Current Pharmaceutical Biotechnology - Volume 22, Issue 14, 2021

Background: Nanotechnology involves the synthesis of materials that are in nanometre sizes (1 to 100 nm). Nanotechnology has appeared as a new area of fundamental science and is getting worldwide consideration due to its extensive applications. They have exceptional physicochemical properties due to their unique size, shape and size distribution. Methods: Conventionally, nanoparticles were synthesized using physical and chemical methods. These methods have various disadvantages; therefore, biological methods possess great interest. Biological synthesis uses bacteria, fungi, algae and plants. Results: Biosynthesis of nanoparticles using plants emerges as nanofactories as they offer nontoxic, clean and eco-friendly methods with various physicochemical properties. Out of all the nanoparticles, silver nanoparticles gain special attention due to their various therapeutical and environmental applications. Conclusion: In this review, a summary of various reports within the last ten years where plants were utilized for silver nanoparticle synthesis has been discussed. Further mechanisms involving synthesis, factors affecting the process of synthesis and therapeutical applications have been discussed.

Background: The COVID-19 pandemic had infected more than 3.5M people around the world and more than 250K people died in 187 countries by May 2020. The causal agent of this disease is a coronavirus whose onset of symptoms to death range from 6 to 41 days with a median of 14 days. This period is dependent on several factors such as the presence of comorbidities, age and the efficiency of the innate or adaptive immune responses. Methods: The effector mechanisms of both types of immune responses depend on the pathogen involved. In the case of a viral infection, the innate immune response may approach the harmful virus through pattern recognition receptors inducing an antiviral state. Results: On the other hand, the adaptive immune response activates antibody production to neutralize or eliminate the virus. Phenolics are plant secondary metabolites with many biological activities for plants and humans against infection. Chemical modification of proteins may enhance their biological properties; thus, a protein of medical interest, for instance, a viral protein can be used as a scaffold to build a biopharmaceutical conjugated or complexated with phenolics exhibiting structural complexity or biological activities to achieve effective phenolic-protein-based therapeutics like vaccine adjuvant complexes, immunogen conjugates, and antiviral conjugates. Conclusion: Pharmaceutical biotechnology applies the principles of biotechnology to develop biopharmaceuticals for protein-based therapeutics; such as adjuvants, recombinant proteins, monoclonal antibodies, and antivirals. As neither a vaccine nor a treatment for COVID-19 is currently available, this manuscript focuses on insights from pharmaceutical biotechnology into phenolic biopharmaceuticals against COVID-19.

Background: Molecular biology tools, such as the detection of Single Nucleotide Polymorphisms (SNPs), have been considered to assist in the management of ovarian stimulation protocols. Purpose: The aim of this study was to evaluate the impact of two polymorphisms, the Asn680Ser polymorphism of the FSHR gene, and the FSH β subunit (FSHβ) gene polymorphism -211 G>T, in a Greek population of women undergoing IVF/ICSI program in our center. In addition, a control group of fertile women was studied to verify whether there are differences in the genotype distribution between fertile and infertile population for both polymorphisms, as the FSHβ gene polymorphism -211 G>T is studied for the first time in the Greek population. Results: The FSH β-211 G>T polymorphism, studied for the first time in the infertile Greek population, appears to be quite rare. When studying the two polymorphisms separately, statistically significant differences were obtained that concerned the LH levels. Discussion: According to the combination analysis of the two polymorphisms by the number of alleles, women with 2-3 polymorphic alleles needed more days of stimulation, but there were no differences in pregnancy rates. Conclusion: This molecular genetic study helps to elucidate whether the polygenic combination of the Asn680Ser and FSH β subunit -211 G>T gene polymorphisms is of additive value in the prediction of ovarian response to exogenous gonadotropins.

