Establishment and characterization of fibroblast lines from the northern tiger cat (Leopardus tigrinus, Schreber, 1775) during extended passage and cryopreservation.

The establishment of fibroblast lines enables several applications from the formation of biobanks for the conservation of biodiversity to the use of these cells in physiological and toxicological assays. Considered a species vulnerable to extinction, the characterization of fibroblastic lines of northern tiger cat would contribute to its conservation. Therefore, we established and characterized fibroblasts derived from northern tiger cat during extended passage (third, seventh, and eleventh passages) and cryopreservation with regard to the morphology, viability, apoptotic classification, metabolism, proliferative activity, and oxidative stress by reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). Initially, we identified four dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the third, seventh, and eleventh passages did not affect the viability, apoptotic classification, and ROS levels. Nevertheless, cells at seventh and eleventh passages featured a reduction in metabolism and an alteration in ΔΨm when compared to third passage cells. Additionally, cells at eleventh passage showed changes in the proliferative activity and morphology when compared to other passages. Regarding cryopreservation, no effect was observed on cryopreserved cells for morphology, viability, apoptotic classification, metabolism, and proliferative activity. Nevertheless, cryopreserved cells had alteration for ROS levels and ΔΨm. In summary, fibroblasts from northern tiger cat were affected by extended passage (seventh and eleventh passages) and cryopreservation. Adjustments to the in vitro culture and cryopreservation are necessary to reduce cellular oxidative stress caused by in vitro conditions.

Calcium polystyrene sulfonate-induced colitis: advanced characterization of crystal nature with infrared spectroscopy.

Classical potassium binders are used in the treatment of hyperkalemia and are widely associated with gastrointestinal side effects, with crystal colonic injury being rare but potentially fatal. In this report, we describe the case of an 82-year-old male with hyperkalemia and calcium polystyrene sulfonate crystal-associated colonic necrosis. Traditionally, this diagnosis has relied on the examination of crystal morphology and polarization through microscopy. Our study enhances crystal identification by incorporating an analysis of the physical characteristics of the crystals using infrared spectroscopy. This is the first description, to our knowledge, of the calcium polystyrene sulfonate infrared spectrum.

Effect of Cyclin-Dependent Kinase 4/6 Inhibitors on Circulating Cells in Patients with Metastatic Breast Cancer.

The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard-of-care for estrogen receptor (ER)-positive, HER2-negative (ER+/HER2- advanced/metastatic breast cancer (mBC). However, the impact of CDK4/6i on circulating immune cells and circulating tumor cells (CTCs) in patients receiving CDK4/6i and ET (CDK4/6i+ET) remains poorly understood. This was a prospective cohort study including 44 patients with ER+/HER2- mBC treated with CDK4/6i+ET in either first or second line. Peripheral blood samples were collected before (baseline) and 3 months (t2) after therapy. Immune cell's subsets were quantified by flow cytometry, and microfluidic-captured CTCs were counted and classified according to the expression of cytokeratin and/or vimentin. Patients were categorized according to response as responders (progression-free survival [PFS] ≥ 6.0 months; 79.1%) and non-responders (PFS < 6.0 months; 20.9%). CDK4/6i+ET resulted in significant changes in the hematological parameters, including decreased hemoglobin levels and increased mean corpuscular volume, as well as reductions in neutrophil, eosinophil, and basophil counts. Specific immune cell subsets, such as early-stage myeloid-derived suppressor cells, central memory CD4+ T cells, and Vδ2+ T cells expressing NKG2D, decreased 3 months after CDK4/6i+ET. Additionally, correlations between the presence of CTCs and immune cell populations were observed, highlighting the interplay between immune dysfunction and tumor dissemination. This study provides insights into the immunomodulatory effects of CDK4/6i+ET, underscoring the importance of considering immune dynamics in the management of ER+/HER2- mBC.

Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells.

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1-10 µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Preparation of Gel Forming Polymer-Based Sprays for First Aid Care of Skin Injuries.

