Integrated transcriptomics and metabolomics reveal the toxic mechanisms of mercury exposure to an endangered species Tachypleus tridentatus.

Mercury (Hg) pollution is threatening the health of endangered Tachypleus tridentatus whereas the toxic mechanism is still unclear. This study combined transcriptomic and metabolomics technology to reveal the toxic mechanisms of mercury (Hg 2+, 0.025 mg/L) exposing to T. tridentatus larvae for 15 days. Mercury induced cellular toxicity and cardiovascular dysfunction by dysregulating the genes related to endocrine system, such as polyubiquitin-A, cathepsin B, atrial natriuretic peptide, etc. Mercury induced lipid metabolic disorder with the abnormal increase of lysoPC, leukotriene D4, and prostaglandin E2. Cytochrome P450 pathway was activated to produce anti-inflammatory substances to reconstruct the homeostasis. Mercury also inhibited arginine generation, which may affect the development of T. tridentatus by disrupting the crucial signaling pathway. The mercury methylation caused enhancement of S-adenosylmethionine to meet the need of methyl donor. The mechanisms described in present study provide new insight into the risk assessment of mercury exposure to T. tridentatus.

Response of moulting genes and gut microbiome to nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus.

Nanoplastics (NPs) and heavy metals are typical marine pollutants, affecting the gut microbiota composition and molting rate of marine organisms. Currently, there is a lack of research on the toxicological effects of combined exposure to horseshoe crabs. In this study, we investigated the effects of NPs and copper on the expression of molt-related genes and gut microbiome in juvenile tri-spine horseshoe crabs Tachypleus tridentatus by exposing them to NPs (100 nm, 104 particles L-1) and/or Cu2+ (10 µgL-1) in seawater for 21 days. Compared with the control group, the relative mRNA expression of ecdysone receptor (EcR), retinoid x receptor (RXR), calmodulin-A-like isoform X1 (CaM X1), and heat shock 70 kDa protein (Hsp70) were significantly increased under the combined stress of NPs and Cu2+. There were no significant differences in the diversity and abundance indices of the gut microbial population of horseshoe crabs between the NPs and/or Cu2+ groups and the control group. According to linear discriminant analysis, Oleobacillus was the most abundant microorganism in the NPs and Cu2+ stress groups. These results indicate that exposure to either NPs stress alone or combined NPs and Cu2+ stress can promote the expression levels of juvenile molting genes. NPs exposure has a greater impact on the gut microbial community structure of juvenile horseshoe crabs compared to Cu2+ exposure. This study is helpful for predicting the growth and development of horseshoe crabs under complex environmental pollution.

Comparative physicochemical analysis of chitin quality of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus.

Recently, chitin biopolymer has received much attention as a wide variety of biomedical application for this and its derivatives have been reported, in fact, the study of non-conventional species as alternative sources of them compounds has taken particular interest. Here, we present a comparative physicochemical survey of the two tagmata in the exoskeleton of the horseshoe crab Limulus polyphemus: the prosoma and the opisthosoma, collected in Yucatán, Mexico. The characterization included CHNSO analysis, FTIR, TGA, DSC, XRD, and SEM. The CHNSO analysis revealed that C is present in the highest proportion (∼45 %) and that chemical composition did not show significant differences (P < 0.05) between the two tagmata. FTIR spectra of two tagmata presented a wide characteristic band of the chitin between 3600 and 3000 cm-1, confirming the presence of this biopolymer in the exoskeleton studied. TGA and DTGA profiles resulted very similar for both tagmata being the residual mass at 650 °C of around 30 % for both samples; these values were associated to the presence of minerals. SEM micrographs showed a porous matrix with infinite large number of irregularly shaped particles. Results show that both tagmata are made up of chitin, and they seem to have a high mineral content.

Gradients of Orientation, Composition, and Hydration of Proteins for Efficient Light Collection by the Cornea of the Horseshoe Crab.

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.

Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review.

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.

Purification and Assays of Tachylectin-2.

