Purification, and characterization of a new pro-coagulant protein from Iranian Echis carinatus venom.

This work aimed to purify the proteins that cause blood coagulation in the venom of the Iranian Echis carinatus snake species in a comprehensive manner. Gel filtration chromatography (GFC), Ion exchange chromatography (IEC), and Size Exclusion High-Performance Liquid Chromatography (SEC-HPLC) were utilized in the purification of the coagulation factors. The prothrombin clotting time (PRCT) and SDS-PAGE electrophoresis were performed to confirm the coagulative fractions. The fraction with the shortest coagulation time was selected. The components of this designated fraction were identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) following thorough purification. Circular dichroism (CD) was employed to determine the second structure of the coagulation factor. The crude venom (CV) was analyzed and had a total protein concentration of 97%. Furthermore, the PRCT of the crude venom solution at a concentration of 1 mg/ml was determined to be 24.19 ± 1.05 s. The dosage administered was found to be a factor in the venom's capacity to induce hemolysis. According to CD analysis, the protein under investigation had a helical structure of 16.7%, a beta structure of 41%, and a turn structure of 9.8%. CHNS proved that the purified coagulant protein had a Carbon content of 77.82%, 5.66% Hydrogen, 3.19% Nitrogen, and 0.49% Sulphur. In the present investigation, a particular type of snake venom metalloproteinase (SVMP) has undergone the process of purification and characterization and has been designated as EC-124. This purified fraction shows significant efficacy as a procoagulant. Our findings have shown that this compound has a function similar to factor X and most likely it can cause blood coagulation by activating factor II (FII).

Critical Aggregation Concentration Can be a Predictor of Doxorubicin Delivery Performance of Self-Assembling Amphiphilic Peptides with Different Hydrophobic Tails.

Amphiphilic peptides hold great potential as drug delivery systems. A popular peptide design approach has been to place amino acids in the peptide sequence based on their known properties. On the other hand, the directed discovery approach aims to screen a sequence space for a desired property. However, screening amphiphilic peptides for desirable drug delivery properties is not possible without a quantity that is predictive of these properties. We studied the predictive power of critical aggregation concentration (CAC) values on the drug delivery performance of a series of amphiphilic peptides with different hydrophobic tails and close CAC values. The CAC values were predicted by our previously developed model and doxorubicin was used as a model hydrophobic drug. All peptides showed close drug loading, entrapment efficiency, and release profile. They also formed similar spherical particles by assembling in reverse ß-sheet arrangements regardless of drug presence. Moreover, the assembled particles were able to accumulate doxorubicin inside ordinary as well as drug-resistant breast cancer cells and enhance its toxicity up to 39 and 17 folds, respectively. It can be concluded that similar drug delivery properties displayed by the peptides can be attributed to their similar hydrophilic-lipophilic balance as reflected in their close CAC values.

Two Birds with One Stone: Drug Regime Targets Viral Pathogenesis Phases and COVID-19 ARDS at the Same Time.

BACKGROUND: Severe COVID-19 or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a kind of viral pneumonia induced by infection with the coronavirus that causes ARDS. It involves symptoms that are a combination of viral pneumonia and ARDS. Antiviral or immunosuppressive medicines are used to treat many COVID-19 patients. Several drugs are now undergoing clinical studies in order to see if they can be repurposed in the future. MATERIAL AND METHODS: In this study, in silico biomarker-targeted methodologies, such as target/molecule virtual screening by docking technique and drug repositioning strategy, as well as data mining approach and meta-analysis of investigational data, were used. RESULTS: In silico findings of used combination of drug repurposing and high-throughput docking methods presented acetaminophen, ursodiol, and ß-carotene as a three-drug therapy regimen to treat ARDS induced by viral pneumonia in addition to inducing direct antiviral effects against COVID-19 viral infection. CONCLUSION: In the current study, drug repurposing and high throughput docking methods have been employed to develop combination drug regimens as multiple-molecule drugs for the therapy of COVID-19 and ARDS based on a multiple-target therapy strategy. This approach offers a promising avenue for the treatment of COVID-19 and ARDS, and highlights the potential benefits of drug repurposing in the fight against the current pandemic.

