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Background: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells., Material and Methods: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment., Results: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells., Conclusion: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage., (Copyright © 2023 Copyright: © 2023 Journal of Cancer Research and Therapeutics.)

Purpose: Surface-guided radiation therapy (SGRT) has been investigated intensively to ensure correct patient positioning during a radiation therapy course. Although the implementation is well defined for photon-beam facilities, only a few analyses have been published for ion-beam therapy centers. To investigate the accuracy, reliability, and efficiency of SGRT used in ion-beam treatments against the conventional skin marks, a retrospective study of a unique SGRT installation in an ion gantry treatment room was conducted, where the environment is quite different to conventional radiation therapy., Methods and Materials: There were 32 patients, divided into 3 cohorts-pelvis, limb, and chest/spine tumors-and treated with ion-beams. Two patient positioning workflows based on 300 fractions were compared: workflow with skin marks and workflow with SGRT. Position verification was followed by planar kilo voltage imaging. After image matching, 6 degrees of freedom corrections were recorded to assess interfraction positioning errors. In addition, the time required for patient positioning, image matching, and the number of repeated kilo voltage imaging also were gathered., Results: SGRT decreased the translational magnitude shifts significantly ( P < .05) by 0.5 ± 1.4 mm for pelvis and 1.9 ± 0.5 mm for limb, whereas for chest/spine, it increased by 0.7 ± 0.3 mm. Rotational corrections were predominantly lowered with SGRT for all cohorts with significant differences in pitch for pelvis ( P  = .002) and chest/spine ( P  = .009). The patient positioning time decreased by 18%, 9%, and 15% for pelvis, limb, and chest/spine, respectively, compared with skin marks. By using SGRT, 53% of all studied patients had faster positioning time, and 87.5% had faster matching time. Repositioning and consequent reimaging decreased from about 7% to 2% with a statistically significant difference of .042., Conclusions: The quality of patient positioning before ion-beam treatments has been optimized by using SGRT without additional imaging dose. SGRT clearly reduced inefficiencies in the patient positioning workflow., (© 2022 The Author(s).)

Background: The management of non-obstructive azoospermia (NOA) disease relies on microdissection testicular sperm extraction (micro-TESE). Few studies have assessed the role of micro-TESE in men with NOA in our country., Objective: The aim of the current study was to investigate the success rate of micro-TESE., Materials and Methods: This retrospective descriptive-analytical study was conducted on 463 men with NOA in Yazd Reproductive Sciences Institute during September 2017 through September 2019. Sperm were retrieved and frozen according to the rapid sperm freezing protocol. After preparing the oocyte of the male partner's spouse, sperms were thawed and then entered the intracytoplasmic sperm injection process. The clinical pregnancy of individuals was confirmed via ultrasound. Demographic data were extracted from medical records., Results: The success rate of micro-TESE was 38% and successful fertilization, biochemical pregnancy, clinical pregnancy, and live birth were observed in 111 (85.4%), 29 (22.3%), 29 (22.3%) and 14 (10.7%) men, respectively. A significant difference was seen between the two groups, regarding age (p = 0.01). In addition, the mean follicle-stimulating hormone in men with positive micro-TESE was significantly lower than in men with negative micro-TESE (p = 0.02)., Conclusion: The success of pregnancy in couples with NOA managed via micro-TESE was significant. The study found that the success rate of micro-TESE was higher in older men and in those with lower follicle-stimulating hormone levels., Competing Interests: There was no conflict of interest., (Copyright © 2021 Vahidi et al.)

Background: The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR)., Methods: Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control., Results: Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells., Conclusion: We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.

