Publications by authors named "Lisa Brennan"

39 Publications

Accomplishing CAPE Domain 3 During APPE Rotations: Student Perceptions.

J Pharm Pract 2021 Mar 19:8971900211000689. Epub 2021 Mar 19.

Wingate University School of Pharmacy, Wingate, NC, USA.

Introduction: The purpose of this study is to examine student perceptions of accomplishment among 6 subdomains of Center for Advancement of Pharmacy Education (CAPE) Domain 3 "Approach to Practice and Care" outcomes in Advanced Pharmacy Practice Experiences (APPE) across distinct geographical regions.

Methods: An 18-item electronic survey was distributed to 88 student pharmacists at a private university completing APPEs in 5 distinct regions and 2 concentrated learning experiences during their penultimate rotation. The survey assessed whether students had at least 1 opportunity to achieve Domain 3 outcomes. Students were prompted to report a percentage of perceived successful accomplishment of outcomes if they stated they had at least 1 opportunity for achievement.

Results: Survey response rate was 52% (n = 46). Respondents reported a median accomplishment of at least 85% for each question. For 2 questions, respondents reported a median accomplishment of 99%. Students perceived successful accomplishment for most of the questions related to communication outcomes, while the lowest completion percentages were noted in outcomes related to patient advocacy (85%) and problem solving (88%). Student perceptions of accomplishment among the 6 subdomains were similar across regions and concentrated learning experiences.

Conclusions: Students felt confident in accomplishing the outcomes associated with CAPE Domain 3. Regional assignments did not impact student perceptions of outcome accomplishment. Preceptors may play a pivotal role in providing students with opportunities to further polish their skills and increase confidence, specifically in the areas of patient advocacy and problem solving.
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http://dx.doi.org/10.1177/08971900211000689DOI Listing
March 2021

Novel mitochondrial derived Nuclear Excisosome degrades nuclei during differentiation of prosimian Galago (bush baby) monkey lenses.

PLoS One 2020 12;15(11):e0241631. Epub 2020 Nov 12.

Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL, United States of America.

The unique cellular organization and transparent function of the ocular lens depend on the continuous differentiation of immature epithelial cells on the lens anterior surface into mature elongated fiber cells within the lens core. A ubiquitous event during lens differentiation is the complete elimination of organelles required for mature lens fiber cell structure and transparency. Distinct pathways have been identified to mediate the elimination of non-nuclear organelles and nuclei. Recently, we reported the discovery of a unique structure in developing fiber cells of the chick embryo lens, called the Nuclear Excisosome, that is intractably associated with degrading nuclei during lens fiber cell differentiation. In the chick lens, the Nuclear Excisosome is derived from projections of adjacent cells contacting the nuclear envelope during nuclear elimination. Here, we demonstrate that, in contrast to the avian model, Nuclear Excisosomes in a primate model, Galago (bush baby) monkeys, are derived through the recruitment of mitochondria to form unique linear assemblies that define a novel primate Nuclear Excisosome. Four lenses from three monkeys aged 2-5 years were fixed in formalin, followed by paraformaldehyde, then processed for Airyscan confocal microscopy or transmission electron microscopy. For confocal imaging, fluorescent dyes labelled membranes, carbohydrate in the extracellular space, filamentous actin and nuclei. Fiber cells from Galago lenses typically displayed prominent linear structures within the cytoplasm with a distinctive cross-section of four membranes and lengths up to 30 μm. The outer membranes of these linear structures were observed to attach to the outer nuclear envelope membrane to initiate degradation near the organelle-free zone. The origin of these unique structures was mitochondria in the equatorial epithelium (not from plasma membranes of adjacent cells as in the chick embryo model). Early changes in mitochondria appeared to be the collapse of the cristae and modification of one side of the mitochondrial outer membrane to promote accumulation of protein in a dense cluster. As a mitochondrion surrounded the dense protein cluster, an outer mitochondrial membrane enclosed the protein to form a core and another outer mitochondrial membrane formed the outermost layer. The paired membranes of irregular texture between the inner core membrane and the outer limiting membrane appeared to be derived from modified mitochondrial cristae. Several mitochondria were involved in the formation and maturation of these unique complexes that apparently migrated around the fulcrum into the cytoplasm of nascent fiber cells where they were stabilized until the nuclear degradation was initiated. Thus, unlike in the chick embryo, the Galago lenses degraded nuclear envelopes with a Nuclear Excisosome derived from multiple mitochondria in the epithelium that formed novel linear assemblies in developing fiber cells. These findings suggest that recruitment of distinct structures is required for Nuclear Excisosome formation in different species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241631PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660580PMC
December 2020

Hypoxia regulates the degradation of non-nuclear organelles during lens differentiation through activation of HIF1a.

Exp Eye Res 2020 09 3;198:108129. Epub 2020 Jul 3.

Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, 33431, USA. Electronic address:

Formation of the eye lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To attain their mature structure and transparent function, nascent lens fiber cells must complete a precise cellular remodeling program hallmarked by the complete elimination of organelles to form the core lens organelle-free zone (OFZ). Lacking a blood supply, the lens resides in a hypoxic environment that results in a decreasing oxygen concentration from the lens surface to the lens core. This oxygen gradient results in a hypoxic microenvironment in the region of the lens where immature lens fiber cells initiate loss of organelles to form the core OFZ. These features of the lens suggest a potential role for low lens oxygen levels in the regulation of organelle degradation and other events critical for mature lens fiber cell formation. Hypoxia activates the master regulator of the hypoxic response, hypoxia-inducible factor 1a (HIF1a) that regulates hypoxia-responsive genes. To identify a potential role for hypoxia and HIF1a in the elimination of organelles during lens fiber cell maturation, we tested the requirement for hypoxia in the degradation of non-nuclear organelles in ex vivo cultured embryonic chick lenses by monitoring the degradation of mitochondria (MT), Golgi apparatus (GA) and endoplasmic reticulum (ER) under conditions of low (1% O) and high (21% O) oxygen. We also examined the requirement for HIF1a activation for elimination of these organelles under the same conditions using a specific HIF1a activator (DMOG) and a specific HIF1a inhibitor (chetomin) and examined the requirements for hypoxia and HIF1a for regulating transcription of BNIP3L that we previously showed to be required for elimination of non-nuclear lens organelles. We used ChIP-qPCR to confirm direct binding of HIF1a to the 5' untranslated region of the BNIP3L gene. Finally, we examined the effects of expressing an oxygen insensitive mutant form of HIF1a (P402A/P565A) and BNIP3L on non-nuclear organelle degradation. Our data demonstrate that hypoxia and HIF1a are required for the degradation of non-nuclear organelles during lens fiber cell formation and that they regulate this process by governing BNIP3L transcription. Our results also provide evidence that hypoxia and HIF1a are essential for achieving mature lens structure.
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http://dx.doi.org/10.1016/j.exer.2020.108129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508769PMC
September 2020

Cultural Sensitivity and Global Pharmacy Engagement in the Arab World.

Am J Pharm Educ 2019 05;83(4):7228

Faculty of Pharmacy, Misr International University, Cairo, Egypt.

To create a resource on cultural sensitivity for schools and colleges of pharmacy that are currently engaged or considering future outreach opportunities in the Arab world. A literature review (2000-2018) of databases and Internet searches with specific keywords and terms were conducted. Authors who had experience in travelling to and hosting students and professionals from the Arab world and authors with local work experience in the Arab world were solicited. General information about the Arab world, including unique aspects of individual countries, is presented. Stereotypes and misconceptions regarding the region and the people are discussed. Specific information about the government and infrastructure of each country, including their health care system is provided, with emphasis given to pharmacy education and practice in the region. In addition, recommendations for culturally sensitive engagement for pharmacy and other health care practitioners are discussed. Finally, recommendations for culturally sensitive engagement when hosting students and/or faculty members from the Arab world are also addressed. Global engagement between schools and colleges of pharmacy in the United States and those in the Arab world is increasing. For an enriching and fruitful engagement, sensitivity toward the cultural and clinical needs of the people, and in particular, the professionals of that region is critical.
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http://dx.doi.org/10.5688/ajpe7228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581338PMC
May 2019

An Introduction to Cultural Sensitivity and Global Pharmacy Engagement.

Am J Pharm Educ 2019 05;83(4):7221

Lloyd L. Gregory School of Pharmacy, Palm Beach Atlantic University, West Palm Beach, Florida.

Global engagement between schools and colleges of pharmacy in the United States and many regions of the world is increasing. For an enriching and fruitful interaction, sensitivity toward the cultural, ethical, educational, religious, historical, political, regulatory, and practice issues is critical. Lack of sensitivity may negatively impact engagement among students, professionals and other people from different regions of the world. In this special issue, eight papers will introduce general information about five regions of the world that have established and increased global engagements with institutions in the United States: Africa, the Arab world, Asia, the Caribbean, and Latin America. In addition, the special issue will include a paper with key information related to global engagement within the United States. For each paper, the specifics provided about the selected countries include: demographics, culture, climate, pharmacy education, and health care systems, as well as common stereotypes and misconceptions held by and about the people of the country. Further, recommendations for pharmacists and other health care professionals on culturally sensitive engagement will be emphasized. Finally, recommendations for culturally sensitive engagement when US schools are hosting students and faculty members from those regions will be summarized. The papers are based on literature reviews of databases from 2000 to 2018 and internet searches with specific keywords or terms, such as and . Additional keywords are identified in individual papers on specific regions. Authors for each paper consist of practitioners with experience in travelling to and hosting students and professionals from the regions; practitioners with local work experience, and professionals from each region. The special issue is intended to serve as a resource for US schools and colleges of pharmacy currently engaged in or considering future outreach opportunities in these regions, and for those seeking opportunities in the United States. The special issue will provide key information to facilitate culturally sensitive engagement in existing or future relationships.
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http://dx.doi.org/10.5688/ajpe7221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581352PMC
May 2019

Cultural Sensitivity and Global Pharmacy Engagement in the United States.

