RESUMO
Marine sponges from the Plakinidae family are well known for hosting cytotoxic secondary metabolites and the Brazilian Atlantic coast and its oceanic islands have been considered as a hotspot for the discovery of new Plakinidae species. Herein, we report the chemical profile among cytotoxic extracts obtained from four species of Plakinidae, collected in Fernando de Noronha Archipelago (PE, Northeastern Brazil). Crude organic extracts of Plakinastrella microspiculifera, Plakortis angulospiculatus, Plakortis insularis, and Plakortis petrupaulensis showed strong antiproliferative effects against two different cancer cell lines (HCT-116: 86.7-100%; MCF-7: 74.9-89.5%) at 50 µg/mL, by the MTT assay. However, at a lower concentration (5 µg/mL), high variability in inhibition of cell growth was observed (HCT-116: 17.3-68.7%; MCF-7: 0.00-55.5%), even within two samples of Plakortis insularis which were collected in the west and east sides of the Archipelago. To discriminate the chemical profile, the samples were investigated by UHPLC-HRMS under positive ionization mode. The produced data was uploaded to the Global Natural Products Social Molecular Networking and organized based on spectral similarities for purposes of comparison and annotation. Compounds such as dipeptides, nucleosides and derivatives, polyketides, and thiazine alkaloids were annotated and metabolomic differences were perceived among the species. To the best of our knowledge, this is the first assessment for cytotoxic activity and chemical profiling for Plakinastrella microspiculifera, Plakortis insularis and Plakortis petrupaulensis, revealing other biotechnologically relevant members of the Plakinidae family.
Assuntos
Metaboloma , Poríferos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Brasil , Proliferação de Células/efeitos dos fármacos , Células HCT116 , Humanos , Ilhas , Células MCF-7 , Metabolômica , Neoplasias/tratamento farmacológico , Plakortis/química , Plakortis/metabolismo , Poríferos/metabolismoRESUMO
The extensive marine biodiversity has proved to be a promising source of substances with biomedical potential. In this study, the cytotoxicity of the Brazilian octocoral Phyllogorgia dilatata (Gorgoniidae) was evaluated against two tumor cell lines and three bacterial strains. The methanol/dichloromethane crude extract presented no antibacterial activity up to the highest concentration tested (512 µg/mL), however it revealed a noteworthy antiproliferative effect against HCT-116 (80%) and MCF-7 (54%) cell lines at 50 µg/mL. Therefore, guided by the cytotoxic activity, a multistep chemical fractionation of the extract provided the subfraction 5 (PDPH2-5) with IC50 values of 3.18 and 17.80 µg/mL against HCT-116 and MCF-7, respectively. The LC-HRMS/MS analysis of PDPH2-5 showed ions of m/z 219.1742 and 219.1743, characterized as (E,E) and (Z,E) germacrone, after a LC-DAD-SPE/NMR analysis of the hexanic fraction and comparisons of NMR data with the literature. Previously reported assessments to the cytotoxic activity of the (E,E)-diastereoisomer disclosed higher IC50 values than that obtained for the PDPH2-5 fraction, suggesting, herein, a potentiated effect of the diastereoisomeric mixture. Such remark encourage further bioactivity studies with stereoisomer mixtures and reduce the urge for compound isolation.
Assuntos
Antozoários , Antineoplásicos , Produtos Biológicos/farmacologia , Animais , Antozoários/química , Antineoplásicos/farmacologia , Células HCT116 , Humanos , Células MCF-7RESUMO
In recent decades, considerable attention has been devoted to endocrine disruptor chemicals (EDC) and studies on fish feminization have increased throughout the years as a key signal for aquatic environmental contamination. The input of domestic sewage into water reservoirs is common in South American countries, especially in cities that experienced rapid population growths and unplanned urbanization. This study aimed at characterizing morphofunctional parameters of the tropical fish Sphoeroides testudineus and investigating the potential occurrence and effects of endocrine disruptors in the Pacoti River (Ceará, Brazil), often considered a reference site. After collection from the field, fish were measure/weighted and desiccated for gender identification (males, females, and undifferentiated), gonadal histology, and vitellogenin expression. From the biometric analysis, undifferentiated fish showed lower weight and length than female and male fish, although no differences in the condition index were observed. The gonadal weight of undifferentiated fish was significantly lower than those of females and males. Although this pattern was observed, gonadosomatic index (GSI) showed a different pattern, with differences being observed just between males and the other two groups (females and undifferentiated). Vitellogenin (VTG) expression was detected in many mature male and undifferentiated fish, indicating endocrine disruption. In addition, several EDCs (estrone, 17α-estradiol, 17ß-estradiol, 17α-ethinylestradiol, diethylstilbestrol, and estriol) were identified and quantified in sediments from the sampling site. These results were unexpected and indicative that the Pacoti River is impaired by estrogenic contamination.
