AAV1.NT-3 gene therapy in the SOD1KO mouse model of accelerated sarcopenia.

BACKGROUND: Sarcopenia, an age-related loss of muscle mass, is a critical factor that affects the health of the older adults. The SOD1KO mouse is deficient of Cu/Zn superoxide dismutase, used as an accelerated aging model. We previously showed that NT-3 improves muscle fibre size by activating the mTOR pathway, suggesting a potential for attenuating age-related muscle loss. This study assessed the therapeutic efficacy of AAV1.NT-3 in this accelerated aging model. METHODS: Twelve 6 months old SOD1KO mice were injected intramuscularly with a 1 × 1011 vg dose of AAV1.tMCK.NT-3, and 13 age-matched SOD1KO mice were used as controls. The treatment effect was evaluated using treadmill, rotarod and gait analyses as well as histological studies assessing changes in muscle fibre, and fibre type switch, in tibialis anterior, gastrocnemius, and triceps muscles, and myelin thickness by calculating G ratio in sciatic and tibial nerves. Molecular studies involved qPCR experiments to analyse the expression levels of mitochondrial and glycolysis markers and western blot experiments to assess the activity of mTORC1 pathway. RESULTS: Treatment resulted in a 36% (154.9 vs. 114.1; P < 0.0001) and 76% increase (154.3 vs. 87.6; P < 0.0001) in meters ran, with treadmill test at 3 and 6 months post gene delivery. In addition, the treated cohort stayed on rotarod 30% (52.7 s vs. 40.4 s; P = 0.0095) and 54% (50.4 s vs. 32.7 s; P = 0.0007) longer, compared with untreated counterparts at 3 and 6 months post injection. Gait analysis, performed at endpoint, showed that stride width was normalized to wild type levels (29.3 mm) by an 11% decrease, compared with untreated cohort (28.6 mm vs. 32.1 mm; P = 0.0014). Compared with wild-type, SOD1KO mice showed 9.4% and 11.4% fibre size decrease in tibialis anterior and gastrocnemius muscles, respectively, which were normalized to wild type levels with treatment. Fibre diameter increase was observed prominently in FTG fibre type. G ratio analysis revealed hypomyelination in the tibial (0.721) and sciatic (0.676) nerves of SOD1KO model, which was reversed in the NT-3 cohort (0.646 and 0.634, respectively). Fibre size increase correlated with the increase in the p-S6 and p-4E-BP1 levels, and in the glycolysis markers in tibialis anterior. Alterations observed in the mitochondrial markers were not rescued with treatment. Overall, response to NT-3 was subdued in gastrocnemius muscle. CONCLUSIONS: This study shows that AAV1.NT-3 gene therapy protected SOD1KO mouse from accelerated aging effects functionally and histologically. We further confirmed that NT-3 has potential to activate the mTOR and glycolytic pathways in muscle.

AAV1.NT-3 gene therapy prevents age-related sarcopenia.

Sarcopenia is progressive loss of muscle mass and strength, occurring during normal aging with significant consequences on the quality of life for elderly. Neurotrophin 3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulating axon regeneration and myelination. NT-3 is involved in the maintenance of neuromuscular junction (NMJ) integrity, restoration of impaired radial growth of muscle fibers through activation of the Akt/mTOR pathway. We tested the efficacy of NT-3 gene transfer therapy in wild type (WT)-aged C57BL/6 mice, a model for natural aging and sarcopenia, via intramuscular injection 1 × 1011 vg AAV1.tMCK.NT-3, at 18 months of age. The treatment efficacy was assessed at 6 months post-injection using run to exhaustion and rotarod tests, in vivo muscle contractility assay, and histopathological studies of the peripheral nervous system, including NMJ connectivity and muscle. AAV1.NT-3 gene therapy in WT-aged C57BL/6 mice resulted in functional and in vivo muscle physiology improvements, supported by quantitative histology from muscle, peripheral nerves and NMJ. Hindlimb and forelimb muscles in the untreated cohort showed the presence of a muscle- and sex-dependent remodeling and fiber size decrease with aging, which was normalized toward values obtained from 10 months old WT mice with treatment. The molecular studies assessing the NT-3 effect on the oxidative state of distal hindlimb muscles, accompanied by western blot analyses for mTORC1 activation were in accordance with the histological findings. Considering the cost and quality of life to the individual, we believe our study has important implications for management of age-related sarcopenia.

