Human plasma ricinine quantification by LC-HRMS after micro-solid-phase elution.

A rapid, sensitive and specific method for ricinine identification and quantification in plasma has been developed by LC-HRMS. Deuterated ricinine was used as the internal standard. From 100 µL of plasma, ricinine was extracted using micro-solid-phase elution, which allows a reduced extraction time, by eliminating the evaporation step. Eluate is directly injected into the LC-HRMS system. Chromatographic separation was performed using a reverse-phase C18 column with a 4.5 min gradient elution. The method was validated according to European Medicines Agency guidelines. Linearity was verified between 0.25 and 500.0 ng/mL; the maximum precision calculated was 19.9% for the lower limit of quantitation and 9.6% for quality control, and accuracy was within ± 5.6% of the nominal concentrations. Selectivity, carryover, matrix effect and stability were also verified according to European Medicines Agency guidelines. The method allows the rapid and reliable identification of ricin-exposed victims in case of terrorist attacks or poisonings: three intoxication cases are reported.

[Forensic chemical and chemicotoxicological examination of ricin poisoning by the HPLC-MS/MS method]. / Sudebno-khimicheskoe i khimiko-toksikologicheskoe issledovanie metodom VEZhKh-MS/MS pri otravlenii ritsinom.

THE AIM OF THE STUDY: Is to suggest the method of ricin determination in biological liquids during forensic medical and chemicotoxicological examination. This research describes the optimal conditions of sample processing of biological liquids, allowing to extract the components (ricinine and ricinoleic acid) of castor seeds. The recommended analysis conditions allow to perform research for 15 minutes by high resolution mass spectrometry method combined with high-value liquid chromatography on a chromato-mass spectrometer to detect ricinine and ricinoleic acid. The chromatographic (retention time) and mass-spectrometric parameters (mass spectra) were established for the exact high-quality determination of ricinine and ricinoleic acid.

Fatal intoxication by intravenous injection of castor bean (Ricinus communis L.) extract-a case study.

A case report of a 25-year-old man who committed suicide by intravenous injection himself of an aqueous home-made castor bean extract is presented. The patient was hospitalized and treated symptomatically and was released at its own request fourth day after intoxication. The next day, the patient's condition deteriorated, and he died 6 days after intoxication even though he was given medical care. Case history, autopsy, and toxicological investigation of ante- and post-mortem collected materials are described. Blood and urine collected from the patient ante-mortem and other several biological materials (namely blood from the upper and lower limb, blood from the right and left ventricle, pericardial fluid, vitreous humour, liver, kidney, and spleen) were collected post-mortem during autopsy. Liquid-liquid extraction procedure followed by high-performance liquid chromatography tandem mass spectrometry analysis for identification and determination of ricinine as a biomarker of ricin/castor seed intoxication was developed and validated. The method was applied on analysis of collected ante- and post-mortem biological materials. The post-mortem contents of ricinine in organs (namely the liver, kidney, and spleen) are firstly reported. The obtained results indicated approximately uniform distribution of ricinine (concentration level about 1 ng mL-1) in the body after death. In addition, the GC-MS method was also applied for the analysis of extract of castor seed and the patient's urine, to demonstrate alternative possibility for identification of ricinine for clinical and forensic purposes.

Ricin Poisoning after Oral Ingestion of Castor Beans: A Case Report and Review of the Literature and Laboratory Testing.

