Augmented Therapeutic Potential of EC-Synthetic Retinoids in Caco-2 Cancer Cells Using an In Vitro Approach.

Colorectal cancer therapies have produced promising clinical responses, but tumor cells rapidly develop resistance to these drugs. It has been previously shown that EC19 and EC23, two EC-synthetic retinoids, have single-agent preclinical anticancer activity in colorectal carcinoma. Here, isobologram analysis revealed that they have synergistic cytotoxicity with retinoic acid receptor (RAR) isoform-selective agonistic retinoids such as AC261066 (RARß2-selective agonist) and CD437 (RARγ-selective agonist) in Caco-2 cells. This synergism was confirmed by calculating the combination index (lower than 1) and the dose reduction index (higher than 1). Flow cytometry of combinatorial IC50 (the concentration causing 50% cell death) confirmed the cell cycle arrest at the SubG0-G1 phase with potentiated apoptotic and necrotic effects. The reported synergistic anticancer activity can be attributed to their ability to reduce the expression of ATP-binding cassette (ABC) transporters including P-glycoprotein (P-gp1), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein-1 (MRP1) and Heat Shock Protein 70 (Hsp70). This adds up to the apoptosis-promoting activity of EC19 and EC23, as shown by the increased Caspase-3/7 activities and DNA fragmentation leading to DNA double-strand breaks. This study sheds the light on the possible use of EC-synthetic retinoids in the rescue of multi-drug resistance in colorectal cancer using Caco-2 as a model and suggests new promising combinations between different synthetic retinoids. The current in vitro results pave the way for future studies on these compounds as possible cures for colorectal carcinoma.

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion.

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89-230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

Chitinase 3-like-1, Tolloid-like protein 1, and intergenic gene polymorphisms are predictors for hepatocellular carcinoma development after hepatitis C virus eradication by direct-acting antivirals.

Hepatocellular carcinoma (HCC) is a major cause of cancer death in Egypt. There is still a risk for HCC development even after eradicating hepatitis C virus (HCV) by direct-acting antivirals (DAAs). Chitinase-3-like-protein-1 (CHI3L1), a biomarker for predicting many diseases, plays an essential role in inflammation, angiogenesis, and antiapoptosis. Tolloid-like protein 1 (TLL1) may be involved in hepatic fibrogenesis and carcinogenesis. This study aimed to determine the role and combined effect of CHI3L1 (rs880633), TLL1 (rs1503298), and an intergenic (rs597533) polymorphisms on the risk of developing HCC in Egyptian patients after achieving sustained virological response (SVR) by DAAs. Blood samples were collected from 68 HCC patients, 77 non-HCC subjects, and 80 healthy controls. The DNA was extracted and analyzed for rs880633, rs1503298, and rs597533 using Genotyping TaqMan™ assay. The result of the present study showed a significant difference in genotypes and alleles frequencies in both (rs880633) and (rs597533) in HCC group as compared to healthy control and also as compared to the non-HCC group. However, regarding to (rs1503298) genotypes and alleles between the HCC and non-HCC groups, there were no significant differences. Combined polymorphism in more than one gene simultaneously showed a higher risk to HCC after SVR than an individual locus. Both allelic and genotypic variations of the CHI3L1 gene (rs880633) and an intergenic (rs597533) seemed to be significant predictors confirming a great risk for HCC susceptibility in Egyptian patients achieved SVR. Patients with a polymorphism in more than one gene showed an increased risk to HCC after SVR rather than individual locus.

Revealing the Potential Application of EC-Synthetic Retinoid Analogues in Anticancer Therapy.

