Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.     

Hydrophobic Residues Confer the Helicity and Membrane Permeability of Ocellatin-1 Antimicrobial Peptide Scaffold Towards Therapeutics

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) are persisting in all living organisms as evolutionarily conserved inherent components of the innate...     

Structural insight into the evolution of a new chemokine family from zebrafish

The mammalian chemokine family is segregated into four families – CC, CXC, CX3C, and XC—based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N‐terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur‐SAD phasing to determine the three‐dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin‐binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family. Proteins 2014; 82:708–716. © 2013 Wiley Periodicals, Inc.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Computational Identification of Significant Missense Mutations in AKT1 Gene

The AKT1 gene is of supreme importance in cell signaling and human cancer. In the present study, we aim to understand the phenotype variations that were believed to have the highest impact in AKT1 gene by different computational approaches. The analysis was initiated with SIFT tool followed by PolyPhen 2.0, I-Mutant 2.0, and SNPs&GO tools with the aid of 22 nonsynonymous (nsSNPs) retrieved from dbSNP. A total of five AKT1 variants such as E17K, E17S, E319G, L357P, and P388T are found to exert deleterious effects on the protein structure and function. Furthermore, the molecular docking study indicates the lesser binding affinity of inhibitor with the mutant structure than the native type. In addition, root mean square deviation and hydrogen bond details were also analyzed in the 10 ns molecular dynamics simulation study. These computational evidences suggested that E17K, E17S, E319G, L357P, and P388T variants of AKT1 could destabilize the protein networks, thus causing functional deviations of protein to some extent. Moreover, the findings strongly indicate that screening for AKT1, E17K, E17S, E319G, L357P, and P388T variants may be useful for disease molecular diagnosis and also to design the potential AKT inhibitors.     

Changes in Soil Microbial Biomass and Residual Indices as Ecological Indicators of Land Use Change in Temperate Permanent Grassland

The relationship between microbial biomass, residues and their contribution to microbial turnover is important to understand ecosystem C storage. The effects of permanent grassland (100 % ryegrass—PG), conversion to modified grassland (mixture of grass and clover—MG) or maize monoculture (MM) on the dynamics of soil organic C (SOC), microbial biomass, fungal ergosterol and microbial residues (bacterial muramic acid and fungal glucosamine) were investigated. Cattle slurry was applied to quantify the effects of fertilisation on microbial residues and functional diversity of microbial community across land use types. Slurry application significantly increased the stocks of microbial biomass C and S and especially led to a shift in microbial residues towards bacterial tissue. The MM treatment decreased the stocks of SOC, microbial biomass C, N and S and microbial residues compared with the PG and MG treatments at 0–40 cm depth. The MM treatment led to a greater accumulation of saprotrophic fungi, as indicated by the higher ergosterol-to-microbial biomass C ratio and lower microbial biomass C/S ratio compared with the grassland treatments. The absence of a white clover population in the PG treatment caused a greater accumulation of fungal residues (presumably arbuscular mycorrhizal fungi (AMF), which do not contain ergosterol but glucosamine), as indicated by the significantly higher fungal C-to-bacterial C ratio and lower ergosterol-to-microbial biomass C ratio compared with the MG treatment. In addition to these microbial biomass and residual indices, the community level physiological profiles (CLPP) demonstrated distinct differences between the PG and MG treatments, suggesting the potential of these measurements to act as an integrative indicator of soil functioning.     

Finding Hamiltonian paths in tournaments on clusters

This paper presents a general methodology for the communication-efficient parallelization of graph algorithms using the divide-and-conquer approach and shows that this class of problems can be solved in cluster environments with good communication efficiency. Specifically, the first practical parallel algorithm, based on a general coarse-grained model, for finding Hamiltonian paths in tournaments is presented. On any such parallel machines, this algorithm uses only (3log p+1), where p is the number of processors, communication rounds, which is independent of the tournament size, and can reuse the existing linear-time algorithm in the sequential setting. For theoretical completeness, the algorithm is revised for fine-grained models, where the ratio of computation and communication throughputs is low or the local memory size, , of each individual processor is extremely limited for any , solving the problem with O(log p) communication rounds, while the hidden constant grows with the scalability factor 1/∊. Experiments have been carried out on a Linux cluster of 32 Sun Ultra5 computers and an SGI Origin 2000 with 32 R10000 processors. The algorithm performance on the Linux Cluster reaches 75% of the performance on the SGI Origin 2000 when the tournament size is about one million. Computational resources and technical support are provided by the Center for Computational Research (CCR) at the State University of New York at Buffalo. Chun-Hsi Huang received his Ph.D. degree in Computer Science from the State University of New York at Buffalo in 2001. His is currently an Assistant Professor of Computer Science and Engineering at the University of Connecticut. His interests include High Performance Parallel Computing, Cluster and Grid Computing, Biomedical and Health Informatics, Algorithm Design and Analysis, Experimental Algorithms and Computational Biology. Sanguthevar Rajasekaran received his Ph.D. degree in Computer Science from Harvard University in 1988. Currently he is the UTC Chair Professor of Computer Science and Engineering at the University of Connecticut and the Director of Booth Engineering Center for Advanced Technologies (BECAT). His research interests include Parallel Algorithms, Bioinformatics, Data Mining, Randomized Computing, Computer Simulations, and Combinatorial Optimization. Laurence Tianruo Yang received is Ph.D. degree in Computer Science from the Oxford University. He is currently a professor of Computer Science of the St. Francis Xavier University in Canada. His research interests include high-performance computing, embedded systems, computer archtecture and high-speed networking. Xin He received his Ph.D. degree in Computer Science from the Ohio State University in 1987. He is currently Professor of Computer Science and Engineering at the State University of New York at Buffalo. His research interests include Algorithms, Data Structures, Combinatorics and Computational Geometry.     

Esterase activity of Candida species isolated from immunocompromised hosts

A total of 149 clinical isolates of Candida species isolated from immunocompromised patients were examined to ascertain their esterase activity by the Tween 80 opacity test, which is a biochemical test used mainly to differentiate between Candida albicans and Candida dubliniensis. Our results showed that C. albicans (92.3%), Candida tropicalis (92.3%), Candida parapsilosis (25%), C. dubliniensis (16.6%), Candida inconspicua (100%), and Candida lipolytica (100%) produced opacity halos through the 10-day post-inoculation period. The remaining Candida species did not produce a positive test response. These findings indicate that Tween 80 opacity test cannot be used as the sole phenotypic trait in the differentiation of C. albicans and C. dubliniensis.