Background: Genetic risk factors for dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and dengue fever (DF) are limited, in particular there are sparse data on genetic risk across diverse populations., Methods: We conducted a genome-wide association study (GWAS) in a derivation and validation sample of 7, 460 participants of Latin American, South Asian, and South East Asian ancestries. We then developed a weighted polygenic risk score (PRS) for each participant in each of the validation cohorts of the three ancestries to predict the risk of DHF/DSS compared to DF, DHF/DSS compared to controls, and, DF compared to controls., Findings: The risk of DHF/DSS was significantly increased, odds ratio [OR] 1.84 (95%CI 1.47 to 2.31) (195 SNPs), compared to DF, fourth PRS quartile versus first quartile, in the validation cohort. The risk of DHF/DSS compared to controls was increased (OR=3.94; 95% CI 2.84 to 5.45) (278 SNPs), as was the risk of DF compared to controls (OR=1.97; 95%CI 1.63 to 2.39) (251 SNPs). Risk increased in a dose-dependent manner with increase in quartiles of PRS across comparisons. Significant associations persisted for PRS built within ancestries and applied to the same or different ancestries as well as for PRS built for one outcome (DHF/DSS or DF) and applied to the other., Interpretation: There is a strong genetic effect that predisposes to risk of DHF/DSS and DF. The genetic risk for DHF/DSS is higher than that for DF when compared to controls, and this effect persists across multiple ancestries., Competing Interests: Declaration of competing interest Dr. Pare reports other from Amgen, other from Sanofi, outside the submitted work; the other authors have no conflicts of interest., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Long non-coding RNAs (lncRNAs) are a class of cellular transcripts, which are involved in various biological processes. There is conflicting data regarding to the origin of these non-coding molecules and lncRNAs are thought to be the origin of viral genome. Here we sought to find the homology between human lncRNAs and viruses. For this purpose, the lncRNAdb database was searched for human lncRNAs. The lncRNAs' sequences were aligned with virus taxa using NCBI's BLAST tool. The phylogenic study was performed with maximum-likelihood based algorithm. The database contains 152 human lncRNAs. As a result, 63 (41.44%) of the lncRNAs have homologies with viruses. Of which, 50 (79.36%) have homology with Stealth virus. Other viruses with homology to lncRNAs were nuclear integrating DNA/RNA viruses. Moreover, 35 of 64 (23.03%) of cancer-associated lncRNAs have sequence homology with the same viruses. In phylogenetic analyses, lncRNAs with no homology to viruses were found to be the ancestor of those with homology to viruses and cancer-irrelevant lncRNAs were found to be the ancestor of cancer-related transcripts. In conclusion, lncRNAs could be the origin of nuclear integrating viruses and the nuclear integrating viruses may evolved from the non-coding regions. The results imply the role of lncRNAs with homology to viruses in human cancers.

There is increasing evidence that oxytocin is involved in learning and memory process. This study investigated the effects of blockade of oxytocin receptors using the selective oxytocin receptor antagonist atosiban (ATO) on contextual fear memory consolidation and reconsolidation in male rats. Post-training injections of different doses of ATO (1, 10, 100 or 1000 µg/kg) impaired the 48 h retention performance in a dose-dependent manner. The same doses of ATO following memory reactivation did not impair subsequent expression of contextual fear memories which formed under low or high shock intensities and tested 24 h or one week following memory reactivation. Also, no effect was found when ATO was administrated in the absence of memory reactivation. Our finding is the first report that indicates endogenous oxytocin released during training play an important role in the consolidation, but not reconsolidation of contextual fear memory in rats., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: West Nile virus emerged as an important human pathogen in North America and continues to pose a risk to public health. It can cause a highly variable range of clinical manifestations ranging from asymptomatic to severe illness. Neuroinvasive disease due to West Nile virus can lead to long-term neurological deficits and psychological impairment. However, these deficits have not been well described. The objective of this study was to characterize the neuropsychological manifestations of West Nile virus infection with a focus on neuroinvasive status and time since infection., Methods: Patients from Ontario Canada with a diagnosis of neuroinvasive disease (meningitis, encephalitis, or acute flaccid paralysis) and non-neuroinvasive disease who had participated in a cohort study were enrolled. Clinical and laboratory were collected, as well as demographics and medical history. Cognitive functioning was assessed using a comprehensive battery of neuropsychological tests., Results: Data from 49 individuals (32 with West Nile fever and 17 with West Nile neuroinvasive disease) were included in the present cross-sectional analysis. Patterns of neuropsychological impairment were comparable across participants with both neuroinvasive and non-neuroinvasive West Nile virus infection on all cognitive measures. Neuropsychiatric impairment was also observed more frequently at two to four years post-infection compared to earlier stages of illness., Conclusions: Our data provide objective evidence for cognitive difficulties among patients who were infected with West Nile virus; these deficits appear to manifest regardless of severity of West Nile virus infection (West Nile fever vs. West Nile neuroinvasive disease), and are more prevalent with increasing illness duration (2-4 years vs. 1 month). Data from this study will help inform patients and healthcare providers about the expected course of recovery, as well as the need to implement effective treatment strategies that include neuropsychological interventions.