BACKGROUND: Ricin is a protein toxin derived from the castor bean plant Ricinus communis. Several cases secondary to its consumption have been published and, more recently, its use as a potential bioterrorism agent has also been reported. Oral absorption of ricin is highly erratic, leading to a wide spectrum of symptoms. In addition, conventional urine drug screening tests will not be able to detect this compound, posing a diagnostic challenge. CASE REPORT: A male teenager intended to die by ingesting 200 castor beans after mixing and blending them with juice. Eight hours later, he presented with weakness, light-headedness, nausea, and vomiting and sought medical treatment. The patient was admitted and treated conservatively. An immune-based standard urine toxicology drug screen panel was reported as negative. A comprehensive untargeted urine drug screen test showed the presence of ricinine, a surrogate marker of ricin intoxication. He was transferred to the psychiatric service 3 days after admission. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: This case highlights the importance of knowing the peculiar pharmacokinetic properties of ricin after oral ingestion of castor beans and toxin release through mastication. Emergency physicians should be aware that oral absorption of ricin is dependent on several factors, such type and size of seeds and the geographic harvesting region, making it extremely difficult to estimate its lethality based solely on the number of ingested beans. Finally, comprehensive untargeted urine drug screening testing is highly valuable as a diagnostic tool in this context.

A novel quality-by-design optimized spectrofluorimetric method for the sensitive determination of ricinine alkaloid in edible oils.

Ricinine is an important biomarker used for detecting the exposure to castor bean products. The current study describes a highly sensitive and selective spectrofluorimetric approach to determine ricinine in various edible oils. It depends on measuring the native fluorescence of ricinine at 365 nm following excitation at 307 nm over a concentration range of 50.0-1200.0 ng/mL. The method displayed high sensitivity with quantitation and detection limits down to 19.56 and 6.46 ng/mL, respectively. The significant factors affecting the fluorescence of ricinine were optimized using 22 full factorial design. The proposed approach was successfully employed for ricinine determination in three types of edible oils with high percent recoveries and low S.D. values. The most important advantage of the developed method is the reduction of sample preparation steps, analysis time, and cost. Hence, it can be better suited for routine analysis and quality control of cooking oils adulterated with castor oil.

Evaluating the release and metabolism of ricinine from castor cake fertilizer in soils using a LC-QTOF/MS coupled with SIRIUS workflow.

Castor cake is a major by-product generated after castor oil extraction and has been widely used as an organic fertilizer. Once applied to soil, a toxic alkaloid ricinine in castor cake may be released into soils and subsequently taken up by crops, which poses a potential threat to food safety and human health. However, the environmental fate of castor cake derived ricinine in agroecosystems remains unclear. In this study, the release and metabolism of ricinine in soils were conducted using soil pot experiments with different castor cake application rates. The analytical methodology of ricinine quantification in soil pore water was first established using solid phase extraction (SPE) coupled with liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). A non-target screening workflow associated with LC-QTOF/MS and SIRIUS platform was further developed to identify ricinine metabolites in soil pore water. After castor cake application, the ricinine concentrations in soil pore water significantly increased to 297-7990 µg L-1 at 1 day and then gradually decreased to 62.1-3460 µg L-1 at 7 days and 1.70-279 µg L-1 at 14 days for the selected two tested soils with castor cake application rates of 2, 10, and 20 g castor cake/kg soil. In addition, two ricinine metabolites R-194 and R-180 were tentatively identified and one ricinine metabolite N-demethyl-ricinin was confirmed through authentic reference standard for the first time by the developed non-target screening workflow. This study highlights the release and metabolism of toxic alkaloid ricinine in soils once applied castor cake as an organic fertilizer. Ricinine could be released into soil pore water in a short-term after castor cake application and then undergo demethylation, hydroxylation, and hydroxylation followed by methylation metabolisms over time in agroecosystems.

Neurological and gastrointestinal manifestations of spontaneous poisoning by Ricinus communis in goats.

An outbreak of Ricinus communis poisoning in goats with neurological and digestive changes was related to the ingestion of different vegetative parts of the plant. Two poisoned animals died within 5 h of the plant intake showing necrotic gastroenteritis and hepatocytes degeneration and necrosis. Toxicological analysis by HPLC-DAD assay demonstrated 21.1-25.1 µg/g of ricinine in samples of ruminal fluids and 10.1-10.9 µg/g in the liver of poisoned goats.

