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1.  Microfabrication of High-Resolution Porous Membranes for Cell Culture 
Journal of membrane science  2013;452:460-469.
Microporous membranes are widely utilized in cell biology to study cell-cell signaling and cell migration. However, the thickness and low porosity of commercial track-etched membranes limit the quality of cell imaging and the degree of cell-cell contact that can be achieved on such devices. We employ photolithography-based microfabrication to achieve porous membranes with pore diameter as small as 0.9 μm, up to 40% porosity, and less than 5% variation in pore size. Through the use of a soap release layer, membranes as thin as 1 μm can be achieved. The thin membranes minimally disrupt contrast enhancement optics, thus allowing good quality imaging of unlabeled cells under white light, unlike commercial membranes. In addition, the polymer membrane materials display low autofluorescence even after patterning, facilitating high quality fluorescence microscopy. Finally, confocal imaging suggests that substantial cell-cell contact is possible through the pores of these thin membranes. This membrane technology can enhance existing uses of porous membranes in cell biology as well as enable new types of experiments.
PMCID: PMC3931465  PMID: 24567663
microporous; cell biology
2.  Cross-modal induction of thalamocortical potentiation leads to enhanced information processing in the auditory cortex 
Neuron  2014;81(3):664-673.
Sensory systems do not work in isolation; instead they show interactions that are specifically uncovered during sensory loss. To identify and characterize these interactions, we investigated whether visual deprivation leads to functional enhancement in primary auditory cortex (A1). We compared sound-evoked responses of A1 neurons in visually-deprived animals to those from normally-reared animals. Here we show that visual deprivation leads to improved frequency selectivity as well as increased frequency and intensity discrimination performance of A1 neurons. Furthermore, we demonstrate in vitro that in adults, visual deprivation strengthens thalamocortical (TC-) synapses in A1, but not in primary visual cortex (V1). Because deafening potentiated TC-synapses in V1 but not A1, cross-modal TC-potentiation seems to be a general property of adult cortex. Our results suggest that adults retain the capability for cross-modal changes while such capability is absent within a sensory modality. Thus multimodal training paradigms might be beneficial in sensory processing disorders.
PMCID: PMC4023256  PMID: 24507197
3.  YAP Regulates Cell Proliferation, Migration, and Steroidogenesis in Adult Granulosa Cell Tumors 
Endocrine-related cancer  2014;21(2):297-310.
The Hippo signaling pathway has been implicated as a conserved regulator of organ size in both Drosophila and mammals. Yes associated protein (YAP), the central component of the Hippo signaling cascade, functions as an oncogene in several malignancies. Ovarian granulosa cell tumors (GCT) are characterized by enlargement of ovary, excess production of estrogen, high frequency of recurrence and potential of malignancy and metastasis. Whether the Hippo pathway plays a role in the pathogenesis of GCT is unknown. This study was conducted to examine the expression of YAP in human adult GCTs and to determine the role of YAP in the proliferation and steroidogenesis of GCT cells. Compared with age-matched normal human ovaries, GCT tissues exhibited higher levels of YAP expression. YAP protein was predominantly expressed in the nucleus of tumor cells, whereas the non-tumor ovarian stromal cells expressed very low levels of YAP. YAP was also expressed in cultured primary human granulosa cells and in KGN and COV434 GCT cell lines. siRNA-mediated knockdown of YAP in KGN cells resulted in a significant reduction in cell proliferation (P<0.001). Conversely, overexpression of wild-type YAP or a constitutively active YAP mutant resulted in a significant increase in KGN cell proliferation and migration. Moreover, YAP knockdown reduced FSH-induced aromatase (CYP19A1) protein expression and estrogen production in KGN cells. These results demonstrate that YAP plays an important role in regulating GCT cell proliferation, migration and steroidogenesis. Targeting the Hippo/YAP pathway may provide a novel therapeutic approach for GCT.