Introduction: Previous studies have demonstrated that the β-adrenergic receptor antagonist propranolol impairs fear memory reconsolidation in experimental animals. There are experimental parameters such as the age and the strength of memory that can interact with pharmacological manipulations of memory reconsolidation. In this study, we investigated the ability of the age and the strength of memory to influence the disrupting effects of propranolol on fear memory reconsolidation in rats., Methods: The rats were trained in a contextual fear conditioning using two (weak training) or five (strong training) footshocks (1mA). Propranolol (10mg/kg) injection was immediately followed retrieval of either a one-day recent (weak or strong) or 36-day remote (weak or strong) contextual fear memories., Results: We found that propranolol induced a long-lasting impairment of subsequent expression of recent and remote memories with either weak or strong strength. We also found no memory recovery after a weak reminder shock. Furthermore, no significant differences were found on the amount of memory deficit induced by propranolol among memories with different age and strength., Discussion: Our data suggest that the efficacy of propranolol in impairing fear memory reconsolidation is not limited to the age or strength of the memory.

Introduction: One of the hallmark symptoms of posttraumatic stress disorder (PTSD) is the impaired extinction of traumatic memory. Single prolonged stress (SPS) has been suggested as an animal model of PTSD, since SPS rats exhibited the impaired fear extinction. Oxytocin (OXT) has been recently suggested as a potential pharmacotherapy for treatment of PTSD. In this study, using SPS rats we investigated the effects of multiple systemic administration of OXT on contextual fear extinction., Methods: SPS WAS CONDUCTED IN THREE STAGES: restraint for 2 h, forced swim for 20 min, and diethyl ether anesthesia, and then left undisturbed in their home cage for 7 days. In the SPS group, 7 days after SPS treatment, contextual fear conditioning was performed (on day 0), and then extinction training was performed on each of four consecutive days following fear conditioning. In the sham group, the procedures were similar except that SPS treatment was not performed., Results: During extinction trial (10 min) freezing behavior was recorded. OXT (1, 10, 100 and 1000µg/kg) was administrated (I.P) immediately after each extinction trial. SPS rats exhibited significant impairment of contextual fear extinction as compared with sham rats. While there was no significant difference in the freezing levels between SPS and Sham rats 24 h after the fear conditioning, the freezing levels in SPS rats were significantly higher than those in sham rats after the second extinction training. Systemic OXT delayed fear extinction in sham rats as compared with sham-saline treated animals. No effect of OXT was found in SPS rats., Discussion: These findings indicate that increasing OXT transmission during fear memory reactivation delays fear extinction, and thus, the recommendation of OXT for PTSD treatment should be considered with caution.

To determine genetic factors predisposing to neurological complications following West Nile virus infection, we analyzed a cohort of 560 neuroinvasive case patients and 950 control patients for 13 371 mostly nonsynonymous single-nucleotide polymorphisms (SNPs). The top 3 SNPs on the basis of statistical significance were also in genes of biological plausibility: rs2066786 in RFC1 (replication factor C1) (P = 1.88 × 10(-5); odds ratio [OR], 0.68 [95% confidence interval {CI}, .56-.81]); rs2298771 in SCN1A (sodium channel, neuronal type I α subunit) (P = 5.87 × 10(-5); OR, 1.47 [95% CI, 1.21-1.77]); and rs25651 in ANPEP (ananyl aminopeptidase) (P = 1.44 × 10(-4); OR, 0.69 [95% CI, .56-.83]). Additional genotyping of these SNPs in a separate sample of 264 case patients and 296 control patients resulted in a lack of significance in the replication cohort; joint significance was as follows: rs2066786, P = .0022; rs2298771, P = .005; rs25651, P = .042. Using mostly nonsynonymous variants, we therefore did not identify genetic variants associated with neuroinvasive disease.

