Spindle assembly checkpoint gene BUB1B is essential in breast cancer cell survival.

PURPOSE: The study aimed to investigate the role of spindle assembly checkpoint (SAC) in cancer cells with compromised genomic integrity. Chromosomal instability (CIN) gives cancer cells an adaptive advantage. However, maintaining the balance of this instability is crucial for the survival of cancer cells as it could lead them to the mitotic catastrophe. Therefore, cancer cells adapt to the detrimental effects of CIN. We hypothesized that changes in SAC might be one such adaptation mechanism. The focus of the study was BUB1B, an integral part of the checkpoint. METHODS: Clinical datasets were analyzed to compare expression levels of SAC genes in normal tissue vs. breast carcinoma. The effects of the reduction of BUB1B expression was examined utilizing RNA interference method with siRNAs. In vitro viability, clonogenicity, apoptosis, and SAC activity levels of a variety of breast cancer (BrCa) cell lines, as well as in vivo tumorigenicity of the triple-negative breast cancer (TNBC) cell line MDA-MB-468, were tested. Additionally, the chromosomal stability of these cells was tested by immunofluorescence staining and flow cytometry. RESULTS: In clinical breast cancer datasets, SAC genes were elevated in BrCa with BUB1B having the highest fold change. BUB1B overexpression was associated with a decreased probability of overall survival. The knockdown of BUB1B resulted in reduced viability and clonogenicity in BrCa cell lines and a significant increase in apoptosis and cell death. However, the viability and apoptosis levels of the normal breast epithelial cell line, MCF12A, were not affected. BUB1B knockdown also impaired chromosome alignment and resulted in acute chromosomal abnormalities. We also showed that BUB1B knockdown on the MDA-MB-468 cell line decreases tumor growth in mice. CONCLUSIONS: A functional spindle assembly checkpoint is essential for the survival of BrCa cells. BUB1B is a critical factor in SAC, and therefore breast cancer cell survival. Impairment of BUB1B has damaging effects on cancer cell viability and tumorigenicity, especially on the more aggressive variants of BrCa.

Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF-cCAF homotypic clusters, and in heterotypic clusters with CTCs. METHODS: In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I-IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. RESULTS: We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is  involved in heterotypic clustering of these cells. CONCLUSION: We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.

Menstrual cycle and reproductive aging alters immune reactivity, NGF expression, antioxidant enzyme activities, and intracellular signaling pathways in the peripheral blood mononuclear cells of healthy women.

Reproductive senescence in women is a process that begins with regular menstrual cycles and culminates in menopause followed by gradual development of diseases such as autoimmune diseases, osteoporosis, neurodegenerative diseases, and hormone-dependent cancers. The age-associated impairment in the functions of neuroendocrine system and immune system results in menopause which contributes to subsequent development of diseases and cancer. The aim of this study is to characterize the alterations in immune responses, compensatory factors such as nerve growth factor (NGF) and antioxidant enzyme activities, and the molecular mechanisms of actions in the peripheral blood mononuclear cells (PBMCs) of young (follicular and luteal phases), middle-aged, and old healthy women. Peripheral blood mononuclear cells were isolated from young women in follicular and luteal phases of the menstrual cycle (n=20; 22.6±2.9 yrs), middle-aged women (n=19; 47.1±3.8 yrs; perimenopausal) and old (n=16; 63.2±4.7 yrs; post-menopausal) women and analyzed for Concanavalin (Con A)-induced proliferation of lymphocytes and cytokine (IL-2 and IFN-γ) production, expression of NGF, p-NF-κB, p-ERK, p-CREB, and p-Akt, antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), glutathione-S-transferase (GST)], extent of lipid peroxidation, and nitric oxide (NO) production. Serum gonadal hormones (17ß-estradiol and progesterone) were also measured. A characteristic age- and menstrual cycle-related change was observed in the serum gonadal hormone secretion (estrogen and progesterone), T lymphocyte proliferation and IFN-γ production. Salient features include the age-related decline observed in target-derived growth factors (lymphocyte NGF expression), signaling molecules (p-ERK/ERK and p-CREB/CREB ratios) and compensatory factors such as the activities of plasma and PBMC antioxidant enzymes (SOD and catalase) and NO production. Further, an age-associated increase in p-NF-κB expression and lipid peroxidation was observed. Also, serum 17ß-estradiol levels were positively correlated with IFN-γ production, SOD activity and NGF expression in the PBMCs. These results suggest that alterations in the levels of gonadal hormones are associated with immunosenescence characterized by decreased IFN-γ production and proliferation of T lymphocytes, decline in NGF expression, SOD and catalase activities, NO production, and signaling mechanisms and thus, may increase the incidence of diseases and cancer in women.

