Photo-Induced Disproportionation-Mediated Photodynamic Therapy: Simultaneous Oxidation of Tetrahydrobiopterin and Generation of Superoxide Radicals.

We herein present an approach of photo-induced disproportionation for preparation of Type-I photodynamic agents. As a proof of concept, BODIPY-based photosensitizers were rationally designed and prepared. The photo-induced intermolecular electron transfer between homotypic chromophores leads to the disproportionation reaction, resulting in the formation of charged intermediates, cationic and anionic radicals. The cationic radicals efficiently oxidize the cellularimportant coenzyme, tetrahydrobiopterin (BH4 ), and the anionic radicals transfer electrons to oxygen to produce superoxide radicals (O2 - ⋅). One of our Type-I photodynamic agents not only self-assembles in water but also effectively targets the endoplasmic reticulum. It displayed excellent photocytotoxicity even in highly hypoxic environments (2 % O2 ), with a half-maximal inhibitory concentration (IC50 ) of 0.96 µM, and demonstrated outstanding antitumor efficacy in murine models bearing HeLa tumors.

Circularly Polarized Lasing from Helical Superstructures of Chiral Organic Molecules.

Chiral supramolecular aggregates have the potential to explore circularly polarized lasing with large dissymmetry factors. However, the controllable assembly of chiral superstructures towards deterministic circularly polarized laser emission remains elusive. Here, we design a pair of chiral organic molecules capable of stacking into a pair of definite helical superstructures in microcrystals, which enables circularly polarized lasing with deterministic chirality and high dissymmetry factors. The microcrystals function as optical cavities and gain media simultaneously for laser oscillations, while the supramolecular helices endow the laser emission with strong and opposite chirality. As a result, the microcrystals of two enantiomers allow for circularly polarized laser emission with opposite chirality and high dissymmetry factors up to ~1.0. This work demonstrates the chiral supramolecular assemblies as an excellent platform for high-performance circularly polarized lasers.

Spontaneous Symmetry Breaking of Achiral Molecules Leading to the Formation of Homochiral Superstructures that Exhibit Mechanoluminescence.

Chirality, with its intrinsic symmetry-breaking feature, is frequently utilized in the creation of acentric crystalline functional materials that exhibit intriguing optoelectronic properties. On the other hand, the development of chiral crystals from achiral molecules offers a solution that bypasses the need for enantiopure motifs, presenting a promising alternative and thereby expanding the possibilities of the self-assembly toolkit. Nevertheless, the rational design of achiral molecules that prefer spontaneous symmetry breaking during crystallization has so far been obscure. In this study, we present a series of six achiral molecules, demonstrating that when these conformationally flexible molecules adopt a cis-conformation and engage in multiple non-covalent interactions along a helical path, they collectively self-assemble into chiral superstructures consisting of single-handed supramolecular columns. When these homochiral supramolecular columns align in parallel, they form polar crystals that exhibit intense luminescence upon grinding or scraping. We therefore demonstrate our molecular design strategy could significantly increase the likelihood of symmetry breaking in achiral molecular synthons during self-assembly, offering a facile access to novel chiral crystalline materials with unique optoelectronic properties.

Intense Circularly Polarized Luminescence Induced by Chiral Supramolecular Assembly: The Importance of Intermolecular Electronic Coupling.

Herein we report on circularly polarized luminescence (CPL) emission originating from supramolecular chirality of organic microcrystals with a |glum| value up to 0.11. The microcrystals were prepared from highly emissive difluoroboron ß-diketonate (BF2dbk) dyes R-1 or S-1 with chiral binaphthol (BINOL) skeletons. R-1 and S-1 exhibit undetectable CPL signals in solution but manifest intense CPL emission in their chiral microcrystals. The chiral superstructures induced by BINOL skeletons were confirmed by single-crystal XRD analysis. Spectral analysis and theoretical calculations indicate that intermolecular electronic coupling, mediated by the asymmetric stacking in the chiral superstructures, effectively alters excited-state electronic structures and facilitates electron transitions perpendicular to BF2bdk planes. The coupling increases cosθµ,m from 0.05 (monomer) to 0.86 (tetramer) and triggers intense optical activity of BF2bdk. The results demonstrate that optical activity of chromophores within assemblies can be regulated by both orientation and extent of intermolecular electronic couplings.