PMCID: PMC4222524  PMID: 24389730
Hippo/YAP pathway; granulosa cell tumor; cell proliferation; steroidogenesis; migration
4.  Virtually No Thoracic Lesion Inaccessible: A Pictorial Case Review 
Access route considerations in percutaneous intrathoracic biopsy or ablation offers its own unique set of challenges, with special consideration toward reducing the rate of pneumothorax. This review highlights several novel and atypical methods to improve access to intrathoracic lesions through a series of representative cases. These methods include patient positioning, curved needles, hydrodissection, induced/artificial pneumothorax, and use of specialized equipment functions. No intrathoracic lesion should be considered “inaccessible” either for biopsy or treatment by percutaneous approaches without consideration of performing these adjunctive techniques.
PMCID: PMC3709986  PMID: 24436538
biopsy; ablation; lung cancer; thoracic; technique; interventional radiology
5.  Effects of Base Polymer Hydrophobicity and End Group Modification on Polymeric Gene Delivery 
Biomacromolecules  2011;12(10):3592-3600.
A new 320 member polymer library of end-modified poly(beta amino) esters was synthesized. This library was chosen such that small differences to the structures of component backbone, side-chain, and end-group monomers could be systematically and simultaneously evaluated. The in vitro transfection efficacy and cytotoxicity of DNA nanoparticles formed from this library was assessed. This library approach enabled us not only to synthesize and test a large variety of structures rapidly, but provided us with a robust dataset to analyze for the effect of small structural permutations to polymer chain structure. Small changes to the side chains, backbones, and end groups within this polymer library produced dramatic results, with transfection efficacy of CMV-Luc varying over 4-orders in a 96-well plate format. Increasing hydrophobicity of the base polymer backbone and side chain tended to increase transfection efficacy, but the most hydrophobic side chains and backbones showed the least requirement for a hydrophobic pair. Optimal PBAE formulations were superior to commercially available non-viral alternatives FuGENE® HD and Lipofectamine™ 2000, enabling ~3-fold increased luminescence (2.2×106 RLU/well vs 8.1×105 RLU/well) and 2-fold increased transfection percentage (76.7% vs 42.9%) as measured by flow cytometry with comparable or reduced toxicity.
PMCID: PMC3959121  PMID: 21888340
Non-viral; Gene delivery; Biodegradable polymer; Combinatorial library; Hydrophobicity
6.  Magnetic resonance frequency shifts during acute MS lesion formation 
Neurology  2013;81(3):211-218.
We investigated the evolution of new multiple sclerosis (MS) lesions over time using frequency shifts of the magnetic resonance (MR) signal.
Twenty patients with relapsing-remitting MS were serially scanned for 6 months at 1-month intervals. Maps of MR frequency shifts were acquired using susceptibility-weighted imaging. New lesions were identified by enhancement with gadolinium (Gd).
Forty new lesions were identified as areas of signal increase on Gd-enhanced scans. Up to 3 months before lesion appearance, the frequency in areas of future Gd enhancement was not detectably different from the frequency in normal-appearing white matter. Rapid increase in MR frequency was observed between 1 month before and 1 month after Gd enhancement. Two months postenhancement and later, the frequency stabilized and remained at a constantly increased level.
These findings suggest that an increase in MR frequency does not simply reflect blood-brain barrier disruption or edema; rather, it reflects a change of tissue architecture as a consequence of new lesion formation. The data demonstrate that the MR frequency of focal MS lesions is increased before the lesions appear on conventional MRI. Unlike many other advanced imaging techniques, the images for frequency mapping can be rapidly acquired at high spatial resolution and standardized on most clinical scanners.
PMCID: PMC3770162  PMID: 23761621
7.  AIPL1 implicated in the pathogenesis of two cases of autosomal recessive retinal degeneration 
Molecular Vision  2014;20:1-14.
To localize and identify the gene and mutations causing autosomal recessive retinal dystrophy in two consanguineous Pakistani families.
Consanguineous families from Pakistan were ascertained to be affected with autosomal recessive retinal degeneration. All affected individuals underwent thorough ophthalmologic examinations. Blood samples were collected, and genomic DNA was extracted using a salting out procedure. Genotyping was performed using microsatellite markers spaced at approximately 10 cM intervals. Two-point linkage analysis was performed with the lod score method. Direct DNA sequencing of amplified genomic DNA was performed for mutation screening of candidate genes.
