The impact of hepatitis B virus and hepatitis C virus infections in patients with Hodgkin's and non-Hodgkin's lymphoma.

BACKGROUND: This study aimed to determine the prevalence of HCV and occult HBV among newly diagnosed pre-treatment Egyptian lymphoma patients and evaluate patients' outcomes based on the presence of the viral infections. METHODS: The study included 80 therapy-naïve lymphoma patients including 71 non-Hodgkin lymphoma (NHL) and 9 Hodgkin lymphoma disease (HD) in addition to 100 healthy volunteers. HBV screening using HBsAg and anti-HBc IgM and HCV using AB/Ag ELISA and real-time RT-PCR were screened in tested and control groups. The diagnosis was confirmed by histopathology. Overall survival (OS) and progression-free survival (PFS) were conducted to diseased patients. RESULTS: Healthy patients showed 4/100, (4%) active HCV infection and 1/100, (1%) active HBV infection and no occult HBV infection. Among NHL patients, 28 were positive for HBV (6 active and 22 occult HBV infection). Occult HBV was also detected in 5/9 HD patients. HCV was detected in (30/71, 42.3%) of NHL patients and in a single HD patient. Ten occult HBV NHL patients showed a mixed infection with HCV. The incidence of both HCV and HBV are higher in NHL than HL patients. After antitumor treatment, complete remission for lymphoma was achieved in 45% of patients. Both overall survival (OS) and progression-free survival (PFS) were correlated and significantly associated with patients' LDH levels. CONCLUSIONS: Our findings claim the suggestive role of HCV and occult HBV infections in NHL but not HL patients in comparison to healthy control, suggesting pre-screening of related factors including occult HBV in for potential better therapy response.

Surf Redfish-Based ZnO-NPs and Their Biological Activity with Reference to Their Non-Target Toxicity.

The marine environment is a rich source of bioactive compounds. Therefore, the sea cucumber was isolated from the Red Sea at the Al-Ain Al-Sokhna coast and it was identified as surf redfish (Actinopyga mauritiana). The aqueous extract of the surf redfish was utilized as an ecofriendly, novel and sustainable approach to fabricate zinc oxide nanoparticles (ZnO-NPs). The biosynthesized ZnO-NPs were physico-chemically characterized and evaluated for their possible antibacterial and insecticidal activities. Additionally, their safety in the non-target organism model (Nile tilapia fish) was also investigated. ZnO-NPs were spherical with an average size of 24.69 ± 11.61 nm and had a peak at 350 nm as shown by TEM and UV-Vis, respectively. XRD analysis indicated a crystalline phase of ZnO-NPs with an average size of 21.7 nm. The FTIR pattern showed biological residues from the surf redfish extract, highlighting their potential role in the biosynthesis process. DLS indicated a negative zeta potential (-19.2 mV) of the ZnO-NPs which is a good preliminary indicator for their stability. ZnO-NPs showed larvicidal activity against mosquito Culex pipiens (LC50 = 15.412 ppm and LC90 = 52.745 ppm) and a potent adulticidal effect to the housefly Musca domestica (LD50 = 21.132 ppm and LD90 = 84.930 ppm). Tested concentrations of ZnO-NPs showed strong activity against the 3rd larval instar. Topical assays revealed dose-dependent adulticidal activity against M. domestica after 24 h of treatment with ZnO-NPs. ZnO-NPs presented a wide antibacterial activity against two fish-pathogen bacteria, Pseudomonas aeruginosa and Aeromonas hydrophila. Histopathological and hematological investigations of the non-target organism, Nile tilapia fish exposed to 75-600 ppm ZnO-NPs provide dose-dependent impacts. Overall, data highlighted the potential applications of surf redfish-mediated ZnO-NPs as an effective and safe way to control mosquitoes, houseflies and fish pathogenic bacteria.

Antifungal Activity of Biosynthesized Silver Nanoparticles (AgNPs) against Aspergilli Causing Aspergillosis: Ultrastructure Study.

