Consistent refinement of submitted models at CASP using a knowledge‐based potential

Protein structure refinement is an important but unsolved problem; it must be solved if we are to predict biological function that is very sensitive to structural details. Specifically, critical assessment of techniques for protein structure prediction (CASP) shows that the accuracy of predictions in the comparative modeling category is often worse than that of the template on which the homology model is based. Here we describe a refinement protocol that is able to consistently refine submitted predictions for all categories at CASP7. The protocol uses direct energy minimization of the knowledge‐based potential of mean force that is based on the interaction statistics of 167 atom types (Summa and Levitt, Proc Natl Acad Sci USA 2007; 104:3177–3182). Our protocol is thus computationally very efficient; it only takes a few minutes of CPU time to run typical protein models (300 residues). We observe an average structural improvement of 1% in GDT_TS, for predictions that have low and medium homology to known PDB structures (Global Distance Test score or GDT_TS between 50 and 80%). We also observe a marked improvement in the stereochemistry of the models. The level of improvement varies amongst the various participants at CASP, but we see large improvements (>10% increase in GDT_TS) even for models predicted by the best performing groups at CASP7. In addition, our protocol consistently improved the best predicted models in the refinement category at CASP7 and CASP8. These improvements in structure and stereochemistry prove the usefulness of our computationally inexpensive, powerful and automatic refinement protocol. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Label-less fluorescence-based method to detect hybridization with applications to DNA micro-array

By coupling scattered light from DNA to excite fluorescence in a polymer, we describe a quantitative, label-free assay for DNA hybridization detection. Since light scattering is intrinsically proportional to number of molecules, the change in (scattering coupled) fluorescence is highly linear with respect to percent binding of single stranded DNA (ssDNA) target with the immobilized ssDNA probes. The coupling is achieved by immobilizing ssDNA on a fluorescent polymer film at optimum thickness in nanoscale. The fluorescence from the underlining polymer increases due to proportionate increase in scattering from double stranded DNA (dsDNA) (i.e., probe-target binding) compared to ssDNA (i.e., probe). Because the scattering is proportional to fourth power of refractive index, the detection of binding is an order of magnitude more sensitive compared to other label-free optical methods, such as, reflectivity, interference, ellipsometry and surface-plasmon resonance. Remarkably, polystyrene film of optimum thickness 30 nm is the best fluorescent agent since its excitation wavelength matches (within 5 nm) with wavelength for the maximum refractive index difference between ssDNA and dsDNA. A quantitative model (with no fitting parameters) explains the observations. Potential dynamic range is 1 in 10(4) at signal-to-noise ratio of 3:1.     

CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.     

A New Player in the Spermiogenesis Pathway of Caenorhabditis elegans

Precise timing of sperm activation ensures the greatest likelihood of fertilization. Precision in Caenorhabditis elegans sperm activation is ensured by external signaling, which induces the spherical spermatid to reorganize and extend a pseudopod for motility. Spermatid activation, also called spermiogenesis, is prevented from occurring prematurely by the activity of SPE-6 and perhaps other proteins, termed “the brake model.” Here, we identify the spe-47 gene from the hc198 mutation that causes premature spermiogenesis. The mutation was isolated in a suppressor screen of spe-27(it132ts), which normally renders worms sterile, due to defective transduction of the activation signal. In a spe-27(+) background, spe-47(hc198) causes a temperature-sensitive reduction of fertility, and in addition to premature spermiogenesis, many mutant sperm fail to activate altogether. The hc198 mutation is semidominant, inducing a more severe loss of fertility than do null alleles generated by CRISPR-associated protein 9 (Cas9) technology. The hc198 mutation affects an major sperm protein (MSP) domain, altering a conserved amino acid residue in a β-strand that mediates MSP–MSP dimerization. Both N- and C-terminal SPE-47 reporters associate with the forming fibrous body (FB)-membranous organelle, a specialized sperm organelle that packages MSP and other components during spermatogenesis. Once the FB is fully formed, the SPE-47 reporters dissociate and disappear. SPE-47 reporter localization is not altered by either the hc198 mutation or a C-terminal truncation deleting the MSP domain. The disappearance of SPE-47 reporters prior to the formation of spermatids requires a reevaluation of the brake model for prevention of premature spermatid activation.     

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.     

IFITM3 inhibits influenza A virus infection by preventing cytosolic entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.