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Ahmed, E. T. M., M. Hassan, R. N. Shamma, Amna Makky, and D. H. Hassan, "Controlling the Evolution of Selective Vancomycin Resistance through Successful Ophthalmic Eye-Drop Preparation of Vancomycin-Loaded Nanoliposomes Using the Active-Loading Method", Pharmaceutics, vol. 15, issue 6, 2023. Abstract

Vancomycin is the front-line defense and drug of choice for the most serious and life-threatening methicillin-resistant Staphylococcus aureus (MRSA) infections. However, poor vancomycin therapeutic practice limits its use, and there is a consequent rise of the threat of vancomycin resistance by complete loss of its antibacterial activity. Nanovesicles as a drug-delivery platform, with their featured capabilities of targeted delivery and cell penetration, are a promising strategy to resolve the shortcomings of vancomycin therapy. However, vancomycin’s physicochemical properties challenge its effective loading. In this study, we used the ammonium sulfate gradient method to enhance vancomycin loading into liposomes. Depending on the pH difference between the extraliposomal vancomycin–Tris buffer solution (pH 9) and the intraliposomal ammonium sulfate solution (pH 5–6), vancomycin was actively and successfully loaded into liposomes (up to 65% entrapment efficiency), while the liposomal size was maintained at 155 nm. Vancomycin-loaded nanoliposomes effectively enhanced the bactericidal effect of vancomycin; the minimum inhibitory concentration (MIC) value for MRSA decreased 4.6-fold. Furthermore, they effectively inhibited and killed heteroresistant vancomycin-intermediate S.aureous (h-VISA) with an MIC of 0.338 μg mL−1. Moreover, MRSA could not develop resistance against vancomycin that was loaded into and delivered by liposomes. Vancomycin-loaded nanoliposomes could be a feasible solution for enhancing vancomycin’s therapeutic use and controlling the emerging vancomycin resistance.

Ali, N. B., R. A. El-Shiekh, R. M. Ashour, S. H. El-Gayed, E. Abdel-Sattar, and M. Hassan, "In Vitro and In Vivo Antibiofilm Activity of Red Onion Scales: An Agro-Food Waste", Molecules, vol. 28, issue 1, 2023. Abstract

Red onion wastes (ROW) are valuable sources of bioactive metabolites with promising antimicrobial effects. Methicillin-resistant Staphylococcus aureus (MRSA) infections are a growing risk in hospitals and communities. This study aims to investigate the in vitro and in vivo antibiofilm activities of the acidified ethanolic extract of red onion scales (RO-T) and its fractions against an MRSA vaginal colonization model. The RO-T extract, as well as its anthocyanin-rich fraction (RO-P) and flavonoid-rich fraction (RO-S), recorded a promising antibacterial activity against highly virulent strains of bacteria (MRSA, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa). RO-S showed the highest antibacterial activity (MBC of 0.33 ± 0.11 mg/mL) against MRSA USA300 and significantly eradicated its biofilm formation with an IC50 of 0.003. Using a rat model, in vivo assessment on all samples, which were formulated as a hydrogel, revealed a significant reduction of MRSA bacterial load recovered from an infected vagina compared to that of the negative control group (NCG). RO-T extract and vancomycin groups recorded the highest antibacterial activity with a bacterial load 2.998 and 3.358 logs lower than the NCG, respectively. The histopathological investigation confirmed our findings. RO-T and RO-S were standardized for their quercetin content. Finally, ROW offers a new potent antibiofilm agent mostly due to its high quercetin content.

