Liposomal nanocarriers containing siRNA as small molecule-based drugs to overcome cancer drug resistance.

This review discusses the application of nanoliposomes containing siRNA/drug to overcome multidrug resistance for all types of cancer treatments. As drug resistance-associated factors are overexpressed in many cancer cell types, pumping chemotherapy drugs out of the cytoplasm leads to an inadequate therapeutic response. The siRNA/drug-loaded nanoliposomes are a promising approach to treating multidrug-resistant cancer, as they can effectively transmit a small-molecule drug into the target cytoplasm, ensuring that the drug binds efficiently. Moreover, nanoliposome-based therapeutics with advances in nanotechnology can effectively deliver siRNA to cancer cells. Overall, nanoliposomes have the potential to effectively deliver siRNA and small-molecule drugs in a targeted manner and are thus a promising tool for the treatment of cancer and other diseases.

Tissue engineering studies in male infertility disorder.

Infertility is an important issue among couples worldwide which is caused by a variety of complex diseases. Male infertility is a problem in 7% of all men. In vitro spermatogenesis (IVS) is the experimental approach that has been developed for mimicking seminiferous tubules-like functional structures in vitro. Currently, various researchers are interested in finding and developing a microenvironmental condition or a bioartificial testis applied for fertility restoration via gamete production in vitro. The tissue engineering (TE) has developed new approaches to treat male fertility preservation through development of functional male germ cells. This makes TE a possible future strategy for restoration of male fertility. Although 3D culture systems supply the perception of the effect of cellular interactions in the process of spermatogenesis, formation of a native gradient of autocrine/paracrine factors in 3D culture systems have not been considered. These results collectively suggest that maintaining the microenvironment of testicular cells even in the form of a 3D-culture system is crucial in achieving spermatogenesis ex vivo. It is also possible to engineer the testicular structures using biomaterials to provide a supporting scaffold for somatic and stem cells. The insemination of these cells with GFs is possible for temporally and spatially adjusted release to mimic the microenvironment of the in situ seminiferous epithelium. This review focuses on recent studies and advances in the application of TE strategies to cell-tissue culture on synthetic or natural scaffolds supplemented with growth factors.

Design of a novel multi-epitope vaccine candidate against Chlamydia trachomatis using structural and nonstructural proteins: an immunoinformatics study.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium which causes eye and sexually transmitted infections. During pregnancy, the bacterium is associated with preterm complications, low weight of neonates, fetal demise and endometritis leading to infertility. The aim of our study was design of a multi-epitope vaccine (MEV) candidate against C. trachomatis. After protein sequence adoption from the NCBI, potential epitopes toxicity, antigenicity, allergenicity, MHC-I and MHC-II binding, cytotoxic T lymphocytes (CTLs), Helper T lymphocytes (HTLs) and interferon-γ (IFN-γ)- induction were predicted. The adopted epitopes were fused together using appropriate linkers. In the next step, the MEV structural mapping and characterization, three-dimensional (3D) structure homology modeling and refinement were also performed. The MEV candidate interaction with the toll-like receptor 4 (TLR4) was also docked. The immune responses simulation was assessed using the C-IMMSIM server. Molecular dynamic (MD) simulation verified the structural stability of the TLR4-MEV complex. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach demonstrated the MEV high affinity of binding to the TLR4, MHC-I and MHC-II. The MEV construct was also stable and water soluble and had enough antigenicity and lacked allergenicity with stimulation of T cells and B cells and INF-γ release. The immune simulation confirmed acceptable responses of both the humoral and cellular arms. It is proposed that in vitro and in vivo studies are needed to evaluate the findings of this study.Communicated by Ramaswamy H. Sarma.

Finding and engineering the newly found bacterial superoxide dismutase enzyme to increase its thermostability and decrease the immunogenicity: a computational and experimental research.

