We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

It is quickly becoming apparent that situating human variation in a pathway context is crucial to understanding its phenotypic significance. Toward this end, we have developed a general method for finding pathways associated with traits that control for pathway size. We have applied this method to a new whole genome survey of coding SNP variation in 187 patients afflicted with Parkinson disease (PD) and 187 controls. We show that our dataset provides an independent replication of the axon guidance association recently reported by Lesnick et al. [PLoS Genet 2007;3:e98], and also indicates that variation in the ubiquitin-mediated proteolysis and T-cell receptor signaling pathways may predict PD susceptibility. Given this result, it is reasonable to hypothesize that pathway associations are more replicable than individual SNP associations in whole genome association studies. However, this hypothesis is complicated by a detailed comparison of our dataset to the second recent PD association study by Fung et al. [Lancet Neurol 2006;5:911-916]. Surprisingly, we find that the axon guidance pathway does not rank at the very top of the Fung dataset after controlling for pathway size. More generally, in comparing the studies, we find that SNP frequencies replicate well despite technologically different assays, but that both SNP and pathway associations are globally uncorrelated across studies. We thus have a situation in which an association between axon guidance pathway variation and PD has been found in 2 out of 3 studies. We conclude by relating this seeming inconsistency to the molecular heterogeneity of PD, and suggest future analyses that may resolve such discrepancies.

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.

Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, and the most common in European populations. Results of genetic analysis and mutation screening of SOD1 in a cohort of 60 Iranian ALS patients are here reported. Initially, linkage analysis in 4 families identified a disease-linked locus that included the known ALS gene, SOD1. Screening of SOD1 identified homozygous p.Asp90Ala causing mutations in all the linked families. Haplotype analysis suggests that the p.Asp90Ala alleles in the Iranian patients might share a common founder with the renowned Scandinavian recessive p.Asp90Ala allele. Subsequent screening in all the patients resulted in identification of 3 other mutations in SOD1, including p.Leu84Phe in the homozygous state. Phenotypic features of the mutation-bearing patients are presented. SOD1 mutations were found in 11.7% of the cohort, 38.5% of the familial ALS probands, and 4.25% of the sporadic ALS cases. SOD1 mutations contribute significantly to ALS among Iranians.

We aimed to identify the genetic cause of coronary artery disease (CAD) in an Iranian pedigree. Genetic linkage analysis identified three loci with an LOD score of 2.2. Twelve sequence variations identified by exome sequencing were tested for segregation with disease. A p.Val99Met causing mutation in ST6GALNAC5 was considered the likely cause of CAD. ST6GALNAC5 encodes sialyltransferase 7e. The variation affects a highly conserved amino acid, was absent in 800 controls, and was predicted to damage protein function. ST6GALNAC5 is positioned within loci previously linked to CAD-associated parameters. While hypercholesterolemia was a prominent feature in the family, clinical and genetic data suggest that this condition is not caused by the mutation in ST6GALNAC5. Sequencing of ST6GALNAC5 in 160 Iranian patients revealed a candidate causative stop-loss mutation in two other patients. The p.Val99Met and stop-loss mutations both caused increased sialyltransferase activity. Sequence data from combined Iranian and US controls and CAD affected individuals provided evidence consistent with potential role of ST6GALNAC5 in CAD. We conclude that ST6GALNAC5 mutations can cause CAD. There is substantial literature suggesting a relation between sialyltransferase and sialic acid levels and coronary disease. Our findings provide strong evidence for the existence of this relation.

The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrum of diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes.

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.

To investigate the role of MYOC and CYP1B1 in Iranian juvenile open angle glaucoma (JOAG) patients.

We developed a glaucoma-on-a-chip model to evaluate the viability of retinal ganglion cells (RGCs) against high pressure and the potential effect of neuroprotection.

We aimed to assess whether the transcription factor PAX6 affects transcription of FMNL2. PAX6 is a transcription factor with significant roles in development of the eye and eye-related functions. FMNL2 encodes a member of the formin family of proteins and has roles in polymerization of actin and features of the cytoskeleton. The state of the cytoskeleton affects the flow of aqueous humor, disruption of which is a cornerstone of glaucoma pathology.