Genome-wide linkage scans yielded a lod score of 3.05 at θ=0 for D17S1832 and 3.82 at θ=0 for D17S938, localizing the disease gene to a 12.22 cM (6.64 Mb) region flanked by D17S1828 and D17S1852 for family 61032 and family 61227, which contains aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), a gene previously implicated in recessive Leber congenital amaurosis and autosomal dominant cone-rod dystrophy. Sequencing of AIPL1 showed a homozygous c.773G>C (p.Arg258Pro) sequence change in all affected individuals of family 61032 and a homozygous c.465G>T (p.(H93_Q155del)) change in all affected members of family 61227.
The results strongly suggest that the c.773G>C (p.R258P) and c.465G>T (p.(H93_Q155del)) mutations in AIPL1 cause autosomal recessive retinal degeneration in these consanguineous Pakistani families.
PMCID: PMC3888496  PMID: 24426771
8.  Stromal modulation of bladder cancer-initiating cells in a subcutaneous tumor model 
The development of new cancer therapeutics would benefit from incorporating efficient tumor models that mimic human disease. We have developed a subcutaneous bladder tumor regeneration system that recapitulates primary human bladder tumor architecture by recombining benign human fetal bladder stromal cells with SW780 bladder carcinoma cells. As a first step, SW780 cells were seeded in ultra low attachment cultures in order to select for sphere-forming cells, the putative cancer stem cell (CSC) phenotype. Spheroids were combined with primary human fetal stromal cells or vehicle control and injected subcutaneously with Matrigel into NSG mice. SW780 bladder tumors that formed in the presence of stroma showed accelerated growth, muscle invasion, epithelial to mesenchymal transition (EMT), decreased differentiation, and greater activation of growth pathways compared to tumors formed in the absence of fetal stroma. Tumors grown with stroma also demonstrated a greater similarity to typical malignant bladder architecture, including the formation of papillary structures. In an effort to determine if cancer cells from primary tumors could form similar structures in vivo using this recombinatorial approach, putative CSCs, sorted based on the CD44+CD49f+ antigenic profile, were collected and recombined with fetal bladder stromal cells and Matrigel prior to subcutaneous implantation. Retrieved grafts contained tumors that exhibited the same structure as the original primary human tumor. Primary bladder tumor regeneration using human fetal bladder stroma may help elucidate the influences of stroma on tumor growth and development, as well as provide an efficient and accessible system for therapeutic testing.
PMCID: PMC3512189  PMID: 23226620
Bladder cancer; cancer stem cell (CSC); subcutaneous tumor model; stroma; sphere
9.  Improving the clinical correlation of multiple sclerosis black hole volume change by paired-scan analysis☆ 
NeuroImage : Clinical  2012;1(1):29-36.
The change in T1-hypointense lesion (“black hole”) volume is an important marker of pathological progression in multiple sclerosis (MS). Black hole boundaries often have low contrast and are difficult to determine accurately and most (semi‐)automated segmentation methods first compute the T2-hyperintense lesions, which are a superset of the black holes and are typically more distinct, to form a search space for the T1w lesions. Two main potential sources of measurement noise in longitudinal black hole volume computation are partial volume and variability in the T2w lesion segmentation. A paired analysis approach is proposed herein that uses registration to equalize partial volume and lesion mask processing to combine T2w lesion segmentations across time. The scans of 247 MS patients are used to compare a selected black hole computation method with an enhanced version incorporating paired analysis, using rank correlation to a clinical variable (MS functional composite) as the primary outcome measure. The comparison is done at nine different levels of intensity as a previous study suggests that darker black holes may yield stronger correlations. The results demonstrate that paired analysis can strongly improve longitudinal correlation (from -0.148 to -0.303 in this sample) and may produce segmentations that are more sensitive to clinically relevant changes.
PMCID: PMC3757731  PMID: 24179734
Multiple sclerosis; Black holes; Image segmentation; Longitudinal analysis; Volume change; Clinical correlation
10.  Use of Magnetic Resonance Imaging as Well as Clinical Disease Activity in the Clinical Classification of Multiple Sclerosis and Assessment of Its Course 
International Journal of MS Care  2012;14(3):105-114.