Assuntos
Disruptores Endócrinos/análise , Monitoramento Ambiental , Tetraodontiformes/fisiologia , Poluentes Químicos da Água/análise , Animais , Brasil , Disruptores Endócrinos/metabolismo , Disruptores Endócrinos/toxicidade , Estradiol/metabolismo , Estrona/metabolismo , Estuários , Etinilestradiol/metabolismo , Feminino , Gônadas/metabolismo , Masculino , Rios/química , Esgotos/química , América do Sul , Vitelogeninas/metabolismo , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.
Assuntos
Autofagia/efeitos dos fármacos , Melanoma/tratamento farmacológico , Plicamicina/análogos & derivados , Brasil , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromomicina A3/metabolismo , Cromomicinas/farmacologia , Células HCT116 , Células HL-60 , Humanos , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Plicamicina/farmacologia , Streptomyces/químicaRESUMO
The tropical ascidian Eudistoma vannamei, endemic to the northeastern coast of Brazil, is considered a prolific source of secondary metabolites and hosts Actinomycetota that produce bioactive compounds. Herein, we used an omics approach to study the ascidian as a holobiont, including the microbial diversity through 16S rRNA gene sequencing and metabolite production using mass spectrometry-based metabolomics. Gene sequencing analysis revealed all samples of E. vannamei shared about 50% of the observed ASVs, and Pseudomonadota (50.7%), Planctomycetota (9.58%), Actinomycetota (10.34%), Bacteroidota (12.05%) were the most abundant bacterial phyla. Analysis of tandem mass spectrometry (MS/MS) data allowed annotation of compounds, including phospholipids, amino acids, and pyrimidine alkaloids, such as staurosporine, a member of a well-known chemical class recognized as a microbial metabolite. Isolated bacteria, mainly belonging to Streptomyces and Micromonospora genera, were cultivated and extracted with ethyl acetate. MS/MS analysis of bacterial extracts allowed annotation of compounds not detected in the ascidian tissue, including marineosin and dihydroergotamine, yielding about 30% overlapped ions between host and isolated bacteria. This study reveals E. vannamei as a rich source of microbial and chemical diversity and, furthermore, highlights the importance of omic tools for a comprehensive investigation of holobiont systems.
Assuntos
Urocordados , Animais , Filogenia , RNA Ribossômico 16S/genética , Espectrometria de Massas em Tandem , Bactérias/genéticaRESUMO
DNA-targeting agents have a significant clinical use, although toxicity remains an issue that plays against their widespread application. Understanding the mechanism of action and DNA damage response elicited by such compounds might contribute to the improvement of their use in anticancer chemotherapy. In a previous study, our research group characterized a new DNA-targeting agent - pradimicin-IRD. Since DNA-targeting agents and DNA repair are close-related subjects, the present study used in silico-modelling and a transcriptomic approach seeking to characterize the DNA repair pathways activated in HCT 116 cells following pradimicin-IRD treatment. Molecular docking analysis showed pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Furthermore, the transcriptomic study highlighted DNA repair functions related to genes modulated by pradimicin-IRD, such as nucleotide excision repair, telomeres maintenance and double-strand break repair. When validating these functions, PCNA protein levels decreased after exposure to pradimicin. Furthermore, molecular docking analysis suggested DNA-pradimicin-PCNA interaction. In addition, hTERT and POLH showed reduced mRNA levels after 6 h of treatment with pradimicin-IRD. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53-independent, unlike doxorubicin, dissimilarities between the mechanism of action and the DNA damage response of pradimicin-IRD and doxorubicin open new insights for further studies of pradimicin-IRD as a new antineoplastic compound.