Brain-derived neurotrophic factor (BDNF) is a well-known neuroprotectant and a potent therapeutic candidate for neurodegenerative diseases. However, there are several clinical concerns about its therapeutic applications. In the current study, we designed and developed BDNF-mimicking small peptides as an alternative to circumvent these problems. A phage-displayed peptide library was screened using BDNF receptor (neurotrophic tyrosine kinase receptor type2 [NTRK2]) and evaluated by ELISA. The peptide sequences showed similarity to loop2 of BDNF, they were recognized as discontinuous epitopes though. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and the surface residues of the NTRK2 protein at the IgC2 domain. A consensus peptide sequence was then synthesized to generate a mimetic construct (named as RNYK). The affinity binding and function of this construct was confirmed by testing against the native structure of NTRK2 in SH-SY5Y cells in vitro using flow-cytometry and MTT assays, respectively. RNYK at 5 ng/mL prevented neuronal degeneration of all- trans-retinoic acid-treated SH-SY5Y with equal efficacy to or even better than BDNF at 50 ng/mL.

To compare trabeculectomy with mitomycin-C (MMC) application before versus after scleral flap dissection in terms of corneal endothelial cell loss and surgical outcomes.

To assess the association of LTBP2 mutations with primary angle closure glaucoma (PACG).

Background: To determine the predictive value of postoperative bleb morphological features and intraocular pressure (IOP) on the success rate of trabeculectomy. Methods: In this prospective interventional case series, we analyzed for one year 80 consecutive primary open angle glaucoma patients who underwent mitomycin-augmented trabeculectomy. Bleb morphology was scored using the Indiana bleb appearance grading scale (IBAGS). Success was defined as IOP ≤15 mmHg at 12 months. We applied a multivariable regression analysis and determined the area under the receiver operating characteristic curve (AUC). Results: The mean age of participants was 62±12.3 years in the success and 63.2±16.3 years in the failure group (P= 0.430) with equal gender distribution (P=0.911). IOPs on day 1, 7 and 30 were similar in both (P= 0.193, 0.639, and 0.238, respectively.) The AUC of IOP at day 1, day 7 and 30 for predicting a successful outcome was 0.355, 0.452, and 0.80, respectively. The AUC for bleb morphology parameters of bleb height, extension, and vascularization, on day 14 were 0.368, 0.408, and 0.549, respectively. Values for day 30 were 0.428, 0.563, and 0.654. IOP change from day 1 to day 30 was a good predictor of failure (AUC=0.838, 95% CI: 0.704 to 0.971) with a change of more than 3 mmHg predicting failure with a sensitivity of 82.5% (95% CI: 68 to 91%) and a specificity of 87.5% (95% CI: 53 to 98%). Conclusions: IOP on day 30 had a fair to good accuracy while bleb features failed to predict success except bleb vascularity that had a poor to fair accuracy.  An IOP increase more than 3 mmHg during the first 30 days was a good predictor of failure.

To assess for the first time the possible contribution of latent transforming growth factor (TGF)-beta binding protein 2 (LTBP2), an extracellular matrix (ECM) protein that associates with fibrillin-1-containing microfibrils, to the etiology of primary open angle glaucoma (POAG) and pseudoexfoliation (PEX) syndrome. Mutations in LTBP2 have previously been shown to be the cause of primary congenital glaucoma (PCG) and other disorders that often manifest as secondary glaucoma.

We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.

Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.

This review article aimed to evaluate ocular biometric changes after trabeculectomy. The PubMed database was searched using the keywords "axial length" (AL), "anterior chamber depth" (ACD), "corneal astigmatism," "corneal topography" and "trabeculectomy." The extracted studies were categorized based on the evaluated parameters and the biometry method (contact and non-contact). Comparable studies with respect to their sample size were combined for statistical analysis. Twenty-five studies including 690 individuals which met the inclusion criteria were selected. After trabeculectomy, a significant and persistent AL reduction, with a range of 0.1-0.19 and 0.1-0.9 mm measured with contact and non-contact methods, respectively, was observed. With respect to topographic changes, 0.38-1.4 diopters (D) with-the-rule (WTR) astigmatism was induced postoperatively. All studies revealed ACD reduction immediately after surgery, which gradually deepened and approximated its preoperative levels on day 14. ACD reduction was not significant after that period in the majority of cases. In conclusion, changes in ACD is of small amount and of short period, thus it can be ignored; however, reported changes in AL and keratometry are of sufficient magnitude and can affect the refractive prediction of combined cataract surgery and trabeculectomy.

To evaluate the effect of orbital steroid injections in patients with active thyroid ophthalmopathy resistant to or dependent on systemic steroids, or with complications related to systemic steroid use.