Occupational risk assessment of glutaraldehyde through personal air monitoring in a hospital setting.

Glutaraldehyde is a high-efficiency disinfectant that has been included in the protocols of some hospitals for controlling the spread of SARS-CoV-2, together with sodium hypochlorite and quaternary ammonium disinfectants. However, exposure has been poorly studied in workplace settings, despite the association between glutaraldehyde and respiratory diseases and skin conditions in exposed workers. This study evaluated the magnitude of exposure associated with the use of glutaraldehyde in healthcare workers across various work areas of a first level of Hospital-Based Care in Colombia. Workers were classified into similar exposure groups (SEGs) based on work areas and tasks performed, and airborne concentrations of glutaraldehyde were measured in different work areas of each SEG through direct monitoring. The 95th percentile of glutaraldehyde concentrations in all SEGs studied exceeded the TLV-C immediately after use. Cleaning workers and nurses had the highest exposures to glutaraldehyde. Results indicate that workers were overexposed and highlight the need to implement controls to reduce exposure. The high-exposure levels also raise the need to consider glutaraldehyde substitution and adequate use of personal protective equipment (PPE).

Oxidative Stress as a Molecular Mechanism of Exposure to Organophosphorus Pesticides: A Review.

Exposure to organophosphorus pesticides is an important public health issue due to a large number of occupationally exposed populations, as well as their effects mainly at the level of the nervous, reproductive, and immune systems. It has been reported that one of the molecular mechanisms by which adverse effects of exposure to organophosphorus pesticides can be explained is oxidative stress, which leads to alterations at the cellular level that, if chronic, could affect the functionality of different organs and tissues. These data constitute the basis of the relevant literature on its toxicity. The induction of oxidative damage, which has been referred to, increases the occurrence of processes such as eryptosis and/or hemolysis in erythrocytes that promote greater susceptibility to clinical conditions such as anemia, dehydration, and chronic kidney disease. Thus, it is mentioned that the determination of oxidative damage parameters could be useful to monitor occupationally exposed people by exploring their oxidative status. This review focuses on presenting the state of knowledge in recent years on the toxicity of organophosphorus pesticides and their relationship with the oxidative damage evaluated in erythrocytes.

Cellular Complexity of Hemochorial Placenta: Stem Cell Populations, Insights from scRNA-seq, and SARS-CoV-2 Susceptibility.

Purpose of Review: The placenta is a transient organ that forms de novo and serves a critical role in supporting fetal growth and development. Placental oxygen, nutrients, and waste are transported through processes that depend on vascular structure and cell type-specific expression and localization of membrane transporters. Understanding how the placenta develops holds great significance for maternal-fetal medicine. The purpose of this review is to examine current information regarding placental progenitor populations. Recent Findings: Recent advancements in single-cell RNA sequencing (scRNA-seq) provide unprecedented depth for the investigation of cell type-specific gene expression patterns in the placenta. Thus far, several mouse placenta scRNA-seq studies have been conducted which produced and analyzed transcriptomes of placental progenitors and cells of the fully developed placenta between embryonic day (E) 7.0 and E12.5. Together with human placenta scRNA-seq data which, in part, has been produced through coordinated research campaigns in the scientific community to understand the potential for SARS-CoV-2 infection, these mammalian studies lend fundamental insight into the cellular and molecular composition of hemochorial placentae found in both mouse and human. Summary: Single-cell placenta research has advanced understanding of tissue-resident stem cells and molecules that are poised to support maternal-fetal communication and nutrient transport. Herein, we provide context for these recent findings by reviewing placental anatomy and cell populations, and discuss recent scRNA-seq mouse placenta findings. Further research is needed to evaluate the utility of placental stem cells in the development of new therapeutic approaches for the treatment of wound healing and disease.

Vaginal Lactobacillus species and inflammatory biomarkers in pregnancy.

