Diosmin alleviates ulcerative colitis in mice by increasing Akkermansia muciniphila abundance, improving intestinal barrier function, and modulating the NF-κB and Nrf2 pathways.

Ulcerative colitis is a common type of inflammatory bowel disease that affects millions of individuals around the world. Traditional UC treatment has focused on suppressing immune responses rather than treating the underlying causes of UC, which include oxidative stress, inflammation, and microbiota dysbiosis. Diosmin (DIO), a naturally occurring flavonoid, possesses antioxidant and anti-inflammatory properties. This study aimed to assess the efficacy of DIO in treating dextran-sulfate sodium (DSS)-induced colitis, and to investigate some of its underlying mechanisms, with an emphasis on Akkermansia muciniphila abundance, inflammatory markers, and intestinal barrier function. C57BL/6 mice were given 4% (w/v) DSS to induce colitis. DSS-induced mice were administered DIO (100 and 200 mg/kg) or sulfasalazine orally for 7 days. Every day, the disease activity index (DAI) was determined by recording body weight, diarrhea, and bloody stool. Changes in fecal A. muciniphila abundance, colonic MUC1 and MUC2 expression, as well as oxidative stress and inflammatory markers were all assessed. Histopathological changes, colonic PIK3PR3 and ZO-1 levels, and immunohistochemical examinations of occludin and claudin-1, were investigated. DIO administration resulted in a dose-dependent decrease in DAI, as well as increase in A. muciniphila abundance and MUC2 expression while decreasing MUC1 expression. DIO also dramatically reduced colonic oxidative stress and inflammation by regulating the NF-κB and Nrf2 cascades, restored intestinal barrier integrity by inhibiting PIK3R3 and inducing ZO-1, and improved occludin/claudin-1 gene expression and immunostaining. This study provides the first evidence that DIO preserves intestinal barrier integrity and increases A. muciniphila abundance in DSS-induced colitis. However, more research is required to explore the impact of DIO on the overall composition and diversity of the gut microbiota. Likewise, it will be important to fully understand the molecular mechanisms by which A. muciniphila maintains intestinal barrier function and its potential use as an adjuvant in the treatment of UC.

Molecular docking, characterization, ADME/toxicity prediction, and anti-ulcer activity of new quercetin derivatives on indomethacin-induced gastric ulcer in mice.

Gastric ulcer (GU) is a serious upper gastrointestinal tract disorder that affects people worldwide. The drugs now available for GU treatment have a high rate of relapses and drug interactions, as well as mild to severe side effects. As a result, new natural therapeutic medications for treating GU with fewer negative side effects are desperately needed. Because of quercetin's (QCT) diverse pharmacological effects and unique structural features, we decided to semi-synthesize new QCT derivatives and test them for antiulcer activity. Docking assays were performed on the synthesized compounds to determine their affinity for TLR-4/MD-2, MyD88/TIR, and NF-κB domains, an important inflammatory pathway involved in GU development and progression. Mice were given oral famotidine (40 mg/kg/day), QCT, QCT pentamethyl (QPM), or QCT pentaacetyl (QPA) (50 mg/kg/day) for 5 days before GU induction by a single intraperitoneal injection of indomethacin (INDO; 18 mg/kg). QPM and QPA have a stronger binding affinity for TLR-4/MD-2, MyD88/TIR and NF-κB domains than QCT. In comparison, they demonstrated the greatest reduction in ulcer score and index, gastric MDA and nitric oxide (NO) contents, MyD88 and NF-κB expressions, and gastric TLR-4 immunostaining. They also enhanced the levels of GSH, CAT, COX-1, and COX-2 in the gastric mucosa, as well as HO-1 and Nrf2 expression, with histological regression in gastric mucosal lesions, with QPA-treated mice demonstrating the best GU healing. QPA is safe against all of the target organs and adverse pathways studied, with good ADME properties. However, further in vitro experiments are necessary to demonstrate the inhibitory effects of QPM and QPA on the protein targets of interest. In addition, preclinical research on its bioavailability and safety is essential before clinical management can be undertaken. Overall, the new QPA derivative could one day serve as the basis for a new class of potential antiulcer drugs.

Molecular Machinery of the Triad Holin, Endolysin, and Spanin: Key Players Orchestrating Bacteriophage-Induced Cell Lysis and their Therapeutic Applications.

