High Fructose Negatively Impacts Proliferation of NSC-34 Motor Neuron Cell Line

Objectives The main aim of this study is to identify the deleterious effects of indiscriminately consumed high fructose on motor neurons that are critically affected in many neurological conditions causing movement disorders including paralysis. Materials and Methods Neuroblastoma x mouse spinal cord motor neuron cell line (NSC-34) motor neuron cell lines were treated with high fructose and oxygen supplementation (18.8%) and assayed for cell proliferation/death, reactive oxygen species (ROS) generation, and oxidative stress response induction Statistical Analysis Mean and standard deviation, significance with and without high fructose (F)-5%, were estimated by t-tests using GraphPad Prism ver. 8.2.1 Results F-5% along with O2 (18.8%) annihilates the cells (∼85%) by day10 and inhibits cell division as observed by the presence of multinucleated cells. Unexpectedly, 1 to 2% of cells that survived, differentiated and displayed progressive neurite extension. Though not healthy, they were viable up to 80 days. F-5% increased ROS levels (∼34%) not accompanied by concomitant enhanced expression of oxidative stress response regulator, the transcription factor, nrf-2, or downstream effector, sod-1. Conclusion High fructose is extremely harmful to NSC-34 motor neuron cell line.

Recurrent herpes zoster with IgD deposits, multinucleated keratinocytes and overexpression of galectin and glypican 3 in a patient with SARS-COVID-19 infection

The novel coronavirus disease (COVID-19) that currently plagues the world and caused by SARS-CoV-2, has spread internationally since late 2019. The dermatologic manifestations of this virus are currently being identified. We describe a 73-year-old Caucasian female who presented to many physicians for recurrent Herpes zoster episodes that persisted, despite treatment with multiple antiviral medications. The patient was diagnosed with COVID-19 before an onset of vesicular pustular lesions. The clinical diagnoses were recurrent herpes zoster and recurrent varicella. A skin biopsy was obtained and stained with hematoxylin and eosin to confirm a diagnosis. Immunohistochemical stains for Ki-67, Phospho-Histone H3, galectin 3, glypican and IgD were positive in multinucleated cells of the skin, where the viral lesions were detected. Recidivated herpes zoster and varicella are currently being clinically associated with COVID- 19; the abnormal immune response in patients with COVID-19 may be due to the overexpression of molecules that facilitate the outbreak of these viruses.

The effect of paclitaxel on apoptosis, autophagy and mitotic catastrophe in AGS cells

Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

Evaluation of genotoxicity in buccal mucosa of patients subjected to X-rays by degenerative nuclear alterations study

The aim of this study is to explore the genotoxicity of cells obtained from the buccal mucosa in patients who were exposed to dental X-rays using micronucleus analysis. All the subjects underwent a routine oral clinical examination and subjects with any visible or symptomatic change in the buccal mucosa were excluded. Subjects who were expose to X rays in the past 6 months were also excluded. Based on the inclusion and exclusion criteria a total of 116 subjects were recruited. The included subjects were all nonsmokers. The genotoxicity was studied by micronucleus assay. There was significant difference in the frequency of multinucleated cell numbers from before exposure to after exposure to OPG. In patients having exposed to CBCT, a higher cell turnover was detected. The number of multinucleated cells gradually increases after panoramic radiographs, hence dental X-rays should be prescribed only when absolutely necessary.

OC_Finder: A deep learning-based software for osteoclast segmentation, counting, and classification

Osteoclasts are multinucleated cells that exclusively resorb bone matrix proteins and minerals on the bone surface. They differentiate from monocyte/macrophage-lineage cells in the presence of osteoclastogenic cytokines such as the receptor activator of nuclear factor-κB ligand (RANKL) and are stained positive for tartrate-resistant acid phosphatase (TRAP). In vitro, osteoclast formation assays are commonly used to assess the capacity of osteoclast precursor cells for differentiating into osteoclasts wherein the number of TRAP-positive multinucleated cells are counted as osteoclasts. Osteoclasts are manually identified on cell culture dishes by human eyes, which is a labor-intensive process. Moreover, the manual procedure is not objective and result in lack of reproducibility. To accelerate the process and reduce the workload for counting the number of osteoclasts, we developed OC_Finder, a fully automated system for identifying osteoclasts in microscopic images. OC_Finder consists of segmentation and classification steps. OC_Finder detected osteoclasts differentiated from wild-type and Sh3bp2KI/+ precursor cells at a 99.4% accuracy for segmentation and at a 98.1% accuracy for classification. The number of osteoclasts classified by OC_Finder was at the same accuracy level with manual counting by a human expert. Together, successful development of OC_Finder suggests that deep learning is a useful tool to perform prompt and accurate unbiased classification and detection of specific cell types in microscopic images.

