Search Results (17)

Morphine addiction is driven in part by persistent reward-associated memory, yet the molecular mechanisms linking translational control to cellular stress responses remain unclear. In the present study, using a mouse morphine-induced conditioned place preference and N2a cell model, we investigated apoptosis-related alterations in the medial prefrontal cortex and the involvement of eIF4E-dependent translational regulation and BDNF/TrkB signaling. Morphine-induced conditioned place preference was associated with an increase in TUNEL-positive cells in the medial prefrontal cortex, accompanied by upregulation of Bax and downregulation of Bcl-2. In N2a cells, morphine induced apoptosis in a dose-dependent manner. Morphine also increased neuronal eIF4E expression in both mPFC tissue and N2a cells, accompanied by upregulation of BDNF and TrkB. Inhibition of the eIF4E/eIF4G interaction with 4EGI-1 significantly affected morphine-induced CPP formation and altered apoptosis-related markers and BDNF/TrkB expression. Notably, intra-mPFC administration of 4EGI-1 suppressed morphine-induced CPP without affecting anxiety-like behavior, locomotor activity, or general learning and memory performance. These findings suggest that eIF4E-dependent translational regulation is functionally associated with morphine-induced reward memory and apoptosis, potentially in association with changes in BDNF/TrkB molecular expression. This study offers novel insight into the molecular basis of morphine addiction and highlights a potentially targetable translational regulatory pathway for therapeutic intervention. Full article

Illumina sequencing of segment 2, which encodes the polymerase basic subunit 2 (PB2) of the RNA-dependent RNA polymerase of a divergent amnoonvirus recently detected in tissues of Nile tilapia farmed in Egypt, revealed the presence of two genetic variants of the same virus: AmnoonvirusEGY1F and -H. The phylogenetic and genetic analyses presented in this study support the inclusion of both variants in the genus Tilapinevirus, family Amnoonviridae, order Articulavirales. The Egyptian strains formed distinct, well-supported clades in both nucleotide- and amino acid-based trees, showing a notable divergence from unclassified amnoonviruses and clustered with members of the genus Tilapinevirus. Within the genus Tilapinevirus, both Egyptian strains were divergent from all tilapia lake virus (TiLV) strains, whose full RNA segment 2 sequences are available in public databases, as well as the newly isolated Tilapinevirus poikilos from the fancy-tailed guppy. The Egyptian strains were also divergent from TiLV strains identified in Israel and Lake Victoria. Although the PB2 proteins of AmnoonvirusEGY1F and -H exhibited striking similarity, several mutations were detected that altered the sequence of their antigenic cell epitopes. Some of these mutations in the AmnoonvirusEGY1H strain were predicted to affect PB2-encoded functions. Collectively, findings of this study aid in the growing understanding of viral diversity and PB2 evolution in emerging amnoonviruses, particularly the role of amino acid substitutions in affecting the encoded protein structure, function, and immunogenicity. Full article

Simulating carbohydrate digestion in physiologically relevant ways remains a challenge for in vitro models. In this study, the Dynamic In vitro Human Stomach (DIVHS) system was applied to investigate cereal digestion and subsequent intestinal cellular responses. Rice, millet, and corn were digested under dynamic and static conditions. Compared with the static model, the dynamic system generated smaller grain fragments, a larger chyme–enzyme contact area (451.2 ± 4.4 cm² vs. 160.4 ± 6.0 cm²), and higher average intragastric pressure (25.0 ± 1.2 kPa vs. 7.2 ± 0.7 kPa). Salivary amylase activity also declined more gradually in the dynamic system. An empirical approach for predicting the glycemic index (eGI) was proposed, which showed improved agreement with reported human GI values compared with earlier in vitro methods. Exposure of Caco-2 cells to digested products significantly altered transcriptional profiles, including protein binding, ATP binding, and glucose transporter activity. Notably, products from the dynamic model induced stronger transcriptional responses than those from the static model, including 421 genes up-regulated and 138 down-regulated. Functional enrichment highlighted pathways related to glucose transport, energy metabolism, and cellular regulation. Overall, this study demonstrates the advantages of dynamic digestion models in replicating gastrointestinal conditions, improving GI prediction, and providing mechanistic insights into intestinal cellular responses to digested carbohydrates. Full article