Phage therapy has attracted attention as a possible alternative treatment for multi-drug resistance (MDR) infections in recent years. The lytic bacteriophages encode proteins for bacterial host envelope rupture. Phages produce endolysins muralytic enzymes, they are phage-encoded peptidoglycan hydrolases (PGHs) that cause enzymatically breakdown of the host bacterium's peptidoglycan (PG) or murein layer at the end of their lytic multiplication cycle. Phage holins regulate endolysin access to the PG, starting the lysis process at a specific moment 'lysis clock'. Phage spanins disrupt the outer membrane. Holin/Endolysin/Spanin can be utilized as novel antimicrobial agents against infections caused by bacteria. These proteins are generating interest across a variety of industries, including those in the pharmaceutical, food, biotechnological, and medical disciplines. In this review, we highlight the importance of these proteins, and their application in animal studies. Moreover, some clinical trials are mentioned.

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.

Human DNA Mutations and their Impact on Genetic Disorders.

DNA is a remarkably precise medium for copying and storing biological information. It serves as a design for cellular machinery that permits cells, organs, and even whole organisms to work. The fidelity of DNA replication results from the action of hundreds of genes involved in proofreading and damage repair. All human cells can acquire genetic changes in their DNA all over life. Genetic mutations are changes to the DNA sequence that happen during cell division when the cells make copies of themselves. Mutations in the DNA can cause genetic illnesses such as cancer, or they could help humans better adapt to their environment over time. The endogenous reactive metabolites, therapeutic medicines, and an excess of environmental mutagens, such as UV rays all continuously damage DNA, compromising its integrity. One or more chromosomal alterations and point mutations at a single site (monogenic mutation) including deletions, duplications, and inversions illustrate such DNA mutations. Genetic conditions can occur when an altered gene is inherited from parents, which increases the risk of developing that particular condition, or some gene alterations can happen randomly. Moreover, symptoms of genetic conditions depend on which gene has a mutation. There are many different diseases and conditions caused by mutations. Some of the most common genetic conditions are Alzheimer's disease, some cancers, cystic fibrosis, Down syndrome, and sickle cell disease. Interestingly, scientists find that DNA mutations are more common than formerly thought. This review outlines the main DNA mutations that occur along the human genome and their influence on human health.

Phages for treatment of Staphylococcus aureus infection.

Combating multi-drug resistant bacterial infections should be a universal urgency. The gram- positive Staphylococcus aureus (S. aureus) bacteria are generally harmless; healthy people frequently have them on their skin and nose. These bacteria, for the most part, produce no difficulties or only minor skin diseases. Antibiotics and cleansing of the affected region are usually the treatments of choice. S. aureus can become virulent causing serious infections that may lead to pustules to sepsis or death. Normally, it is thought that antibiotics may solve problems concerning bacterial infection; but unfortunately, Staphylococci have evolved mechanisms to resist drugs. Methicillin-Resistant Staphylococcus aureus (MRSA); both in hospitals and in the community, infections are evolving into dangerous pathogens. Health care practitioners may need to use antibiotics with more adverse effects to treat antibiotic-resistant S. aureus infections. Amid existing efforts to resolve this problem, phage therapy proposes a hopeful alternate to face Staphylococcal infections. When the majority of antibiotics have failed to treat infections caused by multidrug-resistant bacteria, such as methicillin- and vancomycin-resistant S. aureus, phage therapy may be an option. Here, we appraise the potential efficacy, current knowledge on bacteriophages for S. aureus, experimental research and information on their clinical application, and limitations of phage therapy for S. aureus infections.

Cornu aspersum mucin attenuates indomethacins-induced gastric ulcers in mice via alleviating oxidative stress and inflammation.

In the past three decades, a significant progress has been made in the prevention and treatment of gastric ulcers. The incidence of the disease has decreased, but gastric ulcer is still a medical problem. Currently, the available drugs for gastric ulcer treatment have many side effects; therefore, searching for new and safe therapeutic agents is mandatory. The present study aims to investigate the gastroprotective potential of Cornu aspersum (C. aspersum) mucin against gastric ulcers, and the mechanisms related to oxidative stress and inflammation. C. aspersum mucin was collected from 50 snails. The characteristics of C. aspersum mucin (chemical and microbiological) were evaluated. Mice were pretreated with famotidine and C. aspersum mucin (7.5 and 15 ml/kg b.w.) for 5 days, and then gastric ulcers were induced by indomethacin. Macroscopic examination, biochemical estimations, and Quantitative real-time PCR were carried out. Also, histopathological and immunohistopathological examinations were evaluated. We found that the high dose of the mucin significantly decreased the gastric mucosal malondialdehyde (MDA) and nitric oxide (NO) contents as well as interleukin 1ß (IL-1ß) and nuclear factor kappa ß (NF-Ò¡B) expression, and inducible nitric oxide synthase (iNOS) immunostaining. It also increased the gastric mucosal GSH and catalase contents as well as hemoxygenase-1 (HO-1) and nuclear factor-erythroid 2-related factor 2 (Nrf2) expressions with regressions in gastric mucosal lesions. In conclusion, C. aspersum mucin could be a potential therapeutic candidate to protect against gastric ulceration.