Superoxide dismutase (SOD) is one of the most important antioxidant enzymes that can reduce oxidative stress in the cell environment. Nowadays, bacterial sources of enzyme are commercially applicable in the cosmetics and pharmaceutical industries, but the allergenic effect of proteins from non-human sources has been mentioned as disadvantage of these kinds of enzymes. In this study, to find the suitable bacterial SOD candidate for decreasing immunogenicity, the sequences of five thermophilic bacteria were selected as reference species. Then, linear and conformational B-cell epitopes of the SOD were analyzed by different servers. The stability and immunogenicity of mutant positions were also evaluated. The mutant gene was inserted into the pET-23a expression vector and transformed into E. Coli BL21 (DE3) for expression of the recombinant enzyme. Afterward, the expression of the mutant enzyme was evaluated by SDS-PAGE analysis and the recombinant enzyme activity was assessed. Anoxybacillus gonensis was selected as a reasonable SOD source according to BLAST search, physicochemical properties analysis, and prediction of allergenic features. Regarding our results, five residues including E84, E142, K144, G147, and M148 were predicted as candidates for mutagenesis. Finally, the K144A was chosen as the final modification due to the increase in the stability of the enzyme and decreased immunogenicity of the enzyme as well. The enzyme activity was 240 U/ml at room temperature. Alternation in K144 to alanine caused increased stability of the enzyme. In silico studies confirmed non-antigenic protein after mutation.

Protective role of flavonoids quercetin and silymarin in the viral-associated inflammatory bowel disease: an updated review.

Inflammatory bowel disease (IBD) is a chronic recurrent inflammation of the gastrointestinal tract (GIT). IBD patients are susceptible to various infections such as viral infections due to the long-term consumption of immunosuppressive drugs and biologics. The antiviral and IBD protective traits of flavonoids have not been entirely investigated. This study objective included an overview of the protective role of flavonoids quercetin and silymarin in viral-associated IBD. Several viral agents such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV) and enteric viruses can be reactivated and thus develop or exacerbate the IBD conditions or eventually facilitate the disease remission. Flavonoids such as quercetin and silymarin are non-toxic and safe bioactive compounds with remarkable anti-oxidant, anti-inflammatory and anti-viral effects. Mechanisms of anti-inflammatory and antiviral effects of silymarin and quercetin mainly include immune modulation and inhibition of caspase enzymes, viral binding and replication, RNA synthesis, viral proteases and viral assembly. In the nutraceutical sector, natural flavonoids low bioavailability and solubility necessitate the application of delivery systems to enhance their efficacy. This review study provided an updated understanding of the protective role of quercetin and silymarin against viral-associated IBD.

Antibacterial effects of single phage and phage cocktail against multidrug-resistant Klebsiella pneumoniae isolated from diabetic foot ulcer.

Diabetic foot ulcer (DFU) is associated with long-term hospitalization and amputation. Antibiotic resistance has made the infection eradication more difficult. Hence, seeking alternative therapies such as phage therapy seems necessary. Bacteriophages are viruses targeting specific bacterial species. Klebsiella pneumoniae (K. pneumoniae) is among causative agents of the DFU. In this study, the therapeutic effects of single phage and phage cocktail were investigated against multidrug-resistant (MDR) K. pneumonia isolated from DFU. Bacteriophages were isolated from animal feces and sewage samples, and were enriched and propagated using K. pneumoniae as the host. Thirty K. pneumoniae clinical isolates were collected from hospitalized patients with DFU. The antibiotic susceptibility pattern was determined using agar disk diffusion test. The phages' morphological traits were determined using transmission electron microscopy (TEM). The killing effect of isolated phages was assessed using plaque assay. Four phage types were isolated and recognized including KP1, KP2, KP3, and KP4. The bacterial rapid regrowth was observed following each single phage-host interaction, but not phage cocktail due to the evolution of mutant strains. Phage cocktail demonstrated significantly higher antibacterial activity than each single phage (p < 0.05) without any bacterial regrowth. The employment of phage cocktail was promising for the eradication of MDR-K. pneumoniae isolates. The development of phage therapy in particular, phage cocktail is promising as an efficient approach to eradicate MDR-K. pneumoniae isolated from DFU. The application of a specific phage cocktail can be investigated to try and achieve the eradication of various infections.

Preparation and In Vitro Osteogenic Evaluation of Biomimetic Hybrid Nanocomposite Scaffolds Based on Gelatin/Plasma Rich in Growth Factors (PRGF) and Lithium-Doped 45s5 Bioactive Glass Nanoparticles.

