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.

SERS Investigation of Cancer Cells Treated with PDT: Quantification of Cell Survival and Follow-up.

In this study Surface Enhanced Raman Spectroscopy (SERS) data recorded from mouse mammary glands cancer cells (4T1 cell line) was used to assess information regarding differences between control, death and viable cells after Photodynamic Therapy (PDT) treatment. The treatment used nanoemulsions (NE/PS) loaded with different chloroaluminumphthalocyanine (ClAlP) photosensitizer (PS) contents (5 and 10 µmol × L-1) and illumination (660 nm wavelength) at 10 J × cm-2 (10 minutes). The SERS data revealed significant molecular alterations in proteins and lipids due to the PDT treatment. Principal Component Analysis (PCA) was applied to analyze the data recorded. Three-dimensional and well reproductive PCA scatter plots were obtained, revealing that two clusters of dead cells were well separated from one another and from control cluster. Overlap between two clusters of viable cells was observed, though well separated from control cluster. Moreover, the data analysis also pointed out necrosis as the main cell death mechanism induced by the PDT, in agreement with the literature. Finally, Raman modes peaking at 608 cm-1 (proteins) and 1231 cm-1 (lipids) can be selected for follow up of survival rate of neoplastic cells after PDT. We envisage that this finding is key to contribute to a quick development of quantitative infrared thermography imaging.

Biotransformation of magnetic nanoparticles as a function of coating in a rat model.

Long-term in vivo studies in murine models have shown that DMSA-coated nanoparticles accumulate in spleen, liver and lung tissues during extended periods of time (at least up to 3 months) without any significant signs of toxicity detected. During that time, nanoparticles undergo a process of biotransformation either by reducing the size or the particle aggregation or both. Using a rat model, we have evaluated the transformations of magnetic nanoparticles injected at low doses. Particles with two different coatings, dimercaptosuccinic acid (NP-DMSA) and polyethylene glycol (NP-PEG-(NH2)2) have been administered to animals, to evaluate the role of coating in the degradation of the particles. We have found that low doses of magnetic nanoparticles are quickly metabolized by the animals. In fact, using a nanoparticle dose four times lower than in previous experiments, NP-DMSA were not observed 24 h after the administration either in the liver or in the lungs. Interestingly, an increased amount of ferritin, the iron storage protein, was observed in liver tissues from rats that were treated with the low dose of NP-DMSA in comparison with the control ones, suggesting a rapid metabolization of the particles into ferritin iron. On the other side we have found that, NP-PEG-(NH2)2 are still detectable in several organs 24 h after their administration at low doses. Probably, due to the longer circulation times of the NP-PEG-(NH2)2, there is a delay in the arrival of the particles to the tissue and this is the reason why we are able to see the particles 24 h post-administration. PEG coating could also be protecting the nanoparticles from rapid degradation of the reticuloendothelial system. Knowledge on the biodistribution, circulation time and degradation processes is required to gain a better understanding of the safety evaluation of this kind of nanomaterial for biomedical applications.

Photoacoustic investigation of maghemite-based nanocomposite.

Photoacoustic spectroscopy was used to investigate magnetic nanocomposites incorporating nanosized maghemite particles into styrene-divinylbenzene copolymer template. Typical photoacoustic features were observed in bands C, S and L in the wavelength region of 300-1000 nm. The relative intensity of band-C scaled with the nominal concentration of nanosized maghemite incorporated into the polymeric template whereas the lowest relative intensity of band-S was found in the sample in which the template polymerization took place in the presence of the highest polar-like reaction medium. X-ray diffraction and transmission electron microscopy were used to characterize the magnetic nanosized phase as maghemite, with average particle diameter of 6.9 nm (sample Est34), 7.0 nm (sample H30), and 7.9 nm (sample Em15).

Synthesis, characterization and magnetic properties of polymer-Fe3O4 nanocomposite.

