBH)

While susceptibility to hypersensitive reactions is a common problem amongst humans and animals alike, the population structure of certain animal species and breeds provides a more advantageous route to better understanding the biology underpinning these conditions. The current study uses Exmoor ponies, a highly inbred breed of horse known to frequently suffer from insect bite hypersensitivity, to identify genomic regions associated with a type I and type IV hypersensitive reaction. A total of 110 cases and 170 controls were genotyped on the 670K Axiom Equine Genotyping Array. Quality control resulted in 452,457 SNPs and 268 individuals being tested for association. Genome-wide association analyses were performed using the GenABEL package in R and resulted in the identification of two regions of interest on Chromosome 8. The first region contained the most significant SNP identified, which was located in an intron of the DCC netrin 1 receptor gene. The second region identified contained multiple top SNPs and encompassed the PIGN, KIAA1468, TNFRSF11A, ZCCHC2, and PHLPP1 genes. Although additional studies will be needed to validate the importance of these regions in horses and the relevance of these regions in other species, the knowledge gained from the current study has the potential to be a step forward in unraveling the complex nature of hypersensitive reactions.     

Left Atrial Size and Function in a Canine Model of Chronic Atrial Fibrillation and Heart Failure

Background

Our aim was to assess how atrial fibrillation (AF) induction, chronicity, and RR interval irregularity affect left atrial (LA) function and size in the setting of underlying heart failure (HF), and to determine whether AF effects can be mitigated by vagal nerve stimulation (VNS).

Methods

HF was induced by 4-weeks of rapid ventricular pacing in 24 dogs. Subsequently, AF was induced and maintained by atrial pacing at 600 bpm. Dogs were randomized into control (n = 9) and VNS (n = 15) groups. In the VNS group, atrioventricular node fat pad stimulation (310 μs, 20 Hz, 3–7 mA) was delivered continuously for 6 months. LA volume and LA strain data were calculated from bi-weekly echocardiograms.

Results

RR intervals decreased with HF in both groups (p = 0.001), and decreased further during AF in control group (p = 0.014), with a non-significant increase in the VNS group during AF. LA size increased with HF (p<0.0001), with no additional increase during AF. LA strain decreased with HF (p = 0.025) and further decreased after induction of AF (p = 0.0001). LA strain decreased less (p = 0.001) in the VNS than in the control group. Beat-by-beat analysis showed a curvilinear increase of LA strain with longer preceding RR interval, (r = 0.45, p <0.0001) with LA strain 1.1% higher (p = 0.02) in the VNS-treated animals, independent of preceding RR interval duration. The curvilinear relationship between ratio of preceding and pre-preceding RR intervals, and subsequent LA strain was weaker, (r = 0.28, p = 0.001). However, VNS-treated animals again had higher LA strain (by 2.2%, p = 0.002) independently of the ratio of preceding and pre-preceding RR intervals.

Conclusions

In the underlying presence of pacing-induced HF, AF decreased LA strain, with little impact on LA size. LA strain depends on the preceding RR interval duration.     

Modulation of the lethal effects of cocaine by cholinomimetics

In view of the toxicity of cocaine and recent reports on the antimuscarinic properties of cocaine, the present study evaluated the effects of manipulations in cholinergic neurotransmission on the lethal effects of cocaine. Physostigmine pretreatment significantly altered the lethality of cocaine, increasing the LD 50 from 82.5 mg/kg to 96.9 mg/kg in male F344 rats. Atropine alone did not alter the lethal effects of cocaine at doses that are effective in preventing parasympathetic effects and lethality of oxotremorine. The prophylactic effect of physostigmine was not prevented by atropine. Neostigmine did not significantly affect the cocaine lethality dose-effect function. Both oxotremorine and (-)-nicotine were also devoid of protective actions. At higher doses, all of the cholinomimetics potentiated the lethal effects of cocaine. These results suggest that whereas cocaine lethality may be enhanced by stimulation of muscarinic receptors, low doses of physostigmine protect against lethality through actions at noncholinergic sites.