Fundamental Uses of Peptides as a New Model in Both Treatment and Diagnosis.

An amino acid short chain is known as a peptide. Peptide bonds are the connections that hold the amino acids of a peptide together in a particular order. Characteristically, the shorter length of peptides helps to identify them from proteins. Different ways are used to classify peptides, including chain length, source of peptides, or their biological functions. The fact that peptides serve several purposes suggests that there is a foundation for improvement in peptide production and structure to enhance action. In addition, many patents on peptides for therapeutic and diagnostic approaches have been obtained. This review aims to give an overview of peptides used recently in treatment and diagnosis.

Interdisciplinary gene manipulation, molecular cloning, and recombinant expression of modified human growth hormone isoform-1 in E. coli system.

BACKGROUND: Growth hormone (GH) is a hormone that promotes growth, cell reproduction, and cell restoration in humans and animals. OBJECTIVES: Production of recombinant human growth hormone (rhGH) in Escherichia coli (E. coli) and assessment of its characteristics and proliferation stimulatory activity. METHODS: The hGH gene was cloned into a pET 3a expression vector and transformed into a competent E. coli cell. The refolded hGH was purified, Western blot and batch fermentation were performed. Cell cytotoxicity was tested on Vero cells, and MALDI-TOF and Nano-LC-ESI MS/MS were used for protein and target peptide analysis. RESULTS: Induced rhGH was purified with a concentration of 511.9 mg/ml. Western blot confirmed the molecular identity of rhGH, showing a single 22 kDa band. The bacterial growth at OD600 after 24 h in batch fermentation was 9.78 ± 0.26, and wet cell weight (WCWg/L) was 15.2 ± 0.32. Purified rhGH activity on Vero cells was 0.535 IU/mg. LC-MS/MS analysis revealed a score of 70.51 % and coverage of 60.37 %. CONCLUSION: Biologically active native rhGH protein was successfully expressed in the Prokaryotic system. Our goal is to increase its production on a pilot level in the native form at a high activity effect identical to isoform 1.

A novel antibacterial approach of Cecropin-B peptide loaded on chitosan nanoparticles against MDR Klebsiella pneumoniae isolates.

Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The proper treatment choice for MDR-KP infections is not well determined which renders the problem more complicated, thus making the control of such infections a serious challenge for healthcare professionals. This study aims to encapsulate the cationic antimicrobial peptide; Cecropin-B (Cec-B), to increase its lifetime, drug targeting, and efficacy and study the antimicrobial effect of free and encapsulated recombinant rCec-B peptide on multidrug-resistant K. pneumoniae (MDR-KP) isolates. Fifty isolates were collected from different clinical departments at Theodore Bilharz Research Institute. Minimal inhibitory concentrations (MICs) of rCec-B against MDR-KP isolates were determined by the broth microdilution test. In addition, encapsulation of rCec-B peptide into chitosan nanoparticles and studying its bactericidal effect against MDR-KP isolates were also performed. The relative expression of efflux pump and porin coding genes (ArcrB, TolC, mtdK, and Ompk35) was detected by quantitative PCR in treated MDR-KP bacterial isolates compared to untreated isolates. Out of 60 clinical MDR isolates, 50 were MDR-KP. 60% of the isolates were XDR while 40% were MDR. rCec-B were bactericidal on 21 isolates, then these isolates were subjected to treatment using free nanocapsule in addition to the encapsulated peptide. Free capsules showed a mild cytotoxic effect on MDR-KP at the highest concentration. MIC of encapsulated rCec-B was higher than the free peptide. The expression level of genes encoding efflux and porin (ArcrB, TolC, mtdK, and Ompk35) was downregulated after treatment with encapsulated rCec-B. These findings indicate that encapsulated rCec-B is a promising candidate with potent antibacterial activities against drug-resistant K. pneumoniae.

A multimodality therapeutic application on Toxoplasma gondii encephalitis utilizing Spiramycin and 'de novo' Ferula asafetida in immunodeficient mice.