El-Naggar, M. M., M. A. El-Nabarawi, M. H. Teaima, M. Hassan, M. I. A. Hamed, A. A. Elrashedy, and rofida albash, Integration of terpesomes loaded Levocetrizine dihydrochloride gel as a repurposed cure for Methicillin-Resistant Staphylococcus aureus (MRSA)-Induced skin infection; D-optimal optimization, ex-vivo, in-silico, and in-vivo studies, , vol. 633, pp. 122621, 2023. AbstractWebsite

The intention of this work is to assess the repurposed antimicrobial impact of Levocetirizine dihydrochloride (LVC), which is a well-known antihistaminic drug, in addition, to augment the antimicrobial effect by using terpene-enriched vesicles (TPs). To investigate how various parameters affect TPs aspects, TPs were made employing the ethanol-injection-method and optimized d-optimal design. The TPs were characterized based on their entrapment efficiency percentage (EE%), particle size (PS), polydispersity index (PDI), and zeta potential (ZP). The optimum TP was submitted to more examinations. The optimum TP (TP12) showed a spherical vesicle having an EE% of 66.39 ± 0.12%, PS of 243.3 ± 4.60 nm, PDI of 0.458 ± 0.003, and ZP of 24.2 ± 0.55 mV. The in-vitro release study results demonstrated that LVC is sustainedly liberated from the optimum TP compared to LVC-solution. The ex-vivo assessment showed that LVC was released in a more sustained manner from TPs-gel related to LVC solution, optimum TP, and LVC gel. Ex-vivo visualization by confocal laser scanning microscopy showed good deposition of the fluorescein-labeled TP. Further, the in-vitro anti-bacterial effect and biofilm inhibition and detachment assessment confirmed the potency of LVC against Methicillin-resistant-Staphylococcus-aureus (MRSA). The in-silico study demonstrated that the LVC has excellent stability with other ingredients combined with it in the TPs, further, it proved that LVC is a potential candidate for treating MRSA. In-vivo assessments revealed a good antimicrobial effect toward MRSA infection. Moreover, the histopathological evaluation confirmed the safety of using TPs-gel topically. In conclusion, MRSA-related skin infections may be treated using the LVC loaded TPs-gel as a promising system.

Ibrahim, N. M., S. H. Fahim, M. Hassan, A. E. Farag, and H. H. Georgey, Design and synthesis of ciprofloxacin-sulfonamide hybrids to manipulate ciprofloxacin pharmacological qualities: Potency and side effects, , vol. 228, pp. 114021, 2022. AbstractWebsite

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections. Although being potent, susceptibility to CNS side effects limits their use. It was observed that improvements in absorption, activity and side effects were achieved via modifications at the N atom of the C7 of the side chain. To meet the increasing demand for development of new antibacterial agents, nineteen novel ciprofloxacin-sulfonamide hybrid molecules were designed, synthesized and characterized by IR, 1H NMR and 13C NMR as potential antibacterial agents with dual DNA gyrase/topoisomerase IV inhibitory activity. Most of the synthesized compounds showed significant antibacterial activity that was revealed by testing their inhibitory activity against DNA gyrase, DNA topoisomerase IV as well as their minimum inhibitory concentration against Staphylococcus aureus. Six ciprofloxacin-sulfonamide hybrids (3f, 5d, 7a, 7d, 7e and 9b) showed potent inhibitory activity against DNA topoisomerase IV, compared to ciprofloxacin (IC50: 0.55 μM), with IC50 range: 0.23–0.44 μM. DNA gyrase was also efficiently inhibited by five ciprofloxacin-sulfonamide hybrids (3f, 5d, 5e, 7a and 7d) with IC50 range: 0.43–1.1 μM (IC50 of ciprofloxacin: 0.83 μM). Compounds 3a and 3b showed a marked improvement in the antibacterial activity over ciprofloxacin against both Gram-positive and Gram-negative pathogens, namely, Staphylococcus aureus Newman and Escherichia coli ATCC8739, with MIC = 0.324 and 0.422 μM, respectively, that is 4.2-fold and 3.2-fold lower than ciprofloxacin (MIC = 1.359 μM) against the Gram-positive Staphylococcus aureus, and MIC = 0.025 and 0.013 μM, respectively, that is 10.2-fold and 19.6-fold lower than ciprofloxacin (MIC = 0.255 μM) against the Gram-negative Escherichia coli ATCC8739. Also, the most active compounds showed lower CNS and convulsive side effects compared to ciprofloxacin with a concomitant decrease in GABA expression.

