Identification of Hypoglycemic Glycolipids from Ipomoea murucoides by Affinity-Directed Fractionation, In Vitro, In Silico and Dynamic Light Scattering Analysis.

In the pursuit of identifying the novel resin glycoside modulators glucose-6-phosphatase and α-glucosidase enzymes, associated with blood sugar regulation, methanol-soluble extracts from the flowers of Ipomoea murucoides (cazahuate, Nahuatl), renowned for its abundance of glycolipids, were employed. The methanol-soluble extracts were fractionated by applying the affinity-directed method with glucose-6-phosphatase enzymes from a rat's liver and α-glucosidase enzymes from its intestines. Mass spectrometry and nuclear magnetic resonance were employed to identify the high-affinity compound as a free ligand following the release from the enzymatic complex. Gel permeation through a spin size-exclusion column allowed the separated high-affinity molecules to bind to glucose-6-phosphatase and α-glucosidase enzymes in solution, which led to the identification of some previously reported resin glycosides in the flowers of cazahuate, where a glycolipid mainly structurally related to murucoidin XIV was observed. In vitro studies demonstrated the modulating properties of resin glycosides on the glucose-6-phosphatase enzyme. Dynamic light scattering revealed conformational variations induced by resin glycosides on α-glucosidase enzyme, causing them to become more compact, akin to observations with the positive control, acarbose. These findings suggest that resin glycosides may serve as a potential source for phytotherapeutic agents with antihyperglycemic properties.

An Observation of a Very High Swelling of Bromovirus Members at Specific Ionic Strengths and pH.

Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) are naked plant viruses with similar characteristics; both form a T = 3 icosahedral protein capsid and are members of the bromoviridae family. It is well known that these viruses completely disassemble and liberate their genome at a pH around 7.2 and 1 M ionic strength. However, the 1 M ionic strength condition is not present inside cells, so an important question is how these viruses deliver their genome inside cells for their viral replication. There are some studies reporting the swelling of the CCMV virus using different techniques. For example, it is reported that at a pH~7.2 and low ionic strength, the swelling observed is about 10% of the initial diameter of the virus. Furthermore, different regions within the cell are known to have different pH levels and ionic strengths. In this work, we performed several experiments at low ionic strengths of 0.1, 0.2, and 0.3 and systematically increased the pH in 0.2 increments from 4.6 to 7.4. To determine the change in virus size at the different pHs and ionic strengths, we first used dynamic light scattering (DLS). Most of the experiments agree with a 10% capsid swelling under the conditions reported in previous works, but surprisingly, we found that at some particular conditions, the virus capsid swelling could be as big as 20 to 35% of the original size. These measurements were corroborated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) around the conditions where the big swelling was determined by DLS. Therefore, this big swelling could be an easier mechanism that viruses use inside the cell to deliver their genome to the cell machinery for viral replication.

Cytotoxicity, structural, conformational, and techno-functional properties of α-lactalbumin nanostructured aggregates.

Protein nanostructures can be used in food applications to improve the techno-functional properties of a food formulation. This study aims to find the best conditions for the production and conformational change of α-lactalbumin nanostructured aggregates. The criteria to determine the best operating conditions to produce α-lactalbumin nanostructured aggregates were intensification of foaming and emulsification, techno-functional proprieties, cytotoxic, and antibacterial activity of nanostructures compared with native α-lactalbumin. Conformational alterations occurred in the α-helix and sheet-ß protein structures. The size obtained by dynamic light scattering was 163.84 nm with a polydispersity index of 0.29. The nano protein improved the techno-functional property compared to the native protein. Additionally, nanostructures had no cytotoxic effect and were innocuous to bacterial activity. Thus, this study presents the best conditions to produce α-lactalbumin nanostructured aggregates with improved properties that allow new food industry applications.

Biophysical Parameters of Plasma-Derived Extracellular Vesicles as Potential Biomarkers of Bone Disturbances in Breast Cancer Patients Receiving an Individualized Nutrition Intervention.

Extracellular vesicles (EVs) are implicated in several biological conditions, including bone metabolism disturbances in breast cancer patients (BCPs). These disorders hinder the adjustment of nutrition interventions due to changes in bone mineral density (BMD). The biophysical properties of EVs (e.g., size or electrostatic repulsion) affect their cellular uptake, however, their clinical relevance is unclear. In this study, we aimed to investigate the association between the biophysical properties of the plasma-derived EVs and BMDs in BCPs who received an individualized nutrition intervention during the first six months of antineoplastic treatment. As part of the nutritional assessment before and after the intervention, body composition including bone densitometry and plasma samples were obtained. In 16 BCPs, EVs were isolated using ExoQuick® and their biophysical properties were analyzed using light-scattering techniques. We found that the average hydrodynamic diameter of large EVs was associated with femoral neck bone mineral content, lumbar spine BMD, and neoplasms' molecular subtypes. These results provide evidence that EVs play a role in BCPs' bone disorders and suggest that the biophysical properties of EVs may serve as potential nutritional biomarkers. Further studies are needed to evaluate EVs' biophysical properties as potential nutritional biomarkers in a clinical context.

Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking.

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Characterization and Differential Cytotoxicity of Gramicidin Nanoparticles Combined with Cationic Polymer or Lipid Bilayer.

Gramicidin (Gr) nanoparticles (NPs) and poly (diallyl dimethyl ammonium) chloride (PDDA) water dispersions were characterized and evaluated against Gram-positive and Gram-negative bacteria and fungus. Dynamic light scattering for sizing, zeta potential analysis, polydispersity, and colloidal stability over time characterized Gr NPs/PDDA dispersions, and plating and colony-forming units counting determined their microbicidal activity. Cell viabilities of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in the presence of the combinations were reduced by 6, 7, and 7 logs, respectively, at 10 µM Gr/10 µg·mL-1 PDDA, 0.5 µM Gr/0. 5µg·mL-1 PDDA, and 0.5 µM Gr/0.5 µg·mL-1 PDDA, respectively. In comparison to individual Gr doses, the combinations reduced doses by half (S. aureus) and a quarter (C. albicans); in comparison to individual PDDA doses, the combinations reduced doses by 6 times (P. aeruginosa) and 10 times (C. albicans). Gr in supported or free cationic lipid bilayers reduced Gr activity against S. aureus due to reduced Gr access to the pathogen. Facile Gr NPs/PDDA disassembly favored access of each agent to the pathogen: PDDA suctioned the pathogen cell wall facilitating Gr insertion in the pathogen cell membrane. Gr NPs/PDDA differential cytotoxicity suggested the possibility of novel systemic uses for the combination.

Expression, purification and characterization of the transcription termination factor Rho from Azospirillum brasilense.

The Transcription Termination factor Rho is a ring-shaped, homohexameric protein that causes transcript termination by interaction with specific sites on nascent mRNAs. The process of transcription termination is essential for proper expression and regulation of bacterial genes. Although Rho has been extensively studied in the model bacteria Escherichia coli (EcRho), the properties of other Rho orthologues in other bacteria are poorly characterized. Here we present the heterologous expression and purification of untagged Rho protein from the diazotrophic environmental bacterium Azospirillum brasilense (AbRho). The AbRho protein was purified to >99% through a simple, reproducible and efficient purification protocol, a two-step chromatography procedure (affinity/gel filtration). By using analytical gel filtration and dynamic light scattering (DLS), we found that AbRho is arranged as an homohexamer as observed in the EcRho orthologue. Secondary structure and enzyme activity of AbRho was also evaluate indicating a properly folded and active protein after purification. Enzymatic assays indicate that AbRho is a RNA-dependent NTPase enzyme.

Influence of particle size on the SARS-CoV-2 spike protein detection using IgG-capped gold nanoparticles and dynamic light scattering.

Due to the unprecedented and ongoing nature of the coronavirus outbreak, the development of rapid immunoassays to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its highly contagious variants is an important and challenging task. Here, we report the development of polyclonal antibody-functionalized spherical gold nanoparticle biosensors as well as the influence of the nanoparticle sizes on the immunoassay response to detect the SARS-CoV-2 spike protein by dynamic light scattering. By monitoring the increment in the hydrodynamic diameter (ΔDH) by dynamic light scattering measurements in the antigen-antibody interaction, SARS-CoV-2 S-protein can be detected in only 5 min. The larger the nanoparticles, the larger ΔDH in the presence of spike protein. From adsorption isotherm, the calculated binding constant (K D ) was 83 nM and the estimated limit of detection was 13 ng/mL (30 pM). The biosensor was stable up to 90 days at 4 °C. Therefore, the biosensor developed in this work could be potentially applied as a fast and sensible immunoassay to detect SARS-CoV-2 infection in patient samples.

Cationic and Biocompatible Polymer/Lipid Nanoparticles as Immunoadjuvants.