Ultra-Performance Liquid Chromatography-Mass Spectrometry-Based Untargeted Metabolomics Reveals the Key Potential Biomarkers for Castor Meal-Induced Enteritis in Juvenile Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

The intensification of aquaculture to help kerb global food security issues has led to the quest for more economical new protein-rich ingredients for the feed-based aquaculture since fishmeal (FM, the ingredient with the finest protein and lipid profile) is losing its acceptability due to high cost and demand. Although very high in protein, castor meal (CM), a by-product after oil-extraction, is disposed-off due to the high presence of toxins. Concurrently, the agro-industrial wastes' consistent production and disposal are of utmost concern; however, having better nutritional profiles of these wastes can lead to their adoption. This study was conducted to identify potential biomarkers of CM-induced enteritis in juvenile hybrid-grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) alongside their growth and distal intestinal (DI) health evaluation. A total of 360 fish (initial weight = 9.13 ± 0.01g) were randomly assigned into three groups, namely, fish-meal (FM) (control), 4% CM (CM4), and 20% CM (CM20). After the 56-days feeding-trial, the DI tissues of FM, CM4, and CM20 groups were collected for metabolomics analysis. Principal components analysis and partial least-squares discriminant-analysis (PLS-DA, used to differentiate the CM20 and CM4, from the FM group with satisfactory explanation and predictive ability) were used to analyze the UPLC-MS data. The results revealed a significant improvement in the growth, DI immune responses and digestive enzyme activities, and DI histological examinations in the CM4 group than the others. Nonetheless, CM20 replacement caused DI physiological damage and enteritis in grouper as shown by AB-PAS staining and scanning electron microscopy examinations, respectively. The most influential metabolites in DI contents identified as the potential biomarkers in the positive and negative modes using the metabolomics UPLC-MS profiles were 28 which included five organoheterocyclic compounds, seven lipids, and lipid-like molecules, seven organic oxygen compounds, two benzenoids, five organic acids and derivatives, one phenylpropanoids and polyketides, and one from nucleosides, nucleotides, and analogues superclass. The present study identified a broad array of DI tissue metabolites that differed between FM and CM diets, which provides a valuable reference for further managing fish intestinal health issues. A replacement level of 4% is recommended based on the growth and immunity of fish.

Synthesis of new selective cytotoxic ricinine analogues against oral squamous cell carcinoma.

Sixteen new analogues were synthesized from ricinine and tested alongside with seven known analogues for their cytotoxic activity against oral cancer (SAS cells) and normal epithelial cells (L132 cells). In contrast to 5-FU, the synthesized ricinine analogues did not show toxicity to normal cells. However, some of them inhibited the proliferation of oral cancer cells at 25 µM as evident from the MTT assay results. Ricinine analogue (19) was shown to be the most active derivative (69.22% inhibition). Potential targets involved in the oral cancer inhibitory activity of compound 19 were investigated using in-silico studies and western blot analysis. PTP1B was predicted to be a target for ricinine using reverse docking approach. This prediction was confirmed by western blot analysis that revealed the downregulation of PTP1B protein by compound 19. Moreover, it showed downregulation of COX-2 which is also extensively expressed in oral cancer.

Effect of Salicylic Acid in the Yield of Ricinine in Ricinus communis under Greenhouse Condition.

Castor bean (Ricinus communis) seeds contain ricinine, an alkaloid with insecticidal and insectistatic activities. Elicitation with salicylic acid (SA) has proven to stress R. communis and might modify the ricinine concentration. The aim of this study was to evaluate the concentration of ricinine in the bagasse of seeds from R. communis elicited with exogenous SA under greenhouse conditions. Plants were grown and divided into five groups, which were sprayed with SA and drench with 50 mL 60 days after sowing with concentrations of SA (0, 100, 300, 600 and 900 µM). Clusters were mixed and separated according to the treatment, and dried. The seeds were ground, the oil was extracted by Soxhlet with hexane, and then the bagasse was extracted with methanol. Ricinine was determined by HPLC. Elicitation did not change the plant height or diameter; the control group had 9.17 µg mL-1 of ricinine; and the concentrations followed a hormesis curve with the peak at 300 µM of SA that had a ricinine concentration of 18.25 µg mL-1. Elicitation with SA might be a cost-effective technique to increase ricinine from R. communis bagasse.

