Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells.

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

Genomic instability in long-term culture of human adipose-derived mesenchymal stromal cells

Mesenchymal stromal/stem cells stem (MSC) have been widely studied due to their great potential for application in tissue engineering and regenerative and translational medicine. In MSC-based therapy for human diseases, cell proliferation is required to obtain a large and adequate number of cells to ensure therapeutic efficacy. During in vitro culture, cells are under an artificial environment and manipulative stress that can affect genetic stability. Several regulatory agencies have established guidelines to ensure greater safety in cell-based regenerative and translational medicine, but there is no specific definition about the maximum number of passages that ensure the lowest possible risk in MSC-based regenerative medicine. In this context, the aim of this study was to analyze DNA damage and chromosome alterations in adipose-derived mesenchymal stromal cells (ADMSC) until the eleventh passage and to provide additional subsidies to regulatory agencies related to number of passages in these cells. Thus, two methods in genetic toxicology were adopted: comet assay and micronucleus test. The comet assay results showed an increase in DNA damage from the fifth passage onwards. The micronucleus test showed a statistically significant increase of micronucleus from the seventh passage onwards, indicating a possible mutagenic effect associated with the increase in the number of passages. Based on these results, it is important to emphasize the need to assess genetic toxicology and inclusion of new guidelines by regulatory agencies to guarantee the safety of MSC-based therapies for human diseases.

APPLICATION OF THE MONTE CARLO METHOD FOR THE EVALUATION OF SCATTERED RADIATION DOSE DUE TO THE USE OF HANDHELD X-RAY IN DENTISTRY.

The object of this study was to evaluate the dose of scattered radiation in the organs of the operator and assistant located in different positions within a dental room when acquiring intraoral images with a portable handheld X-ray device, using Monte Carlo simulations for recommended and traditional techniques. A typical dental installation was modeled, where the operator and assistant were placed. The beam is represented by 60-kV spectrum. Ten scenarios were simulated, representing different positions and use of the lead apron. The results of the simulations were carried out with typical parameters of the annual workload, showing significant increases in dose in the organs of the operator due to the angulation. The minimum dose in the organs of the assistant occurred when he was located 2-m away and 45° from the direction of the beam. The dose received by the operator is significantly reduced with the use apron.

Experimental study on needle insertion force to minimize tissue deformation in tongue tissue.

This study reports on the effects of insertion velocity, needle tip geometry and needle diameter on tissue deformation and maximum insertion force. Moreover, the effect of multiple insertions with the same needle on the maximum insertion force is reported. The tissue deformation and maximum insertion force strongly depend on the insertion velocity and the tip geometry. No correlation was found between the outer diameter and the maximum insertion force for small needles (30G - 32G). The endurance experiments showed no remarkable difference in the maximum insertion force during 100 insertions.

Quantitative proteomic analysis of the tizoxanide effect in vero cells.

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.

Fluctuating potentials in GaAs:Si nanowires: critical reduction of the influence of polytypism on the electronic structure.

In this work, the effects of Si doping in GaAs nanowires (NWs) grown on GaAs (111)B by molecular beam epitaxy with different Si doping levels (nominal free carrier concentrations of 1 × 1016, 8 × 1016, 1 × 1018 and 5 × 1018 cm-3) are deeply investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), grazing incidence X-ray diffraction (GID), photoluminescence (PL) and cathadoluminescence (CL). TEM results reveal a mixture of wurtzite (WZ) and zinc-blende (ZB) segments along the NW axis independently of the Si doping levels. GID measurements suggest a slight increase of the ZB fraction with the Si doping. Low temperature PL and CL spectra exhibit sharp lines in the energy range 1.41-1.48 eV, for the samples with lower Si doping levels. However, the emission intensity increases and is accompanied by a clear broadening of the observed lines for the samples with higher Si doping levels. The staggered type-II band alignment only determines the optical properties of the lower doping levels in GaAs:Si NWs. For the higher Si doping levels, the electronic energy level structure of the NWs is determined by electrostatic fluctuating potentials intimately related to the amphoteric behavior of the Si dopant in GaAs. For the heavily doped NWs, the estimated depth of the potential wells is ∼96-117 meV. Our results reveal that the occurrence of the fluctuating potentials is not dependent on the crystalline phase and shows that the limitation imposed by the polytypism can be overcome.

Upconversion luminescence and blackbody radiation in tetragonal YSZ co-doped with Tm(3+) and Yb(3+).

