Analysis Of Histological Sections Research Articles

Large Hypertrophy but Unmodified Specific Tension of Single Fibers of Body Builders

IntroductionMuscle hypertrophy is the main outcome of training for body building. Previous studies showed a discrepancy between the functional (force/tension) and structural (cross‐sectional area, CSA) proprieties of leg extensor muscles measured in vivo and the corresponding parameters measured in single muscle fibers [1] [2]. In particular, a puzzling finding from these studies was that of a lower specific tension of skinned fibers of body builders (BB) compared to a control (C) population.In this study we aimed to investigate the possible reasons behind the alleged lower specific tension in single fibers of a strictly diet‐and‐training‐controlled BB population.MethodsFive male body builders (26±4.7 yr; 87.2±9.7 kg; 178.4±7.5 cm) with a training history of at least 5 years were recruited for this study. Five age‐matched recreationally active males (24.6±3.6 yr; 84.4±14.9 kg; 186.4±4.4 cm) were recruited as controls. After ethical approval, biopsies from the vastus lateralis (VL) muscle were collected together with in‐vivo quadriceps maximum isometric voluntary contraction (MVC) and architecture and CSA. Data analysis included (i) single skinned fiber mechanics (n=200 fibers; maximum force, CSA, specific tension and calcium diffusion), (ii) single fiber myosin quantification (n=100 fibers), (iii) slow and fast fiber CSA via histological section analysis. In addition, swelling ratio (skinned fibers CSA/histological CSA) was calculated for each fiber type and for each subject. A general linear mixed model was used to assess statistically significant differences.ResultsQuadriceps MVC and CSA were 64% and 33% higher in BB than C (p<0.05), VL pennation angle (PA) and thickness (MT) were 16% and 15% higher in BB than C (p<0.05). Quadriceps specific force was 25% higher in BB than C (n.s.). Single skinned type 2 CSA and force were 48% and 28% higher in BB than C (p<0.05), while specific tension was 19% lower (p<0.05). Type 1 fibers specific tension was 32% higher in BB than C (p<0.05). Myosin content and calcium diffusion were similar among populations. CSA determined in histological sections was 10% and 27% higher in BB than C (p<0.05) for type1 and 2 fibers, respectively. When type 1 and type 2 fiber tensions were corrected for individual swelling ratio, the difference between BB and C disappeared.ConclusionsAlthough at first sight these findings seem to confirm that in type 2 skinned fibers of BB specific tension is lower than in C, once accounting for fiber swelling, this difference disappears. This is further confirmed by the lack of difference in myosin concentration and calcium diffusion kinetics.In contrast, the results point to the existence of a differential swelling response to the skinning process by BB and C fibers, possibly due to adaptations of the fiber cytoskeleton proteins or specific permeability of the membrane of slow and fast fibers.

Sex hormones in Strombus pugilis (Mollusca: Gastropoda) in different gonadal stages

Sex hormones play an essential role in sexual differentiation, maintenance of sexual characteristics, gamete maturation, and mating behavior. However, very little is known about their dynamics in molluscs. We conducted a study on sex hormone (17β-estradiol, testosterone, and progesterone) concentrations in male and female Strombus pugilis to identify their variations at different gonadal stages. A total of 90 organisms (30 per month) were collected in February, September, and November 2016. The gonadal digestive gland complex of each specimen was dissected and divided into two sections. One section was set in alcoholic Bouin's fluid and processed with classic histological techniques; the second was macerated with 80% ethanol to extract steroids and analyzed by enzyme immunoassays. Histological section analysis was used to classify gonadal development into three stages: gametogenesis, mature, and undifferentiated. Mature females were observed in September. Testosterone and 17β-estradiol concentrations in both sexes were highest in the mature stage. In S. pugilis, 17β-estradiol, progesterone, and testosterone were all present, with higher concentrations associated with reproductive activity.

Comparison of the effect of sodium bicarbonate and glycine air polishing systems on tooth surface roughness: An atomic force microscopic analysis.

