Phenotypic and molecular investigation of circulating tumor cells (CTCs) isolated from breast cancer patients at single cell resolution

Despite the paramount advances in cancer research, breast cancer (BC) still ranks one of the leading causes of cancer-related death worldwide. Thanks to the screening campaign started in developed countries, BC is often diagnosed at early stages (non-metastatic BC, nmBC), but disease relapse occurrence even after decades and at distant sites is not an uncommon phenomenon. Conversely, metastatic BC (mBC) is considered an incurable disease. The major perpetrators of tumor spread to secondary organs are circulating tumor cells (CTCs), a rare population of cells detectable in the peripheral blood of oncologic patients. In this study, CTCs from patients diagnosed with luminal nmBC and mBC (hormone receptor positive, Human Epidermal Growth Factor Receptor 2 (HER2) negative) were characterized at both phenotypic and molecular levels. To better understand the molecular mechanisms underlying their biology and their metastatic potential, next-generation sequencing (NGS) analyses were performed at single-cell resolution to assess copy number aberrations (CNAs), single nucleotide variants (SNVs) and gene expression profiling. The findings of this study arise hints in CTC detection, and pave the way to new application in CTC research.

Effect Of Aloe Vera Application On The Content And Molecular Arrangement Of Glycosaminoglycans During Calcaneal Tendon Healing.

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7(th) , 14(th) , or 21(st) day post-surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera-treated group on the 14(th) day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera-treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-β1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera-treated group on the 21(st) day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins.

Solid Lipid Nanoparticles As Nucleic Acid Delivery System: Properties And Molecular Mechanisms.

Solid lipid nanoparticles (SLNs) have been proposed in the 1990s as appropriate drug delivery systems, and ever since they have been applied in a wide variety of cosmetic and pharmaceutical applications. In addition, SLNs are considered suitable alternatives as carriers in gene delivery. Although important advances have been made in this particular field, fundamental knowledge of the underlying mechanisms of SLN-mediated gene delivery is conspicuously lacking, an imperative requirement in efforts aimed at further improving their efficiency. Here, we address recent advances in the use of SLNs as platform for delivery of nucleic acids as therapeutic agents. In addition, we will discuss available technology for conveniently producing SLNs. In particular, we will focus on underlying molecular mechanisms by which SLNs and nucleic acids assemble into complexes and how the nucleic acid cargo may be released intracellularly. In discussing underlying mechanisms, we will, when appropriate, refer to analogous studies carried out with systems based on cationic lipids and polymers, that have proven useful in the assessment of structure-function relationships. Finally, we will give suggestions for improving SLN-based gene delivery systems, by pointing to alternative methods for SLNplex assembly, focusing on the realization of a sustained nucleic acid release.

Chemical And Structural Analyses Of Titanium Plates Retrieved From Patients.

The aim of this study was to evaluate the microscopic structure and chemical composition of titanium bone plates and screws retrieved from patients with a clinical indication and to relate the results to the clinical conditions associated with the removal of these devices. Osteosynthesis plates and screws retrieved from 30 patients between January 2010 and September 2013 were studied by metallographic, gas, and energy dispersive X-ray (EDX) analyses and the medical records of these patients were reviewed. Forty-eight plates and 238 screws were retrieved. The time elapsed between plate and screw insertion and removal ranged between 11 days and 10 years. Metallographic analysis revealed that all the plates were manufactured from commercially pure titanium (CP-Ti). The screw samples analyzed consisted of Ti-6Al-4V alloy, except four samples, which consisted of CP-Ti. Titanium plates studied by EDX analysis presented greater than 99.7% titanium by mass. On gas analysis of Ti-6Al-4V screws, three samples were outside the standard values. One CP-Ti screw sample and one plate sample also presented an oxygen analysis value above the standard. The results indicated that the physical properties and chemical compositions of the plates and screws did not correspond with the need to remove these devices or the time of retention.

Two Novel Mutations In The Thyroid Hormone Receptor β In Patients With Resistance To Thyroid Hormone (rth β): Clinical, Biochemical, And Molecular Data.

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-l-triiodothyronine (T3), with persistent elevation of free-circulating T3 (FT3) and free thyroxine (FT4) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods.

Micromorphological and hardness analyses of human and bovine sclerotic dentin: a comparative study

The purpose of this study was to test the hypothesis that both human and bovine sclerotic dentin have similar hardness properties, in addition to similar micromorphological characteristics. Sixteen teeth (8 human and 8 bovine) exhibiting exposed dentin in the incisal edge and showing characteristics typical of sclerosis were used. Vickers surface microhardness testing was conducted. Three areas of the dentin surface of each specimen were selected. All teeth were processed for scanning electron microscopy in order to estimate the amount (in percentage) of solid dentin on the sclerotic dentin surface. The data were compared by Student's t test (α = 0.05). The micromorphological and microhardness data were compared by Pearson's linear correlation test (α = 0.05). The mean percentages of solid dentin of human and bovine sclerotic dentin were similar (human 90.71 ± 0.83 and bovine 89.08 ± 0.81, p = 0.18). The mean microhardness value (VHN) of human sclerotic dentin was significantly higher than that of bovine sclerotic dentin (human 45.26 ± 2.92 and bovine 29.93 ± 3.83, p = 0.006). No correlation was found between the microhardness values and the amount of solid dentin in the sclerotic dentin, irrespective of the species considered (human R² = 0.0240, p = 0.714; bovine R² = 0.0017, p = 0.923; and combined R² = 0.038, p = 0.46). We concluded that although both bovine and human sclerotic dentin present a similar amount of solid tissue, human sclerotic dentin presents higher microhardness than bovine sclerotic dentin.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).

Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Recovery and upgrading bovine rumen protein by extrusion: effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9 per cent or 3.8 per cent) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9 per cent residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8 per cent in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end

First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

Determination and Molecular Analysis of the Complete Genome Sequence of Two Wild-Type Rabies Viruses Isolated from a Haematophagous Bat and a Frugivorous Bat in Brazil

The complete genome sequences of two Brazilian wild-type rabies viruses (RABV), a BR-DR1 isolate from a haematophagous bat (Desmodus rotundus) and a BR-AL1 isolate from a frugivorous bat (Artibeus lituratus), were determined. The genomes of the BR-DR1 and RR-AL1 had 11,923 and 11,922 nt, respectively, and both encoded the five standard genes of rhabdoviruses. The complete nucleotide sequence identity between the BR-DR1 and BR-AL1 isolates was 97%. The BR-DR1 and BR-AL1 isolates had some conserved functional sites revealed by the fixed isolates, whereas both isolates had unique amino acid substitutions in the antigenic region IV of the nucleocapsid gene. Therefore, it is speculated that both isolates were nearly identical in virologic character. According to our phylogenetic analysis based on the complete genomes, both isolates belonged to genotype 1, and to the previously defined ""vampire bat-related RABV lineage"" which consisted of mainly D. rotundus- and A. lituratus- isolates; however, a branch pattern with high bootstrap values suggested that BR-DR1 was more closely related to the 9001FRA isolate, which was collected from a dog bitten by a bat in French Guiana, than to BR-AL1. This result suggests that the vampire bat-related RABV lineage includes Brazilian vampire bat and Brazilian frugivorous bat RABV and is further divided into Brazilian vampire bat and Brazilian frugivorous bat RABV sub-lineages. The phylogenetic analysis based on the complete genomes was valuable in discriminating among very closely related isolates.

Yellow Fever Virus in Haemagogus leucocelaenus and Aedes serratus Mosquitoes, Southern Brazil, 2008

Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil In October 2008 a yellow fever outbreak was reported there with nonhuman primate deaths and human cases This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths Mosquitoes were collected at ground level identified and processed for virus isolation and molecular analyses Eight YFV strains were isolated (7 from pools of Hg leucocelaenus mosquitoes and another from Aedes serratus mosquitoes) 6 were sequenced and they grouped in the YFV South American genotype I The results confirmed the role of Hg leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae serratus mosquitoes may have a potential role as a secondary vector

Syphilis at the Crossroad of Phylogenetics and Paleopathology

The origin of syphilis is still controversial. Different research avenues explore its fascinating history. Here we employed a new integrative approach, where paleopathology and molecular analyses are combined. As an exercise to test the validity of this approach we examined different hypotheses on the origin of syphilis and other human diseases caused by treponemes (treponematoses). Initially, we constructed a worldwide map containing all accessible reports on palaeopathological evidences of treponematoses before Columbus's return to Europe. Then, we selected the oldest ones to calibrate the time of the most recent common ancestor of Treponema pallidum subsp. pallidum, T. pallidum subsp. endemicum and T. pallidum subsp. pertenue in phylogenetic analyses with 21 genetic regions of different T. pallidum strains previously reported. Finally, we estimated the treponemes' evolutionary rate to test three scenarios: A) if treponematoses accompanied human evolution since Homo erectus; B) if venereal syphilis arose very recently from less virulent strains caught in the New World about 500 years ago, and C) if it emerged in the Americas between 16,500 and 5,000 years ago. Two of the resulting evolutionary rates were unlikely and do not explain the existent osseous evidence. Thus, treponematoses, as we know them today, did not emerge with H. erectus, nor did venereal syphilis appear only five centuries ago. However, considering 16,500 years before present (yBP) as the time of the first colonization of the Americas, and approximately 5,000 yBP as the oldest probable evidence of venereal syphilis in the world, we could not entirely reject hypothesis C. We confirm that syphilis seems to have emerged in this time span, since the resulting evolutionary rate is compatible with those observed in other bacteria. In contrast, if the claims of precolumbian venereal syphilis outside the Americas are taken into account, the place of origin remains unsolved. Finally, the endeavor of joining paleopathology and phylogenetics proved to be a fruitful and promising approach for the study of infectious diseases.