Currently, there are several types of materials for the treatment of wounds, burns, and other topical injuries available on the market. The most used are gauzes and compresses due to their fluid absorption capacity; however, these materials adhere to the surface of the lesions, which can lead to further bleeding and tissue damage upon removal. In the present study, the development of a polymer-based gel that can be applied as a spray provides a new vision in injury protection, respecting the requirements of safety, ease, and quickness of both applicability and removal. The following polymeric sprays were developed to further obtain gels based on different polymers: hydroxypropyl cellulose (HPC), polyvinyl pyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) using polyethylene glycol (PEG) as a plasticizer. The developed sprays revealed suitable properties for use in topical injuries. A protective film was obtained when sprayed on a surface through a casting mechanism. The obtained films adhered to the surface of biological tissue (pig muscle), turning into a gel when the exudate was absorbed, and proved to be washable with saline solution and contribute to the clotting process. Moreover, biocompatibility results showed that all materials were biocompatible, as cell viability was over 90% for all the materials.

Prophylactic use of standardized extract of propolis of Apis mellifera (EPP-AF®) reduces lung inflammation and improves survival in experimental lethal sepsis.

ETHNOPHARMACOLOGICAL RELEVANCE: Sepsis poses one of the biggest public health problems, necessitating the search for new therapeutic alternatives. For centuries, propolis has been widely used in folk medicine to treat various inflammatory and infectious diseases. Given its extensive use, it has excellent potential as an adjuvant treatment for patients with sepsis. OBJECTIVE: This study evaluated prophylactic treatment with standardized propolis extract (EPP-AF®) and followed the prognosis of sepsis induced by ligation and cecal ligation and puncture (CLP). METHODS: Initially, for survival assessment, Swiss mice were separated into five groups: Sham (false operated), control (PBS), ATB (received antibiotic, 8 mg/kg), P10 (received EPP-AF®, 10 mg/kg), and P100 (received EPP-AF®, 100 mg/kg). The animals received PBS, antibiotic, or EPP-AF® by the subcutaneous route 6 h before the CLP procedure. Animal survival was assessed every 12 h for five days when all of them were euthanized. RESULTS: We show that the treatment with EPP-AF® significantly increased the life expectancy of animals with sepsis compared to the control group. Interestingly, prophylactic treatment with EPP-AF® showed no effect on the number of colony-forming units in the peritoneum, blood, or lung. However, there was a decrease in cellular influx in the peritoneum. This alteration was unrelated to the number of bone marrow cells or the differential counting of peripheral blood cells. The coagulogram remained unchanged, including the number of platelets and prothrombin time-activated partial thromboplastin time. However, the inflammatory infiltrate and bleeding in the lung tissue were lower in the animals that received EPP-AF®. CONCLUSION: Thus, it was possible to conclude that prophylactic treatment with EPP-AF® preserved the lung parenchyma, resulting in an increased lifespan of mice with sepsis. It can be a helpful adjuvant in prophylactic treatment with antibiotics in presurgical conditions.

Miniaturization of hiPSC-derived 3D neural cultures in stirred-tank bioreactors for parallelized preclinical assessment of rAAV.

Introduction: Engineered 3D models employing human induced pluripotent stem cell (hiPSC) derivatives have the potential to recapitulate the cell diversity and structure found in the human central nervous system (CNS). Therefore, these complex cellular systems offer promising human models to address the safety and potency of advanced therapy medicinal products (ATMPs), such as gene therapies. Specifically, recombinant adeno-associated viruses (rAAVs) are currently considered highly attractive for CNS gene therapy due to their broad tropism, low toxicity, and moderate immunogenicity. To accelerate the clinical translation of rAAVs, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. The integration of hiPSC-derived CNS models in rAAV development will require, amongst other factors, robust, small-scale, high-throughput culture platforms that can feed the preclinical trials. Methods: Herein, we pioneer the miniaturization and parallelization of a 200 mL stirred-tank bioreactor-based 3D brain cell culture derived from hiPSCs. We demonstrate the applicability of the automated miniaturized Ambr® 15 Cell Culture system for the maintenance of hiPSC-derived neurospheroids (iNSpheroids), composed of neuronal and glial cells. Critical process parameters were optimized, namely, cell density and agitation mode. Results: Under optimized conditions, stable iNSpheroid cultures were attained in the microbioreactors for at least 15 days, with high cell viability and astrocytic and neuronal phenotype maintenance. This culture setup allowed the parallelization of different rAAVs, in different multiplicity of infections (MOIs), to address rAAV-host interactions at a preclinical scale. The iNSpheroids were exposed to rAAV2- and rAAV9-eGFP in the microbioreactors. Transgene expression was detected 14 days post-transduction, revealing different astrocyte/neuron tropism of the two serotypes. Discussion: We advocate that the iNSpheroid cultures in miniaturized bioreactors are reliable and reproducible screening tools for addressing rAAV transduction and tropism, compatible with preclinical demands.