Mehana, N. A., H. R. ghaiad, M. Hassan, Y. A. Elsabagh, S. Labib, and M. A. Abd-Elmawla, LncRNA MEG3 regulates the interplay between Th17 and Treg cells in Behçet's disease and systemic lupus erythematosus, , vol. 309, pp. 120965, 2022. AbstractWebsite

BackgroundBehçet's disease (BD) and systemic lupus erythematosus (SLE) are two autoimmune inflammatory diseases of indefinite etiology. However, up till now, no study has explored the exact regulatory mechanisms of lncRNA maternally expressed gene-3 (MEG3) over the balance between regulatory T-cells (Treg) and T helper-17 (Th17) cells in BD and SLE.
The current study aimed to investigate the role of lncRNA MEG3 in the interplay between the anti-inflammatory Treg/transcription factor forkhead box P3 (FOXP3) axis versus the pro-inflammatory Th17/retinoic acid orphan receptor-γt (RORγt) axis.
Main methods
100 subjects, 35 with BD and 35 with SLE in addition to 30 healthy participants were included in the study. Gene expression analysis was performed and ShinyGO database was utilized for in-depth analysis and graphical visualization of the gene ontology (GO) and pathway enrichment analysis for lncRNA and the other target genes.
Key findings
The current results demonstrate the upregulation of lncRNA MEG3 in BD but not SLE patients. Moreover, significant differences in RORγt and FOXP3 were found between BD and SLE patients. The present findings linked lncRNA MEG3 to BD activity scores as well as CRP levels. Finally, lncRNA MEG3 showed excellent diagnostic power for BD, in addition to adequate discriminative power that can be used to differentiate between BD and SLE.
The current study objectively elucidated a framework for the involvement of Treg/Th17 through transcription factors RORγt and FOXP3, in addition to their links to the downstream cytokines network including TGF-ꞵ, IL-10, IL-17 and IL-23 in BD and SLE pathogenesis and activity.

Sedeek, M. S., S. M. Afifi, M. K. Mansour, M. Hassan, F. M. Mehaya, I. A. Naguib, M. A. S. Abourehab, and M. A. Farag, "Unveiling Antimicrobial and Antioxidant Compositional Differences between Dukkah and Za’atar via SPME-GCMS and HPLC-DAD", Molecules, vol. 27, issue 19, 2022. Abstract

Interest in plant-based diets has been on the rise in recent years owing to the potential health benefits of their individual components and the notion that plant-based diets might reduce the incidence of several diseases. Egyptian dukkah and Syrian za’atar are two of the most historic and famous Middle Eastern herbal blends used for their anti-inflammatory, hypolipidemic, and antidiabetic effects. Headspace SPME-GCMS and HPLC-DAD were adopted for characterizing the aroma profile and phenolic compounds of both herbal blends, respectively. Further, vapor-phase minimum inhibitory concentration was employed for assessing each blend’s antibacterial potential, while their antioxidant potential was estimated via in vitro antioxidant assays. SPME headspace analysis indicated the abundance of ethers and monoterpene hydrocarbons, while HPLC revealed the presence of several phenolics including rosmarinic acid, ferulic acid, and rutin. Biological investigations affirmed that vapor-phase of the tested blends exhibited antibacterial activities against Gram-positive and Gram-negative pathogens, while the antioxidant potential of the blends was investigated and expressed as Trolox (125.15 ± 5.92 to 337.26 ± 13.84 μM T eq/mg) and EDTA (18.08 ± 1.62 to 51.69 41 ± 5.33 μM EDTA eq/mg) equivalent. The presented study offers the first insight into the chemical profile and biological activities of both dukkah and za’atar.