It has recently been suggested that the Lublin-Reingold clinical classification of multiple sclerosis (MS) be modified to include the use of magnetic resonance imaging (MRI). An international consensus conference sponsored by the Consortium of Multiple Sclerosis Centers (CMSC) was held from March 5 to 7, 2010, to review the available evidence on the need for such modification of the Lublin-Reingold criteria and whether the addition of MRI or other biomarkers might lead to a better understanding of MS pathophysiology and disease course over time. The conference participants concluded that evidence of new MRI gadolinium-enhancing (Gd+) T1-weighted lesions and unequivocally new or enlarging T2-weighted lesions (subclinical activity, subclinical relapses) should be added to the clinical classification of MS in distinguishing relapsing inflammatory from progressive forms of the disease. The consensus was that these changes to the classification system would provide more rigorous definitions and categorization of MS course, leading to better insights as to the evolution and treatment of MS.
PMCID: PMC3882992  PMID: 24453741
11.  Nonsense mutation in MERTK causes autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family 
The British Journal of Ophthalmology  2010;94(8):1094-1099.
Retinitis pigmentosa (RP) is one of the most common ophthalmic disorders affecting one in approximately 5000 people worldwide. A nuclear family was recruited from the Punjab province of Pakistan to study the genetic basis of autosomal recessive RP.
All affected individuals underwent a thorough ophthalmic examination and the disease was characterised based upon results for fundus photographs and electroretinogram recordings. Genomic DNA was extracted from peripheral leucocytes. Exclusion studies were performed with short tandem repeat (STR) markers flanking reported autosomal recessive RP loci. Haplotypes were constructed and results were statistically evaluated.
The results of exclusion analyses suggested that family PKRP173 was linked to chromosome 2q harbouring mer tyrosine kinase protooncogene (MERTK), a gene previously associated with autosomal recessive RP. Additional STR markers refined the critical interval and placed it in a 13.4 cM (17 Mb) region flanked by D2S293 proximally and D2S347 distally. Significant logarithm of odds (LOD) scores of 3.2, 3.25 and 3.18 at θ=0 were obtained with markers D2S1896, D2S2269 and D2S160. Sequencing of the coding exons of MERTK identified a mutation, c.718G→T in exon 4, which results in a premature termination of p.E240X that segregates with the disease phenotype in the family.
Our results strongly suggest that the nonsense mutation in MERTK, leading to premature termination of the protein, is responsible for RP phenotype in the affected individuals of the Pakistani family.
PMCID: PMC3393880  PMID: 20538656
12.  Knockdown of Akt1 Promotes Akt2 Upregulation and Resistance to Oxidative-Stress-Induced Apoptosis Through Control of Multiple Signaling Pathways 
The Akt signaling pathway plays a key role in promoting the survival of various types of cells from stress-induced apoptosis, and different members of the Akt family display distinct physiological roles. Previous studies have shown that in response to UV irradiation, Akt2 is sensitized to counteract the induced apoptosis. However, in response to oxidative stress such as hydrogen peroxide, it remains to be elucidated what member of the Akt family would be activated to initiate the signaling cascades leading to resistance of the induced apoptosis. In the present study, we present the first evidence that knockdown of Akt1 enhances cell survival under exposure to 50 μM H2O2. This survival is derived from selective upregulation and activation of Akt2 but not Akt3, which initiates 3 major signaling cascades. First, murine double minute 2 (MDM2) is hyperphosphorylated, which promotes p53 degradation and attenuates its Ser-15 phosphorylation, significantly attenuating Bcl-2 homologous antagonist killer (Bak) upregulation. Second, Akt2 activation inactivates glycogen synthase kinase 3 beta (GSK-3β) to promote stability of myeloid leukemia cell differentiation protein 1 (MCL-1). Finally, Akt2 activation promotes phosphorylation of FOXO3A toward cytosolic export and thus downregulates Bim expression. Overexpression of Bim enhances H2O2-induced apoptosis. Together, our results demonstrate that among the Akt family members, Akt2 is an essential kinase in counteracting oxidative-stress-induced apoptosis through multiple signaling pathways. Antioxid. Redox Signal. 15, 1–17.