Propagated Circulating Tumor Cells Uncover the Potential Role of NFκB, EMT, and TGFß Signaling Pathways and COP1 in Metastasis.

Circulating tumor cells (CTCs), a population of cancer cells that represent the seeds of metastatic nodules, are a promising model system for studying metastasis. However, the expansion of patient-derived CTCs ex vivo is challenging and dependent on the collection of high numbers of CTCs, which are ultra-rare. Here we report the development of a combined CTC and cultured CTC-derived xenograft (CDX) platform for expanding and studying patient-derived CTCs from metastatic colon, lung, and pancreatic cancers. The propagated CTCs yielded a highly aggressive population of cells that could be used to routinely and robustly establish primary tumors and metastatic lesions in CDXs. Differential gene analysis of the resultant CTC models emphasized a role for NF-κB, EMT, and TGFß signaling as pan-cancer signaling pathways involved in metastasis. Furthermore, metastatic CTCs were identified through a prospective five-gene signature (BCAR1, COL1A1, IGSF3, RRAD, and TFPI2). Whole-exome sequencing of CDX models and metastases further identified mutations in constitutive photomorphogenesis protein 1 (COP1) as a potential driver of metastasis. These findings illustrate the utility of the combined patient-derived CTC model and provide a glimpse of the promise of CTCs in identifying drivers of cancer metastasis.

Investigating methods of normalization for X-ray fluorescence measurements of zinc in nail clippings using the TOPAS Monte Carlo code.

Development of portable X-ray fluorescence devices has made it easier to quickly assess trace elements such as zinc in human tissue. A TOPAS Monte Carlo code was used to investigate the use of a portable X-ray fluorescence system for detecting zinc in nail clippings. The obtained energy spectra from different nail thicknesses were analyzed and three different normalization techniques (coherent, Compton, and entire spectrum) were introduced. The simulation results suggested that coherent normalization can be a particularly robust normalization procedure.

Stress and Anxiety Among Parents of Transition-Aged Children with Autism Spectrum Disorder: a Systematic Review of Interventions and Scales.

The period between adolescence to young adulthood can be stressful for parents of transition-aged children (14 to 22 years old) with autism spectrum disorder. A systematic review was undertaken to examine if existing interventions address the unique parental stressors of this phase and if the scales used to measure parental stress and anxiety are suitable for this group. Of the 9813 studies screened, only 13 studies met the inclusion criteria. Findings indicated that interventions focused on mindfulness, social functioning, or multiple components, of which only two addressed the transition period and only three specifically targeted parents of this age group. Moreover, of the six scales which assessed stress or anxiety, none were designed for these parents. Findings highlight the urgent need for more suitable scales and targeted interventions.

Knowledge, attitude, and practice of general dental practitioners toward following proper standards of endodontic practice and use of latest technology in Dehradun: A cross-sectional study.