Establishment of pseudorabies virus latency and reactivation model in mice dorsal root ganglia culture.

Pseudorabies virus (PRV) belongs to the alpha herpesvirus family and is responsible for Aujeszky's disease in pigs. Similar to other alpha herpesviruses, PRV establishes a lifelong latent infection in trigeminal ganglion. These latently infected pigs serve as a reservoir for recurrent infections when reactivation is triggered, making the eradication of PRV a challenging task. However, the molecular mechanism underlying PRV latency and reactivation in neurons is still poorly understood due to limitations in the in vitro model. To establish a pseudorabies virus latency and reactivation model in primary neuron cultures, we isolated dorsal root ganglion (DRG) from newborn Kunming mice using a method named epineurium-pulling for DRG collection (EPDC) and cultured primary neurons in vitro. A dual-colour recombinant PRV BAC mRuby-VP16 was constructed and 0.5 multiplicity of infection (MOI) was found as an appropriate dose in the presence of aciclovir to establish latency. Reactivation was induced using UV-inactivated herpesviruses or a series of chemical inhibitors. Interestingly, we found that not only UV-PRV, but also UV-HSV-1 and UV-BHoV-5 were able to induce rapid PRV reactivation. The efficiency of reactivation for LY294002, forskolin, etoposide, dexamethasone, and acetylcholine was found to be dependent on their concentration. In conclusion, we developed a valuable model of PRV latency and reactivation, which provides a basis for future mechanism research.

Circularly Polarized Laser Emission from Homochiral Superstructures based on Achiral Molecules with Conformal Flexibility.

Without external chiral intervention, it is a challenge to form homochirality from achiral molecules with conformational flexibility. We here report on a rational strategy that uses multivalent noncovalent interactions to clamp the molecular conformations of achiral D-A molecules. These interactions overcome the otherwise dominant dipole-dipole interactions and thus disfavor their symmetric antiparallel stacking. It in turn facilitates parallel packing, leading to spontaneous symmetry breaking during crystallization and thus the formation of homochiral conglomerates. When this emergent homochirality is coupled with optical gain characteristics of the molecules, the homochiral crystals are explored as excellent circularly polarized micro-lasers with low lasing threshold (16.4 µJ cm-2 ) and high dissymmetry factor glum (0.9). This study therefore provides a facile design strategy for supramolecular chiral materials and active laser ones without the necessity of intrinsic chiral element.

Feedback inhibition of bovine herpesvirus 5 replication by dual-copy bhv5-miR-B10-3p.

Bovine herpesvirus 5 (BoHV-5) is a pathogen of cattle responsible for fatal meningoencephalitis. Like alpha herpesvirus subfamily members, BoHV-5 also encodes microRNA in lytic infections of epithelial cells. BoHV-5-miR-B10 was the most abundant miRNA detected in a high-throughput sequencing study. Here, we evaluated the kinetics of miR-B10 expression after BoHV-5 productive infection by stem-loop real-time quantitative PCR. miR-B10 candidate target sites in the virus were predicted, and BoHV-5 UL39 was confirmed as a target gene by dual-luciferase assay with the design of an miR-B10 tough decoy (TuD). The UL39 gene encoding ribonucleotide reductase (RR) large subunit plays an important role in the early stage of BoHV-5 lytic infection. As BoHV-5-miR-B10 is located in internal and terminal repeat regions, we generated a TuD gene-integrated BoHV-5 strain, which effectively down-regulated miR-B10-3p. Strikingly, the suppression of miR-B10-3p significantly improved BoHV-5 replication. Taking these findings together, our study established an efficient method to deliver and express TuD RNA for viral miRNA suppression, and demonstrated that virus-encoded miRNA suppresses viral-genome biogenesis with a feedback mode, which might serve as a brake for viral replication. Herpesviruses infect humans and a variety of animals. Almost all herpesviruses can encode miRNAs, but the functions of these miRNAs remain to be elucidated. Most herpesvirus-encoded miRNA harbours dual copies, which is difficult to be deleted by current genetic modulation. Here, we developed an efficient method to deliver and express TuD RNA to efficiently suppress viral miRNA with multiple copies. Using this method, we demonstrated for the first time that viral miRNA feedback regulates viral replication by suppressing the expression of RR.