Characterization of Caerulomycin A as a dual-targeting anticancer agent.

Caerulomycin A (CaeA), isolated from actinomycetes, has a featured 2,2'-bipyridine core structure. Based on the results of in silico drug-protein docking analysis, CaeA shows potential ligands for interacting with both tubulin and DNA topoisomerase I (Topo-1). The result was confirmed by cell-free tubulin polymerization assay and Topo-1 activity assay. In vitro assays also demonstrated that CaeA increases the polymerization of tubulin and increases cell size. In addition, CaeA inhibits cell viability and growth of various cancer cells, yet exhibits low cytotoxicity. CaeA also affects paclitaxel-resistant cancer cells and synergizes the effect with paclitaxel in reducing cancer cell colony formation rate. In vivo experiments confirm the effect of CaeA on reducing tumor size and weight in nude mouse inoculated with tumor cells with no noticeable side effects. Taken together, our data demonstrate that CaeA is a potential potent agent for cancer treatment through tubulin and Topo-1 dual-targeting with little side effects.

UVB-induced eIF2α phosphorylation in keratinocytes depends on decreased ATF4, GADD34 and CReP expression levels.

AIM: To elucidate the mechanism behind the sustained high levels of phosphorylated eIF2α in HaCaT cells post-UVB. MAIN METHODS: In this study, expression levels of the machinery involved in the dephosphorylation of eIF2α (GADD34, CReP and PP1), as well as the PERK-eIF2α-ATF4-CHOP, IRE1α/XBP1s and ATF6α signaling cascades, were analyzed by western blot and fluorescence microscope. KEY FINDINGS: Our data showed that UVB induces the phosphorylation of eIF2α, which induces the translation of ATF4 and consequently the expression of CHOP and GADD34. Nevertheless, UVB also suppresses the translation of ATF4 and GADD34 in HaCaT cells via a p-eIF2α independent mechanism. Therefore, the lack of ATF4, GADD34 and CReP is responsible for the sustained phosphorylation of eIF2α. Finally, our data also showed that UVB selectively modifies PERK and downregulates ATF6α expression but does not induce activation of the IRE1α/XBP1s pathway in HaCaT cells. SIGNIFICANCE: Novel mechanism to explain the prolonged phosphorylation of eIF2α post-UVB irradiation.

Mathermycin, an anti-cancer molecule that targets cell surface phospholipids.

Mathermycin, a lantipeptide isolated from marine actinomycete Marinactinospora thermotolerans, is an antibiotic that has been shown to disrupt bacterial plasma membrane. We now provide evidences that mathermycin can also disrupt cancer, but not normal, cell plasma membranes through targeting phosphatidylethanolamine (PE), which is located only in the inner leaflet of the plasma membrane in normal cells but in both the inner and outer leaflets of the membrane in tumor cells. Our data shows that mathermycin inhibits the metabolic activity and induces mainly necrotic death of all cancer cell lines with EC50 between 4.2 and 16.9 µM, while normal cell lines have EC50 between 113 and 129 µM. The cytotoxicity of mathermycin could be inhibited by exogenous PE, but not phosphoserine and phosphocholine. The formation of mathermycin-PE complexes was confirmed by in silico analysis, HPLC and MS spectrometer. Furthermore, mathermycin exhibited similar cytotoxicity toward cancer and multidrug resistant cancer cells, which could be due to its ability to inhibit mitochondrial function, as shown by our data from the Seahorse™ metabolic analyzer. This study demonstrates that mathermycin is a potentially effective class of anti-tumor chemotherapeutics that do not easily develop resistance due to a mechanism of action targeting PE.

CIRP Sensitizes Cancer Cell Responses to Ionizing Radiation.