The chemical stability of magnetic particles is of great importance for their applications in medicine and biotechnology. The most challenging problem in physics of disordered systems of magnetic nanoparticles is the investigation of their dynamic properties. The chemical coprecipitation process was used to synthesize spherical magnetite nanoparticles of 14 nm. The as-prepared magnetite nanoparticles have been aged in the matrix. Magnetic properties and aging effect were studied by Mössbauer spectroscopy at temperatures ranging from 77 to 300 K, and X-ray diffraction. At room temperature, the Mössbauer spectrum showed superparamagnetic behavior of the particles, while well-defined sextets were observed at 77K, indicating a blocked regime. The superparamagnetic magnetite nanoparticles can be used as microbead biosensors.

Photoacoustic spectroscopy study of Blepharocalyx salicifolius (Kunt) O. Berg.

Photoacoustic spectroscopy (PAS) has revolutionized the fields of biological, environmental, and agricultural sciences. It is a very simple, sensitive, and non-destructive technique that allows the determination of optical properties of bio-samples. The in vivo chlorophylls of the leaf have a recorded maximum absorption peak at 675 nm as against 665 nm of the in vitro chlorophylls. The intensity of purple pigmentation in leaves of Blepharocalyx salicifolius (Kunt) O. Berg, is inversely correlated to the soil moisture levels, leaf water content and leaf water potentials. The applicability of PAS to biological samples was discussed. It allows the validation of existing emission models which are important for atmospheric process. A portable device for photoacoustic spectroscopy of plants and other photosynthetic tissues, cells and organelles is provided. Further, there is provided a method to measure photosynthesis of such tissues, cells and organelles.

Ex vivo evaluation of caries infiltration after different application times in primary molars.

Low viscosity resins (infiltrants) have been shown to penetrate the lesion body of natural caries lesions almost completely in vitro. However, penetration depths (PD) have not been evaluated in vivo. Therefore, the aim of the present study was to evaluate the penetration of an infiltrant into proximal caries lesions in primary molars after different application times using an ex vivo model. 59 proximal lesions from 34 children were randomly allocated to one of the application times and were infiltrated under clinical conditions for 1, 3, or 5 min. After extraction or exfoliation (n = 48), teeth were sectioned perpendicular to their surfaces and lesion depths (LD) as well as lesion areas (LA) were evaluated using polarized light microscopy. PD and penetration areas (PA) were measured on scanning electron microscopic images. Percentage penetration depth (PPD) and percentage penetration area (PPA) were calculated. The mean (±SD) LD and LA were 596 ± 203 µm and 4.03 ± 2.75 × 10(5) µm(2), respectively. PPD ranged from 70 to 80% and PPA from 54 to 60%. Longer application times did not result in significantly deeper or more complete penetration (p > 0.05; ANOVA). In conclusion, proximal caries lesions in primary molars can be infiltrated in vivo to a similar extent as observed previously in vitro. Moreover, 1-min application of the infiltrant led to PD and homogeneity similar to those observed with longer application times up to 5 min.

New magnetic fluid developed with natural organic compounds biocompatible.

This work was developed with an aqueous suspension of maghemite nanoparticles and colloidal emulsions with nanoparticles of magnetite. The nanoparticles were synthesized by co-precipitation method. The first was the magnetic emulsion nanoparticles of maghemite dispersed in the aqueous extract obtained from the leaf embauba (Cecropia Obtusifolia), whose tree is native to Central and South America. Thereby achieving the magnetic fluid extract embauba stabilized with ionic buffer solution pH 7.4. A second emulsion was prepared with colloidal magnetite nanoparticles with surfaces previously coated with oleic acid as a means of dispersing and using the oil extracted from in nature seed Andiroba (Carapa Guianensis), tree of the Brazilian Amazon. These new magnetic fluids the nanoparticles were characterized by Photoacoustic spectroscopy (PAS) to determine the coating layer of molecules on the surfaces of nanoparticles. In aqueous ionic magnetic fluid Cecropia Obtusifolia (MFCO) chlorogenic acid contributes to the electron density in the presence of four groups alcohols, a ketone group and a carboxylic group. In magnetic fluid-based oil andiroba MFAD PAS spectra show that oleic acid molecules are tightly linked on the surface of the nanoparticles.

