Chaperone-Mediated Autophagy Regulates Hypoxic Pathology in Cardiomyocytes.

Tight regulation of protein degradation pathways is essential for maintaining cardiac homeostasis. The goal of this work was to define the role of chaperone-mediated autophagy (CMA), in cardiomyocytes. CMA acts as a selective degradation pathway of proteins using a cytosolic and lysosomal co-chaperone, HSPA8/HSC70, and the CMA-specific LAMP2A (lysosomal-associated membrane protein 2A) receptor.

Comprehensive Analysis of Proteasomal Complexes in Mouse Brain Regions Detects ENO2 as a Potential Partner of the Proteasome in the Striatum.

Defects in the activity of the proteasome or its regulators are linked to several pathologies, including neurodegenerative diseases. We hypothesize that proteasome heterogeneity and its selective partners vary across brain regions and have a significant impact on proteasomal catalytic activities. Using neuronal cell cultures and brain tissues obtained from mice, we compared proteasomal activities from two distinct brain regions affected in neurodegenerative diseases, the striatum and the hippocampus.

Promoting evaluation capacity building in a complex adaptive system.

This study provides results from an NSF funded, four year, case study about evaluation capacity building in a complex adaptive system, the Nanoscale Informal Science Education Network (NISE Net). The results of the Complex Adaptive Systems as a Model for Network Evaluations (CASNET) project indicate that complex adaptive system concepts help to explain evaluation capacity building in a network. The NISE Network was found to be a complex learning system that was supportive of evaluation capacity building through feedback loops that provided for information sharing and interaction.

Macroautophagy and Chaperone-Mediated Autophagy in Heart Failure: The Known and the Unknown.

Cardiac diseases including hypertrophic and ischemic cardiomyopathies are increasingly being reported to accumulate misfolded proteins and damaged organelles. These findings have led to an increasing interest in protein degradation pathways, like autophagy, which are essential not only for normal protein turnover but also in the removal of misfolded and damaged proteins. Emerging evidence suggests a previously unprecedented role for autophagic processes in cardiac physiology and pathology.

Safety and activity of microRNA-loaded minicells in patients with recurrent malignant pleural mesothelioma: a first-in-man, phase 1, open-label, dose-escalation study.

Background: TargomiRs are minicells (EnGeneIC Dream Vectors) loaded with miR-16-based mimic microRNA (miRNA) and targeted to EGFR that are designed to counteract the loss of the miR-15 and miR-16 family miRNAs, which is associated with unsuppressed tumour growth in preclinical models of malignant pleural mesothelioma. We aimed to assess the safety, optimal dosing, and activity of TargomiRs in patients with malignant pleural mesothelioma.

Methods: In this first-in-man, open-label, dose-escalation phase 1 trial at three major cancer centres in Sydney (NSW, Australia), we recruited adults (aged ≥18 years) with a confirmed diagnosis of malignant pleural mesothelioma, measurable disease, radiological signs of progression after previous chemotherapy, Eastern Cooperative Oncology Group performance status of 0 or 1, life expectancy of 3 months or more, immunohistochemical evidence of tumour EGFR expression, and adequate bone marrow, liver, and renal function.

Targeted Doxorubicin Delivery to Brain Tumors via Minicells: Proof of Principle Using Dogs with Spontaneously Occurring Tumors as a Model.

Background: Cytotoxic chemotherapy can be very effective for the treatment of cancer but toxicity on normal tissues often limits patient tolerance and often causes long-term adverse effects. The objective of this study was to assist in the preclinical development of using modified, non-living bacterially-derived minicells to deliver the potent chemotherapeutic doxorubicin via epidermal growth factor receptor (EGFR) targeting. Specifically, this study sought to evaluate the safety and efficacy of EGFR targeted, doxorubicin loaded minicells (designated EGFRminicellsDox) to deliver doxorubicin to spontaneous brain tumors in 17 companion dogs; a comparative oncology model of human brain cancers.

A First-Time-In-Human Phase I Clinical Trial of Bispecific Antibody-Targeted, Paclitaxel-Packaged Bacterial Minicells.

Background: We have harnessed a novel biological system, the bacterial minicell, to deliver cancer therapeutics to cancer cells. Preclinical studies showed that epidermal growth factor receptor (EGFR)-targeted, paclitaxel-loaded minicells (EGFRminicellsPac) have antitumor effects in xenograft models. To examine the safety of the minicell delivery system, we initiated a first-time-in-human, open-label, phase I clinical study of EGFRminicellsPac in patients with advanced solid tumors.

Enhanced autophagy ameliorates cardiac proteinopathy.

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein.

Posttranslational modification and quality control.

Protein quality control functions to minimize the level and toxicity of misfolded proteins in the cell. Protein quality control is performed by intricate collaboration among chaperones and target protein degradation. The latter is performed primarily by the ubiquitin-proteasome system and perhaps autophagy.

Targeted nanoparticle delivery of doxorubicin into placental tissues to treat ectopic pregnancies.

Abnormal trophoblast growth can cause life-threatening disorders such as ectopic pregnancy, choriocarcinoma, and placenta accreta. EnGeneIC Delivery Vehicles (EDVs) are nanocells that can promote tissue-specific delivery of drugs and may be useful to medically treat such disorders. The objective of this study was to determine whether EDVs loaded with the chemotherapeutic doxorubicin and targeting the epidermal growth factor receptor (EGFR, very highly expressed on the placental surface) can regress placental cells in vitro, ex vivo, and in vivo.

Macrophage autophagy plays a protective role in advanced atherosclerosis.