BACKGROUND: The aim of this study was to identify vaginal Lactobacillus spp. and quantify vaginal inflammatory cytokines in primigravida vs. multigravida women and pregnant vs. non-pregnant women. METHODS: Vaginal swabs were obtained from four groups of patients. A real-time PCR was carried out to identify the Lactobacillus spp. Multiplex immunoassays were performed to quantify a total of 27 cytokines using the Bio-Plex MAGPIX multiplex reader and MesoQuick Plex SQ 120 (Meso Scale Diagnostics LLC, Rockville, MD, USA). Inferential statistics using hypothesis tests were applied to detect differences in cytokine levels. RESULTS: Significant differences in cytokines and chemokines exist among the four populations of women studied. IP-10 is significantly higher in multigravida women as compared to primigravida women. IFN-γ, MCP-1, MIP-1ß, IL-2 and IL-10 are significantly higher in non-pregnant women compared to pregnant women. L. iners was the most abundant species in multigravida, pregnant and non-pregnant patients, while L. crispatus was the most abundant species in primigravida patients. Significant differences in the levels of MIP-1ß, TNF-α, PDGF-BB, VEGF-A, IL-12, and IL-10 exist between women identified with Lactobacillus species and women not identified with Lactobacillus species. CONCLUSIONS: There were significant differences regarding cytokines, chemokines, and Lactobacillus spp. among four groups of studied patients. With these results, we increase our understanding of the role that vaginal cytokines and Lactobacillus species have during pregnancy, with the goal that this novel research will be useful for examining vaginal biomarkers in obstetrical conditions.

Nanoparticle mediated increased insulin-like growth factor 1 expression enhances human placenta syncytium function.

INTRODUCTION: Placental dysfunction is an underlying cause of many major obstetric diseases and treatment options for complications like fetal growth restriction (FGR) are limited .We previously demonstrated nanoparticle delivery of the human insulin-like growth factor 1 (hIGF1) transgene under control of the trophoblast-specific PLAC1 promoter maintains normal fetal growth in a surgically-induced FGR mouse model. However, uptake by human placental syncytiotrophoblast has yet to be determined. METHODS: An ex vivo human placenta perfusion model, term placenta villous fragments, and other in vitro syncytiotrophoblast models were used to determine nanoparticle uptake, transgene expression, and functional responses under oxidative stress conditions. RESULTS: In the ex vivo perfusion, fluorescence from a Texas-Red conjugated nanoparticle increased in maternal perfusate upon nanoparticle addition and declined by the conclusion of the experiment (P < 0.001. Fluorescent histology confirmed localization in the syncytiotrophoblasts. No Texas-Red fluorescence was detected in the fetal perfusate. Transgene expression of hIGF1 in differentiated BeWo cells, isolated primary trophoblasts and fragments was increased compared to untreated (55,000-fold, P = 0.0003; 95-fold, P = 0.003; 400-fold, P < 0.001, respectively). Functionally, increased hIGF1 expression in villous fragments resulted in translocation of glucose transporter 1 to the syncytiotrophoblast cell membrane and under conditions of oxidative stress in BeWo cells, protected against increased cell death (P < 0.01) and decreased mitochondrial activity (P < 0.01). CONCLUSION: The current study confirms that our nanoparticle is capable of uptake in human placental syncytium which results in enhanced transgene expression, functional changes to cellular activity and protection against increased oxidative stress.

Microbiological and cytological characterization of coelomic fluid from three captive endangered amphibian Gastrotheca species with edema syndrome: preliminary analysis.

OBJECTIVE: Edema syndrome is highly prevalent but under researched in captive frogs around the world. The objective of the present study was to characterize at a basic microbiological and cytological level of the bacteria of the edema fluid of 20 individuals of the genus Gastrotheca to determine the presence of possible anaerobic and aerobic bacteria. RESULTS: Fourteen types of bacteria were identified in the edema fluid, 12 of them at the species level (Pasteurella haemolytica, Hafnia alvei, Enterobacter agglomerans, Aeromonas hydrophila, Pseudomonas fluorescens, Burkholderia pseudomallei, Salmonella arizonae, Enterobacter gergoviae, Enterobacter sakazakii, Yersinia enterocolitica, Klebsiella oxytoca, and Klebsiella ozaenae) and two at the genus level (Enterococcus spp. and Streptococcus spp.). The most frequently identified cells were lymphocytes (37.7% in females and 46.4% in males), erythrocytes (23.5% in females and 17.5% in males) and neutrophils (4.2% in females and 2.8% in males). Finally, no relationship was found between the data obtained and the sex of the individuals studied.