This study investigated a 'de Novo' medicinal herb, Ferula asafetida (FA), against toxoplasma encephalitis either alone or combined with spiramycin (SP). Female Swiss-Webster mice (n = 72) were divided into three batches. Batch-I received no DMS to serve as an immunocompetent control, batch-II was immune-suppressed with the DMS (0.25 mg/g/day) for 14 days pre-infection, whilst batch-III was immune-suppressed with the DMS on the same day of infection. All experimental mice were inoculated with Toxoplasma gondii ME49 cysts (n = 75). Each batch was split into four subgroups: Mono-SP, mono-FA, combined drug (SP + FA), or neither. Therapies were administered on day zero of infection in batches (I and II) and 35 days post-infection in batch (III). Treatments lasted for 14 days, and mice were sacrificed 60 days post-infection. Histopathological changes, cysts load, and CD4 and CD8 T-cells were counted in brain tissues. The cyst-load count in mice receiving SP + FA was significantly (p < .0001) the least compared to the mono treatments in all protocols. Interestingly, the combined therapy demolished the T-cell subsets to zero in immunocompetent and immunocompromised infected mice. In conclusion, F. asafetida might be a powerfully natural, safe vehicle of SP in the digestive system and/or across the brain-blood barrier to control toxoplasmosis even through immunodeficient conditions.

Investigation of novel HCV therapies: Boscia angustifalia & Boscia senegalensis extracts loaded on galactosylated chitosan nanoparticles synthesized by eco-friendly method for HCV treatment.

Hepatitis C virus (HCV) is a major causative agent of chronic liver diseases including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Treatment of HCV has evolved from early interferon monotherapy to the current all-oral regimens using direct-acting antivirals. However, antiviral resistance has become a critical issue in the treatment of chronic hepatitis C after receiving therapy with direct-acting antivirals (DAA) with a 0.5 % chance of the hepatitis C virus recurrence, similar to other chronic viral infections. So, retreatment options following treatment failure have become crucial issues. Hence, this study aims to investigate a new promising therapy for HCV. In the field of nanomedicine, chitosan nanoparticles are well-known delivery systems that are frequently used as polymeric carriers. Galactosylated chitosan nanoparticles have been widely applied in HCV treatment. In this study, we have modified galactosylation by an eco-friendly method using l-ascorbic instead of hazardous reagents and we have loaded it with newly tested two Boscia extracts each in three different concentrations. The synthesized chitosan nanoparticles showed two dispersion peaks, at 196 ± 29 nm and 1.33 ± 0.36 µm, with a zeta potential of +3.3 ± 0.4mV with high stability in a range of 40.7 mV. The percentage of encapsulation of Boscia angustifalia extract was found to be 46.58 ± 1.33 % and for Boscia senegalensis extract was 9.77 ± 0.33 %. The release of Boscia angustifalia extract from the nanoparticles was about 40 % in acidic media after 180 min and about 60 % in normal pH. However, the release of Boscia senegalensis extract was 20 % in acidic media and 56 % in normal media after 24 h. Testing of these two newly developed composites against HCV was carried out using an in vitro system for the production of hepatitis C virus (HCV) which was established by infection of human hepatoma cells. Evidence for persistent virus production was monitored by the ELISA technique using an anti-HCV-specific antibody. Results obtained showed that all samples had an anti-HCV activity that increased by increasing concentration, and Boscia angustifalia had remarkable anti-HCV activity compared to Boscia senegalensis.

Metabolite Profiling of Alocasia gigantea Leaf Extract and Its Potential Anticancer Effect through Autophagy in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a poor-prognosis type of cancer with high resistance to chemotherapy, making the search for safe drugs a mandatory issue. Plant-derived products have potential to reduce negative side effects of cancer treatments. In this work, ability of a defatted methanolic extract of Alocasia gigantea leaves to fight HCC was evaluated in an animal model. Overall, treatment of HCC-induced mice with the methanolic extract at 150 mg/kg body weight for four consecutive weeks caused induction of autophagy through silencing of the relative expression of autophagy suppressor (mTOR) and inducement of autophagy markers (AMPK, Beclin-1, and LC-3). Moreover, it improved preservation of the hepatic histological architecture of the animals, with minor hepatocytic changes but scattered foci of hepatocytic apoptosis. Chemical profiling of the methanolic extract via ultra-high-performance liquid chromatography coupled to a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI-MS/MS) allowed identification of di-C-glycosyl flavones, mostly represented by 6-C-hexosyl-8-C-pentosyl apigenin isomers, which may possibly be associated with inducement of the autophagy pathway in HCC. Overall, these outcomes gave an initial visualization of the operative effect of some compounds in A. gigantea leaves that are potential treatment for HCC.