Thieno[2,3-c]isoquinolines: A novel chemotype of antiproliferative agents inducing cellular apoptosis while targeting the G2/M phase and Tubulin.

In the era of modern synthetic methodology and advanced bio-evaluation techniques and considering the notorious history of hepatocellular carcinoma (HCC), hopeful expectations regarding novel bioactive chemotypes have grown dramatically. Among the widely versatile motifs in drug discovery studies are isoquinoline and thieno[2,3-b]pyridine. Herein, the molecular merging of both motifs evoked thieno[2,3-c]isoquinoline as a novel antiproliferative chemotype being hardly studied against HCC. Accordingly, compound series 4, 5, 7 and 8 were synthesized and bioevaluated against the HepG2 cell line. The role of C7-Ac/C8-OH substituents, C8-C9 unsaturation, 1H-pyrrol-1-yl ring closure at C1-NH2 and C6-Ph p-halo-substitution were biologically studied and successfully furnished the lead 5b while showing safe profile against Vero cells. Further, flow cytometric and Annexin V-FITC/PI apoptotic bio-investigations of 5b unveiled remarkable cell cycle arrest at the G2/M phase besides a 60-fold increase in apoptosis. The use of a DFT conformational study followed by Molecular docking and molecular mechanics/generalized born surface area scoring evoked potential tubulin-targeting activity of 5b at colchicine-binding site, which was confirmed by experimental evidence (Tub Inhib IC50 = 71 µM vs. 14 µM for colchicine). Accordingly, preserving C7-acetyl and optimizing halogen position while preserving [6S,7R]-stereochemistry is crucial for optimum binding to colchicine binding site of tubulin.

Basic Guidelines for Bacteriophage Isolation and Characterization.

The world is on the cusp of a post-antibiotic period. A century ago, before the advent of antibiotics, bacteriophage therapy was the treatment of choice for bacterial infections. Although bacteriophages have yet to be approved as a treatment in Western medicine, researchers and clinicians have begun to anticipate phage therapy. Bacteriophages are viruses that depend on bacterial cell metabolism to multiply. They offer a promising alternative to the use of antibiotics and an excellent antibacterial option for combating multidrug resistance in bacteria. However, not every phage is suitable for phage therapy. In particular, prophages should not be used because they can lysogenize host cells instead of lysing them. To offer adequate therapeutic options for patients suffering from various infectious diseases, a wide selection of different phages is needed. While there is no evidence of direct toxicity induced by phage particles, it is crucial to study mammalian cell-phage interactions. This requires phage preparations to be free of bacterial cells, toxins and other compounds to avoid skewing host responses. Negative staining of purified viruses and electron microscopy remain the gold standard in the identification of bacteriophages. Interestingly, genomics has greatly changed our understanding of phage biology. Bacteriophage genome sequencing is essential to obtain a complete understanding of the bacteriophages' biology and to obtain confirmation of their lifestyle. Full genetic sequencing of bacteriophage will enable a better understanding of the phage-encoded proteins and biomolecules (especially phage lytic enzymes) involved in the process of bacterial cell lysis and death. Mass spectrometry can be used for the identification of phage structural proteins. The use of lytic phages as biocontrol agents requires the most appropriate and standard methods to ensure application safety. This review pursues recent research and methods in molecular biology for the isolation and characterization of phages to facilitate follow-up and implementation of work for other researchers. Patents related to this topic have been mentioned in the text.

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.