A variety of prophylactic materials are used in the dental office for the removal of stains and calculus. To evaluate tooth surface changes caused by the application of air abrasive powders (sodium bicarbonate, SBAP and glycine air powder, GPAP) along with scaling and root planing (SRP), under atomic force microscope (AFM) and to analyze the histological soft tissue changes caused by these agents, using light microscopy. This study was conducted in two phases: in vitro and in vivo. In the in vitro phase, hard tissue analysis was done under AFM following air powder polishing. Eighteen extracted teeth were chosen. SRP and tooth sectioning were carried out. Subsequently, each section of the tooth was mounted on a glass plate with self-cure acrylic resin and air polished using SBAP and GPAP. In the vivo phase, the soft tissue was analyzed under a light microscope for surface roughness. A biopsy specimen was taken from patients who had received phase I therapy, and flap surgery was planned using a modified Widman flap technique. This study compared surface changes in enamel and cementum, under AFM, as indicated by RA after SRP, SRP and SBAP, and SRP and GPAP; comparisons were then drawn across the three groups. The mean AFM values were 108.5 and 144.7, 102.7 and 81.7, and 95.6 and 7.4, at the crown and root, for SRP, SRP and SBAP, and SRP and GPAP interventions, respectively. GPAP was the least rough on soft tissues. SBAP and GPAP were better than hand instrumentation as indicated by AFM and histological section analysis.

Tissue quantification of radioiodine thyroid uptake in humans by an isotopic imaging technique on slides.

Thyroid metabolism involves iodine, which allows us to use radioactive iodine for diagnostic and therapy purposes. The efficiency of radioiodine therapy depends on several parameters; the ability of thyroid tissue to uptake radioactive iodine is one of them. The objective of this work is to quantify the radioactive iodine uptake on thyroid tissue. In this work, we developed a method to quantify the in vivo uptake of iodine-131 on sections of thyroid glands removed by thyroidectomies. We performed an analysis of histological sections of the thyroid tissue by beta imaging. We had the opportunity to quantify the fixed radioactivity and to analyze its distribution in the thyroid gland, thanks to the good spatial resolution available with the type of detector used. The results gave a high image resolution showing the heterogeneity of iodine-131 fixation by the thyroid tissue. We were able to quantify the tissue radioactivity in mega Becquerel (MBq) per volume unit. This work has shown that the direct quantification of the thyroid tissue uptake is possible using the beta imaging system.