Bone tissue engineering is an emerging technique for repairing large bone lesions. Biomimetic techniques expand the use of organic-inorganic spongy-like nanocomposite scaffolds and platelet concentrates. In this study, a biomimetic nanocomposite scaffold was prepared using lithium-doped bioactive-glass nanoparticles and gelatin/PRGF. First, sol-gel method was used to prepare bioactive-glass nanoparticles that contain 0, 1, 3, and 5%wt lithium. The lithium content was then optimized based on antibacterial and MTT testing. By freeze-drying, hybrid scaffolds comprising 5, 10, and 20% bioglass were made. On the scaffolds, human endometrial stem cells (hEnSCs) were cultured for adhesion (SEM), survival, and osteogenic differentiation. Alkaline phosphatase activity and osteopontin, osteocalcin, and Runx2 gene expression were measured. The effect of bioactive-glass nanoparticles and PRGF on nanocomposites' mechanical characteristics and glass-transition temperature (T g) was also studied. An optimal lithium content in bioactive glass structure was found to be 3% wt. Nanoparticle SEM examination indicated grain deformation due to different sizes of lithium and sodium ions. Results showed up to 10% wt bioactive-glass and PRGF increased survival and cell adhesion. Also, Hybrid scaffolds revealed higher ALP-activity and OP, OC, and Runx2 gene expression. Furthermore, bioactive-glass has mainly increased ALP-activity and Runx2 expression, whereas PRGF increases the expression of OP and OC genes. Bioactive-glass increases scaffold modulus and T g continuously. Hence, the presence of both bioactive-glass and nanocomposite scaffold improves the expression of osteogenic differentiation biomarkers. Subsequently, it seems that hybrid scaffolds based on biopolymers, Li-doped bioactive-glass, and platelet extracts can be a good strategy for bone repair.

Advanced simulations and screening to repurposing a 3C protease inhibitor against the rupintrivir-resistant human norovirus-induced gastroenteritis.

Human norovirus (HuNoV) causes acute viral gastroenteritis in all age groups, and dehydration and severe diarrhea in the elderly. The World Health Organization reports ∼1.45 million deaths from acute gastroenteritis annually in the world. Rupintrivir, an inhibitory medicine against the human rhinovirus C3 protease, has been reported to inhibit HuNoV 3C protease. However, several HuNoV 3C protease mutations have been revealed to reduce the susceptibility of HuNoV to rupintrivir. The structural details behind rupintrivir-resistance of these single-point mutations (A105V and I109V) are not still clear. Hence, in this study, a combination of computational techniques were used to determine the rupintrivir-resistance mechanism and to propose an inhibitor against wild-type and mutant HuNoV 3C protease through structure-based virtual screening. Dynamic structural results indicated the unstable binding of rupintrivir at the cleft binding site of the wild-type and mutant 3C proteases, leading to its detachment. Our findings presented that the domain II of the HuNoV 3C protease had a critical role in binding of inhibitory molecules. Binding energy computations, steered molecular dynamics and umbrella sampling simulations confirmed that amentoflavone, the novel suggested inhibitor, strongly binds to the cleft site of all protease models and has a good structural stability in the complex system along the molecular dynamic simulations. Our in silico study proposed the selected compound as a potential inhibitor against the HuNoV 3C protease. However, additional experimental and clinical studies are required to corroborate the therapeutic efficacy of the compound.

The Antibacterial Effects of Cocktail and Single Forms of Lytic Phages Belonging to Podoviridae and Myoviridae Families from Sewage against Shigella sonnei and Shigella flexneri.

Background: Diarrhea caused by bacterial pathogens such as Shigella spp. is one of the prominent public health concerns. The evolution of vast antibiotic resistance by these pathogens, leading to failure in the infections eradication, has made an impetus to seek and develop novel approaches. Recently, some alternative therapies such as phage therapy have been investigated. Bacteriophages are viruses that target specific bacterial species. The objective of this study was to assess the therapeutic effect of phages obtained from hospital sewage against Shigella sonnei (S. sonnei) ATCC® 9290 and S. flexneri ATCC 12022 standard and clinical strains. Methods: Four various lytic bacteriophages were isolated from animal fecal and sewage samples and propagated using S. sonnei and S. flexneri as host organisms. The phages' morphology was determined using transmission electron microscopy (TEM). The lytic potential and host specificity of isolated phages were evaluated using double layer plaque assay and spot test. Moreover, bacterial turbidity values were evaluated in coculture with phages in the Luria Bertani (LB) medium for 24 hours at time intervals of 30 min. Results: Phage cocktails (Shs1, Shs2, Shf1, and Shf2) exhibited higher antimicrobial activity than single phage application against S. sonnei and S. flexneri standard strains. The phages belonged to Podoviridae and Myoviridae families according to TEM-assisted morphological features analysis. In addition, the phages exhibited host specificity using the spot test against 18 Shigella clinical isolates. Conclusion: In this study, phage cocktail of Podoviridae and Myoviridae families from sewage conferred substantial antibacterial effects against S. sonnei and S. flexneri. However, single phage effects were unstable in the LB coculture. Moreover, the phages had host specificity using the spot test performed against Shigella spp. clinical isolates.