Métodos de identificación polifásica aplicados a la búsqueda de microorganismos con potencial industrial / Polyphasic identification methods applied in searching for microorganisms with industrial potential

Introducción: La bioprospección de metabolitos de interés antropogénico emplea métodos de recolección de microorganismos en ecosistemas extremófilos o endémicos. La microbiota aislada en estos lugares puede o no incluir microorganismos patógenos. Es imprescindible un enfoque interdisciplinario que permita abordar la búsqueda de las especies de interés mientras se preserva la buena salud de los investigadores. Objetivo: Identificar molecular, bioquímica y morfológicamente microorganismos patógenos humanos en cepas celulolíticas de importancia industrial almacenadas en el banco de cepas del Laboratorio de Investigación de la Facultad de Ingeniería Química de la Universidad Central del Ecuador, procedentes del Yasuní, la Antártida y Balzapamba. Métodos: Se realizó un estudio de bioprospección de bacterias celulolíticas empleando técnicas de microbiología ambiental. Se evaluaron las características morfológicas mediante tinciones, como por ejemplo Gram. Además, se realizaron pruebas bioquímicas y antibiogramas para bacterias Gram-negativas y Gram-positivas. Las pruebas moleculares utilizaron extracción de ADN bacteriano para la secuenciación Sanger del gen 16S. Resultados: Se encontraron las especies Klebsiella pneumoniae (Y2 y Y3r) y Nocardia asteroides (Y1 y Y3p) en las muestras de material lignocelulósico recolectadas en Yasuní, mientras que las especies aisladas en la Antártida y en Balzapamba corresponden a Bacillus subtillis. Conclusiones: Se identificaron cepas pertenecientes a diferentes géneros bacterianos. Las bacterias del género Klebsiella, en las muestras colectadas en Yasuní, podrían tener un potencial patógeno. Eso se puede corroborar con técnicas de genotipificación. Por lo tanto, puede existir riesgo para los seres humanos que realizan bioprospección en ese ecosistema y se deben tomar medidas de bioseguridad.

Abstract Background: The bioprospection of metabolites of anthropogenic interests employs methods of collecting microorganisms in extremophile or endemic ecosystems. The microbiota isolated in these places may or may not include pathogenic microorganisms. Therefore, an interdisciplinary approach is essential to address the search of the species of interest while the good health of the researchers is preserved. Objective: To identify in molecular, biochemical and morphologically ways some human pathogenic microorganisms in cellulolytic strains of industrial importance stored in the strain bank of the Research Laboratory of the Faculty of Chemical Engineering at the Central University of Ecuador, from Yasuní, Antarctic and Balzapamba. Methods: IA bioprospecting study of cellulolytic bacteria was performed using environmental microbiology techniques. Morphological characteristics were assessed by Gram staining. In addition, biochemical tests and antibiograms were performed for Gram-negative and Gram-positive bacteria. The molecular tests used extraction of bacterial ADN for 16S gene Sanger sequencing. Results: The species Klebsiella pneumoniae (Y2 and Y3r) and Nocardia asteroides (Y1 and Y3p) were found in samples of lignocellulosic material collected in Yasuni, while the isolated species in Antarctica and Balzapamba correspond to Bacillus subtillis. Conclusions: Strains belonging to different bacterial genera were identified. The bacteria of the genus Klebsiella from the samples collected in Yasuní could have a potential pathogen. This can be corroborated with genotyping techniques. Therefore, there could be a risk to humans who perform bioprospecting in that ecosystem and biosecurity measures should be taken.

A first case of hepatocellular carcinoma in the baboon (Papio spp.) placenta.

We present a case of hepatocellular carcinoma (HCC) in the placenta of healthy baboon (Papio spp.). Grossly, the fetal, maternal, and placental tissues were unremarkable. Histologically, the placenta contained an unencapsulated, poorly demarcated, infiltrative, solidly cellular neoplasm composed of cells that resembled hepatocytes. The neoplastic cells were diffusely positive for vimentin and focally positive for Ae1/Ae3, Arginase -1, glutamine synthetase, and CD10, and negative for ER, vascular markers (CD31 and D240), S100, glypican, C-reactive protein, FABP, desmin, and beta-catenin; INI1 positivity was similar to non-neoplastic tissues. The case likely represents a unique subtype of HCC.

Optical tissue clearing in combination with perfusion and immunofluorescence for placental vascular imaging.

