Manufacturing and <Emphasis Type="Italic">in vivo</Emphasis> inner ear visualization of MRI traceable liposome nanoparticles encapsulating gadolinium

Background  

Treatment of inner ear diseases remains a problem because of limited passage through the blood-inner ear barriers and lack of control with the delivery of treatment agents by intravenous or oral administration. As a minimally-invasive approach, intratympanic delivery of multifunctional nanoparticles (MFNPs) carrying genes or drugs to the inner ear is a future therapy for treating inner ear diseases, including sensorineural hearing loss (SNHL) and Meniere's disease. In an attempt to track the dynamics and distribution of nanoparticles in vivo, here we describe manufacturing MRI traceable liposome nanoparticles by encapsulating gadolinium-tetra-azacyclo-dodecane-tetra-acetic acid (Gd-DOTA) (abbreviated as LPS+Gd-DOTA) and their distribution in the inner ear after either intratympanic or intracochlear administration.     

The role of hair cell regeneration in an avian model of inner ear injury and repair from acoustic trauma

The auditory system of young chicks (Gallus domesticus)is an important model for studying the structure and function of the inner ear. For over 20 years this model has gained interest because of the discovery that birds, and perhaps lower vertebrates in general, are capable of generating new hair cells to replace those lost to ototrauma, a capacity that is absent along the mammalian organ of Corti. Accompanying this remarkable capacity is the nearly complete restoration of auditory function to the chick peripheral ear. This article presents a review of findings on the toxic effect of exposure to extremely loud sound on the young chick ear, and the subsequent recovery from inner ear structural damage and accompanying recovery of auditory function. The evidence, surprisingly, suggests that the role of the regenerated hair cells in the latter may be minimal and that multiple other factors play more important roles. There is also a section on the unique problems encountered in using chicks as laboratory animal subjects in experiments designed to understand the consequences of acoustic trauma. The conclusion summarizes some of the issues that need to be addressed in future research.     

EDD mediates DNA damage-induced activation of CHK2

EDD, the human orthologue of Drosophila melanogaster "hyperplastic discs," is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.     

Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3

The endothelial cell (EC)-derived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following EC-pericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. EC-pericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in EC-pericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)-, MMP-10-, and ADAM-15 (a disintegrin and metalloproteinase-15)-dependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events.     

Rapid vasoregulatory mechanisms in exercising human skeletal muscle: dynamic response to repeated changes in contraction intensity

We tested the hypothesis that vasoregulatory mechanisms exist in humans that can rapidly adjust muscle blood flow to repeated increases and decreases in exercise intensity. Six men and seven women (age, 24.4+/-1.3 yr) performed continuous dynamic forearm handgrip contractions (1- to 2-s contraction-to-relaxation duty cycle) during repeated step increases and decreases in contraction intensity. Three step change oscillation protocols were examined: Slow (7 contractions per contraction intensityx10 steps); Fast (2 contractions per contraction intensityx15 steps); and Very Fast (1 contraction per contraction intensityx15 steps). Forearm blood flow (FBF; Doppler and echo ultrasonography), heart rate (ECG), and mean arterial pressure (arterial tonometry) were examined for the equivalent of a cardiac cycle during each relaxation phase (FBFrelax). Mean arterial pressure and heart rate did not change during repeated step changes (P=0.352 and P=0.190). For both Slow and Fast conditions, relaxation phase FBFrelax adjusted immediately and repeatedly to both increases and decreases in contraction intensity, and the magnitude and time course of FBFrelax changes were virtually identical. For the Very Fast condition, FBFrelax increased with the first contraction and thereafter slowly increased over the course of repeated contraction intensity oscillations. We conclude that vasoregulatory mechanisms exist in human skeletal muscle that are capable of rapidly and repeatedly adjusting muscle blood flow with ongoing step changes in contraction intensity. Importantly, they demonstrate symmetry in response magnitude and time course with increasing versus decreasing contraction intensity but cannot adjust to very fast exercise intensity oscillations.     

Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA

MicroRNAs are an extensive family of approximately 22-nucleotide-long noncoding RNAs expressed in a wide range of eukaryotes, including humans, and they are important in development and disease. We found that microRNA Mir-17-5p has extensive complementarity to the mRNA of AIB1 (named for "amplified in breast cancer 1"). Cell culture experiments showed that AIB1 expression was downregulated by Mir-17-5p, primarily through translational inhibition. Expression of Mir-17-5p was low in breast cancer cell lines. We also found that downregulation of AIB1 by Mir-17-5p resulted in decreased estrogen receptor-mediated, as well as estrogen receptor-independent, gene expression and decreased proliferation of breast cancer cells. Mir-17-5p also completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancer cells. Our results reveal that Mir-17-5p has a role as a tumor suppressor in breast cancer cells.     

Decoupling the direct and indirect effects of nitrogen deposition on ecosystem function

Elevated nitrogen (N) inputs into terrestrial ecosystems are causing major changes to the composition and functioning of ecosystems. Understanding these changes is challenging because there are complex interactions between 'direct' effects of N on plant physiology and soil biogeochemistry, and 'indirect' effects caused by changes in plant species composition. By planting high N and low N plant community compositions into high and low N deposition model terrestrial ecosystems we experimentally decoupled direct and indirect effects and quantified their contribution to changes in carbon, N and water cycling. Our results show that direct effects on plant growth dominate ecosystem response to N deposition, although long-term carbon storage is reduced under high N plant-species composition. These findings suggest that direct effects of N deposition on ecosystem function could be relatively strong in comparison with the indirect effects of plant community change.     

Proteomic and computational analysis of secreted proteins with type I signal peptides from the Antarctic archaeon Methanococcoides burtonii

LC-MS/MS was used to identify secreted proteins in the Antarctic archaeon Methanococcoides burtonii. Seven proteins possessing a classical class 1 signal peptide were identified in the supernatant from cultures grown at 4 and 23 degrees C. The proteins included a putative S-layer cell surface protein, cell surface protein involved with cell adhesion, and trypsin-like serine protease. Protease activity was detected in the secreted fraction, and the signal peptide cleavage site of the protease was confirmed using Edman sequencing. The expression profile of putative cell surface proteins suggests a requirement for cell interactions during growth at low temperature. Sequences of the secreted proteins were used to compile a dataset containing a further 32 predicted secreted proteins from the Methanosarcinaceae. Many of these proteins were also S-layer cell surface proteins with a variety of predicted roles, particularly in cell-cell interaction. Computational analysis of signal peptides revealed a preference for lysine in the n-region, leucine in the h-region, and a eucaryal-type cleavage site, highlighting the mosaic nature of signal peptides in Archaea. This is the first study to experimentally characterize secreted proteins from a cold-adapted archaeon and provides new insight and a functional dataset for studying secretion in Archaea.