Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R), and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R) protein.CEH and KIM acknowledge funding support from NH&MRC Project Grant #471421

HETDEX pilot survey for emission-line galaxies - I. Survey design, performance, and catalog

We present a catalog of emission-line galaxies selected solely by their emission-line fluxes using a wide-field integral field spectrograph. This work is partially motivated as a pilot survey for the upcoming Hobby-Eberly Telescope Dark Energy Experiment (HETDEX). We describe the observations, reductions, detections, redshift classifications, line fluxes, and counterpart information for 397 emission-line galaxies detected over 169 sq.arcmin with a 3500-5800 Ang. bandpass under 5 Ang. full-width-half-maximum (FWHM) spectral resolution. The survey's best sensitivity for unresolved objects under photometric conditions is between 4-20 E-17 erg/s/sq.cm depending on the wavelength, and Ly-alpha luminosities between 3-6 E42 erg/s are detectable. This survey method complements narrowband and color-selection techniques in the search for high redshift galaxies with its different selection properties and large volume probed. The four survey fields within the COSMOS, GOODS-N, MUNICS, and XMM-LSS areas are rich with existing, complementary data. We find 104 galaxies via their high redshift Ly-alpha emission at 1.9<z<3.8, and the majority of the remainder objects are low redshift [OII]3727 emitters at z<0.56. The classification between low and high redshift objects depends on rest frame equivalent width, as well as other indicators, where available. Based on matches to X-ray catalogs, the active galactic nuclei (AGN) fraction amongst the Ly-alpha emitters (LAEs) is 6%. We also analyze the survey's completeness and contamination properties through simulations. We find five high-z, highly-significant, resolved objects with full-width-half-maximum sizes >44 sq.arcsec which appear to be extended Ly-alpha nebulae. We also find three high-z objects with rest frame Ly-alpha equivalent widths above the level believed to be achievable with normal star formation, EW(rest)>240 Ang.Comment: 45 pages, 36 figures, 5 tables, submitted to ApJ

Strategies and challenges associated with recruiting retirement village communities and residents into a group exercise intervention

Background: Randomized controlled trials (RCTs) provide the highest level of scientific evidence, but successful participant recruitment is critical to ensure the external and internal validity of results. This study describes the strategies associated with recruiting older adults at increased falls risk residing in retirement villages into an 18-month cluster RCT designed to evaluate the effects of a dual-task exercise program on falls and physical and cognitive function. Methods: Recruitment of adults aged ≥65 at increased falls risk residing within retirement villages (size 60–350 residents) was initially designed to occur over 12 months using two distinct cohorts (C). Recruitment occurred via a three-stage approach that included liaising with: 1) village operators, 2) independent village managers, and 3) residents. To recruit residents, a variety of different approaches were used, including distribution of information pack, on-site presentations, free muscle and functional testing, and posters displayed in common areas. Results: Due to challenges with recruitment, three cohorts were established between February 2014 and April 2015 (14 months). Sixty retirement villages were initially invited, of which 32 declined or did not respond, leaving 28 villages that expressed interest. A total of 3947 individual letters of invitation were subsequently distributed to residents of these villages, from which 517 (13.1%) expressions of interest (EOI) were received. Across three cohorts with different recruitment strategies adopted there were only modest differences in the number of EOI received (10.5 to 15.3%), which suggests that no particular recruitment approach was most effective. Following the initial screening of these residents, 398 (77.0%) participants were deemed eligible to participate, but a final sample of 300 (58.0% of the 517 EOI) consented and was randomized; 7.6% of the 3947 residents invited. Principal reasons for not participating, despite being eligible, were poor health, lack of time and no GP approval. Conclusion: This study highlights that there are significant challenges associated with recruiting sufficient numbers of older adults from independent living retirement villages into an exercise intervention designed to improve health and well-being. Trial registration: Australian New Zealand Clinical Trials Registry: ACTRN12613001 161718. Date registered 23rd October 2013