Am J Pharm Educ 2019 05;83(4):7220

School of Pharmacy, Loma Linda University, Loma Linda, California.

To provide a resource for schools and colleges of pharmacy in different regions of the world that are considering sending their students and faculty members to the United States for training. A literature review (2000-2018) was conducted that involved database and Internet searches using specific keywords and terms. Information was also solicited from authors in different regions of the United States who have hosted international students and faculty members. Recommendations for pharmacists and other health care practitioners on culturally sensitive engagement were formulated. Global engagement between schools and colleges of pharmacy from different regions of the world and the United States is increasing. In addition to various cultural aspects, general information about the US health care system, pharmacy education programs, and pharmacy practice were found to be available to individuals who are charged with organizing and facilitating these exchanges. Common stereotypes and misconceptions about the United States were also identified. For international learners to have an enriching and fruitful engagement while in the United States, an understanding of American culture in general as well as the unique cultural aspects of different regions of the country as provided in this paper is critical.
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http://dx.doi.org/10.5688/ajpe7220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581351PMC
May 2019

Lens differentiation is characterized by stage-specific changes in chromatin accessibility correlating with differentiation state-specific gene expression.

Dev Biol 2019 09 25;453(1):86-104. Epub 2019 May 25.

Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA. Electronic address:

Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during lens cell differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of lens cell differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 lens genes obtained by high-throughput RNA sequencing at the same stages of lens cell differentiation. Approximately 90,000 regions of the lens genome exhibited distinct changes in chromatin accessibility at one or more stages of lens differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r ​> ​0.7) between altered expression levels at specific stages of lens cell differentiation and changes in chromatin accessibility in potential promoter (-7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 ​kb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and lens differentiation and they identify a wide-variety of lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the lens differentiation program.
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http://dx.doi.org/10.1016/j.ydbio.2019.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667291PMC
September 2019

BNIP3L/NIX is required for elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during eye lens organelle-free zone formation.

Exp Eye Res 2018 09 4;174:173-184. Epub 2018 Jun 4.

Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, 33431, USA. Electronic address:

The formation and life-long growth of the ocular lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To achieve their mature structure and transparent function, newly formed lens fiber cells undergo a series of cellular remodeling events including the complete elimination of cellular organelles to form the lens organelle-free zone (OFZ). To date, the mechanisms and requirements for organelle elimination by lens fiber cells remain to be fully elucidated. In previous studies, we detected the presence of mitochondria contained within autophagolysosomes throughout human and chick lenses suggesting that proteins targeting mitochondria for degradation by mitophagy could be required for the elimination of mitochondria during OFZ formation. Consistently, high-throughput RNA sequencing of microdissected embryonic chick lenses revealed that expression of a protein that targets mitochondria for elimination during erythrocyte formation, called BCL2 interacting protein 3-like (BNIP3L/NIX), peaks in the region of lens where organelle elimination occurs. To examine the potential role for BNIP3L in the elimination of mitochondria during lens fiber cell remodeling, we analyzed the expression pattern of BNIP3L in newborn mouse lenses, the effect of its deletion on organelle elimination and its co-localization with lens organelles. We demonstrate that the expression pattern of BNIP3L in the mouse lens is consistent with it playing an important role in the elimination of mitochondria during lens fiber cell organelle elimination. Importantly, we demonstrate that deletion of BNIP3L results in retention of mitochondria during lens fiber cell remodeling, and, surprisingly, that deletion of BNIP3L also results in the retention of endoplasmic reticulum and Golgi apparatus but not nuclei. Finally, we show that BNIP3L localizes to the endoplasmic reticulum and Golgi apparatus of wild-type newborn mouse lenses and is contained within mitochondria, endoplasmic reticulum and Golgi apparatus isolated from adult mouse liver. These data identify BNIP3L as a novel requirement for the elimination of mitochondria, endoplasmic reticulum and Golgi apparatus during lens fiber cell remodeling and they suggest a novel function for BNIP3L in the regulation of endoplasmic reticulum and Golgi apparatus populations in the lens and non-lens tissues.
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http://dx.doi.org/10.1016/j.exer.2018.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110959PMC
September 2018

Infusion of Alloanergized Donor Lymphocytes after CD34-selected Haploidentical Myeloablative Hematopoietic Stem Cell Transplantation.

Clin Cancer Res 2018 09 16;24(17):4098-4109. Epub 2018 May 16.

Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Allogeneic hematopoietic stem-cell transplantation (HSCT) is a curative treatment for many hematologic cancers. Use of haploidentical (mismatched) donors increases HSCT availability but is limited by severe graft-versus-host disease (GvHD) and delayed immune reconstitution. Alloanergization of donor T cells is a simple approach to rebuild immunity while limiting GvHD after haploidentical HSCT, but the optimal T-cell dose and impact on immune reconstitution remain unknown. We performed a multicenter phase I trial of alloanergized donor lymphocyte infusion (aDLI) after CD34-selected myeloablative haploidentical HSCT. The primary aim was feasibility and safety with secondary aims of assessing the less frequently addressed issue of impact on immune reconstitution. Nineteen patients with high-risk acute leukemia or myelodysplasia were enrolled. Engraftment occurred in 18 of 19 patients (95%). Pre-aDLI, 12 patients (63%) had bacteremia, nine of 17 at-risk patients (53%) reactivated CMV, and one developed acute GvHD. Sixteen patients received aDLI at dose levels 1 (10 T cells/kg, = 4), 2 (10, = 8), and 3 (10, = 4). After aDLI, five patients developed clinically significant acute GvHD, and four of 14 at-risk patients (29%) reactivated CMV. T-cell recovery was significantly greater, and functional virus- and tumor-associated antigen-specific T cells were detectable earlier in patients receiving dose level 2 or 3 versus dose level 1/no aDLI. Alloanergization of donor cells expanded the CD4 T-regulatory cell frequency within aDLI, which increased further without impeding expansion of virus- and tumor-associated antigen-specific T cells. These data demonstrate safety and a potential role for aDLI in contributing to immune reconstitution and expanding tolerogenic regulatory T cells after CD34-selected myeloablative haploidentical HSCT. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125184PMC
September 2018

Heart failure in the elderly: ten peculiar management considerations.

Heart Fail Rev 2017 03;22(2):219-228

Cleveland Clinic Abu Dhabi, United Arab Emirates, Heart and Vascular Institute, Al Maryah Island, PO Box 112412, Abu Dhabi, UAE.

Chronic heart failure (HF) is a disease with significantly higher prevalence in the elderly or patients older than 65 years old. Typically, older patients have more risk factors for HF, more comorbidities, and are more likely to have recurrent admissions for acute decompensations. With HF burden on health care systems primarily related to hospital and nursing home costs, it is critical that elderly patients are approached with a clear understanding of certain unique clinical, laboratory, imaging, and pharmacokinetic differences that can alter their management and outcomes. Psychosocial factors have major implications on adherence to therapy as well as decisions on advanced care for elderly HF patients. In this article, we highlight ten peculiar management considerations when approaching older patients with HF. We discuss issues related to epidemiology, diagnostic challenges, pharmacotherapy, and palliative care; all of which can impact this unique population and, more importantly, the disease burden as a whole.
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http://dx.doi.org/10.1007/s10741-017-9598-3DOI Listing
March 2017

Parkin elimination of mitochondria is important for maintenance of lens epithelial cell ROS levels and survival upon oxidative stress exposure.

Biochim Biophys Acta Mol Basis Dis 2017 01 1;1863(1):21-32. Epub 2016 Oct 1.

Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL 33431, USA. Electronic address:

Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to HO-oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency.
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http://dx.doi.org/10.1016/j.bbadis.2016.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154830PMC
January 2017

Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.

PLoS One 2016 18;11(8):e0160785. Epub 2016 Aug 18.

Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL, United States of America.

An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12-15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial-like projections appear to be initiated with a clathrin-like coat and driven by an internal actin network. In summary, a specialized cellular organelle, the nuclear excisosome, generated in part by adjacent fiber cells degrades nuclei during fiber cell differentiation and maturation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160785PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990417PMC
August 2017

Chromatin remodeling enzyme Snf2h regulates embryonic lens differentiation and denucleation.

Development 2016 06;143(11):1937-47

Department of Ophthalmology & Visual Sciences and Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIβ, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.
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http://dx.doi.org/10.1242/dev.135285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920164PMC
June 2016

Pilot experience with opebacan/rBPI 21 in myeloablative hematopoietic cell transplantation.

F1000Res 2015 21;4:1480. Epub 2015 Dec 21.

Harvard Medical School, Boston, USA; Boston Children's Hospital, Boston, USA.

Bacterial infection and inflammation contribute significantly to the morbidity and mortality of myeloablative allogeneic hematopoietic cell transplantation (HCT). Endotoxin, a component of the outer membrane of Gram-negative bacteria, is a potent inflammatory stimulus in humans. Bactericidal/permeability increasing protein (BPI), a constituent of human neutrophil granules, binds endotoxin thereby precluding endotoxin-induced inflammation and also has direct anti-infective properties against bacteria. As a consequence of myeloablative therapy used in preparation for hematopoietic cell infusion, patients experience gastrointestinal leak of bacteria and bacterial toxins into the systemic circulation and a period of inflammatory cytokine elevation associated with subsequent regimen-related toxicities.  Patients frequently become endotoxemic and febrile as well as BPI-deficient due to sustained neutropenia. To examine whether enhancing endotoxin-neutralizing and anti-infective activity by exogenous administration of a recombinant N-terminal fragment of BPI (rBPI 21, generic name opebacan) might ameliorate regimen-related toxicities including infection, we recruited patients scheduled to undergo myeloablative HCT to participate in a proof-of-concept prospective phase I/II trial. After the HCT preparative regimen was completed, opebacan was initiated 18-36 hours prior to administration of allogeneic hematopoietic stem cells (defined as Day 0) and continued for 72 hours. The trial was to have included escalation of rBPI 21 dose and duration but was stopped prematurely due to lack of further drug availability.  Therefore, to better understand the clinical course of opebacan-treated patients (n=6), we compared their outcomes with a comparable cohort meeting the same eligibility criteria and enrolled in a non-interventional myeloablative HCT observational study (n = 35).  Opebacan-treated participants had earlier platelet engraftment (p=0.005), mirroring beneficial effects of rBPI 21 previously observed in irradiated mice, fewer documented infections (p=0.03) and appeared less likely to experience significant regimen-related toxicities (p=0.05). This small pilot experience supports the potential utility of rBPI 21 in ameliorating HCT-related morbidity and merits further exploration.
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http://dx.doi.org/10.12688/f1000research.7558.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722698PMC
February 2016