Near-fatal poisoning after ricin injection.

OBJECTIVE: To report a near-fatal poisoning after intentional injection of ricin from a castor bean (Ricinus communis) extract. CASE REPORT: A 21 year-old man self-injected ∼3 mL of a castor bean extract intramuscularly and subcutaneously in the left antecubital fossa. Upon admission to our ED (1 h post-exposure; day 1, D1) he was awake and alert, but complained of mild local pain and showed slight local edema and erythema. He evolved to refractory shock (∼24 h post-exposure) that required the administration of a large volume of fluids and high doses of norepinephrine and vasopressin, mainly from D2 to D4. During this period, he developed clinical and laboratory features compatible with systemic inflammatory response syndrome, multiple organ dysfunction, capillary leak syndrome, rhabdomyolysis, necrotizing fasciitis and possible compartment syndrome. The patient underwent forearm fasciotomy on D4 and there was progressive improvement of the hemodynamic status from D7 onwards. Wound management involved several debridements, broad-spectrum antibiotics and two skin grafts. Major laboratory findings within 12 days post-exposure revealed hypoalbuminemia, proteinuria, thrombocytopenia, leukocytosis and increases in cytokines (IL-6, IL-10 and TNF-α), troponin and creatine kinase. Ricin A-chain (ELISA) was detected in serum up to D3 (peak at 24 h post-exposure), with ∼79% being excreted in the urine within 64 h post-exposure. Ricinine was detected in serum and urine by LC-MS up to D5. A ricin A-chain concentration of 246 µg/mL was found in the seed extract, corresponding to the injection of ∼738 µg of ricin A-chain (∼10.5 µg/kg). The patient was discharged on D71, with limited range of motion and function of the left forearm and hand. CONCLUSION: Ricin injection resulted in a near-fatal poisoning that evolved with septic shock-like syndrome, multiple organ dysfunction and necrotizing fasciitis, all of which were successfully treated with supportive care.

Contrasting effects of the alkaloid ricinine on the capacity of Anopheles gambiae and Anopheles coluzzii to transmit Plasmodium falciparum.

BACKGROUND: Besides feeding on blood, females of the malaria vector Anopheles gambiae sensu lato readily feed on natural sources of plant sugars. The impact of toxic secondary phytochemicals contained in plant-derived sugars on mosquito physiology and the development of Plasmodium parasites remains elusive. The focus of this study was to explore the influence of the alkaloid ricinine, found in the nectar of the castor bean Ricinus communis, on the ability of mosquitoes to transmit Plasmodium falciparum. METHODS: Females of Anopheles gambiae and its sibling species Anopheles coluzzii were exposed to ricinine through sugar feeding assays to assess the effect of this phytochemical on mosquito survival, level of P. falciparum infection and growth rate of the parasite. RESULTS: Ricinine induced a significant reduction in the longevity of both Anopheles species. Ricinine caused acceleration in the parasite growth rate with an earlier invasion of the salivary glands in both species. At a concentration of 0.04 g l-1 in An. coluzzii, ricinine had no effect on mosquito infection, while 0.08 g l-1 ricinine-5% glucose solution induced a 14% increase in An. gambiae infection rate. CONCLUSIONS: Overall, our findings reveal that consumption of certain nectar phytochemicals can have unexpected and contrasting effects on key phenotypic traits that govern the intensity of malaria transmission. Further studies will be required before concluding on the putative role of ricinine as a novel control agent, including the development of ricinine-based toxic and transmission-blocking sugar baits. Testing other secondary phytochemicals in plant nectar will provide a broader understanding of the impact which plants can have on the transmission of vector-borne diseases.

