Effects of doxorubicin on autophagy in fibroblasts.

Objectives: Doxorubicin (DOX) is a highly effective chemotherapeutic used to treat many adult and pediatric cancers, such as solid tumors, leukemia, lymphomas and breast cancer. It can also cause injuries to multiple organs, including the heart, liver, and brain or kidney, although cardiotoxicity is the most prominent side effect of DOX. In this study, we examined the potential effects of DOX on autophagy activity in two different mouse fibroblasts.Methods: Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX to assess changes in the expression of two commonly used autophagy protein markers, LC3II and p62. We also examined the effects of DOX the on expression of key genes that encode components of the molecular machinery and regulators modulating autophagy in response to both extracellular and intracellular signals.Results: We observed that LC3II levels increased and p62 levels decreased following the DOX treatment in NIH3T3 cells. However, similar effects were not observed in primary cardiac fibroblasts. In addition, DOX treatment induced the upregulation of a significant number of genes involved in autophagy in NIH3T3 cells, but not in primary cardiac fibroblasts.Conclusions: Taken together, these results indicate that DOX upregulates autophagy in fibroblasts in a cell-specific manner.

Age-dependent changes in collagen crosslinks reduce the mechanical toughness of human meniscus.

The mechanical resilience of the knee meniscus is provided by a group of structural proteins in the extracellular matrix. Aging can alter the quantity and molecular structure of these proteins making the meniscus more susceptible to debilitating tears. In this study, we determined the effect of aging on the quantity of structural proteins and collagen crosslinks in human lateral meniscus, and examined whether the quantity of these molecules was predictive of tensile toughness (area under the stress-strain curve). Two age groups were tested: a young group under 40 and an older group over 65 years old. Using mass spectrometry, we quantified the abundance of proteins and collagen crosslinks in meniscal tissue that was adjacent to the dumbbell-shaped specimens used to measure uniaxial tensile toughness parallel or perpendicular to the circumferential fiber orientation. We found that the enzymatic collagen crosslink deoxypyridinoline had a significant positive correlation with toughness, and reductions in the quantity of this crosslink with aging were associated with a loss of toughness in the ground substance and fibers. The non-enzymatic collagen crosslink carboxymethyl-lysine increased in quantity with aging, and these increases corresponded to reductions in ground substance toughness. For the collagenous (Types I, II, IV, VI, VIII) and non-collagenous structural proteins (elastin, decorin, biglycan, prolargin) analyzed in this study, only the quantity of collagen VIII was predictive of toughness. This study provides valuable insights on the structure-function relationships of the human meniscus, and how aging causes structural adaptations that weaken the tissue's mechanical integrity.

DockoMatic 2.0: high throughput inverse virtual screening and homology modeling.

DockoMatic is a free and open source application that unifies a suite of software programs within a user-friendly graphical user interface (GUI) to facilitate molecular docking experiments. Here we describe the release of DockoMatic 2.0; significant software advances include the ability to (1) conduct high throughput inverse virtual screening (IVS); (2) construct 3D homology models; and (3) customize the user interface. Users can now efficiently setup, start, and manage IVS experiments through the DockoMatic GUI by specifying receptor(s), ligand(s), grid parameter file(s), and docking engine (either AutoDock or AutoDock Vina). DockoMatic automatically generates the needed experiment input files and output directories and allows the user to manage and monitor job progress. Upon job completion, a summary of results is generated by Dockomatic to facilitate interpretation by the user. DockoMatic functionality has also been expanded to facilitate the construction of 3D protein homology models using the Timely Integrated Modeler (TIM) wizard. The wizard TIM provides an interface that accesses the basic local alignment search tool (BLAST) and MODELER programs and guides the user through the necessary steps to easily and efficiently create 3D homology models for biomacromolecular structures. The DockoMatic GUI can be customized by the user, and the software design makes it relatively easy to integrate additional docking engines, scoring functions, or third party programs. DockoMatic is a free comprehensive molecular docking software program for all levels of scientists in both research and education.

Osteochondritis dissecans knee histology studies have variable findings and theories of etiology.