Effectiveness of dual-task functional power training for preventing falls in older people: Study protocol for a cluster randomised controlled trial

Background: Falls are a major public health concern with at least one third of people aged 65 years and over falling at least once per year, and half of these will fall repeatedly, which can lead to injury, pain, loss of function and independence, reduced quality of life and even death. Although the causes of falls are varied and complex, the age-related loss in muscle power has emerged as a useful predictor of disability and falls in older people. In this population, the requirements to produce explosive and rapid movements often occurs whilst simultaneously performing other attention-demanding cognitive or motor tasks, such as walking while talking or carrying an object. The primary aim of this study is to determine whether dual-task functional power training (DT-FPT) can reduce the rate of falls in community-dwelling older people. Methods/Design: The study design is an 18-month cluster randomised controlled trial in which 280 adults aged =65 years residing in retirement villages, who are at increased risk of falling, will be randomly allocated to: 1) an exercise programme involving DT-FPT, or 2) a usual care control group. The intervention is divided into 3 distinct phases: 6 months of supervised DT-FPT, a 6-month 'step down' maintenance programme, and a 6-month follow-up. The primary outcome will be the number of falls after 6, 12 and 18 months. Secondary outcomes will include: lower extremity muscle power and strength, grip strength, functional assessments of gait, reaction time and dynamic balance under single- and dual-task conditions, activities of daily living, quality of life, cognitive function and falls-related self-efficacy. We will also evaluate the cost-effectiveness of the programme for preventing falls. Discussion: The study offers a novel approach that may guide the development and implementation of future community-based falls prevention programmes that specifically focus on optimising muscle power and dual-task performance to reduce falls risk under 'real life' conditions in older adults. In addition, the 'step down' programme will provide new information about the efficacy of a less intensive maintenance programme for reducing the risk of falls over an extended period. Trial registration: Australian New Zealand Clinical Trials Registry: ACTRN12613001161718. Date registered 23 October 2013

Sympathetic overdrive in obesity involves purinergic hyperactivity in the resistance vasculature

While a close correlation exists in obese humans between sympathetic, adrenergic hyperactivity and structural and functional organ damage, a role for the co-transmitter, ATP, in vascular function remains unexplored. We therefore studied sympathetic nerve-mediated responses of pressurised small mesenteric arteries from control and obese rats. Diet-induced obesity significantly increased the amplitude of vasoconstriction to transmural nerve stimulation (1-10 Hz; P < 0.05). At 1 and 5 Hz, both adrenergic and purinergic responses were significantly augmented, while only the purinergic component was increased at 10 Hz (P < 0.05). Nerve stimulation at 1 Hz evoked contractions and underlying excitatory junction potentials (EJPs), which were both significantly increased in amplitude during obesity (P < 0.05) and abolished by αβ-methylene ATP (1 μm; desensitises purinergic receptors). The rise time and rate of decay of these EJPs were significant decreased (P < 0.05), without change in resting membrane potential. Amplitude and frequency of spontaneous EJPs and the density of perivascular sympathetic nerves were also significantly increased (P < 0.05). Inhibition of sensory neurotransmitter release (capsaicin; 10 μm) significantly increased the amplitude of nerve-mediated contraction (P < 0.05), with a greater effect in control than obese animals, although the density of sensory nerves was unaffected by obesity. We demonstrate that sympathetic nerve-mediated vasoconstriction is enhanced by diet-induced obesity due to upregulation of purinergic, in addition to adrenergic, neurotransmission. Changes result from increased perivascular sympathetic innervation and release of ATP. We conclude that augmented sympathetic control of vasoconstriction induced by obesity could contribute directly to hypertension and global organ damage. A decrease in sensitivity to sensory vasodilatory neurotransmitters may also affect these processes

Specialised sympathetic neuroeffector associations in immature rat iris arterioles