Development and application of a strategy for analyzing eight biomarkers in human urine to verify toxic mushroom or ricinus communis ingestions by means of hydrophilic interaction LC coupled to HRMS/MS.

The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and ß-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and ß-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and ß-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.

Antimicrobial and antiquorum-sensing activity of Ricinus communis extracts and ricinine derivatives.

Ricinine (1), a known major alkaloid in Ricinus communis plant, was used as a starting compound for the synthesis of six ricinine derivatives; two new and four known compounds. The new derivatives; 3-amino-5-methyl-1H-pyrazolo[4,3-c]pyridin-4(5H)-one (2), and 3-amino-5-methyl-1-(phenylsulfonyl)-1H-pyrazolo[4,3-c]pyridin-4(5H)-one (3), as well as the previously prepared derivatives (4-7) were subjected for antimicrobial and antiquorum-sensing evaluation in comparison to different R. communis extracts. Acetyl ricininic acid derivative (5) showed the highest antimicrobial activity among all tested derivatives against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeuroginosa and Candida albicans. However, compound 7 (4-methoxy-1-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide) showed the highest antiquorum-sensing activity among all tested compounds and extracts. These findings proved the usefulness of ricinine as a good scaffold for the synthesis of new antimicrobial and antiquorum-sensing derivatives in spite of its poor contribution to the antimicrobial activity of the plant extracts.

Microwave-Assisted Extraction of Ricinine from Ricinus communis Leaves.

The alkaloid ricinine (3-cyano-4-methoxy-N-methyl-2-pyridone) is found in different parts of the Ricinus communis plant and is known to possess several bioactive properties, including strong antioxidant activity. In this study, a new microwave-assisted extraction (MAE) method was developed for the recovery of ricinine from R. communis leaves. The extraction variables studied were extraction temperature (between 125 °C and 175 °C), microwave power (between 500 W and 1000 W), extraction time (between 5 min and 15 min), extraction solvent (between 10% and 90% of EtOAc in MeOH), and solvent-to-sample ratio (between 25:1 mL and 50:1 mL of solvent per gram of the sample). On studying the effects of extraction variables, both solvent and liquid-to-solid ratio were found to exhibit the highest effects on ricinine recovery. A fast (15 min) microwave-assisted extraction method was developed (high temperatures can be applied because the stability of ricinine is proven in the literature), allowing for the recovery of ricinine from R. communis leaves. The study revealed that R. communis leaves had almost 1.5 mg g-1 (dried weight) of ricinine.

Ricin Toxicity: Clinical and Molecular Aspects.

Seeds of the castor bean plant Ricinuscommunis L (CB) contain ricin toxin (RT), one of the most poisonous naturally-occurring substances known. Ricin toxin, a water-soluble glycoprotein that does not partition into the oil extract, is a ribosome-inactivating toxin composed of two chains, labeled A and B. Severity of the toxicity varies depending on the route of exposure to the toxin. Inhalational is the most toxic route, followed by oral ingestion. Orally-ingested RT accumulates in the liver and spleen but other cells are also affected. The main clinical manifestations are also related to the administration route. Oral ingestion of CB or RT results in abdominal pain, vomiting, diarrhea, and various types of gastrointestinal bleeding that leading to volume depletion, hypovolemic shock, and renal failure. Inhalation of the toxin presents with non-cardiogenic pulmonary edema, diffuse necrotizing pneumonia, interstitial and alveolar inflammation, and edema. Local injection of RT induces indurations at the injection site, swelling of regional lymph nodes, hypotension, and death. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect RT in animal tissues and fluids. Ricinine, an alkaloid of CB, can be detected in rat urine within 48 h of RT exposure. Supportive care is the basic treatment and standard biowarfare decontamination protocols are used for RT intoxication. Dexamethasone and difluoromethylornithine might be effective treatments. This review examines the clinical and molecular aspects of ricin toxicity.