Currently, nanoparticles and nanomaterials are widely used for biomedical applications. In the present study, silver nanoparticles (AgNPs) were successfully biosynthesized using a cell-free extract (CFE) of Bacillus thuringiensis MAE 6 through a green and ecofriendly method. The size of the biosynthesized AgNPs was 32.7 nm, and their crystalline nature was confirmed by XRD, according to characterization results. A surface plasmon resonance spectrum of AgNPs was obtained at 420 nm. Nanoparticles were further characterized using DLS and FTIR analyses, which provided information on their size, stability, and functional groups. AgNPs revealed less cytotoxicity against normal Vero cell line [IC50 = 155 µg/mL]. Moreover, the biosynthesized AgNPs exhibited promising antifungal activity against four most common Aspergillus, including Aspergillus niger, A. terreus, A. flavus, and A. fumigatus at concentrations of 500 µg/mL where inhibition zones were 16, 20, 26, and 19 mm, respectively. In addition, MICs of AgNPs against A. niger, A. terreus, A. flavus, and A. fumigatus were 125, 62.5, 15.62, and 62.5 µg/mL, respectively. Furthermore, the ultrastructural study confirmed the antifungal effect of AgNPs, where the cell wall's integrity and homogeneity were lost; the cell membrane had separated from the cell wall and had intruded into the cytoplasm. In conclusion, the biosynthesized AgNPs using a CFE of B. thuringiensis can be used as a promising antifungal agent against Aspergillus species causing Aspergillosis.

First report on isolation of Mucor bainieri from honeybees, Apis mellifera: Characterization and biological activities.

Fungi are potential biocontrol agents and rich sources of secondary metabolites with demonstrated biological activities. This study aimed to isolate and identify fungi from surface-sterilized honeybees (Apis mellifera), as well as to evaluate their biological activities. One fungal isolate was obtained and identified morphologically and genetically as Mucor bainieri MK-Bee-2. Gas chromatography-mass spectroscopy (GC-MS) analysis of fungus crude extract, showed the existence of six major metabolites representing 92.48% of the total peak area. The crude extract of Mucor bainieri MK-Bee-2 was tested for antimicrobial, antioxidant, and antitumor activities. It demonstrated wide antimicrobial activities against human pathogenic Gram-positive and Gram-negative bacterial strains, as well as Candida albicans, with MIC values ranged from 62.5 to 250 µg/ml. The results revealed that the extract exhibited considerable antioxidant activities indicated by strong inhibition of both DPPH and ABTS free radicals. Additionally, the extract exhibited greater potential anticancer activity against both adenocarcinomic human non-small cell lung cancer cells (A549) [IC50 = 6.45 µg/ml], and immortal cell line hepatoma G2 (HepG2) human liver cancer cells [IC50 = 27.48 µg/ml] and higher selectivity in cancer cells than normal cell lines. Furthermore, the extract showed less cytotoxic activity against normal cells with higher IC50 values of 106.99 and 132.57 µg/ml against human lung fibroblast Wistar-38 (Wi-38) and oral epithelial cells (OEC), respectively. Taken together, the Mucor bainieri MK-Bee-2 extract comprises bioactive compounds as promising potential therapeutic candidates for the treatment of lung cancer. Strikingly, the extract sensitizes the lung cancer cells A549  to the ionizing radiation through the pro-apoptotic pathway as indicated by the annexin V flow cytometry analysis which showed that the extract reduced the apoptosis of lung cancer cells.

Photocatalytic and Antimicrobial Activities of Biosynthesized Silver Nanoparticles Using Cytobacillus firmus.

The toxicity of the ecosystem has increased recently as a result of the increased industrial wastewater loaded with organic contaminants, including methylene blue (MB), which exerts serious damage to the environment. Thus, the present work aims to green the synthesis of silver nanoparticles (Ag-NPs) and to evaluate their degradability of notorious MB dye, as well as their antimicrobial activities. Ag-NPs were synthesized by Cytobacillus firmus extract fully characterized by UV-vis, TEM, DLS, XRD, and FTIR. Ag-NPs showed good antibacterial and antifungal activities against Escherichia coli ATCC 25922, Enterococcus feacalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Candida albicans ATCC 90028. Moreover, Ag-NPs exhibited a high biodegradability level (98%) of MB dye after 8 h of co-incubation in the presence of sunlight. Additionally, the phytotoxicity of treated MB dye-contaminated water sample showed good germination of Vicia faba as compared with non-treated MB dye-contaminated solution. In conclusion, the herein biosynthesized Ag-NPs demonstrated its feasibility of the purification of contaminated water from microbes and methylene blue dye and the probability of reusing purified water for agricultural purposes.