Lab-scale Preparation of Recombinant Human Insulin-like Growth Factor-1 in Escherichia coli and its Potential Safety on Normal Human Lung Cell Line.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) is structurally similar to insulin and acts as an endocrine hormone secreted by the liver. OBJECTIVE: Production of recombinant human IGF-1 (rhIGF-1) in Escherichia coli (E.coli) and evaluation of its proliferation stimulatory activity. METHODS: hIGF-1 gene cloned into pBSK (+) simple vector was transformed into TOP 10 chemically competent cells of E. coli. Polymerase chain reaction (PCR) was achieved using specific hIGF-1 gene primers to confirm the successful transformation. To express the rhIGF-1 in E. coli (Rosetta (DE3) pLysS); the hIGF-1 gene was cloned into the pET-15b expression vector and then the recombinant pET-15b/IGF-1 vector was transformed into a chemically prepared competent expression bacterial cells; Rosetta (DE3) pLysS. The rhIGF-1 was expressed as insoluble aggregates called inclusion bodies (IBs) using a 2 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) inducer. IBs were solubilized in a denatured form using 6 M guanidinium hydrochloride (GdmCl), followed by in vitro protein refolding using the rapid dilution method. The refolded hIGF-1 was purified using the HiTrap- ANX anion exchange column. Western blot and ELISA using rabbit polyvalent anti-hIGF- 1 were performed to confirm the protein antigenic identity. Cell proliferation activity of rhIGF-1 was testified on normal human lung cell line (WI-38). RESULTS: rhIGF-1 was purified from the HiTrap-ANX column at a concentration of 300 µg/ml. Western blot showed a single 7.6 kDa band obtained in the induced Rosetta (DE3) pLYsS. ELISA confirmed the molecular identity of the rhIGF-1 epitope, the concentration of purified rhIGF-1 obtained from the ELISA standard curve using rhIGF-1 reference protein as a standard was 300 µg/ml, and activity on WI-38 cells was 2604.17I U/mg. CONCLUSION: Biologically active native rhIGF-1 protein was successfully expressed. Patents related to the preparation of IGF-1 were mentioned along the text.

High Rates of Aminoglycoside Methyltransferases Associated with Metallo-Beta-Lactamases in Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa Clinical Isolates from a Tertiary Care Hospital in Egypt.

BACKGROUND: Multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Pseudomonas aeruginosa are the leading cause of healthcare-associated infections worldwide. OBJECTIVE: The aim was to identify the resistant phenotypes among P. aeruginosa and to characterize different aminoglycosides and carbapenem resistance genes as major mechanisms of resistance in these isolates, in Theodor Bilharz Research Institute (TBRI), a tertiary care hospital in Cairo, Egypt. METHODS: During a period of 11 months, 42 P. aeruginosa clinical isolates were collected from the microbiology laboratory by routine culture. Antimicrobial sensitivity testing to the aminoglycosides gentamicin and amikacin, and other classes of antibiotics, was performed by a disk diffusion method. Isolates were tested for aminoglycoside resistance genes, aac(6')-lb, aac-(3)-lla, rmtB, rmtC, armA, rmtD, and rmtF, and carbapenemase resistance genes bla NDM, bla VIM, and bla IMP, using conventional PCR. RESULTS: Thirty-three (78.5%) of the clinical P. aeruginosa isolates showed MDR and XDR phenotypes at 42.4% and 57.65%, respectively, and these were included in the study. Aminoglycoside resistance was found in 97%, whereas carbapenem resistance was found in 81% of the isolates phenotypically. Only 59.4% (19/26) of the aminoglycoside-resistant isolates harbored resistance genes; none of the amikacin-susceptible isolates harbored any of the tested aminoglycoside resistance genes. Aminoglycoside resistance genes rmtB, armA, aac(6')-lb, and rmtF were found at rates of 17/33 (51.5%), 3/33 (9%), 2/33 (6%), and 2/33 (6%), respectively, whereas rmtD, acc(3)-II, and rmtC were not detected. Only 40.7% (11/27) of the carbapenem-resistant isolates harbored resistance genes. Carbapenem resistance genes, bla NDM andbla VIM, were found at rates of 7/33 (21.2%) and 6/33 (18.1%), respectively, and bla IMP was not detected. CONCLUSION: Rates of MDR and XDR P. aeruginosa and resistance to aminoglycosides and carbapenems in our setting are high. Methyltransferases and metallo-beta-lactamases are the main mechanisms of resistance to aminoglycosides and carbapenems, respectively. The presence of bla NDM and rmtF in the strains confirms their rapid dissemination in the Egyptian environment.

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.

Assessment of eugenol inhibitory effect on biofilm formation and biofilm gene expression in methicillin resistant Staphylococcus aureus clinical isolates in Egypt.

