Apoptosis.

Caenorhabditis elegans optionally enter into a dauer diapause phase that results
in a prolonged life in a semi-dormant state. Entry into and recovery from dauer diapause
includes many physical changes in body structure, physiology, and gene expression.
Entry into dauer diapause is regulated by several signaling pathways including
transforming growth factor (TGF-β). Autophagy plays an important role in dauer
formation and recover. During dauer transformation autophagy is up-regulated and may
play a role in remodeling the molecular structure for long term survival during dauer
diapause. This research helps determine the role of autophagy in dauer development and
recovery mediated through the TGF-β signaling pathway. This research also determines
in which tissue autophagy is necessary for dauer formation and recovery through TGF-β signaling. This research is shedding light on the function of autophagy in the TGF-β
signaling pathway, both processes of which have been linked to tumorigenesis, heart
disease and cancer.

Marine sponges interact and coexist with many different organisms. A two-sponge
association between Amphimedon erina and Geodia gibberosa commonly occurs in the
Florida Keys. Previous studies have only focused on the ecological influence of the
association; they did not examine the cellular basis of the association. This association
between A. erina and G. gibberosa was used in the development of an in vitro model to
further the understanding of the cellular basis of natural sponge-sponge associations. In
this study, sponge cells were cultured individually and in co-cultures and their responses
related to apoptosis, cell death, and proliferation were monitored using high content
imaging. Co-cultured cells of species that form sponge-sponge associations did not have
the same cellular responses compared to co-cultured cells of species that do not form
sponge-sponge associations. Protein expression analyses demonstrated that the model that
was established does not mimic the cellular response of the association in nature, but this
model can be used to test in vitro cellular interactions of sponge species that do not form
associations in nature. In addition, the protein expression data that were obtained revealed that sponges use similar apoptotic pathways as humans and suggest that sponge cells may
shut down cell cycling in order to repair damaged DNA. This research is a small piece to
the puzzle that is sponge cell culture research.

Studies have shown that tumor cells are susceptible to pharmacological targeting
of their altered glycolytic metabolism with a variety of compounds that result in
apoptosis. One such compound, 3-bromopyruvate (3-BP), has been shown to eradicate
cancer in an animal model. However, no studies have shown whether the apoptotic
fragments resulting from 3-BP treatment have the capacity to elicit an immunogenic cell
death that activates dendritic cells, the primary antigen presenting cell in the immune
system. Immunogenic cell death is critical to eliciting an effective adaptive immune
response that selectively kills additional target cells and generates immunological
memory. We demonstrated that 3-bromopyruvate induced apoptosis in a number of
different murine breast cancer cell lines, including the highly metastatic 4T1 line. The
dying tumor cells stimulated immature dendritic cells (DCs) of the immortal JAWS II
cell line to produce high levels of the pro-inflammatory cytokine IL-12, and increased their expression of key co-stimulatory molecules CD80 and CD86. The activated
dendritic cells showed increased uptake of fragments from dying tumor cells that
correlated with the increased levels of calreticulin on the surface and release of high
group motility box 1 (HMGB1) of the latter following 3-BP treatment. Additionally, the
anti-phagocytic signal CD47 present on breast cancer cells was reduced by treatment with
3-bromopyruvate when compared to the levels on untreated 4T1 cells. 3-BP treated breast
cancer cells were able to activate dendritic cells through TLR4 signaling. Signaling was
dependent on both the expression of surface calreticulin and on the extracellular release
of high mobility group box 1 protein (HMGB1) during the process of immunogenic cell
death. Killing by 3-BP was compared to mitoxantrone and doxorubicin, among the few
chemotherapeutics that induce immunogenic cell death. 3-BP killing was likewise
compared to camptothecin, a compound that fails to induce immunogenic cell death.
Importantly, 3-BP did not markedly decrease the levels of the key peptide presenting
molecule MHC I on DCs that were co-cultivated with dying tumor cells. Treatment of the
highly aggressive triple negative BT-20 human breast cancer cell line with 3-BP also
induced an immunogenic cell death, activating human dendritic cells in vitro.

In the round worm C. elegans, it has recently been shown that autophagy, a highly
conserved lysosomal degradation pathway that is present in all eukaryotic cells, is
required for maintaining healthspan and for increasing the adult lifespan of worms fed
under dietary restriction conditions or with reduced IGF signaling. It is currently
unknown how extracellular signals regulate autophagy activity within different tissues
during these processes and whether autophagy functions cell-autonomously or nonautonomously.
We have data that for the first time shows autophagy activity in the
neurons and intestinal cells plays a major role in regulating adult lifespan and the
longevity conferred by altered IGF signaling and dietary restriction, suggesting
autophagy can control these phenotypes cell non-autonomously. We hypothesize that
autophagy in the neurons and intestinal cells is an essential cellular process regulated by
different signaling pathways to control wild type adult lifespan, IGF mediated longevity and dietary restriction induced longevity. Excitingly we also have found that in animals
with reduced IGF signaling autophagy can control longevity in only a small subset of
neurons alone. Autophagy in either specific individual chemosensory neurons or a small
group of them is completely sufficient to control IGF mediated longevity. This work
provides novel insight to the function and regulation of autophagy which will help shed
light on understanding this essential process in higher organisms, including mammals.