The Carboxyl Terminus of Tegument Protein pUL21 Contributes to Pseudorabies Virus Neuroinvasion.

Following its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21's contribution to PRV neuroinvasion in vivo Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transport in vitro and in vivo Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion.IMPORTANCE Herpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

Typical gene expression profile of pseudorabies virus reactivation from latency in swine trigeminal ganglion.

Pseudorabies virus (PRV) establishes a lifelong latent infection in swine trigeminal ganglion (TG) following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Muscle injection combined with intravenous deliver of the synthetic corticosteroid dexamethasone (DEX) consistently induces reactivation from latency in pigs. In this study, PRV-free piglets were infected with PRV. Viral shedding in nasal and ocular swabs demonstrated that PRV infection entered the latent period. The anti-PRV antibody was detected by enzyme-linked immunosorbent assay and the serum neutralization test, which suggested that the PRV could establish latent infection in the presence of humoral immunity. Immunohistochemistry and viral genome detection of TG neurons suggested that PRV was reactivated from latency. Viral gene expressions of IE180, EP0, VP16, and LLT-intron were readily detected at 3-h post-DEX treatment, but gB, a γ1 gene, was not detectable. The differentially expressed phosphorylated proteins of TG neurons were analyzed by ITRAQ coupled with LC-MS/MS, and p-EIF2S2 differentially expression was confirmed by western blot assay. Taken together, our study provides the evidence that typical gene expression in PRV reactivation from latency in TG is disordered compared with known lytic infection in epithelial cells.

G2-quadruplex in the 3'UTR of IE180 regulates Pseudorabies virus replication by enhancing gene expression.

RNA secondary structure elements in the mRNA 3'-untranslated regions (3'UTR) play important roles in post-transcriptional regulation. RNA structure elements in the viral RNA provide valuable model for studying diverse regulation mechanisms. Herpesvirus genomes are double-stranded DNA with GC-rich sequences, which can be transcribed into abundant GC-rich RNAs. It is valuable to explore the structures and function of those GC-rich RNAs. We identified a G2-quadruplex-forming sequence named PQS18-1 in the 3'UTR of the unique immediate early gene of Pseudorabies virus (PRV), an important member of Alphaherpesvirinae subfamily. The RNA PQS18-1 was folded into parallel G-quadruplex structure, enhancing gene expression. Both non-G-quadruplex mutant and G3-quadruplex mutant in the 3'UTR showed lower gene expression level than the wildtype G2-quadruplex. TMPyP4 destroyed PQS18-1 G2-quadruplex and suppressed gene expression, accordingly reducing PRV replication by one titre in the PK15 cells at 24 h post infection. Our findings indicated that the RNA G2-quadruplex in 3'UTR was essential for high expression of IE180 gene, and it could be a specific post-transcription regulation element in response to small molecules or other macromolecules. This study discovers a novel RNA G2-quadruplex in the 3'UTR of an immediate early gene of alphaherpesvirus and provides a new nucleic acid target for anti-virus drug design.

The Effect of Electron Donation and Intermolecular Interactions on Ultralong Phosphorescence Lifetime of 4-Carnoyl Phenylboronic Acids.