The pharmacological treatment of cholangiocarcinoma (CCA) is often hampered by tumor resistance. Improving our understanding of this issue is crucial for developing strategies that can overcome drug refractoriness. We have established and characterized two novel human cell sublines derived from extrahepatic CCA EGI-1 cells that are resistant to cisplatin and 5-fluorouracil (5-FU). Migration and proliferation were analyzed using holographic microscopy. The expression of genes involved in drug uptake and efflux was determined by RT-qPCR. Cross-resistance to commonly used antitumor drugs was assayed using the MTT test. EGI-1 sublines resistant to cisplatin (CR) or 5-FU (FR) exhibited more than a three-fold increase in resistance to cisplatin and 5-FU, respectively, and showed reduced proliferation, migration, and colony-formation rates, along with an altered cell cycle compared to wild-type cells, while retaining tumorigenic capacity. The analysis of the transportome showed downregulation of uptake transporters and upregulation of the export pumps MRP3/4. EGI-1 cells with acquired resistance to 5-FU demonstrated cross-resistance to irinotecan and gemcitabine, while cisplatin-resistant cells showed decreased sensitivity to 5-FU and platinum derivatives. These resistant cell lines offer valuable models for investigating the molecular basis of chemoresistance in CCA, providing a robust platform for the development and evaluation of novel therapeutic strategies. Full article

Cereals are among the foods rich in myo-inositol hexakisphosphate (phytic acid, IP6), lower myo-inositol phosphates (IPx), a wide range of phenolic compounds, as well as vitamins, minerals, oligosaccharides, phytosterols and para-aminobenzoic acid, and are attributed with multiple bioactivities, particularly associated with the prevention of metabolic syndrome and colon cancer. The bran fraction of wheat, maize, brown rice and other cereals contains high levels of phytate, free and total phenolics, and endogenous enzymes such as amylases, phytase, xylanase, β-glucanase and feruloyl esterase, whose activities can be increased by germination. The preliminary steps of digestion begin in the oral cavity where substrates for the action of endogenous cereal and salivary enzymes start to be released from the food matrix. IP6 released from phytate complexes with arabinoxylans, starch and protein bodies would eventually enhance the absorption of nutrients, including phenolics, by regulating tight junctions and, together with ferulic acid (FA), would maintain cell barrier integrity and epithelial antibacterial immunity. In addition, both IP6 and FA exert potent and complementary antioxidant effects, while FA together with IPx generated through advanced hydrolysis of IP6 by endogenous and microbial phytases may affect digestive enzyme activity and incretin secretion, resulting in modulated insulin and glucagon release and prevention of various diabetic complications. Contrary to widespread negative attitudes towards phytate, in this review, we present the strategy of selecting cereals with high phytate and phenolic content, as well as high endogenous phytase, feruloyl esterase and endoxylanase activities, to produce value-added health-promoting foods. The advanced hydrolysis of phytate and phenolic compounds by cereal and/or microbial enzymes would generate substantial amounts of “enzymatically generated inositol” (EGI), including IP6, IPx and myo-inositol, the compounds that, together with free FA, provide enhanced bioavailability of cereal nutrients through multiple synergistic effects not previously realised. Full article

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial–mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression. Full article

FMRP is a multifunctional protein encoded by the Fragile X Messenger Ribonucleoprotein 1 gene (FMR1). The inactivation of the FMR1 gene results in fragile X syndrome (FXS), a serious neurodevelopmental disorder. FMRP deficiency causes abnormal neurite outgrowth, which is likely to lead to abnormal learning and memory capabilities. However, the mechanism of FMRP in modulating neuronal development remains unknown. We found that FMRP enhances the translation of 4EBP2, a neuron-specific form of 4EBPs that inactivates eIF4E by inhibiting the interaction between eIF4E and eIF4G. Depletion of 4EBP2 results in abnormal neurite outgrowth. Moreover, the impairment of neurite outgrowth upon FMRP depletion was overcome by the ectopic expression of 4EBP2. These results suggest that FMRP controls neuronal development by enhancing 4EBP2 expression at the translational level. In addition, treatment with 4EGI-1, a chemical that blocks eIF4E activity, restored neurite length in FMRP-depleted and 4EBP2-depleted cells. In conclusion, we discovered that 4EBP2 functions as a key downstream regulator of FMRP activity in neuronal development and that FMRP represses eIF4E activity by enhancing 4EBP2 translation. Full article

The epithelial–mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC. Full article