Comparison of stem cell properties of cells isolated from normal and inflamed dental pulps.

AIM: To compare cells from normal and inflamed human dental pulps regarding the presence of stem cells, their proliferation and differentiation potential. METHODOLOGY: Human dental pulp stem cells (hDPSCs) were isolated from normal (DPSC-N) and inflamed dental pulps (DPSC-I). They were compared in respect to proliferation (MTT assay), morphology and STRO-1 expression. STRO-1-positive cells were subject to proliferation (MTT and CFU counting) and morphological analyses and then submitted to odonto-osteogenic, adipogenic and condrogenic differentiation. Differentiated cells were evaluated concerning morphology and the expression, by qRT-PCR, of BSP, LPL and SOX-9 genes. The amount of mineralized matrix produced after odonto-osteogenic differentiation was compared with quantitative Alizarin Red staining. RESULTS: No difference was observed in the morphology and in the proliferation rate of DPSC-N and DPSC-I either before or after separation of STRO-1-positive cells. These cells represented 0.46% (±0.14) and 0.43% (±0.19) of the cell population from normal and inflamed dental pulps, respectively. Both DPSC-N and DPSC-I were capable of differentiating under the three assayed conditions and presented similar patterns for BSP, LPL and SOX-9 expression. Mineralized matrix production was also compatible. In all the quantitative experiments, differences were found between cells from each patient, either from normal or from inflamed pulps. Nonetheless, there was no statistical difference between these two groups. CONCLUSION: The morphology, proliferation rate and differentiation potential of DPSC-I were similar to the observed in DPSC-N, thus demonstrating that the inflammatory process did not affect the stem cell properties that were assessed.

Validity of MicroCT for in vitro detection of proximal carious lesions in primary molars.

OBJECTIVE: This study aimed to validate the MicroCT for detection of proximal carious lesions in primary molars, using histology as the gold standard. METHODS: Forty-eight proximal surfaces of primary molars were examined. Two calibrated examiners conducted the examinations independently. Proximal surfaces were visually scored, using ICDAS. Bitewing radiographs, Micro-CT and histological analyses used caries scores: 0=sound; 1=outer enamel; 2=inner enamel; 3=not spread dentine; 4=outer dentine; 5=inner dentine. Axial and sagital images were used for Micro-CT analysis, whilst for histology, tooth sections (400µm) were analyzed stereomicroscopically (×15). RESULTS: Inter-examiner agreement ranged from 0.87 to 0.93 kappa coefficient (k). Histological analysis revealed a frequency of sound tooth surfaces (18.8%) enamel carious lesions (E1) (48%) and dentine carious lesions (D1) (33.3%). MicroCT showed high correlation with histology (r(s)0.88). At both diagnostic thresholds (E1 and D1), sensitivity and accuracy were higher for MicroCT. Inter-device agreement between MicroCT and histology was k=0.81. No difference was found between MicroCT and histology as gold standards for detecting carious lesions using ICDAS. CONCLUSION: MicroCT can be used as a gold standard for detecting carious lesions in proximal surfaces in primary molars.

Liposomal photosensitizers: potential platforms for anticancer photodynamic therapy.

Photodynamic therapy is a well-established and clinically approved treatment for several types of cancer. Antineoplastic photodynamic therapy is based on photosensitizers, i.e., drugs that absorb photons translating light energy into a chemical potential that damages tumor tissues. Despite the encouraging clinical results with the approved photosensitizers available today, the prolonged skin phototoxicity, poor selectivity for diseased tissues, hydrophobic nature, and extended retention in the host organism shown by these drugs have stimulated researchers to develop new formulations for photodynamic therapy. In this context, due to their amphiphilic characteristic (compatibility with both hydrophobic and hydrophilic substances), liposomes have proven to be suitable carriers for photosensitizers, improving the photophysical properties of the photosensitizers. Moreover, as nanostructured drug delivery systems, liposomes improve the efficiency and safety of antineoplastic photodynamic therapy, mainly by the classical phenomenon of extended permeation and retention. Therefore, the association of photosensitizers with liposomes has been extensively studied. In this review, both current knowledge and future perspectives on liposomal carriers for antineoplastic photodynamic therapy are critically discussed.