Phycocyanin stimulates ulcerative colitis healing via selective activation of cannabinoid receptor-2, intestinal mucosal healing, Treg accumulation, and p38MAPK/MK2 signaling inhibition.

Ulcerative colitis (UC) is a chronic inflammatory condition that until this date, lacks curative treatments. Previously, synthetic selective CB2 receptor (CB2R) agonists demonstrated effective preclinical anti-inflammatory activities in UC. Phycocyanin (PC), photosynthetic assistant protein isolated from Microcystis aeruginosa Kützing blue green algae, has multiple pharmacological effects, however, it's effect against UC remains unexplored. Our study aimed at investigating the therapeutic effectiveness of PC against UC, and correlating its mechanisms with CB2R agonistic activities. In silico; PC showed structural similarity with endocannabinoid receptors' ligand "Δ9-tetrahydrocannabinol", target prediction studies suggested high affinity for G-coupled protein family-receptors, and molecular docking affirmed preferable affinity towards CB2R vs CB1R. In LPS-exposed-Caco-2 cell line; PC demonstrated comparable interaction with CB2R, and downregulation of CB2R, p38 and MK2 gene expressions with reference agonist "6d", and exhibited preferred selectivity towards CB2R over CB1R. In DSS-induced mice; PC-treatment ameliorated DSS-induced colon shortening, elevated disease activity index, and colonic pathological alterations. PC showed effective CB2R activation through potent anti-inflammatory activities, Treg-cell accumulation, suppression in p38MAPK/MK2 signaling, and tight junction barrier restoration as indicated by ultrastructural examinations, elevated ZO-1 and occludin protein expressions, and Ki67 immunohistochemical expression in colonic tissues. Additionally, PC alleviated intestinal dysbiosis via downregulating LPS/TLR4/NF-κB signaling and gut microbiota maintenance. Notably, PC-protective activities were abolished when co-administered with SR144528 (selective CB2 antagonist) except for gut microbiota maintenance, which was independent from CB2R activation. Our findings provide evidence of PC effectiveness against UC through acting as CB2R agonist, thus expanding its possible therapeutic application against other inflammatory diseases.

Isolation and characterization of lytic bacteriophages from sewage at an egyptian tertiary care hospital against methicillin-resistant Staphylococcus aureus clinical isolates.

Background: Methicillin resistant Staphylococcus aureus (MRSA) is a pathogen to humans causing life-threatening infections. MRSA have the capability to grow resistance to many antibiotics, and phage therapy is one treatment option for this infection. Objectives: The aim of the present study was to isolate and characterize the lytic bacteriophages specific to MRSA from domestic sewage water at a tertiary care hospital in Egypt. Methods: Thirty MRSA strains were isolated from different clinical samples admitted to the microbiology lab at Theodor Bilharz Research institute (TBRI) hospital, Giza, Egypt. They were confirmed to be MRSA through phenotypic detection and conventional PCR for mecA gene. They were used for the isolation of phages from sewage water of TBRI hospital. Plaque assay was applied to purify and quantify the titer of the isolated phages. The host range of the isolated phages was detected using the spot test assay. The morphology of phages was confirmed using transmission electron microscope (TEM). Digestion of DNA extracted from phages with endonuclease enzymes including EcoRI and SmaI was performed. SDS-PAGE was performed to analyze MRSA specific phage proteins. As a positive control prophages were isolated from a mitomycin C (MitC) treated culture of S. aureus strain ATCC25923. Further characterization using conventional polymerase chain reaction (PCR) was used to select three known Staphylophages by detecting the endolysin gene of phage K, the polymerase gene of phage 44AHJD, and the minor tail gene of phage P68. Results: Isolated phages in this research displayed a wide host range against MRSA using the spot test, out of thirty tested MRSA isolates 24 were sensitive and got lysed (80%). The titer of the phages was estimated to be 1.04 × 106 pfu/ml using plaque test. Identification of head and tail morphology of the phages was achieved using TEM and they were designated to tailed phages of order Caudovirales, they composed an icosahedral capsid. Prophages were isolated through MitC induction. DNA of phages was digested by endonuclease enzymes. Conventional PCR yielded 341 bp of phage K endolysin gene and phage P68 minor tail protein gene 501 bp. Protein analysis using SDS-PAGE showed 4 proteins of sizes between 42 kDa and 140 kDa. Conclusion: Phages isolated here are alike to others mentioned in previous studies. The high broad host range of the isolated phages is promising to control MRSA and can be in the future commercially suitable for treatment as lysate preparations. Animal models of phage-bacterial interaction will be our next step that may help in resolving the multidrug resistant crisis of MRSA in Egypt.