Tachylectin-2, a 27-kDa protein consisting of a five-bladed ß-propeller structure, is purified by three steps of chromatography, including dextran sulfate-Sepharose CL-6B, CM-Sepharose CL-6B, and Mono S. Three isolectins of tachylectin-2 including tachylectin-2a, -2b, and -2c are purified. These isolectins exhibit hemagglutinating activity against human A-type erythrocytes in a Ca2+-independent manner with tachylectin-2b showing the highest activity. Tachylectin-2b specifically agglutinates Staphylococcus saprophyticus KD. The tachylectin-2b-mediated hemagglutination is inhibited in the presence of GlcNAc and GalNAc. The association constants for GlcNAc and GalNAc are Ka = 1.95 × 104 M-1 and Ka = 1.11 × 103 M-1, respectively. Ultracentrifugation analysis shows that tachylectin-2b is present in monomer form in solution.

Cyclic Analogues of Horseshoe Crab Peptide Tachyplesin I with Anticancer and Cell Penetrating Properties.

Tachyplesin-I (TI) is a host defense peptide from the horseshoe crab Tachypleus tridentatus that has outstanding potential as an anticancer therapeutic lead. Backbone cyclized TI (cTI) has similar anticancer properties to TI but has higher stability and lower hemolytic activity. We designed and synthesized cTI analogues to further improve anticancer potential and investigated structure-activity relationships based on peptide-membrane interactions, cellular uptake, and anticancer activity. The membrane-binding affinity and cytotoxic activity of cTI were found to be highly dependent on peptide hydrophobicity and charge. We describe two analogues with increased selectivity toward melanoma cells and one analogue with the ability to enter cells with high efficacy and low toxicity. Overall, the structure-activity relationship study shows that cTI can be developed as a membrane-active antimelanoma lead, or be employed as a cell penetrating peptide scaffold that can target and enter cells without damaging their integrity.

The Ameliorating Effect of Plasma Protein from Tachypleus tridentatus on Cyclophosphamide-Induced Acute Kidney Injury in Mice.

In the study, the protective effect of plasma protein from Tachypleus tridentatus (PPTT) on acute kidney injury (AKI) and the related molecular mechanisms were first investigated by Western blotting analyses, TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and immunohistochemistry. It was found that PPTT had an obviously inhibitory effect on Reactive oxygen species (ROS) in cyclophosphamide (CTX)-exposed mice. Furthermore, results demonstrated that the renal cell death mode is due to inducing apoptosis and autophagy inhibited by dose-dependent PPTT in mice treated with CTX by decreasing the protein expression of bax, beclin-1, and LC3 and increasing the expression of bcl-2. Moreover, the p38 MAPK and PI3K/Akt signaling pathways were observed to take part in the PPTT-induced renal cell growth effect by enhancing the upregulation of the expression of Akt and p-Akt as well as the downregulation of the expression of p38 and p-p38, which indicated a PPTT ameliorating effect on AKI CTX-induced in mice through p38 MAPK and PI3K/Akt signaling pathways. Briefly, this article preliminarily studies the mechanism of the PPTT ameliorating effect on AKI CTX-induced in mice, which helps to provide a reference for PPTT clinical application in AKI therapy.

Toxin and toxicity identification of mangrove horseshoe crab Carcinoscorpius rotundicauda collected from South China.

The presence of paralytic shellfish poisoning (PSP), diarrhetic Shellfish Poisoning (DSP), tetrodotoxin (TTX) and its analogues (11-oxoTTX, 4.9-anhydro-11-oxoTTX, 4.9-anhydroTTX, 5-deoxyTTX, 5.11-dideoxyTTX, 5.6.11-trideoxyTTX and 4.9-anhydro-5.6.11-trideoxy TTX) were initially investigated in Carcinoscorpius rotundicauda collected from south China with Liquid chromatography-tandem mass spectrometry (LC-MS-MS) and mouse bioassay. The TTX toxicity was 10.8 ±â€¯3.9 MU/g muscle, 6.3 ±â€¯0.6 MU/g viscera and 6.3 ±â€¯0.6 MU/g eggs in mean value. Merely dcGTX2 and dcSTX were detected in ten Specimens, ranging from 0.01 to 0.77 µg/g. Analyses suggested that these Carcinoscorpius rotundicauda contain TTX and its analogues as the major toxin and PSPs as the minor, respectively. Besides, no DSPs were found.

An engineered arginine-rich α-helical antimicrobial peptide exhibits broad-spectrum bactericidal activity against pathogenic bacteria and reduces bacterial infections in mice.