Cold inducible RNA binding protein (CIRP), also named A18 hnRNP or CIRBP, is a cold-shock RNA-binding protein which can be induced upon various cellular stresses. Its expression level is induced in various cancer tissues relative to adjacent normal tissues; this is believed to play a critical role in cancer development and progression. In this study, we investigated the role of CIRP in cells exposed to ionizing radiation. Our data show that CIRP reduction causes cell colony formation and cell viability reduction after irradiation. In addition, CIRP knockdown cells demonstrated a higher DNA damage rate but less cell cycle arrest after irradiation. As a result, the induced DNA damage with less DNA repair processes led to an increased cell apoptosis rate in CIRP knockdown cells postirradiation. These findings suggest that CIRP is a critical protein in irradiated cells and can be used as a potential target for sensitizing cancer cells to radiation therapy.

The Mechanism of CIRP in Regulation of STAT3 Phosphorylation and Bag-1/S Expression Upon UVB Radiation.

Cold-inducible RNA binding protein (CIRP) is a stress-inducible protein, which could be activated by various cellular stresses, such as hypothermia, hypoxia and UV irradiation. Our previous study indicated that UVB radiation (3 mJ cm-2 ) induces CIRP expression, which promotes keratinocytes growth, survival and eventually transformation via activation of STAT3-Bag-1/S signaling cascade. However, the mechanism(s) of CIRP in regulating p-STAT3 activation and Bag-1/S expression have not been fully elucidated. In this study, we demonstrate that repeated exposure of UVB radiation (3 mJ cm-2 ) or overexpression of CIRP could lead to an elevation of the phosphorylation of Janus kinase (JAK) family proteins (JAK2 and JAK3) in HaCaT cells. The increased phosphorylation of the JAKs correlates to an increased phosphorylation of STAT3 (p-STAT3) in the cells; inhibiting JAKs using JAK inhibitor I lead to a reduction of STAT3 phosphorylation and Bag-1/S expression in wild type HaCaT and CIRP stably transfected HaCaT cells with or without UVB exposure. Furthermore, our data indicated that inhibiting the downstream factor of CIRP, NF-κB, using BAY 11-7085 could also decrease the p-STAT3. These results lead us to propose that CIRP mediates the activation of STAT3-Bag-1/S signaling cascade via activating the JAKs and NF-κB signaling pathways.

The Mechanisms of Carnosol in Chemoprevention of Ultraviolet B-Light-Induced Non-Melanoma Skin Cancer Formation.

Carnosol is a natural compound extracted from rosemary and sage, which has been demonstrated to have anti-inflammatory, anti-oxidant, and anti-cancer properties. In this report, we evaluated the therapeutic potential and elucidated the potential mechanism of action of carnosol in chemoprevention of ultraviolet B-light (UVB) induced non-melanoma skin cancer formation. Our data indicated that carnosol could partially reduce UVB-induced reactive oxygen species (ROS) elevation and thus reduce DNA damage. It could also reduce UVB-induced formation of cyclobutane pyrimidine dimers (CDP) in keratinocytes possibly through its ability in absorbing UVB radiation. In addition, carnosol could inhibit the UVB-induced activation of NF-κB and also reduce UVB-induced transformation of keratinocytes. Taken together, the results indicate the role of carnosol as a potential chemopreventive agent upon UVB radiation.

Quantitative Evaluation of Enzyme-Induced Porcine Articular Cartilage Degeneration Based on Observation of Entire Cartilage Layer Using Ultrasound.

Enzyme-induced articular cartilage degeneration resembling osteoarthritis was evaluated using a newly defined acoustic parameter, the "averaged magnitude ratio" (AMR), which has been suggested as an indicator of articular cartilage degeneration. In vitro experiments were conducted on porcine cartilage samples digested with trypsin for 2 h (n = 10) and 4 h (n = 13) and healthy control samples (n = 13). AMR was determined with 15- and 25-MHz ultrasound, and the integrated reflection coefficient (IRC) and apparent integrated backscattering coefficient (AIB) were also calculated for comparison. The Young's modulus of superficial cartilage was measured using atomic force microscopy. Performance of the AMR differs between 15 and 25 MHz, possibly because of frequency-related attenuation and resolution of ultrasound. At the proper settings, AMR exhibited a competence similar to that of IRC and AIB in detecting cartilage degeneration and could also detect differences in deeper positions. Furthermore, AMR has the advantages of being easy to measure and requiring no reference material.

