Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P1, P4-di(2\',5\'-dideoxy-5\'-selenouridinyl)-tetraphosphate (P4-SedU2), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P4-SedU2 and the corresponding selenothymidine analogue P4-SeT2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P4-SedU2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma Cmax than the equivalent injection of free P4-SedU2, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.

Cluster of differentiation 36 (CD36) is a scavenger receptor (SR), recognizing diverse extracellular ligands in various types of mammalian cells. Long-chain fatty acids (FAs), which are important constituents of phospholipids and triglycerides, also utilize CD36 as a predominant membrane transporter, being incorporated from the circulation across the plasma membrane in several cell types, including cardiac and skeletal myocytes and adipocytes. CD36 is localized in intracellular vesicles as well as the plasma membrane, and its distribution is modulated by extracellular stimuli. Herein, we aimed to clarify the molecular basis of insulin-stimulated translocation of CD36, which leads to the enhanced uptake of long-chain FAs, in adipocytes. To this end, we developed a novel exofacial epitope-tagged reporter to specifically detect cell surface-localized CD36. By employing this reporter, we demonstrate that the small GTPase Rac1 plays a pivotal role in insulin-stimulated translocation of CD36 to the plasma membrane in 3T3-L1 adipocytes. Additionally, phosphoinositide 3-kinase and the protein kinase Akt2 are shown to be involved in the regulation of Rac1. Downstream of Rac1, another small GTPase RalA directs CD36 translocation. Collectively, these results suggest that CD36 is translocated to the plasma membrane by insulin through mechanisms similar to those for the glucose transporter GLUT4 in adipocytes.

During kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis.
Our study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs.
Our study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process. Video Abstract.

Papillary renal neoplasm with reverse polarity (PRNRP) is a renal tumor with frequent KRAS mutations. In this study, we aimed to report the clinical, histological, and immunohistochemical characteristics of PRNRP and the protein expression of various KRAS signaling pathway downstream effectors in PRNRP. PRNRP samples from patients who underwent surgical resection at Seoul National University Hospital over an 11-year period (January 2011 to December 2021) were analyzed. We identified 43 PRNRPs, defined as papillary renal tumors with a thin papillary architecture, eosinophilic finely granular cytoplasm, and apical nuclear position. Immunohistochemistry revealed typical characteristics of PRNRP, including exclusively positive GATA3 (43/43); highly positive L1CAM (43/43), PAX8 (43/43), and EMA (43/43); and low positive AMACR (4/43), RCC (1/43), and vimentin (1/43). KRAS signaling pathway effectors, such as p-ERK, RalA, and RalB, were highly expressed in PRNRP compared to papillary renal cell carcinoma (pRCC) with low or high nuclear grade (P < .001, all). Compared to pRCC with high nuclear grade, patients with PRNRP exhibited significantly longer progression-free survival (P < .001). PRNRP showed the best clinical outcome, with no disease progression in any of the cases. Our study analyzed the largest number of PRNRP cases and is the first to analyze the association between PRNRP and the KRAS downstream signaling pathway. PRNRP was found at a high frequency among all papillary renal tumors (43/207) and demonstrated a very good prognosis. PRNRP showed high GATA3, L1CAM, PAX8, and EMA protein expression as well as high p-ERK, RalA, and RalB protein expression.

Lipid nanoparticles (LNP) have been instrumental in the success of mRNA vaccines and have opened up the field to a new wave of therapeutics. However, what is ahead beyond the LNP? The approach herein used a nanoparticle containing a blend of Spike, Membrane and Envelope antigens complexed for the first time with the RALA peptide (RALA-SME). The physicochemical characteristics and functionality of RALA-SME were assessed. With >99% encapsulation, RALA-SME was administered via intradermal injection in vivo, and all three antigen-specific IgG antibodies were highly significant. The IgG2a:IgG1 ratio were all >1.2, indicating a robust TH1 response, and this was further confirmed with the T-Cell response in mice. A complete safety panel of markers from mice were all within normal range, supported by safety data in hamsters. Vaccination of Syrian Golden hamsters with RALA-SME derivatives produced functional antibodies capable of neutralising SARS-CoV-2 from both Wuhan-Hu-1 and Omicron BA.1 lineages after two doses. Antibody levels increased over the study period and provided protection from disease-specific weight loss, with inhibition of viral migration down the respiratory tract. This peptide technology enables the flexibility to interchange and add antigens as required, which is essential for the next generation of adaptable mRNA vaccines.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