(1) Background and Aim: All-trans retinoic acid (ATRA) induces differentiation and inhibits growth of many cancer cells. However, resistance develops rapidly prompting the urgent need for new synthetic and potent derivatives. EC19 and EC23 are two synthetic retinoids with potent stem cell neuro-differentiation activity. Here, these compounds were screened for their in vitro antiproliferative and cytotoxic activity using an array of different cancer cell lines. (2) Methods: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, AV/PI (annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)), cell cycle analysis, immunocytochemistry, gene expression analysis, Western blotting, measurement of glutamate and total antioxidant concentrations were recruited. (3) Results: HepG2, Caco-2, and MCF-7 were the most sensitive cell lines; HepG2 (ATRA; 36.2, EC19; 42.2 and EC23; 0.74 µM), Caco-2 (ATRA; 58.0, EC19; 10.8 and EC23; 14.7 µM) and MCF-7 (ATRA; 99.0, EC19; 9.4 and EC23; 5.56 µM). Caco-2 cells were selected for further biochemical investigations. Isobologram analysis revealed the combined synergistic effects with 5-fluorouracil with substantial reduction in IC50. All retinoids induced apoptosis but EC19 had higher potency, with significant cell cycle arrest at subG0-G1, -S and G2/M phases, than ATRA and EC23. Moreover, EC19 reduced cellular metastasis in a transwell invasion assay due to overexpression of E-cadherin, retinoic acid-induced 2 (RAI2) and Werner (WRN) genes. (4) Conclusion: The present study suggests that EC-synthetic retinoids, particularly EC19, can be effective, alone or in combinations, for potential anticancer activity to colorectal cancer. Further in vivo studies are recommended to pave the way for clinical applications.

Role of Diosmin in protection against the oxidative stress induced damage by gamma-radiation in Wistar albino rats.

The benchmark of this study is to evaluate the radio protective efficiency of diosmin, a natural citrus flavone of hesperidin derivative on radiation-induced damage in wistar albino rats. Rats orally administered two diosmin doses (100 and 200 mg/kg body wt.) for a month (every other day) prior to exposure to high gamma radiation single dose (8Gy) or cumulative dose (10Gy). To evaluate the radio protective efficiency of diosmin various biochemical estimations, histopathological alterations as well as comet assay and caspase-3 activity for assessment of apoptosis were performed. Results indicated that radiation-induced decline in the levels of antioxidant parameters (SOD and GSH), increased lipid peroxidation, DNA damage and apoptosis were improved by pre-administration of diosmin. Diosmin dose (200 mg/kg body wt.) restored the antioxidant status to near normal and reduced lipid peroxidation, DNA and tissue damage. These results were confirmed by histopathological examinations, which showed that pre-administration of diosmin protected the liver and kidney of albino rats against gamma-irradiation induced damage. Hence, it has been illustrated that diosmin might be an effective radio protector against radiation-induced damage in rats. Moreover, diosmin alone pretreated group did not show any biochemical alterations or DNA damage indicating the protective nature of the drug.

Inflammasome Genes' Polymorphisms in Egyptian Chronic Hepatitis C Patients: Influence on Vulnerability to Infection and Response to Treatment.

Chronic inflammation is a pivotal contributor to the liver damage mediated by hepatitis C virus (HCV). The NOD-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome is activated by HCV in both hepatocytes and Kupffer cells. The aim of our study was to investigate the association of nine single-nucleotide polymorphisms in four inflammasome genes (NLRP3, CARD8, IL-1ß, and IL-18) with the susceptibility to HCV infection and outcome of interferon treatment in 201 Egyptian chronic hepatitis C patients and 95 healthy controls. The genotyping was conducted using TaqMan predesigned SNP assay. In the comparative analysis, the CC genotype of the NLRP3 rs1539019 was found to be associated with the lower risk to chronic HCV infection (OR: 0.33, 95% CI: 0.17-0.62). This association was also found for the CA genotype and the A allele of the NLRP3 rs35829419 (OR: 0.18 and 0.22, respectively), in addition to the GG genotype and G allele of IL-18 rs1946518 (OR: 0.55 and 0.61, respectively). In contrast, the AA genotype of the IL-1ß rs1143629 was significantly more frequent in HCV patients (OR: 1.7, 95% CI: 1-2.86). Notably, the frequency of the AA genotype of NLRP3 rs1539019 was significantly higher in patients with lack of response (NR) to the interferon treatment (OR: 1.95, 95% CI: 1-3.7). A similar association was found for both the CC genotype and C allele of the NLRP3 rs35829419 (OR: 2.78 and 2.73, respectively) and for the TT genotype and T allele of CARD8 rs2043211 (OR: 2.64 and 1.54, respectively). Yet, the IL-1ß (rs1143629, rs1143634) and IL-18 (rs187238, rs1946518) polymorphisms did not show any significant association with response to interferon treatment. In conclusion, this study reports, for the first time, the association of genetic variations in NLRP3 with hepatitis C susceptibility and response to treatment in Egyptian patients. However, further large-scale studies are recommended to confirm our findings.

