Upf proteins: highly conserved factors involved in nonsense mRNA mediated decay

Over 10% of genetic diseases are caused by mutations that introduce a premature termination codon in protein-coding mRNA. Nonsense-mediated mRNA decay (NMD) is an essential cellular pathway that degrades these mRNAs to prevent the accumulation of harmful partial protein products. NMD machinery is also increasingly appreciated to play a role in other essential cellular functions, including telomere homeostasis and the regulation of normal mRNA turnover, and is misregulated in numerous cancers. Hence, understanding and designing therapeutics targeting NMD is an important goal in biomedical science. The central regulator of NMD, the Upf1 protein, interacts with translation termination factors and contextual factors to initiate NMD specifically on mRNAs containing PTCs. The molecular details of how these contextual factors affect Upf1 function remain poorly understood. Here, we review plausible models for the NMD pathway and the evidence for the variety of roles NMD machinery may play in different cellular processes.     

A phylogenomic tree inferred with an inexpensive PCR-generated probe kit resolves higher-level relationships among Neptis butterflies (Nymphalidae: Limenitidinae)

Recent advances in obtaining reduced representation libraries for next-generation sequencing permit phylogenomic analysis of species-rich, recently diverged taxa. In this study, we performed sequence capture with homemade PCR-generated probes to study diversification among closely related species in a large insect genus to examine the utility of this method. We reconstructed the phylogeny of Neptis Fabricius, a large and poorly studied nymphalid butterfly genus distributed throughout the Old World. We inferred relationships among 108 Neptis samples using 89 loci totaling up to 84 519 bp per specimen. Our taxon sample focused on Palearctic, Oriental and Australasian species, but included 8 African species and outgroups from 5 related genera. Maximum likelihood and Bayesian analyses yielded identical trees with full support for almost all nodes. We confirmed that Neptis is not monophyletic because Lasippa heliodore (Fabricius) and Phaedyma amphion (Linnaeus) are nested within the genus, and we redefine species groups for Neptis found outside of Africa. The statistical support of our results demonstrates that the probe set we employed is useful for inferring phylogenetic relationships among Neptis species and likely has great value for intrageneric phylogenetic reconstruction of Lepidoptera. Based on our results, we revise the following two taxa: Neptis heliodore comb. rev. and Neptis amphion comb. rev.     

Challenges in calculation of the gamma index in radiotherapy – Towards good practice

The gamma index (γ) is one of the most commonly used metrics for the verification of complex modulated radiotherapy. The mathematical definition of the γ is computationally expensive and various techniques have been reported to speed up the calculation either by mathematically refining the γ or employing various computational techniques. These techniques can cause variation in output with different software implementations. The γ has traditionally been used to compare a 2D measured plane against a 2D or 3D dose distribution. Recently, software algorithm and hardware improvements have led to the possibility of using measured 2D data from commercial detector arrays to reconstruct a 3D-dose distribution and perform a volumetric comparison against the treatment planning system (TPS). A limitation in this approach is that commercial detector arrays have so far been limited by their spatial resolution which may affect the accuracy of the reconstructed 3D volume and subsequently the γ calculation. Additionally, 3D versus 3D γ comparison adds a layer of complication in the calculation of the γ given the increase in the number of calculation points and the result cannot be as easily interpreted in the same way as 2D comparison. This review summarises and highlights the computational challenges of the γ calculation and sheds light on some of these issues by means of a bespoke MATLAB software to demonstrate the impact of interpolation, γ search distance, resolution and 2D and 3D calculations. Finally, a recommendation is made on the minimum information that should be reported when publishing γ results.     

Integrating Biosystem Models Using Waveform Relaxation

Modelling in systems biology often involves the integration of component models into larger composite models. How to do this systematically and efficiently is a significant challenge: coupling of components can be unidirectional or bidirectional, and of variable strengths. We adapt the waveform relaxation (WR) method for parallel computation of ODEs as a general methodology for computing systems of linked submodels. Four test cases are presented: (i) a cascade of unidirectionally and bidirectionally coupled harmonic oscillators, (ii) deterministic and stochastic simulations of calcium oscillations, (iii) single cell calcium oscillations showing complex behaviour such as periodic and chaotic bursting, and (iv) a multicellular calcium model for a cell plate of hepatocytes. We conclude that WR provides a flexible means to deal with multitime-scale computation and model heterogeneity. Global solutions over time can be captured independently of the solution techniques for the individual components, which may be distributed in different computing environments.     

Synthesis,molecular docking and anti-inflammatory screening of novel quinoline incorporated pyrazole derivatives using the Pfitzinger reaction II

In continuation of our study of novel quinolines with anti-inflammatory activity using the Pfitzinger reaction, several new quinoline derivatives were synthesized and tested for their anti-inflammatory and ulcerogenic effect. A docking study on the COX-2 binding pocket was carried out for the target compounds to rationalize the possible selectivity of them against COX-2 enzyme. The most active compounds (5a, 8a and 11a) were found to be superior to celecoxib. Compound 11a demonstrated the highest anti-inflammatory activity as well as the best binding profiles into the COX-2 binding site. Moreover, compounds 9c, 9e, 10a and 11a were devoid of ulcerogenic activity.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.     

Structure of the Human TELO2-TTI1-TTI2 Complex

Phosphatidylinositol 3-kinase-related protein kinases (PIKKs) play critical roles in various metabolic pathways related to cell proliferation and survival. The TELO2-TTI1-TTI2 (TTT) complex has been proposed to recognize newly synthesized PIKKs and to deliver them to the R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) and the heat shock protein 90 chaperone, thereby supporting their folding and assembly. Here, we determined the cryo-EM structure of the TTT complex at an average resolution of 4.2 Å. We describe the full-length structures of TTI1 and TELO2, and a partial structure of TTI2. All three proteins form elongated helical repeat structures. TTI1 provides a platform on which TELO2 and TTI2 bind to its central region and C-terminal end, respectively. The TELO2 C-terminal domain (CTD) is required for the interaction with TTI1 and recruitment of Ataxia-telangiectasia mutated (ATM). The N- and C-terminal segments of TTI1 recognize the FRAP-ATM-TRRAP (FAT) domain and the N-terminal HEAT repeats of ATM, respectively. The TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival in response to ionizing radiation.