Liposomal photosensitizers: potential platforms for anticancer photodynamic therapy

Photodynamic therapy is a well-established and clinically approved treatment for several types of cancer. Antineoplastic photodynamic therapy is based on photosensitizers, i.e., drugs that absorb photons translating light energy into a chemical potential that damages tumor tissues. Despite the encouraging clinical results with the approved photosensitizers available today, the prolonged skin phototoxicity, poor selectivity for diseased tissues, hydrophobic nature, and extended retention in the host organism shown by these drugs have stimulated researchers to develop new formulations for photodynamic therapy. In this context, due to their amphiphilic characteristic (compatibility with both hydrophobic and hydrophilic substances), liposomes have proven to be suitable carriers for photosensitizers, improving the photophysical properties of the photosensitizers. Moreover, as nanostructured drug delivery systems, liposomes improve the efficiency and safety of antineoplastic photodynamic therapy, mainly by the classical phenomenon of extended permeation and retention. Therefore, the association of photosensitizers with liposomes has been extensively studied. In this review, both current knowledge and future perspectives on liposomal carriers for antineoplastic photodynamic therapy are critically discussed.

In vitro photodynamic therapy on human oral keratinocytes using chloroaluminum-phthalocyanine.

In this study, oral carcinoma cells were used to evaluate chloroaluminum-phthalocyanine encapsulated in liposomes as the photosensitizer agent in support of photodynamic therapy (PDT). The genotoxicity and cytotoxicity behavior of the encapsulated photosensitizer in both dark and under irradiation using the 670-nm laser were investigated with the classical trypan blue cell viability test, the acridine orange/ethidium bromide staining organelles test, micronucleus formation frequency, DNA fragmentation, and cell morphology. The cell morphology investigation was carried out using light and electronic microscopes. Our findings after PDT include reduction in cell viability (95%) associated with morphologic alterations. The neoplastic cell destruction was predominantly started by a necrotic process, according to the assay with acridine orange and ethidium bromide, and this was confirmed by electronic microscopy analysis. Neither the PDT agent nor laser irradiation alone showed cytotoxicity, genotoxicity, or even morphologic alterations. Our results reinforce the efficiency of light-irradiated chloroaluminum-phthalocyanine in inducing a positive effect of PDT.

Cell toxicity studies of albumin-based nanosized magnetic beads.

The aim of this study was to prepare bovine serum albumin-based beads containing maghemite nanoparticles incorporated via ionic magnetic fluid and to evaluate the cell toxicity of this biocompatible system using the J774-A1 cell line. Transmission electron micrographs obtained from the magnetic fluid sample were used to estimate the average particle diameter around 7.6 nm and diameter dispersion of 0.22. The BSA-based magnetic beads were prepared using the heat protein denaturation route. The nanoparticle concentration in the magnetic fluid sample used for the synthesis of the magnetic beads was in the range of 1.2 x 10(16) to 2.3 x 10(17) particle/ml. The methodology used to investigate the cell toxicity of the magnetic beads was the classical MTT assay. Our observation showed that the toxicity against the J774-A1 cell line depends upon the amount of magnetic material incorporated into the magnetic nanobeads and was found to be 14, 11, 9, 5, and 3% for 2.3 x 10(17), 1.2 x 10(17), 4.6 x 10(16), 2.3 x 10(16), and 1.2 x 10(16) particle/ml, respectively.

Light microscopical and ultrastructural characterization of black howler monkey (Alouatta caraya) ovarian follicles.

