Understanding Structural Stability of Pharmaceutically Relevant Macromolecular Complexes: A Biophysical and Biochemical Approach

Macromolecules, due to their large size and complexity are prone to a variety of physical and chemical degradations. Development of stable formulations that retain the macromolecule's stability and activity over its designated shelf life is therefore a crucial step in the production of such complexes as safe and effective therapeutics. One rapid and systematic formulation tool is the utility of the empirical phase diagrams (EPDs) which have enhanced our understanding of the response of the structure and stability of proteins to environmental perturbations. In the case of more complex macromolecular systems (e.g., multi-domain fusion proteins, large recombinant proteins, and viruses), however, the measured stability is presumably the sum of all components. This makes interpretations at the molecular level difficult. Herein, we have investigated the structural stability of three macromolecular systems of varying complexity to see if their structural stability can at least be partially understood in terms of the behavior of their individual domains and components. We have examined the effect of the observed structural alterations on the losses in activity. We have discussed how this information can be used in designing high throughput screening assays for identification of solution stabilizers for development of optimal formulations. The utility of the obtained information in interpretation of the biological functions of these systems in vivo is also evaluated. Empirical Phase Diagrams constructed based on techniques sensitive to transitions due to alterations of protein motions have been shown to provide information above and beyond that obtained by the static approach. Therefore, integration of techniques that detect extensive and subtle conformational alterations of proteins, as well as structural fluctuations into an EPD appears to be crucial. Herein, we show that plots of the temperature dependent 2nd derivative peak positions of aromatic residues have measurable slopes prior to protein unfolding and that these slopes are sensitive to the dielectric properties of the surrounding microenvironment. We further demonstrate that these slopes correlate with hydration of the buried aromatic residues in protein cores and can therefore be used as qualitative probes of protein dynamics

Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions

Formulation and Immunogencity studies of Type III Secretion System needle antigens as Vaccine Candidates

Bacterial infections caused by Shigella flexneri, Salmonella typhimurium and Burkholderia pseudomallei are currently difficult to prevent due to the lack of a licensed vaccine. Here we present formulation and immunogenicity studies for the three type III secretion system (TTSS) needle proteins MxiHΔ5, PrgIΔ5 and BsaLΔ5 (each truncated by five residues at its C terminus) as potential candidates for vaccine development. These antigens are found to be thermally stabilized by the presence of carbohydrates and polyols. Additionally, all adsorb readily to aluminum hydroxide apparently through a combination of hydrogen bonds and/or Van der Waals forces. The interaction of these proteins with the aluminum-based adjuvant changes with time to resulting in varying degrees of irreversible binding. Peptide maps of desorbed protein, however, suggest that chemical changes are not responsible for this irreversible association. The ability of MxiHΔ5 and PrgIΔ5 to elicit strong humoral immune responses was tested in a murine model. When administered intramuscularly as monomers, the needle components exhibited dose dependent immunogenic behavior. The polymerized version of MxiH was exceptionally immunogenic even at low doses. The responses of both monomeric and polymerized forms were boosted by adsorption to an aluminum salt adjuvant

Evidence for an association of TP53 codon 72 polymorphism with sporadic colorectal cancer risk in Isfahan

<ul> <li><strong>BACKGROUND</strong>: A common polymorphism at codon 72 of TP53 gene has been associated with increased risk for many human cancers. We studied this TP53 polymorphism in colorectal adenocarcinomas in small population selected from Isfahan city.</li> <li><strong>METHODS</strong>: Samples: We undertook a case-control study on 180 controls and 180 paraffin block specimens of sporadic colorectal adenocarcinomas. PCR amplification of TP53 codon 72 polymorphism: TP53 codon 72 genotypes were detected by PCR using specific primer pairs for amplifying the Proline or the Arginine alleles.</li> <li><strong>STATISTICAL ANALYSES:</strong> The 42-test was used to assess the significance of any difference in the prevalence of TP53 codon 72 polymorphism between colorectal cancer patients and controls.</li> <li><strong>RESULTS</strong>: In control samples, the genotype distribution for TP53 polymorphism showed 28.3%, 48.9% and 22.8% for the Arginine/Arginine, Arginine/Proline and Proline/Proline genotypes, respectively. In the cancer group 40% of the cases were Arginine/Arginine, 42.2% were Arginine/Proline and 17.8% were Proline/Proline. A significant difference between cases and controls was found for the Arginine/Arginine genotype compared with (grouped) Arginine/Proline and Proline/Proline genotypes (Odds Ratio = 1.686 (1.085-2.620), P = 0.02).</li> <li><strong>CONCLUSIONS</strong>: TP53 codon 72 polymorphism may be a genetic predisposing factor for colorectal adenocarcinomas in Isfahan city.</li> <li><strong>KEYWORDS</strong>: Colorectal adenocarcinoma, TP53, Arginine, Proline, Polymorphism.</li> </ul&gt