Integrin αVβ5-mediated Removal of Apoptotic Cell Debris by the Eye Lens and Its Inhibition by UV Light Exposure.

J Biol Chem 2015 Dec 2;290(51):30253-66. Epub 2015 Nov 2.

From the Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida 33431

Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin αVβ5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVβ5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVβ5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the αVβ5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVβ5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.
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http://dx.doi.org/10.1074/jbc.M115.688390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683250PMC
December 2015

Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens.

G3 (Bethesda) 2014 Jun 13;4(8):1515-27. Epub 2014 Jun 13.

Department of Biomedical Science, Florida Atlantic University, Boca Raton, Florida 33431

The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells.
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http://dx.doi.org/10.1534/g3.114.012120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132181PMC
June 2014

Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin.

Innate Immun 2014 Oct 9;20(7):697-711. Epub 2013 Oct 9.

Boston Children's Hospital, Boston, MA, USA Harvard Medical School, Boston, MA, USA

Hematopoietic cell transplant (HCT) is a life-saving therapy for many malignant and non-malignant bone marrow diseases. Associated morbidities are often due to transplant-related toxicities and infections, exacerbated by regimen-induced immune suppression and systemic incursion of bacterial products. Patients undergoing myeloablative conditioning for HCT become endotoxemic and display blood/plasma changes consistent with lipopolysaccharide (LPS)-induced systemic innate immune activation. Herein, we addressed whether patients scheduled for HCT display differences in recognition/response to LPS ex vivo traceable to specific single nucleotide polymorphisms (SNPs). Two SNPs of LPS binding protein (LBP) were associated with changes in plasma LBP levels, with one LBP SNP also associating with differences in efficiency of extraction and transfer of endotoxin to myeloid differentiation factor-2 (MD-2), a step needed for activation of TLR4. None of the examined SNPs of CD14, bactericidal/permeability-increasing protein (BPI), TLR4 or MD-2 were associated with corresponding protein plasma levels or endotoxin delivery to MD-2, but CD14 and BPI SNPs significantly associated with differences in LPS-induced TNF-α release ex vivo and infection frequency, respectively. These findings suggest that specific LBP, CD14 and BPI SNPs might be contributory assessments in studies where clinical outcome may be affected by host response to endotoxin and bacterial infection.
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http://dx.doi.org/10.1177/1753425913505122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128912PMC
October 2014

Autophagy and mitophagy participate in ocular lens organelle degradation.

Exp Eye Res 2013 Nov 4;116:141-50. Epub 2013 Sep 4.

Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL.

The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation.
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http://dx.doi.org/10.1016/j.exer.2013.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856666PMC
November 2013

Chaperone-independent mitochondrial translocation and protection by αB-crystallin in RPE cells.

Exp Eye Res 2013 May 4;110:10-7. Epub 2013 Mar 4.

Biomedical Sciences Department, Charles E. Schmidt College of Medicine, Florida Atlantic University, 777 Glades Rd, Boca Raton, FL 33431, USA.

αB-crystallin is a small heat shock protein that exhibits chaperone activity and can protect multiple cell types against oxidative stress damage. Altered levels and specific mutations of αB-crystallin are associated with multiple degenerative diseases. We previously found that αB-crystallin translocates to lens and retinal cell mitochondria upon oxidative stress exposure where it provides protection against oxidative stress damage. To date, the role of the chaperone function of αB-crystallin in mitochondrial translocation and protection has not been established. Here, we sought to determine the relationship between the chaperone activity of αB-crystallin and its ability to translocate to and protect retinal cell mitochondria against oxidative stress damage. Our data provide evidence that three forms of αB-crystallin exhibiting different chaperone activity levels including wild-type, R120G (decreased chaperone activity) and M68A (increased chaperone activity) provide comparable levels of mitochondrial translocation and protection to retinal cells exposed to oxidative stress. The results provide evidence that mitochondrial translocation and protection by αB-crystallin is independent of its chaperone activity and that other functions of αB-crystallin may also be independent of its chaperone activity.
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http://dx.doi.org/10.1016/j.exer.2013.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3634887PMC
May 2013

Effect of weight on outcomes of children undergoing hematopoietic cell transplantation.