Identification of new potential candidates to inhibit EGF via machine learning algorithm.

One of the cost-effective alternative methods to find new inhibitors has been the repositioning approach of existing drugs. The advantage of computational drug repositioning method is saving time and cost to remove the pre-clinical step and accelerate the drug discovery process. Hence, an ensemble computational-experimental approach, consisting of three steps, a machine learning model, simulation of drug-target interaction and experimental characterization, was developed. The machine learning type used here was a different tree classification method, which is one of the best randomize machine learning model to identify potential inhibitors from weak inhibitors. This model was trained more than one-hundred times, and forty top trained models were extracted for the drug repositioning step. The machine learning step aimed to discover the approved drugs with the highest possible success rate in the experimental step. Therefore, among all the identified molecules with more than 0.9 probability in more than 70% of the models, nine compounds, were selected. Besides, out of the nine chosen drugs, seven compounds have been confirmed to inhibit EGF in the published articles since 2019. Hence, two identified compounds, in addition to gefitinib, as a positive control, five weak-inhibitors and one neutral, were considered via molecular docking study. Finally, the eight proposed drugs, including gefitinib, were investigated using MTT assay and In-Cell ELISA to characterize the drugs' effect on A431 cell growth and EGF-signaling. From our experiments, we could conclude that salicylic acid and piperazine could play an EGF-inhibitor role like gefitinib.

Bromelain-loaded nanocomposites decrease inflammatory and cytotoxicity effects of gliadin on Caco-2 cells and peripheral blood mononuclear cells of celiac patients.

Enzyme therapy can be an appropriate treatment option for celiac disease (CeD). Here, we developed Bromelain-Loaded Nanocomposites (BLNCs) to improve the stability and retention of bromelain enzyme activity. After the characterization of BLNCs, the cytotoxicity of BLNCs was determined on the Caco-2 cell line. The effect of BLNCs on gliadin degradation and the production of pro-inflammatory cytokines and anti-inflammatory molecules in peripheral blood mononuclear cells (PBMCs) obtained from celiac patients were assessed. Furthermore, the expression of CXCR3 and CCR5 genes was measured in CaCo-2 cells treated with gliadin, gliadin-digested with BLNCs, and bromelain. Our study demonstrated that the Bromelain entrapment efficiency in these nanoparticles was acceptable, and BLNCs have no toxic effect on cells. SDS-PAGE confirmed the digestion effect of bromelain released from nanocomposites. When Caco-2 cells were treated with gliadin digested by free bromelain and BLNCs, the expression of CXCR3 and CCR5 genes was significantly decreased. PBMCs of celiac patients treated with Bromelain and BLNCs decreased inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) production compared to untreated PBMCs. This treatment also increased IL-10 and CTLA-4 in PBMCs of CeD patients. According to the promising results of this study, we can hope for the therapeutic potential of BLNCs for CeD.

Bromelain and ficin proteolytic effects on gliadin cytotoxicity and expression of genes involved in cell-tight junctions in Caco-2 cells.

Enzyme therapy for celiac disease (CeD), which digests gliadin into non-immunogenic and non-toxic peptides, can be an appropriate treatment option for CeD. Here, we have investigated the effectiveness of bromelain and ficin on gliadin digestion using in vitro, such as SDS-PAGE, HPLC, and circular dichroism (CD). Furthermore, the cytotoxicity of gliadin and 19-mer peptide before and after digestion with these enzymes was evaluated using the MTT assay in the Caco-2 cell line. Finally, we examined the effect of these treatments along with Larazotide Acetate on the expression of genes involved in cell-tight junctions, such as Occludin, Claudin 3, tight junction protein-1, and Zonulin in the Caco-2 cell line. Our study demonstrated bromelain and ficin digestion effects on the commercial and wheat-extracted gliadin by SDS-PAGE, HPLC, and CD. Also, the cytotoxicity results on Caco-2 showed that toxicity of the gliadin and synthetic 19-mer peptide was decreased by adding bromelain and ficin. Furthermore, the proteolytic effects of bromelain and ficin on gliadin indicated the expression of genes involved in cell-tight junctions was improved. This study confirms that bromelain and ficin mixture could be effective in improving the symptoms of CeD.

Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures.

Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1 (replication factor C subunit 1), encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS neither is known nor is it understood why some, but not all expanded, motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, whereas (AAAAG)n•(CTTTT)n is the most common nonpathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic forms unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Nonpathogenic repeats did not form G-quadruplexes. Triple-stranded structures are formed by the pathogenic motifs but not by the nonpathogenic motifs. G- and C-richness of the pathogenic strands favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, stabilized by increased hydrogen bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the nonpathogenic motif. The ligand, TMPyP4, binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.

N-unsubstituted Imidazoles: Design, Synthesis, and Antimicrobial Evaluation.

BACKGROUND: All the current antifungal azoles have one substituted nitrogen atom in their imidazole or triazole rings. In this study, eleven imine and amine derivatives of imidazole, in which both nitrogen atoms of the imidazole ring are unsubstituted, were designed and synthesized. MATERIALS AND METHODS: Imine derivatives were prepared by condensation of imidazole-4-carboxaldehyde with appropriate amines, and then in the next step, using sodium borohydride, the imines were reduced to amine derivatives. Docking studies reveal unsubstituted nitrogen atom of the imidazole ring coordinated well with the heme molecule of the receptor. In vitro, antimicrobial evaluation was tested on Candida albicans, E. coli, and Staphylococcus aureus. RESULTS: Based on the results of the antimicrobial study, compound 10, which contains 4-chlorobenzyl moiety, proved to be the most potent compound against Candida albicans, and it was more active than the reference drug fluconazole and showed comparable activity to amphotericin B. Compounds 10 and 11 and compounds 8, 10 and 11 showed significant responses against E. coli and Staphylococcus aureus respectively. CONCLUSION: It is concluded that compound 10 can be acted as a new lead compound to find new azoles antifungal.

Trastuzumab Charge Variants: a Study on Physicochemical and Pharmacokinetic Properties.

Background: Post-translational modifications in bioprocessing and storage of recombinant mAbs are the main sources of charge variants. While the profile of these kinds of variants is considered an important attribute for the therapeutic mAbs, there is controversy about their direct role in safety and efficacy. In this study, the physicochemical and pharmacokinetic (PK) properties of the separated charge variants belonging to a trastuzumab potential biosimilar, were examined. Methods: The acidic peaks, basic peaks, and main variants of trastuzumab were separated and enriched by semi-preparative weak cation exchange. A panel of analytical techniques was utilized to characterize the physicochemical properties of these variants. The binding affinity to HER2 and FcγRs and the PK parameters were evaluated for each variant. Results: Based on the results, the charge variants of the proposed biosimilar had no significant influence on the examined efficacy and PK parameters. Conclusion: During the development and production of biosimilar monoclonal antibodies, evaluating the effect of their charge variants on efficacy and PK parameters is needed.

Effect of metformin alone and in combination with etoposide and epirubicin on proliferation, apoptosis, necrosis, and migration of B-CPAP and SW cells as thyroid cancer cell lines.