BACKGROUND: Choosing latest technology for the treatment improves the chances of favorable prognosis and saves the time of the clinician; hence, the aim of the study was to explore their knowledge, attitude, and practice (KAP) toward following proper standards of endodontic practice and use of latest technology. MATERIALS AND METHODS: The present study was a cross-sectional, descriptive questionnaire study conducted among general dental practitioners (GDPs). The survey was conducted among 156 GDPs. In the present study, a close-ended interview schedule was prepared to test the KAP of GDPs. RESULTS: For diagnosis, most of the study participants (58 [37.08%]) relied on case history and radiograph. Apex locator was used by 71 (45.51%) of the study subjects. Among all the study participants, 58 (37.17%) dental practitioners used rotary nickel-titanium (NiTi) files with normal saline and preheated disinfectants for cleaning and shaping of root canal. It was observed that the knowledge of majority of the dental practitioners was fair (58 [37.17%]). However, the attitude and practice toward following proper standards of endodontic practice and use of latest technology were poor. CONCLUSION: It was concluded that very few general practitioners used the latest technology in endodontics. Knowledge was fair while attitude and practice regarding following proper standards of endodontic practice and use of latest technology were poor among study participants.

Adaptation of Single-Cone Gutta-Percha in Curved Canals Prepared and Obturated with Protaper and Heroshaper Systems by Using Cone Beam Computed Tomography.

OBJECTIVE: The aim of this study is to prepare and obturate the curved canals of the mesiobuccal root of maxillary first molar with four different file systems that is protaper hand files, rotary pro taper (RP) files, heroshaper hand files, and rotary hero shapers files and to evaluate the adaptation of their single-cone Gutta-percha by cone beam computed tomography (CBCT). MATERIALS AND METHODS: Eighty teeth were selected and were divided into two groups (G1A1, G1A2 and G2A1, G2A2 as hand and RP file system, G1A3, G1A4 and G2A3, G2A4 as hand and rotary hero shaper files system) of fourth teeth each. After access opening working length of the mesiobuccal canal was established. The distobuccal and palatal roots of the samples were removed using the diamond disc at the furcation level. "Endoanalyser" software was used to measure Schneider's angle on the preoperative radiograph. This angle was measured by drawing two lines-one parallel to the long axis of the canal, in the coronal third, and the second line from the apical foramen to intersect the point where the first line left the long axis of the canal. The canals of each group were then prepared according to the manufacturer's instructions for protaper hand files, RP files, heroshaper hand files, and rotary hero shapers files systems. Direct digital radiography image of all the samples was obtained. And then, the samples were exposed to CBCT to evaluate their single-cone adaptations. The data were analyzed using SPSS 20, IBM, Armonk, NY, United States of America. RESULTS: The null hypothesis that there will not be any gap area in the adaptation of their single-cone of different file system was rejected. Minimal gap area was seen in rotary hero shapers file system (0.001 mm2) and was maximum in hand protaper (HP) file system (0.015 mm2). CONCLUSION: CBCT is a useful tool in detecting the gap area after obturation in curved canals. The HP single-cone adaptation showed the maximum gap area and rotary heroshaper single-cone adaptation showed the minimum gap area.

An in vitro Comparative Evaluation of Volume of Removed Dentin, Canal Transportation, and Centering Ratio of 2Shape, WaveOne Gold, and ProTaper Gold Files Using Cone-Beam Computed Tomography.

OBJECTIVES: The aim of this study was to compare the shaping ability of three file systems - 2Shape (2S), WaveOne Gold (WOG), and ProTaper Gold - using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: Forty-five freshly extracted mandibular teeth were chosen and instrumented using the following brands of NiTi files: 2S, WOG, and ProTaper Gold. Pre- and post-instrumentation CBCT imaging was performed to measure mesial and distal distance of the dentin walls and calculate the volume of removed dentin, apical transportation, and centering ratio. A statistical analysis of the data was performed using independent t-test. Statistical significance was set at 5%. RESULTS: There were no significant differences between the 2S and WOG in terms of the total volume of removed dentin, apical transportation, and centering ratio, whereas ProTaper showed a greater significant difference when compared to 2S and WOG. CONCLUSION: Both 2S and WOG maintained the original canal anatomy better and did not remove excess dentin while chemomechanical preparation as compared to ProTaper Gold. Instruments that use rotary movement achieved an effect similar to that of the reciprocating instruments in relation to change in angle. 2S which is a two-file system and WOG which is a single-file system might perform better clinically and might show enhanced shaping ability with less canal transportation and more centered preparation when compared to ProTaper Gold that is a multiple file-system.