The increase in the prevalence of antibiotic-resistant bacteria has become a major public health concern. Antimicrobial peptides (AMPs) are emerging as promising candidates addressing this issue. In this study, we designed several AMPs by increasing α-helical contents and positive charges and optimizing hydrophobicity and amphipathicity in the Sushi 1 peptide from horseshoe crabs. A neural network-based bioinformatic prediction tool was used for the first stage evaluations of peptide properties. Among the peptides designed, Sushi-replacement peptide (SRP)-2, an arginine-rich and highly α-helical peptide, showed broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and multidrug-resistant Acinetobacter baumannii; nevertheless, it showed little hemolytic and cytotoxic activity against mammalian cells. Atomic force microscopy results indicated that SRP-2 should interact directly with cell membrane components, resulting in bacterial cell death. SRP-2 also neutralized LPS-induced macrophage activation. Moreover, in an intraperitoneal multidrug-resistant A. baumannii infection mouse model, SRP-2 successfully reduced the bacterial number in ascitic fluid and tumor necrosis factor-α production. Our study findings demonstrate that bioinformatic calculations can be powerful tools to help design potent AMPs and that arginine is superior to lysine for providing positive charges for AMPs to exhibit better bactericidal activity and selectivity against bacterial cells.

Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection.

Horseshoe crabs have been integral to the safe production of vaccines and injectable medications for the past 40 years. The bleeding of live horseshoe crabs, a process that leaves thousands dead annually, is an ecologically unsustainable practice for all four species of horseshoe crab and the shorebirds that rely on their eggs as a primary food source during spring migration. Populations of both horseshoe crabs and shorebirds are in decline. This study confirms the efficacy of recombinant Factor C (rFC), a synthetic alternative that eliminates the need for animal products in endotoxin detection. Furthermore, our findings confirm that the biomedical industry can achieve a 90% reduction in the use of reagents derived from horseshoe crabs by using the synthetic alternative for the testing of water and other common materials used in the manufacturing process. This represents an extraordinary opportunity for the biomedical and pharmaceutical industries to significantly contribute to the conservation of horseshoe crabs and the birds that depend on them.

Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide.

Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, α-factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737-Ile738 bond (Phe737 site) of factor C required for the conversion to α-factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site-uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein-protein interactions between factor C* molecules.

Antimicrobial Peptides of Marine Crustaceans: The Potential and Challenges of Developing Therapeutic Agents.

The alarming increase of antimicrobial resistance has led to a growing number of studies aiming to develop novel antimicrobial therapeutics. Natural antimicrobial peptides possess a potent and broad-spectrum antimicrobial activity combined with diverse and unique structural motifs, which confer their different mechanisms of action. These peptides are ubiquitous in organisms and are integral to the innate immune system. Recently, identification of antimicrobial peptides from marine crustaceans has become the centre of attention of many researchers. This increasing interest stems from the remarkable diversity in the structural and genetic composition of these peptides compared to terrestrial counterparts. Thus, peptides from marine crustaceans can serve as future templates for novel antimicrobial agents. Here, we provide an overview of various antimicrobial peptides from the marine crustaceans, their antimicrobial activity and structure- activity relationships. We also discuss the potential and challenges of their development as new antimicrobial agents.

Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon.

Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called "Low Endotoxin Recovery (LER)". LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration's (FDA's) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.

The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase.

Arginine kinase provides a model for functional dynamics, studied through crystallography, enzymology, and nuclear magnetic resonance. Structures are now solved, at ambient temperature, for the transition state analog (TSA) complex. Analysis of quasi-rigid sub-domain displacements show that differences between the two TSA structures average about 5% of changes between substrate-free and TSA forms, and they are nearly co-linear. Small backbone hinge rotations map to sites that also flex on substrate binding. Anisotropic atomic displacement parameters (ADPs) are refined using rigid-body TLS constraints. Consistency between crystal forms shows that they reflect intrinsic molecular properties more than crystal lattice effects. In many regions, the favored directions of thermal/static displacement are appreciably correlated with movements on substrate binding. Correlation between ADPs and larger substrate-associated movements implies that the latter approximately follow paths of low-energy intrinsic motions.

De Novo Assembly and Characterization of Early Embryonic Transcriptome of the Horseshoe Crab Tachypleus tridentatus.