Pediatr Hematol Oncol 2013 Mar 28;30(2):116-30. Epub 2012 Nov 28.

Department of Internal Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

Chemotherapy dosing in hematopoietic cell therapy (HCT) conditioning regimens is based on patient weight. We hypothesized that potential underdosing or overdosing of patients with significant deviation of weight from normal might alter HCT outcomes, such as early mortality, overall or organ-specific toxicity, and/or relapse. We therefore conducted a retrospective analysis of 400 children between the ages of 2 and 18 years who underwent HCT for malignant or nonmalignant disease at Boston Children's Hospital over a 10-year period. Using the Centers for Disease Control and Prevention standard weight classification schema, we found no evidence to suggest a difference in survival or in time to engraftment or in relapse in patients with malignant disease. In the subgroups of patients either receiving autologous HCT or with underlying malignancy, combined overweight and obese patients had a higher rate of any organ, but not organ-specific, Grade 3-5 toxicity compared with the normal weight group. The study was not powered to detect a difference between underweight and normal weight patients. These data suggest that multiple outcome measures over the first year after HCT are unaffected by weight.
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http://dx.doi.org/10.3109/08880018.2012.743201DOI Listing
March 2013

Spatial expression patterns of autophagy genes in the eye lens and induction of autophagy in lens cells.

Mol Vis 2012 30;18:1773-86. Epub 2012 Jun 30.

Department of Biomedical Science, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA.

Purpose: Mutation of the autophagy gene FYVE (named after the four cysteine-rich proteins: Fab 1 [yeast orthologue of PIKfyve], YOTB, Vac 1 [vesicle transport protein], and EEA1) and coiled coil containing 1 (fyco1) causes human cataract suggesting a role for autophagy in lens function. Here, we analyzed the range and spatial expression patterns of lens autophagy genes and we evaluated whether autophagy could be induced in lens cells exposed to stress.

Methods: Autophagy gene expression levels and their spatial distribution patterns were evaluated between microdissected human lens epithelium and fibers at the mRNA and protein levels by microarray data analysis, real-time PCR and western blot analysis. Selected autophagy protein spatial expression patterns were also examined in newborn mouse lenses by immunohistochemistry. The autophagosomal content of cultured human lens epithelial cells was determined by counting the number of microtubule-associated protein 1 light chain 3B (LC3B)-positive puncta in cells cultured in the presence or absence of serum.

Results: A total of 42 autophagy genes were detected as being expressed by human lens epithelium and fibers. The autophagosomal markers LC3B and FYCO1 were detected throughout the newborn mouse lens. Consistently, the autophagy active form of LC3B (LC3B II) was detected in microdissected human lens fibers. An increased number of LC3B-positive puncta was detected in cultured lens cells upon serum starvation suggesting induction of autophagy in lens cells under stress conditions.

Conclusions: The data provide evidence that autophagy is an important component for the function of lens epithelial and fiber cells. The data are consistent with the notion that disruption of lens autophagy through mutation or inactivation of specific autophagy proteins could lead to loss of lens resistance to stress and/or loss of lens differentiation resulting in cataract formation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398491PMC
November 2012

αB-crystallin/sHSP protects cytochrome c and mitochondrial function against oxidative stress in lens and retinal cells.

Biochim Biophys Acta 2012 Jul 12;1820(7):921-30. Epub 2012 Apr 12.

Biomedical Sciences Department, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA.

Background: αB-crystallin/sHSP protects cells against oxidative stress damage. Here, we mechanistically examined its ability to preserve mitochondrial function in lens and retinal cells and protect cytochrome c under oxidative stress conditions.

Methods: αB-crystallin/sHSP was localized in human lens (HLE-B3) and retinal (ARPE-19) cells. αB-crystallin/sHSP was stably over-expressed and its ability to preserve mitochondrial membrane potential under oxidative stress conditions was monitored. Interactions between αB-crystallin/sHSP and cytochrome c were examined by fluorescent resonance energy transfer (FRET) and by co-immune precipitation. The ability of αB-crystallin/sHSP to protect cytochrome c against methionine-80 oxidation was monitored.

Results: αB-crystallin/sHSP is present in the mitochondria of lens and retinal cells and is translocated to the mitochondria under oxidative conditions. αB-crystallin/sHSP specifically interacts with cytochrome c in vitro and in vivo and its overexpression preserves mitochondrial membrane potential under oxidative stress conditions. αB-crystallin/sHSP directly protects cytochrome c against oxidation.

General Significance: These data demonstrate that αB-crystallin/sHSP maintains lens and retinal cells under oxidative stress conditions at least in part by preserving mitochondrial function and by protecting cytochrome c against oxidation. Since oxidative stress and loss of mitochondrial function are associated with eye lens cataract and age-related macular degeneration, loss of these αB-crystallin/sHSP functions likely plays a key role in the development of these diseases. αB-crystallin/sHSP is expressed throughout the body and its ability to maintain mitochondrial function is likely important for the prevention of multiple degenerative diseases.
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http://dx.doi.org/10.1016/j.bbagen.2012.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362689PMC
July 2012

Oxidative stress defense and repair systems of the ocular lens.