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm infection is a major threat in Healthcare facilities. The search for biofilm inhibitors is essential to overcome the antibiotic resistance. Eugenol is a phyto-compound that possesses many biological properties. In this study, the aim was to estimate the effect of eugenol on biofilms of MRSA through quantifying the level of gene expression of three genes (IcaA, IcaD and SarA) involved in biofilm development.. Fifty MRSA biofilm producers collected from the microbiology lab at Theodor Bilharz Research Institute were incubated with different concentrations of eugenol for 24 h. The minimum inhibitory concentration of eugenol (MIC) that eradicates the biofilms growth was detected. mRNA was extracted from all isolates before and after the application of eugenol at 0.5 x MIC, and then subjected to quantitative real-time PCR (qPCR). Results showed that fourteen isolates out of 50 (28%) exhibited intermediate biofilm formation ability, and 36 out of 50 (72%) were strong biofilm producers. The MIC values of eugenol for MRSA ranged from 3.125% to 0.01%. The mean values of MIC in both strong and intermediate biofilm forming MRSA isolates were statistically comparable (p = 0.202). qPCR results revealed that the levels of expression of the studied genes IcaA, IcaD, and SarA were decreased after eugenol treatment when compared with their corresponding values before treatment (p = 0.001). Eugenol inhibited the formation of biofilm of MRSA isolates, indicating it could be used to control infections associated with MRSA biofilms.

Bacteriophages as Therapeutic Agents: Alternatives to Antibiotics.

Bacteriophages are bacterio-specific viruses that constitute the main portion of the environment. Bacteriophages inject their genome into the targeted bacterial cells and some of them can disrupt the metabolism of bacteria and cause bacterial cell disintegration. The application of bacteriophages to kill bacteria is known as bacteriophage therapy. Since bacteriophages target bacteria and are strain-specific, every bacteriophage/bacterial host pair is unique. They are believed to cause no harm to humans. An additional advantage of the strain-specific nature of bacteriophages is that they do not disrupt the beneficial natural flora in the body. Bacteriophage therapy in the West is not a recognized medicine at this time, and no products are registered. Some clinicians are turning to bacteriophage therapy for the treatment of antibiotic-resistant infections. Lack of adverse effects makes bacteriophage therapy ideal for use. Funding research, media attention, and the increased publication of articles helped in a widespread understanding of its therapeutic potential. The first prerequisite for the use of bacteriophage therapy is simply the availability of bacteriophages for treatment, which is often complicated at this stage of bacteriophage production. This includes providing access to all biologically active bacteriophages against the bacterial isolate of the patient and meeting regulatory criteria of purity, traceability, and characterization. A monophage preparation, which is a single bacteriophage, or a phage cocktail, which consists of a number of combined bacteriophages against one or more bacterial species may be used. Accordingly, the antibiotic resistance crisis brought back bacteriophage therapy as a potential complementary or alternative treatment. Bacteriophages are promising cheap antibacterials.

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.

Molecular and Biological Characterization of a Prepared Recombinant Human Interferon Alpha 2b Isoform.

Recombinant human interferon alpha2b (rhIFN-α2b) protein is FDA approved for treatment of many tumors and viral diseases. A rhIFN-α2b isoform has been produced and purified from the refolding reaction using high-resolution anion ion exchange chromatography. This isoform has a proper MW (19 kDa) and high purity and homogeneity. The conservation of native linear and conformational epitopes in this isoform was immunologically confirmed by Western blot and ELISA. Mass spectrometry assessment of its intact mass showed average mass (19,337 Da) equivalent to that of the expressed rhIFN-α2b protein without any chemical modification and without the first methionine. Peptide mapping of rhIFN-α2b through tryptic digestion of reductive/alkylated protein using urea as a denaturing agent gave the best pattern. The rhIFN-α2b had a high specific antiviral activity (2.5 × 108 ± 1.1 × 108IU/mg protein). In vivo clearance study of rhIFN-α2b in female SD rats (500 µg/kg, intramuscularly) revealed rapid clearance (elimination half-life 0.54 h with a maximum plasma concentration of 33,792 pg/ml) compared with the commercial rhIFN-α2 (elimination half-life 0.75-0.96 h). In conclusion, the prepared rhIFN-α2b isoform has high purity, homogeneity, native like chemical and structural composition, high antiviral activity, and proper biological stability, which reduce its immunogenicity and raise its therapeutic efficiency.