BACKGROUND: Although many etiological theories have been proposed for osteochondritis dissecans (OCD), its etiology remains unclear. Histological analysis of the articular cartilage and subchondral bone tissues of OCD lesions can provide useful information about the cellular changes and progression of OCD. Previous research is predominantly comprised of retrospective clinical studies from which limited conclusions can be drawn. QUESTIONS/PURPOSES: The purposes of this study were threefold: (1) Is osteonecrosis a consistent finding in OCD biopsy specimens? (2) Is normal articular cartilage a consistent finding in OCD biopsy specimens? (3) Do histological studies propose an etiology for OCD based on the tissue findings? METHODS: We searched the PubMed, Embase, and CINAHL databases for studies that conducted histological analyses of OCD lesions of the knee and identified 1560 articles. Of these, 11 met our inclusion criteria: a study of OCD lesions about the knee, published in the English language, and performed a histological analysis of subchondral bone and articular cartilage. These 11 studies were assessed for an etiology proposed in the study based on the study findings. RESULTS: Seven of 11 studies reported subchondral bone necrosis. Four studies reported normal articular cartilage, two studies reported degenerated or irregular articular cartilage, and five studies found a combination of normal and degenerated or irregular articular cartilage. Five studies proposed trauma or repetitive stress and two studies proposed poor blood supply as possible etiologies. CONCLUSIONS: We found limited research on histological analysis of OCD lesions of the knee. Future studies with consistent methodology are necessary to draw major conclusions about the histology and progression of OCD lesions. Inconsistent histologic findings have resulted in a lack of consensus regarding the presence of osteonecrosis, whether the necrosis is primary or secondary, the association of cartilage degeneration, and the etiology of OCD. Such studies could use a standardized grading system to allow better comparison of findings.

Structure-Property-Processing Correlations of Graphene Bioscaffolds for Proliferation and Differentiation of C2C12 Cells.

Graphene - an atomically thin layer of carbon atoms arranged in a hexagonal lattice - has gained interest as a bioscaffold for tissue engineering due to its exceptional mechanical, electrical, and thermal properties. Graphene's structure and properties are tightly coupled to synthesis and processing conditions, yet their influence on biomolecular interactions at the graphene-cell interface remains unclear. In this study, C2C12 cells were grown on graphene bioscaffolds with specific structure-property- processing-performance (SP3) correlations. Bioscaffolds were prepared using three different methods - chemical vapor deposition (CVD), sublimation of silicon carbide (SiC), and printing of liquid phase exfoliated graphene. To investigate the biocompatibility of each scaffold, cellular morphology and gene expression patterns were investigated using the bipotential mouse C2C12 cell line. Using a combination of fluorescence microscopy and qRT-PCR, we demonstrate that graphene production methods determine the structural and mechanical properties of the resulting bioscaffold, which in turn determine cell morphology, gene expression patterns, and cell differentiation fate. Therefore, production methods and resultant structure and properties of graphene bioscaffolds must be chosen carefully when considering graphene as a bioscaffold for musculoskeletal tissue engineering.

An amino-terminal fragment of apolipoprotein E4 leads to behavioral deficits, increased PHF-1 immunoreactivity, and mortality in zebrafish.

Although the increased risk of developing sporadic Alzheimer's disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE41-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE41-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity and inflammation. In the present study, we extend these findings to zebrafish (Danio rerio), an in vivo model system to assess the toxicity of nApoE41-151. Exogenous treatment of nApoE41-151 to 24-hour post-fertilization for 24 hours resulted in significant mortality. In addition, developmental abnormalities were observed following treatment with nApoE41-151 including improper folding of the hindbrain, delay in ear development, deformed yolk sac, enlarged cardiac cavity, and significantly lower heart rates. A similar nApoE31-151 fragment that differs by a single amino acid change (C>R) at position 112 had no effects on these parameters under identical treatment conditions. Decreased presence of pigmentation was noted for both nApoE31-151- and nApoE41-151-treated larvae compared with controls. Behaviorally, touch-evoked responses to stimulus were negatively impacted by treatment with nApoE41-151 but did not reach statistical significance. Additionally, triple-labeling confocal microscopy not only confirmed the nuclear localization of the nApoE41-151 fragment within neuronal populations following exogenous treatment, but also identified the presence of tau pathology, one of the hallmark features of AD. Collectively, these in vivo data demonstrating toxicity as well as sublethal effects on organ and tissue development support a novel pathophysiological function of this AD associated-risk factor.

Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion.

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated ß-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging.

A Cold Atmospheric Pressure Plasma Discharge Device Exerts Antimicrobial Effects.

A cold atmospheric pressure plasma device was developed using two parallel plates of Low Temperature Co-fired Ceramic with embedded electrodes. The 2.4 cm wide by 1 mm deep plasma discharge operates at 20 kHz with a 2-5 kV AC drive signal across a 0.25 mm gap. Mixed Argon/oxygen plasmas were directed between the plates to flow toward a bacterial biofilm sample for treatment. Results showed that at 4-5 kV the plasma etched away a bacterial biofilm on glass in 10 minutes. In addition, we showed that short plasma treatments rapidly killed biofilm resident bacteria with ED90 values of <15 s.