Lanthanide doped inorganic nanoparticles with upconversion luminescence are of utmost importance for biomedical applications, solid state lighting and photovoltaics. In this work we studied the downshifted luminescence, upconversion luminescence (UCL) and blackbody radiation of tetragonal yttrium stabilized zirconia co-doped with Tm(3+) and Yb(3+) single crystals and nanoparticles produced by laser floating zone and laser ablation in liquids, respectively. The photoluminescence (PL) and PL excitation (PLE) were investigated at room temperature (RT). PL spectra exhibit the characteristic lines in UV, blue/green, red and NIR regions of the Tm(3+) (4f(12)) under resonant excitation into the high energy (2S+1)LJ multiplets. Under NIR excitation (980 nm), the samples placed in air display an intense NIR at ∼800 nm due to the (1)G4→(3)H5/(3)H4→(3)H6 transitions. Additionally, red, blue/green and ultraviolet UCL is observed arising from higher excited (1)G4 and (1)D2 multiplets. The power excitation dependence of the UCL intensity indicated that 2-3 low energy absorbed photons are involved in the UCL for low power levels, while for high powers, the identified saturation is dependent on the material size with a enhanced effect on the NPs. The temperature dependence of the UCL was investigated for single crystals and targets used in the ablation. An overall increase of the integrated intensity was found to occur between 12 K and the RT. The thermally activated process is described by activation energies of 10 meV and 30 meV for single crystals and targets, respectively. For the NPs, the UCL was found to be strongly sensitive to pressure conditions. Under vacuum conditions, instead of the narrow lines of the Tm(3+), a wide blackbody radiation was detected, responsible for the change in the emission colour from blue to orange. This phenomenon is totally reversible when the NPs are placed at ambient pressure. The UCL/blackbody radiation in the nanosized material exhibits non-contact pressure colour-based sensor characteristics. Moreover, tuning the color of the blackbody radiation in the nanoparticles by harvesting the low energy photons into the visible spectral region was found to be possible by adjusting the excitation power, paving the way for further developments of these nanoparticles for lighting and photovoltaic applications.

Analysis of the salivary proteome in gingivitis patients.

BACKGROUND AND OBJECTIVE: Gingivitis is a disease that is characterized by inflammation of the gingival tissue, which can progress to periodontitis and tooth loss. Although many studies have attempted to identify salivary proteins that are associated with the disease, this is the first study to use a proteomic approach to analyze and compare the proteomic profile of whole saliva from gingivitis patients and healthy controls. MATERIAL AND METHOD: To analyze the saliva proteome, two-dimensional gel electrophoresis and liquid chromatography were used, followed by mass spectrometry. RESULTS: The analyses showed that gingival inflammation was associated with increased amounts of blood proteins (serum albumin and hemoglobin), immunoglobulin peptides and keratins. In the control group, salivary cystatins, which were detected using capillary Liquid Chromatography on line to electrospray ionization Quadrupole Time-of-flight mass spectrometry, appeared to be more abundant. CONCLUSION: This approach provides novel insight into profiles of the salivary proteome during gingival inflammation, which may contribute to improvements in diagnosis.

Investigation of IGF2/ApaI and H19/RsaI polymorphisms in patients with cutaneous melanoma.

OBJECTIVE: The etiology of cutaneous melanoma is complex, involving both heterogeneous genetic and environmental components. The aim of our study was to verify if single polymorphic sites within IGF2 and H19 genes and their consequent haplotypes influence risk and/or prognosis of familial melanoma. DESIGN: Twenty one patients with clinical criteria of hereditary melanoma (early onset, presence of multiple primary melanoma, and/or one or more affected first- or second-degree relatives) and previously screened for CDKN2A mutations were genotyped by IGF2/ApaI and H19/RsaI PCR-RFLPs. Data were compared between patients and a control group (100 healthy young individuals) using Chi-square and Fisher's exact tests. We also investigated if these polymorphic sites could be microRNAs potential targets, using RegRNA software. RESULTS: Although the IGF2 and H19 genotypes/haplotypes were not significantly associated with melanoma, two of the most severe cases (very early onset or multiple melanomas) showed to be heterozygous for both genes. We found an overlap between IGF2/ApaI and miR-615-5p, and between H19/RsaI and miR-574-3p. CONCLUSIONS: Some studies have shown H19, and IGF2 genes (or related genes or protein, for example, IGF2R and IMP-3) differential expression in melanoma. However, no study has attempted to examine markers across this cluster in relation to melanoma until now. Since the base change may impair the pairing of microRNA and its binding site, our results suggest a new window for future studies of IGF2 and H19 genetic variability and posttranscriptional regulation. Due to the importance and based on the present results, we suggest that the genotype/haplotype analysis of IGF2 and H19 polymorphisms should be better investigated in large populations with cutaneous melanoma, attempting to tie the association with progression of the disease.

Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A2 and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and Mr of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.

Rats exposed to Solanum lycocarpum fruit in utero and during lactation: neurochemical, behavioral and histopathological effects.