DNA-nanostructure-templated assembly of planar and curved lipid-bilayer membranes.

Lipid-bilayer nanodiscs and liposomes have been developed to stabilize membrane proteins in order to study their structures and functions. Nanodiscs are detergent-free, water-soluble, and size-controlled planar phospholipid-bilayer platforms. On the other hand, liposomes are curved phospholipid-bilayer spheres with an aqueous core used as drug delivery systems and model membrane platforms for studying cellular activities. A long-standing challenge is the generation of a homogenous and monodispersed lipid-bilayer system with a very wide range of dimensions and curvatures (elongation, bending, and twisting). A DNA-origami template provides a way to control the shapes, sizes, and arrangements of lipid bilayers via enforcing the assembly of lipid bilayers within the cavities created by DNA nanostructures. Here, we provide a concise overview and discuss how to design planar and curved lipid-bilayer membranes by using DNA-origami nanostructures as templates. Finally, we will discuss the potential applications of DNA-origami nanostructures in the structural and functional studies of large membrane proteins and their complexes.

Ecofriendly preparation of silver nanoparticles-based nanocomposite stabilized by polysaccharides with antibacterial, antifungal and antiviral activities.

In the present work, sustainable and green method was used to prepare silver nanoparticles (Ag-NPs), followed with incorporation into tertiary nanocomposite consisted of starch, oxidized cellulose and ethyl cellulose. The prepared tertiary silver-nanocomposite (Ag-NC) was fully characterized via instrumental analysis (UV-vis, FT-IR, XRD, SEM, EDX and TEM) and evaluated for antibacterial, antifungal, and antiviral activities. Ag-NC significantly suppressed growth of tested bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis)  as compared with controls. Antifungal activity revealed that the prepared tertiary Ag-NC has a promising antifungal activity towards unicellular (Candida albicans) and multicellular fungi  ( Aspergillus niger, A. terreus, A. flavus and A. fumigatus). In same line, both Ag-NC and free Ag-NPs have shown a dose-dependent reduction in Vero cell line with maximum non-toxic dose at 6.25 and 12.5 µg/mL, respectively. Both Ag-NPs and Ag-NC exhibited antiviral effects against Herpes simplex virus, Adenovirus and Coxsackie B virus in a dose-dependent manner. Combined treatment of Ag-NPs incorporated into tertiary nanocomposite based on starch, oxidized cellulose and ethyl cellulose opens new possibilities to be more efficient nanomaterials for preventing microbial growth. In conclusion, the prepared tertiary Ag-NC has a promising antibacterial, antifungal as well as antiviral activities.

Interferon Regulatory Factor 5 siRNA-Loaded Folate-Modified Cationic Liposomes for Acute Lung Injury Therapy.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is an overwhelming pulmonary inflammation with limited clinical treatment strategies. Interferon regulatory factor 5 (IRF5) is a crucial regulator of inflammation factors, which can be upregulated under an inflammatory state and related to the efferocytosis of macrophages. Herein, IRF5 was knockdown by small interfering RNA (siIRF5) to promote the anti-inflammatory effect of macrophages. Macrophage-targeting cationic liposome modified by folate (FA-LP) was developed to deliver siIRF5 (FA-LP/siIRF5). Liposomes were characterized for their particle size, zeta potential, protein adsorption and hemolysis of red blood cells. The amount of IRF5 mRNA and the expression of IRF5 were measured using quantitative reverse transcription PCR (RT-qPCR) and western blot, respectively. The phenotype and efferocytosis of macrophages and the regulatory pathway of efferocytosis and biodistribution of liposomes in the ALI mice model were investigated. Data revealed that FA-LP/siIRF5 could obviously downregulate the expression of IRF5 in macrophages, skewing the polarization of macrophages to M2 phenotype (anti-inflammatory state) and thus improving their efferocytosis. Moreover, regulation of efferocytosis of macrophages by siIRF5 is related to the NF- B pathway. The in vivo biodistribution of FA-LP exhibited higher accumulation in the inflammatory lungs, suggesting that FA-LP could be considered as a promising gene delivery system and FA-LP/siIRF5 is an alternative strategy for the treatment of ALI/ARDS. To the best of our knowledge, this is the first study reporting that siIRF5 can be used for the treatment of ALI/ARDS.

