Journal of Applied Biotechnology Reports
Volume:9 Issue: 2, Spring 2022

Acne vulgaris affects ~85% of young adults aged 12–25 years. Regarding the critical role of Propionibacterium acnes in the pathogenesis of acne and current therapeutic failures, developing efficient acne vaccines are appreciated. The purpose of this study was to design a chimeric protein from CAMP and sialidase parts of Propionibacterium acnes and evaluating its immunogenicity in a mice model as an acne vaccine candidate. CAMP-Sialidase recombinant gene expression was carried out through cloning in the vector pET28a and transferring to E.coli BL21DE3. The protein was purified using the Ni-NTA column and its concentration was measured. The recombinant protein was injected in test and control mice groups. Then, antibody titration and challenge tests were made to determine the immunogenicity. After successful expression and purification, the protein band was observed at a molecular weight of 65 kDa. Western blotting confirmed the purified protein. The results of serum ELISA indicated the IgG titer was 1:204800 and 1:1600 against the recombinant protein and inactivated P. acne, respectively. Although there was no change in test mice, inflammation happened in 50% of the control group. The current study demonstrated that the CAMP-Sialidase recombinant protein can appropriately induce humoral antibodies. However, more evaluations need to introduce it as an acne vaccine candidate.

Lactobacillus rhamnosus GG is a probiotic bacterium with anti-cancer and anti-microbial characteristics. In addition, Extracellular Vesicles (EVs) from Lactobacillus rhamnosus GG showed apoptotic impacts on colorectal cancer cells. In this study, we aimed to isolate EVs from Lactobacillus rhamnosus GG (EVL) and identify its effect on cell adhesion and expression of mmp2 and mmp9 genes.

HT29 cells were exposed to 16, 32, 64, 128, 256, 512, 1024, and 2048 µg/ml EVL, and the cell viability, cell adhesion, and mRNA gene expression were investigated using the MTT assay, cell adhesion assay, and real-time PCR.

We found that 128, 256, 512, 1024, and 2048 µg/ml of EVL led to attenuated cell viability of HT29 cells (p<0.05). Moreover, the expression of both mmp2 and mmp9 genes and cell adhesion significantly reduced at 1024 and 2048 µg/ml EVL compared to untreated cells (p<0.05).

According to the findings of the present study, it is suggested that EVL could be effective in cancer cell cytotoxicity.

Somatostatin (SST) is a versatile hormone that plays a key role in inhibiting the secretion of several pituitary hormones. It is well known that SST participates in the regulation of other essential proteins throughout the body. The abnormal function of the SST protein is still not fully understood. In this study, the disease susceptible Single Nucleotide Polymorphisms (SNPs) in SST were classified by using different computational algorithms.

Sequence-based and structure-based computational tools were employed to classify the most disease susceptible nsSNPs that would have the most harmful effects on SST protein. Docking and molecular dynamic simulations were performed to compare the ability of the normal SST and its most deleterious mutants to bind with corresponding SSTRs to assess the potential role of these nsSNPs to alter protein-protein interactions.

Two nsSNPs, namely L13P, and G104S, were considered to have the most severe functional consequences on the 3D structure of SST. These results were confirmed by molecular dynamic simulations. Docking of SST and its mutant models with SST receptors (SSTR1-SSTR5) showed remarkable roles of both mutant L13P and G104S in altering the binding of SST with SSTR2 and SSTR5.

The findings of the present study provide the first comprehensive in silico prediction for assessing the damaging effects of nsSNPs on SST, which may help in a better understanding of how the altered SST would impact the overall health of the body. This study may provide a platform to conduct large-scale experiments on the genetic polymorphism of SST.