Alterations in the pro-resolving lipid mediator machinery within first trimester maternal tissue: Implications in decidualization and miscarriage risk.

A pivotal event in uterine receptivity and human reproduction is the differentiation of endometrial stromal cells into decidual cells, known as decidualization. Decidualization is interlinked with its inflammatory environment. Our study aimed to investigate the presence and role of pro-resolving lipid mediators in first trimester maternal tissue. We assessed the levels of LXA4 and RvD1, along with their metabolic LOX enzymes, in elective (control) and sporadic miscarriage samples. We investigated the effects of LXA4 and RvD1 on decidualization using primary endometrial stromal cells and the immortalized endometrial stromal St-T1b cell line. The upregulation of 12- and 15-LOX expression was observed in pregnancy tissue after sporadic miscarriage, suggesting an inflammatory imbalance. Furthermore, incubation with these lipid mediators led to a decrease in decidualization biomarkers PRL and IGFBP-1, accompanied by morphological changes indicative of aberrant differentiation. The expression of LOX enzymes in decidual natural killer cells suggests their involvement in regulating the inflammatory surroundings and the extent of decidualization.

SWATH-MS as a strategy for CHO host cell protein identification and quantification supporting the characterization of mAb purification platforms.

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2 ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance.

Exploring Bacterial Cellulose and a Biosurfactant as Eco-Friendly Strategies for Addressing Pharmaceutical Contaminants.

Aquatic environments face contamination by pharmaceuticals, prompting concerns due to their toxicity even at low concentrations. To combat this, we developed an ecologically sustainable biosurfactant derived from a microorganism and integrated it into bacterial cellulose (BC). This study aimed to evaluate BC's efficacy, with and without the biosurfactant, as a sorbent for paracetamol and 17α-ethinylestradiol (EE2) in water. We cultivated BC membranes using Gluconacetobacter xylinus ATCC 53582 and synthesized the biosurfactant through pre-inoculation of Bacillus subtilis in a synthetic medium. Subsequently, BC membranes were immersed in the biosurfactant solution for incorporation. Experiments were conducted using contaminated water, analyzing paracetamol concentrations via spectrophotometry and EE2 levels through high-performance liquid chromatography. Results indicated BC's superior adsorption for EE2 over paracetamol. Incorporating the biosurfactant reduced hormone adsorption but enhanced paracetamol sorption. Notably, original and freeze-dried BC exhibited better adsorption efficacy than biosurfactant-infused BC. In conclusion, BC showed promise in mitigating EE2 contamination, suggesting its potential for environmental remediation. Future research could focus on optimizing biosurfactant concentrations to enhance sorption capabilities without compromising BC's inherent effectiveness.

Identification of Mispairing Omic Signatures in Chinese Hamster Ovary (CHO) Cells Producing a Tri-Specific Antibody.

Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity. Moreover, mispairing carries significant challenges for downstream purification, decreasing yields and increasing the cost of bioprocess development. In this study, quantitative transcriptomics and proteomics analyses were employed to investigate which signaling pathways correlated with low and high mispairing clone signatures. Gene and protein expression profiles of Chinese hamster ovary (CHO) clones producing an tsAb were analyzed in the exponential growth and stationary (tsAb production) phase of fed-batch culture. Functional analysis revealed activated endoplasmic reticulum stress in high mispairing clones in both culture phases, while low mispairing clones exhibited expression profiles indicative of activated protein translation, as well as higher endocytosis and target protein degradation, suggesting the clearance of unfolded proteins through ubiquitin-mediated mechanisms. In addition, through transcriptomic profiling, we identified a group of genes that have the potential to be used as a biomarker panel tool for identifying high mispairing levels in the early stages of bioprocess development.

Effects of a combination of cannabidiol and delta-9-tetrahydrocannabinol on key biological functions of HTR-8/SVneo extravillous trophoblast cells.

In recent years, cannabis use has increased among pregnant women. In addition, the phytocannabinoids cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) alone or in combination are being used for therapeutical applications. THC and CBD are able to cross the placenta and a lot remains unknown concerning their impact on angiogenesis and extravillous trophoblasts' (EVTs) migration and invasion, which are essential processes for placentation. Thus, in this study, the HTR-8/SVneo cell line was employed to evaluate the effects of CBD, THC and of their combination (1:1, 2 µM). Cannabinoids affected epithelial-mesenchymal transition, as showed by increased expression of the epithelial protein marker E-cadherin for CBD and CBD plus THC treatments, and decrease of mesenchymal intermediate filament vimentin for all treatments. The gene expression of the metalloproteinases MMP2 and MMP9, and of their inhibitors TIMP1 and TIMP2 was increased, except the latter for THC treatment. Moreover, CBD reduced cell migration and invasion, an effect that was enhanced by its combination with THC. CBD with or without THC also upregulated the gene expression of PGF, while the anti-angiogenic factor sFLT1 was increased for all treatments. VEGFA and FLT1 were not affected. Alone or combined CBD and THC also decreased tube segments' length. Additionally, ERK1/2 and STAT3 phosphorylation was increased in the CBD and CBD plus THC-treated cells, while THC only activated STAT3. AKT activation was only affected by CBD. This work demonstrates that the exposure to cannabinoid-based products containing CBD and/or THC, may interfere with key processes of EVTs differentiation. Therefore, crucial phases of placental development can be affected, compromising pregnancy success.

The Immunomodulatory Activity of Punica granatum L. Peel Extract Increases the Lifespan of Mice with Lethal Sepsis.

Sepsis is an organ dysfunction syndrome associated with high mortality. To date, no effective treatment is available to combat this disease. Punica granatum L. is a potential alternative treatment due to its anti-inflammatory, antimicrobial, and antioxidant properties. Thus, this study aimed to evaluate the effects of a hydroalcoholic crude extract from the peels of P. granatum (HCEPg) in mice with lethal sepsis. Lethal polymicrobial sepsis was induced in female Swiss mice via cecal ligation and puncture (CLP). Initially, the animals were divided into three groups: Sham (false-operated), CLP-control (phosphate-buffered saline), and CLP-HCEPg (single dose, 5 mg/kg, subcutaneous administration). Treatment was initiated immediately after the induction of sepsis, and survival was evaluated every 12 hr for 5 days. Those who survived were euthanized. Serum cytokine levels were measured using a cytometric bead array Mouse Inflammatory Cytokine Kit. The number of colony-forming units, as well as the number of cells in the lymphoid organs and their activation markers, were analyzed. Results showed that treatment with HCEPg increased lifespan and reduced bacterial counts in the peritoneum, bloodstream, and spleen. HCEPg also decreased hydrogen peroxide secretion by phagocytes and augmented serum IL-10 levels, indicating its systemic anti-inflammatory effects. Additionally, treatment with HCEPg attenuated infection-induced lung hemorrhage. Overall, P. granatum extract improved the lifespan of septic mice, possibly due to its antimicrobial, anti-inflammatory, and immunomodulatory effects, thereby regulating bacterial load and translocation, as well as controlling the systemic inflammation induced by sepsis.

The M 1 -muscarinic acetylcholine receptor subtype may play a role in learning and memory performance in the hippocampus of neonatal monosodium glutamate-obese rats.