Inflammatory response and immunohistochemical characterization of experimental calcium silicate-based perforation repair material.

This study compares the immunohistochemical reaction of a new experimental tricalcium silicate perforation repair material to mineral trioxide aggregate (MTA) and Biodentine. A total of 162 mature premolar teeth from 12 dogs were divided into three experimental groups (n = 54 teeth each) according to the evaluation period: 1, 2 and 3 months. Each group was further divided into two equal subgroups (n = 27 teeth each) according to the time of repair: immediate repair and delayed repair. Each subgroup was subdivided according to the material used into three experimental subdivisions (n = 8 teeth each): MTA, Biodentine (Septodont) and experimental material, and two control subdivisions: positive control (n = 2 teeth) and negative control (one tooth). Under general anaesthesia, access cavity was done. Cleaning and shaping were performed using ProTaper universal rotary instruments. The canals were obturated using cold lateral compaction technique with Gutta percha and Adseal sealer. Furcation perforations were created then randomly sealed using the three materials either immediately or after one month (delayed repair). Inflammatory cell count and immunohistochemical analysis of osteopontin-positive area fraction were digitally analysed using the ImageJ software. Delayed furcal perforation repair showed significantly higher inflammatory cell count than immediate repair. No significant difference in inflammatory cell count and immunohistochemical analysis was detected between the three tested materials. The immunohistochemical analysis revealed the highest immunopositive area fraction in the 3-month evaluation period. The experimental tricalcium silicate cement performed similarly to Biodentine and MTA regarding the osteopontin expression during perforation repair, suggesting it is a suitable alternative with favourable handling characters.

Purified recombinant human Chromogranin A N46 peptide with remarkable anticancer effect on human colon cancer cells.

Human Chromogranin A N46 (CGA-N46) is a weak cationic α-helical peptide with wide-spectrum antibacterial, fungal, and anticancer activities. In this study, the recombinant human CGA-N46 peptide was expressed successfully in Escherichia coli. The gene of CGA-N46 was cloned into the expression vector pET-15b without a fusion tag at the N terminus and the peptide was expressed using Isopropyl-ß-d-thiogalactoside (IPTG) as an inducer. Using 8 M guanidinium HCl, inclusion bodies containing the peptide were purified and solubilized. The rhCGA-N46 peptide was purified using Q-FF anion exchange column. The cytotoxicity of the purified rhCGA-N46 peptide was investigated on WI-38 human lung normal cell line. The anticancer activity was studied on human colon cancer cell line; HCT-116 cell line. Besides, the possible involvement of rhCGA-N46 peptide in regulating apoptotic signal pathways was analyzed by detecting the expression levels of BCL2, BID, and CAS-8 in the treated cells. The results concluded that the active peptide recovery was up to 41.98%. The purified rhCGA-N46 was safe on normal cells with IC50 = 227.74 µg/ml (40.67 µM) and had an obvious anticancer effect on colon cancer cells with IC50 = 1.997 µg/ml (356.6 nM). The expression level of BCL2 was down-regulated and BID and CAS-8 were up-regulated significantly in treated HCT-116 cells compared to untreated. In conclusion, the rhCGA-N46 peptide was produced successfully in the native form with promising anti-colon cancer activity.

Metallothionein expression in Aspergillus exposed to environmentally relevant concentrations of heavy metals at different pH levels.

Heavy metal pollution represents a health threat. Many fungal species are capable of tolerating various heavy metals, especially if they are isolated from a contaminated watercourse. One of the mechanisms by which fungi can sequester certain heavy metals is synthesizing stress proteins. The aim of this study is to investigate the production of metallothioneins in Aspergillus oryzae and Aspergillus clavatus exposed to environmentally relevant concentrations of Cd, Cu, Fe, and Zn at neutral, alkaline, and acidic pH conditions within 10 days. We determined the concentrations of these heavy metals in certain watercourses representing Behira and Giza governorates; also, we identified the most prevalent fungal species. We carried out a statistical correlation between different heavy metals and the isolated fungi. Then, in the laboratory, we exposed two of the most prevalent fungal species to the environmentally detected concentrations of the heavy metals and their doubles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that in A. oryzae, the metallothionein bands appeared in neutral medium containing Cd and Cu and in alkaline medium containing Cd and Zn, while in A. clavatus, no metallothionein bands appeared at all. In conclusion, metallothionein is a good indicator of pollution with Cd, Cu, and Zn in Aspergillus oryzae, and pH plays a central role in metallothionein production.

