Circumventricular organ apelin receptor knockdown decreases blood pressure and sympathetic drive responses in the spontaneously hypertensive rat

The central site(s) mediating the cardiovascular actions of the apelin-apelin receptor (APJ) system remains a major question. We hypothesized that the sensory circumventricular organs (CVOs), interfacing between the circulation and deeper brain structures, are sites where circulating apelin acts as a signal in the central nervous system to decrease blood pressure (BP). We show that APJ gene (aplnr) expression was elevated in the CVOs of spontaneously hypertensive rats (SHRs) compared to normotensive Wistar Kyoto (WKY) controls, and that there was a greater mean arterial BP (MABP) decrease following microinjection of [Pyr(1)]apelin-13 to the CVOs of SHRs compared to WKY rats. Lentiviral APJ-specific-shRNA (LV-APJ-shRNA) was used to knockdown aplnr expression, both collectively in three CVOs and discretely in individual CVOs, of rats implanted with radiotelemeters to measure arterial pressure. LV-APJ-shRNA-injection decreased aplnr expression in the CVOs and abolished MABP responses to microinjection of [Pyr(1)]apelin-13. Chronic knockdown of aplnr in any of the CVOs, collectively or individually, did not affect basal MABP in SHR or WKY rats. Moreover, knockdown of aplnr in any of the CVOs individually did not affect the depressor response to systemic [Pyr(1)]apelin-13. By contrast, multiple knockdown of aplnr in the three CVOs reduced acute cardiovascular responses to peripheral [Pyr(1)]apelin-13 administration in SHR but not WKY rats. These results suggest that endogenous APJ activity in the CVOs has no effect on basal BP but that functional APJ in the CVOs is required for an intact cardiovascular response to peripherally administered apelin in the SHR

Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Using cell-type-specific promoters to restrict gene expression to particular cells is an attractive approach for gene therapy, but often hampered by insufficient transcriptional activity of these promoters. Previous studies have shown that transcriptional amplification strategy (TAS) can be used to enhance the activity of such promoters without loss of cell type specificity. Originally TAS involved the use of two copies of a cell-specific promoter leading to generation of large expression cassettes, which can be hard to use given the space limitations of the conventional viral gene expression vectors.</p> <p>Results</p> <p>We have now developed a new bidirectional lentiviral vector system, based on TAS that can enhance the transcriptional activity of human synapsin-1 (SYN) promoter and the compact glial fibrillary acidic protein (GfaABC₁D) promoter. In the opposite orientation, a minimal core promoter (65 bp) derived from the human cytomegalovirus (CMV) was joined upstream of the SYN promoter or GfaABC₁D promoter. This led to the formation of synthetic bidirectional promoters which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator. The downstream cassette drove the synthesis of the gene of interest. Studies in both cell cultures and <it>in vivo </it>showed that the new bidirectional promoters greatly increased the expression level of the reporter gene. <it>In vivo </it>studies also showed that transgene expression was enhanced without loss of cell specificity of both SYN and GfaABC₁D promoters.</p> <p>Conclusion</p> <p>This work establishes a novel approach for creating compact TAS-amplified cell-specific promoters, a feature important for their use in viral backbones. This improved approach should prove useful for the development of powerful gene expression systems based on weak cell-specific promoters.</p

Distinguishing low frequency mutations from RT-PCR and sequence errors in viral deep sequencing data

There is a high prevalence of coronary artery disease (CAD) in patients with left bundle branch block (LBBB); however there are many other causes for this electrocardiographic abnormality. Non-invasive assessment of these patients remains difficult, and all commonly used modalities exhibit several drawbacks. This often leads to these patients undergoing invasive coronary angiography which may not have been necessary. In this review, we examine the uses and limitations of commonly performed non-invasive tests for diagnosis of CAD in patients with LBBB

Mapping the cellular electrophysiology of rat sympathetic preganglionic neurones to their roles in cardiorespiratory reflex integration:A whole cell recording study in situ

Sympathetic preganglionic neurones (SPNs) convey sympathetic activity flowing from the CNS to the periphery to reach the target organs. Although previous in vivo and in vitro cell recording studies have explored their electrophysiological characteristics, it has not been possible to relate these characteristics to their roles in cardiorespiratory reflex integration. We used the working heart–brainstem preparation to make whole cell patch clamp recordings from T3–4 SPNs (n = 98). These SPNs were classified by their distinct responses to activation of the peripheral chemoreflex, diving response and arterial baroreflex, allowing the discrimination of muscle vasoconstrictor-like (MVC(like), 39%) from cutaneous vasoconstrictor-like (CVC(like), 28%) SPNs. The MVC(like) SPNs have higher baseline firing frequencies (2.52 ± 0.33 Hz vs. CVC(like) 1.34 ± 0.17 Hz, P = 0.007). The CVC(like) have longer after-hyperpolarisations (314 ± 36 ms vs. MVC(like) 191 ± 13 ms, P < 0.001) and lower input resistance (346 ± 49  MΩ vs. MVC(like) 496 ± 41 MΩ, P < 0.05). MVC(like) firing was respiratory-modulated with peak discharge in the late inspiratory/early expiratory phase and this activity was generated by both a tonic and respiratory-modulated barrage of synaptic events that were blocked by intrathecal kynurenate. In contrast, the activity of CVC(like) SPNs was underpinned by rhythmical membrane potential oscillations suggestive of gap junctional coupling. Thus, we have related the intrinsic electrophysiological properties of two classes of SPNs in situ to their roles in cardiorespiratory reflex integration and have shown that they deploy different cellular mechanisms that are likely to influence how they integrate and shape the distinctive sympathetic outputs

Microstructural penetrometry of asteroid regolith analogues and Titan’s surface

We investigate essential aspects of penetrometer design required to measure particle properties on asteroids using a combination of laboratory analogue regoliths and spaceflight data returned by the Huygens probe from Titan. Penetrometry in granular material is complicated due to multiple and interdependent mechanical processes that occur during penetration. A numerical impact model is developed that simulates the behaviour of a penetrometer and its force sensor in a granular medium. The model is based on the Huygens ACC-E instrument that successfully returned penetrometry data from the surface of Titan. Penetrometry measurements are made in analogue asteroid regoliths using a laboratory copy of ACC-E. The average particle size in the targets ranged from 0.1 to 0.9 of the penetrometer tip diameter. To describe the structure seen in the data a number of metrics are defined to characterise the peaks seen in the returned signal. The significance of the variation of the metrics (such as peak height or width) with particle mass and radius are analysed in terms of penetrometer properties such as impact velocity, elastic properties and data logging parameters. We find the penetrometer can be used to measure average particle radius and mass adequately for a mid-range of particle radii. Electronic noise effects mostly the results from very small and very large particles. For high mass particles there is evidence that particle–particle impacts, within the target are being felt by the tip that make any straight forward interpretations using peak frequency a challenge. Using our numerical model the Huygens penetrometry data from Titan is analysed. A particle diameter, of around half a centimetre, is found to be consistent with the penetrometry data returned by Huygens. Recommendations and lessons learned, regarding data interpretation techniques are made for asteroid penetrometry (or any other extraterrestrial surface) when using this instrument