Imaging of placental tissues is a difficult task, because of specific for this organ complex multicellular and 3D tissue structure. The tissue clearing systems (X-CLARITY) system is a valuable tool for the examining the expression of molecular pathways in whole tissues and organs, originally developed for brain imaging.In the present report, we utilized this technology for the examination of placental vasculature and protein expression in perfused human placental tissue.The placental tissue was sufficiently cleared with preservation of endothelial staining and fluorescent markers, allowing visualization using confocal microscopy. The CLARITY method and X-CLARITY system is a valuable tool in placental imaging.

Effect of maternal high-fat diet on key components of the placental and hepatic endocannabinoid system.

Maternal obesity in pregnancy has been linked to a spectrum of adverse developmental changes. Involvement of eCBs in obesity is well characterized. However, information regarding eCB physiology in obesity associated with pregnancy is sparse. This study evaluated fetomaternal hepatic, systemic, and placental eCB molecular changes in response to maternal consumption of a HFD. From ≥9 mo before conception, nonpregnant baboons ( Papio spp.) were fed a diet of either 45 (HFD; n = 11) or 12% fat or a control diet (CTR; n = 11), and dietary intervention continued through pregnancy. Maternal and fetal venous plasma samples were evaluated using liquid chromatography-mass spectrometry to quantify AEA and 2-AG. Placental, maternal and fetal hepatic tissues were analyzed using RT-PCR, Western blot, and immunohistochemistry. mRNA and protein expression of endocannabinoid receptors (CB1R and CB2R), FAAH, DAGL, MAGL, and COX-2 were determined. Statistical analyses were performed with the nonparametric Scheirer-Ray-Hare extension of the Kruskal-Wallis test to analyze the effects of diet (HFD vs. CTR), fetal sex (male vs. female), and the diet × sex interaction. Fetal weight was influenced by fetal sex but not by maternal diet. The increase in maternal weight in animals fed the HFD vs. the CTR diet approached significance ( P = 0.055). Maternal circulating 2-AG concentrations increased, and fetal circulating concentrations decreased in the HFD group, independently of fetal sex. CB1R receptor expression was detected in syncytiotrophoblasts (HFD) and the fetal endothelium (CTR and HFD). Placental CB2R protein expression was higher in males and lower in female fetuses in the HFD group. Fetal hepatic CB2R, FAAH, COX-2 (for both fetal sexes), and DAGLα (in male fetuses) protein expression decreased in the HFD group compared with the CTR group. We conclude that consumption of a HFD during pregnancy results in fetal systemic 2-AG and hepatic eCB deficiency.

Serum Vitamin D Concentrations in Baboons (Papio spp.) during Pregnancy and Obesity.

Obesity is associated with vitamin D deficiency, which can lead to serious problems during pregnancy. However, the mechanisms of the deficiency and guidelines for vitamin D supplementation during pregnancy are not established yet, and variations in environmental exposures combined with the difficulties of performing research in pregnant women are obstacles in the evaluation of vitamin D metabolism. Baboons (Papio spp.) are an excellent, well-established model for reproductive research and represent a unique opportunity to study vitamin D metabolism in a controlled environment. This study used secondary data and specimen analysis as well as a novel experimental design to evaluate pregnant and nonpregnant baboons that were or were not exposed to sunlight while they were obese and after weight reduction. Daily D3 intake was 71% higher in nonpregnant obese baboons than in their nonobese counterparts, but serum vitamin D concentrations did not differ between these populations. In addition, serum 25-hydroxyvitamin D concentrations correlated negatively with the obesity index. This report is the first to show the effect of obesity and pregnancy on vitamin D concentrations in a NHP population. These data underline the importance of adequate vitamin D supplementation in obese animals.

Coccidioidomycosis in pregnancy: Case report and literature review of associated placental lesions.

