Development of Natural Cyclic Peptide Inhibitors of XRCC4/XLF Interaction for Radio-Sensitization of Breast Tumor Cells

Breast cancer is the second leading cause of cancer death in women according to the American Cancer Society. The standard treatment regimen for breast cancer involves ionizing radiation combined with surgery and chemotherapy. Ionizing radiation induces a complex signaling response in cells resulting in either growth arrest, senescence or cell death, and the cell killing after exposure to radiation results largely from DNA double-strand breaks (DSBs). There are two main mechanisms in mammalian cells responsible for repairing the DSBs; the primary mechanism is non-homologous end joining (NHEJ) and the secondary mechanism is homologous recombination (HRR). Previous studies showed that breast tumor cells depend mainly on NHEJ for repairing induced DNA damage. XRCC4 and XLF are two essential proteins in the NHEJ process. The interaction between XRCC4 and XLF (also called Cernunnos) is responsible for stimulating ligase IV for rejoining DNA ends. A single mutation on the XLF-binding interface of XRCC4 at M61, F106, M59 or D58 has been shown to disrupt its interaction with XLF and thus inhibiting NHEJ. Therefore, it is proposed that small natural cyclic peptides that bind to the XLF interface of XRCC4 near M61 and F106 can be identified through an mRNA display in vitro selection, and these peptides will inhibit NHEJ and thereby radiosensitize breast tumor cells. We have synthesized five DNA libraries that produced mRNA-peptide fusions containing a trillion unique peptide sequences that will be used for the selection of peptide inhibitors of the XRCC4/XLF interaction, and we have verified their randomness. Tagged wild-type and mutant versions of the head domain of XRCC4 protein, containing the XLF binding site, were successfully purified, and the wild-type version was applied to initial stages of selection of inhibitory peptides by mRNA display. The percentage of the mRNA-peptide fusions that bound to the XRCC4157 after the first round was 2.1%. The recovery after the second and third rounds was 1.14% and 2%, respectively. Results obtained thus far, although preliminary, suggest that the mRNA display method can be successfully applied to the XLF/XRCC4 interaction

XLF-Dependent Nonhomologous End Joining of Complex DNA Double-Strand Breaks with Proximal Thymine Glycol and Screening for XRCC4-XLF Interaction Inhibitors

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol (Tg) at the first (Tg1) , second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3’ terminus was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (\u3e10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. XLFL115D mutant completely eliminates ligation of all five substrates and previous X‑ray crystallography shows that XLF binds to XRCC4 via a “leucine lock” motif wherein L115 of XLF slips into a hydrophobic pocket in XRCC4. This makes the XRCC4-XLF interaction a good target to develop peptide inhibitors in order to radiosensitize breast tumor cells that are dependent on NHEJ to repair their DSBs after ionizing radiation exposure. Using mRNA display, we created a diverse library of 870 billion unique peptide sequences. After seven rounds of in vitro selection, the eluted fusions were cloned and sequenced. The results showed homology of sequences of five main families. We have selected representative peptides from those families (Pep 7.1-7.5), and several were chemically synthesized. However, none of these significantly inhibited XLF-dependent end joining in whole-cell extracts. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. The effectiveness of XLF-XLRCC4 inhibitors in blocking nonhomologous end joining remains to be determined

Nanotechnology integration for sars-cov-2 diagnosis and treatment: An approach to preventing pandemic

The SARS-CoV-2 outbreak is the COVID-19 disease, which has caused massive health devastation, prompting the World Health Organization to declare a worldwide health emergency. The corona virus infected millions of people worldwide, and many died as a result of a lack of particular medications. The current emergency necessitates extensive therapy in order to stop the spread of the coronavirus. There are various vaccinations available, but no validated COVID-19 treatments. Since its outbreak, many therapeutics have been tested, including the use of repurposed medications, nucleoside inhibitors, protease inhibitors, broad spectrum antivirals, convalescence plasma therapies, immune-modulators, and monoclonal antibodies. However, these approaches have not yielded any outcomes and are mostly used to alleviate symptoms associated with potentially fatal adverse drug reactions. Nanoparticles, on the other hand, may prove to be an effective treatment for COVID-19. They can be designed to boost the efficacy of currently available antiviral medications or to trigger a rapid immune response against COVID-19. In the last decade, there has been significant progress in nanotechnology. This review focuses on the virus’s basic structure, pathogenesis, and current treatment options for COVID-19. This study addresses nanotechnology and its applications in diagnosis, prevention, treatment, and targeted vaccine delivery, laying the groundwork for a successful pandemic fight