Establishment of a New Cell Line of Canine Mammary Tumor CMT-1026

Canine mammary tumors (CMTs) have histopathological, epidemiologic and clinical characteristics similar to those in humans and are known to be one of the best models for human breast cancer (HBC). This research aimed to describe a newly established canine cell line, CMT-1026. Tumor samples were collected from a female dog exhibiting clinical mammary neoplasm, and the adherent cells were cultured. Both the histology and immunohistochemistry (IHC) of tumor samples were estimated. Cell growth, ultrastructural, cytological and immunocytochemistry (ICC) features of CMT-1026 were examined. CMT-1026 cells were inoculated into 10 female BALB/c nude mice to evaluate oncogenicity and metastatic ability. Hematoxylin-eosin (H.E.) staining of the tumors revealed an epithelial morphology. Electron microscopy was used to detect histological and cytological of smears, and ultrathin sections showed that CMT-1026 cells were polygonal and characterized by atypia and high mitotic index in the tumor, with prominent nucleoli and multinucleated cells. IHC characterization of CMT-1026 indicated ER-, PR-, HER-2, p63+, CK5/6+, and α-SMA+ epithelial cells. ICC characterization of CMT-1026 showed high expression of Claudin-1, Delta-catenin, SOX-2, and KI-67. At 2 weeks after inoculation of the CMT-1026 cells, phyma was found in 100% of the mice. The xenograft cancers showed conservation of the original H.E. features of the female dog cancer. In conclusion, CMT-1026 may be a model of canine mammary cancer that can be used in research on the pathogenesis of both CMT and HBC.

In Silico and Cellular Differences Related to the Cell Division Process between the A and B Races of the Colonial Microalga Botryococcus braunii

Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.

Morphological features of bone tissue regenerates in experimental animals when using osteotropic drugs for recovery bone defects

The aim of the study was to determine the effectiveness using of osteotropic drugs for the restoration of bone defects. Materials and methods. The model experiment was performing on male Wistar rats. The animals has been simulated a bone defect into which was implanted:"mp3 Osteobiol" (1st experimental group - 14 animals), PRP coated with fibrin glue (2nd experimental group - 13 animals) and PRP + "mp3 Osteobiol" (3rd experimental group - 15 animals). The histological material were staining with hematoxylin-eosin (according to Van – Gizon). Microscopic studies were performing on a Leica MDE optical microscope at a magnification of x 100, x 200. Results. At the 60th day of observation, after implantation of "mp3 OsteoBiol" + PRP, a pronounced growth of fibrous, loose, not quite mature connective tissue was visualized, with the proliferation of giant multinucleated cells, macrophages, osteoblasts and the formation of osteoids. At the end of the experiment (90th day) in the bone regenerates of animals of group III noted the active formation of the newly formed bone lamellar structure, the formation of osteons, the bone marrow canal was filled with bone marrow. Conclusion. According to histological studies, it was found that our proposed composition, containing "mp3 OsteoBiol" and PRP, accelerated the process of bone regeneration, compared with the groups where the above agents were used alone, without inflammation, with the formation of bone, which has mature in nature.

GPR120 Inhibits RANKL-Induced Osteoclast Formation and Resorption by Attenuating Reactive Oxygen Species Production in RAW264.7 Murine Macrophages

Osteoclasts are large, multinucleated cells that are responsible for the resorption of bone. Bone degenerative diseases, such as osteoporosis, are characterized by overactive osteoclasts. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) binding to its receptor on osteoclast precursors will trigger osteoclast formation and resorption. The production of reactive oxygen species (ROS) is known to play a crucial role in RANKL-induced osteoclast formation and resorption. G-protein coupled receptor 120 (GPR120) signalling has been shown to affect osteoclast formation, but the exact mechanisms of action require further investigation. RAW264.7 murine macrophages were seeded into culture plates and exposed to the GPR120 agonist, TUG-891, at varying concentrations (20–100 µM) and RANKL to induce osteoclast formation. TUG-891 was shown to inhibit osteoclast formation and resorption without affecting cell viability in RAW264.7 macrophages. TUG-891 further decreased ROS production when compared to RANKL only cells. Antioxidant proteins, Nrf2, HO-1 and NQO1 were shown to be upregulated while the ROS inducing protein, Nox1, was downregulated by TUG-891. Gene silencing revealed that TUG-891 exerted its effects specifically through GPR120. This study reveals that GPR120 signalling may inhibit osteoclast formation and resorption through inhibition on ROS production.