The horseshoe crab Tachypleus tridentatus is a unique marine species and a potential model for marine invertebrate. Limited genomic and transcriptional data are currently available to understand the molecular mechanisms underlying the embryonic development of T. tridentatus. Here, we reported for the first time the de novo transcriptome assembly for T. tridentatus at embryonic developmental stage using Illumina RNA-seq platform. Approximate 38 million reads were obtained and further assembled into 133,212 unigenes. Sequence homology analysis against public databases revealed that 33,796 unigenes could be annotated with gene descriptions. Of the annotated unigenes, we identified a number of key components of several conserved metazoan signaling pathways (Hedgehog, Wnt, TGF-beta and Notch pathways) and other important regulatory genes involved in embryonic development. Targeted searching of Pax family genes which play critical roles in the formation of tissue and organ during embryonic development identified a complete set of Pax family genes. Moreover, the full length T. tridentatus Pax1/9a (TtPax1/9a) and Pax1/9b (TtPax1/9b) cDNA sequences were determined based on the transcriptome, demonstrating the immediate application of our database. Using quantitative real time PCR, we analyzed the expression patterns of TtPax1/9a and TtPax1/9b in different tissues of horseshoe crab. Taking advantage of Drosophila model, we further found that TtPax1/9b, but not TtPax1/9a, can partly rescue the Drosophila homolog Poxm dysfunction-caused lethality at the larval stage. Our study provides the embryonic transcriptome of T. tridentatus which could be immediately used for gene discovery and characterization, functional genomics studies in T. tridentatus. This transcriptome database will also facilitate the investigations of molecular mechanisms underlying embryonic development of T. tridentatus and other marine arthropods as well.

Use of Natural Antimicrobial Peptides and Bacterial Biopolymers for Cultured Pearl Production.

Cultured pearls are the product of grafting and rearing of Pinctada margaritifera pearl oysters in their natural environment. Nucleus rejections and oyster mortality appear to result from bacterial infections or from an inappropriate grafting practice. To reduce the impact of bacterial infections, synthetic antibiotics have been applied during the grafting practice. However, the use of such antibiotics presents a number of problems associated with their incomplete biodegradability, limited efficacy in some cases, and an increased risk of selecting for antimicrobial resistant bacteria. We investigated the application of a marine antimicrobial peptide, tachyplesin, which is present in the Japanese horseshoe crab Tachypleus tridentatus, in combination with two marine bacterial exopolymers as alternative treatment agents. In field studies, the combination treatment resulted in a significant reduction in graft failures vs. untreated controls. The combination of tachyplesin (73 mg/L) with two bacterial exopolysaccharides (0.5% w/w) acting as filming agents, reduces graft-associated bacterial contamination. The survival data were similar to that reported for antibiotic treatments. These data suggest that non-antibiotic treatments of pearl oysters may provide an effective means of improving oyster survival following grafting procedures.

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate.

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

Opsin expression in Limulus eyes: a UV opsin is expressed in each eye type and co-expressed with a visible light-sensitive opsin in ventral larval eyes.

The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors.

Quantum mechanics/molecular mechanics study of oxygen binding in hemocyanin.

We report a combined quantum mechanics/molecular mechanics (QM/MM) study on the mechanism of reversible dioxygen binding in the active site of hemocyanin (Hc). The QM region is treated by broken-symmetry density functional theory (DFT) with spin projection corrections. The X-ray structures of deoxygenated (deoxyHc) and oxygenated (oxyHc) hemocyanin are well reproduced by QM/MM geometry optimizations. The computed relative energies strongly depend on the chosen density functional. They are consistent with the available thermodynamic data for oxygen binding in hemocyanin and in synthetic model complexes when the BH&HLYP hybrid functional with 50% Hartree-Fock exchange is used. According to the QM(BH&HLYP)/MM results, the reaction proceeds stepwise with two sequential electron transfer (ET) processes in the triplet state followed by an intersystem crossing to the singlet product. The first ET step leads to a nonbridged superoxo CuB(II)-O2(•-) intermediate via a low-barrier transition state. The second ET step is even more facile and yields a side-on oxyHc complex with the characteristic Cu2O2 butterfly core, accompanied by triplet-singlet intersystem crossing. The computed barriers are very small so that the two ET processes are expected to very rapid and nearly simultaneous.