It is well established that Cholangiocarcioma (CCA) drug resistance plays a crucial role in the spread and survival of cancer cells. The major enzyme in the nicotinamide-adenine dinucleotide (NAD+)-mediated pathways, nicotinamide phosphoribosyltransferase (NAMPT), is essential for cancer cell survival and metastasis. Previous research has shown that the targeted NAMPT inhibitor FK866 reduces cancer cell viability and triggers cancer cell death; however, whether FK866 affects CCA cell survival has not been addressed before. We show herein that NAMPT is expressed in CCA cells, and FK866 suppresses the capacity of CCA cells to grow in a dose-dependent manner. Furthermore, by preventing NAMPT activity, FK866 significantly reduced the amount of NAD+ and adenosine 5′-triphosphate (ATP) in HuCCT1, KMCH, and EGI cells. The present study’s findings further show that FK866 causes changes in mitochondrial metabolism in CCA cells. Additionally, FK866 enhances the anticancer effects of cisplatin in vitro. Taken together, the results of the current study suggest that the NAMPT/NAD+ pathway may be a possible therapeutic target for CCA, and FK866 may be a useful medication targeting CCA in combination with cisplatin. Full article

Avian influenza virus (AIV) H9N2 was declared to be endemic in birds of the Middle East, in particular in Egypt, with multiple cases of human infections. Despite concerns about the pandemic threat posed by H9N2 AIV, due to the fact that its receptor specificity is similar to that of human influenza viruses, its morbidity and mortality rates in humans are so far negligible. However, the acquisition of specific adaptive amino acid (aa) mutations in the viral polymerase can enhance cross-species transmission of the virus itself or of reassortants, which gained these changes. The polymerase basic protein 2 (PB2) is one of the key determinants for AIV adaptation towards mammals. Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different AIVs, the specific effect of single or multiple mutations on viral fitness has not been compared so far. Here, we studied the effect of the aa K at position 591, which was frequently reported in the PB2 of Egyptian H9N2 isolates, on the proliferation efficiency and polymerase activity of an H5N1 (clade 2.2.1.2) AIV already carrying the mammalian adaptive mutation 627K. Using reverse genetics, we generated a set of recombinant parental strains and H5N1 variants carrying the avian-like 591Q/627E or mammalian-like adaptive mutations 591K/627K (H5N1_EGY, H9N2_EGY, H5N1_PB2-H9N2EGY, H5N1_{H9N2_PB2_K591Q}, H5N1_{PB2_K627E}, H5N1_{PB2_K627E/591K}, H5N1_{PB2_627K/591K}). Regardless of the avian-like 627E or the mammalian-adaptive 627K, both variants carrying the 591K (H5N1_{PB2_K627E/591K}, H5N1_{PB2_627K/591K}) and the reassortant H5N1_PB2-H9N2EGY replicated to significantly higher levels in mammalian continuous MDCK and Calu-3 cell lines and primary normal human bronchial epithelial cells than the parental H5N1_EGY virus (carrying solely the 627K adaptive mutation). Expectedly, the H5N1 variants carrying avian-like PB2 mutations (H5N1_{H9N2_PB2_K591Q}, H5N1_{PB2_K627E}) replicated to significantly lower levels than the parental H5N1_EGY virus in the predefined primary and continuous mammalian cell line systems. Consistently, the activity of H5N1 subtype AIV polymerase complexes comprising PB2 segments with singular 591K or combined with 627K was significantly enhanced when compared to parental H5N1_EGY and H9N2_EGY. This study emphasizes the significant impact of 591K containing PB2 segments in the background of H5N1 polymerase on viral fitness in addition to the well-known MPM 627K in vitro. Full article