Colorectal cancer remains a leading cause of cancer-related mortality worldwide. Cancer immunotherapy involves autologous tumor-derived antigen-loaded Dendritic Cells (DCs) that activate potent antitumor immunity. Cytokine-Induced Killer (CIK) cells are a heterogeneous population of anti-tumor cytotoxic CD8+ T-cells. Cancer immunotherapy using a combination of CIK cells with DCs vaccines is a promising strategy. We investigated some of the latest developments in the DCs vaccination field, with a special emphasis on strategies to prepare highly immunogenic tumor cell antigens to load and to activate DCs. In this context, we applied the effects of immunogenic treatmentmodalities (heat shock) and four potent inducers of immunogenic cell death and apoptosis (mitoxantrone, oxaliplatin, 5 fluorouracil, and staurosporine) on DCs biology and their employment in DC-based activation of CIK cells. DCs were generated from bone marrow cells using granulocyte-macrophage colony-stimulating factor and Interleukin (IL)-4. CIK cells were expanded by interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody, and IL-2 stimulation. The cytotoxic activity of CIK cells against CMT-93 cancer cells was assessed by MTT assay. IFN-γ production of CIK cells was examined by ELISA. Coincubation of untreated DCs with CIK cells significantly induces antitumor immunity of CIK cells. Mature DCs loaded by polyethylene glycol with mitoxantrone-oxaliplatin-induced apoptotic tumor cells stimulate greater cytotoxicity of CIK cells compared to DCs loaded with staurosporine, oxaliplatin- and 5-fluorouracil-inactivated tumor cell antigens, whole tumor lysates, and total tumor RNA in CMT-93 cells. Heat shock and mitoxantrone-oxaliplatin-treatment are the best approaches for cancer cell antigens preparation for DC-induced CIK cells activation.

The current study aimed to purify antifungal compounds from Chlorella vulgaris extracts, fractions, sub-fractions and pure compounds against different strains of mycotoxigenic fungi. Antifungal activity was conducted using disc diffusion assay, TLC-bioautography and Minimum Inhibitory Concentration (MIC). Isolation, purification and structure elucidation of antifungal compounds were carried out using column chromatography, Thin Layer Chromatography (TLC), UV-Vis spectrophotometer, Gas Chromatography–Mass Spectrometry (GC-MS), and Nuclear Magnetic Resonance (NMR). C. vulgaris Diethyl Ether Extract (DEE) showed the highest antifungal activity against all tested fungi with inhibition zone from 11.5 to 21.9 mm. By fractionation of DEE, Fraction F3 (chloroform:methanol, 50:50) and F5 (methanol 100%); sub-fraction CF3-10 and CF5-10 exhibited antifungal activity against all tested fungi. Two pure compounds, hydroxyphenophytin B and hexadecanoic acid methyl ester, with antifungal activity were isolated from CF3-10 and CF5-10, respectively. C. vulgaris DEE and isolated compounds can be used as promising antifungal agents from natural sources against mycotoxigenic fungi at post-harvest or storage stages.

Recently, Spirulina platensis has scientifically become popular because of its importance as food, feed, and a natural producer of pigments with specific nutritional and functional characteristics. In this study, the effect of various environmental factors affecting growth conditions of Spirulina platensis, including primary inoculation, light-dark cycle, cultivation time, Light-Emitting Diode (LED) composition, nitrogen source, carbon source, and NaCl concentration, on biomass, C-phycocyanin (C-PC), Allophycocyanin (APC) and chlorophyll-a contents were assessed using Placket-Burman Design (PBD). Results showed that out of the seven screened factors, four factors of carbon source, LED composition, light-dark cycle and NaCl concentration significantly affected biomass production (p<0.01). Among the investigated factors, nitrogen source, light-dark cycle, and NaCl concentration had significant effects on phycocyanin production (p<0.05). Results showed that cultivation time, light-dark cycle, and NaCl concentration significantly affected the production of allophycocyanin (p<0.05). Furthermore, NaCl concentration, carbon source, LED composition, cultivation time, and initial inoculation included significant effects on chlorophyll-a production (p<0.05). The present study screened variables affecting biomass, phycocyanin, allophycocyanin, and chlorophyll-a production as the first step in optimizing Spirulina platensis growth condition. Briefly, NaCl concentration was one of the factors which had a significant impact on all responses. The dark cycle also had an effect on three dependent variables except for chlorophyll-a production.

Heat-labile enterotoxin (LT) of Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin (Stx) of Shigella dysenteriae serotype 1 are two important toxins of food-borne pathogens associated with diarrheal disease. These agents have been recognized as the first leading causes of neonatal diarrhea and the Hemolytic-Uremic Syndrome (HUS). These toxins have two subunits, A and B, that B subunit can be used as a diagnostic tool. In this study, the LTB and STXB genes were amplified by using the Polymerase Chain Reaction (PCR) technique and cloned into the prokaryotic expression vector. Following the expression of recombinant LTB and STXB proteins, mouse polyclonal anti recombinant LT enterotoxin and Shiga toxin B subunits were produced for immunological detection. An Enzyme-Linked Immunosorbent Assay (ELISA) was developed for detecting toxins using clinical samples. Our results showed that the competitive ELISA has high specificity. In addition, the detection limits for LTB and STXB were 20 ng and 90 ng, respectively. Our findings revealed that the B subunit of LT and STX can be of a great help in detecting these agents.