FoxM1 Haploinsufficiency Drives Clonal Hematopoiesis

Hematopoiesis is an orchestrated process in which hematopoietic stem cells (HSCs) can self-renew and produce all lineages of blood cells. Majority of HSCs are in a quiescent state with a low growth rate. However, some genetic mutations that occur in HSCs impel HSCs to exit the quiescent state and to proliferate excessively, which enables mutant HSCs to outcompete normal HSCs and leads to clonal expansion of mutant HSCs. Myelodysplastic syndromes (MDSs) as a clonal disease, arise from the expansion of mutant HSCs and are characterized by morphologic dysplasia, ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia. FoxM1 is one of transcription factors in the family of Fox ('Forkhead box') proteins. Analysis of public database revealed that the expression level of FOXM1 was decreased significantly in CD34 + cells from a subset of patients with MDS as compared to healthy individuals. Thus, we sought to determine whether haploinsufficiency of FOXM1 contributes to the development of MDS in mice. Our study showed that haploinsufficiency of Foxm1 led to an expansion of hematopoietic stem/progenitor cells in mice. Since FoxM1 has previously been implicated in regulation of cell cycle, we determined the cell cycle status of Foxm1 heterozygous HSCs. By BrdU incorporation assay, we showed that Foxm1 heterozygous HSCs have an increased S phase and G2/M phase as compared to control HSCs from wildtype mice. Additional analysis with Hochest33342/Pyronin-Y staining and Ki67/DAPI staining showed a significant decrease in the number of quiescent (G0) cells in Foxm1 heterozygous HSCs as compared to control HSCs. These results suggest that FoxM1 haploinsufficiency promotes HSCs to exit quiescence and to enter cell cycle, thereby leading to exhaustion of HSCs. To further assess the function of Foxm1 heterozygous HSCs in vivo, we performed competitive repopulation assay. We found that Foxm1 haploinsufficiency HSCs exhibited competitive repopulation advantage in the first and secondary recipient mice, but displayed significantly decreased capacity of repopulation in tertiary recipient mice as compared to control HSCs, suggesting that Foxm1 haploinsufficiency promoted clonal expansion of HSCs, which leads to an exhaustion of HSCs eventually. HSC proliferation can be induced by some specific immune effectors such as Toll-like receptor 4 (TLR4). Lipopolysaccharide (LPS) stimulates HSC proliferation by activating TLR4 signaling pathway. Low dose of LPS treatment over time accelerated the development of MDS in mice. Interestingly, low dose of LPS injection chronically induced defects in hematopoiesis in Foxm1 haploinsufficient mice but not the control wildtype mice. Recipient mice transplanted with Foxm1 heterozygous BM cells but not the control BM cells developed MDS-like disease with cytopenia and a decreased number of hematopoietic stem/progenitor cells after LPS stimulation. Moreover, we found that nearly half of aged Foxm1 haploinsufficient mice (20 months) developed splenomegaly. Analysis of histologic sections in Foxm1 haploinsufficient mice showed that the mice developed hematopoietic dysplasia including dysplastic megakaryocytes with bizarre-shaped nuclei in bone marrow and extramedullary hematopoiesis with accumulation of myeloid cells in spleen. RNA-seq analysis indicated that haploinsufficiency of Foxm1 perturbed multiple stem cell-maintenance mechanisms especially in metabolic processes. Taken together, our studies suggest that Foxm1 haploinsufficiency in mice causes clonal expansion of HSCs and promotes MDS-like disease, which underscores the significant role of FOXM1 downregulation in the initiation and development of human MDS. Disclosures No relevant conflicts of interest to declare.

The Effect of Oxytocin on Reparative Histogenesis in the Periodontium and Mucous Membrane of the Airways (Experimental Study)

The aim of the study was to elucidate the role and significance of oxytocin in reparative histogenesis of tissues of various genesis using the example of periodontal tissues and extrapulmonary airways.Material and methods. Periodontitis and tracheobronchitis were simulated on sexually mature white outbred male rats. Periodontitis was simulated by traumatic injury to the periodontium, followed by the wound infection with Staphylococcus aureus. Introduction of oxytocin as an additional tool was used in the treatment of periodontitis. Experimental tracheobronchitis was simulated in rats by intratracheal infection with S. aureus (strains with and without antilactoferrin activity). Paraventricular large-cell nuclei of the hypothalamus were additionally destroyed in the simulated experimental tracheobronchitis (in order to reduce the production of endogenous oxytocin). The resulting material (the periodontium, alveolar processes, trachea and extrapulmonary bronchi) was examined using histological methods of investigation.Results. Analysis of histological sections demonstrated that when oxytocin was used in the complex therapy of experimental periodontitis, the proliferation and cyto-differentiation of fibroblasts, osteoblasts and cementoblasts was intensified, followed by their activation; this resulted in the optimization of reparative histogenesis in the structures of the periodontium and alveolar ridge. In animals with destruction of the paraventricular nuclei of the hypothalamus (against the background of an oxytocin deficiency in the body), a decrease in the reparative potential of the tracheal and bronchial mucosa was observed. Moreover, the severity of the inflammatory process in the mucous membrane of the trachea and bronchi with the introduction of staphylococci with antilactoferrin activity was significantly higher than that of animals that were administered staphylococci without antilactoferrin activity.Conclusion. The results obtained indicate the optimizing and correcting effect of the hypothalamic nonapeptide oxytocin on the reparative regeneration of the studied periodontal and the trachea and bronchi structures.