Nanostructures have been of paramount importance for developing immunoadjuvants. They must be cationic and non-cytotoxic, easily assembling with usually oppositely charged antigens such as proteins, haptens or nucleic acids for use in vaccines. We obtained optimal hybrid nanoparticles (NPs) from the biocompatible polymer poly(methyl methacrylate) (PMMA) and the cationic lipid dioctadecyl dimethyl ammonium bromide (DODAB) by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB. NPs adsorbed ovalbumin (OVA) as a model antigen and we determined their adjuvant properties. Interestingly, they elicited high double immune responses of the cellular and humoral types overcoming the poor biocompatibility of DODAB-based adjuvants of the bilayer type. The results suggested that the novel adjuvant would be possibly of use in a variety of vaccines.

A Low Energy Approach for the Preparation of Nano-Emulsions with a High Citral-Content Essential Oil.

Pectis elongata is found in the northern and northeastern regions of Brazil. It is considered a lemongrass due to its citric scent. The remarkable citral content and the wide antimicrobial properties and bioactive features of this terpene make this essential oil (EO) eligible for several industrial purposes, especially in cosmetics and phytotherapics. However, to address the problems regarding citral solubility, nano-emulsification is considered a promising strategy thanks to its improved dispersability. Thus, in this paper we propose a low-energy approach for the development of citral-based nano-emulsions prepared with P. elongata EO. The plant was hydrodistillated to produce the EO, which was characterized with a gas chromatograph coupled to mass spectrometry. The nano-emulsion prepared by a non-heated water titrating (low-energy) method was composed of 5% (w/w) EO, 5% (w/w) non-ionic surfactants and 90% (w/w) deionized water and was analyzed by dynamic light scattering. Levels of citral of around 90% (neral:geranial-4:5) were detected in the EO and no major alteration in the ratio of citral was observed after the nano-emulsification. The nano-emulsion was stable until the 14th day (size around 115 nm and polydispersity index around 0.2) and no major alteration in droplet size was observed within 30 days of storage. Understanding the droplet size distribution as a function of time and correlating it to concepts of compositional ripening, as opposing forces to the conventional Ostwald ripening destabilization mechanism, may open interesting approaches for further industrial application of novel, low-energy, ecofriendly approaches to high citral essential oil-based nano-emulsions based on lemongrass plants.

Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin.

BACKGROUND: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. OBJECTIVE: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. METHODS: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. RESULTS: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25°C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45°C and exhibited one transition state with a melting temperature of 76.8°C. CONCLUSION: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

Cationic and biocompatible polymer/lipid nanoparticles as immunoadjuvants

Nanostructures have been of paramount importance for developing immunoadjuvants. They must be cationic and non-cytotoxic, easily assembling with usually oppositely charged antigens such as proteins, haptens or nucleic acids for use in vaccines. We obtained optimal hybrid nanoparticles (NPs) from the biocompatible polymer poly(methyl methacrylate) (PMMA) and the cationic lipid dioctadecyl dimethyl ammonium bromide (DODAB) by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB. NPs adsorbed ovalbumin (OVA) as a model antigen and we determined their adjuvant properties. Interestingly, they elicited high double immune responses of the cellular and humoral types overcoming the poor biocompatibility of DODAB-based adjuvants of the bilayer type. The results suggested that the novel adjuvant would be possibly of use in a variety of vaccines.

Nano-emulsification of Aeollanthus suaveolens Mart. Ex Spreng essential oil modifies its neuroeffects?

Oil in water nano-emulsions are drug delivery systems constituted by liquid lipophilic nano-droplets dispersed through the external aqueous phase, often reaching the kinetic stability with surfactant as stabilizers. Essential oils can be the oily phase or the source of bioactive compounds. In this study, the essential oil of Aeollanthus suaveolens-a plant used in folk medicine due to its psychopharmacological effects-was used for preparation of fine nano-emulsions by a low-energy titrating method. Monodisperse small nano-droplets (ca. 70 nm; PdI 0.200) were assembled by using blends of non-ionic surfactants, indicating modulation on surfactant system lead to altering the physical property. In a separate set of experiments, we investigated the role of this modulation on biological properties of the optimal nano-emulsion. The zebrafish embryos were more susceptible to the nano-emulsion than the bulk essential oil, showing the improved bioactivity due to nano-sizing. Therefore, adult zebrafish was treated, and paralysis was observed in the groups treated with the nano-emulsion, being this finding in accordance with hypnosis. At the same essential oil dose, another behavior was observed, suggesting that expected dose-dependent effects associated to sedative-hypnotics can be achieved by nano-sizing of psychoactive essential oils. This paper contributes to the state-of-art drug delivery systems by opening perspectives for novel sedative-hypnotics nano-emulsified essentials oils that can reach hypnotic effects at considerably lower dose, when compared with bulk materials, being useful for further completed dose-response studies.Graphical abstract.

Extracellular vesicles containing the transferrin receptor as nanocarriers of apotransferrin.