Efflux dynamics of the antiseizure drug, levetiracetam, through the P-glycoprotein channel revealed by advanced comparative molecular simulations.

Understanding the precise mechanistic details of the possible binding and transport of antiseizure medications (ASMs) through the P-glycoprotein (P-gp) efflux pump is essential to find strategies for the treatment of patients with epilepsy resistant to ASMs. In the present work, conventional molecular dynamics, binding free energy calculations, steered molecular dynamics and umbrella sampling were applied to study the interactions of levetiracetam and brivaracetam with P-gp and their possible egress path from the binding site. Comparative results for the control drugs, zosuquidar and verapamil, confirmed their established P-gp inhibitory activity. Brivaracetam, a non-substrate of P-gp, demonstrated stronger static and dynamic interactions with the exporter protein, than levetiracetam. The potential of mean force calculations indicated that the energy barriers through the ligand export were the lowest for levetiracetam, suggesting the drug as a P-gp substrate with facile passage through the transporter channel. Our findings also stressed the contribution of nonpolar interactions with P-gp channel lining as well as with membrane lipid molecules to hamper the ASM efflux by the transmembrane exporter. Appropriate structural engineering of the ASMs is thus recommended to address drug-resistant epilepsy.

Design of a novel multi-epitope vaccine candidate against hepatitis C virus using structural and nonstructural proteins: An immunoinformatics approach.

Hepatitis C virus (HCV) infects the liver and causes chronic infection. Several mutations in the viral genome have been associated with drug resistance development. Currently, there is no approved vaccine against the HCV. The employment of computational biology is the primary and crucial step for vaccine design or antiviral therapy which can substantially reduce the duration and cost of studies. Therefore, in this study, we designed a multi-epitope vaccine using various immunoinformatics tools to elicit the efficient human immune responses against the HCV. Initially, various potential (antigenic, immunogenic, non-toxic and non-allergenic) epitope segments were extracted from viral structural and non-structural protein sequences using multiple screening methods. The selected epitopes were linked to each other properly. Then, toll-like receptors (TLRs) 3 and 4 agonists (50S ribosomal protein L7/L12 and human ß-defensin 2, respectively) were added to the N-terminus of the final vaccine sequence to increase its immunogenicity. The 3D structure of the vaccine was modeled. Molecular dynamics simulations studies verified the high stability of final free vaccines and in complex with TLR3 and TLR4. These constructs were also antigenic, non-allergenic, nontoxic and immunogenic. Although the designed vaccine traits were promising as a potential candidate against the HCV infection, experimental studies and clinical trials are required to verify the protective traits and safety of the designed vaccine.

Evaluating Five Escherichia coli Derivative Strains as a Platform for Arginine Deiminase Overproduction.

AIMS: This study attempted to evaluate the five host strains, including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE, and SHuffle, in terms of arginine deiminase (ADI) production and enzyme activity. BACKGROUND: Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors, and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. Various E. coli cells have been examined in different patent applications. METHODS: ADI was chosen as a bacterial enzyme that degrades L-arginine. It is effective in the treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC), which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through the CaCl2 method. It was then transformed with the construct of pET3a-ADI using the heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for the expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. RESULTS: All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein, followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. CONCLUSION: There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

Application of Platelet Rich Fibrin in Tissue Engineering: Focus on Bone Regeneration.

Bone tissue engineering (BTE) is a strategy for reconstructing bone lesions, which is rapidly developing in response to higher demands for bone repairing. Recently, this method, along with the emergence of functionally graded, biocompatible and biodegradable materials, has been expanded. Moreover, scaffolds with chemical, physical and external patterns have induced bone regeneration. However, the maintenance of healthy bone and its regeneration in the human body needs a series of complex and accurate processes. Hence, many studies have been accompanied for reconstructing bone by using blood-derived biomaterials, especially platelet-rich fabricates. The most important reason for using platelet-rich formulations in bone regeneration is based on releasing growth factors from alpha granules in platelets, which can induce osteogenesis. Moreover, the presence of fibrin nano-fiber structures as a constituent can provide a good substrate for cell attachments. This study attempts to review the history, structure, and biology of platelet-rich fibrin (PRF) as well as in vitro, pre-clinical, and clinical studies on the use of PRF for bone regeneration.