Al-mahallawi, A. M., D. Ahmed, M. Hassan, and D. A. El-Setouhy, Enhanced ocular delivery of clotrimazole via loading into mucoadhesive microemulsion system: In vitro characterization and in vivo assessment, , vol. 64, pp. 102561, 2021. AbstractWebsite

This work aimed to formulate clotrimazole (CLZ), a water-insoluble antifungal drug, into a chitosan-coated microemulsion system for achieving enhanced ocular delivery. In this study, CLZ loaded microemulsions were firstly prepared according to 22 × 31 full factorial design in order to investigate the influence of different formulation variables on microemulsion properties. The selected microemulsion formulation (F4: oleic acid, Cremophor EL: Transcutol HP (1:1) and water (20, 70 and 10%, w/w, respectively)) showed nanosized spherical globules with a droplet size of 229.1 ± 0.989 nm, polydispersity index of 0.5085 ± 0.0095, and zeta potential of −33.3 ± 0.98 mV. The selected microemulsion was then coated with low molecular weight chitosan to increase the contact time with the eye surface. In vivo studies in albino rabbits demonstrated the superiority of chitosan-coated microemulsion over the uncoated microemulsion and drug suspension formulation concerning sustainment of antifungal activity over the eye surface. Moreover, the in vivo, ocular tolerance and histopathological studies conducted using male albino rabbits proved the safety of the prepared microemulsions after topical ocular application. Generally, the obtained results confirmed that CLZ chitosan-coated microemulsion could be promising for ocular CLZ delivery.

Elzeini, H. M., A. R. A. A. Ali, M. Hassan, N. F. Nasr, A. A. M. Hassan, and Y. E. Elenany, " Probiotic capability of novel lactic acid bacteria isolated from worker honey bees gut microbiota", FEMS Microbiology Letters, 2021.
Ali, S., M. Hassan, T. Essam, M. Ibrahim, and K. Elamry, "Biodegradation of aflatoxin by bacterial species isolated from poultry farms", Toxicon, vol. 195, pp. 7-16, 2021.
El-Shiekh, R. A., M. Hassan, R. A. Hashem, and E. Abd-Elsattar, "Bioguided Isolation of Antibiofilm and Antibacterial Pregnane Glycosides from Caralluma quadrangula: Disarming Multidrug-Resistant Pathogens", Antibiotics, vol. 10, issue 8, pp. 1-12, 2021.
Albash, R., A. M. Al-mahallawi, M. Hassan, and A. A. Alaa-Eldin, "Development and Optimization of Terpene-Enriched Vesicles (Terpesomes) for Effective Ocular Delivery of Fenticonazole Nitrate: In vitro Characterization and in vivo Assessment", International Journal of Nanomedicine, vol. 16, pp. 609—621, 2021.
Ismail, M. M., M. Hassan, S. S. Moawad, M. O. N. A. M. OKBA, R. M. Ashour, N. m Fayek, and F. R. Saber, "Exploring the Antivirulence Activity of Pulverulentone A, a Phloroglucinol-Derivative from Callistemon citrinus Leaf Extract, against Multi-Drug Resistant Pseudomonas aeruginosa", Antibiotics, vol. 10, issue 8, pp. 1-12, 2021.
Ezzat, M. I., M. Hassan, M. A. Abdelhalim, A. M. El-Desoky, S. O. Mohamed, and S. M. Ezzat, "Immunomodulatory effect of Noni fruit and its isolates: insights into cell-mediated immune response and inhibition of LPS-induced THP-1 macrophage inflammation", Food & Function, 2021. d0fo03402a.pdf
Salem, M. A., R. A. El-Shiekh, R. A. Hashem, and M. Hassan, "In vivo Antibacterial Activity of Star Anise (Illicium verum Hook.) Extract Using Murine MRSA Skin Infection Model in Relation to Its Metabolite Profile", Infection and Drug Resistance, vol. 14, pp. 33—48, 2021. Website
Fahmy, A. M., M. Hassan, D. A. El-Setouhy, S. A. Tayel, and A. M. Al-mahallawi, "Statistical optimization of hyaluronic acid enriched ultradeformable elastosomes for ocular delivery of voriconazole via Box-Behnken design: in vitro characterization and in vivo evaluation.", Drug Delivery, vol. 28, issue 1, pp. 77-86, 2021.
Albash, R., M. M. Abdellatif, M. Hassan, and N. M. Badawi, "Tailoring Terpesomes and Leciplex for the Effective Ocular Conveyance of Moxifloxacin Hydrochloride (Comparative Assessment): In-vitro, Ex-vivo, and In-vivo Evaluation", International Journal of Nanomedicine, vol. 16, pp. 5247—5263, 2021.
Fahmy, A. M., M. Hassan, D. A. El-Setouhy, S. A. Tayel, and A. M. Al-mahallawi, "Voriconazole Ternary Micellar Systems for the Treatment of Ocular Mycosis: Statistical Optimization and In Vivo Evaluation", Journal of Pharmaceutical Sciences , vol. 17, 2021.
Hassan, A., M. Hassan, N. F. Nasr, A. Ali, Y. Elenany, and H. Elzeini, "Probiotic capabilities of lactic acid bacteria isolated from the gut microbiota of honey bee Apis mellifera L", NGU - Biologics & Biosimilars Symposium, Giza, Egypt, March, 2020.
Abd-Elmawla, M. A., M. Hassan, Y. A. Elsabagh, A. R. L. R. Alnaggar, and M. A. Senousy, "Deregulation of long noncoding RNAs ANCR, TINCR, HOTTIP and SPRY4-IT1 in plasma of systemic sclerosis patients: SPRY4-IT1 as a novel biomarker of scleroderma and its subtypes", Cytokine, vol. 133, pp. 155124, 2020. AbstractWebsite