Background: Amplification of several genes that map to a region of chromosome 17p11.2, including COPS3, was observed in high-grade osteosarcoma. These genes were also shown to be overexpressed and may be involved in osteosarcoma tumorigenesis. COPS3 encodes a subunit of the COP9 signalosome implicated in the ubiquitination and ultimately degradation of the P53 tumor suppressor. To determine the relation between COPS3 amplification, P53 mutation, and patient outcome in osteosarcoma, tumors from a large cohort of patients with high-grade osteosarcoma and long-term clinical follow-up were examined., Methods: Quantitative real-time polymerase chain reaction (PCR) was performed to detect copy number changes for COPS3, as well as additional genes (NCOR1, TOM1L2, and PMP22) from the 17p11.2 amplicon, in 155 osteosarcomas from a prospective collection of tumors with corresponding clinical data. Univariate and multivariate analyses were performed to assess differences in survival between groups., Results: Amplification of COPS3, detected in 31% of the osteosarcomas, was strongly associated with large tumor size (P=.0009), but was not associated with age at diagnosis, site, sex, and tumor necrosis. COPS3 amplification was significantly correlated with a shorter time to metastasis with an estimated hazard ratio (HR) of 1.61 (95% confidence interval [CI], 1.02-2.55) in univariate analysis (log-rank test, P=.042). However, in an a priori multivariate Cox model including the other clinical parameters, the HR for COPS3 amplification decreased to 1.32 (95% CI, 0.82-2.13, P=.25), mainly due to the strong correlation with tumor size. COPS3 amplification and P53 mutation frequently occurred in the same tumors, suggesting that these are not mutually exclusive events in osteosarcoma. Although not statistically significant, patients whose tumors exhibited both molecular alterations tended to be more likely to develop metastasis compared with patients with either COPS3 amplification or P53 mutation alone., Conclusions: COPS3 is the likely target of the 17p11.2 amplicon. COPS3 may function as an oncogene in osteosarcoma, and an increased copy number may lead to an unfavorable prognosis., (Copyright (c) 2007 American Cancer Society)

Purpose: Mutations of the TP53 gene have been associated with resistance to chemotherapy as well as poor prognosis in many different malignancies. This is the first prospective study of the prognostic value of somatic TP53 mutations in patients with newly diagnosed extremity osteosarcoma., Patients and Methods: One hundred ninety-six patients with high-grade, nonmetastatic osteosarcoma of the extremities were enrolled from seven tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 44 months). All patients received neoadjuvant or adjuvant chemotherapy and surgery. Tumors were analyzed for the presence of TP53 mutations by polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing. The association of the status of the TP53 gene with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors., Results: Patient age was the only factor that varied with TP53 gene status (P = .05). No relationship was identified between TP53 status and systemic relapse (relative risk, 1.24; P = .41). Analyses based on missense or nonsense mutations gave similar results (P > .10). In multivariate analysis, large (> 9 cm) tumor size (relative risk, 1.9; P = .006) and poor histologic response (< or = 90% necrosis) to chemotherapy (relative risk, 2.14; P = .02) were the only significant independent predictors of systemic outcome., Conclusion: We found no evidence that TP53 mutations predict for development of metastases in patients with high-grade osteosarcoma. Identification of other genes that influence chemotherapy response and clinical outcome in osteosarcoma is needed to facilitate further improvements in patient outcomes.

Background: To understand the role of sporadic mutations in the tumor suppressor gene p53 (also known as TP53) in the pathogenesis of breast cancer, it is important to identify at which histologic stage such mutations first occur. We previously showed that a p53 mutation present in invasive breast cancer was found in all surrounding areas of ductal carcinoma in situ (DCIS) but not in areas of hyperplasia or normal breast epithelium. In the present investigation, we studied patients with DCIS, but without invasive breast cancer, to determine the spectrum of DCIS types that can harbor a p53 mutation., Methods: Formalin-fixed, paraffin-embedded tissues from 94 patients with DCIS were evaluated histologically for the predominant cellular architectural pattern, degree of necrosis, and nuclear grade. Each specimen was also assigned an overall histologic grade (with the use of the Van Nuys Prognostic Index pathologic classification). Tissue specimens were stained immunohistochemically with an anti-p53 antibody. Positively stained tissue areas were analyzed for the presence of p53 mutations by single-strand conformation polymorphism and direct sequencing. All statistical tests were two-sided., Results: DCIS from 10 of 94 patients were found to contain p53 missense mutations. All 10 were of a solid or a comedo histologic pattern and contained cells of nuclear grade 2 or 3 (i.e., more abnormal nuclei). The frequency of p53 missense mutations was statistically significantly different among the three overall histologic grade categories (zero [0%] of 49 with low-grade DCIS, one [4.35%] of 23 with intermediate-grade DCIS, and nine [40.9%] of 22 with high-grade DCIS; df = 2 and P<.0001)., Conclusion: The DCIS types in patients in this series are representative of clinically detected DCIS. Our finding that p53 mutations can occur before the development of invasive breast cancer, particularly in DCIS of high histologic grade, has potentially important implications for prevention and treatment.