Ischemic stroke has a multiplicity of pathophysiological mechanisms.
Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that
exerts a diverse range of neuroprotection against ischemic stroke. Several lines of
evidence demonstrated the contribution of endoplasmic reticulum (ER) in apoptotic cell
death involving ischemia. Cell culture of undifferentiated PC12 cells were subjected to
10mM glutamate and selected doses of G-CSF (25ng/ml, 50ng/ml, 100ng/ml and
250ng/ml) for 24 hours. Cell viability, expression of the G-CSF receptor and expression
level of CHOP were assessed in vitro. Sprague-Dawley rats were subjected to middle
cerebral artery occlusion (MCAO). Rats were subcutaneously injected with G-CSF (n=
15; 50ug/kg body weight) 24 hours post-MCAO for 4 days. Vehicle treated rats were
administered 5% dextrose for 1 day (n=4) or 4 days (n=16). Sham-operated rats (n=9)
were not subjected to MCAO. Neurological deficit and infarct volume were measured while expression levels of pAKT, Bcl2, Bax, Bak, cleaved caspase-3, GRP78, ATF4,
ATF6, p-p38MAPK, pJNK, CHOP and HSP27 were analyzed by western blotting. In
vitro G-CSF receptor was expressed on undifferentiated PC12 cell, and an optimal dose
of 50 ng/ml G-CSF significantly protected these cells against glutamate-induced
cytotoxicity (P < 0.05). G-CSF significantly down-regulated (P < 0.01) the ER stressinduced
pro-apoptotic marker CHOP in vitro. In vivo, G-CSF reduced infarct volume to
50% while significantly improved neurological deficit compared to vehicle rats. G-CSF
significantly (P < 0.05) up-regulated pro-survival proteins pAKT and Bcl2 while downregulating
pro-apoptotic proteins Bax, Bak and cleaved caspase 3 in the ischemic brain.
It also significantly (P < 0.05) downregulated the ER intraluminal stress sensor GRP78,
proteins of ER stress induced intracellular pathway; ATF4, ATF6, p-p38MAPK, pJNK
and the ER stress induced apoptotic marker CHOP, which suggests that ER stress is
being ameliorated by G-CSF treatment. G-CSF also reduced the level of HSP27,
providing additional evidence of cellular stress reduction. G-CSF treatment increased
cell survival by attenuating both general pro-apoptotic proteins and specific effector
proteins in the ER stress induced apoptotic pathways. Our data has provided new insight
into the anti-apoptotic mechanism of G-CSF, especially as it relates to ER stress induced
apoptosis in ischemia.

The vertebrate eye lens functions to focus light onto the retina to produce vision.
The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a
core of organelle free fiber cells. The lens develops and grows throughout life by the
successive layering of lens fiber cells via their differentiation from lens epithelial cells.
Lens developmental defect and damage to the lens are associated with cataract formation,
an opacity of the lens that is a leading cause of visual impairment worldwide. The only
treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms
that regulate the development and lifelong protection of the lens is critical toward the
development of future therapies to prevent or treat cataract. To determine those
molecules and mechanisms that may be important for these lens requirements we
employed high-throughput RNA sequencing of microdissected differentiation statespecific
lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified
differentiation state-specific molecules that regulate mitochondrial populations between
lens epithelial cells that require the maintenance of a functional population of
mitochondria and lens fiber cells that must eliminate their mitochondria for their
maturation. In addition, we discovered a novel mechanism for how lens epithelial cells
clear apoptotic cell debris that could arise from damage to the lens and found that UVlight
likely compromises this system. Moreover, the data herein provide a framework to
determine novel lens cell differentiation state-specific mechanisms. Future studies are
required to determine the requirements of the identified molecules and mechanisms
during lens development, lens defense against damage, and cataract formation.

Matrix metalloproteinases (MMPs) constitute the major class of enzymes capable
of degrading all protein components of extracellular matrix (ECM) and have important
roles in normal physiologic processes of maintaining tissue integrity and remodeling.
However, excess MMP activities are associated with many diseases including rheumatoid
arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of
MMPs is regulated by their endogenous protein inhibitors, the tissue inhibitors of
metalloproteinases (TIMPs) which are avid broad-spectrum inhibitors of numerous
human matrixins (MMPs and ADAMs). Uncontrolled matrix degradation occurs when
the balance between TIMPs and MMPs is disrupted, resulting in serious diseases such as
cancer, arthritis and chronic tissue ulcers. Thus, the engineering of TIMPs to produce
highly selective and efficacious inhibitors of individual MMPs may be utilized for future
treatment of diseases. Such engineering requires detailed analysis for the structural and
biophysical information of MMP-TIMP interaction. Changes in the dynamics of proteins and solvent that accompany their
associations with different binding partners, influence the specificity of binding through
entropic effects. From the current studies it appears that the interactions of the inhibitory
domains of TIMPs-1 and -2 (N-TIMPs) with MT1-MMP are driven by entropy increases
that are partitioned between solvent and conformational entropy (ΔSsolv and ΔSconf), and a
large conformational entropy penalty is responsible for the weak inhibition of MT1-MMP
by NT1.We investigated how mutations that modify N-TIMP selectivity affect the
thermodynamics of interactions with MMP1, MMP3 and MT1-MMP. The weak
inhibition of MT1-MMP by N-TIMP-1 is enhanced by mutation of threonine 98, on the
edge of the binding ridge, to leucine. This mutation increases the large ΔSconf cost for
binding to MT1-MMP but this is offset by a greater increase in ΔSsolv. In contrast, this
mutation enhances binding to MMP3 by increasing ΔSconf for the interaction. ΔSsolv and
ΔSconf show mutual compensation for all interactions, with characteristic ranges for each
MMP. Distinct electrostatic and dynamic features of MMPs are key factors in their
selective inhibition.