Detection of depth-depend changes in porcine cartilage after wear test using Raman spectroscopy.

Cartilage damage and wear can lead to severe diseases, such as osteoarthritis, thus, many studies on the cartilage wear process have already been performed to better understand the cartilage wear mechanism. However, most characterization methods focus on the cartilage surface or the total wear extent. With the advantages of high spatial resolution and easy characterization, Raman microspectroscopy was employed for the first time to characterize full-depth changes in the cartilage extracellular matrix (ECM) after wear test. Sections from the cartilage samples after wear were compared with sections from the control group. Univariate and multivariate analyses both indicated that collagen content loss at certain depths (20%-30% relative to the cartilage surface) is possibly the dominating alteration during wear rather than changes in collagen fiber orientation or proteoglycan content. These findings are consistent with the observations obtained by scanning electron microscopy and histological staining. This study successfully used Raman microspectroscopy efficiently assess full-depth changes in cartilage ECM after wear test, thus providing new insight into cartilage damage and wear.

The role of cold-inducible RNA binding protein in cell stress response.

Cold-inducible RNA binding protein (CIRP) was discovered after the cells were exposed to a moderate cold shock because its production was induced. Other cellular stresses such as ultraviolet light radiation and hypoxia also could increase its expression. Under stress conditions, CIRP could up regulate its own expression by self-transcriptional activation of alternative promoters. After relocating into cytoplasm from nucleus, CIRP assists cells in adapting to novel environmental conditions via stabilizing specific mRNAs and facilitating their translation. It not only participates in anti-apoptosis processes under mild hypothermia condition, but also protects cells from ultraviolet radiation and hypoxia induced senescence process. This article focuses on the possible mechanisms of its inducible expression, cytoprotective functions and carcinogenesis. In addition, extracellular CIRP has been shown to be a novel danger-associated molecular patter (DAMP) member and is able to induce inflammatory response. Finally, based on the distinct roles of CIRP in intracellular and extracellular conditions, a possible model of CIRP-mediated cell fate has been proposed.

The collagen microstructural changes of rat menisci and tibiofemoral cartilages under the influence of mechanical loading: An in vitro wear test of whole joints.

BACKGROUND: Knee osteoarthritis (OA) is suggested to be induced by multi-factors, and mechanical environment is regarded as a risky factor. OBJECTIVE: To investigate the effect of isolated mechanical factor on cartilage. METHODS: An active wear test system was designed to perform parameters-controlled in vitro wear tests on rat knee joints with specific load magnitude, flexion-extension angle, and movement frequency. Six hind limbs of 9-month-old male Sprague-Dawley rats, with an additional spring on the medial side, were worn by using the custom-designed apparatus. Researchers observed both the menisci and tibial cartilages of these hind limbs using multiphoton laser scanning microscopy to analyze the change of the collagen microstructure caused by wear. RESULTS: Collagen microstructure of both the medial and lateral meniscus became disordered under cyclic load. Some tissues on the surface of the medial tibial cartilage were removed and the middle layer of the medial compartment displayed cracks. On the contrary, the lateral tibial cartilage was intact. CONCLUSIONS: The results implied that cyclic load caused menisci microstructure disarrangement prior to tibial cartilage damage and the collagen structure of mid-layer tibial cartilage failed before that of the superficial layer under the kinematics adopted in the study.

The Involvement of Splicing Factor hnRNP A1 in UVB-induced Alternative Splicing of hdm2.

Human homolog double minute 2 (hdm2), an oncoprotein, which binds to tumor suppressor p53 to facilitate its degradation, has been known to contribute to tumorigenesis. Its splicing variants are reported to be highly expressed in many cancers and can be induced by ultraviolet B light (UVB). However, the mechanisms of how UVB radiation induces hdm2 alternative splicing still remain unclear. In this study, we investigated the roles of two common splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and serine/arginine-rich splicing factor 1 (SRSF1), in regulating UVB-induced hdm2 splicing. Our study indicated that while the expression of both hnRNP A1 and SRSF1 are induced, only hnRNP A1 is involved in hdm2 alternative splicing upon UVB irradiation. Overexpression of hnRNP A1 resulted in decrease of full-length hdm2 (hdm2-FL) and increase of hdm2B, one of hdm2 alternate-splicing forms; while down-regulated hnRNP A1 expression led to the decrease of the hdm2-FL and hdm2B in HaCaT cells. Protein-mRNA binding assay confirmed that UVB irradiation could increase the binding of hnRNP A1 to hdm2 pre-mRNA. In conclusion, we elucidated that UVB induces alternative splicing of hdm2 by increasing the expression and the binding of hnRNP A1 to hdm2 full-length mRNA.

microRNA and NF-kappa B.