Previous work by our group has shown the pro-differentiating effects of apotransferrin (aTf) on oligodendroglial cells in vivo and in vitro. Further studies showed the remyelinating effect of aTf in animal demyelination models such as hypoxia/ischemia, where the intranasal administration of human aTf provided brain neuroprotection and reduced white matter damage, neuronal loss, and astrogliosis in different brain regions. These data led us to search for a less invasive and controlled technique to deliver aTf to the CNS. To such end, we isolated extracellular vesicles (EVs) from human and mouse plasma and different neuron and glia conditioned media and characterized them based on their quality, quantity, identity, and structural integrity by western blot, dynamic light scattering, and scanning electron microscopy. All sources yielded highly pure vesicles whose size and structures were in keeping with previous literary evidence. Given that, remarkably, EVs from all sources analyzed contained Tf receptor 1 (TfR1) in their composition, we employed two passive cargo-loading strategies which rendered successful EV loading with aTf, specifically through binding to TfR1. These results unveil EVs as potential nanovehicles of aTf to be delivered into the CNS parenchyma, and pave the way for further studies into their possible clinical application in the treatment of demyelinating diseases.

Hybrid Nanoparticles of Poly (Methyl Methacrylate) and Antimicrobial Quaternary Ammonium Surfactants.

Quaternary ammonium surfactants (QACs) are microbicides, whereas poly (acrylates) are biocompatible polymers. Here, the physical and antimicrobial properties of two QACs, cetyl trimethyl ammonium bromide (CTAB) or dioctadecyl dimethyl ammonium bromide (DODAB) in poly (methyl methacrylate) (PMMA) nanoparticles (NPs) are compared to those of QACs alone. Methyl methacrylate (MMA) polymerization using DODAB or CTAB as emulsifiers and initiator azobisisobutyronitrile (AIBN) yielded cationic, nanometric, homodisperse, and stable NPs. NPs' physical and antimicrobial properties were assessed from dynamic light scattering (DLS), scanning electron microscopy, and viability curves of Escherichia coli, Staphylococcus aureus, or Candida albicans determined as log(colony-forming unities counting) over a range of [QACs]. NPs were spherical and homodisperse but activity for free QACs was higher than those for QACs in NPs. Inhibition halos against bacteria and yeast were observed only for free or incorporated CTAB in NPs because PMMA/CTAB NPs controlled the CTAB release. DODAB displayed fungicidal activity against C. albicans since DODAB bilayer disks could penetrate the outer glycoproteins fungus layer. The physical properties and stability of the cationic NPs highlighted their potential to combine with other bioactive molecules for further applications in drug and vaccine delivery.

Simple Nanoparticles from the Assembly of Cationic Polymer and Antigen as Immunoadjuvants.

Since antigens are negatively charged, they combine well with positively charged adjuvants. Here, ovalbumin (OVA) (0.1 mg·mL-1) and poly (diallyldimethylammonium chloride) (PDDA) (0.01 mg·mL-1) yielded PDDA/OVA assemblies characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) as spherical nanoparticles (NPs) of 170 ± 4 nm hydrodynamic diameter, 30 ± 2 mV of zeta-potential and 0.11 ± 0.01 of polydispersity. Mice immunization with the NPs elicited high OVA-specific IgG1 and low OVA-specific IgG2a production, indicating a Th-2 response. Delayed-type hypersensitivity reaction (DTH) was low and comparable to the one elicited by Al(OH)3/OVA, suggesting again a Th-2 response. PDDA advantages as an adjuvant were simplicity (a single-component adjuvant), low concentration needed (0.01 mg·mL-1 PDDA) combined with antigen yielding neglectable cytotoxicity, and high stability of PDDA/OVA dispersions. The NPs elicited much higher OVA-specific antibodies production than Al(OH)3/OVA. In vivo, the nano-metric size possibly assured antigen presentation by antigen-presenting cells (APC) at the lymph nodes, in contrast to the location of Al(OH)3/OVA microparticles at the site of injection for longer periods with stimulation of local dendritic cells. In the future, it will be interesting to evaluate combinations of the antigen with NPs carrying both PDDA and elicitors of the Th-1 response.