Assuntos
Antineoplásicos , Humanos , Simulação de Acoplamento Molecular , Antígeno Nuclear de Célula em Proliferação , Antineoplásicos/farmacologia , Reparo do DNA , DNA , Doxorrubicina/farmacologia , Dano ao DNARESUMO
BACKGROUND: Previous studies have experimentally validated and reported that chemical constituents of marine sponges are a source of natural anti-inflammatory substances with the biotechnological potential to develop novel drugs. AIMS: Therefore, the aim of this study was to perform a systematic review to provide an overview of the anti-inflammatory substances isolated from marine sponges with therapeutic potential. METHODS: This systematic review was performed on the Embase, PubMed, Scopus and Web of Science electronic databases. In total, 613 were found, but 340 duplicate studies were excluded, only 100 manuscripts were eligible, and 83 were included. RESULTS: The results were based on in vivo and in vitro assays, and the anti-inflammatory effects of 251 bioactive compounds extracted from marine sponges were investigated. Their anti-inflammatory activities include inhibition of pro-inflammatory mediators, such as tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), nitrite or nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1ß (IL-1ß), prostaglandin E2 (PGE2), phospholipase A2 (PLA2), nuclear transcription factor-kappa B (NF-κB), leukotriene B4 (LTB4), cyclooxygenase- 1 (COX-1), and superoxide radicals. CONCLUSION: In conclusion, data suggest (approximately 98% of articles) that substances obtained from marine sponges may be promising for the development of novel anti-inflammatory drugs for the treatment of different pathological conditions.
Assuntos
NF-kappa B , Poríferos , Animais , NF-kappa B/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Poríferos/metabolismo , Lipopolissacarídeos/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Ciclo-Oxigenase 2/metabolismo , Óxido Nítrico/metabolismoRESUMO
The present study reports the identification of two new staurosporine derivatives, 2-hydroxy-7-oxostaurosporine (1) and 3-hydroxy-7-oxostaurosporine (2), obtained from mid-polar fractions of an aqueous methanol extract of the tunicate Eudistoma vannamei, endemic to the northeast coast of Brazil. The mixture of 1 and 2 displayed IC50 values in the nM range and was up to 14 times more cytotoxic than staurosporine across a panel of tumor cell lines, as evaluated using the MTT assay.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Estaurosporina/análogos & derivados , Urocordados/química , Animais , Antineoplásicos/isolamento & purificação , Organismos Aquáticos/química , Brasil , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HL-60 , Humanos , Concentração Inibidora 50 , Células Jurkat , Células K562 , Espectroscopia de Ressonância Magnética/métodos , Estaurosporina/química , Estaurosporina/isolamento & purificação , Estaurosporina/farmacologiaRESUMO
While the use of biodegradable polymers is recognized as a global strategy to minimize plastic pollution, the technical standards (TS) used to attest their biodegradability may not be in compliance with most environmental parameters observed aquatic ecosystems. Indeed, through a careful assessment of the TS currently in use, this study evidenced that these guidelines cover only a fraction of the biogeochemical parameters seen in nature and largely disregard those that occur in the deep-sea. Thus, these TS may not be able to ensure the degradation of such polymers in natural environments, where microbial activity, pH, temperature, salinity, UV radiation and pressure are highly variable. This raises environmental concern, since relevant parcel of plastic ends up in the oceans reaching deep zones. Therefore, there is an urgent need to revise these TS, which must consider the actual fate of most plastic debris and include assessments under the challenging conditions found at these types of environment, alongside microplastic formation and ecotoxicology effects. Moreover, the next generation of biodegradability tests must be designed to enable a cost-effective implementation and incorporate accurate analytical techniques to assess polymer transformation. Furthermore, certification should provide information on time scale and degradation rates and, preferably, be globally harmonized.
Assuntos
Plásticos Biodegradáveis , Poluentes Químicos da Água , Ecossistema , Ecotoxicologia , Monitoramento Ambiental , Poluição Ambiental , Oceanos e Mares , Plásticos , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation.