In advanced atherosclerosis, macrophage apoptosis coupled with defective phagocytic clearance of the apoptotic cells (efferocytosis) promotes plaque necrosis, which precipitates acute atherothrombotic cardiovascular events. Oxidative and endoplasmic reticulum (ER) stress in macrophages are important causes of advanced lesional macrophage apoptosis. We now show that proapoptotic oxidative/ER stress inducers trigger another stress reaction in macrophages, autophagy.

Autophagy and proteotoxicity in cardiomyocytes.

Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. We wished to determine if autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes. We used loss- and gain-of-function approaches in cultured cardiomyocytes to determine the effects of ATG7 knockdown and Atg7 overexpression in protein conformation-based toxicity induced by expression of a mutant aB crystallin (CryAB (R120G) ) known to cause human heart disease.

Atg7 induces basal autophagy and rescues autophagic deficiency in CryABR120G cardiomyocytes.

Rationale: Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. Several cardiomyopathies, including the αB-crystallin R120G mutation (CryAB(R120G)) model of desmin-related cardiomyopathy, accumulate cytotoxic misfolded proteins in the form of preamyloid oligomers and aggresomes. Impaired autophagic function is a potential cause of misfolded protein accumulations, cytoplasmic aggregate loads, and cardiac disease.

Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug.

The dose-limiting toxicity of chemotherapeutics, heterogeneity and drug resistance of cancer cells, and difficulties of targeted delivery to tumors all pose daunting challenges to effective cancer therapy. We report that small interfering RNA (siRNA) duplexes readily penetrate intact bacterially derived minicells previously shown to cause tumor stabilization and regression when packaged with chemotherapeutics. When targeted via antibodies to tumor-cell-surface receptors, minicells can specifically and sequentially deliver to tumor xenografts first siRNAs or short hairpin RNA (shRNA)-encoding plasmids to compromise drug resistance by knocking down a multidrug resistance protein.

Protein misfolding and cardiac disease: establishing cause and effect.

Numerous neurodegenerative diseases are characterized by the accumulation of misfolded amyloidogenic proteins. Recent data indicate that a soluble pre-amyloid oligomer (PAO) may be the toxic entity in these diseases and the visible amyloid plaques, rather than causing the disease, may simply mark the terminal pathology. In prior studies, we observed PAO in the cardiomyocytes of many human heart failure samples.

Cardiomyocyte expression of a polyglutamine preamyloid oligomer causes heart failure.

Background: To determine whether soluble preamyloid oligomers (PAOs) are toxic when expressed internally in the cardiomyocyte, we tested the hypothesis that cardiomyocyte-restricted expression and accumulation of a known PAO is cytotoxic and sufficient to cause heart failure.

Methods And Results: Intracellular PAOs, the entities believed to cause toxicity in many neurodegenerative diseases, have been observed in cardiomyocytes derived from mouse and human heart failure samples. Long (>50) polyglutamine (PQ) repeats form PAOs and cause neurotoxicity in Huntington disease and other neurodegenerative diseases, whereas shorter PQ peptides are benign.

Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics.

Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of chemotherapeutics in bacterially derived 400 nm minicells. We discovered that minicells can be packaged with therapeutically significant concentrations of chemotherapeutics of differing charge, hydrophobicity, and solubility.

Expression profiling identifies dysregulation of myosin heavy chains IIb and IIx during limb immobilization in the soleus muscles of old rats.

Aged individuals suffer from multiple dysfunctions during skeletal muscle atrophy. The purpose of this study was to determine differential changes in gene expression in atrophied soleus muscle induced by hindlimb immobilization in young (3-4 months) and old (30-31 months) rats. The hypothesis was that differentially expressed mRNAs with age-atrophy interactions would reveal candidates that induce loss of function responses in aged animals.

Selected Contribution: Identification of differentially expressed genes between young and old rat soleus muscle during recovery from immobilization-induced atrophy.

After cessation of hindlimb immobilization, which resulted in a 27-37% loss in soleus mass, the atrophied soleus muscle of young but not old rats regrows to its mass before treatment. We hypothesized that during remobilization the mRNA levels of growth potentiating factor(s) would be present in the soleus muscle of young (3- to 4-mo-old) but absent in old (30- to 31-mo-old) Fischer 344 x Brown Norway rats or that mRNAs for growth inhibitory factor(s) would be absent in young but present in old. Gene expression levels of >24,000 transcripts were determined by using Affymetrix RGU34A-C high-density oligonucleotide microarrays in soleus muscles at 3, 6, 10, and 30 days of remobilization after cessation of a 10-day period of hindlimb immobilization.

Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle.

The direction of change in skeletal muscle mass differs between young and old individuals, growing in young animals and atrophying in old animals. The purpose of the experiment was to develop a statistically conservative list of genes whose expression differed significantly between young growing and old atrophying (sarcopenic) skeletal muscles, which may be contributing to physical frailty. Gene expression levels of >24,000 transcripts were determined in soleus muscle samples from young (3-4 mo) and old (30-31 mo) rats.

In vivo ligand-inducible regulation of gene expression in a gutless adenoviral vector system.

Transcriptional regulation that is rapid, reversible, and repeatedly inducible would greatly enhance the safety and efficacy of many gene therapy strategies. We developed a chimeric ligand-inducible regulation system based on the human estrogen receptor. This system has two components, the responsive promoter driving expression of the transgene of interest, and the ligand-inducible chimeric transcription factor.

Optimization of the generation and propagation of gutless adenoviral vectors.

Adenoviral vectors devoid of all viral coding regions are referred to by many names, including gutless vectors. Gutless vectors display reduced toxicity and immunogenicity, increased duration of transgene expression, and increased coding capacity compared to early generation vectors, which contain the majority of the viral backbone genes. However, the production of gutless vectors at a scale and purity suitable for clinical use has limited the utility of this technology.