NRBF2 is involved in the autophagic degradation process of APP-CTFs in Alzheimer disease models

Alzheimer disease (AD) is the most common neurodegenerative disease characterized by the deposition of amyloid plaque in the brain. The autophagy-associated PIK3C3-containing phosphatidylinositol 3-kinase (PtdIns3K) complex has been shown to interfere with APP metabolism and amyloid beta peptide (Aβ) homeostasis via poorly understood mechanisms. Here we report that NRBF2 (nuclear receptor binding factor 2), a key component and regulator of the PtdIns3K, is involved in APP-CTFs homeostasis in AD cell models. We found that NRBF2 interacts with APP in vivo and its expression levels are reduced in hippocampus of 5XFAD AD mice; we further demonstrated that NRBF2 overexpression promotes degradation of APP C-terminal fragments (APP-CTFs), and reduces Aβ_1–40 and Aβ_1-42 levels in human mutant APP-overexpressing cells. Conversely, APP-CTFs, Aβ_1–40 and Aβ_1-42 levels were increased in Nrbf2 knockdown or nrbf2 knockout cells. Furthermore, NRBF2 positively regulates autophagy in neuronal cells and NRBF2-mediated reduction of APP-CTFs levels is autophagy dependent. Importantly, nrbf2 knockout attenuates the recruitment of APP and APP-CTFs into phagophores and the sorting of APP and APP-CTFs into endosomal intralumenal vesicles, which is accompanied by the accumulation of the APP and APP-CTFs into RAB5-positive early endosomes. Collectively, our results reveal the potential connection between NRBF2 and the AD-associated protein APP by showing that NRBF2 plays an important role in regulating degradation of APP-CTFs through modulating autophagy.     

Physical mapping of DNA markers linked to stem rust resistance gene <Emphasis Type="Italic">Sr47</Emphasis> in durum wheat

Key message

Markers linked to stem rust resistance gene Sr47 were physically mapped in three small Aegilops speltoides chromosomal bins. Five markers, including two PCR-based SNP markers, were validated for marker-assisted selection.

Abstract

In durum wheat (Triticum turgidum subsp. durum), the gene Sr47 derived from Aegilops speltoides conditions resistance to race TTKSK (Ug99) of the stem rust pathogen (Puccinia graminis f. sp. tritici). Sr47 is carried on small interstitial translocation chromosomes (Ti2BL-2SL-2BL·2BS) in which the Ae. speltoides chromosome 2S segments are divided into four bins in genetic stocks RWG35, RWG36, and RWG37. Our objective was to physically map molecular markers to bins and to determine if any of the molecular markers would be useful in marker-assisted selection (MAS). Durum cultivar Joppa was used as the recurrent parent to produce three BC₂F₂ populations. Each BC₂F₂ plant was genotyped with markers to detect the segment carrying Sr47, and stem rust testing of BC₂F₃ progeny with race TTKSK confirmed the genotyping. Forty-nine markers from published sources, four new SSR markers, and five new STARP (semi-thermal asymmetric reverse PCR) markers, were evaluated in BC₂F₂ populations for assignment of markers to bins. Sr47 was mapped to bin 3 along with 13 markers. No markers were assigned to bin 1; however, 7 and 13 markers were assigned to bins 2 and 4, respectively. Markers Xrwgs38a, Xmag1729, Xwmc41, Xtnac3119, Xrwgsnp1, and Xrwgsnp4 were found to be useful for MAS of Sr47. However, STARP markers Xrwgsnp1 and Xrwgsnp4 can be used in gel-free systems, and are the preferred markers for high-throughput MAS. The physical mapping data from this study will also be useful for pyramiding Sr47 with other Sr genes on chromosome 2B.

     

A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

Tissue specific genes that contain high GC segments are difficult to amplify by standard PCR. We report an improved method for successful amplification of gene segment that has >70% GC base pairs. This new method of touch down PCR differed by having an initial annealing temperature (Ta) 1.5°C below the primers melting temperature that descended 0.2°C per cycle for 20 cycles and continued thereafter at fixed Ta for next 15 cycles. Different co-solvents were tested with this method to improve the result and betaine proved better than the other co-solvents. This new method is economical, fast and specific in amplifying GC rich region of other genes also.     

Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation

The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H(2)O(2) cytotoxicity. Treatment with 30 microm H(2)O(2) for 48 h or 40 microm PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H(2)O(2) treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H(2)O(2) suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.     