Purely organic phosphors with persistent room-temperature phosphorescence (RTP) demonstrate promising potential applications in optoelectronic area, bioimaging, and chemical sensing. However, it is still a formidable challenge to further design new organic phosphors due to the unclear mechanism to produce ultralong phosphorescence lifetimes. This paper investigates the correlation between the ultralong phosphorescence lifetime and structure of a series of 4-carbonylphenylboronic acid derivatives in the crystal state. Experimental and calculation results reveal that the electron-donating effect of substituents makes the phosphorescence lifetime longer by not only weakening the vibration relaxation of the excited triplet state but also increasing the energy of T1. Moreover, numerous intermolecular interactions for reducing nonradiative relaxation and the degree of the π-π stacking for stabilizing the triplet state are beneficial to the persistent RTP. The work is conducted to clarify the structure-property correlation of phosphorescent materials and design new persistent phosphors. Finally, an attempt is completed using phosphorescent materials to design two-dimensional or three-dimensional codes and anticounterfeiting applications.

Capping-RACE: a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Rapid amplification of cDNA ends (RACE) is a prevalent technique used to obtain the 5' ends of transcripts. Several different 5' RACE methods have been developed, and one particularly simple and efficient approach called CapFinder relies on the 5' cap-dependent template-switching that occurs in eukaryotes. However, most prokaryotic transcripts lack a 5' cap structure. Here, we report a procedure to capture primary transcripts based on capping the 5' triphosphorylated RNA in prokaryotes. Primary transcripts were first treated with vaccinia capping enzyme to add a 5' cap structure. First-strand cDNA was then synthesized using Moloney murine leukaemia virus reverse transcriptase. Finally, a template-switching oligonucleotide with a tail containing three ribonucleic acid guanines was hybridized to the cDNA 3' poly(C) and further used as template for reverse transcriptase. It is oligonucleotide sequence independent and is more sensitive compared to RLM-RACE. This approach specifically identified the transcription start sites of ompA, sodB and shiA in Escherichia coli and of ompA, rne and rppH in Brucella melitensis. Furthermore, we also successfully identified the transcription start sites of small noncoding genes ryhB and micC in E. coli and bsnc135 and bsnc149 in B. melitensis. Our findings suggest that Capping-RACE is a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Expression of pseudorabies virus-encoded long noncoding RNAs in epithelial cells and neurons.

Long noncoding RNAs (lncRNAs) play important roles in regulating eukaryotic genome replication and gene expression in diverse biological systems. Here, we identified lncRNAs transcribed from pseudorabies virus (PRV)-infected PK-15 cells. Based on high-throughput sequencing data, we obtained 87,263,926 and 93,947,628 clean reads from mock-infected and PRV-infected PK-15 cells, respectively. Through a normalized analytic protocol, we identified three novel viral lncRNAs. According to an analysis of differential expression between the mock-infected and PRV-infected cells, 4151 host lncRNAs were significantly upregulated and 2327 host lncRNAs were significantly downregulated in the latter group. Viral lncRNAs and several host lncRNAs were verified by northern blotting and real-time PCR. The findings showed that the viral lncRNA LDI might regulate the expression of IE180, a potent transcriptional activator of viral genes. Furthermore, we characterized the expression of viral lncRNAs in a culture of infected primary chicken dorsal root ganglia (DRG). Collectively, the obtained data suggest that PRV generates lncRNAs in both epithelial cells and chick DRG neurons.

In Situ Polymerized PAN-Assisted S/C Nanosphere with Enhanced High-Power Performance as Cathode for Lithium/Sulfur Batteries.