The present study describes the morphological characteristics of black howler monkey (Alouatta caraya) ovarian follicles. One ovary of an adult healthy black howler monkey was collected and processed for light and electron microscopy. Primordial, primary, secondary, tertiary and pre-ovulatory follicles were evaluated for their morphometrical aspects. The ovary of black howler monkey presented a distinct conformation with a uniform distribution of the follicles mostly in the peripheric cortex. This black howler monkey ovary presented a total of 59 921 ovarian follicles. From this amount, 71.1% were classified as primordial, 18.9% as primary, 8.1% as secondary, 1.4% as tertiary and 0.5% as pre-ovulatory follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells are described. Moreover, primordial, primary and secondary follicles have been observed by electron microscopy.

Distraction osteogenesis may promote periodontal bone regeneration.

Distraction osteogenesis has numerous applications in the treatment of conditions affecting the dentofacial complex, from midface advancement to orthodontic tooth movement produced by transversal distraction of the periodontal ligament. The purpose of this study was to test the hypothesis that periodontium can be consistently distracted toward the tooth crown to promote periodontal bone regeneration. After the surgical production of periodontal defects in maxillary canines of 5 mongrel dogs, periodontal bone distraction was performed. Light microscopy was used for histopathological and morphometric analysis. Periodontal bone regeneration occurred in all animals. Periodontal bone regeneration in the distraction sites (Mean +/- SD: 5.45 +/- 2.01 mm) differed from that in control sites (0.008 +/- 0.67 mm; p < 0.0001). Periodontal bone distraction resulted in periodontal bone regeneration. This finding may establish periodontal bone distraction as a new treatment alternative for periodontal defects.

Effects of 2% chlorhexidine and 5.25% sodium hypochlorite on gutta-percha cones studied by atomic force microscopy.

AIM: To compare the effects of 2% chlorhexidine (CHX) and 5.25% sodium hypochlorite (NaOCl) on gutta-percha (GP) cone structure using atomic force microscopy (AFM). METHODOLOGY: Two standardized GP cones were sectioned 3 mm from the tip, attached to a glass base and immersed in 2% CHX or 5.25% NaOCl for 1, 5, 10, 20 and 30 min. Untreated GP cones were used as control. Topography and elasticity analyses were performed on 12 different regions located between 1 and 2 mm from the tip. Root mean square (RMS) parameters for contact mode imaging and force modulation microscopy variations were measured. The differences between RMS values were tested by anova with Fisher's protected LSD test for multiple comparisons. RESULTS: There was no deterioration in the topography and physical properties studied when 2% CHX was used in comparison with the control (P < 0.05). The RMS parameter for topography increased after 10 min of 5.25% NaOCl exposure in comparison with the control (P < 0.05). In addition, 5.25% NaOCl increased the elasticity of the GP cone after an immersion time of 1 min in comparison with the control (P < 0.05). CONCLUSIONS: Two per cent CHX did not change GP cone structure following up to 30 min exposure. Conversely, 5.25% NaOCl caused elastic changes after 1 min exposure.

Effect of bullfrog (Rana catesbeiana) oil administered by gavage on the fatty acid composition and oxidative stress of mouse liver

The aim of the present study was to investigate the effects of daily intragastric administration of bullfrog oil (oleic, linoleic and palmitoleic acid-rich oil), corresponding to 0.4 percent of body weight for four weeks, on fatty acid composition and oxidative stress (lipid peroxidation and catalase activity) in mouse liver. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), biomarkers of tissue injury, were determined in liver homogenates and serum. The proportions of 18:2n-6, 20:4n-6, 20:5n-3, and 22:6n-3 (polyunsaturated fatty acids, from 37 to 60 percent) in the total fatty acid content were increased in the liver of the bullfrog oil-treated group (P < 0.05) compared to control. At the same time, a significant decrease in the relative abundance of 14:0, 16:0, and 18:0 (saturated fatty acids, from 49 to 25 percent) was observed. The hepatic content of thiobarbituric acid reactive substances (TBARS) was increased from 2.3 ± 0.2 to 12.3 ± 0.3 nmol TBA-MDA/mg protein and catalase activity was increased from 840 ± 32 to 1110 ± 45 æmol reduced H2O2 min-1 mg protein-1 in the treated group. Bullfrog oil administration increased AST and ALP activities in the liver (from 234.10 ± 0.12 to 342.84 ± 0.13 and 9.38 ± 0.60 to 20.06 ± 0.27 U/g, respectively) and in serum (from 95.41 ± 6.13 to 120.32 ± 3.15 and 234.75 ± 11.5 to 254.41 ± 2.73 U/l, respectively), suggesting that this treatment induced tissue damage. ALT activity was increased from 287.28 ± 0.29 to 315.98 ± 0.34 U/g in the liver but remained unchanged in serum, whereas the GGT activity was not affected by bullfrog oil treatment. Therefore, despite the interesting modulation of fatty acids by bullfrog oil, a possible therapeutic use requires care since some adverse effects were observed in liver.