Assuntos
Aminoácidos Neutros/farmacologia , Antozoários/metabolismo , Antineoplásicos/química , Aminoácidos Neutros/química , Aminoácidos Neutros/isolamento & purificação , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , CamundongosRESUMO
Seriniquinone is a secondary metabolite isolated from a rare marine bacterium of the genus Serinicoccus. This natural quinone is highlighted for its selective cytotoxic activity toward melanoma cancer cells, in which rapid metastatic properties are still a challenge for clinical treatment of malignant melanoma. The progress of seriniquinone as a promising bioactive molecule for drug development requires the assessment of its clinical interaction potential with other drugs. This study aimed to investigate the in vitro inhibitory effects of seriniquinone on the main human CYP450 isoforms involved in drug metabolism. The results showed strong inhibition of CYP1A2, CYP2E1 and CYP3A, with IC50 values up to 1.4 µM, and moderate inhibition of CYP2C19, with IC50 value >15 µM. Detailed experiments performed with human liver microsomes showed that the inhibition of CYP450 isoforms can be explained by competitive and non-competitive inhibition mechanisms. In addition, seriniquinone demonstrated to be an irreversible and time-dependent inhibitor of CYP1A2 and CYP3A. The low inhibition constants values obtained experimentally suggest that concomitant intake of seriniquinone with drug metabolized by these isoforms should be carefully monitored for adverse effects or therapeutic failure.
Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Quinonas/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Microssomos Hepáticos/metabolismoRESUMO
Chemical investigation of the methanolic extract of the ascidian Didemnum psammatodes has led to the identification of fourteen known compounds: three methyl esters (methyl myristate, methyl palmitate and methyl stearate), four steroids (cholesterol, campesterol, stigmasterol and beta-sitosterol), two fatty acids (palmitic acid and stearic acid), three glyceryl ethers {(1,2-propanediol, 3-(heptadecyloxy), batyl alcohol and 1,2-propanediol, 3-[(methyloctadecyl)oxy]} and two nucleosides (thymidine and 2'-deoxyguanosine). Their structures were proposed by NMR and comparison with literature data and GC analysis in comparison with authentic sample. The cytotoxic activity of these compounds was evaluated against human leukemia cell line panel using the MTT assay. The mixture of the three methyl esters was the most active group of compounds, showing antiproliferative and cytotoxic effects. Further studies on their mode of action suggest that these activities are connected with inhibition of DNA synthesis and induction of both necrosis and apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Citotoxinas/farmacologia , Leucemia/tratamento farmacológico , Urocordados/química , Animais , Divisão Celular/efeitos dos fármacos , Citotoxinas/química , Citotoxinas/isolamento & purificação , Ésteres/química , Ésteres/isolamento & purificação , Ésteres/farmacologia , Células HL-60 , Humanos , Células K562 , Leucemia/patologia , Leucemia de Células T , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células PrecursorasRESUMO
This review will discuss the contributions of marine natural molecules, a source only recently found to have pharmaceutical prospects, to the development of anticancer drugs. Of the seven clinically utilized compounds with a marine origin, four are used for the treatment of cancer. The development of these drugs has afforded valuable knowledge and crucial insights to meet the most common challenges in this endeavor, such as toxicity and supply. In this context, the development of these compounds will be discussed herein to illustrate, with successful examples provided by cytarabine, trabectedin, eribulin and brentuximab vedotin, the steps involved in this process as well as the scientific advances and technological innovation potential associated with developing a new drug from marine resources.