MicroRNAs Profiling in HIV, HCV, and HIV/HCV Co-Infected Patients.

BACKGROUND: Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections are important public health issues. OBJECTIVE: This study aimed to assess the association between microRNAs expression leveland immunological and viral markers in HIV, HCV, and HIV/HCV co-infected patients. METHODS: The expression level of miR-29, miR-149, miR-199, miR-let7, miR-223, miR-155, miR-122, and miR-150 was evaluated in 20 HIV, 20 HCV, 20 co-infected patients, and 20 healthy controls using real-time PCR assay. HIV and HCVviral loads were measuredby real-time PCR, and also, CD4+ T-lymphocyte count was measuredby the PIMA CD4 analyzer. RESULTS: The miRNA expression pattern in each mentioned group showed significantly different expression profiles, but some miRNA species were shared between the groups. MiR-122 and miR-155 were upregulated, while miR-29 and miR-223 were downregulated in three patients groups compared to healthy controls. A significant positive correlation was observed between the expression of miR-122 and HIV/HCV loads. But, miR-29 and let-7 were negatively correlated with HIV load, and miR-149 and let-7 were negatively correlated with HCV load. Also, miR-155 was positively correlated with HCV load. MiR-122 and miR-199 were negative while others were positively correlated with CD4+ T cell count. CONCLUSION: These miRNAs are probably involved in the clinical progression and pathogenesis of HIV and HCV infections. Therefore, determining and manipulating these miRNAs can lead to opening a new gate to control these important infections.

Colorimetric Sensor Based on ß-Cyclodextrin-Functionalized Silver Nanoparticles for Zidovudine Sensitive Determination.

Zidovudine (ZDV) is an antiviral drug against HIV that was approved by the FDA on March, 1987. It is a reverse transcriptase inhibitor. This type of drug stops the reproduction of DNA and decreases the amount of the virus in the patients' blood. Due to the ability of forming various molecular bonds, silver nanoparticles (AgNPs) are widely used for the detection of large range of agents, including drugs. In this study, we synthesized AgNP-modified ß-cyclodextrin (ß-CD) using green synthesis for the sensitive and selective zidovudine (ZDV) determination. Characterization of nanoparticles was done using different methods including infrared (IR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and also, X-ray diffraction (XRD) patterns. The AgNP λ-max peak was at approximately 405 nm. In the presence of ZDV, yellow solution was turned to red color, and surface plasmon absorption band was dramatically centered at 560 nm. ZDV was determined in the range of 50-500 µM, and the detection limit value was obtained as 42 µM. The sensor was used to determine ZDV in tablets with good recovery.

Amino Acids Sequence-based Analysis of Arginine Deiminase from Different Prokaryotic Organisms: An In Silico Approach.

BACKGROUND: Arginine deiminase is a bacterial enzyme, which degrades L-arginine. Some human cancers such as hepatocellular carcinoma (HCC) and melanoma are auxotrophic for arginine. Therefore, PEGylated arginine deiminase (ADI-PEG20) is a good anticancer candidate with antitumor effects. It causes local depletion of L-arginine and growth inhibition in arginineauxotrophic tumor cells. The FDA and EMA have granted orphan status to this drug. Some recently published patents have dealt with this enzyme or its PEGylated form. OBJECTIVE: Due to increasing attention to it, we aimed to evaluate and compare 30 arginine deiminase proteins from different bacterial species through in silico analysis. METHODS: The exploited analyses included the investigation of physicochemical properties, multiple sequence alignment (MSA), motif, superfamily, phylogenetic and 3D comparative analyses of arginine deiminase proteins thorough various bioinformatics tools. RESULTS: The most abundant amino acid in the arginine deiminase proteins is leucine (10.13%) while the least amino acid ratio is cysteine (0.98%). Multiple sequence alignment showed 47 conserved patterns between 30 arginine deiminase amino acid sequences. The results of sequence homology among 30 different groups of arginine deiminase enzymes revealed that all the studied sequences located in amidinotransferase superfamily. Based on the phylogenetic analysis, two major clusters were identified. Considering the results of various in silico studies; we selected the five best candidates for further investigations. The 3D structures of the best five arginine deiminase proteins were generated by the I-TASSER server and PyMOL. The RAMPAGE analysis revealed that 81.4%-91.4%, of the selected sequences, were located in the favored region of arginine deiminase proteins. CONCLUSION: The results of this study shed light on the basic physicochemical properties of thirty major arginine deiminase sequences. The obtained data could be employed for further in vivo and clinical studies and also for developing the related therapeutic enzymes.