Solanum lycocarpum St. Hil (Solanaceae) is a native shrub very common in the Brazilian savannah. This plant contains steroidal glycoalkaloids that can be transformed into an intermediate for steroidal drug production. In this way, it is very possible that these glycoalkaloids and its aglycone, once in the body by ingestion of S. lycocarpum fruits, may act by disrupting the endocrine system. Because its fruits may be consumed by pregnant animals in the fields, the present study determined the possible toxic effects of exposure to S. lycocarpum fruit (10% added in the diet) from gestation day (GD) 6 to postnatal day (PND) 07 in rat dams. The unripe fruits contained 0.6% of solamargine and 0.9% of solasonine. S. lycocarpum, 10% in the diet, during gestation and the beginning of lactation reduced intrauterine growth. In addition, 20% of the treated dams showed some dead pups at birth. Reduced body weight was observed from birth through adulthood in male and female offspring exposed to 10% S. lycocarpum unripe fruits. During adulthood, female offspring showed impaired sexual behavior and male offspring showed prominent degeneration of testis germinative cells, characterized by a reduced number of germ cells and vacuolation. Also, the exposed offspring showed reduced hypothalamic norepinephrine (NOR), vanillylmandelic acid (VMA), 3-methoxy-4-hydrophenylglycol (MHPG) and homovanillic acid (HVA) levels, and reduced striatum NOR, HVA, VMA, MHPG, dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels. These results suggest that the fruit may act as an estrogen, with a long-term effect, impairing the receptive lordosis behavior of female offspring and promoting testis abnormalities in male offspring at adulthood. Finally, it appears to disrupt brain organization since important central monoamine level alterations were also observed.

Association between birth weight, body mass index and IGF2/ApaI polymorphism.

Insulin-like growth factor II (IGF-II) is a polypeptide that plays a key role in mammalian growth, influencing fetal cell division, differentiation, and possibly metabolic regulation. In adult humans, polymorphisms of the IGF2 gene have been associated with predisposition to obesity. In the present study, we tested the association between IGF2/ApaI genotype and Body Mass Index (BMI) in 294 healthy volunteers (95 men and 199 women; 18-30y) and correlated the results with their birth weights (BW) in order to investigate the relationship between this polymorphic site, fetal life and adult BMI. Blood samples were obtained for DNA extraction, PCR and genotyping. The statistical analyses were performed by the Chi-square, Kolmogorov-Smirnov normality, and Tukey post hoc tests. Although the IGF2 genotype was not significantly associated with BMI and/or BW, we observed a statistically significant correlation of 0.33 (p < 0.023) between BW and BMI in GG subjects whose BW was higher than 3.5 kg (n = 47). We hypothesize that high BWs associated with homozygosis for the G allele of IGF2/ApaI is not a null factor and might be associated with predisposition to high BMI in young adults.

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis.

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas' disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2beta protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.

Embryotoxic effects of Solanum lycocarpum St. Hill fruits consumption during preimplantation and organogenesis in rats.

Solanum lycocarpum St. Hill is a common plant in the Brazilian savanna. This plant has an alkaloid with stereospecific configuration to the synthesis of steroid hormones. Since the plant may be consumed by pregnant animals, the present study was undertaken to determine the possible embryotoxic effects of S. lycocarpum fruit ingestion (3% added to the diet) during preimplantation (from the first to the sixth days of gestation) or during organogenesis in rats (from the sixth to the sixteenth days of gestation). Few differences were observed in food and water consumption without biological importance. The placental weight in the group that received the plant during the organogenesis period was decreased. An increase in sternebra abnormalities was observed in animals treated with the plant during organogenesis. Olfactory bulb hemorrhage was increased in the group that received the plant during preimplantation when compared to the control group. These results indicate that consumption of S. lycocarpum at 3% in diet during pregnancy cause slight toxicological effects. Other studies have to be conducted to better investigate the causes of toxicity and other toxic effects of higher levels of exposure to this plant.

[An evaluation of municipal actions in the control of dengue vectors in the region of São José do Rio Prêto, São Paulo, 1989 to 1995]. / Avaliação de ações municipais de combate a vetores da dengue na região de São José do Rio Prêto, São Paulo, 1989 a 1995.

The aim of this study was to determine the coverage of municipal activities in terms of the control of Aedes aegypti and/or Aedes albopictus by routine house-to-house visits and by emergency activity, carried out between 1989 and 1995 in the area of São José do Rio Prêto, São Paulo State, and to evaluate the cross-correlation between them and the Breteau index (BI). For towns with up to 50,000 real estate properties, the joint coverage by routine and emergency activities was mostly appropriate and the routine activities showed a negative cross-correlation with the BI. For the county seat (more than 50,000 real estate properties), the coverage provided by the above activities was not correlated with the BI. In general, the coverage was inversely proportional to town size. Emergency activities did not show a correlation with the BI in any town size range, proving to be ineffective.

[Description of the colonization of Aedes albopictus (Diptera: culicidae) in the region of Saõ José do Rio Preto, SP, 1991-1994]. / Descrição da colonização de Aedes albopictus (Diptera: culicidae) na região de São José do Rio Preto, SP, 1991-1994.

This study aims to describe and analyze the colonization of Aedes albopictus whose presence was detected in 1991 in the area of Sào José do Rio. Preto already colonized by the Aedes aegypti. Year and month of occurrence, counties, composition and location of larval samples, kinds of containers, average number of larvae an Breteau Index bare been analyzed from the information obtained in measurements of larval density by Superintendência de Controle de Endemias (SUCEN). The presence of Aedes albopictus was ascertained in 34 towns up to December 1994. The colonization of the area by the mosquito is still reduced showing some differences in relation to Aedes aegypti such as greater ratio outdoors, occupying container in different proportions. The average number of Aedes albopictus larvae has had influence of larvae of another species. It has showed a seasonal behavior similar to Aedes aegypti and it has moved from east to west direction.