Analyzing the performance of ArcCHECK diode array detector for VMAT plan

Aim

The aim of this study is to evaluate performance of ArcCHECK diode array detector for the volumetric modulated arc therapy (VMAT) patient specific quality assurance (QA). VMAT patient specific QA results were correlated with ion chamber measurement. Dose response of the ArcCHECK detector was studied.

Background

VMAT delivery technique improves the dose distribution. It is complex in nature and requires proper QA before its clinical implementation. ArcCHECK is a novel three dimensional dosimetry system.

Materials and methods

Twelve retrospective VMAT plans were calculated on ArcCHECK phantom. Point dose and dose map were measured simultaneously with ion chamber (IC-15) and ArcCHECK diode array detector, respectively. These measurements were compared with their respective TPS calculated values.

Results

The ion chamber measurements are in good agreement with TPS calculated doses. Mean difference between them is 0.50% with standard deviation of 0.51%. Concordance correlation coefficient (CCC) obtained for ion chamber measurements is 0.9996. These results demonstrate a strong correlation between the absolute dose predicted by our TPS and the measured dose. The CCC between ArcCHECK doses and TPS predictions on the CAX was found to be 0.9978. In gamma analysis of dose map, the mean passing rate was 98.53% for 3% dose difference and 3 mm distance to agreement.

Conclusions

The VMAT patient specific QA with an ion chamber and ArcCHECK phantom are consistent with the TPS calculated dose. Statistically good agreement was observed between ArcCHECK measured and TPS calculated. Hence, it can be used for routine VMAT QA.     

Effects of Photophase and Altitude on Oviposition Rhythm of the Himalayan Strains of Drosophila Ananassae

The effects of varying photophase and altitude of origin on the phase angle difference (Ψ) of the circadian rhythm of oviposition during entrainment to light‐dark (LD) cycles and the aftereffects of such photophases on the period of the free‐running rhythm (τ) in constant darkness (DD) were evaluated in two Himalayan strains of Drosophila ananassae, the high‐altitude (HA) strain from Badrinath (5,123 m above sea level=ASL) and the low‐altitude (LA) strain from Firozpur (179 m ASL). The Ψ (i.e., the hours from lights‐on of the LD cycle to oviposition median) of both strains was determined in LD cycles in which the photophase at 100 lux varied from 6 to 18 h/24 h. The HA strain was entrained by all LD cycles except the one with 6 h photophase in which it was weakly rhythmic, but the LA strain was entrained by only three LD cycles with photophases of 10, 12, and 14 h, but photophases of 6, 8, 16, and 18 h rendered it arrhythmic. Lights‐off transition of LD cycles was the phase‐determining signal for both strains as oviposition medians of the HA strain occurred～6 h prior to lights‐off, while those of the LA strain occurred～1 h after lights‐off. The Ψ of the HA strain increased from～2 h in 8 h photophase to～11 h in 18 h photophase, while that of the LA strain increased from～11 h in 10 h photophase to～15 h in 14 h photophase. The aftereffects of photophase of the prior entraining LD cycles on τ in DD were determined by transferring flies from LD cycles to DD. The τ of the HA strain increased from～19 to～25 h when transferred to DD from LD 8:16 and LD 18:6 cycles, respectively, whereas the τ of the LA strain increased from～26 to～28 h when transferred to DD from LD 10:14 and LD 14:10 cycles, respectively. Thus, these results demonstrate that the photophases of entraining LD cycles and the altitude of origin affected several parameters of entrainment and the period of the free‐running rhythm of these strains.     

An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

DESICCATION AND STARVATION TOLERANCE OF ADULT DROSOPHILA: OPPOSITE LATITUDINAL CLINES IN NATURAL POPULATIONS OF THREE DIFFERENT SPECIES

Desiccation and starvation tolerance were measured along latitudinal transects in three Drosophilid species (Drosophila ananassae, D. melanogaster, and Zaprionus indianus) of the Indian subcontinent. In each case, significant latitudinal clines were observed; desiccation tolerance increased with latitude while starvation tolerance decreased. Such field observations suggest that desiccation and starvation tolerance are fitness related traits that are independently selected in nature and genetically independent. It was, however, difficult to relate these genetic changes with precise climatic variables, except winter temperature. The overall negative correlation between the two traits, which was evidenced in natural populations, contrasts with a positive correlation generally observed in various laboratory selection experiments and that also seems to exist between different species. These observations point to the difficulty of interpreting correlations among fitness-related traits when different evolutionary levels are compared, and also different sets of data, that is, field versus laboratory studies.