Search Results (10)

Binary translation, as an important bridge for application compatibility between different instruction set architectures (ISAs), has attracted much attention in the industry. However, due to hardware resource limitations of the target ISA, the translation efficiency and the practicability are poor. Recently, Apple has made it possible to run x86 programs on ARM through a translation technology called Rosetta based on software-hardware collaboration. In this paper, we proposed a hardware non-invasive mapping method for condition bits (HNIMCB) in binary translation, which innovatively implements the setting and referencing operations of the condition bits without changing the original instruction encoding and function of the target processor. This method is applicable for binary translation from source architectures with condition bit operations to target architectures without condition bit operations. It eliminates the difference of conditional bit resources between the source and target ISAs, reduces the computational instructions and memory access operations after translation from the source to the target ISA, and dramatically improves the translation efficiency. We conducted this experiment on a functional simulation level using the QEMU binary translator from ARM to RISC-V. A series of benchmark tests revealed that the total number of instructions decreased by 41%, while the number of memory access instructions decreased by 37% after the translation applying with the HNIMCB. Full article

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture. Computational modeling techniques can complement sparse NMR datasets (<1 restraint per residue) with additional structural information to elucidate protein structures in these difficult cases. The Rosetta software for protein structure modeling and design is used by structural biologists for structure determination tasks in which limited experimental data is available. This review gives an overview of the computational protocols available in the Rosetta framework for modeling protein structures from NMR data. We explain the computational algorithms used for the integration of different NMR data types in Rosetta. We also highlight new developments, including modeling tools for data from paramagnetic NMR and hydrogen–deuterium exchange, as well as chemical shifts in CS-Rosetta. Furthermore, strategies are discussed to complement and improve structure predictions made by the current state-of-the-art AlphaFold2 program using NMR-guided Rosetta modeling. Full article

Starch content is one of the major quality criteria targeted by potato breeding programs. Traditional potato breeding is a laborious duty due to the tetraploid nature and immense heterozygosity of potato genomes. In addition, screening for functional genetic variations in wild relatives is slow and strenuous. Moreover, genetic diversity, which is the raw material for breeding programs, is limited due to vegetative propagation used in the potato industry. Somaclonal variation provides a time-efficient tool to breeders for obtaining genetic variability, which is essential for breeding programs, at a reasonable cost and independent of sophisticated technology. The present investigation aimed to create potato somaclones with an improved potential for starch accumulation. Based on the weight and starch content of tubers, the somaclonal variant Ros 119, among 105 callus-sourced clones, recorded a higher tuberization potential than the parent cv Lady Rosetta in a field experiment. Although this somaclone was similar to the parent in the number of tubers produced, it exhibited tubers with 42 and 61% higher fresh and dry weights, respectively. Additionally, this clone recorded 10 and 75% increases in starch content based on the dry weight and average content per plant, respectively. The enhanced starch accumulation was associated with the upregulation of six starch-synthesis-related genes, namely, the AGPase, GBSS I, SBE I, SBE II, SS II and SS III genes. AGPase affords the glycosyl moieties required for the synthesis of amylose and amylopectin. GBSS is required for amylose elongation, while SBE I, SBE II, SS II and SS III are responsible for amylopectin. Full article

Membrane proteins are broadly classified as transmembrane (TM) or peripheral, with functions that pertain to only a single bilayer at a given time. Here, we explicate a class of proteins that contain both transmembrane and peripheral domains, which we dub transmembrane membrane readers (TMMRs). Their transmembrane and peripheral elements anchor them to one bilayer and reversibly attach them to another section of bilayer, respectively, positioning them to tether and fuse membranes while recognizing signals such as phosphoinositides (PIs) and modifying lipid chemistries in proximity to their transmembrane domains. Here, we analyze full-length models from AlphaFold2 and Rosetta, as well as structures from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, using the Membrane Optimal Docking Area (MODA) program to map their membrane-binding surfaces. Eukaryotic TMMRs include phospholipid-binding C1, C2, CRAL-TRIO, FYVE, GRAM, GTPase, MATH, PDZ, PH, PX, SMP, StART and WD domains within proteins including protrudin, sorting nexins and synaptotagmins. The spike proteins of SARS-CoV-2 as well as other viruses are also TMMRs, seeing as they are anchored into the viral membrane while mediating fusion with host cell membranes. As such, TMMRs have key roles in cell biology and membrane trafficking, and include drug targets for diseases such as COVID-19. Full article