Investigation of multiphasic 3D-bioplotted scaffolds for site-specific chondrogenic and osteogenic differentiation of human adipose-derived stem cells for osteochondral tissue engineering applications.

Osteoarthritis is a degenerative joint disease that limits mobility of the affected joint due to the degradation of articular cartilage and subchondral bone. The limited regenerative capacity of cartilage presents significant challenges when attempting to repair or reverse the effects of cartilage degradation. Tissue engineered medical products are a promising alternative to treat osteochondral degeneration due to their potential to integrate into the patient's existing tissue. The goal of this study was to create a scaffold that would induce site-specific osteogenic and chondrogenic differentiation of human adipose-derived stem cells (hASC) to generate a full osteochondral implant. Scaffolds were fabricated using 3D-bioplotting of biodegradable polycraprolactone (PCL) with either ß-tricalcium phosphate (TCP) or decellularized bovine cartilage extracellular matrix (dECM) to drive site-specific hASC osteogenesis and chondrogenesis, respectively. PCL-dECM scaffolds demonstrated elevated matrix deposition and organization in scaffolds seeded with hASC as well as a reduction in collagen I gene expression. 3D-bioplotted PCL scaffolds with 20% TCP demonstrated elevated calcium deposition, endogenous alkaline phosphatase activity, and osteopontin gene expression. Osteochondral scaffolds comprised of hASC-seeded 3D-bioplotted PCL-TCP, electrospun PCL, and 3D-bioplotted PCL-dECM phases were evaluated and demonstrated site-specific osteochondral tissue characteristics. This technique holds great promise as cartilage morbidity is minimized since autologous cartilage harvest is not required, tissue rejection is minimized via use of an abundant and accessible source of autologous stem cells, and biofabrication techniques allow for a precise, customizable methodology to rapidly produce the scaffold.

AKT2 maintains brain endothelial claudin-5 expression and selective activation of IR/AKT2/FOXO1-signaling reverses barrier dysfunction.

Inflammation-induced blood-brain barrier (BBB) dysfunction and microvascular leakage are associated with a host of neurological disorders. The tight junction protein claudin-5 (CLDN5) is a crucial protein necessary for BBB integrity and maintenance. CLDN5 is negatively regulated by the transcriptional repressor FOXO1, whose activity increases during impaired insulin/AKT signaling. Owing to an incomplete understanding of the mechanisms that regulate CLDN5 expression in BBB maintenance and dysfunction, therapeutic interventions remain underdeveloped. Here, we show a novel isoform-specific function for AKT2 in maintenance of BBB integrity. We identified that AKT2 during homeostasis specifically regulates CLDN5-dependent barrier integrity in brain microvascular endothelial cells (BMVECs) and that intervention with a selective insulin-receptor (IR) agonist, demethylasterriquinone B1 (DMAQ-B1), rescued IL-1ß-induced AKT2 inactivation, FOXO1 nuclear accumulation, and loss of CLDN5-dependent barrier integrity. Moreover, DMAQ-B1 attenuated preclinical CLDN5-dependent BBB dysfunction in mice subjected to experimental autoimmune encephalomyelitis. Taken together, the data suggest a regulatory role for IR/AKT2/FOXO1-signaling in CLDN5 expression and BBB integrity during neuroinflammation.

Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation.

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, both of which are characterized by craniofacial abnormalities, nearsightedness and hearing deficiencies. Despite its link to human diseases, few studies have described factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the beta-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted suppression of Col11a1 with an antisense morpholino accelerated osteoblastic differentiation and mineralization in C2C12 cells, similar to what was observed in Lef1-suppressed MC3T3 cells. Moreover incubation with a purified Col11a1 N-terminal fragment, V1B, prevented alkaline phosphatase expression in MC3T3 and C2C12 cells. These results suggest that Lef1 is an activator of the Col11a1 promoter and that Col11a1 suppresses terminal osteoblast differentiation.

Collagen alpha1(XI) in normal and malignant breast tissue.