Wheat crop is moderately tolerant to salt stress and is considered as an excellent system to study salt stress tolerance despite its genetic complexity. In the present study, the top ten biological processes in a root proteome were mapped for Protein-Protein Interaction (PPI) networks and analyzed to examine the effect of salt stress on wheat cultivars KH-65 and PBW-373. NaCl salinity treatment 0 and 300 mM NaCl was performed on a three-leaf stage plant. Roots proteins were analyzed by liquid chromatography-mass spectroscopy. Proteins were grouped according to GENE ontology terms for biological processes and arranged in descending order. Interactome analysis was done through the STRING database. Interacting root proteins of tolerant line KH-65 show a comparatively higher number of nodes, edges, and interacting proteins than the sensitive line, PBW-373. The number of proteins whose expression was positively induced upon salinity stress was significantly higher in the roots of salinity-tolerant KH-65 than that of the PBW-373 roots. Moreover, the fold induction too was also high in the tolerant line. Similarly, the number of participant proteins in an interaction network of the KH-65 roots was higher than that of the PBW-373 cultivar. This analysis provides valuable information in elucidating the molecular mechanism associated with salt stress response in wheat seedlings’ roots. The observation is correlated with the efficient salt tolerance capacity of KH-65. The higher expressing proteins in interaction networks may be seen under the increased salt tolerance capabilities of salt-tolerant KH-65 line.

Innate immunity cytokines conduct significant functions in the stimulation and induction of liver complications in Hepatitis B infected patients. It has been reported that Royal Jelly (RJ) has important roles in decreasing the pro-inflammatory cytokines in both in vitro and in vivo conditions. This project aimed to investigate the impacts of 1 month RJ administration on interleukin 1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) serum levels in the Hepatitis B patients. In this research, 30 Hepatitis B cases (patients) were entered in order to be under the treatment of RJ for 1 month. Before and after treatment with RJ, Hepatitis B Virus (HBV) copy numbers were evaluated using Real-Time PCR and IL-1β, TNF-α, and IL-6 serum levels were evaluated using the ELISA technique. The RJ treatment significantly reduced the number of HBV-DNA copies and led to down-regulation of TNF-α and IL-6, which were not substantial. The IL-1β, TNF-α, and IL-6 serum levels were not changed after RJ treatment in both men and women. Based on the findings of the present study, it seems that RJ plays anti-viral and anti-inflammatory roles in the in vivo conditions in infected patients with HBV.

Among all the different types of cancer, Gastric Carcinoma (GC) has become the most frequently diagnosed and has continued to be a major public health issue in the last few decades worldwide. Association between the polymorphisms of the (T/C) -1031 TNF-α promoter gene sequence (rs1799964) and the incidence of gastric carcinoma was tested in Iraqi patients undergoing 5-FU plus Cisplatin, patients without chemotherapy, and healthy controls.

Blood samples were collected from patients and control groups to carry out the molecular and immunological diagnostic tests. Two ml of blood were collected in EDTA tubes for (T/C) -1031 TNF-α genotyping by using the RFLP technique. The serum part was used for the purpose of immunological tests via ELISA technique.

Findings revealed that the homozygous wild genotype (T/T) was more abundant than other genotypes (T/C and C/C) in different groups of this study (82, 73, and 72% in control, patients under treatment, and patients without treatment respectively). From the Chi-square (data of the p value), there were no significant differences between genotypes in the different groups. TNF-α concentration increased significantly in heterozygous (T/C) and homozygous (T/T) genotypes in patients without treatment (p = 0.0143) and was highly significant in healthy control samples (p = 0.0003). The results showed there were non-substantial differences (p = 0.1083) in the TNF-α concentrations between different genotypes in patients treated with chemotherapy.

The genotyping study through the RFLP Technique and allele frequency measurement revealed that the homozygous wild genotype (T/T) was more frequent compared with the other genotypes in different groups of this study. However, TNF-α concentration significantly increased in heterozygous (T/C) genotypes. Non-significant differences in TNF-α concentration were detected among different genotypes in gastric carcinoma patients under treatment of chemotherapy.