BACKGROUND: Coccidioidomycosis is an endemic fungal infection found most commonly in the Southwestern United States, Northwestern Mexico, and parts of Central and South America. Although infection is relatively uncommon during pregnancy, it is imperative to have an index of suspicion in order to diagnose and begin timely treatment to prevent dissemination and dire consequences. CASE REPORT: A 33-year-old Hispanic female was evaluated after she was involved in an automobile accident. Radiographic evaluation showed a 3.2 × 3.2 cm cavitary thick-walled lesion. A biopsy was negative for malignancy. Evaluation was positive for coccidioidomycosis by complement fixation reaction. Four months later, the patient presented 7 weeks into a pregnancy with massive hemoptysis. Bronchoscopy revealed bleeding from the right upper lobe and emergency embolization was performed. The patient had a spontaneous abortion 9 days after admission. The right upper and middle lobes of the lung were resected due to continuous bleeding. A subsequent pregnancy was un-eventful. Coccidioidomycosis titers remained negative throughout the second pregnancy. DISCUSSION: This case demonstrates the potential for severe pulmonary coccidioidomycosis and vascular strain of pregnancy-associated vascular expansion in the first trimester of pregnancy and the possibility of a favorable pregnancy outcome in subsequent pregnancies after appropriate treatment. The route of feto-maternal transmission and placental lesions in coccidioidomycosis are discussed.

Role of zebrafish thrombocyte and non-thrombocyte microparticles in hemostasis.

Hemostasis is a defense mechanism that protects an organism from bleeding in the event of injury. We have previously demonstrated the utility of the zebrafish as a model to study human hemostasis. However, there are no studies on the role of microparticles in hemostasis in early vertebrates. Studying microparticles in zebrafish may provide insight into the evolution of microparticle function in hemostasis and may lead to direct observation of these microparticles in zebrafish larvae due to transparency of the vessels. In this investigation we demonstrate the presence of cellular microparticles in fish blood by both immunostaining as well as by using zebrafish whose thrombocytes are labeled with green fluorescent protein. Further investigation showed that microparticles were also labeled by fluorescein isothiocyanate annexin V, suggesting that these particles are derived via apoptosis. A portion of the fluorescein isothiocyanate annexin V labeled microparticles was also labeled by DiI-C18. Labeling by DiI-C18 suggests that some microparticles are derived from young thrombocytes. Additionally, GpIIb antibody labels almost all thrombocyte-derived microparticles and a greater percentage of microparticles are labeled by GpIIb antibody than by DiI-C18. This suggests that thrombocyte microparticles are derived from both young and mature thrombocytes. Furthermore, the increase of microparticles by adding excessive microparticles into blood in vitro and through intravenous injections led to an increased hemostatic response. In addition, treatment with tumor necrosis factor alpha resulted in an increased number of thrombocyte microparticles and enhanced hemostasis; in contrast, treatment with zVAD-FMK, a caspase inhibitor, resulted in a decrease in thrombocyte microparticles and decreased hemostasis. We also found that thrombocyte microparticles agglutinate, along with other cells and cellular microparticles, in the presence of an excess of either ristocetin or ultra-large von Willebrand factor. Also, stimulation of von Willebrand factor release in vivo resulted in clusters of thrombocyte microparticles in the veins. Moreover, thrombocyte microparticles were the first to appear at the site of arterial injury. We found that thrombocyte microparticles are functionally equivalent to platelet microparticles. The microparticles initiate arterial thrombus formation in a von Willebrand factor-dependent manner and further enhance thrombus formation by forming clusters of microparticles in venous thrombosis. This finding may have applications for understanding the role of platelet microparticles in humans and may have diagnostic applications.

Laser-induced thrombosis in zebrafish.

In the event of injury to the vasculature in vertebrate organisms bleeding is stopped by a defense mechanism called hemostasis. Even though biochemical studies characterized a number of factors, classical genetic methods have not been applied to study hemostasis. We introduced zebrafish as an animal model to study genetics of hemostasis. To conduct genetic studies of hemostasis, we required a global screening method to address all the factors of hemostasis such as those present in plasma, in platelets or those present in the endothelium. Therefore, we developed a global laser induced thrombosis method which can assay all these components. In this paper, we describe the principle of this method as well as provide the detailed protocol so this could be used as a screening tool to measure hemostasis in any laboratory.