Background: With the improvements in living standards, height is getting more attention. Malnutrition is one of the main causes of children's short stature; therefore, nutritional intervention in adolescence is the key to prevent short stature. The peptides from Antarctic Krill (AKPs), the ideal protein model, act in bone formation and anti-osteoporosis. However, the studies on promoting longitudinal bone growth by AKPs have not been reported. Methods: Three-week-old male ICR mice, to construct the adolescent mice model, randomly divided into three groups: normal group, casein group (casein, 300 mg/kg·BW), and AKPs group (AKPs, 300 mg/kg·BW). After 21 days of drug administration, the effects of AKPs on serum biochemical indexes and femur histomorphology of mice, and the mechanism of AKPs promoting longitudinal bone growth was discussed. Results: AKPs significantly increased longitudinal bone growth and improved bone strength. In addition, AKPs remarkably promoted proliferation and hypertrophy of chondrocytes in the growth plate. The further mechanism revealed that AKPs increased serum Growth Hormone (GH) and Insulin-Like Growth Factors-1(IGF-1) contents, which activated the downstream GH/IGF-1 axis signaling pathways. Moreover, AKPs induced the secretion and expression of bone morphogenetic protein 2 (BMP- 2) and triggered the activation of BMP2-dependent Smads signaling. AKPs also activated Wnt/ β-catenin signaling, and synergistically activated the expression of Runt-related transcription factor 2 (Runx 2) and Osterix (OSX). Conclusion: AKPs promoted longitudinal bone growth by activating GH/IGF-1 axis, BMP-2/Smads and Wnt/β-catenin pathways, suggesting AKPs to be a potential nutrient fortifier for longitudinal bone growth.

Background: Restoration of blood flow during ischemic stroke leads to Cerebral Ischemia- Reperfusion Injury (CIRI) by activating neuroinflammatory cascades. Pomelo Peel Volatile Oil (PPVO) extracted from Citrus maxima (Burm.) from the genus Rutaceae, comprises some antiinflammatory ingredients, such as limonene and β-myrcene. Objective: The present study aimed to investigate the potential effect of PPVO on alleviating CIRI related to the Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-ΚB) pathway. Methods: Transient middle cerebral artery occlusion/reperfusion (tMCAO/R) was performed on 65 rats, which were then distributed into five groups (n = 13/group) depending on the intervention they received: Normal Saline (NS) group, normal Glycerin (GL) group, low-dose PPVO (LP, 10mg/kg) group, high-dose PPVO (HP, 30 mg/kg) group, and Sham-operated (SH) group. Neurological Deficit Scores (NDSs) and histological changes were evaluated. Infarct volumes were measured by 2,3,5- Triphenyltetrazolium Chloride (TTC) staining. The expression of TLR4 and neutrophil infiltration were detected by Immunofluorescence (IF) staining. Moreover, the downstream molecules of the TLR4/NF-ΚB signaling pathway, such as IL-6, IL-1β, TNF-α, p-IΚB/IΚB, and p-NF-ΚB p65/NF-ΚB p65 were analyzed by Western Blot (WB). Results and Discussion: The results showed that PPVO (30 mg/kg) significantly decreased infarct volumes, improved neurological deficits and pathologic changes, inhibited TLR4/NF-ΚB signaling pathway suppressed neutrophil infiltration, and suppressed pro-inflammatory cytokine release. Conclusion: It can be concluded that PPVO may alleviate neuroinflammation and protect against CIRI via inhibiting the TLR4/NF-ΚB signaling pathway.

Background: The ability of pathogenic bacteria to survive Antimicrobial Peptides (AMPs) in various host niches may contribute to their virulence. Polymyxin B is a cationic AMP, and polymyxin drugs are considered to be the "last line of defense" in the clinical treatment of bacterial infections. Objective: The objectives of this study were to comprehensively study the response of Brucella melitensis strain NI to polymyxin B treatment and to identify the target genes in Brucella induced by polymyxin B stimulation. Methods: Following treatment with polymyxin B, differentially expressed genes in Brucella were detected using RNA-seq and validated using qRT-PCR. Results: In total, 874 differentially expressed genes were identified, including 560 up-regulated and 314 down-regulated genes. Functional annotation and KEGG pathway analysis revealed that many of these genes are involved in metabolism, two-component systems, transcriptional regulation, transport/ membrane proteins, and virulence factors. Expression of genes involved in T4SS and flagellar biosynthesis and assembly, which are important virulence factors in Brucella, were up-regulated by polymyxin B treatment. Discussion: Additionally, genes encoding the ABC transporters YejABEF and the cold-shock protein CspA were also up-regulated. These genes confer resistance to AMPs and contribute to the virulence of Brucella. The NIΔsufC, NIΔsufD, NIΔompW, NIΔexbB, NIΔtetR, and NIΔcspA mutants were also more sensitive than B. melitensis NI to polymyxin B. Conclusion: The results of this study provide important insights into the comprehensive response of Brucella in response to polymyxin B stimulation.