Novel effects of Rosa damascena extract on patients with neurocognitive disorder and depression: A clinical trial study

Background: Dementia as a major cognitive neurological disorder is defined as impairment in one or more cognitive territories compared with the former level of performance. This disorder disrupts patient's independence, and the patient would need others aid in order of doing daily and complex activities. The aim of this study was to evaluate the efficacy of Rosa damascena extract in the improvement of cognitive function in patients with dementia. Methods: This study is a randomized double-blind, placebo-controlled clinical trial on 40 patients older than 55 years with dementia referred to Specialized Elderly Patients Clinic in 2015–2016. Patients were divided randomly into two groups (control and intervention). The intervention group used donepezil and R. damascena capsules, and in control group, placebo capsule instead of R. damascena added on donepezil. Four test was filled three times at the study initiation, after month one and also after month three: Mini–Mental State Examination (MMSE) and Addenbrooke's Cognitive Examination Revised (ACE-R) were used for cognition evaluation, for depression assessment, Geriatric Depression Scale was administered, and checklist of memory and behavioral disorders were filled. Results: The results showed add-on donepezil and R. damascena versus placebo improved cognitive impairment based on MMSE with P = 0.002, ACE-R with total P = 0.001, depression (P = 0.012), behavioral disorders (P < 0.001), and daily activity (P < 0.001). Conclusions: The R. damascena extract affected cognitive impairment of dementia patients significantly and also have significant effects on improving depression and behavioral problems

Lactobacilli Modulate Hypoxia-Inducible Factor (HIF)-1 Regulatory Pathway in Triple Negative Breast Cancer Cell Line

Objective Hypoxia-Inducible Factor (HIF)-1 plays an essential role in the body’s response to low oxygen concentrations and regulates expression of several genes implicated in homeostasis, vascularization, anaerobic metabolism as well as immunological responses. Increased levels of HIF-1α are associated with increased proliferation and more aggressive breast tumor development. Lactobacilli have been shown to exert anti-cancer effects on several malignancies including breast cancer. However, the exact mechanism of such effect is not clear yet. The aim of this study was to analyze the expression of selected genes from HIF pathway in a triple negative breast cancer cell line (expressing no estrogen and progesterone receptors as well as HER-2/Neu), MDA-MB-231, following treatment with two lactobacilli culture supernatants. Materials and Methods In this experimental study, we analyzed the expression of HIF-1α, SLC2A1, VHL, HSP90, XBP1 and SHARP1 genes from HIF pathway in MDA-MB-231 cells, before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants (LCS and LRS, respectively) by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results Both LRS and LCS had cytotoxic effects on MDA-MB-231 cells, while the former type was more cytotoxic. LRS dramatically down-regulated expression levels of the HIF-1α, HSP90 and SLC2A1 in the MDA-MB-231 cells. LCS had similar effect on the expression of HSP90, to what was observed in the LRS treatment. The expression level of tumor suppressor genes VHL and SHARP1 were also decreased in LCS treated cells. Conclusion Although both LCS and LRS had cytotoxic effects on the MDA-MB-231 cells, it is proposed that LRS could be more appropriate for pathway directed treatment modalities, as it did not decrease expression of tumor suppressor genes involved in HIF pathway. Down-regulation of HIF pathway mediated oncogenes by LRS suggests that the cytotoxic effects of this Lactobacillus may at least be partly caused by this mechanism. As previous studies have shown that inhibition of HIF-1α and HSP90 expressions have therapeutic impact on cancer treatment, the inhibitory effect of LRS on expression of these genes implies that this Lactobacillus can be used in treatment strategies