Faculty Bibliography

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 microg/ml (EC50 0.5-1 mug/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/beta-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.-Attur, M., Yang, Q., Shimada, K., Tachida, Y., Nagase, H., Mignatti, P., Statman, L., Palmer, G., Thorsten, K., Beier, F., Abramson, A. B. Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

Background: Data on TDF use during pregnancy for preventing mother-to-child transmission (MTCT) of hepatitis B virus (HBV) are scarce. Methods: Hepatitis B E antigen (HBeAg)-positive mothers with HBV DNA levels >200,000 IU/mL were randomized 1:1 to receive either TDF from gestation week 30-32 to postpartum week 4 or no treatment, and were followed-up until postpartum week 28. All infants received immunoprophylaxis. The primary measurement was the MTCT rate, while endpoints included TDF safety, maternal HBV DNA reduction at delivery, and HBeAg or hepatitis B s antigen loss/seroconversion at postpartum week 28. Results: Among the 200 mothers enrolled in 5 regions of the country, 180 completed the study. At postpartum week 28, the MTCT rate was significantly lower in infants from TDF-treated mothers when compared to those from non-treated mothers, both on per-protocol analysis (0% vs. 6.82%, P = 0.013) and intention-to-treat analysis (5.16% vs. 18.0%, P = 0.007). The safety profile was similar between groups, with no difference in birth defect rates (2.11% with TDF exposure vs. 1.14% without exposure, P = 1.00). HBV DNA levels decreased to <200,000 IU/mL in 68% (66/97) of TDFtreated mothers before delivery compared to 2.0% (2/100) of non-treated mothers (P < 0.001). The HBV serologic outcome did not differ between groups. Conclusions: TDF therapy in late pregnancy for highly viremic mothers effectively reduced MTCT. The treatment was well tolerated, and no safety concerns were identified. TDF therapy should be strongly considered for mothers whose HBV DNA levels exceeded 200,000 IU/mL and started at gestation week 30-32. (Table Presented)

A comprehensive analysis of the effect of lesion in-painting on the estimation of cortical thickness using magnetic resonance imaging was performed on a large cohort of 918 relapsing-remitting multiple sclerosis patients who participated in a phase III multicenter clinical trial. An automatic lesion in-painting algorithm was developed and implemented. Cortical thickness was measured using the FreeSurfer pipeline with and without in-painting. The effect of in-painting was evaluated using FreeSurfer's paired analysis pipeline. Multivariate regression analysis was also performed with field strength and lesion load as additional factors. Overall, the estimated cortical thickness was different with in-painting than without. The effect of in-painting was observed to be region dependent, more significant in the left hemisphere compared to the right, was more prominent at 1.5 T relative to 3 T, and was greater at higher lesion volumes. Our results show that even for data acquired at 1.5 T in patients with high lesion load, the mean cortical thickness difference with and without in-painting is ∼2%. Based on these results, it appears that in-painting has only a small effect on the estimated regional and global cortical thickness. Hum Brain Mapp 36:3749-3760, 2015. © 2015 Wiley Periodicals, Inc.

Stem cells and progenitor cells are integral to tissue homeostasis and repair. They contribute to health through their ability to self-renew and commit to specialized effector cells. Recently, defects in a variety of progenitor cell populations have been described in both preclinical and human diabetes. These deficits affect multiple aspects of stem cell biology, including quiescence, renewal, and differentiation, as well as homing, cytokine production, and neovascularization, through mechanisms that are still unclear. More important, stem cell aberrations resulting from diabetes have direct implications on tissue function and seem to persist even after return to normoglycemia. Understanding how diabetes alters stem cell signaling and homeostasis is critical for understanding the complex pathophysiology of many diabetic complications. Moreover, the success of cell-based therapies will depend on a more comprehensive understanding of these deficiencies. This review has three goals: to analyze stem cell pathways dysregulated during diabetes, to highlight the effects of hyperglycemic memory on stem cells, and to define ways of using stem cell therapy to overcome diabetic complications.

During normal tissue development, the accumulation of unrepaired cellular and genomic damage can impair growth and ultimately leads to death. To preserve cellular integrity, cells employ a number of defense mechanisms including molecular checkpoints, during which development is halted while dedicated pathways attempt repair. This process is most critical in germline tissues where cellular damage directly threatens an organism's reproductive capacity and offspring viability. In the fruit fly, Drosophila melanogaster, germline development has been extensively studied for over a century and the breadth of our knowledge has flourished in the genomics age. Intriguingly, several peculiar phenomena that trigger catastrophic germline damage described decades ago, still endure only a partial understanding of the underlying molecular causes. A deeper reexamination using new molecular and genetic tools may greatly benefit our understanding of host system biology. Among these, and the focus of this concise review, are hybrid dysgenesis and an intragenomic conflict that pits the X and Y sex chromosomes against each other.

Multiple lines of evidence corroborate impaired signaling pathways as relevant to the underpinnings of schizophrenia. There has been an interest in neurotrophins, since they are crucial mediators of neurodevelopment and in synaptic connectivity in the adult brain. Neurotrophins and their receptors demonstrate aberrant expression patterns in cortical areas for schizophrenia cases in comparison to control subjects. There is little known about the contribution of neurotrophin genes in psychiatric disorders. To begin to address this issue, we conducted high-coverage targeted exome capture in a subset of neurotrophin genes in 48 comprehensively characterized cases with schizophrenia-related psychosis. We herein report rare missense polymorphisms and novel missense mutations in neurotrophin receptor signaling pathway genes. Furthermore, we observed that several genes have a higher propensity to harbor missense coding variants than others. Based on this initial analysis we suggest that rare variants and missense mutations in neurotrophin genes might represent genetic contributions involved across psychiatric disorders.

Mammalian cells are widely used for the production of therapeutic recombinant proteins, as these cells facilitate accurate folding and posttranslational modifications often essential for optimum activity. Targeted insertion of a plasmid harboring a gene of interest into the genome of mammalian cells for the expression of a desired protein is a key step in production of such biologics. Here we show that a site specific double strand break (DSB) generated both in the genome and the donor plasmid using the CRISPR-Cas9 system can be efficiently used to target approximately 5 kb plasmids into mammalian genomes via nonhomologous end joining (NHEJ). We were able to achieve efficiencies of up to 0.17% in HEK293 cells and 0.45% in CHO cells. This technique holds promise for quick and efficient insertion of a large foreign DNA sequence into a predetermined genomic site in mammalian cells

The origin of the eukaryotes is a fundamental scientific question that for over 30 years has generated a spirited debate between the competing Archaea (or three domains) tree and the eocyte tree. As eukaryotes ourselves, humans have a personal interest in our origins. Eukaryotes contain their defining organelle, the nucleus, after which they are named. They have a complex evolutionary history, over time acquiring multiple organelles, including mitochondria, chloroplasts, smooth and rough endoplasmic reticula, and other organelles all of which may hint at their origins. It is the evolutionary history of the nucleus and their other organelles that have intrigued molecular evolutionists, myself included, for the past 30 years and which continues to hold our interest as increasingly compelling evidence favours the eocyte tree. As with any orthodoxy, it takes time to embrace new concepts and techniques.

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix-fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3'UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3'UTRs, and provide structural insights into a novel protein-RNA interaction motif involving a hydrolase-related domain.