HBVsvp-Pulsed Dendritic Cell Immunotherapy Induces Th1 Polarization and Hepatitis B Virus-Specific Cytotoxic T Lymphocytes Production.

BACKGROUND: In chronic hepatitis B virus (CHB) patients, both dendritic cells (DCs) and T cells are functionally impaired and consequently the HBV-specific cellular immune responses are downregulated. The present study aims to investigate whether monocyte-derived DC (MoDCs)-pulsed-HBV subviral particles (HBVsvp) can polarize Th1 cells to induce HBV-specific cytotoxic T-lymphocytes (CTL) responses in CHB patients. METHODS AND MATERIALS: To this end, the human hepatoma HepG2.2.15 cell line was used to produce HBVsvp as a culturing system, and HBVsvp were concentrated for highly virus titer using the polyethylene glycol protocol. Peripheral blood mononuclear cells (PBMCs), collected from CHB patients and healthy donors, were differentiated into MoDCs and T cells. PBMCs-derived MoDCs were first pulsed with HBVsvp and then cultured with PBMCs-derived T cells. MoDCs and/or T subsets cells were identified for phenotypic activation by FACS analysis. The cytokine secretion of IL-4, IL-12, and IFN-γ in the culture supernatants was detected. RESULTS: The MoDCs were restored for their activation upon pulsing with HBVsvp in vitro, as identified by significantly overexpression of both CD86 and HLA-DR, and overproduction of IL-4 and IL-12. Furthermore, MoDCs-pulsed-HBVsvp induced Th1 frequencies and activated HBV-specific CTL to produce significantly highest amount of IFN-γ. Enhanced HBV-specific CTL led to strong cytolytic capacity against HepG2.2.15. CONCLUSION: Overall, our data suggest that in vitro activation of MoDCs with HBVsvp overcomes the functionally impaired DCs and T cells in CHB patients offering a promising tool for therapeutic or vaccine-based approaches against HBV.

Cloning, expression and nanodiscs assemble of recombinant HIV-1 gp41.

Structural studies of membrane proteins have been hurdled by their difficulty for expression in heterogeneous expression systems due to their intrinsically strong hydrophobicity and requirements for association with other cellular membranes. This study aims to design a construct for expression of membrane proteins. Because of its outstanding interest in HIV-1 vaccine design, transmembrane gp41 amino acid residue 662-723 was chosen as a representative membrane protein. Therefore, we constructed expression vectors for expression of gp41(662-723) alone (pET28a-gp41(662-723)) or coupled with a fusion partner: GB1 (pET30a-GB1-gp41(662-723)) and Trx (pET32a-Trx-gp41(662-723)). For enhancing protein expression, the expression plasmids were transformed into E. coli BL-21 (DE3), E. coli T7 Express lysY/Iq and E. coli Lemo21 (DE3). Interestingly, HIV-1 gp41(662-723) was expressed as a C-terminus fusion to the fusion partner Trx (Trx-gp41(662-723)) with an apparent molecular mass of 21.8 kDa. Trx-gp41(662-723) was overexpressed into E. coli T7 Express lysY/Iq by early induction as OD600 ~0.5 followed by incubation at 20 °C/overnight. Our data demonstrated that almost all recombinant Trx-gp41(662-723) was incorporated into lipid nanodiscs by slowing down the nanodiscs assembly process. Negative-stained electron micrographs revealed homogenous 10 nm Trx-gp41(662-723)-nanodiscs. While the neutralizing epitopes in the purified Trx-gp41(662-723) were accessible and recognizable by anti-MPER bNAbs, these epitopes became less accessibly exposed, particularly in the C-terminal region of MPER, after incorporation of Trx-gp41(662-723) into nanodiscs.