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine.

BACKGROUND: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. METHODS: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. RESULTS: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund's adjuvant group. CONCLUSION: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

Structural vaccinology considerations for in silico designing of a multi-epitope vaccine.

Multi-epitope peptide vaccines, as a kind of fusion proteins, usually possess a string-of-beads structure, consisting of several peptidic epitopes, probably adjuvants and linkers. Very numerous options are possible in selecting the order of different segments and linkers. Such factors can affect the vaccine efficacy through impacting physicochemical characteristics and protein tertiary structure. To investigate such relations, eleven different constructs were designed and studied as a multi-epitope prophylaxis vaccine for human papilloma virus (HPV). The vaccine contained two epitopes from the minor protein of virus capsid (L2) of HPV16, two TLR agonists as adjuvants (flagellin and RS09, as TLR5 and TLR4 agonists, respectively), and two universal T-helper epitopes. Since the used TLR4 agonist was inserted in the middle of the construct, its appropriate interaction with the bulky TLR4 was a serious concern. Thus, beyond evaluating the physicochemical properties, secondary and tertiary structures, and conformational B-cell epitopes of the designed constructs, TLR4 agonist exposability was also studied. Besides, the interaction between TLR4 and its agonist was investigated through docking and MD studies. Consequently, one structure ("D") with proper physicochemical features, a high frequency of conformational B-cell epitopes, and appropriate interactions with TLR4 and TLR5 in docking and MD studies, was selected as a proper candidate. Accordingly, for in silico designing of multi-epitope vaccines, structural concerns should be considered, and the linkers and arrangement of epitopes and adjuvants should be optimized. Considering the diversity of the possible structures, devising computational tools for such investigations would be very valuable.

Bioconversion of shrimp waste Penaeus merguiensis using lactic acid fermentation: An alternative procedure for chemical extraction of chitin and chitosan.

Chitin extraction from shrimp wastes by biological treatment, using the Pseudomonas aeruginosa was a positive and simple method. In order to look for the optimal conditions, the wastes were incubated at 30°C and 100rpm in different glucose (0%, 10%, 15% and 20%) and inoculation (10%, 15% and 20%) concentrations for 4 and 6days. At the end of fermentation, Protease activity was investigated at different temperatures and temperature 50°C was considered as the optimum. The results obtained also showed a direct relationship between the concentration of different parameters and deproteinization and demineralization rates, so that the optimal conditions were 20% glucose, 20% inoculation and 6days fermentation. These conditions led to 82% demineralization, 92% deproteinization and chitin yield of 47%. Then, chitin was converted to chitosan using microwave, autoclave and traditional methods. The highest yield (87%) was obtained with autoclave method. At the end, the chitin and chitosan were characterized by elemental analysis and FTIR.

Chitin from Penaeus merguiensis via microbial fermentation processing and antioxidant activity.

The ability of three proteases producing bacteria such as Pseudomonas aeruginosa, Serratia marcescens, and Bacillus pumilus on the demineralization and deproteinzation efficiency of shrimp waste, for chitin extraction, was investigated. Statistical analysis of data was showed a significant difference between the percentage of demineralization and deproteinization in different bacteria species (p<0.05). The highest deproteinization (74.76%) and demineralization rate (78.46%) was obtained with P. aeruginosa and the lowest was observed in the treatment of S. marcescens. Then, chitin was converted to chitosan by deacetylation in the presence of NaOH 50%. The antioxidant activity of chitosan solution was determined using different tests. The highest activity (DO 700 nm=0.74, DO 695 nm=0.31) was observed for chitosan sample at concentration of 1,000 µg/ml. The antioxidant potential of the hydrolysates was also evaluated. The highest reducing power in a volume of 400 µl hydrolysate of S. marcescens and the highest total antioxidant capacity in a volume 100 µl hydrolysate of B. pumilus were observed. These results indicated that the P. aeruginosa bacterium in comparison with other bacterial strains, higher ability to remove proteins and mineral from shrimp waste. Therefore, the use of this bacterium is recommended for protein and mineral removal from marine crustaceans.