Design, synthesis, assessment, and molecular docking of novel pyrrolopyrimidine (7-deazapurine) derivatives as non-nucleoside hepatitis C virus NS5B polymerase inhibitors.

Hepatitis C virus (HCV) infection is highly persistent and presents an unmet medical need requiring more effective treatment options. This has spurred intensive efforts to discover novel anti-HCV agents. The RNA-dependent RNA polymerase (RdRp), NS5B of HCV, constitutes a selective target for drug discovery due to its absence in human cells; also, it is the centerpiece for viral replication. Here, we synthesized novel pyrrole, pyrrolo[2,3-d]pyrimidine and pyrrolo[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives. The non-toxic doses of these compounds on Huh 7.5 cell line were determined and their antiviral activity against HCVcc genotype 4a was examined. Compounds 7j, 7f, 5c, 12i and 12f showed significant anti HCV activity. The percent of reduction for the non-toxic doses of 7j, 7f, 5c, 12i and 12f were 90%, 76.7±5.8%, 73.3±5.8%, 70% and 63.3±5.8%, respectively. The activity of these compounds was interpreted by molecular docking against HCV NS5B polymerase enzyme.

Novel antiviral compounds against gastroenteric viral infections.

Viral gastroenteritis is a serious viral infection which affects a large number of individuals around the world, most of them being children. The infection may occur due to different viruses, for example, coxsackievirus, adenovirus, and rotavirus. There is no available cure for such infections, and the treatment mainly depends on hospitalization and administration of nutritional supports. A new antiviral agent against gastroenteritis viral infection will be a breakthrough in healthcare. Pyrrole and pyrrolopyrimidine derivatives are well known for their biological activity as antibacterial, antifungal, and anticancer agents. These compounds also proved to possess antiviral activity. Here, we synthesized novel pyrrole and pyrrolopyrimidine compounds and examined their antiviral activity. We synthesized several new pyrrole, pyrrolo[2,3-d]pyrimidine, and pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. The characterization of all synthesized compounds was based on microanalysis and spectral data. Moreover, we determined the non-toxic doses of these compounds on BGM, Hep-2, and MA-104 cells. We tested all the synthesized compounds for their antiviral activities against coxsackievirus B4, adenovirus type 7, and rotavirus Wa strain. Several compounds exhibited significant activities as antiviral agents.

Probing the N-terminal ß-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.

Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP(C), into its misfolded oligomeric form, known as prion or PrP(Sc). The canonical human PrP(C) (HuPrP) fold features an unstructured N-terminal part (residues 23-124) and a well-defined C-terminal globular domain (residues 125-231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113-120) plays an important role in the conversion to PrP(Sc). The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel ß-sheet with the ß1 and ß2 strands, demonstrating that the full-length HuPrP(C) can adopt a more elaborate ß0-ß1-α1-ß2-α2-α3 structural organization than the canonical ß1-α1-ß2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates ß-enrichment in the PrP(C) monomer as one of the early events in the conversion of PrP(C) into PrP(Sc).

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

Egypt Genome: Towards an African new genomic era.

BACKGROUND: Studying the human genome is crucial to embrace precision medicine through tailoring treatment and prevention strategies to the unique genetic makeup and molecular information of individuals. After human genome project (1990-2003) generated the first full sequence of a human genome, there have been concerns towards Northern bias due to underrepresentation of other populations. Multiple countries have now established national genome projects aiming at the genomic knowledge that can be harnessed from their populations, which in turn can serve as a basis for their health care policies in the near future. AIM OF REVIEW: The intention is to introduce the recently established Egypt Genome (EG) to delineate the genomics and genetics of both the modern and Ancient Egyptian populations. Leveraging genomic medicine to improve precision medicine strategies while building a solid foundation for large-scale genomic research capacity is the fundamental focus of EG. KEY SCIENTIFIC CONCEPTS: EG generated genomic knowledge is predicted to enrich the existing human genome and to expand its diversity by studying the underrepresented African/Middle Eastern populations. The insightful impact of EG goes beyond Egypt and Africa as it fills the knowledge gaps in health and disease genomics towards improved and sustainable genomic-driven healthcare systems for better outcomes. Promoting the integration of genomics into clinical practice and spearheading the implementation of genomic-driven healthcare and precision medicine is therefore a key focus of EG. Mining into the wealth of Ancient Egyptian Genomics to delineate the genetic bridge between the contemporary and Ancient Egyptian populations is another excitingly unique area of EG to realize the global vision of human genome.

Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy.

High-resolution structure of a papaya plant-defense barwin-like protein solved by in-house sulfur-SAD phasing.

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Šwavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central ß-barrel of six strands surrounded by several α-helices and loops. The ß-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

A novel expression system for production of soluble prion proteins in E. coli.

Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

High dose methotrexate in adult Egyptian patients with hematological malignancies: impact of ABCB1 3435C > T rs1045642 and MTHFR 677C > T rs1801133 polymorphisms on toxicities and delayed elimination.

High dose methotrexate (HDMTX) is an essential agent in chemotherapeutic regimens used in various hematological malignancies in Egyptian adults. The research for the impact of gene polymorphism on HDMTX induced toxicities and delayed elimination is an important ongoing objective in many studies, variable and conflicting results produced in the past years to clarify that impact. This study aimed to investigate the role of ABCB1 3435 C > T rs1045642 and MTHFR 677 C > T rs1801133 polymorphisms on HDMTX induced toxicity outcomes and delayed elimination in Egyptian adult patients with hematological malignancies. A prospective, observational cohort study was conducted on a total of 62 Egyptian adult patients with hematological malignancies age ≥ 18-years-old. All demographic, medical, and laboratory data were continuously collected from the patients' medical files in an up-to-date follow-up in selected clinics during the period from April 2018 to March 2020. Venous blood samples were collected for the purpose of genotyping, DNA extraction, and measurement of MTX levels. All the relevant data were statistically analyzed. The studied patients' median age was 25 years old with a range of (18-62) years. Forty-six patients were males with about 74%, and 16 were females with about 26%. Eighty-nine percent of the patients diagnosed with acute lymphoblastic leukemia 'ALL', 5% of the patients had B cell non-hodgkin lymphoma 'B-NHL' and 3% diagnosed with primary central nervous system lymphoma 'PCNSL' and Burkitt's lymphoma 'BL' Hematological, hepatic, renal and gastrointestinal toxicities observed post-HDMTX were recorded with the hematological toxicities toping on all the others, also patients with delayed elimination at 72 hours post the HDMTX dose were determined. Statistical analysis revealed a significant association between ABCB1 3435 C > T rs1045642 and HDMTX delayed elimination with about 10 times higher risk among the minor allele 'T' carriers (p-value = 0.006) (odds ratio [OR]: 10.470; 95% CI: 1.961-55.904). No significant association observed between the studied gene polymorphisms: MTHFR 677 C > T rs1801133, ABCB1 3435 C > T rs1045642, and different toxicity outcomes. According to our best knowledge, this study is the first to conclude a significant association between ABCB1 3435 C > T rs1045642 gene polymorphism and HDMTX delayed elimination at 72 hours post HDMTX infusion; also, it is the first study to analyze the association between ABCB1 3435 C > T rs1045642 polymorphism with HDMTX toxicity and delayed elimination in adult Egyptian patients with hematological malignancies.

Crystallization and preliminary X-ray diffraction analysis of kanamycin-binding ß-lactamase in complex with its ligand.

TEM-1 ß-lactamase is a highly efficient enzyme that is involved in bacterial resistance against ß-lactam antibiotics such as penicillin. It is also a robust scaffold protein which can be engineered by molecular-evolution techniques to bind a variety of targets. One such ß-lactamase variant (BlaKr) has been constructed to bind kanamycin (kan) and other aminoglycoside antibiotics, which are neither substrates nor ligands of native ß-lactamases. In addition to recognizing kan, BlaKr activity is up-regulated by its binding via an activation mechanism which is not yet understood at the molecular level. In order to fill this gap, determination of the structure of the BlaKr-kan complex was embarked upon. A crystallization condition for BlaKr-kan was identified using high-throughput screening, and crystal growth was further optimized using streak-seeding and hanging-drop methods. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.01, b = 72.33, c = 74.62 Å, and diffracted to 1.67 Šresolution using synchrotron radiation. The X-ray structure of BlaKr with its ligand kanamycin should provide the molecular-level details necessary for understanding the activation mechanism of the engineered enzyme.