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications. Full article

A key parameter for the design of soil drainage and irrigation facilities and for the modelling of surface runoff and erosion phenomena in land-formed areas is the saturated hydraulic conductivity (Ks). There are many methods for determining its value. In situ and laboratory measurements are commonly regarded as the most accurate and direct methods; however, they are costly and time-consuming. Alternatives can be found in the increasingly popular models of pedotransfer functions (PTFs), which can be used for rapid determination of soil hydrophysical parameters. This study presents an analysis of the Ks values obtained from in situ measurements conducted using a double-ring infiltrometer (DRI). The measurements were conducted using a laboratory permeability meter (LPM) and were estimated using five PTFs in the Rosetta program, based on easily accessible input data, i.e., the soil type, content of various grain sizes in %, density, and water content at 2.5 and 4.2 pF, respectively. The degrees of matching between the results from the PTF models and the values obtained from the in situ and laboratory measurements were investigated based on the root-mean-square deviation (RMSD), Nash–Sutcliffe efficiency (NSE), and determination coefficient (R2). The statistical relationships between the tested variables tested were confirmed using Spearman’s rank correlation coefficient (rho). Data analysis showed that in situ measurements of Ks were only significantly correlated with the laboratory tests conducted on intact samples; the values obtained in situ were much higher. The high sensitivity of Ks to biotic and abiotic factors, especially in the upper soil horizons, did not allow for a satisfactory match between the values from the in situ measurements and those obtained from the PTFs. In contrast, the laboratory measurements, showed a significant correlation with the Ks values, as estimated by the models PTF-2 to PTF-5; the best match was found for PTF-2. Full article

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter globiformis (AgUox) has been reported to be thermostable and less immunogenic than the Aspergillus-derived urate oxidase. Conjugation of human serum albumin (HSA) to therapeutic proteins has become a promising strategy to prolong circulation time in vivo. To develop a thermostable and long-circulating urate oxidase, we investigated the site-specific conjugation of HSA to AgUox based on site-specific incorporation of a clickable non-natural amino acid (frTet) and an inverse electron demand Diels–Alder reaction. We selected 14 sites for frTet incorporation using the ROSETTA design, a computational stability prediction program, among which AgUox containing frTet at position 196 (Ag12) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, Ag12 exhibited a high HSA conjugation yield without compromising the enzymatic activity, generating well-defined HSA-conjugated AgUox (Ag12-HSA). In mice, the serum half-life of Ag12-HSA was approximately 29 h, which was roughly 17-fold longer than that of wild-type AgUox. Altogether, this novel formulated AgUox may hold enhanced therapeutic efficacy for several diseases. Full article

Prolactin-releasing Peptide (PrRP) is a neuropeptide whose receptor is GPR10. Recently, the regulatory role of PrRP in the neuroendocrine field has attracted increasing attention. However, the influence of PrRP on macrophages, the critical housekeeper in the neuroendocrine field, has not yet been fully elucidated. Here, we investigated the effect of PrRP on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) with RNA sequencing, bioinformatics, and molecular simulation. BMDMs were exposed to PrRP (18 h) and were subjected to RNA sequencing. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. Eight qPCR-validated DEGs were chosen as hub genes. Next, the three-dimensional structures of the proteins encoded by these hub genes were modeled by Rosetta and Modeller, followed by molecular dynamics simulation by the Gromacs program. Finally, the binding modes between PrRP and hub proteins were investigated with the Rosetta program. PrRP showed no noticeable effect on the morphology of macrophages. A total of 410 DEGs were acquired, and PrRP regulated multiple BMDM-mediated functional pathways. Besides, the possible docking modes between PrRP and hub proteins were investigated. Moreover, GPR10 was expressed on the cell membrane of BMDMs, which increased after PrRP exposure. Collectively, PrRP significantly changed the transcriptome profile of BMDMs, implying that PrRP may be involved in various physiological activities mastered by macrophages. Full article

Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency. Full article

Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in “Brugia Rapid”. However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics. Full article