Background and purpose: There has not been a comprehensive study on the simultaneous effects of metformin, etoposide, and epirubicin on thyroid cancer cells. Hence, the current research proposed the in vitro study on the effect of metformin alone and in combination with etoposide and epirubicin on the rate of proliferation, apoptosis, necrosis, and migration against B-CPAP and SW-1736 cells as thyroid cancer cell lines. Experimental approach: MTT-based proliferation assay, combination index method, flow cytometry, and scratch wound healing assays were used to evaluate the simultaneous effects of the three approved drugs against thyroid cancer cells. Findings/Results: This study showed that the toxic concentration of metformin on normal Hu02 cells was more than 10 folds higher than B-CPAP and SW cancerous cells. Metformin in combination with epirubicin and etoposide could increase percentages of B-CPAP and SW cells in early and late apoptosis and necrosis phases in comparison with their single concentrations, significantly. Metformin in combination with epirubicin and etoposide could arrest the S phase in B-CPAP and SW cells, significantly. Metformin in combination with epirubicin and etoposide could reduce ~100% migration rate, whereas single concentrations of epirubicin and etoposide could reduce ~50% migration rate. Conclusion and implication: Combined treatment of metformin with anticancer drugs epirubicin and etoposide can increase the mortality in thyroid cancer cell lines and reduce the toxicity of these drugs on the normal cell line, which could be the starting point for proposing a new combination strategy in the therapy of thyroid cancer to induce more potency and reduce acute toxicity.

Combined in silico strategy for repurposing DrugBank entries towards introducing potential anti-SARS-CoV-2 drugs.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from China in December 2019 led to the coronavirus disorder 2019 pandemic, which has affected tens of millions of humans worldwide. Various in silico research via bio-cheminformatics methods were performed to examine the efficiency of a range of repurposed approved drugs with a new role as anti-SARS-CoV-2 drugs. The current study has been performed to screen the approved drugs in the DrugBank database based on a novel bioinformatics/cheminformatics strategy to repurpose available approved drugs towards introducing them as a possible anti-SARS-CoV-2 drug. As a result, 96 approved drugs with the best docking scores passed through several relevant filters were presented as the candidate drugs with potential novel antiviral activities against the SARS-CoV-2 virus.

Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP.

BACKGROUND: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli). METHODS: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum. RESULTS: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability. CONCLUSION: We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.

Recent Patents and FDA-Approved Drugs Based on Antiviral Peptides and Other Peptide-Related Antivirals.

In spite of existing cases of severe viral infections with a high mortality rate, there are not enough antiviral drugs and vaccines available for the prevention and treatment of such diseases. In addition, the increasing reports of the emergence of viral epidemics highlight, the need for novel molecules with antiviral potential. Antimicrobial peptides (AMPs) with antiviral activity or antiviral peptides (AVPs) have turned into a research hotspot and already show tremendous potential to become pharmaceutically available antiviral medicines. AMPs, a diverse group of bioactive peptides act as a part of our first line of defense against pathogen inactivation. Although most of the currently reported AMPs are either antibacterial or antifungal peptides, the number of antiviral peptides is gradually increasing. Some of the AMPs that are shown as effective antivirals have been deployed against viruses such as influenza A virus, severe acute respiratory syndrome coronavirus (SARS-CoV), HIV, HSV, West Nile Virus (WNV), and other viruses. This review offers an overview of AVPs that have been approved within the past few years and will set out a few of the most essential patents and their usage within the context mentioned above during 2000-2020. Moreover, the present study will explain some of the progress in antiviral drugs based on peptides and peptide-related antivirals.

Gefitinib-loaded polydopamine-coated hollow mesoporous silica nanoparticle for gastric cancer application.