Little is known about collagen XI expression in normal and malignant breast tissue. Tissue microarrays, constructed from 72 patients with breast carcinoma and matched normal tissue, were immunohistochemically stained with five antisera against isoform-specific regions of collagen alpha1(XI) N-terminal domain. Staining intensity was graded on a 0-3 scale in epithelial cytoplasm, stroma, and endothelial staining of the vasculature of each tissue core. The staining was compared to known pathologic parameters: age, tumor size, overall tumor grade, nuclear grade, tubule formation, mitotic counts, angiolymphatic invasion, node status, estrogen receptor status, progesterone receptor status, and HER-2/neu status. Estrogen and progesterone receptor status were used as a control for comparison. With antisera V1a and amino propeptide (Npp), stroma surrounding cancerous cells was found to have decreased collagen alpha1(XI) staining compared to stroma adjacent to normal epithelium (P=0.0006, P<0.0001). Collagen alpha1(XI) staining with V1a antiserum in cytoplasm of cancer cells demonstrated decreased intensity in metastasized primary tumors when compared to nonmetastasized primary tumors (P=0.009). Cytoplasmic staining with Npp antiserum in cancer demonstrated an inverse relationship to positive estrogen receptor status in cancer (P=0.012) and to progesterone receptor status (P=0.044). Stromal staining for Npp in cancerous tissue demonstrated an inverse relationship with tubule formation score (P=0.015). This is the first study to localize collagen XI within normal and malignant breast tissue. Collagen alpha1(XI) appears to be downregulated in stroma surrounding breast cancer. Detection of collagen XI in breast tissue may help predict women who have lymph node metastases.

Mechanical Properties of Graphene Foam and Graphene Foam - Tissue Composites.

Graphene foam (GF), a 3-dimensional derivative of graphene, has received much attention recently for applications in tissue engineering due to its unique mechanical, electrical, and thermal properties. Although GF is an appealing material for cartilage tissue engineering, the mechanical properties of GF - tissue composites under dynamic compressive loads have not yet been reported. The objective of this study was to measure the elastic and viscoelastic properties of GF and GF-tissue composites under unconfined compression when quasi-static and dynamic loads are applied at strain magnitudes below 20%. The mechanical tests demonstrate a 46% increase in the elastic modulus and a 29% increase in the equilibrium modulus after 28-days of cell culture as compared to GF soaked in tissue culture medium for 24h. There was no significant difference in the amount of stress relaxation, however, the phase shift demonstrated a significant increase between pure GF and GF that had been soaked in tissue culture medium for 24h. Furthermore, we have shown that ATDC5 chondrocyte progenitor cells are viable on graphene foam and have identified the cellular contribution to the mechanical strength and viscoelastic properties of GF - tissue composites, with important implications for cartilage tissue engineering.

Clinical significance of interleukin (IL)-6 in cancer metastasis to bone: potential of anti-IL-6 therapies.

Metastatic events to the bone occur frequently in numerous cancer types such as breast, prostate, lung, and renal carcinomas, melanoma, neuroblastoma, and multiple myeloma. Accumulating evidence suggests that the inflammatory cytokine interleukin (IL)-6 is frequently upregulated and is implicated in the ability of cancer cells to metastasize to bone. IL-6 is able to activate various cell signaling cascades that include the STAT (signal transducer and activator of transcription) pathway, the PI3K (phosphatidylinositol-3 kinase) pathway, and the MAPK (mitogen-activated protein kinase) pathway. Activation of these pathways may explain the ability of IL-6 to mediate various aspects of normal and pathogenic bone remodeling, inflammation, cell survival, proliferation, and pro-tumorigenic effects. This review article will discuss the role of IL-6: 1) in bone metabolism, 2) in cancer metastasis to bone, 3) in cancer prognosis, and 4) as potential therapies for metastatic bone cancer.

Calpain-cleavage of alpha-synuclein: connecting proteolytic processing to disease-linked aggregation.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of alpha-synuclein (alpha-Syn), and although it is now recognized that alpha-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of alpha-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a beta-sheet structure. To determine whether calpain-cleavage of alpha-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to alpha-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved alpha-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved alpha-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved alpha-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of alpha-Syn in various alpha-synucleinopathies.

Early events in cartilage repair after subchondral bone microfracture.

The current study investigated healing of large full-thickness articular cartilage defects during the first 8 weeks with and without penetration of the subchondral bone using microfracture in an established equine model of cartilage healing. Chondral defects in the weightbearing portion of the medial femoral condyle were made bilaterally; one defect in each horse was microfractured whereas the contralateral leg served as the control. The expression of cartilage extracellular matrix components (Types I and II collagen and aggrecan) was evaluated using histologic techniques, reverse transcription coupled polymerase chain reaction, in situ hybridization, and immunohistochemistry. This study confirms an increase in Type II collagen mRNA expression in repair tissue as early as 6 weeks after microfracture. Although other matrix mRNA and protein levels changed in concentration and tissue location over the course of the study, no significant differences were seen in microfractured defects. Although the microfracture techniques appear to improve clinical functionality, volume of repair tissue, and augment Type II collagen content, aggrecan content is less than ideal. Therefore, methods to enhance key matrix components such as aggrecan after microfracture may additionally improve repair tissue observed after the procedure.