Here, we investigate the effects of obesity induced by monosodium glutamate (MSG) on cognitive impairment and whether this model induces any alteration in the affinity, density, and subtypes of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Healthy rats were used as controls, and MSG-obese rats were selected via the Lee index > 0.300. The effects of MSG-induced obesity on hippocampal spatial learning and memory processes were evaluated by using the working memory versions of the Morris' water maze task and the evaluation of mAChRs by binding assay and their subtypes by immunoprecipitation assays. [ 3 H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (K D ) did not differ between control and MSG, indicating that affinity is not affected by obesity induced by MSG. The maximum number of binding sites (B max ) obtained in MSG subjects was lower than that obtained from control rats, indicating a decrease in the expression of total mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M 1 subtype of MSG when compared with control rats (M 2 to M 5 subtypes did not differ between control and MSG). We also observed that MSG promotes a disruption of the spatial working memory which was accompanied by a decrease in the M 1 mAChR subtype in rat hippocampus, thus suggesting deleterious long-term effects besides the obesity. In conclusion, these findings provide new insights into how obesity can influence spatial learning and memory that is hippocampal-dependent. The data suggest that the M 1 mAChR subtype protein expression is a potential therapeutic target.

Silica Aerogel-Polycaprolactone Scaffolds for Bone Tissue Engineering.

Silica aerogel is a material composed of SiO2 that has exceptional physical properties when utilized for tissue engineering applications. Poly-ε-caprolactone (PCL) is a biodegradable polyester that has been widely used for biomedical applications, namely as sutures, drug carriers, and implantable scaffolds. Herein, a hybrid composite of silica aerogel, prepared with two different silica precursors, tetraethoxysilane (TEOS) or methyltrimethoxysilane (MTMS), and PCL was synthesized to fulfil bone regeneration requirements. The developed porous hybrid biocomposite scaffolds were extensively characterized, regarding their physical, morphological, and mechanical features. The results showed that their properties were relevant, leading to composites with different properties. The water absorption capacity and mass loss were evaluated as well as the influence of the different hybrid scaffolds on osteoblasts' viability and morphology. Both hybrid scaffolds showed a hydrophobic character (with water contact angles higher than 90°), low swelling (maximum of 14%), and low mass loss (1-7%). hOB cells exposed to the different silica aerogel-PCL scaffolds remained highly viable, even for long periods of incubation (7 days). Considering the obtained results, the produced hybrid scaffolds may be good candidates for future application in bone tissue engineering.

Group recommender systems for tourism: how does personality predict preferences for attractions, travel motivations, preferences and concerns?

To travel in leisure is an emotional experience, and therefore, the more the information about the tourist is known, the more the personalized recommendations of places and attractions can be made. But if to provide recommendations to a tourist is complex, to provide them to a group is even more. The emergence of personality computing and personality-aware recommender systems (RS) brought a new solution for the cold-start problem inherent to the conventional RS and can be the leverage needed to solve conflicting preferences in heterogenous groups and to make more precise and personalized recommendations to tourists, as it has been evidenced that personality is strongly related to preferences in many domains, including tourism. Although many studies on psychology of tourism can be found, not many predict the tourists' preferences based on the Big Five personality dimensions. This work aims to find how personality relates to the choice of a wide range of tourist attractions, traveling motivations, and travel-related preferences and concerns, hoping to provide a solid base for researchers in the tourism RS area to automatically model tourists in the system without the need for tedious configurations, and solve the cold-start problem and conflicting preferences. By performing Exploratory and Confirmatory Factor Analysis on the data gathered from an online questionnaire, sent to Portuguese individuals from different areas of formation and age groups (n = 1035), we show all five personality dimensions can help predict the choice of tourist attractions and travel-related preferences and concerns, and that only neuroticism and openness predict traveling motivations.

Corrigendum to "Biomedical and health informatics teaching in Portugal: Current status" [Heliyon 9(3) (March 2023) e14163].

[This corrects the article DOI: 10.1016/j.heliyon.2023.e14163.].

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.