PMCID: PMC3110099  PMID: 21303257
13.  Time-Domain Fluorescence Lifetime Imaging Techniques Suitable for Solid-State Imaging Sensor Arrays 
Sensors (Basel, Switzerland)  2012;12(5):5650-5669.
We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD)-based cameras for fluorescence lifetime imaging microscopy (FLIM) by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 μm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber) are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast.
PMCID: PMC3386705  PMID: 22778606
fluorescence lifetime imaging microscopy; FLIM; CMOS; single-photon avalanche diode; single-decay; multi-decay; time-domain FLIM; lifetime based sensing
14.  Establishing Long-Term Efficacy in Chronic Disease: Use of Recursive Partitioning and Propensity Score Adjustment to Estimate Outcome in MS 
PLoS ONE  2011;6(11):e22444.
Establishing the long-term benefit of therapy in chronic diseases has been challenging. Long-term studies require non-randomized designs and, thus, are often confounded by biases. For example, although disease-modifying therapy in MS has a convincing benefit on several short-term outcome-measures in randomized trials, its impact on long-term function remains uncertain.
Data from the 16-year Long-Term Follow-up study of interferon-beta-1b is used to assess the relationship between drug-exposure and long-term disability in MS patients.
To mitigate the bias of outcome-dependent exposure variation in non-randomized long-term studies, drug-exposure was measured as the medication-possession-ratio, adjusted up or down according to multiple different weighting-schemes based on MS severity and MS duration at treatment initiation. A recursive-partitioning algorithm assessed whether exposure (using any weighing scheme) affected long-term outcome. The optimal cut-point that was used to define “high” or “low” exposure-groups was chosen by the algorithm. Subsequent to verification of an exposure-impact that included all predictor variables, the two groups were compared using a weighted propensity-stratified analysis in order to mitigate any treatment-selection bias that may have been present. Finally, multiple sensitivity-analyses were undertaken using different definitions of long-term outcome and different assumptions about the data.
Main Outcome Measure
Long-Term Disability.
In these analyses, the same weighting-scheme was consistently selected by the recursive-partitioning algorithm. This scheme reduced (down-weighted) the effectiveness of drug exposure as either disease duration or disability at treatment-onset increased. Applying this scheme and using propensity-stratification to further mitigate bias, high-exposure had a consistently better clinical outcome compared to low-exposure (Cox proportional hazard ratio = 0.30–0.42; p<0.0001).
Early initiation and sustained use of interferon-beta-1b has a beneficial impact on long-term outcome in MS. Our analysis strategy provides a methodological framework for bias-mitigation in the analysis of non-randomized clinical data.
Trial Registration NCT00206635
PMCID: PMC3227563  PMID: 22140424
15.  Structural brain imaging abnormalities associated with schizophrenia and partial trisomy of chromosome 5 
Psychological medicine  1992;22(2):519-524.
Chromosomal abnormalities occurring in association with mental illness provide a unique opportunity to study the interaction of genetic abnormalities and the brain in mental illness. Four individuals from a family in which schizophrenia was found to cosegregate with a partial trisomy of chromosome 5 were studied with computed tomography and magnetic resonance imaging. Temporal lobe atrophy was found in the two trisomic males and in the asymptomatic balanced translocation female. In addition, a large cavum septum pellucidum and a cavum vergae were found in the older trisomic individual. Scans from the normal male were free of abnormalities. These results suggest that molecular studies of the translocation breakpoints in this chromosomal abnormality may be of interest, and encourage further studies of brain structure in other chromosomal abnormalities associated with psychosis.
PMCID: PMC3154172  PMID: 1615118 CAMSID: cams1823
16.  Patterns of Genetic Variation Within and Between Gibbon Species 
Molecular Biology and Evolution  2011;28(8):2211-2218.