Zebrafish von Willebrand factor.

von Willebrand factor (vWF) is a large protein involved in primary hemostasis. A dysfunction in this protein or an insufficient production of the protein leads to improper platelet adhesion/aggregation, resulting in a bleeding phenotype known as von Willebrand disease (vWD). To gain a better understanding of vWF interactions in vivo, the use of zebrafish as a model is ideal because of the transparency of the embryos and larvae. In this article, we examined the presence and function of vWF in hemostasis of zebrafish utilizing a variety of molecular methods. Using RT-PCR and antibody staining, we have shown that vWF mRNA is present in thrombocytes. Through antibody staining, we demonstrated vWF is synthesized in blood vessels. The role of zebrafish vWF in hemostasis was established through knockdown methods using vWF morpholino (vWF MO) antisense oligonucleotides. Embryos injected with vWF MO at the one to four cell stages resulted in a bleeding phenotype. Injection of embryos with vWF MO also caused an increase in time to occlusion within arteries in larvae upon laser induced injury. We then used vWF-specific Vivo-morpholinos (VMO) to induce vWF knockdown in adult zebrafish by targeting the exon homologous to the human exon 28 of the vWF gene. The reduced ristocetin-mediated agglutination of thrombocytes in a plate tilting assay, using blood from adult zebrafish injected with VMO, provided evidence that vWF is involved in the hemostatic process. We also administered desmopressin acetate to larvae and adults which resulted in enhanced aggregation/agglutination of thrombocytes. Zebrafish genome database analysis revealed the presence of GPIbß gene. It also revealed the exon of zebrafish vWF gene corresponding to exon 28 of human vWF gene is highly similar to the exon 28 of human vWF gene, except that it has an insertion that leads to a translated peptide sequence that separates the two A domains coded by this exon. This exon is also conserved in other fishes. In summary, we established that zebrafish vWF has a role similar to that of vWF found in humans, thus, making zebrafish a useful model for studying the cell biology of vWF in vivo.

Evolution of primary hemostasis in early vertebrates.

Hemostasis is a defense mechanism which protects the organism in the event of injury to stop bleeding. Recently, we established that all the known major mammalian hemostatic factors are conserved in early vertebrates. However, since their highly vascularized gills experience high blood pressure and are exposed to the environment, even very small injuries could be fatal to fish. Since trypsins are forerunners for coagulation proteases and are expressed by many extrapancreatic cells such as endothelial cells and epithelial cells, we hypothesized that trypsin or trypsin-like proteases from gill epithelial cells may protect these animals from gill bleeding following injuries. In this paper we identified the release of three different trypsins from fish gills into water under stress or injury, which have tenfold greater serine protease activity compared to bovine trypsin. We found that these trypsins activate the thrombocytes and protect the fish from gill bleeding. We found 27 protease-activated receptors (PARs) by analyzing zebrafish genome and classified them into five groups, based on tethering peptides, and two families, PAR1 and PAR2, based on homologies. We also found a canonical member of PAR2 family, PAR2-21A which is activated more readily by trypsin, and PAR2-21A tethering peptide stops gill bleeding just as trypsin. This finding provides evidence that trypsin cleaves a PAR2 member on thrombocyte surface. In conclusion, we believe that the gills are evolutionarily selected to produce trypsin to activate PAR2 on thrombocyte surface and protect the gills from bleeding. We also speculate that trypsin may also protect the fish from bleeding from other body injuries due to quick contact with the thrombocytes. Thus, this finding provides evidence for the role of trypsins in primary hemostasis in early vertebrates.

Isomers of C20: an energy profile III.

Semiempirical calculations, at the PM3 level provided within the Winmopac v2.0 software package, are used to geometrically optimize and determine the absolute energies (heats of formation) of a variety of C(20) isomers that are predicted to exist in and around the ring and cage isomers. Using the optimized Cartesian coordinates for the ring and the cage isomers, a saddle-point calculation was performed. The resulting energy profile, consisting of a series of peaks and valleys, is used as a starting point for the identification and location of fifteen additional isomers of C(20) that are predicted to be energetically stable, both via geometry optimizations and force constant analysis. These additional isomers were subsequently determined to lie adjacent to one another on the potential surface and establish a step-wise transformation between the ring and the cage. Transition-state optimization of the Cartesian coordinates at the saddle point between adjacent isomers was performed to quantify the energy of the transition state. The step-wise process from one isomer to another, which extends out over the three-dimensional surface, is predicted to require approximately 15% less energy than that of the direct, two-dimensional transformation predicted in the bowl-cage profile. However, the net atomic rearrangement for the step-wise process is about four times greater than that of the direct process. Although less in energy, the amount of atomic rearrangement in the step-wise process would make the occurrence of such a route prohibitive. Utilizing the direct distance separating the three primary isomers (ring, bowl, cage), the method of triangulation is performed to quantitatively position other C(20) structures on the potential surface, relative to the ring, bowl, and cage isomers.