Background: Streptococcus pneumoniae is the leading cause of pneumonia, mostly in children less than five years and elderly people. Although the Pneumoniae Polysaccharide Vaccine (PPV) and Pneumonia Conjugate Vaccines (PCV) are the efficient pneumococcal vaccine in adults and children, the serotype replacement of S. pneumoniae strains causes the reduction in the efficacy of PPV and PCV vaccines. Epitope-based vaccines are a promising alternative to the present capsular antigen vaccines. Methods: In this study, we evaluated cellular and humoral immune responses induced by our novel designed multi-epitope vaccine in BALB/c mice. CD8+ Cytolytic T Lymphocytes (CTLs) epitopes were selected from PspA and CbpA antigens, and CD4+ Helper T Lymphocytes (HTLs) epitopes were chosen from PhtD and PiuA antigens. PorB, the TLR2 agonist, as an adjuvant, was employed to increase the immunogenicity of the vaccine. Results and Conclusion: The high levels of specific anti-peptide vaccine IgG and an increase in the level of IgG2 in the vaccinated group demonstrated our vaccine could elicit robust antibody production. The significant increase in IFN-γ, IL-2, TNF-α, IL-4, IL-6, and decrease in IL-10 showed that the designed vaccine could be proposed as the efficient preventative pneumococcal vaccine in the mouse model.

Background: Influenza is a contagious respiratory illness caused by an acute infection of influenza viruses, among which influenza A virus causes seasonal epidemic infections nearly every year. Due to the unpredictability of the evolving influenza A virus and time-consuming vaccine development cycles, novel universal influenza vaccines designed to induce broadly cross-reactive immune responses against frequently mutant influenza A virus strains are urgently required. Objective: The aim of this study was to synthesize a novel vaccine through the dual-site specific conjugation of the constant epitope of 23 amino acids (M2e) of the influenza A virus with a highly immunogenic carrier protein of Cross-Reacting Material (CRM197) under denaturation and to evaluate its primary immunogenicity in mice. Methods: The antigen (M2e) and the carrier protein (CRM197) were linked with different types of hetero-functionalized linkers, α-Maleimide-ε-Hydrazide Polyethylene Glycol 2k (MAL-PEG-HZ) and N-β-Maleimidopropionic Acid Hydrazide (BMPH) separately. The immunogenicity of the M2e-CRM197 conjugates with different types of linkers was evaluated in mice, and the M2especific total IgG and IgG-isotypes were determined by ELISA. Results: Immunogenicity studies revealed that anti-M2e antibody could be induced by the conjugate products, M2e-PEG-CRM197 and M2e-BMPH-CRM197, by approximately 30 and 90-fold higher than that of the M2e group. In addition, the anti-M2e antibody level induced by M2e-PEG-CRM197 conjugate was three times higher than that of M2e-BMPH-CRM197 conjugate, and the former could simultaneously activate both cellular and humoral immune responses. Conclusion: The M2e-CRM197 conjugated vaccines we synthesized in this study are highly immunogenic compared with M2e alone. Besides, evidence presented here indicated that the hydrophilic, non-immunogenic and biocompatible chain of the cross-linker might be a better choice for the development of a conjugate vaccine.

Background: Curcumin is claimed as a potent protectant against Gastric Ulcer (GU) induced by strong necrotizing agents, including NSAIDs through its antioxidant, anti-inflammatory and gastroprotective activities. However, it was found to exert opposite effects to either delay ulcer healing or exacerbate ulcer inflammation through some curative mechanisms differently modified by curcumin dosage. Its ability to inhibit the expression of COX-2 may also delay the healing of NSAIDs-induced GU. Recently, a topical chitosan-curcumin solution has been found to be a safe and potential alternative agent in treating oral ulcer. Therefore, an oral chitosan-curcumin mixture was developed and determined for its efficacy in treating NSAIDs-induced GU in the rat. Methods: A chitosan (150 mg)-curcumin (20 mg) mixture with optimal gastric pH was developed. Indomethacin (30 mg/kg) was given orally to the rat and test preparations were administered orally at 5 h later and then every 24 h for two consecutive days. The sum of all gastric ulcerated areas (mm2) for each stomach was used as ulcer index. Gastric pro-inflammatory mediators and cytoprotective factors were determined. Results: An oral administration of a chitosan-curcumin mixture exerted a superior efficacy than curcumin, chitosan or lansoprazole (a standard antiulcer agent) in healing indomethacin-induced GU. It was revealed that the mixture exhibited the highest anti-oxidant, anti-inflammatory and gastric mucus producing activities including the high potency in down-regulating pro-inflammatory COX-2 and iNOS expression but up-regulating cytoprotective COX-1, nNOS and eNOS expression. Conclusion: The present findings indicated the benefit of a chitosan-curcumin mixture as a potential alternative agent in treating NSAIDs-induced gastric ulcers.