Nuclear Factor kappa B (NF-κB) plays important roles in regulation of countless cellular functions, including cell cycle and apoptosis. As a versatile transcription factor, NF-κB is a target of a large amount of miRNAs. Abnormal NF-κB activity is frequently associated with an abnormal level of miRNAs, which is found to play critical roles in disease progression including cancer. While the expression and activity of NF-κB can be directly or indirectly up-regulated or downregulated by various miRNAs, NF-κB can also regulate the expression of many miRNAs. Intriguingly, reciprocal regulation between miRNAs and NF-κB, which exists in the form of positive and negative feedback loops, is often observed in various cancers. In this chapter, the mechanisms and roles of miRNA-regulated NF-κB and NF-κB-regulated miRNAs in a variety of cancers will be discussed. The potential therapeutic use of miRNAs that are up- and down-stream of NF-κB signaling pathways as targets for cancer treatment will also be accessed.

An update on the constitutive relation of ligament tissues with the effects of collagen types.

The musculoskeletal ligament is a kind of multiscale composite material with collagen fibers embedded in a ground matrix. As the major constituent in ligaments to bear external loads, collagens are composed mainly of two collagen contents with different mechanical properties, i.e., types I and III collagen. The constitutive relation of ligaments plays a critical role in the stability and normal function of human joints. However, collagen types have not been distinguished in the previous constitutive relations. In this paper a constitutive relation for ligament tissues was modified based on the previous constitutive relation by considering the effects of collagen types. Both the collagen contents and the mechanical properties of sixteen ligament specimens from four cadaveric human knee joints were measured for determining their material coefficients in the constitutive relation. The mechanical behaviors of ligaments were obtained from both the uniaxial tensile and simple shear tests. A linear regression between joint kinematic results from in vitro and in silico experiments was made to validate the accuracy of this constitutive relation. The high correlation coefficient (R(2)=0.93) and significance (P<0.0001) of the regression equation revealed that this modified constitutive relation of ligaments was accurate to be used in studying joint biomechanics. Another finite element analysis with collagen contents changing demonstrated that the effect of variations in collagen ratios on both joint kinematics and ligament biomechanics could be simulated by this constitutive relation.

Reactive oxygen species in redox cancer therapy.

The role of reactive oxygen species (ROS) in cancer cells has been intensively studied for the past two decades. Cancer cells mostly have higher basal ROS levels than their normal counterparts. The induction of ROS has been shown to be associated with cancer development, metastasis, progression, and survival. Various therapeutic approaches targeting intracellular ROS levels have yielded mixed results. As widely accepted dietary supplements, antioxidants demonstrate both ROS scavenging ability and anti-cancer characteristics. However, antioxidants may not always be safe to use since excessive intake of antioxidants could lead to serious health concerns. In this review, we have evaluated the production and scavenging systems of ROS in cells, as well as the beneficial and harmful roles of ROS in cancer cells. We also examine the effect of antioxidants in cancer treatment, the effect of combined treatment of antioxidants with traditional cancer therapies, and the side effects of excessive antioxidant intake.

Therapeutic microRNAs targeting the NF-kappa B signaling circuits of cancers.

MicroRNAs (miRNAs) not only directly regulate NF-κB expression, but also up- or down-regulate NF-κB activity via upstream and downstream signaling pathways of NF-κB. In many cancer cells, miRNA expressions are altered accompanied with an elevation of NF-κB activity, which often plays a role in promoting cancer development and progression as well as hindering the effectiveness of chemo and radiation therapies. Thus NF-κB-targeting miRNAs have been identified and characterized as potential therapeutics for cancer treatment and sensitizers of chemo and radiotherapies. However, due to cross-targeting and instability of miRNAs, some limitations of using miRNA as cancer therapeutics still exist. In this review, the mechanisms for miRNA-mediated alteration of NF-κB expression and activation in different types of cancers will be discussed. The results of therapeutic use of NF-κB-targeting miRNA for cancer treatment will be examined. Some limitations, challenges and potential strategies in future development of miRNA as cancer therapeutics are also assessed.