Gibbons are small, arboreal, highly endangered apes that are understudied compared with other hominoids. At present, there are four recognized genera and approximately 17 species, all likely to have diverged from each other within the last 5–6 My. Although the gibbon phylogeny has been investigated using various approaches (i.e., vocalization, morphology, mitochondrial DNA, karyotype, etc.), the precise taxonomic relationships are still highly debated. Here, we present the first survey of nuclear sequence variation within and between gibbon species with the goal of estimating basic population genetic parameters. We gathered ∼60 kb of sequence data from a panel of 19 gibbons representing nine species and all four genera. We observe high levels of nucleotide diversity within species, indicative of large historical population sizes. In addition, we find low levels of genetic differentiation between species within a genus comparable to what has been estimated for human populations. This is likely due to ongoing or episodic gene flow between species, and we estimate a migration rate between Nomascus leucogenys and N. gabriellae of roughly one migrant every two generations. Together, our findings suggest that gibbons have had a complex demographic history involving hybridization or mixing between diverged populations.
PMCID: PMC3144381  PMID: 21368318
population history; chromosomal rearrangements; genetic diversity; gibbon; population genetics
17.  Impact of exposure to interferon beta-1a on outcomes in patients with relapsing–remitting multiple sclerosis: exploratory analyses from the PRISMS long-term follow-up study 
Objective: To explore the effects of exposure to subcutaneous (sc) interferon (IFN) beta-1a on efficacy in patients with relapsing–remitting multiple sclerosis (RRMS) enrolled in the PRISMS (Prevention of Relapses and disability by Interferon beta-1a Subcutaneously in Multiple Sclerosis) study.
Methods: Patients with RRMS received IFN beta-1a, 44 or 22 µg sc three times weekly (tiw), or placebo, for 2 years, at which point placebo recipients were re-randomized to IFN beta-1a, 44 or 22 µg sc tiw, for a further 2–4 years. Long-term follow-up visits occurred 7–8 years after enrolment and allowed participation of patients who had previously discontinued treatment. Post hoc descriptive analyses were conducted within the lower (MIN) and upper (MAX) quartiles of patients divided according to cumulative dose of IFN beta-1a and cumulative time on treatment. Outcomes were explored in patients initially randomized to IFN beta-1a, 44 µg sc tiw, who had received continuous or noncontinuous therapy during the study.
Results: For both cumulative dose and time analyses, the MIN and MAX groups comprised 96 and 95 patients, respectively. The continuous and noncontinuous groups included 45 and 91 patients, respectively. The MAX DOSE and MAX TIME groups had lower annualized relapse rates, lower rates of conversion to secondary progressive MS, lower percentages of patients with Expanded Disability Status Scale progression, higher percentages of relapse-free patients, and less T2 burden of disease than the MIN groups. The continuous therapy group had a lower annualized relapse rate and lower percentages of patients with Expanded Disability Status Scale progression or conversion to secondary progressive MS than the noncontinuous therapy group.
Conclusions: The findings of these post hoc analyses suggest that high exposure to sc IFN beta-1a may be associated with better clinical outcomes than low exposure, and also highlight the importance of maximizing adherence. Additional prospective investigation is warranted to evaluate further the effects of treatment exposure on outcomes and to determine the benefits of interventions to improve adherence.
PMCID: PMC3036958  PMID: 21339904
adherence; interferon beta-1a; long-term follow up; PRISMS; relapsing–remitting multiple sclerosis
18.  Molecular Cloning of the Genes Encoding the PR55/Bβ/δ Regulatory Subunits for PP-2A and Analysis of Their Functions in Regulating Development of Goldfish, Carassius auratus 
The protein phosphatase-2A (PP-2A), one of the major phosphatases in eukaryotes, is a heterotrimer, consisting of a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits encoded by more than 16 different genes, may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the PR55/B family regulatory subunits: β and δ, analyzed their tissue specific and developmental expression patterns in Goldfish ( Carassius auratus). Our results revealed that the full-length cDNA for PR55/Bβ consists of 1940 bp with an open reading frame of 1332 nucleotides coding for a deduced protein of 443 amino acids. The full length PR55/Bδ cDNA is 2163 bp containing an open reading frame of 1347 nucleotides encoding a deduced protein of 448 amino acids. The two isoforms of PR55/B display high levels of sequence identity with their counterparts in other species. The PR55/Bβ mRNA and protein are detected in brain and heart. In contrast, the PR55/Bδ is expressed in all 9 tissues examined at both mRNA and protein levels. During development of goldfish, the mRNAs for PR55/Bβ and PR55/Bδ show distinct patterns. At the protein level, PR55/Bδ is expressed at all developmental stages examined, suggesting its important role in regulating goldfish development. Expression of the PR55/Bδ anti-sense RNA leads to significant downregulation of PR55/Bδ proteins and caused severe abnormality in goldfish trunk and eye development. Together, our results suggested that PR55/Bδ plays an important role in governing normal trunk and eye formation during goldfish development.
