A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy)

FMDV replicons encoding green fluorescent protein are replication competent

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays

LED-based photometric stereo-imaging employing frequency-division multiple access

Among the many techniques developed for 3D imaging, photometric stereo-imaging excels through the simplicity of the hardware required. It can be used stand-alone or in fusion with other 3D imaging techniques for applications such as 3D scanners, face recognition or surveillance applications [1], [2][2], [3][3], [4][4], [5]. In photometric stereo-imaging, the subject is illuminated by different light sources from different angles, and the surface shades created by each light source allow reconstruction of 3D information

Lighting as a service that provides simultaneous 3D imaging and optical wireless connectivity

Light-emitting diodes enable optical wireless data transmission and advanced imaging methods such as photometric stereo-imaging. Both, wireless communications into a scene and 3D imaging of that scene is enabled in parallel using the same set of LEDs thus providing lighting-based infrastructure e.g. for automated agents

Design, fabrication and application of GaN-based micro-LED arrays with individual addressing by n-electrodes

We demonstrate the development, performance and application of a GaN-based micro-light emitting diode (μLED) array sharing a common p-electrode (anode), and with individually addressable nelectrodes (cathodes). Compared to conventional GaN-based LED arrays, this array design employs a reversed structure of common and individual electrodes, which makes it innovative and compatible with n-type metal-oxide-semiconductor (NMOS) transistor-based drivers for faster modulation. Excellent performance characteristics are illustrated by an example array emitting at 450 nm. At a current density of 17.7 kA/cm2 in direct-current operation, the optical power and small signal electrical-to-optical modulation bandwidth of a single LED element with 24 μm diameter are over 2.0 mW and 440 MHz, respectively. The optimized fabrication process also ensures a high yield of working μLED elements per array, and excellent element-to-element uniformity of electrical/optical characteristics. Results on visible light communication are presented as an application of an array integrated with an NMOS driver. Data transmission at several hundred Mbps without bit error is achieved for single and multiple-μLED-element operations, under an on-off-keying modulation scheme. Transmission of stepped sawtooth waveforms is also demonstrated to confirm that the μLED elements can transmit discrete multi-level signals

Design, fabrication and application of GaN-based micro-LED arrays with individual addressing by n-electrodes

We demonstrate the development, performance and application of a GaN-based micro-light emitting diode (μLED) array sharing a common p-electrode (anode), and with individually addressable nelectrodes (cathodes). Compared to conventional GaN-based LED arrays, this array design employs a reversed structure of common and individual electrodes, which makes it innovative and compatible with n-type metal-oxide-semiconductor (NMOS) transistor-based drivers for faster modulation. Excellent performance characteristics are illustrated by an example array emitting at 450 nm. At a current density of 17.7 kA/cm2 in direct-current operation, the optical power and small signal electrical-to-optical modulation bandwidth of a single LED element with 24 μm diameter are over 2.0 mW and 440 MHz, respectively. The optimized fabrication process also ensures a high yield of working μLED elements per array, and excellent element-to-element uniformity of electrical/optical characteristics. Results on visible light communication are presented as an application of an array integrated with an NMOS driver. Data transmission at several hundred Mbps without bit error is achieved for single and multiple-μLED-element operations, under an on-off-keying modulation scheme. Transmission of stepped sawtooth waveforms is also demonstrated to confirm that the μLED elements can transmit discrete multi-level signals

Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation.We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilized virus-like particles (VLPs) in Pichia pastoris.The stabilized VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilization, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralize virus in vitro. Therefore, anti-EVA71 neutralizing antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies

Development, performance and application of novel GaN-based micro-LED arrays with individually addressable n-electrodes

We demonstrate the development, performance and application of a GaN-based micro-light emitting diode array sharing a common p-electrode with individual-addressed n-electrodes. These individually-addressed n-electrodes minimize the series-resistance difference from conductive paths, and offer compatibility with n-type metal-oxide-semiconductor transistor-based drivers for faster modulation

American marsupials chromosomes: Why study them?

Marsupials, one of the three main groups of mammals, are only found in Australia and in the American continent. Studies performed in Australian marsupials have demonstrated the great potential provided by the group for the understanding of basic genetic mechanisms and chromosome evolution in mammals. Genetic studies in American marsupials are relatively scarce and cytogenetic data of most species are restricted to karyotype descriptions, usually without banding patterns. Nevertheless, the first marsupial genome sequenced was that of Monodelphis domestica, a South American species. The knowledge about mammalian genome evolution and function that resulted from studies on M. domestica is in sharp contrast with the lack of genetic data on most American marsupial species. Here, we present an overview of the chromosome studies performed in marsupials with emphasis on the South American species

Reconstructing the history of ocean wildlife around Ascension Island

1. In 2016, the UK government announced plans for a large-scale Marine Protected Area around Ascension Island, a UK Overseas Territory in the South Atlantic. 2. To improve baselines for marine life to support ambitious conservation and assess change over time, archives were searched for historical accounts of wildlife from Ascension's discovery in 1501 to the present. For more recent changes, 139 interviews with past and present inhabitants were conducted. 3. Ascension's marine life has, from first discovery to the present, been consistently remarked upon for its exceptional abundance. Historical sources indicate declines in seabird and turtle populations from human exploitation and introduction of rats and cats. They are recovering with good management, although still below pre-settlement abundance. 4. Interviews with residents indicate more recent changes, notably declines in catch per unit of fishing effort at popular shore angling sites, a decline in yellowfin tuna (Thunnus albacares) and increase in Galapagos sharks (Carcharhinus galapagensis). 5. What is very notable, however, based on the interviews, was that there was no temporal signal suggestive of recent systemic decline, in marked contrast to many parts of the world where recent wildlife declines have been pervasive and steep. Ascension represents a remarkable and immensely important centre of abundance in a sea of depletion and change, warranting full protection for all the island's waters