Background: Astaxanthin is a natural active substance with a plurality of biological activities, such as anti-oxidation, anti-inflammatory and anti-cardio-cerebrovascular diseases. However, there is less research on the effects of astaxanthin on obesity. Astaxanthin with different structural forms affect the corresponding biological activity. Objective: The study aimed to compare Astaxanthin-octanoic acid diester (C8-AST) and Free Astaxanthin (F-AST) to explore the effect on lipogenesis in vitro. Methods: 3T3-L1 preadipocytes were cultured under astaxanthin treatment. Cell proliferation and differentiation were evaluated by MTT assay and oil red O staining, respectively. The synthesis of metabolic mechanism of intracellular fatty acids and triglycerides was examined by qRT-PCR and Western blotting. Results: C8-AST and F-AST had no effect on adipocyte proliferation at low concentration, but inhibited adipocyte differentiation. The treatment of astaxanthin could inhibit the expression of PPARγ, C/EBPα and SREBP-1c in adipocytes, up-regulate the expression of Wnt10b, LRP6, FZ in Wnt/ β-catenin signaling pathway and increase the β-catenin entry into nucleus, suggesting that the activation of Wnt/β-catenin signaling was involved in axtaxanthin-regulated lipogenesis. Notably, inhibition effect of lipogenesis on C8-AST was better than F-AST overall. Conclusion: At the cellular level, both two kinds of astaxanthins inhibit the synthesis of intracellular fatty acids and triglycerides. Notably, the inhibition effect of C8-AST is better than F-AST overall.

Background: Several studies have shown that plant saponins promoted osteoblast differentiation and improved osteoporosis. In the current study, Sea Cucumber Saponin (SCS) with a purity of 80% was extracted from Filipino sea cucumber, with a similar structure to plant saponins. Objective: This study aims to investigate the effects of SCS on bone formation in vitro and ex vivo. Results: SCS significantly promoted osteogenic differentiation and mineralization of MC3T3-E1 cells, as well as new osteoid formation in neonatal mouse calvarias ex vivo. qRT-PCR results indicated that SCS markedly down-regulated the expression of C/EBPα* and PPARγ at the levels of transcription, which demonstrate that SCS inhibits the trans-differentiation of MC3T3-E1 cells to an adipocytic phenotype. Moreover, further studies revealed that SCS increased the expression levels of Runx2 and OSX. The mechanism revealed that SCS induced the expression of BMP2 and p-Smad1/5, which indicated that SCS facilitated osteogenesis via activating the BMP2/Smads signaling pathway. Conclusion: SCS promoted osteogenic differentiation of pre-osteoblasts by activating the BMP2/ Smads molecular pathway, providing a theoretical basis for the development of sea cucumber saponins for the treatment to bone loss diseases such as osteoporosis.

Background: Asthenozoospermia, also known as lack of sperm motility, accounts for about 27.8% of male infertility as a separate factor, and is often associated with abnormal quantity and morphology of spermatozoa. Therefore, oligozoospermia has become one of the most important factors affecting male infertility. Methods: Ursolic Acid (UA), also known as wusu acid, is the main active component isolated from Prunella vulgaris L. and has a variety of pharmacological effects. However, the protective effect of UA on asthenozoospermia disease has not been reported. In the current study, the purpose of this study was to investigate the regulatory effect of UA in rats with LPS-induced asthenozoospermia disease. SD rats were treated with 5 mg/kg LPS, respectively. Results: After different concentrations of UA were infused into the stomach of SD rats, microscopy, flow cytometry, Enzyme-Linked Immunosorbent Assay (ELISA), qRT-PCR and western blot were used to detect sperm motility, apoptosis, the levels of TNF-α, IL-1β and IL-6, and Bcl-2/Bax apoptosis pathway related proteins in rat serum and epididymis tissues. Discussion: Compared with the normal group, the sperm motility and Bcl-2 level in LPS group decreased significantly, while the expression of inflammatory factors and Bax proteins increased significantly (P<0.05). Compared with LPS group, UA intervention group has the opposite result and dose dependence. Conclusion: This study shows that UA can protect LPS-induced asthenozoospermia of rats by increasing sperm density and motility, regulating Bcl-2/Bax apoptosis pathway and reducing inflammatory apoptosis response. This experiment provides ideas for improving the clinical treatment of infertile patients with oligoasthenospermia.