Assuntos
Antineoplásicos/uso terapêutico , Organismos Aquáticos/química , Biotecnologia/métodos , Desenvolvimento de Medicamentos/métodos , Neoplasias/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacologia , Brentuximab Vedotin , Citarabina/química , Descoberta de Drogas , Furanos/química , Imunoconjugados/química , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Cetonas/química , Oceanos e Mares , Trabectedina/químicaRESUMO
The present study evaluated the anticancer potential of 11 plants used in Bangladeshi folk medicine. The extracts were tested for cytotoxicity using the brine shrimp lethality assay, sea urchin eggs assay, hemolysis assay and MTT assay using tumor cell lines. The extract of Oroxylum indicum showed the highest toxicity on all tumor cell lines tested, with an IC(50) of 19.6 microg/ml for CEM, 14.2 microg/ml for HL-60, 17.2 microg/ml for B-16 and 32.5 microg/ml for HCT-8. On the sea urchin eggs, it inhibited the progression of cell cycle since the frist cleavage (IC(50)=13.5 microg/ml). The extract of Aegle marmelos exhibited toxicity on all used assays, but in a lower potency than Oroxylum indicum. In conclusion, among all tested extracts, only the extracts of Oroxylum indicum, Moringa oleifera and Aegles marmelos could be considered as potential sources of anticancer compounds. Further studies are necessary for chemical characterization of the active principles and more extensive biological evaluations.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Plantas Medicinais/química , Animais , Artemia , Bangladesh , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Medicina Tradicional , Camundongos , Óvulo/efeitos dos fármacos , Casca de Planta/química , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Raízes de Plantas/química , Ouriços-do-Mar , Sais de Tetrazólio , Tiazóis , Células Tumorais CultivadasRESUMO
The MeOH extract of the ascidian Eudistoma vannamei was found to contain three novel compounds, the adenine alkaloid derivatives 9-[N-(leucyl)-isoleucyl]- adenine (1) and 8-hydroxy-8-isopentyl-7,8-dihydroadenine (2), and the phenylalanine peptide derivative N-[N-(leucyl)-isoleucyl]phenethylamine (3). Other previously related compounds isolated from this extract include thymidine, 2'-deoxyuridine and phenylalanine. The structures of the new compounds were elucidated through the use of NMR and mass spectrometry.
Assuntos
Alcaloides/isolamento & purificação , Urocordados/química , Alcaloides/química , AnimaisRESUMO
This review will discuss the contributions of marine natural molecules, a source only recently found to have pharmaceutical prospects, to the development of anticancer drugs. Of the seven clinically utilized compounds with a marine origin, four are used for the treatment of cancer. The development of these drugs has afforded valuable knowledge and crucial insights to meet the most common challenges in this endeavor, such as toxicity and supply. In this context, the development of these compounds will be discussed herein to illustrate, with successful examples provided by cytarabine, trabectedin, eribulin and brentuximab vedotin, the steps involved in this process as well as the scientific advances and technological innovation potential associated with developing a new drug from marine resources.
Assuntos
Biotecnologia/métodos , Organismos Aquáticos/química , Desenvolvimento de Medicamentos/métodos , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Oceanos e Mares , Imunoconjugados/uso terapêutico , Imunoconjugados/farmacologia , Imunoconjugados/química , Citarabina/química , Descoberta de Drogas , Trabectedina/química , Furanos/química , Brentuximab Vedotin , Cetonas/química , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
Abstract Methanolic extracts of the Brazilian endemic ascidian Eudistoma vannamei have been extensively studied for their cytotoxic activity against several human cancer cell lines. Previous work reported the occurrence of purine derivatives and staurosporine alkaloids as the major nitrogen-containing compounds. In this study, we report the occurrence of three pyrimidine alkaloids in addition to cholesterol, sitosterol and stigmasterol.
RESUMO
This study reports the antimitotic effects on sea urchin eggs of five known pterocarpans: (+)-3,10-dihydroxy-9-methoxypterocarpan, (+)-3,9-dimethoxypterocarpan [(+)-homopterocarpin], (+)-2,3,9-trimethoxypterocarpan, (+)-3,4-dihydroxy-9-methoxypterocarpan [(+)-vesticarpan] and (+)-3-hydroxy-9-methoxypterocarpan [(+)-medicarpin], isolated from the trunk of Platymiscium floribundum, a native tree from Brazil. All tested compounds showed strong activity in this assay, with 2,3,9-trimethoxypterocarpan being the most active (log IC50 of -8.10 +/- 0.02; -7.91 +/- 0.01; -7.97 +/- 0.02 M for first and third cleavages, and blastulae stages, respectively). These data suggest that the 2-methoxy substituent can be an important pharmacophoric unit.