Carbonaceous and polymer materials are extensively employed as conductor and container to encapsulate sulfur particles and limit polysulfide dissolution. Even so, high-power performance is still far from satisfaction due to the expansion and collapse of the electrode materials during thousands of charge-discharge process. Herein, it is found that colloidal carbon sphere with high elastic coefficient can be utilized as a framework to load sulfur, which can trap soluble polysulfides species in the pores within the sphere and efficaciously improve the electronic conductivity of the cathode. After modified by polyaniline (PAN) through in situ polymerization, PAN-assisted S/C nanosphere (PSCs-73, with 73 wt % sulfur) effectively minimize polysulfide diffusion, enhance the electron transfer rate and overcome the problem of volume expansion. The fabricated PSCs-73 cell shows outstanding long high-power cycling capability over 2500 charge/discharge cycles with a capacity decay of 0.01% per cycle at 5 C. Substantially, this composite can drive 2.28 W white indicators of LED robustly after minutes of charging by three lithium batteries in series, showing a promising potential application in the future.

Bovine herpesvirus 5 encodes a unique pattern of microRNAs compared with bovine herpesvirus 1.

Bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus 1 (BoHV-1) are two closely related viruses. However, BoHV-5 is responsible for fatal meningitis in calves, while BoHV-1 is associated with infectious rhinotracheitis in cattle, and the mechanism by which the two viruses cause different symptoms is not well understood. In this study, we identified 11 microRNA (miRNA) genes, encoded by the BoHV-5 genome, that were processed into 16 detectable mature miRNAs in productive infection as determined by deep sequencing. We found that 6 out of 16 miRNA genes were present as two copies in the internal repeat and terminal repeat regions, resulting in a total of 17 miRNA-encoding loci distributed in both DNA strands. Surprisingly, BoHV-5 shared only one conservative miRNA with BoHV-1, which was located upstream of the origin of replication. Furthermore, in contrast to BoHV-1, no miRNAs were detected in the unique short region and locus within or near the bovine infected-cell protein 0 and latency-related genes. Variations in both the 5' and 3' ends of the reference sequence were observed, resulting in more than one isoform for each miRNA. All of the 16 miRNAs were detectable by stem-loop reverse transcriptase-PCR. The miRNAs with high read numbers were subjected to Northern blot analysis, and all pre-miRNAs and one mature miRNA were detected. Collectively, the data suggest that BoHV-5 encodes a different pattern of miRNAs, which may regulate the life cycle of BoHV-5 and might account for the different pathogenesis of this virus compared with BoHV-1.

Reduced plasma corticotropin-releasing hormone levels during late gestation in patients with intrahepatic cholestasis of pregnancy.

OBJECTIVE: Intrahepatic cholestasis of pregnancy (ICP) may lead to sudden onset of stillbirth, which most likely is related to uteroplacental insufficiency and dysregulation of the fetal blood supply. The relaxing effect of corticotropin-releasing hormone (CRH) on blood vessels was measured to examine the role of CRH in the pathogenesis of ICP. METHODS: Eighty normal pregnant women and 80 ICP patients were divided into four groups of 20 cases, respectively, each based on gestational age from week 34 to 37. Radioimmunoassay was used to measure CRH in plasma samples collected from all of the subjects. RESULTS: Plasma CRH increased markedly from week 34 to 37 in both ICP and healthy patients, but the increase was lower in the ICP group. Plasma CRH was 322 ± 61 pg/ml in mild ICP cases at 37 weeks' compared to 1,066 ± 173 pg/ml in controls (p < 0.05), but only 218 ± 128 pg/ml in severe ICP (p < 0.05). Plasma CRH was significantly lower at all other measured time points in patients with severe ICP. In ICP patients, there was a negative correlation between plasma CRH and total bile acid (TBA). CONCLUSION: A limited increasing CRH level and negative correlation of CRH with TBA were unveiled in ICP patients.

Pseudorabies virus UL13 primes inflammatory response through downregulating heat shock factor 1.

