The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.     

Transforming Growth Factor Beta (TGF-β) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression

We recently identified Smads1, 5 and 8 as muscle biomarkers in human ALS. In the ALS mouse, these markers are elevated and track disease progression. Smads are signal transducers and become activated upon receptor engagement of ligands from the TGF-β superfamily. Here, we sought to characterize ligands linked to activation of Smads in ALS muscle and their role as biomarkers of disease progression. RNA sequencing data of ALS muscle samples were mined for TGF-β superfamily ligands. Candidate targets were validated by qRT-PCR in a large cohort of human ALS muscle biopsy samples and in the G93A SOD1 mouse. Protein expression was evaluated by Western blot, ELISA and immunohistochemistry. C2C12 muscle cells were used to assess Smad activation and induction. TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8. In the G93A SOD1 mouse, the temporal pattern of TGF-β expression paralleled the Smads and increased with disease progression. TGF-β1 immunoreactivity was detected in mononuclear cells surrounding muscle fibers in ALS samples. In muscle cells, TGF-β ligands were capable of activating Smads. In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle. Their correlation with weakness in human ALS and their progressive increase with advancing disease in the ALS mouse suggest that they, as with the Smads, can track disease progression. These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.     

Alginate Microspheres Containing Temperature Sensitive Liposomes (TSL) for MR-Guided Embolization and Triggered Release of Doxorubicin

Objective

The objective of this study was to develop and characterize alginate microspheres suitable for embolization with on-demand triggered doxorubicin (DOX) release and whereby the microspheres as well as the drug releasing process can be visualized in vivo using MRI.

Methods and Findings

For this purpose, barium crosslinked alginate microspheres were loaded with temperature sensitive liposomes (TSL/TSL-Ba-ms), which release their payload upon mild hyperthermia. These TSL contained DOX and [Gd(HPDO3A)(H₂O)], a T₁ MRI contrast agent, for real time visualization of the release. Empty alginate microspheres crosslinked with holmium ions (T₂* MRI contrast agent, Ho-ms) were mixed with TSL-Ba-ms to allow microsphere visualization. TSL-Ba-ms and Ho-ms were prepared with a homemade spray device and sized by sieving. Encapsulation of TSL in barium crosslinked microspheres changed the triggered release properties only slightly: 95% of the loaded DOX was released from free TSL vs. 86% release for TSL-Ba-ms within 30 seconds in 50% FBS at 42°C. TSL-Ba-ms (76 ± 41 μm) and Ho-ms (64 ± 29 μm) had a comparable size, which most likely will result in a similar in vivo tissue distribution after an i.v. co-injection and therefore Ho-ms can be used as tracer for the TSL-Ba-ms. MR imaging of a TSL-Ba-ms and Ho-ms mixture (ratio 95:5) before and after hyperthermia allowed in vitro and in vivo visualization of microsphere deposition (T₂*-weighted images) as well as temperature-triggered release (T₁-weighted images). The [Gd(HPDO3A)(H₂O)] release and clusters of microspheres containing holmium ions were visualized in a VX₂ tumor model in a rabbit using MRI.

Conclusions

In conclusion, these TSL-Ba-ms and Ho-ms are promising systems for real-time, MR-guided embolization and triggered release of drugs in vivo.     

The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.     

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1β, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.     

Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis,Neurodegeneration, and Impaired Behavior

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.A variety of both exogenous and endogenous reactive compounds present a constant threat to the integrity of DNA in living cells. DNA damage introduced by such compounds can lead to high and deleterious mutation rates as well as DNA cytotoxicity, both to the nuclear and the mitochondrial genome. This has triggered the evolution of several different DNA repair pathways (28). One is the base excision repair (BER) pathway, which repairs small base alterations that do not distort the DNA helix. Repair of such highly abundant lesions by BER is performed by a multistep process that is initiated by a damage-specific DNA glycosylase, which removes the damaged base. One of these glycosylases is uracil-DNA glycosylase (UDG), which acts to preserve the genome by removing mutagenic uracil residues from the DNA. This glycosylase, as well as the OGG1 glycosylase that is specialized for the removal of oxidized bases, exists in a nuclear and mitochondrial splice form (1, 11, 37, 45). Accordingly, BER of a variety of lesions has been observed in mitochondria (26, 31).Damage to the mitochondrial DNA (mtDNA) can cause respiratory chain deficiency and lead to disorders that have varied phenotypes (35, 41). Many involve neurological features that are often associated with cell loss within specific brain regions. These pathologies, along with the increasing evidence of a decline in mitochondrial function with aging, have raised speculation that key changes in mitochondrial DNA sequences and functions could have a vital role in age-related neurodegenerative diseases (41). This has also been studied in several model organisms. Mouse models with respiratory chain deficient dopamine neurons have demonstrated adult onset Parkinsonism phenotype (16), and cell death induced by mitochondrial toxicity is likely to underlie Alzheimer disease (32). Mitochondrial oxidative stress and accumulation of mtDNA damage are believed to be particularly devastating to postmitotic differentiated tissue, including neurons (30). The mtDNA contains genetic information for 13 polypeptides that are a part of the electron transport chain and for rRNAs and tRNAs that are necessary for mitochondrial protein synthesis. Thus, damage to the mtDNA genome will affect the energetic capacities of the mitochondria and also influence the level of reactive oxygen species (ROS) and ultimately the susceptibility to apoptosis (30, 35).Some recent influential studies have assessed the effect of mtDNA mutagenesis, including small base-pair substitutions and larger mtDNA deletions, on the life span of mice. It was concluded that a massive increase in the frequency of mtDNA base-pair substitutions are required for inducing premature aging, whereas the number of mtDNA deletions coincides better with natural aging (25, 47-49).In the present study, we have combined two novel transgenic mouse models, which allow the induction of a high number of apyrimidinic (AP) sites specifically to the mitochondrial genome in adults simply by the addition of doxycycline to the diet. Such AP sites are created by the expression of a mutated version of mitochondrion-targeted human UDG (abbreviated here as mutUNG1), whereby an amino acid substitution results in an enzyme that removes thymine, in addition to uracil, from DNA (23). The CaMKIIα promoter restricts expression of the mutUNG1 to forebrain neurons (34). We demonstrate that a continuous generation of AP sites leads to apoptosis, accelerated neurodegeneration, and impaired behavior.