Sympathetic nerve-mediated vasoconstriction in iris arterioles of mature rats occurs via the activation of α(1B)-adrenoceptors alone, while in immature rat iris arterioles, vasoconstriction occurs via activation of both α(1)- and α(2)-adrenoceptors. In mature rats the vast majority of sympathetic varicosities form close neuroeffector junctions. Serial section electron microscopy of 14 d iris arterioles has been used to determine whether restriction in physiological receptor types with age may result from the establishment of these close neuroeffector junctions. Ninety varicosities which lay within 4 μm of arteriolar smooth muscle were followed for their entire length. Varicosities rarely contained dense cored vesicles even after treatment with 5-hydroxydopamine. 47% of varicosities formed close associations with muscle cells and 88% formed close associations with muscle cells or melanocytes. Varicosities in bundles were as likely as single varicosities to form close associations with vascular smooth muscle cells, although the distribution of synaptic vesicles in single varicosities did not show the asymmetric accumulation towards the smooth muscle cells seen in the varicosities in bundles which were frequently clustered together. We conclude that restriction of physiological receptor types during development does not appear to correlate with the establishment of close neuroeffector junctions, although changes in presynaptic structures may contribute to the refinement of postsynaptic responses

Specialised sympathetic neuroeffector associations in rat iris arterioles

Vascular sympathetic neuroeffector associations have been examined in rat iris arterioles using serial section electron microscopy and reconstruction techniques. Examination of random sections showed that, of all profiles of varicosities (199) seen to lie closer than 4 μm to vascular smooth muscle cells, only a small proportion (29/199) were found in close association with vascular smooth muscle cells, where adjacent membranes were separated by less than 100 nm. However, serial section examination, from intervaricose region to intervaricose region, of 79 varicosities similarly observed lying within 4 μm of vascular smooth muscle cells showed that 54 formed close associations with vascular smooth muscle cells. In serial sections, all these varicosities were also closely associated with melanocytes and of the 25 remaining varicosities, 22 formed close associations with melanocytes alone, whilst 3 did not come into close association with any effector cell. The increased observation of close associations with vascular smooth muscle cells in serial sections, compared with random sections, is consistent with the demonstration that the area of contact only occupies, on average, a small percentage (5%) of the total surface area of the varicosity as seen in the 3-dimensional reconstructions. In both random and serial sections, close associations were observed between varicosities and vascular smooth muscle cells or melanocytes irrespective of whether fibres were present singly or in small nerve bundles. Three-dimensional reconstruction of associations of varicosities and vascular smooth muscle cells demonstrated several common features, such as accumulations of synaptic vesicles and loss of Schwann cell covering at the region of membrane facing the effector cell. The similarity in the appearance of the neuroeffector association seen in this study and those described in previous studies provides evidence for the existence of a common sympathetic neuroeffector association, irrespective of the receptor subtype involved in neurotransmission

T-type calcium channels and vascular function: the new kid on the block?

While L-type voltage-dependent calcium channels have long been considered the predominant source of calcium for myogenic constriction, recent studies of both cerebral and systemic circulations have provided evidence for the prominent expression of other members of the voltage-dependent calcium channel family, in particular the low voltage activated T-type channels. Although physiological studies have not supported the involvement of a classical low voltage activated, T-type channel in vascular function, evidence is accumulating that points to the involvement of a non-L-type, high voltage activated channel with sensitivity to T-type channel antagonists. We propose that this may arise due to expression of a T-type channel splice variant with unique biophysical characteristics resulting in a more depolarised profile. Expression of these channels in smooth muscle cells would broaden the voltage range over which sustained calcium influx occurs, while expression of T-type channels in endothelial cells could provide a feedback mechanism to prevent excessive vasoconstriction. Perturbation of this balance during pathophysiological conditions by upregulation of channel expression and endothelial dysfunction could contribute to vasospastic conditions and therapy-refractory hypertension