PMCID: PMC3020040  PMID: 21245947
protein phosphatase; PP-2A; PR55/Bβ/δ; eye; lens; gene expression; developmental regulation; phosphorylation
19.  A Student Team in a University of Michigan Biomedical Engineering Design Course Constructs a Microfluidic Bioreactor for Studies of Zebrafish Development 
Zebrafish  2009;6(2):201-213.
The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008).
PMCID: PMC2777541  PMID: 19292670
20.  A Student Team in a University of Michigan Biomedical Engineering Design Course Constructs a Microfluidic Bioreactor for Studies of Zebrafish Development 
Zebrafish  2009;6(2):201-213.
The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008).
PMCID: PMC2777541  PMID: 19292670
21.  Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial 
Lancet  2008;372(9653):1881-1893.
Observational data and non-human primate challenge studies suggest that cell-mediated immune (CMI) responses may provide control of HIV replication. The Step Study is the first direct assessment of the efficacy of a CMI vaccine to protect against HIV infection or alter early plasma HIV levels in humans.
HIV-seronegative participants (3000) were randomized (1:1) to receive 3 injections of MRKAd5 HIV-1 gag/pol/nef vaccine or placebo. Randomization was pre-stratified by gender, baseline adenovirus type 5 (Ad5) titer, and study site. Participants were tested ~every 6 months for HIV acquisition; early plasma HIV RNA was measured ~3 months post-HIV diagnosis.
The vaccine elicited IFN-γ ELISPOT responses in 75% of vaccinees. In a pre-specified interim analysis among participants with baseline Ad5 ≤200, 24 of 741 vaccinees became HIV infected, versus 21 of 762 placebo recipients. All but one infection occurred in men. The early geometric mean plasma HIV RNA was comparable in infected vaccine and placebo recipients. In exploratory multivariate analyses, HIV incidence was higher in vaccinees versus placebo recipients among Ad5 seropositive men (5.1% versus 2.2% per year, respectively) and uncircumcised men (5.2% versus 1.4% per year, respectively). HIV incidence was similar in vaccinees versus placebo recipients among Ad5 seronegative men and circumcised men.
This CMI vaccine did not prevent HIV infection or lower early viral level. Mechanisms for failure of the vaccine to protect and for the increased HIV infection rates in subgroups of vaccinees are being explored. Additional follow-up will determine if elevated HIV incidence in vaccinee subgroups persists.
PMCID: PMC2721012  PMID: 19012954
HIV vaccine; efficacy; adenovirus; HIV acquisition; viral load; male circumcision; test of concept
22.  Transcriptional Regulation of PP2A-Aα Is Mediated by Multiple Factors Including AP-2α, CREB, ETS-1, and SP-1 
PLoS ONE  2009;4(9):e7019.
Protein phosphatases-2A (PP-2A) is a major serine/threonine phosphatase and accounts for more than 50% serine/threonine phosphatase activity in eukaryotes. The holoenzyme of PP-2A consists of the scaffold A subunit, the catalytic C subunit and the regulatory B subunit. The scaffold subunits, PP2A-Aα/β, provide a platform for both C and B subunits to bind, thus playing a crucial role in providing specific PP-2A activity. Mutation of the two genes encoding PP2A-Aα/β leads to carcinogenesis and likely other human diseases. Regulation of these genes by various factors, both extracellular and intracellular, remains largely unknown. In the present study, we have conducted functional dissection of the promoter of the mouse PP2A-Aα gene. Our results demonstrate that the proximal promoter of the mouse PP2A-Aα gene contains numerous cis-elements for the binding of CREB, ETS-1, AP-2α, SP-1 besides the putative TFIIB binding site (BRE) and the downstream promoter element (DPE). Gel mobility shifting assays revealed that CREB, ETS-1, AP-2α, and SP-1 all bind to PP2A-Aα gene promoter. In vitro mutagenesis and reporter gene activity assays reveal that while SP-1 displays negative regulation, CREB, ETS-1 and AP-2Aα all positively regulate the promoter of the PP2A-Aα gene. ChIP assays further confirm that all the above transcription factors participate the regulation of PP2A-Aα gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aα gene.