Systemic sclerosis or systemic scleroderma (SSc) is an inflammatory autoimmune disease whose pathogenesis remains ambiguous; however, epigenetics, including long noncoding RNAs (lncRNAs) is an emerging paradigm. To date, the expression, role and clinical significance of most lncRNAs in SSc remain unelucidated. Herein, we investigated the plasma expression profiles of lncRNAs; ANCR, TINCR, HOTTIP, and SPRY4-IT1, which were linked to skin biology, in SSc patients and its subtypes, their potential as diagnostic tools and their correlations with autoantibodies and disease manifestations. Sixty-three SSc patients and thirty-five healthy volunteers were recruited. Autoantibody profile (anti-Scl-70, anti-centromere, anti-RNA polymeraseIII, anti-ribonucleoprotein, antinuclear, and anti-phospholipid antibodies) was determined. lncRNAs analysis was conducted using RT-qPCR. Plasma TINCR, HOTTIP, and SPRY4-IT1 upregulation and ANCR downregulation were observed in SSc patients compared with controls. SPRY4-IT1 was superior in SSc diagnosis in ROC analysis and predicted its risk in multivariate logistic analysis. Plasma SPRT4-IT1 was higher in diffuse than limited SSc. SPRY4-IT1 and HOTTIP were positively correlated with modified Rodnan skin score while ANCR showed a negative correlation only in limited SSc. ANCR and TINCR were positively correlated with disease duration and ESR, respectively. ANCR and SPRY4-IT1 were positively correlated with pulmonary hypertension. HOTTIP was positively correlated with antinuclear antibody. SPRY4-IT1 was positively correlated with HOTTIP in the whole group, and with TINCR only in diffuse SSc. We introduce plasma SPRY4-IT1, HOTTIP, ANCR and TINCR as novel candidate biomarkers for SSc, with SPRY4-IT1 could predict SSc diagnosis and discriminate its subtypes. Our findings widen the epigenetic landscape of SSc and provide surrogates for future predictive studies.