Pseudorabies virus is a swine alpha-herpesvirus. We demonstrated that alpha-herpesvirus infection downregulates HSF1, a master transcription factor in the heat shock response. The serine/threonine protein kinase activity of late viral protein UL13 is indispensable for HSF1 depletion and phosphorylation, and UL13 does not degrade HSF1 posttranslationally but inhibits the HSF1 mRNA level. Importantly, UL13 increased HSF1 activity even though it reduced HSF1 mRNA. Furthermore, viral replication markedly decreased in the HSF1 knockout cell line or in the presence of an HSF1-specific inhibitor. Interestingly, HSF1 knockout accelerated the activation of NF-κB and p38MAPK. The K96 loci of UL13 are important to induce high levels of IL-6, TNF-α, and IL-ß cytokines while playing a crucial role in promoting mild interstitial pneumonia, liver necrosis, and severe inflammatory cell infiltration in the footpad. Thus, UL13 steers the heat shock response to promote viral replication and the inflammatory response. IMPORTANCE: PRV is a ubiquitous pathogen that infects a variety of mammals, such as pigs, ruminants, carnivores, and rodents as well as human beings, causing enormous economic losses in the swine industry. Here, we employed PRV as a model to determine the relationship between α-herpesvirus and the inflammatory response. Overall, our findings indicated that PRV infection inhibits the level of HSF1 mRNA via the serine/threonine protein kinase activity of UL13. Additionally, we discovered that HSF1 was involved in NF-κB activation upon PRV infection. PRV UL13 orchestrates the level of HSF1 mRNA, HSF1 protein phosphorylation, and priming of the inflammatory response. Our study reveals a novel mechanism employed by UL13 serine/threonine protein kinase activity to promote the inflammatory response, providing novel clues for therapy against alpha-herpesvirus infection.

A donor-acceptor cage for circularly polarized TADF emission.

Herein, we report the first example of chiral donor-acceptor cage DA-2 displaying efficient circularly polarized thermally activated delayed fluorescence (CP-TADF) with |glum| values up to 2.1 × 10-3 and PLQY of 32%. A small ΔEST of 0.051 eV and quasi-parallel (θ = 6°) transition electric and magnetic dipole moments were realized from the through-space charge transfer interaction between the parallelly aligned donor and acceptor in DA-2. This D-A cage configuration has provided a novel design strategy for discovering potential efficient CP-TADF emitters.

[Expression of corticotropin-releasing hormone receptor type-1 in intrahepatic cholestatic placental tissue].

OBJECTIVE: To explore the expression level of corticotropin-releasing hormone receptor type-1 (CRHR-1) in intrahepatic cholestatic placental (ICP) tissue. METHODS: Human placental samples were collected from 10 ICP patients and 10 healthy controls after parturition at 37-40 weeks of gestation. CRHR-1 protein and mRNA expression was assessed by western blotting and nested-real-time fluorescence quantitative PCR, respectively. Normally distributed data were summarized as mean +/- standard deviation, and intergroup comparisons were made by two-tailed Student's t-test. Non-normally distributed data were presented as median with interquartile range, and intergroup comparisons were made by Wilcoxon test. For all statistical analyses, a two-tailed P-value of less than 0.05 was considered statistically significant. RESULTS: The CRHR-1 fluorescence intensity was lower in ICP tissues (1.55 +/- 0.28) than in placental tissues from healthy controls (1.60 +/- 0.37), but the difference did not reach statistical significance (t = 0.349, P = 0.732). The CRHR-1 mRNA content was slightly higher in the ICP tissues [0.139(0.268)] than in the placental tissues from healthy controls [0.031(0.245)], but the difference did not reach statistical significance (t = 1.504, P = 0.136). CONCLUSION: CRHR-1 expression is decreased in ICP tissues, which may lead to a smaller volume of placental lobular villi vessels and restrict the CRH positive feedback loop, ultimately promoting acute hypoxic stress and possible harm to the fetus.

Rotors tailoring molecular stacking for constructing multi-stimuli-responsive luminescent materials.

We report a multi-stimuli-responsive luminescent material containing rotor moieties. It forms two types of crystals, G and O. The emission of G can be modulated by multiple external stimuli, whereas O does not show such responsiveness. The X-ray structure analysis reveals that the rotors are critical for the polymorphic emission and stimuli response properties.