Cartography of the mechanical properties of the human amniotic membrane

Because of its low immunogenicity, biological properties, and high availability, the Human Amniotic Membrane (HAM) is widely used in the clinic and in tissue engineering research. However, while its biological characteristics are well described, its mechanical properties remain understudied especially in terms of inter- and intra-HAM variability. To guide bioengineers in the use of this natural biomaterial, a detailed cartography of the HAM’s mechanical properties was performed. Maximal force (Fmax) and strain at break (Smax) were identified as the relevant mechanical criteria for this study after a combined analysis of histological sections, thickness measurements after dehydration, and uniaxial tensile tests. Eight HAMs were studied by mechanical cartography using a standardized cutting protocol and sampling pattern. On average, 103 ± 10 samples were retrieved and tested per HAM. Intra-tissue variability highlighted the fact that there were two mechanically distinct areas (placental and peripheral) in each HAM. For all HAMs, placental HAM was significantly stronger by 82 ± 45% and more stretchable by 19 ± 6% than their peripheral counterparts. Our results also demonstrated that placental, but not peripheral, HAM presented isotropic mechanical properties. Thus, placental HAM can be a raw material of choice that could be favored especially in the development of tissue engineering products where mechanical properties play a key role.

Analysis of Bone Mineral Profile After Prolonged Every-Other-Day Feeding in C57BL/6J Male and Female Mice

Intermitted fasting or every-other-day feeding (EOD) has many positive effects in rodents and humans. Our goal was to describe how EOD influences bone mineral composition in female and male mice under prolonged EOD feeding. Male and female adult mice were fed EOD for 9 months. After this time, we used a direct method of measurement of mineral components in ashes of long bones (humerus and radius) to estimate the content of calcium (Ca), phosphorus (P), potassium (K), magnesium (Mg), and sodium (Na). We also performed histological analysis of sections of long bones. We found no significant changes in mineral composition between ad libitum and EOD fed males and females. We noted higher Ca and P contents in control males vs. females and lower content of Mg in control males vs. females. We observed the presence of marrow adipose tissue (MAT) in sections of EOD-fed females. EOD without supplementation during feeding days did not increase loss of mineral content of bones in C57BL/6J mice, but the presence of MAT only in EOD females indicates a gender-dependent response to EOD treatment in C57BL/6J mice.

Deep fusion of contextual and object-based representations for delineation of multiple nuclear phenotypes.

Nuclear delineation and phenotypic profiling are important steps in the automated analysis of histology sections. However, these are challenging problems due to (i) technical variations (e.g. fixation, staining) that originate as a result of sample preparation; (ii) biological heterogeneity (e.g. vesicular versus high chromatin phenotypes, nuclear atypia) and (iii) overlapping nuclei. This Application-Note couples contextual information about the cellular organization with the individual signature of nuclei to improve performance. As a result, routine delineation of nuclei in H&E stained histology sections is enabled for either computer-aided pathology or integration with genome-wide molecular data. The method has been evaluated on two independent datasets. One dataset originates from our lab and includes H&E stained sections of brain and breast samples. The second dataset is publicly available through IEEE with a focus on gland-based tissue architecture. We report an approximate AJI of 0.592 and an F1-score 0.93 on both datasets. The code-base, modified dataset and results are publicly available. Supplementary data are available at Bioinformatics online.

Structural-functional state of kidneys of rats of two generations when using glyphosate-resistant genetically modified soy and herbicide Roundup

Due to the widespread use of transgenic soy and the herbicide, we conducted histological studies of the kidneys of two generations of rats that received genetically modified soybean Ready, not treated with a herbicide, the same genetically modified soybean, treated with a herbicide and the Roundup herbicide together with drinking water.Materials and methods. The subjects of the study were male rats of the Wistar line at the age of 4 months, weighing 180-200 g. The experimental animals were grouped into 5 groups of 12 rats each (6 females and 6 males): Group I - the intact animals (kept on a standard vivarium diet); Group II – the animals with up to 26% of standard diet nutritional value, replaced by traditional soybean; Group III - the rats with up to 26% of the standard diet, replaced by genetically modified soybeans, not treated with the herbicide Roundup; Group ІV – the rats, fed with feed containing up to 26% of genetically modified soybean, treated with the herbicide Roundup; Group V – the rats, received the herbicide with drinking water at a concentration (0.1 μg / L). After 42 days, all groups of rats were paired, and thus the second generation was obtained. At the age of 12 months, the rats of all groups were decapitated, and pieces, selected from the middle part of the kidneys (indicate which ones), then these pieces were fixed in 10 % neutral solution of formalin for 48 hours. At performing morphological studies, commonly used histological techniques were used.Results. The evaluation of histopathological changes in the kidneys of the experimental animals, fed with genetically modified soybean, not treated with the herbicide (Group III), the ones, fed with transgenic soybean, treated with a herbicide (Group IV), and the ones received the herbicide with water (Group V) was realized. The analysis of histological sections of the kidneys in all rat groups showed changes in the structure of the kidneys in the IV and V groups. As a result of the experiment, reversible swelling of the renal tubule epithelium in these groups and signs of necrosis of individual cells in the first generation of rats were noted, and in the second generation there was a destruction of the tubule cells and a violation of their functions.Conclusions. The use of genetically modified soybean, treated with the herbicide «Roundup» and herbicide, results in the loss of the structure of renal glomeruli and in the disruption of their functions, both in the first and second generation. Such results indicate a negative effect of the herbicide on the rat's organism and its ability to accumulate in soybean seeds