ROS and p53 in regulation of UVB-induced HDM2 alternative splicing.

Alternative splicing plays an important role in proteasome diversity and gene expression regulation in eukaryotic cells. Hdm2, the human homolog of mdm2 (murine double minute oncogene 2), is known to be an oncogene as its role in suppression of p53. Hdm2 alternative splicing, occurs in both tumor and normal tissues, is believed to be a response of cells for cellular stress, and thus modulate p53 activity. Therefore, understanding the regulation of hdm2 splicing is critical in elucidating the mechanisms of tumor development and progression. In this study, we determined the effect of ultraviolet B light (UVB) on alternative splicing of hdm2. Our data indicated that UVB (50 mJ cm(-2)) alone is not a good inducer of alternative splicing of hdm2. The less effectiveness could be due to the induction of ROS and p53 by UVB because removing ROS by L-NAC (10 mm) in p53 null cells could lead to alternative splicing of hdm2 upon UVB irradiation.

The role of constitutive nitric-oxide synthase in ultraviolet B light-induced nuclear factor κB activity.

NF-κB is a transcription factor involved in many signaling pathways that also plays an important role in UV-induced skin tumorigenesis. UV radiation can activate NF-κB, but the detailed mechanism remains unclear. In this study, we provided evidence that the activation of constitutive nitric-oxide synthase plays a role in regulation of IκB reduction and NF-κB activation in human keratinocyte HaCaT cells in early phase (within 6 h) post-UVB. Treating the cells with l-NAME, a selective inhibitor of constitutive nitric-oxide synthase (cNOS), can partially reverse the IκB reduction and inhibit the DNA binding activity as well as nuclear translocation of NF-κB after UVB radiation. A luciferase reporter assay indicates that UVB-induced NF-κB activation is totally diminished in cNOS null cells. The cNOS-mediated reduction of IκB is likely due to the imbalance of nitric oxide/peroxynitrite because treating the cells with lower (50 µm), but not higher (100-500 µm), concentration of S-nitroso-N-acetylpenicillamine (SNAP) can reverse the effect of l-NAME in partial restore IκB level post-UVB. Our data also showed that NF-κB activity was required for maintaining a stable IκB kinase α subunit (IKKα) level because treating the cells with NF-κB or cNOS inhibitors could reduce IKKα level upon UVB radiation. In addition, our data demonstrated that although NF-κB protects cells from UVB-induced death, its pro-survival activity was likely neutralized by the pro-death activity of peroxynitrite after UVB radiation.

Cross-linking electrochemical mass spectrometry for probing protein three-dimensional structures.

Chemical cross-linking combined with mass spectrometry (MS) is powerful to provide protein three-dimensional structure information but difficulties in identifying cross-linked peptides and elucidating their structures limit its usefulness. To tackle these challenges, this study presents a novel cross-linking MS in conjunction with electrochemistry using disulfide-bond-containing dithiobis[succinimidyl propionate] (DSP) as the cross-linker. In our approach, electrolysis of DSP-bridged protein/peptide products, as online monitored by desorption electrospray ionization mass spectrometry is highly informative. First, as disulfide bonds are electrochemically reducible, the cross-links are subject to pronounced intensity decrease upon electrolytic reduction, suggesting a new way to identify cross-links. Also, mass shift before and after electrolysis suggests the linkage pattern of cross-links. Electrochemical reduction removes disulfide bond constraints, possibly increasing sequence coverage for tandem MS analysis and yielding linear peptides whose structures are more easily determined than their cross-linked precursor peptides. Furthermore, this cross-linking electrochemical MS method is rapid, due to the fast nature of electrochemical conversion (much faster than traditional chemical reduction) and no need for chromatographic separation, which would be of high value for structural proteomics research.