Dispersive solid-phase extraction followed by high-performance liquid chromatography/tandem mass spectrometry for the determination of ricinine in cooking oil.

A rapid and accurate method by liquid chromatography/tandem mass spectrometry (LC-MS/MS) using positive electrospray was established for the determination of ricinine in cooking oils. The homogenized samples, spiked with (13)C6-labelled ricinine as an internal standard, were extracted using ethanol/water (20:80, v/v) and purified by dispersive solid-phase extraction (dSPE) using primary-secondary amine (PSA) and C18 as adsorbents. The extract was separated in a short C18 reversed-phase column using methanol/water (25:75, v/v) as the mobile phase and detected in multiple reaction monitoring (MRM) mode with the absolute matrix effect of 93.2-102.2%. The alkali-metal adduct ions were discussed and the mass/mass fragmentation pathway was explained. Ricinine showed good linearity in the range of 0.5-50.0 µg/kg with the limit of quantitation 0.5 µg/kg. The recoveries were between 86.0% and 98.3% with the intra- and inter-day RSDs of 2.6-7.0%, 5.5-10.8%, respectively. This method could be applied to the rapid quantification of ricinine in cooking oils.

Torta de mamona destoxificada para alimentação de poedeiras comerciais / Detoxified castor cake feed for laying hens

Objetivou-se com este experimento avaliar o efeito da inclusão de torta de mamona destoxificada (TMD) na ração de poedeiras comerciais sobre o desempenho e a qualidade interna e externa dos ovos. Foram utilizadas 200 poedeiras comerciais de 40 a 50 semanas de idade, da linhagem Hy-Line W-36®, com 1543±34g de peso corporal, que foram distribuídas em um delineamento inteiramente casualizado com cinco tratamentos (0, 5, 10, 15 e 20% de torta de mamona destoxificada na ração) com cinco repetições de oito aves cada. Foram avaliados o consumo de ração, a produção, o peso e a massa de ovos e a conversão alimentar. A qualidade interna foi avaliada por meio da unidade de Haugh, percentual de albúmen e de gema. A qualidade da casca foi medida pela espessura, densidade específica e percentual de casca. As variáveis de desempenho foram afetadas pela inclusão da TMD com resposta linear negativa para o consumo de ração e quadrática para produção de ovos, peso do ovo, massa do ovo e conversão alimentar, com os melhores níveis de 10,5, 5,7, 9,2 e 10,3% respectivamente. Não houve efeito significativo da inclusão da TMD para as variáveis de qualidade interna e externa dos ovos. Concluiu-se que a TMD pode ser incluída na ração de poedeiras em até 5,7% para otimizar o desempenho e não alterar a qualidade interna e externa dos ovos.

This experiment aimed to evaluate the use of detoxified castor cake (DCC) in the diet of laying hens on performance and internal and external quality of eggs. A total of 200 laying hens with 40-50 weeks of age, of Hy-Line W-36® line, with 1543±34g body weight, were distributed in a completely randomized design with five treatments (0, 5, 10, 15 and 20% of DCC in diet) with five replicates of eight birds each. The feed intake, egg production, egg weight, egg mass and feed conversion were evaluated. The egg internal quality was evaluated by Haugh unit, percentage of albumen and yolk. The shell quality was evaluated by the thickness, density and specific percentage of eggshell. The performance variables were affected by the inclusion of DCC with a negative linear response to food intake and quadratic for egg production, egg weight, egg mass and feed conversion, with the best levels of 10.5, 5.7, 9.2 and 10.3% respectively. There was no significant effect of DCC inclusion for the variables of egg quality. It is conclude that the DCC can be included in hens diets up to 5.7% to optimize performance and do not alter the internal and external quality of the eggs.