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1.  Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research 
Annals of Biomedical Engineering  2012;40(2):277-291.
Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality.
PMCID: PMC3342697  PMID: 22258888
Two-photon microscopy; Cancer; Second harmonic generation; Collagen; SHG; Endoscopy
2.  Mitochondrial membrane potential probes and the proton gradient: a practical usage guide 
BioTechniques  2011;50(2):98-115.
Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.
PMCID: PMC3115691  PMID: 21486251
Mitochondria; hyperpolarization; depolarization; membrane potential; calcium; proton; cationic; fluorescent; probe; dye; pH
3.  HIV-1 Tat activates calpain proteases via the ryanodine receptor, to enhance surface DA transporter levels and increase transporter-specific uptake and Vmax 
HIV-associated neurologic disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy (cART). The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, “reversible” DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging HIV-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson’s Disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT Vmax, without changes in Km or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by TIRF microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3β. We conclude that Tat activates RyRs via a calcium and calpain mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3β inhibition as clinical therapeutic approaches for HAND. Finally we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients, and demonstrating similar findings in Tat-expressing transgenic mice.
PMCID: PMC2972730  PMID: 20962236
HIV; AIDS; brain; dopamine; transporter; Tat
4.  Tumor-associated macrophages and stromal TNF-α regulate collagen structure in a breast tumor model as visualized by second harmonic generation 
Journal of Biomedical Optics  2013;18(8):086003.
Collagen fibers can be imaged with second harmonic generation (SHG) and are associated with efficient tumor cell locomotion. Preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype, and changes in SHG emission properties accompany changes in metastatic outcome. We therefore attempted to elucidate the cellular and molecular machinery that influences SHG in order to understand how the microstructure of tumor collagen fibers is regulated. By quantifying SHG and immunofluorescence (IF) from tumors grown in mice with and without stromal tumor necrosis factor (TNF)-α and in the presence or absence of tumor-associated macrophages (TAMs), we determined that depletion of TAMs alters tumor collagen fibrillar microstructure as quantified by SHG and IF. Furthermore, we determined that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar microstructure and that subsequent depletion of TAMs has no further effect. In each case, metastatic burden correlated with optical readouts of collagen microstructure. Our results implicate TAMs and stromal TNF-α as regulators of breast tumor collagen microstructure and suggest that this regulation plays a role in tumor metastasis. Furthermore, these results indicate that quantification of SHG represents a useful strategy for evaluating the cells and molecular pathways responsible for manipulating fibrillar collagen in breast tumor models.
PMCID: PMC3731198  PMID: 23912760
second harmonic generation; cancer; extracellular matrix; collagen; metastasis; multiphoton microscopy
5.  Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells 
BMC Cancer  2013;13:411.
Matrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen’s organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer.
Tumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann–Whitney tests as appropriate.
We found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs.
These results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast cancer pathology under some conditions, a finding with important implications for development of chemotherapies directed against matrix metalloproteases.
PMCID: PMC3766650  PMID: 24010522
Two-photon; Microscopy; Cancer; Second harmonic generation; Collagen; SHG; Tumor; MMP; Matrix metalloprotease; MMP-13; Intravital; Imaging; In vivo; Multiphoton; Intrinsic; Fluorophore
6.  Measuring intranodal pressure and lymph viscosity to elucidate mechanisms of arthritic flare and therapeutic outcomes 
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints, whose etiology is largely unknown. Recent studies in mice demonstrated alterations in lymphatics from affected joints precede flares. Thus, we aimed to develop novel methods for measuring lymph node pressure and lymph viscosity in limbs of mice. Pressure measurements were performed by inserting a glass micropipette connected to a pressure transducer into popliteal lymph nodes (PLN) or axillary lymph nodes (ALN) of mice and determined that the lymphatic pressures were 9 and 12 cm of water, respectively. We are also developing methods for measuring lymph viscosity in lymphatic vessels afferent to PLN, which can be measured by multi-photon fluorescence recovery after photobleaching (MP-FRAP) of FITC-BSA injected into the hind footpad. These results demonstrate the potential of lymph node pressure and lymph viscosity measurements, and warrant future studies to test these outcomes as biomarkers of arthritic flare.
PMCID: PMC3334848  PMID: 22172039
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
7.  Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production 
PLoS ONE  2010;5(7):e11875.
Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorder (HAND) is likely neuroinflammatory in origin, believed to be triggered by inflammatory and oxidative stress responses to cytokines and HIV protein gene products such as the HIV transactivator of transcription (Tat). Here we demonstrate increased messenger RNA for nuclear factor-kappa B (NF-κB) family member, transcription factor RelB, in the brain of doxycycline-induced Tat transgenic mice, and increased RelB synthesis in Tat-exposed microglial cells. Since genetic ablation of RelB in mice leads to multi-organ inflammation, we hypothesized that Tat-induced, newly synthesized RelB inhibits cytokine production by microglial cells, possibly through the formation of transcriptionally inactive RelB/RelA complexes. Indeed, tumor necrosis factor-alpha (TNFα) production in monocytes isolated from RelB deficient mice was significantly higher than in monocytes isolated from RelB expressing controls. Moreover, RelB overexpression in microglial cells inhibited Tat-induced TNFα synthesis in a manner that involved transcriptional repression of the TNFα promoter, and increased phosphorylation of RelA at serine 276, a prerequisite for increased RelB/RelA protein interactions. The Rel-homology-domain within RelB was necessary for this interaction. Overexpression of RelA itself, in turn, significantly increased TNFα promoter activity, an effect that was completely blocked by RelB overexpression. We conclude that RelB regulates TNFα cytokine synthesis by competitive interference binding with RelA, which leads to downregulation of TNFα production. Moreover, because Tat activates both RelB and TNFα in microglia, and because Tat induces inflammatory TNFα synthesis via NF-κB, we posit that RelB serves as a cryoprotective, anti-inflammatory, counter-regulatory mechanism for pathogenic NF-κB activation. These findings identify a novel regulatory pathway for controlling HIV-induced microglial activation and cytokine production that may have important therapeutic implications for the management of HAND.
PMCID: PMC2912378  PMID: 20686703
8.  HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization 
PLoS ONE  2008;3(11):e3731.
Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.
PMCID: PMC2579580  PMID: 19009018

Results 1-8 (8)