Simple Nanoparticles from the Assembly of Cationic Polymer and Antigen as Immunoadjuvants

Since antigens are negatively charged, they combine well with positively charged adjuvants. Here, ovalbumin (OVA) (0.1 mg·mL-1) and poly (diallyldimethylammonium chloride) (PDDA) (0.01 mg·mL-1) yielded PDDA/OVA assemblies characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) as spherical nanoparticles (NPs) of 170 ± 4 nm hydrodynamic diameter, 30 ± 2 mV of zeta-potential and 0.11 ± 0.01 of polydispersity. Mice immunization with the NPs elicited high OVA-specific IgG1 and low OVA-specific IgG2a production, indicating a Th-2 response. Delayed-type hypersensitivity reaction (DTH) was low and comparable to the one elicited by Al(OH)3/OVA, suggesting again a Th-2 response. PDDA advantages as an adjuvant were simplicity (a single-component adjuvant), low concentration needed (0.01 mg·mL-1 PDDA) combined with antigen yielding neglectable cytotoxicity, and high stability of PDDA/OVA dispersions. The NPs elicited much higher OVA-specific antibodies production than Al(OH)3/OVA. In vivo, the nano-metric size possibly assured antigen presentation by antigen-presenting cells (APC) at the lymph nodes, in contrast to the location of Al(OH)3/OVA microparticles at the site of injection for longer periods with stimulation of local dendritic cells. In the future, it will be interesting to evaluate combinations of the antigen with NPs carrying both PDDA and elicitors of the Th-1 response

Microbicidal Dispersions and Coatings from Hybrid Nanoparticles of Poly (Methyl Methacrylate), Poly (Diallyl Dimethyl Ammonium) Chloride, Lipids, and Surfactants.

Hybrid and antimicrobial nanoparticles (NPs) of poly (methyl methacrylate) (PMMA) in the presence of poly (diallyl dimethyl ammonium) chloride (PDDA) were previously obtained by emulsion polymerization in absence of surfactant with low conversion. In the presence of amphiphiles such as cetyl trimethyl ammonium bromide (CTAB), dioctadecyl dimethyl ammonium bromide (DODAB) or soybean lecithin, we found that conversion increased substantially. In this work, the effect of the amphiphiles on the NPs core-shell structure and on the antimicrobial activity of the NPs was evaluated. NPs dispersions casted on silicon wafers, glass coverslips or polystyrene substrates were also used to obtain antimicrobial coatings. Methods for characterizing the dispersions and coatings were based on scanning electron microscopy, dynamic light scattering, determination of thickness, rugosity, and wettability for the coatings and determination of colony-forming unities (log CFU/mL) of microbia after 1 h interaction with the coatings or dispersions. The amphiphiles used during PMMA/PDDA/amphiphile NPs synthesis reduced the thickness of the NPs PDDA shell surrounding each particle. The antimicrobial activity of the dispersions and coatings were due to PDDA-the amphiphiles were either washed out by dialysis or remained in the PMMA polymeric core of the NPs. The most active NPs and coatings were those of PMMA/PDDA/CTAB-the corresponding coatings showed the highest rugosity and total surface area to interact with the microbes. The dispersions and coatings obtained by casting of the NPs dispersions onto silicon wafers were hydrophilic and exhibited microbicidal activity against Escherichia coli, Staphylococcus aureus, and Candida albicans. In addition, a major effect of reduction in particle size revealed the suitability of nanometric and cationic NPs (sizes below 100 nm) represented by PMMA/PDDA/CTAB NPs to yield maximal microbicidal activity from films and dispersions against all microbia tested. The reduction of cell viability by coatings and dispersions amounted to 6-8 logs from [PDDA] ≥ minimal microbicidal concentration.

Gold@Silica Nanoparticles Functionalized with Oligonucleotides: A Prominent Tool for the Detection of the Methylated Reprimo Gene in Gastric Cancer by Dynamic Light Scattering.

Reprimo (RPRM) is a tumor suppressor gene involved in the development of gastric cancer. Hypermethylation of the RPRM promoter region has been found in tumor tissue and plasma samples from patients with gastric cancer. These findings suggest that circulating methylated DNA of RPRM could be a candidate for a noninvasive detection of gastric cancer. We designed a nanosystem based on the functionalization of silica coated gold nanoparticles with oligonucleotides that recognize a specific DNA fragment of the RPRM promoter region. The functionality of the oligonucleotide on the surface of the nanoparticle was confirmed by polymerase chain reaction (PCR). The nanoparticles were incubated with a synthetic DNA fragment of methylated DNA of RPRM and changes in the size distribution after hybridization were evaluated by dynamic light scattering (DLS). A difference in the size distribution of nanoparticles hybridized with genomic DNA from the KATO III gastric cancer cell line was observed when was compared with DNA from the GES-1 normal cell line. These results showed that this nanosystem may be a useful tool for the specific and sensitive detection of methylated DNA of RPRM in patients at risk of developing gastric cancer.

Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases.

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M-1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10-100 µM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.