Engyodontium album, Fusarium venenatum, and Phanerochaete chrysosporium fungi have an important role in the production of proteinase K, Quorn mycoprotein, and bioremediation, respectively. There are several techniques for the detection of fungi in soil. The purpose of this study was to detect and identify E. album, F. venenatum, and P. chrysosporium, directly from soil using the Polymerase Chain Reaction (PCR) method.

A total of 240 soil samples were collected from different regions of Iran, including Tehran, Zanjan, Hamadan, Kermanshah, Kurdistan, and Hormozgan Provinces. The DNA was extracted and purified directly from soil samples with the modified phenol-chloroform method and by PVPP (Polyvinylpolypyrrolidone) column, respectively. The PCR method was performed using designed specific primers. The PCR products of the E. album, F. venenatum and P. chrysosporium with an approximate size of 248, 202, and 502 bp were sequenced, respectively.

In this study two isolates of P. chrysosporium, 1 isolate of F. venenatum, and 1 isolate of E. album were identified. The fungi detected with specific primers in soil samples were compatible with the results of sequencing.

This investigation described a reliable method that can be used to detect important fungi in the industries and biotechnology directly in soil using specific primers. The results can provide an appropriate platform for next applied research and mass production of valuable fungal products in industries and biotechnology.

Gene expression profiling has high potential in the identification of diagnostic, predictive, and therapeutic gene targets in human cancers such as colorectal cancer (CRC).  Accordingly, in this study, an integrated systems biology analysis was done on several microarray datasets to identify key genes involved in CRC chemoresistance and also to differentiate peoples who benefit from chemotherapy. Subsequently, the findings were validated experimentally.  Datasets were retrieved from Gene Expression Omnibus (GEO). Gene expression analysis was performed using the ExAtlas software. Gene enrichment analysis was done using g: profiler. Protein-Protein Interaction Network (PPIN) was constructed in STRING and visualized/analyzed by Cytoscape 3.8.0. Significant modules were identified using the MCODE plugin in Cytoscape. The clinical importance of candidate genes was evaluated using ROC analysis and immunohistochemistry. Key candidate genes were validated using Real-Time  PCR.  According to findings, 26 datasets were selected. Gene expression analysis revealed 6463 Differentially Expressed Genes (DEGs), among which 4323 were unique and 2140 were related to overlapping DEGs between datasets. The overlapping DEGs with at least four shared datasets (n=217 DEGs) were selected for further analysis. Overlapping DEGs were mainly enriched in the cellular process of response to chemicals stimulus. Most selected DEGs were enriched in KEGG pathways of cancer Benzo(a)pyrene metabolism and glucocorticoid receptor signaling. Fourteen hub genes and two significant modules were identified. Six hub genes (candidate genes) were contributed in significant modules. Among candidate genes, LCN2, CXCL8, and EGR1 expression were significantly associated with chemotherapy response of CRC patients and chemosensitivity of CRC cell lines (P<0.05).  This study revealed three genes signature for predicting chemotherapy responsiveness and treatment decision-making in CRC patients and also for therapeutic purposes.

This paper aims to analyze the medicinal uses of Ocimum basilicum L.var Genovese (basil) in western Algeria and its effectiveness. For the experiments, 154 structured questionnaires were collected to list the medicinal uses of basil. The essential oil of O. basilicum (EOB) obtained by hydro-distillation was analyzed by the GC/MS. The ethanolic and aqueous extracts (EEB and AEB) were analyzed by HPLC. The antioxidant activity was measured by DPPH assays and the antimicrobial activity was measured against five microbes. For the in vivo study, Swiss albinos mice were used to determine the toxicity using Lorke’s method. The anti-inflammatory activity was determined using the Carrageenan method. The experimental doses were converted from mice to humans using the Km factor.  The ethnobotanical study indicates that local people use basil to treat diseases and health problems (50% for inflammation and 38.11% for microbial diseases). The results also show that EOB contains 41.3% linalool, whereas ethanolic extract contains benzoic acid (50.86 mg/g). The IC50 value is 556, 878.7, and 962.3 µg/ml for EOB, EEB, and AEB, respectively. The EOB and AEB inhibit the positive Gram bacteria and yeast; the EEA inhibits the negative Gram. The LD50 is 400, 470, and >5000 mg/kg for AEB, EOB, and EEB respectively. The results of the anti-inflammatory test highlight 76.33, 71.0, and 60.43% inhibition of edema at a 100 mg/kg dose for EOB, AEB, and EEB, respectively. The Algerian basil can be considered as an antioxidant, antimicrobial and anti-inflammatory.