In Silico Analysis of Honeybee Venom Protein Interaction with Wild Type and Mutant (A82V + P375S) <em>Ebola</em> Virus Spike Protein

Venom from different organisms was used in ancient times to treat a wide range of diseases, and to combat a variety of enveloped and non-enveloped viruses. The aim of this in silico research was to investigate the impact of honeybee venom proteins and peptides against Ebola virus. In the current in silico study, different online and offline tools were used. RaptorX (protein 3D modeling) and PatchDock (protein–protein docking) were used as online tools, while Chimera and LigPlot + v2.1 were used for visualizing protein–protein interactions. We screened nine venom proteins and peptides against the normal Ebola virus spike protein and found that melittin, MCD and phospholipase A2 showed a strong interaction. We then screened these peptides and proteins against mutated strains of Ebola virus and found that the enzyme phospholipase A2 showed a strong interaction. According to the findings, phospholipase A2 found in honeybee venom may be an effective source of antiviral therapy against the deadly Ebola virus. Although the antiviral potency of phospholipase A2 has been recorded previously, this is the first in silico analysis of honeybee phospholipase A2 against the Ebola viral spike protein and its more lethal mutant strain

The Effective Strategies to Avoid Medication Errors and Improving Reporting Systems

Background: Population-based studies from several countries have constantly shown excessively high rates of medication errors and avoidable deaths. An efficient medication error reporting system is the backbone of reliable practice and a measure of progress towards achieving safety. Improvement efforts and system changes of medication error reporting systems should be targeted towards reductions in the likelihood of injury to future patients. However, the aim of this review is to provide a summary of medication errors reporting culture, incidence reporting systems, creating effective reporting methods, analysis of medication error reports, and recommendations to improve medication errors reporting systems. Methods: Electronic databases (PubMed, Ovid, EBSCOhost, EMBASE, and ProQuest) were examined from 1 January 1998 to 30 June 2020. 180 articles were found and 60 papers were ultimately included in the review. Data were mined by two reviewers and verified by two other reviewers. The search yielded 684 articles, which were then reduced to 60 after the deletion of duplicates via vetting of titles, abstracts, and full-text papers. Results: Studies were principally from the United States of America and the United Kingdom. Limited studies were from Canada, Australia, New Zealand, Korea, Japan, Greece, France, Saudi Arabia, and Egypt. Detection, measurement, and analysis of medication errors require an active rather than a passive approach. Efforts are needed to encourage medication error reporting, including involving staff in opportunities for improvement and the determination of root cause(s). The National Coordinating Council for Medication Error Reporting and Prevention taxonomy is a classification system to describe and analyze the details around individual medication error events. Conclusion: A successful medication error reporting program should be safe for the reporter, result in constructive and useful recommendations and effective changes while being inclusive of everyone and supported with required resources. Health organizations need to adopt an effectual reporting environment for the medication use process in order to advance into a sounder practice

Formulation and Characterization of Chitosan-Decorated Multiple Nanoemulsion for Topical Delivery In Vitro and Ex Vivo

In the present study, chitosan-decorated multiple nanoemulsion (MNE) was formulated using a two-step emulsification process. The formulated multiple nanoemuslion was evaluated physiochemically for its size and zeta potential, surface morphology, creaming and cracking, viscosity and pH. A Franz diffusion cell apparatus was used to carry out in vitro drug-release and permeation studies. The formulated nanoemulsion showed uniform droplet size and zeta potential. The pH and viscosity of the formulated emulsion were in the range of and suitable for topical delivery. The drug contents of the simple nanoemulsion (SNE), the chitosan-decorated nanoemulsion (CNE) and the MNE were 71 &plusmn; 2%, 82 &plusmn; 2% and 90 &plusmn; 2%, respectively. The formulated MNE showed controlled release of itraconazole as compared with that of the SNE and CNE. This was attributed to the chitosan decoration as well as to formulating multiple emulsions. The significant permeation and skin drug retention profile of the MNE were attributed to using the surfactants tween 80 and span 20 and the co-surfactant PEG 400. ATR-FTIR analysis confirmed that the MNE mainly affects the lipids and proteins of the skin, particularly the stratum corneum, which results in significantly higher permeation and retention of the drug. It was concluded that the proposed MNE formulation delivers drug to the target site of the skin and can be therapeutically used for various cutaneous fungal infections

Insights into the Protective Effects of Thymoquinone against Toxicities Induced by Chemotherapeutic Agents