Evaluation of laser fluorescence in combination with photosensitizers for detection of demineralized lesions.

Early detection of caries plays an important role in its prognosis. This study sought to assess the efficacy of laser fluorescence alone and in combination with photosensitizers for detection of demineralized smooth-surface lesions of permanent teeth. This in vitro experimental study was conducted on 60 extracted sound premolars. Four windows measuring 2 x 2 mm were created on each tooth. Forty out of 60 teeth were subjected to demineralization cycle. All teeth were then sectioned and subjected to DIAGNOdent Pen (Kavo, Biberach, Germany) alone and in combination with 0.2-mM tetrakis N-methylpyridyl porphyrin (TMPyP) fluorescent dye, 2% methylene blue and 2% new methylene blue photosensitizers. The results of histological analysis of sections served as the gold standard. The correlation of the results with the gold standard was assessed using the Pearson's correlation coefficient and receiver operating characteristic (ROC) curve. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the four techniques were evaluated as well. The correlation between the results of DIAGNOdent laser with TMPyP dye and the gold standard was significant (P < 0.05). Also, this technique had the highest sensitivity while DIAGNOdent laser alone had the highest specificity. Laser fluorescence with TMPyP fluorescent dye has suitable efficacy for early detection of initial enamel lesions.

Heterologous expression of nifA or nodD genes improves chickpea-Mesorhizobium symbiotic performance

The aim of this study was to investigate whether the overexpression of NifA and NodD regulators contribute to the symbiotic improvement of chickpea mesorhizobia. The native strains V-15b, ST-2, and PMI-6 were transformed with extra copies of nifA or nodD genes and several plants trial were performed. Plant growth assays showed that nifA overexpression was able to improve the symbiotic effectiveness of V-15b, while nodD overexpression lead to the improvement of ST-2 and PMI-6. Hydroponic assays showed that plants inoculated with V15bnifA+ and PMI6nodD+ started developing nodules earlier than those inoculated with the corresponding control strains. In addition, the number of nodules was always higher in plants inoculated with the strains overexpressing the symbiotic genes. Analysis of histological sections of nodules formed by V15bnifA+ showed a more developed fixation zone when compared with control. On the other hand, nodules induced by PMI6nodD+ did not show a senescent zone, which was observed in nodules from plants inoculated with the control strain. Plants inoculated with PMI6nodD+ and ST2nodD+ showed a higher number of infection threads than the corresponding control inoculations. These results indicate that overexpressing nifA and nodD may be an important tool to achieve the improvement of the symbiotic performance of mesorhizobia.

Upconversion-Magnetic Carbon Sphere for Near Infrared Light-Triggered Bioimaging and Photothermal Therapy.