Artisanal Minas cheese (QMA) is traditionally elaborate using raw milk and endogenous ferment (pingo - whey or rala - grated ripened cheese). In the present study, 91 yeast strains were isolated and identified from pingo and rala. Eight yeast species were identified by the MALDI-TOF mass spectrometry and confirmed by sequencing of the ITS region. The yeasts\' protease and lipase activities were evaluated in addition to probiotic properties such as tolerance to low pH and bile salts, hydrophobicity, autoaggregation, co-aggregation with pathogens, and antimicrobial susceptibility. The rala ferment showed a greater variety of species. Yarrowia lipolytica was the dominant specie (52.7% of isolates), followed by the Kluyveromyces lactis and Kodamaea ohmeri (9.9 and 6.6%, respectively). From the total yeasts evaluated, 74 strains showed positive enzymatic activity: 52 strains showed lipolytic (51 Y. lipolytica and one Trichosporon japonicum) and 44 proteolytic activities (18 Y. lipolytica, 13 K. ohmeri, 11 K. lactis, and 2 Wickerhamiella sp.). All evaluated isolates demonstrated tolerance to pH 2.0, and 69 isolates supported the presence of bile salts. From them, 12 isolates showed the capacity of autoaggregation (> 30%) and hydrophobicity (> 90.0%) and were then selected for co-aggregation and antibiotic resistance assays. All selected isolates showed co-aggregation with Salmonella Enteritidis, Escherichia coli, and Listeria monocytogenes greater than 30%. None of the yeast showed sensibility to the evaluated antibiotics and antagonistic activity against the evaluated pathogens. The results demonstrated that pingo and rala have different yeast composition with different enzymatic activity, which may affect the characteristics of the cheese. Furthermore, some yeast strains: Y. lipolytica (9 strains isolated from rala) and K. ohmeri (3 strains isolated from pingo) demonstrated attractive probiotic potential.

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.

White adipocytes act as lipid storage, and play an important role in energy homeostasis. The small GTPase Rac1 has been implicated in the regulation of insulin-stimulated glucose uptake in white adipocytes. Adipocyte-specific rac1-knockout (adipo-rac1-KO) mice exhibit atrophy of subcutaneous and epididymal white adipose tissue (WAT); white adipocytes in these mice are significantly smaller than controls. Here, we aimed to investigate the mechanisms underlying the aberrations in the development of Rac1-deficient white adipocytes by employing in vitro differentiation systems. Cell fractions containing adipose progenitor cells were obtained from WAT and subjected to treatments that induced differentiation into adipocytes. In concordance with observations in vivo, the generation of lipid droplets was significantly attenuated in Rac1-deficient adipocytes. Notably, the induction of various enzymes responsible for de novo synthesis of fatty acids and triacylglycerol in the late stage of adipogenic differentiation was almost completely suppressed in Rac1-deficient adipocytes. Furthermore, the expression and activation of transcription factors, such as the CCAAT/enhancer-binding protein (C/EBP) β, which is required for the induction of lipogenic enzymes, were largely inhibited in Rac1-deficient cells in both early and late stages of differentiation. Altogether, Rac1 is responsible for adipogenic differentiation, including lipogenesis, through the regulation of differentiation-related transcription.

RALA is a member of the small GTPase Ras superfamily and has been shown to play a role in promoting cell proliferation and migration in most tumors, and increase the resistance of anticancer drugs such as imatinib and cisplatin. Although many literatures have studied the cancer-promoting mechanism of RALA, there is a lack of relevant pan-cancer analysis.
This study systematically analyzed the differential expression and mutation of RALA in pan-cancer, including different tissues and cancer cell lines, and studied the prognosis and immune infiltration associated with RALA in various cancers. Next, based on the genes co-expressed with RALA in pan-cancer, we selected 241 genes with high correlation for enrichment analysis. In terms of pan-cancer, we also analyzed the protein-protein interaction pathway of RALA and the application of small molecule drug Guanosine-5\'-Diphosphate. We screened hepatocellular cancer (HCC) to further study RALA.
The results indicated that RALA was highly expressed in most cancers. RALA was significantly correlated with the infiltration of B cells and macrophages, as well as the expression of immune checkpoint molecules such as CD274, CTLA4, HAVCR2 and LAG3, suggesting that RALA can be used as a kind of new pan-cancer immune marker. The main functions of 241 genes are mitosis and protein localization to nucleosome, which are related to cell cycle. For HCC, the results displayed that RALA was positively correlated with common intracellular signaling pathways such as angiogenesis and apoptosis.
In summary, RALA was closely related to the clinical prognosis and immune infiltration of various tumors, and RALA was expected to become a broad-spectrum molecular immune therapeutic target and prognostic marker for pan-cancer.