PMCID: PMC2736573  PMID: 19750005
23.  The Goldfish SG2NA Gene Encodes Two α-Type Regulatory Subunits for PP-2A and Displays Distinct Developmental Expression Pattern 
SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NAα and SG2NAβ. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the α-type, and are named SG2NAα and SG2NAα+. RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues.
PMCID: PMC2758282  PMID: 19838339
goldfish; PP-2A; SG2NA; gene expression; molecular cloning; development; dephosphorylation; retina; lens
24.  Differential expression of the catalytic subunits for PP-1 and PP-2A and the regulatory subunits for PP–2A in mouse eye 
Molecular Vision  2008;14:762-773.
Reversible protein phosphorylation is a fundamental regulatory mechanism in all biologic processes. Protein serine/threonine phosphatases-1 (PP-1) and 2A (PP-2A) account for 90% of serine/threonine phosphatase activity in eukaryote cells and play distinct roles in regulating multiple cellular processes and activities. Our previous studies have established the expression patterns of the catalytic subunits for PP-1 (PP-1cs) and PP-2A (PP-2Acs) in bovine and rat lenses. In the present study, we have determined the expression patterns of PP-1cs (PP-1α and PP-1β) and PP-2Acs (PP-2Aα and PP-2Aβ) in the retina and cornea along with the ocular lens of the mouse eye. Moreover, since the function of PP-2A is largely relied on its regulatory subunits, we have also analyzed the expression patterns of the genes encoding the scaffold A subunits of PP-2A, PP2A-Aα and PP2A-Aβ, and the regulatory B family subunits of PP-2A, PP2A-Bα, PP2A-Bβ, and PP2A-Bγ. In addition, we have also demonstrated the differential protections of PP-1 and PP-2A in mouse lens epithelial cell line, αTN4–1, against oxidative stress-induced apoptosis.
Total RNAs and proteins were extracted from the retina, lens epithelium, lens fiber cells, and cornea of the mouse eye. Reverse transcription polymerase chain reaction (RT–PCR) and real time PCR were used to detect the mRNA expression. Western blot and immunohistochemistry analysis were applied to examine the protein expression and distribution. Stable clones of αTN4–1 cells expressing either PP-1α or PP-2Aα were used to analyze the differential protections against oxidative stress-induced apoptosis.
PP-1 is more abundant than PP-2A in the mouse eye. The catalytic subunits for PP-1 and PP-2A display similar expression patterns in the retina and cornea but much reduced in the lens. The mRNAs for all five isoforms of PP2A-A and PP2A-B subunits are highly expressed in the retina, but only three out of the five mRNAs are expressed in the cornea. In the ocular lens, only PP2A-Aβ and PP2A-Bγ mRNAs are clearly detectable. The A and B subunit proteins of PP-2A are highly expressed in the retina and cornea but are much reduced in the ocular lens. PP2A-Aα/β are differentially distributed in the mouse retina.When transfected into mouse lens epithelial cells, αTN4–1, PP-1α and PP-2Aα display differential protection against oxidative stress-induced apoptosis.
Our results lead to the following conclusions regarding PP-1 and PP-2A in mouse eye: 1) PP1 is a more abundant phosphatase than PP-2A; 2) both PP-1 and PP-2A may play important roles, and the functions of PP-2A appear to be highly regulated by various regulatory subunits; and 3) the genes encoding PP-1α/β, PP-2Aα/β, PP-2A-Aα/β, and PP-2A-B α/β/γ are all differentially expressed.
PMCID: PMC2324119  PMID: 18432318
25.  Developmental expression of three small GTPases in the mouse eye 
Molecular Vision  2007;13:1144-1153.
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
PMCID: PMC2779149  PMID: 17653061

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