Front Biosci (Elite Ed) 2012 Jan 1;4:141-55. Epub 2012 Jan 1.

Biomedical Sciences Department, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL 33431, USA.

It is well accepted that reactive oxygen species (ROS) play a critical role in many biological processes including disease and longevity. Oxidation of proteins has been linked to many disease states and even the aging process itself. This was first proposed as "The free radical theory of aging" in 1956 by Denham Harman which suggests that free radicals causes cumulative and irreversible damage to macromolecules, loss of cellular function and cell death over time directly impacting health and lifespan. Cellular damage from ROS exposure has been termed oxidative stress, which is an imbalance between cellular ROS production and the ability of the cell to regulate ROS levels and repair damage caused by ROS. This review focuses on the role of oxidative stress in the eye lens as a model for understanding the role of oxidative stress systems in age-related human disease.
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http://dx.doi.org/10.2741/365DOI Listing
January 2012

Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.

Sci Transl Med 2011 Apr;3(80):80ra34

Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA.

Although advanced-stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of antitumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred antitumor CD8(+) T lymphocytes (CTLs) have had limited life spans unless the host has been manipulated. To generate CTLs that have an intrinsic capacity to persist in vivo, we developed a human artificial antigen-presenting cell system that can educate antitumor CTLs to acquire both a central memory and an effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. We examined whether antitumor CTLs generated using this system could function and persist in patients. We showed that MART1-specific CTLs, educated and expanded using our artificial antigen-presenting cell system, could survive for prolonged periods in advanced-stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTLs trafficked to the tumor, mediated biological and clinical responses, and established antitumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.
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http://dx.doi.org/10.1126/scitranslmed.3002207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861895PMC
April 2011

TXNL6 is a novel oxidative stress-induced reducing system for methionine sulfoxide reductase a repair of α-crystallin and cytochrome C in the eye lens.

PLoS One 2010 Nov 4;5(11):e15421. Epub 2010 Nov 4.

Biomedical Sciences Department, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, United States of America.

A key feature of many age-related diseases is the oxidative stress-induced accumulation of protein methionine sulfoxide (PMSO) which causes lost protein function and cell death. Proteins whose functions are lost upon PMSO formation can be repaired by the enzyme methionine sulfoxide reductase A (MsrA) which is a key regulator of longevity. One disease intimately associated with PMSO formation and loss of MsrA activity is age-related human cataract. PMSO levels increase in the eye lens upon aging and in age-related human cataract as much as 70% of total lens protein is converted to PMSO. MsrA is required for lens cell maintenance, defense against oxidative stress damage, mitochondrial function and prevention of lens cataract formation. Essential for MsrA action in the lens and other tissues is the availability of a reducing system sufficient to catalytically regenerate active MsrA. To date, the lens reducing system(s) required for MsrA activity has not been defined. Here, we provide evidence that a novel thioredoxin-like protein called thioredoxin-like 6 (TXNL6) can serve as a reducing system for MsrA repair of the essential lens chaperone α-crystallin/sHSP and mitochondrial cytochrome c. We also show that TXNL6 is induced at high levels in human lens epithelial cells exposed to H(2)O(2)-induced oxidative stress. Collectively, these data suggest a critical role for TXNL6 in MsrA repair of essential lens proteins under oxidative stress conditions and that TXNL6 is important for MsrA defense protection against cataract. They also suggest that MsrA uses multiple reducing systems for its repair activity that may augment its function under different cellular conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015421PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2973970PMC
November 2010

Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions.

FASEB J 2010 Sep 21;24(9):3274-83. Epub 2010 Apr 21.

Department of Ophthalmology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.

The eye lens is an encapsulated avascular organ whose function is to focus light on the retina. Lens comprises a single progenitor cell lineage in multiple states of differentiation. Disruption of lens function leading to protein aggregation and opacity results in age-onset cataract. Cataract is a complex disease involving genetic and environmental factors. Here, we report the development of a new 3-stage system that differentiates human embryonic stem cells (hESCs) into large quantities of lens progenitor-like cells and differentiated 3-dimensional lentoid bodies. Inhibition of BMP signaling by noggin triggered differentiation of hESCs toward neuroectoderm. Subsequent reactivation of BMP and activation of FGF signaling stimulated formation of lens progenitor cells marked by the expression of PAX6 and alpha-crystallins. The formation of lentoid bodies was most efficient in the presence of FGF2 and Wnt-3a, yielding approximately 1000 lentoid bodies/30-mm well. Lentoid bodies expressed and accumulated lens-specific markers including alphaA-, alphaB-, beta-, and gamma-crystallins, filensin, CP49, and MIP/aquaporin 0. Collectively, these studies identify a novel procedure to generate lens cells from hESCs that can be applied for studies of lens differentiation and cataractogenesis using induced pluripotent stem (iPS) cells derived from various cataract patients.
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http://dx.doi.org/10.1096/fj.10-157255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923359PMC
September 2010

Methionine sulfoxide reductase A (MsrA) restores alpha-crystallin chaperone activity lost upon methionine oxidation.