Nanoparticle-based theranostics combines tumor imaging and cancer therapy in one platform, but the synthesis of theranostic agents is impeded by chemical groups on the surface and the size and morphology of the components. Strategies to construct a multifunctional platform for bioimaging and photothermal therapy (PTT) are urgently needed. A new upconversion-magnetic agent (FeCUPs) based on hollow carbon spheres, which is both a photothermal agent and a dual carrier of luminescent and magnetic nanoparticles, provides an effective approach for tumor elimination.Methods: The morphology of FeCUPs was characterized for the construction and size adjustment of the theranostic agent using transmission electron microscopy, high-resolution transmission electron microscopy, energy dispersive spectroscopy and high angle annular dark field scanning transmission electron microscopy. The distribution of FeCUPs was tracked under in-situ upconversion luminescence (UCL) imaging and magnetic resonance imaging (MRI) in vivo. Photothermal therapy was carried out on tumor-bearing mice, after which the toxicity of PTT was evaluated by a blood biochemistry test and histological section analysis.Results: Stable and uniform loading of luminescent nanocomposites on three-dimensional carbon materials is reported for the first time. Based on the mechanism of synthesis, the size of the hybrid particles was adjusted from micrometers to nanometers. External magnetic field-enhanced photothermal therapy with multi-model imaging was accomplished using FeCUPs. Moreover, no cancer recurrence was found during 14 days of recovery without PTT.Conclusions: Hollow carbon spheres, photothermal agents loaded with upconversion nanoparticles inside and magnetic nanoparticles outside were prepared for photothermal therapy. The aggregation of FeCUPs in tumors by the local magnetic field was verified by MRI and UCL imaging, and PTT was enhanced.

Anti-Inflammatory Effect of the &amp;lt;i&amp;gt;Isatis tinctoria&amp;lt;/i&amp;gt; L. Root Extract on Lipopolysaccharide-Induced Periodontitis in Rats

Objectives: Isatis tinctoria L., clary, is an herbal plant traditionally used in folk medicine for the treatment of various diseases and conditions. Although it has been primarily used as an antimicrobial and antifungal, there are data on traditional use of I. tinctoria as an agent against antiallergic, anti-thrombocytosis. The aim of the study was to examine the effect of the I. tinctoria root extract on the lipopolysaccharide (LPS)-induced periodontitis in rats on osteoclast associated bone resorptive activity, cell death including apoptosis, and inflammation in a rat of disease model of periodontitis. Materials and Methods: Periodontitis, acute or chronic inflammatory status in periodontal tissue in rats could be induced by repeated injections of LPS from Escherichia coli into the periodontal pocket area between the first and second right maxillary molars. Eighteen male rats were distributed among the following treatment groups: 1) I. tinctorial root extract (Antifect) 200 mg/kg body weight, 2) acetylsalicylic acid (ASA), 20 mg/kg body weight and 3) Phosphate buffered saline (PBS) treatment used as a control. After 15 days, maxilla, alveolar bone, molar teeth and associated periodontal tissues were harvested. Inflammatory alveolar bone resorption was analyzed by microcomputerized tomography (μCT) (microcomputer tomography). Tissues fixed with paraformaldehyde and formalin for 2 days, after that paraffin embedded histological sections were stained with haematoxylin and eosin (H/E) for the assessment of histopathological changes or tested to immunohistochemistry for detecting TRAP (tartrate resistant acid phosphatase) positive cells and caspase 3. Cell death and Apoptosis were analyzed in the periodontal tissues by tunnel assay. The inflammatory status was assessed by the measurements of proinflammatory cytokines interleukin-Iβ (IL-Iβ), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of gingival tissues and descriptive analysis of histological sections of periodontal. Results: Treatment with Antifect, compared to the control group, significantly diminished the process of inflammation decreasing the levels of IL-Iβ, IL-6 and TNF-α, reducing the gingival tissue lesions and preserving bone alveolar resorption. Considerably a smaller number of inflammatory cells and a larger number of fibroblasts were noticed. Also, μCT analysis showed that only Antifect treated group reduced bone resorption and the number of TRAP-positive multinucleated cells (osteoclasts), also, significantly reduced the number of apoptotic cells in the gingival tissues and of osteocytes in the alveolar bone crest. Conclusion: Antifect manifested anti-inflammatory elect and reducing alveolar bone resorption in LPS-induced periodontitis suggest that it may have a role as a therapeutic agent in periodontal diseases.

Surgical Simulation of a Posttraumatic Spinal Cord Glial Scar in Rats.