Gastric cancer is a common malignancy that is the second cancer-associated mortality worldwide. This study aimed to develop a pH-sensitive drug delivery system including hollow mesoporous silica nanoparticles (HMSNs) loaded with gefitinib (GB) and encapsulated with mussel-inspired polydopamine (PDA) (HMSNs-GB-PDA) for the treatment of gastric cancer; where the HMSNs mainly function as drug storage platforms, and GB interrupts signaling through the epidermal growth factor receptor (EGFR) in cancer cells. In addition, PDA was used as an anticancer factor, mucoadhesive enhancing agent, stimuli, and gatekeeper to mediate the GB release. The drug delivery kinetics (in vitro), mucoadhesive properties (ex vivo), and cytocompatibility in both healthy (HGF) and gastric cancer (AGS) cell lines of this formulation were also investigated. The results showed that HMSNs-GB-PDA not only selectively killed AGS cells but also had no toxic effect on HGF cells, in such a way that more than 70% of AGS cells were eliminated at a GB concentration of 150 ug/ml, whereas only about 15% of HGF cells were killed at the same concentration. In addition, the PDA coating served as a gatekeeper, inhibited burst release, and resulted in a sustained release that lasted for a long time. The ex vivo mucoadhesiveness evaluation revealed the high mucoadhesive property (93.88%) of PDA-coated nanocarriers. According to the results, the suggested HMSNs-GB-PDA could potentially be used to treat gastric cancer.

Determination of antifungal activity and action mechanism of the modified Aurein 1.2 peptide derivatives.

BACKGROUND: With the emergence of drug-resistant fungi and the increased population prone to fungal infections, more effective antifungal drugs are needed. Aurein 1.2 is a potent antimicrobial peptide. Here, we designed a novel derivative of Aurein 1.2, called Aurein N3, which is a modified form of Aurein N2 (another Aurein 1.2 derivative), in which Lys 8 residue was replaced with Leu 13, and was also modified by creating two other mutations. METHODS: Aurein N3 was designed using several algorithms and docking studies. All peptides were synthesized and some of their bio-activity indices such as antifungal properties on 11 fungi, cytotoxicity, hemolysis, and time of the killing were investigated. Electron microscopy, lived/dead staining, and ergosterol binding assay were performed to study their mechanism of action. RESULTS: In comparison to Aurein 1.2 and N2, the docking studies showed that Aurein N3 has reduced binding energy toward ergosterol. The antifungal assessments showed that both Aurein N2 and N3 had strong activity against many fungi. Aurein N3 had lower cytotoxicity and higher binding capability to ergosterol. The hemolytic activity of Aurein N2 and N3 was less than parental Aurein 1.2. All peptides were able to attack the cell wall/membrane and enter the fungi cells. CONCLUSION: Here we introduced a novel derivative of Aurein 1.2 which has lower cytotoxicity, higher ergosterol-binding capability, and comparable antifungal activity compared to the original peptides. It can bind to ergosterol and can also attack the cell wall/membrane of fungi, although more studies are required to find its accurate mechanism of action.

Design and development of a self-assembling protein nanoparticle displaying PfHAP2 antigenic determinants recognized by natural acquired antibodies.

BACKGROUNDS: In order to move towards the elimination and eradication of malaria in the world, the development of vaccines is inevitable. Many modern vaccines are based on recombinant technology; however, they may not provide a fully protective, long-lasting immune response. One of the strategies to improve recombinant vaccines is designing the nanovaccines such as self-assembling protein nanoparticles (SAPNs). Hence, the presentation of epitopes in a repeat array and correct conformation should be considered. P. falciparum generative cell-specific 1 (PfGCS1) is a main transmission-blocking vaccine candidate with two highly conserved fragments, HAP2-GCS1 and cd loop, inducing partial malaria transmission inhibitory antibodies. Therefore, to design an effective malaria vaccine, we used cd loop and HAP2-GCS1 fragments at the amino and carboxy terminuses of the SAPN-forming amino acid sequence, respectively. METHODOLOGY/PRINCIPAL FINDINGS: The SAPN monomer (PfGCS1-SAPN) sequence was designed, and the three-dimensional (3D) structure was predicted. The result of this prediction ensured the presence of antigens on the SAPN surface. Then the accuracy of the predicted 3D structure and its stability were confirmed by 100 ns molecular dynamics (MD) simulation. The designed SAPN substructure sequence was synthesized, cloned, and expressed in Escherichia coli. With a gradual decrease in urea concentration in dialysis solutions, the purified proteins progressed to the final desired structure of the SAPN, which then was confirmed by Dynamic Light Scattering (DLS) and Field Emission Scanning Electron Microscopy (FESEM) tests. According to the Enzyme-Linked Immunosorbent Assay (ELISA), antigenic determinants were presented on the SAPN surface and interacted with antibodies in the serum of malaria patients. CONCLUSIONS/SIGNIFICANCE: Our results show that the SAPN formed by PfGCS1-SAPN has produced the correct shape and size, and the antigenic determinants are presented on the surface of the SAPN, which indicates that the designed SAPN has great potential to be used in the future as a malaria vaccine.

