Dimension- and shape-dependent thermal transport in nano-patterned thin films investigated by scanning thermal microscopy

Scanning thermal microscopy (SThM) is a technique which is often used for the measurement of the thermal conductivity of materials at the nanometre scale. The impact of nano-scale feature size and shape on apparent thermal conductivity, as measured using SThM, has been investigated. To achieve this, our recently developed topography-free samples with 200 and 400 nm wide gold wires (50 nm thick) of length of 400–2500 nm were fabricated and their thermal resistance measured and analysed. This data was used in the development and validation of a rigorous but simple heat transfer model that describes a nanoscopic contact to an object with finite shape and size. This model, in combination with a recently proposed thermal resistance network, was then used to calculate the SThM probe signal obtained by measuring these features. These calculated values closely matched the experimental results obtained from the topography-free sample. By using the model to analyse the dimensional dependence of thermal resistance, we demonstrate that feature size and shape has a significant impact on measured thermal properties that can result in a misinterpretation of material thermal conductivity. In the case of a gold nanowire embedded within a silicon nitride matrix it is found that the apparent thermal conductivity of the wire appears to be depressed by a factor of twenty from the true value. These results clearly demonstrate the importance of knowing both probe-sample thermal interactions and feature dimensions as well as shape when using SThM to quantify material thermal properties. Finally, the new model is used to identify the heat flux sensitivity, as well as the effective contact size of the conventional SThM system used in this study

The changing role of beta-blocker therapy in patients with cirrhosis

SummaryCirrhosis is a leading cause of death in the United States and worldwide. Beta-blockers have been established in numerous studies as part of the cornerstone of the medical management of cirrhosis, particularly in the primary and secondary prevention of variceal hemorrhage. However, new evidence has cautioned the use of beta-blockers in patients with end-stage cirrhosis and refractory ascites. In this article, we review the beneficial effects of beta-blocker therapy, the potential harms of aggressive beta-blocker therapy, and provide suggestions regarding the appropriate use of this class of medications in patients with cirrhosis

In vitro analysis of promoter activity in Müller cells

PurposeRational modification of promoter architecture is necessary for manipulation of transgene activity and requires accurate deciphering of regulatory control elements. Identification of minimally sized promoters is critical to the design of viral vectors for gene therapy. To this end, we evaluated computational methods for predicting short DNA sequences capable of driving gene expression in Müller cells.MethodsWe measured enhanced green fluorescent protein (eGFP) expression levels driven by "full-length" promoters, and compared these data with computationally identified shorter promoter elements from the same genes. We cloned and screened over 90 sequences from nine Müller cell-associated genes: CAR2, CD44, GFAP, GLUL, PDGFRA, RLBP1, S100B, SLC1A3, and vimentin (VIM). We PCR-amplified the "full-length" promoter (~1500 bp), the proximal promoter (~500 bp), and the most proximal evolutionarily conserved region (ECR; 95-871 bp) for each gene, both with and without their respective 5' untranslated regions (UTRs), from C57BL/6J mouse genomic DNA. We selected and cloned additional ECRs from more distal genomic regions (both 5' and 3') of the VIM and CD44 genes, using both mouse and rat (Sprague-Dawley) genomic DNA as templates. PCR products were cloned into the pFTMGW or pFTM3GW lentiviral transfer vectors. Plasmid constructs were transfected into rat (wMC) or human (MIO-M1) Müller cells, and eGFP expression levels were evaluated by fluorescence microscopy and flow cytometry. Selected constructs were also examined in NIH/3T3 and Neuro-2a cells.ResultsSeveral ECRs from the nine Müller cell-associated genes were able to drive reporter gene expression as well as their longer counterparts. Preliminary comparisons of ECRs from the VIM and CD44 genes suggested that inclusion of UTRs in promoter constructs resulted in increased transgene expression levels. Systematic comparison of promoter activity from nine Müller cell-expressed genes supported this finding, and characteristic regulation profiles were evident among the different genes tested. Importantly, individual cloned promoter sequences were capable of driving distinct levels of transgene expression, resulting in up to eightfold more cells expressing eGFP with up to 3.8-fold higher mean fluorescence intensity (MFI). Furthermore, combining constructs into single regulatory "units" modulated transgene expression, suggesting that secondary gene sequences provided in cis may be used to fine-tune gene expression levels.ConclusionsIn this study, we demonstrate that computational and empirical methods, when used in combination, can efficiently identify short promoters that are active in cultured Müller cells. In addition, the pFTM3GW vector can be used to study the effects of combined promoter elements. We anticipate that these methods will expedite the design and testing of synthetic/chimeric promoter constructs that should be useful for both in vitro and in vivo applications

Topography-free sample for thermal spatial response measurement of scanning thermal microscopy

A novel fabrication technique is described for the production of multimaterial, lithographically defined, topography-free samples for use in experiments to investigate the nature of contrast in scanning probe microscopy (SPM). The approach uses a flat sacrificial substrate as the base for fabrication, which is deleted in the final step. This leaves an exposed, flat surface with patterns of materials contrast defined during the lithography stages. In the example application presented, these are designed to challenge the detection ability of a scanning thermal microscopy (SThM) probe, although many other applications can be envisioned. There are many instances in SPM where images can exhibit topographically induced artifacts. In SThM, these can result in a change of the thermal signal which can easily be misinterpreted as changes in the sample thermal conductivity or temperature. The elimination of these artifacts through postprocessing requires a knowledge of how the probe responds thermal features of differing sizes. The complete sample fabrication process, followed by successful topographic/thermal scanning is demonstrated, showing sub-1.5 nm topography with a clear artifact-free thermal signal from sub-100 nm gold wires. The thermal spatial resolution is determined for the sample materials and probe used in this study to be in the range of 35–75 nm