We developed and verified an original, minimally invasive method for surgical simulation of a posttraumatic spinal cord glial scar in rats. The model is intended for use as a biological platform for testing the stimulation of regenerative processes in the central nervous system. Unification of the model enables one to achieve versatility both for implantation techniques and for the development of system-action approaches. Faced with a standard structural defect of the spinal cord, researchers will have the unique opportunity to test in vivo promising methods for spinal function recovery in the posttraumatic period. We developed anesthetic support, surgical tactics, and a set of rehabilitation measures for the chronic postoperative period. Experimental exposure effects were preliminarily assessed in vivo using a standard technique for recording the motor activity of rats in the postoperative period of spinal cord injury. Our final conclusions were drawn based on an analysis of histological sections of the rat spinal cord glial scar in three mutually perpendicular planes.

Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro.

Background: The ribosomal protein S6 kinase 1 (S6K1) is one of the main components of the mTOR/S6K signal transduction pathway, which controls cellular metabolism, autophagy, growth, and proliferation. Overexpression of S6K1 was detected in tumors of different origin including breast cancer, and correlated with the worse disease outcome. In addition, significant accumulation of S6K1 was found in the nuclei of breast carcinoma cells suggesting the implication of kinase nuclear substrates in tumor progression. However, this aspect of S6K1 functioning is still poorly understood. The main aim of the present work was to study the subcellular localization of S6K1 in breast cancer cells with the focus on cell migration. Methods: Multicellular spheroids of MCF-7 cells were generated using agarose-coated Petri dishes. Cell migration was induced by spheroids seeding onto adhesive growth surface and subsequent cultivation for 24 to 72 hours. The subcellular localization of S6K1 was studied in human normal breast and cancer tissue samples, 2D and 3D MCF-7 cell cultures using immunofluorescence analysis and confocal microscopy. Results: Analysis of histological sections of human breast tissue samples revealed predominantly nuclear localization of S6K1 in breast malignant cells and its mainly cytoplasmic localization in conditionally normal cells. In vitro studies of MCF-7 cells demonstrated that the subcellular localization of S6K1 depends on the cell density in the monolayer culture. S6K1 relocalization from the cytoplasm into the nucleus was detected in MCF-7 cells migrating from multicellular spheroids onto growth surface. Immunofluorescence analysis of S6K1 and immunocoprecipitation assay revealed the colocalization and interaction between S6K1 and transcription factor TBR2 (T-box brain protein 2) in MCF-7 cells. Conclusions: Subcellular localization of S6K1 depends on the density and locomotor activity of the MCF-7 cells.

Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro

Background: The ribosomal protein S6 kinase 1 (S6K1) is one of the main components of the mTOR/S6K signal transduction pathway, which controls cellular metabolism, autophagy, growth, and proliferation. Overexpression of S6K1 was detected in tumors of different origin including breast cancer, and correlated with the worse disease outcome. In addition, significant accumulation of S6K1 was found in the nuclei of breast carcinoma cells suggesting the implication of kinase nuclear substrates in tumor progression. However, this aspect of S6K1 functioning is still poorly understood. The main aim of the present work was to study the subcellular localization of S6K1 in breast cancer cells with the focus on cell migration. Methods: Multicellular spheroids of MCF-7 cells were generated using agarose-coated Petri dishes. Cell migration was induced by spheroids seeding onto adhesive growth surface and subsequent cultivation for 24 to 72 hours. The subcellular localization of S6K1 was studied in human normal breast and cancer tissue samples, 2D and 3D MCF-7 cell cultures using immunofluorescence analysis and confocal microscopy. Results: Analysis of histological sections of human breast tissue samples revealed predominantly nuclear localization of S6K1 in breast malignant cells and its mainly cytoplasmic localization in conditionally normal cells. In vitro studies of MCF-7 cells demonstrated that the subcellular localization of S6K1 depends on the cell density in the monolayer culture. S6K1 relocalization from the cytoplasm into the nucleus was detected in MCF-7 cells migrating from multicellular spheroids onto growth surface. Immunofluorescence analysis of S6K1 and immunocoprecipitation assay revealed the colocalization and interaction between S6K1 and transcription factor TBR2 (T-box brain protein 2) in MCF-7 cells. Conclusions: Subcellular localization of S6K1 depends on the density and locomotor activity of the MCF-7 cells.