The drugs used to treat cancer not only kill fast-growing cancer cells, but also kill or slow the growth of healthy cells, causing systemic toxicities that lead to altered functioning of normal cells. Most chemotherapeutic agents have serious toxicities associated with their use, necessitating extreme caution and attention. There is a growing interest in herbal remedies because of their pharmacological activities, minimal side effects, and low cost. Thymoquinone, a major component of the volatile oil of Nigella sativa Linn, also known as black cumin or black seeds, is commonly used in Middle Eastern countries as a condiment. It is also utilized for medicinal purposes and possesses antidiabetic, anti-cancer, anti-inflammatory, hepatoprotective, anti-microbial, immunomodulatory, and antioxidant properties. This review attempts to compile the published literature demonstrating thymoquinone’s protective effect against chemotherapeutic drug-induced toxicities

Tacrolimus-Loaded Solid Lipid Nanoparticle Gel: Formulation Development and In Vitro Assessment for Topical Applications

The currently available topical formulations of tacrolimus have minimal and variable absorption, elevated mean disposition half-life, and skin irritation effects resulting in patient noncompliance. In our study, we fabricated tacrolimus-loaded solid lipid nanoparticles (SLNs) that were converted into a gel for improved topical applications. The SLNs were prepared using a solvent evaporation method and characterized for their physicochemical properties. The particle size of the SLNs was in the range of 439 nm to 669 nm with a PDI of &le;0.4, indicating a monodispersed system. The Zeta potential of uncoated SLNs (F1&ndash;F5) ranged from &minus;25.80 to &minus;15.40 mV. Those values reverted to positive values for chitosan-decorated formulation (F6). The drug content and entrapment efficiency ranged between 0.86 &plusmn; 0.03 and 0.91 &plusmn; 0.03 mg/mL and 68.95 &plusmn; 0.03 and 83.68 &plusmn; 0.04%, respectively. The pH values of 5.45 to 5.53 depict their compatibility for skin application. The surface tension of the SLNs decreased with increasing surfactant concentration that could increase the adherence of the SLNs to the skin. The release of drug from gel formulations was significantly retarded in comparison to their corresponding SLN counterparts (p &le; 0.05). Both SLNs and their corresponding gel achieved the same level of drug permeation, but the retention of the drug was significantly improved with the conversion of SLNs into their corresponding gel formulation (p &le; 0.05) due to its higher bioadhesive properties

In Vitro and Ex Vivo Evaluation of Fluocinolone Acetonide&ndash;Acitretin-Coloaded Nanostructured Lipid Carriers for Topical Treatment of Psoriasis

Psoriasis is chronic autoimmune disease that affects 2&ndash;5% of the global population. Fluocinolone acetonide (FLU) and acitretin (ACT) are widely used antipsoriatic drugs that belong to BCS classes II and IV, respectively. FLU exhibits side effects, such as skin irritation and a burning sensation. ACT also shows adverse effects, such as gingivitis, teratogenic effects and xerophthalmia. In the present study, topical nanostructured lipid carriers (NLCs) were fabricated to reduce the side effects and enhance the therapeutic efficacy. FLU&ndash;ACT-coloaded NLCs were prepared by the modified microemulsion method and optimized by the Box&ndash;Behnken model of Design Expert&reg; version 12. The optimization was based on the particle size (PS), zeta potential (ZP) and percentage of encapsulation efficiency (%EE). The physicochemical analyses were performed by TEM, FTIR, XRD and DSC to assess the morphology, chemical interactions between excipients, crystallinity and thermal behavior of the optimized FLU&ndash;ACT-coloaded NLCs. The FLU&ndash;ACT-coloaded NLCs were successfully loaded into gel and characterized appropriately. The dialysis bag method and Franz diffusion cells were used for the in vitro release and ex vivo permeation studies, respectively. The optimized FLU&ndash;ACT-coloaded NLCs had the desired particle size of 288.2 &plusmn; 2.3 nm, ZP of &minus;34.2 &plusmn; 1.0 mV and %EE values of 81.6 &plusmn; 1.1% for ACT and 75 &plusmn; 1.3% for FLU. The TEM results confirmed the spherical morphology, while the FTIR results showed the absence of chemical interactions of any type among the ingredients of the FLU&ndash;ACT-coloaded NLCs. The XRD and DSC analyses confirmed the amorphous nature and thermal behavior. The in vitro study showed the sustained release of the FLU and ACT from the optimized FLU&ndash;ACT-coloaded NLCs and FLU&ndash;ACT-coloaded NLC gel compared with the FLU&ndash;ACT suspension and conventional gel. The ex vivo study confirmed the minimal permeation of both drugs from the FLU&ndash;ACT-coloaded NLC gel