Functional promoter testing using a modified lentiviral transfer vector

PurposeThe importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.MethodsA synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.ResultsTransfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.ConclusionsThe TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues

Single Molecule Conformational Memory Extraction: P5ab RNA Hairpin

Extracting kinetic models from single molecule data is an important route to mechanistic insight in biophysics, chemistry, and biology. Data collected from force spectroscopy can probe discrete hops of a single molecule between different conformational states. Model extraction from such data is a challenging inverse problem because single molecule data are noisy and rich in structure. Standard modeling methods normally assume (i) a prespecified number of discrete states and (ii) that transitions between states are Markovian. The data set is then fit to this predetermined model to find a handful of rates describing the transitions between states. We show that it is unnecessary to assume either (i) or (ii) and focus our analysis on the zipping/unzipping transitions of an RNA hairpin. The key is in starting with a very broad class of non-Markov models in order to let the data guide us toward the best model from this very broad class. Our method suggests that there exists a folding intermediate for the P5ab RNA hairpin whose zipping/unzipping is monitored by force spectroscopy experiments. This intermediate would not have been resolved if a Markov model had been assumed from the onset. We compare the merits of our method with those of others

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane

Na+/K+-ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

BACKGROUND:The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+)/K(+)-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase. METHODOLOGY/PRINCIPAL FINDINGS:Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma. CONCLUSIONS/SIGNIFICANCE:The results presented here provide a novel method for capillary isolation from the inner ear and the first database on protein components in the blood-labyrinth-barrier. Additionally, we found that ATP1A1 interaction with PKCη and occludin was involved in the integrity of the blood-labyrinth-barrier

Leber Congenital Amaurosis Associated with AIPL1: Challenges in Ascribing Disease Causation, Clinical Findings, and Implications for Gene Therapy

Leber Congenital Amaurosis (LCA) and Early Childhood Onset Severe Retinal Dystrophy are clinically and genetically heterogeneous retinal disorders characterised by visual impairment and nystagmus from birth or early infancy. We investigated the prevalence of sequence variants in AIPL1 in a large cohort of such patients (n = 392) and probed the likelihood of disease-causation of the identified variants, subsequently undertaking a detailed assessment of the phenotype of patients with disease-causing mutations. Genomic DNA samples were screened for known variants in the AIPL1 gene using a microarray LCA chip, with 153 of these cases then being directly sequenced. The assessment of disease-causation of identified AIPL1 variants included segregation testing, assessing evolutionary conservation and in silico predictions of pathogenicity. The chip identified AIPL1 variants in 12 patients. Sequencing of AIPL1 in 153 patients and 96 controls found a total of 46 variants, with 29 being novel. In silico analysis suggested that only 6 of these variants are likely to be disease-causing, indicating a previously unrecognized high degree of polymorphism. Seven patients were identified with biallelic changes in AIPL1 likely to be disease-causing. In the youngest subject, electroretinography revealed reduced cone photoreceptor function, but rod responses were within normal limits, with no measurable ERG in other patients. An increasing degree and extent of peripheral retinal pigmentation and degree of maculopathy was noted with increasing age in our series. AIPL1 is significantly polymorphic in both controls and patients, thereby complicating the establishment of disease-causation of identified variants. Despite the associated phenotype being characterised by early-onset severe visual loss in our patient series, there was some evidence of a degree of retinal structural and functional preservation, which was most marked in the youngest patient in our cohort. This data suggests that there are patients who have a reasonable window of opportunity for gene therapy in childhood

Nuclear Factor Kappa B Activation Occurs in the Amnion Prior to Labour Onset and Modulates the Expression of Numerous Labour Associated Genes

Background: Prior to the onset of human labour there is an increase in the synthesis of prostaglandins, cytokines and chemokines in the fetal membranes, particular the amnion. This is associated with activation of the transcription factor nuclear factor kappa B (NFkB). In this study we characterised the level of NFkB activity in amnion epithelial cells as a measure of amnion activation in samples collected from women undergoing caesarean section at 39 weeks gestation prior to the onset of labour. Methodology/Principal Findings: We found that a proportion of women exhibit low or moderate NFkB activity while other women exhibit high levels of NFkB activity (n = 12). This activation process does not appear to involve classical pathways of NFkB activation but rather is correlated with an increase in nuclear p65-Rel-B dimers. To identify the full range of genes upregulated in association with amnion activation, microarray analysis was performed on carefully characterised nonactivated amnion (n = 3) samples and compared to activated samples (n = 3). A total of 919 genes were upregulated in response to amnion activation including numerous inflammatory genes such cyclooxygenase-2 (COX-2, 44-fold), interleukin 8 (IL-8, 6-fold), IL-1 receptor accessory protein (IL-1RAP, 4.5-fold), thrombospondin 1 (TSP-1, 3-fold) and, unexpectedly, oxytocin receptor (OTR, 24-fold). Ingenuity Pathway Analysis of the microarray data reveal the two main gene networks activated concurrently with amnion activation are i) cell death, cancer and morphology and ii) cell cycle, embryoni