A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction.

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca(2+)-activated gelsolin (K (d) approximately 10 nM) while GsnVHH 11 binds gelsolin irrespective of Ca(2+) (K (d) approximately 5 nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.

Interrupted Crosstalk between Natural Killer Cells and Anti-epidermal Growth Factor Receptor: A Possible Role in Hepatocellular Carcinoma Treatment Failure.

Hepatocellular carcinoma (HCC) is a major health problem worldwide. Most patients are diagnosed for the first time at late stages, which leads to very poor prognosis. It is challenging to discover strategies for treatment at these advanced stages. Recently, monoclonal antibodies (mAbs) targeting specific cellular signaling pathways in HCC have been developed. Unfortunately, they still have a low survival rate, and some of them failed clinically to produce effective responses even if they showed very good results against HCC in preclinical studies. This review focuses on and discusses the possible causes for the failure of mAbs, precisely anti-Epidermal Growth Factor Receptor (EGFR) mAb and the crosstalk between this mAb and patients' NK cells.

Restoring NK cells functionality via cytokine activation enhances cetuximab-mediated NK-cell ADCC: A promising therapeutic tool for HCC patients.

Natural Killer (NK) cells are considered the first line of defense against viral infections and tumors. Several factors affect NK cytotoxic activity rendering it dysfunctional and thereby impeding the ability to scavenge abnormal cells as a part of immune escaping mechanisms induced by different types of cancers. NK cells play a crucial role augmenting the activity of various types of anticancer mAb since dysfunctional NK cells are the main reason for the low response to these therapies. To this light, we examined the phenotypic characters of the circulating NK cells isolated from HCC patients compared to healthy controls. Then, dysfunctional NK cells, from HCC patients, were reactivated with cytokines cocktail and their cytotoxic activity with the anti-EGFR mAb "cetuximab" was investigated. This showed a downregulation of patients NK cells activating receptors (NKP30, NKP46, NKG2D and CD16) as well as CD56 and up-regulation of NKG2A inhibitory receptor. We also reported an increase in aberrant CD56- NK cells subset in peripheral blood of HCC patients compared to healthy controls. Thus, confirming the dysfunctionality of peripheral NK cells isolated from HCC patients. Cytokines re-activation of those NK cells lead to upregulation of NK activating receptors and downregulation of inhibitory receptor. Moreover, the percentage of aberrant CD56- NK cells subset was reduced. Here, we proved that advanced HCC patients have an increased percentage of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for the low cytotoxicity noticed in those patients. A significant improvement in the cytotoxicity against HCC was noticed upon using reactivated NK cells combined with cetuximab. Therefore, this study highlights the potential recruitment of NK immune cells along with cetuximab to enhance cytotoxicity against HCC.

Funding for health policy and systems research in the Eastern Mediterranean region: amount, source and key determinants.

OBJECTIVES: To provide an overview of the current state of funding for health policy and systems research (HPSR) on a national level across the Eastern Mediterranean region (EMR), and to examine the key factors influencing funding for HPSR in the region. METHODS: A multistep approach was employed, involving a documentation review, secondary data analysis and key informant interviews with 30 stakeholders from five countries in the EMR. Findings are presented narratively (and where applicable as percentages). RESULTS: National funding for research and development (R&D) in general, and for health research in particular, has been low in comparative terms and lagging behind at the global scale, while funding for HPSR has been lacking on a national level. None of the 22 EMR countries studied had explicit national funding or a budget line for HPSR. Analysis of funding sources of 1821 published HPSR articles in the EMR (2010-2019) showed that the most notable source was external/international grants (45.6%), followed by university/academia (35.1%), and government (9.5%). Although HPSR publications have been increasing over time, this still falls short of the scale needed for strengthening health systems and informing current transformations in the region. Findings from the interviews identified several factors influencing investment in or funding for HPSR in the EMR. CONCLUSIONS: Many of the EMR's policy priorities are related to health systems, however our research finds that overall investment in health research and HPSR is still low, with limited recognition of the importance of HPSR in the EMR.