Recombinant Expression of Cec-B Peptide in Escherichia coli with a Significant Anticancer Effect on Hepatocellular Carcinoma.

BACKGROUND: Cecropin-B (Cec-B) is an Antimicrobial Peptide (AMP) found in insects. OBJECTIVES: Recombinant production of Cec-B peptide in Escherichia coli (Rosetta™ DE3), and studying its anticancer effect on Hepatocellular Carcinoma Cell line (HCC). METHODS: The Cec-B gene of Drosophila melanogaster was synthesized by PCR assembly using the Simplified Gene Synthesis (SGS) method. To express the recombinant peptide in E. coli (Rosetta™ DE3); the synthesized gene was cloned into pET-15b expression vector. The recombinant peptide was expressed as insoluble aggregates called Inclusion Bodies (IBs) using 2mM lactose inducer. IBs were solubilized in a denatured form using 8 M urea followed by in-vitro protein refolding using rapid dilution method. The refolded Cec-B was purified using cation-exchange SP-FF column. Cytotoxicity of recombinant Cec-B (rCec-B) was reported on normal human lung cell line (WI-38), and Hepatocellular carcinoma cell line (HepG2). RESULTS: The Cec-B gene was expressed and purified at concentration 1.212±0.1 mg/ml which represents 48.49±4% of the total proteins injected to the column (2.5±0.2 mg/ml). The safe dose of purified rCec-B on normal WI-38 cells was calculated to be 1.57 mg/ml. The half-maximal Inhibitory Concentration (IC50) of rCec-B on HepG2 cell line was calculated to be 25 µg/ml. Scanning Electron Microscope (SEM) showed that untreated and treated HepG2 cells had cell diameters from 11-12.92 µm and 14.18-21.58 µm, respectively. CONCLUSION: The results of this study revealed a successful expression of the rCec-B peptide using a pET-based expression system with a simple purification step. The purified peptide could be considered as a hopeful anticancer drug against HCC.

A Novel Cell-based In vitro Assay for Antiviral Activity of Interferons α, ß, and γ by qPCR of MxA Gene Expression.

BACKGROUND: Human MxA gene is related to the class of interferon (IFN)-stimulated genes (ISGs) that plays a role in antiviral resistance. OBJECTIVE: Implementation of standard curves obtained from designing a procedure for data processing in relative qPCR between MxA expression and interferon's antiviral activity (IU/ml). These standard curves can be used to detect the antiviral activity of any new compound rapidly and safely. METHODS: To detect the optimum incubation time for maximum MxA gene expression in human peripheral blood mononuclear cells (PBMC), the isolated human PBMCs (1x106 cells) were incubated with a concentration of 1000 IU/ml of each IFN at different time intervals; 2 h, 4 h, 6 h, and 24 h post-treatment. A standard curve was performed for each IFN (α, ß, and γ) at different concentrations (250, 500, 750, 1000, 1500, and 2000 IU/ml). RESULTS: As observed at 4 h incubation time of 1000 IU/ml concentration, IFN-γ provided a higher expression of MxA compared to IFN-α and IFN-ß. Correlation analyses between IFN-α and IFN-ß, IFN-ß and IFN-γ were non-significant. However, there was a significant correlation between IFN-α and IFN-γ (p<0.01). Receiver operator characteristic (ROC) analysis revealed that cut-off values of IFN- γ, IFN-ß, and IFN-α were 58.14 > 7.31 and > 3.33, respectively. CONCLUSIONS: The relative expression of MxA is a biomarker for IFN-α, ß, and γ, especially IFN-α. It has compiled and validated a standard curve-based protocol for PCR data processing. It shows that the standard curve is an easy alternative tool to assess antiviral activity. We revised all patents relating to the antiviral assays of the used interferons.