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

Optimization of the Linker Length of Mannose-Cholesterol Conjugates for Enhanced mRNA Delivery to Dendritic Cells by Liposomes.

Liposomes (LPs) as commonly used mRNA delivery systems remain to be rationally designed and optimized to ameliorate the antigen expression of mRNA vaccine in dendritic cells (DCs). In this study, we synthesized mannose-cholesterol conjugates (MPn-CHs) by click reaction using different PEG units (PEG100, PEG1000, and PEG2000) as linker molecules. MPn-CHs were fully characterized and subsequently used to prepare DC-targeting liposomes (MPn-LPs) by a thin-film dispersion method. MPn-LPs loaded with mRNA (MPn-LPX) were finally prepared by a simple self-assembly method. MPn-LPX displayed bigger diameter (about 135 nm) and lower zeta potential (about 40 mV) compared to MPn-LPs. The in vitro transfection experiment on DC2.4 cells demonstrated that the PEG length of mannose derivatives had significant effect on the expression of GFP-encoding mRNA. MP1000-LPX containing MP1000-CH can achieve the highest transfection efficiency (52.09 ± 4.85%), which was significantly superior to the commercial transfection reagent Lipo 3K (11.47 ± 2.31%). The optimal DC-targeting MP1000-LPX showed an average size of 132.93 ± 4.93 nm and zeta potential of 37.93 ± 2.95 mV with nearly spherical shape. Moreover, MP1000-LPX can protect mRNA against degradation in serum with high efficacy. The uptake study indicated that MP1000-LPX enhanced mRNA expression mainly through the over-expressing mannose receptor (CD206) on the surface of DCs. In conclusion, mannose modified LPs might be a potential DC-targeting delivery system for mRNA vaccine after rational design and deserve further study on the in vivo delivery profile and anti-tumor efficacy.

Co-delivery of a CD4 T cell helper epitope via covalent liposome attachment with a surface-arrayed B cell target antigen fosters higher affinity antibody responses.

Liposomal vaccines incorporating adjuvant and CD4 T cell helper peptides enhance antibody responses against weakly immunogenic B cell epitopes such as found in the membrane proximal external region (MPER) of the HIV-1 gp41 subunit. While the inclusion of exogenous helper peptides in vaccine formulations facilitates stronger and more durable antibody responses, the helper peptide incorporation strategy per se may influence the overall magnitude and quality of B cell target antigen immunogenicity. Both variability in individual peptide encapsulation as well as the potential for liposome surface-associated helper peptides to misdirect the humoral response are potential parameters impacting outcome. In this study, we used MPER/liposome vaccines as a model system to examine how the mode of the potent LACK T helper peptide formulation modulates antibody responses against the MPER antigen. We directly compared liposome surface-arrayed palmitoyl LACK (pLACK) versus soluble LACK (sLACK) encapsulated in the liposomes and free in solution. Independent of LACK formulation methods, dendritic cell activation and LACK presentation were equivalent in vivo. The frequency of MPER-specific GC B cells promoted by sLACK was higher than that stimulated by pLACK formulation, a finding associated with a significantly greater frequency of LACK-specific GC B cells induced by pLACK. While there were no significant differences in the quantity of MPER-specific serological responses, the MPER-specific antibody titer trended higher with sLACK formulated vaccines at the lower dose of LACK. However, pLACK generated relatively greater MPER-specific antibody affinities than those induced by sLACK-formulated vaccines. Overall, the results suggest that liposomal surface-associated LACK enhances immunogenicity of LACK through better engagement of LACK-specific B cells. Of note, this is not detrimental to the induction of MPER-specific immune responses; rather, the elicitation of higher affinity anti-MPER antibodies benefits from augmented help delivered via covalent linkage of the pLACK CD4 T cell epitope in conjunction with MPER/liposome presentation.