Intermolecular Interactions of Nucleoside Antibiotic Tunicamycin with On-Target MraYCB-TUN and Off-Target DPAGT1-TUN in the Active Sites Delineated by Quantum Mechanics/Molecular Mechanics Calculations.

Tunicamycin (TUN) is a nucleoside antibiotic with a complex structure comprising uracil, tunicamine sugar, N-acetylglucosamine (GlcNAc), and fatty acyl tail moieties. TUN, known as a canonical inhibitor, blocks vital functions of certain transmembrane protein families, for example, the insect enzyme dolichyl phosphate α-N-acetylglucosaminylphosphotransferase (DPAGT1) of Spodoptera frugiperda and the bacterial enzyme phospho-N-acetylmuramoylpentapeptide translocase (MraYCB) of Clostridium bolteae. Accurate description of protein-drug interactions has an immense impact on structure-based drug design, while the main challenge is to create proper topology and parameter entries for TUN in modeling protein-TUN interactions given the structural complexity. Starting from DPAGT1-TUN and MraYCB-TUN crystal structures, we first sketched these structural complexes on the basis of the CHARMM36 force field and optimized each of them using quantum mechanics/molecular mechanics (QM/MM) calculations. By continuing calculations on the active site (QM region) of each optimized structure, we specified the characteristics of intermolecular interactions contributing to the binding of TUN to each active site by quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses at the M06-2X/6-31G** level. The results outlined that TUN insertion into each active site requires multiple weak, moderate, and strong hydrogen bonds accompanying charge-dipole, dipole-dipole, and hydrophobic interactions among different TUN moieties and adjacent residues. The water-mediated interactions also play central roles in situating the uracil and tunicamine moieties of TUN within the DPAGT1 active site as well as in preserving the uracil-binding pocket in the MraYCB active site. The TUN binds more strongly to DPAGT1 than to MraYCB. The information garnered here is valuable particularly for better understanding mode of action at the molecular level, as it is conducive to developing next generations of nucleoside antibiotics.

Extension of human GCSF serum half-life by the fusion of albumin binding domain.

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

In Silico Design and In Vitro Evaluation of Some Novel AMPs Derived From Human LL-37 as Potential Antimicrobial Agents for Keratitis.

The human body produces two classes of antimicrobial peptides (AMPs), namely defensins and cathelicidins. In this study, a novel decapeptide (Catoid) and its dimer (Dicatoid) based on human cathelicidin (LL-37) have been designed by bioinformatics tools to be used in the treatment of bacterial keratitis. After the selection and synthesis of peptide sequences, their antimicrobial activities against the standard and resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus were evaluated. This test was performed with LL-37, gentamicin, ciprofloxacin, amikacin, and penicillin for a more accurate comparison. Furthermore, the cytotoxicity levels of the specified compounds on fibroblast cells and bovine corneal endothelial cells were investigated. The results demonstrated that the designed peptides had a superior antimicrobial activity on P. aeruginosa, compared to LL-37; however, Catoid had a better effect on the S. aureus strain. Additionally, a significant achievement is the very low toxicity level of Catoid and Dicatoid on the human skin fibroblast cell line and bovine corneal endothelial cells, compared to that of LL-37 as the initial design model.

Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate.

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.