Effect of bullfrog (Rana catesbeiana) oil administered by gavage on the fatty acid composition and oxidative stress of mouse liver.

The aim of the present study was to investigate the effects of daily intragastric administration of bullfrog oil (oleic, linoleic and palmitoleic acid-rich oil), corresponding to 0.4% of body weight for four weeks, on fatty acid composition and oxidative stress (lipid peroxidation and catalase activity) in mouse liver. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), biomarkers of tissue injury, were determined in liver homogenates and serum. The proportions of 18:2n-6, 20:4n-6, 20:5n-3, and 22:6n-3 (polyunsaturated fatty acids, from 37 to 60%) in the total fatty acid content were increased in the liver of the bullfrog oil-treated group (P < 0.05) compared to control. At the same time, a significant decrease in the relative abundance of 14:0, 16:0, and 18:0 (saturated fatty acids, from 49 to 25%) was observed. The hepatic content of thiobarbituric acid reactive substances (TBARS) was increased from 2.3 +/- 0.2 to 12.3 +/- 0.3 nmol TBA-MDA/mg protein and catalase activity was increased from 840 +/- 32 to 1110 +/- 45 micromol reduced H2O2 min-1 mg protein-1 in the treated group. Bullfrog oil administration increased AST and ALP activities in the liver (from 234.10 +/- 0.12 to 342.84 +/- 0.13 and 9.38 +/- 0.60 to 20.06 +/- 0.27 U/g, respectively) and in serum (from 95.41 +/- 6.13 to 120.32 +/- 3.15 and 234.75 +/- 11.5 to 254.41 +/- 2.73 U/l, respectively), suggesting that this treatment induced tissue damage. ALT activity was increased from 287.28 +/- 0.29 to 315.98 +/- 0.34 U/g in the liver but remained unchanged in serum, whereas the GGT activity was not affected by bullfrog oil treatment. Therefore, despite the interesting modulation of fatty acids by bullfrog oil, a possible therapeutic use requires care since some adverse effects were observed in liver.

ALES: cell lineage analysis and mapping of developmental events.

MOTIVATION: Animals build their bodies by altering the fates of cells. The way in which they do so is reflected in the topology of cell lineages and the fates of terminal cells. Cell lineages should, therefore, contain information about the molecular events that determined them. Here we introduce new tools for visualizing, manipulating, and extracting the information contained in cell lineages. Our tools enable us to analyze very large cell lineages, where previously analyses have only been carried out on cell lineages no larger than a few dozen cells. RESULTS: Ales (A Lineage Evaluation System) allows the display, evaluation and comparison of cell lineages with the aim of identifying molecular and cellular events underlying development. Ales introduces a series of algorithms that locate putative developmental events. The distribution of these predicted events can then be compared to gene expression patterns or other cellular characteristics. In addition, artificial lineages can be generated, or existing lineages modified, according to a range of models, in order to test hypotheses about lineage evolution. AVAILABILITY: The program can run on any operating system with a compliant Java 2 environment. Ales is free for academic use and can be downloaded from http://mbi.dkfz-heidelberg.de/mbi/research/cellsim/ales.