Ali, S., M. Hassan, T. Essam, M. Ibrahim, and K. Elamry, "Coumarin degrading microorganisms isolated from Egyptian soil", Veterinary Medical Journal , vol. 66, issue 1, pp. 89-97, 2020. Abstract


Hassan, M., T. Essam, A. Mira, and S. Megahed, "Biomonitoring detoxification efficiency of an algal-bacterial microcosm system for treatment of coking wastewater: Harmonization between Chlorella vulgaris microalgae and wastewater microbiome", Science of The Total Environment, vol. 677, pp. 120 - 130, 2019. AbstractWebsite

Nowadays, due to worldwide water shortage, water utilities are forced to re-evaluate treated wastewater. Consequently, wastewater treatment plants need to conduct biomonitoring. Coking wastewater (CWW) has toxic, mutative and carcinogenic components with threatening effect on the environment. CWW was selected as a model for complex highly toxic industrial wastewater that should be treated. CWW from Egypt was treated in a nine-liter photobioreactor using an algal-bacterial system. The photobioreactor was operated for 154 days changing different parameters (toxic load and light duration) for optimization. Optimized conditions achieved significant reduction (45%) in the operation cost. The algal-bacterial system was monitored using chemical assays (chemical oxygen demand and phenol analysis), bioassays (phytotoxicity, Artemia-toxicity, cytotoxicity, algal-bacterial ratio and settleability) and Illumina-MiSeq sequencing of 16S rRNA gene. The algal-bacterial system detoxified (in terms of phytotoxicity, cytotoxicity and Artemia-toxicity) CWW introduced as influent through all phases. A significant difference was recorded in the microbial diversity between influent and effluent samples. Four phyla dominated influent samples; Proteobacteria (77%), Firmicutes (11%), Bacteroidetes (5%) and Deferribacteres (3%) compared to only two in effluent samples; Proteobacteria (66%) and Bacteroidetes (26%). The significant relative-abundance of versatile aromatic degraders (Comamonadaceae and Pseudomonadaceae families) in influent samples conformed to the nature of CWW. Microbial community shifted and promoted the activity of catabolically versatile and xenobiotics degrading families (Chitinophagaceae and Xanthomonadaceae). Co-culture of microalgae had a positive effect on the biodegrading bacteria that was reflected by enhanced treatment efficiency, significant increase in relative abundance of bacterial genera with cyanide-decomposing potential and negative effect on waterborne pathogens.

Hassan, M., T. Essam, and S. Megahed, "Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples", Brazilian Journal of Microbiology, vol. 49, issue 1, pp. 1-8, 2018.
Ismail, M. M., M. Hassan, and T. M. Essam, "Biological Testing and Toxicity Bioassays in Biodegradation: Toward Better Process Control", Toxicity and Biodegradation Testing, New York, NY, Springer New York, pp. 185 - 205, 2018, 2017. Abstract

The main purpose of bioremediation processes is to eliminate or even to reduce the risk of the toxic pollutants to reach safe and acceptable limits. Although environmental pollutants have complex nature and composition, most of the performed studies reported the application of methods for biodegradation of pollutants and xenobiotics in environment, especially wastewater, as a black box without considering sequestered risks. In this sense, chemical analysis alone is not sufficient for complete assessment of water quality while biological toxicity assays can estimate the effect of wastewater on the biota and assess the actual direct and sequestered environmental risks. This was the main driving force to teamwork in our lab to adopt, develop, and optimize biological testing methods and bioassays for better process control and real monitoring of the bioremediation efficiency. Several methods and bioassays were used such as BOD5 and algal toxicity (to assess the degree of wastewater toxicity), Artemia toxicity (to assess impact on the aquatic creatures), phytotoxicity (to assess the impact on terrestrial plants). Also endocrine disruptors’ assays and cytotoxicity (to assess the impact on mammalians and humans) were optimized. The authors discuss and transfer the knowledge and the step by step experience gained with these methods and bioassays. Moreover, this chapter elaborates all necessary practical tricks and precautions required to achieve accurate and reproducible measurements.

Hassan, M., T. Essam, and S. Megahed, "Community shift of water microbiome in an algal-bacterial based photo- bioreactor for wastewater treatment", 9th Probiotics, Prebiotics & New Foods, Nutraceuticals and Botanicals for Nutrition & Human and Microbiota Health, Rome, Italy, 10 September , 2017.