Biochim Biophys Acta 2009 Dec 3;1790(12):1665-72. Epub 2009 Sep 3.

Biomedical Sciences Department, Charles E. Schmidt College of Biomedical Science, Florida Atlantic University, Boca Raton, FL, USA.

Background: Lens cataract is associated with protein oxidation and aggregation. Two proteins that cause cataract when deleted from the lens are methionine sulfoxide reductase A (MsrA) that repairs protein methionine sulfoxide (PMSO) oxidized proteins and alpha-crystallin which is a two-subunit (alphaA and alphaB) chaperone. Here, we tested whether PMSO formation damages alpha-crystallin chaperone function and whether MsrA could repair PMSO-alpha-crystallin.

Methods: Total alpha-crystallin was oxidized to PMSO and evaluated by CNBr-cleavage and mass spectrometry. Chaperone activity was measured by light scattering using lysozyme as target. PMSO-alpha-crystallin was treated with MsrA, and repair was assessed by CNBr cleavage, mass spectrometry and recovery of chaperone function. The levels of alpha-crystallin-PMSO in the lenses of MsrA-knockout relative to wild-type mice were determined.

Results: PMSO oxidation of total alpha-crystallin (met 138 of alphaA and met 68 of alphaB) resulted in loss of alpha-crystallin chaperone activity. MsrA treatment of PMSO-alpha-crystallin repaired its chaperone activity through reduction of PMSO. Deletion of MsrA in mice resulted in increased levels of PMSO-alpha-crystallin.

Conclusions: Methionine oxidation damages alpha-crystallin chaperone function and MsrA can repair PMSO-alpha-crystallin restoring its chaperone function. MsrA is required for maintaining the reduced state of alpha-crystallin methionines in the lens.

Significance: Methionine oxidation of alpha-crystallin in combination with loss of MsrA repair causes loss of alpha-crystallin chaperone function. Since increased PMSO levels and loss of alpha-crystallin function are hallmarks of cataract, these results provide insight into the mechanisms of cataract development and likely those of other age-related diseases.
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http://dx.doi.org/10.1016/j.bbagen.2009.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783866PMC
December 2009

Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies.

Blood 2008 Sep 10;112(6):2232-41. Epub 2008 Jul 10.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

We report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 x10(6)/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.
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http://dx.doi.org/10.1182/blood-2008-03-143636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2532801PMC
September 2008

Mitochondrial function and redox control in the aging eye: role of MsrA and other repair systems in cataract and macular degenerations.

Exp Eye Res 2009 Feb 7;88(2):195-203. Epub 2008 Jun 7.

Biomedical Sciences Department, Charles E. Schmidt College of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USA.

Oxidative stress occurs when the level of prooxidants exceeds the level of antioxidants in cells resulting in oxidation of cellular components and consequent loss of cellular function. Oxidative stress is implicated in wide range of age-related disorders including Alzheimer's disease, Parkinson's disease amyotrophic lateral sclerosis (ALS), Huntington's disease and the aging process itself. In the anterior segment of the eye, oxidative stress has been linked to lens cataract and glaucoma while in the posterior segment of the eye oxidative stress has been associated with macular degeneration. Key to many oxidative stress conditions are alterations in the efficiency of mitochondrial respiration resulting in superoxide (O(2)(-)) production. Superoxide production precedes subsequent reactions that form potentially more dangerous reactive oxygen species (ROS) species such as the hydroxyl radical (OH), hydrogen peroxide (H(2)O(2)) and peroxynitrite (OONO(-)). The major source of ROS in the mitochondria, and in the cell overall, is leakage of electrons from complexes I and III of the electron transport chain. It is estimated that 0.2-2% of oxygen taken up by cells is converted to ROS, through mitochondrial superoxide generation, by the mitochondria. Generation of superoxide at complexes I and III has been shown to occur at both the matrix side of the inner mitochondrial membrane and the cytosolic side of the membrane. While exogenous sources of ROS such as UV light, visible light, ionizing radiation, chemotherapeutics, and environmental toxins may contribute to the oxidative milieu, mitochondria are perhaps the most significant contribution to ROS production affecting the aging process. In addition to producing ROS, mitochondria are also a target for ROS which in turn reduces mitochondrial efficiency and leads to the generation of more ROS in a vicious self-destructive cycle. Consequently, the mitochondria have evolved a number of antioxidant and key repair systems to limit the damaging potential of free oxygen radicals and to repair damaged proteins (Fig. 1). The aging eye appears to be at considerable risk from oxidative stress. This review will outline the potential role of mitochondrial function and redox balance in age-related eye diseases, and detail how the methionine sulfoxide reductase (Msr) protein repair system and other redox systems play key roles in the function and maintenance of the aging eye.
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http://dx.doi.org/10.1016/j.exer.2008.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683477PMC
February 2009