Recently, Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The accelerated dissemination of bla_CTX-M genes among these MDR K. pneumoniae, particularly bla_CTX-M-14 and bla_CTX-M-15, have been noted. In this study, we investigated the occurrence of bla_CTX-M-IV among K. pneumoniae recovered from the laboratory of a major hospital in Alexandria. The 23 tested isolates showed an MDR phenotype and the bla_CTX-M-IV gene was detected in ≈22% of the isolates. The transformation of plasmids harboring bla_CTX-M-IV to chemically competent cells of Escherichia coli DH5α was successful in three out of five of the tested bla_CTX-M-IV-positive isolates. Whole genome sequencing of K22 indicated that the isolate belonged to the high-risk clone ST383, showing a simultaneous carriage of bla_CTX-M-14 on IncL/M plasmid, i.e., pEGY22_CTX-M-14, and bla_CTX-M-15 on a hybrid IncHI1B/IncFIB plasmid, pEGY22_CTX-M-15. Alignment of both plasmids revealed high similarity with those originating in the UK, Germany, Australia, Russia, China, Saudi Arabia, and Morocco. pEGY22_CTX-M-15 was a mosaic plasmid that demonstrated convergence of MDR and virulence genes. The emergence of such a plasmid with enhanced genetic plasticity constitutes the perfect path for the evolution of K. pneumoniae isolates causing invasive untreatable infections especially in a country with a high burden of infectious diseases such as Egypt. Therefore there is an imperative need for countrywide surveillances to monitor the prevalence of these superbugs with limited therapeutic options. Full article

Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viability Full article

Cholangiocarcinoma is the first most common cancer of the biliary tract. To date, surgical resection is the only potentially curative option, but it is possible only for a limited percentage of patients, and in any case survival rate is quite low. Moreover, cholangiocarcinoma is often chemotherapy-resistant, and the only drug with a significant benefit for patient’s survival is Gemcitabine. It is necessary to find new drugs or combination therapies to treat nonresectable cholangiocarcinoma and improve the overall survival rate of patients. In this work, we evaluate in vitro the antitumoral effects of Rigosertib, a multi-kinase inhibitor in clinical development, against cholangiocarcinoma EGI-1 cell lines. Rigosertib impairs EGI-1 cell viability in a dose- and time-dependent manner, reversibility is dose-dependent, and significant morphological and nuclear alterations occur. Moreover, Rigosertib induces the arrest of the cell cycle in the G2/M phase, increases autophagy, and inhibits proteasome, cell migration, and invasion. Lastly, Rigosertib shows to be a stronger radiosensitizer than Gemcitabine and 5-Fluorouracil. In conclusion, Rigosertib could be a potential therapeutic option, alone or in combination with radiations, for nonresectable patients with cholangiocarcinoma. Full article

The estimated glycemic index (eGI) value of adzuki bean powder prepared by steamed cooking (SC), extruded cooking (EC) and roller cooking (RC) was studied comparatively. Results showed that RC had the highest eGI, with 80.1, and both EC and SC resulted in a lower eGI value of 70.0 and 49.7, respectively. Compared with the EC and RC methods, the SC method provided a more intact physical barrier for starch digestion, resulting in a less destroyed cell structure. As the essential components that form the cell wall, the study further investigated the effects of protein and fiber on physicochemical properties, in vitro starch digestibility and the eGI of adzuki bean powder processed with the SC method. Viscozyme and Protamax were used to obtain the deprotein and defiber samples. Results showed that the SC treatment with Viscozyme and Protamax, respectively, had significant effects on in vitro starch digestibility. The eGI of different samples were given as follows: steamed cooking adzuki bean powder (49.7) < deproteined adzuki bean powder (60.5) < defibered adzuki bean powder (83.1), which indicates that fiber may have a greater influence on the eGI than protein. Full article

In Arabidopsis thaliana, the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) gene encodes a thylakoid membrane-localized protease involved in chloroplast development in leaf mesophyll cells. Recently, EGY1 was also found to be crucial for the maintenance of grana in mesophyll chloroplasts. To further explore the function of EGY1 in leaf tissues, we examined the phenotype of chloroplasts in the leaf epidermal guard cells and pavement cells of two ⁴⁰Ar¹⁷⁺ irradiation-derived mutants, Ar50-33-pg1 and egy1-4. Fluorescence microscopy revealed that fully expanded leaves of both egy1 mutants showed severe chlorophyll deficiency in both epidermal cell types. Guard cells in the egy1 mutant exhibited permanent defects in chloroplast formation during leaf expansion. Labeling of plastids with CaMV35S or Protodermal Factor1 (PDF1) promoter-driven stroma-targeted fluorescent proteins revealed that egy1 guard cells contained the normal number of plastids, but with moderately reduced size, compared with wild-type guard cells. Transmission electron microscopy further revealed that the development of thylakoids was impaired in the plastids of egy1 mutant guard mother cells, guard cells, and pavement cells. Collectively, these observations demonstrate that EGY1 is involved in chloroplast formation in the leaf epidermis and is particularly critical for chloroplast differentiation in guard cells. Full article