Coconut milk regulates the allergic response in Balb/c mice sensitized with whey proteins

In this work the effect of coconut milk on mice sensitized to cow's milk proteins was evaluated. Balb/c mice treated orally for twenty eight days with coconut milk were sensitized intraperitoneally with β-lactoglobulin (β-Lg) or α-Lactoglobulin (α-Lac). We used Ussing chamber to analyze ex vivo electrical parameters characterizing the intestinal tissue of mice by measuring the variations of the short current circuit Isc (μA/cm2) as well as the epithelial conductance (G). Jejunal fragments of sensitized and treated mice were mounted in Ussing chamber and stimulated by the deposit of β-Lg or α-Lac. Symptom scores were determined after in vivo challenge to β-Lg or α-Lac. Intestinal damage was assessed by histological analysis. Coconut milk influ-enced the electrophysiological parameters by significantly decreasing the short-circuit current (Isc) (p &lt; 0.001) and the epithelial conductance ((p&lt; 0.01 and (p &lt; 0.001, respectively). Moreover, in coconut milk-treated mice, no significant clinical symptoms were observed. Analysis of histological sections revealed that coconut milk reduced the microscopic lesions induced by β-Lg or α-Lac sensitization. We speculate that the administration of coconut milk could prevent the systemic and anaphylactic responses in sensitized mice.

Preclinical quantitative in-vivo assessment of skin tissue vascularity in radiation-induced fibrosis with optical coherence tomography.

Radiation therapy (RT) is widely and effectively used for cancer treatment but can also cause deleterious side effects, such as a late-toxicity complication called radiation-induced fibrosis (RIF). Accurate diagnosis of RIF requires analysis of histological sections to assess extracellular matrix infiltration. This is invasive, prone to sampling limitations, and thus rarely used; instead, current practice relies on subjective clinical surrogates, including visual observation, palpation, and patient symptomatology questionnaires. This preclinical study demonstrates that functional optical coherence tomography (OCT) is a useful tool for objective noninvasive in-vivo assessment and quantification of fibrosis-associated microvascular changes in tissue. Data were collected from murine hind limbs 6 months after 40-Gy single-dose irradiation and compared with nonirradiated contralateral tissues of the same animals. OCT-derived vascular density and average vessel diameter metrics were compared to quantitative vascular analysis of stained histological slides. Results indicate that RIF manifests significant microvascular changes at this time point posttreatment. Abnormal microvascular changes visualized by OCT in this preclinical setting suggest the potential of this label-free high-resolution noninvasive functional imaging methodology for RIF diagnosis and assessment in the context of clinical RT.

A novel computational method for automatic segmentation, quantification and comparative analysis of immunohistochemically labeled tissue sections

BackgroundIn the clinical practice, the objective quantification of histological results is essential not only to define objective and well-established protocols for diagnosis, treatment, and assessment, but also to ameliorate disease comprehension.SoftwareThe software MIAQuant_Learn presented in this work segments, quantifies and analyzes markers in histochemical and immunohistochemical images obtained by different biological procedures and imaging tools. MIAQuant_Learn employs supervised learning techniques to customize the marker segmentation process with respect to any marker color appearance. Our software expresses the location of the segmented markers with respect to regions of interest by mean-distance histograms, which are numerically compared by measuring their intersection. When contiguous tissue sections stained by different markers are available, MIAQuant_Learn aligns them and overlaps the segmented markers in a unique image enabling a visual comparative analysis of the spatial distribution of each marker (markers’ relative location). Additionally, it computes novel measures of markers’ co-existence in tissue volumes depending on their density.ConclusionsApplications of MIAQuant_Learn in clinical research studies have proven its effectiveness as a fast and efficient tool for the automatic extraction, quantification and analysis of histological sections. It is robust with respect to several deficits caused by image acquisition systems and produces objective and reproducible results. Thanks to its flexibility, MIAQuant_Learn represents an important tool to be exploited in basic research where needs are constantly changing.