Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

Identification of the Interaction between Minichromosome Maintenance Proteins and the Core Protein of Hepatitis B Virus.

Chronic HBV infection is a major cause of cirrhosis and hepatocellular carcinoma. Finding host factors involved in the viral life cycle and elucidating their mechanisms is essential for developing innovative strategies for treating HBV. The HBV core protein has pleiotropic roles in HBV replication; thus, finding the interactions between the core protein and host factors is important in clarifying the mechanism of viral infection and proliferation. Recent studies have revealed that core proteins are involved in cccDNA formation, transcriptional regulation, and RNA metabolism, in addition to their primary functions of capsid formation and pgRNA packaging. Here, we report the interaction of the core protein with MCMs, which have an essential role in host DNA replication. The knockdown of MCM2 led to increased viral replication during infection, suggesting that MCM2 serves as a restriction factor for HBV proliferation. This study opens the possibility of elucidating the relationship between core proteins and host factors and their function in viral proliferation.

Bearing-Fault Diagnosis with Signal-to-RGB Image Mapping and Multichannel Multiscale Convolutional Neural Network.

Deep learning bearing-fault diagnosis has shown strong vitality in recent years. In industrial practice, the running state of bearings is monitored by collecting data from multiple sensors, for instance, the drive end, the fan end, and the base. Given the complexity of the operating conditions and the limited number of bearing-fault samples, obtaining complementary fault features using the traditional fault-diagnosis method, which uses statistical characteristic in time or frequency, is difficult and relies heavily on prior knowledge. In addition, intelligent bearing-fault diagnosis based on a convolutional neural network (CNN) has several deficiencies, such as single-scale fixed convolutional kernels, excessive dependence on experts' experience, and a limited capacity for learning a small training dataset. Considering these drawbacks, a novel intelligent bearing-fault-diagnosis method based on signal-to-RGB image mapping (STRIM) and multichannel multiscale CNN (MCMS-CNN) is proposed. First, the signals from three different sensors are converted into RGB images by the STRIM method to achieve feature fusion. To extract RGB image features effectively, the proposed MCMS-CNN is established, which can automatically learn complementary and abundant features at different scales. By increasing the width and decreasing the depth of the network, the overfitting caused by the complex network for a small dataset is eliminated, and the fault classification capability is guaranteed simultaneously. The performance of the method is verified through the Case Western Reserve University's (CWRU) bearing dataset. Compared with different DL approaches, the proposed approach can effectively realize fault diagnosis and substantially outperform other methods.

The Strategic National Stockpile: identification, support, and acquisition of medical countermeasures for CBRN incidents.

The Strategic National Stockpile (SNS) serves as a repository of materiel, including medical countermeasures (MCMs), that would be used to support the national health security response to a chemical, biological, radiological, or nuclear (CBRN) incident, either natural or terrorism-related. To support and advance the SNS, the National Institutes of Health (NIH) manages targeted investigatory research portfolios, such as Countermeasures Against Chemical Terrorism (CounterACT) for chemical agents, that coordinate projects covering basic research, drug discovery, and preclinical studies. Project BioShield, managed by the Biomedical Advanced Research and Development Agency (BARDA), guides and supports academia and industry with potential MCMs through the Food & Drug Administration's approval process and ultimately supports the acquisition of successful products into the SNS. Public health emergencies such as the COVID-19 pandemic and the ever-increasing number of MCMs in the SNS present logistical and financial challenges to its maintenance. While MCMs for biological agents have been readily adopted, those for chemical agents have required sustained investments. This paper reviews the methods by which MCMs are identified and supported for inclusion in the SNS, the current status of MCMs for CBRN threats, and challenges with SNS maintenance as well as identifies persistent obstacles for MCM development and acquisition, particularly for ones focused on chemical weapons.

Minichromosome Maintenance Proteins Cooperate with LANA during the G1/S Phase of the Cell Cycle To Support Viral DNA Replication.

Latency-associated nuclear antigen (LANA) is essential for maintaining the viral genome by regulating replication and segregation of the viral episomes. The virus maintains 50 to 100 episomal copies during latency and replicates in synchrony with the cellular DNA of the infected cells. Since virus lacks its own replication machinery, it utilizes the cellular proteins for replication and maintenance, and LANA has been shown to make many of these proteins available for replication by directly recruiting them to the viral origin of replication within the terminal repeat (TR) region. Our studies identified members of the minichromosome maintenance (MCM) complex as potential LANA-interacting proteins. Here, we show that LANA specifically interacts with the components of the MCM complex, primarily during the G1/S phase of the cell cycle. MCM3 and -4 of the MCM complex specifically bound to the amino-terminal domain, while MCM6 bound to both the amino- and carboxyl-terminal domains of LANA. The MCM binding region in the N-terminal domain mapped to the chromatin binding domain (CBD). LANA with point mutations in the carboxyl-terminal domain identified an MCM6 binding domain, and overexpression of that domain (amino acids [aa] 1100 to 1150) abolished TR replication. Introduction of a peptide encompassing the LANA aa 1104 to 1123 reduced MCM6 association with LANA and TR replication. Moreover, a recombinant Kaposi's sarcoma-associated herpesvirus (KSHV) expressing LANA with a deletion of aa 1100 to 1150 (BAC16Δ1100-1150, where BAC is bacmid) showed reduced replication and persistence of viral genome copies compared to levels with the wild-type BAC16. Additionally, the role of MCMs in viral replication was confirmed by depleting MCMs and assaying transient and long-term maintenance of the viral episomes. The recruitment of MCMs to the replication origins through LANA was demonstrated through chromatin immunoprecipitation and isolation of proteins on nascent replicated DNA (iPOND). These data clearly show the role of MCMs in latent DNA replication and the potential for targeting the C-terminal domain of LANA to block viral persistence.IMPORTANCE LANA-mediated latent DNA replication is essential for efficient maintenance of KSHV episomes in the host. During latency, virus relies on the host cellular machinery for replication, which occurs in synchrony with the cellular DNA. LANA interacts with the components of multiple cellular pathways, including cellular replication machinery, and recruits them to the viral origin for DNA replication. In this study, we characterize the interactions between LANA and minichromosome maintenance (MCM) proteins, members of the cellular replication complex. We demonstrated a cell cycle-dependent interaction between LANA and MCMs and determined their importance for viral genome replication and maintenance through biochemical assays. In addition, we mapped a 50-amino acid region in LANA which was capable of abrogating the association of MCM6 with LANA and blocking DNA replication. We also detected LANA along with MCMs at the replication forks using a novel approach, isolation of proteins on nascent DNA (iPOND).

Kaposi's Sarcoma-Associated Herpesvirus Deregulates Host Cellular Replication during Lytic Reactivation by Disrupting the MCM Complex through ORF59.

Minichromosome maintenance proteins (MCMs) play an important role in DNA replication by binding to the origins as helicase and recruiting polymerases for DNA synthesis. During the S phase, MCM complex is loaded to limit DNA replication once per cell cycle. We identified MCMs as ORF59 binding partners in our protein pulldown assays, which led us to hypothesize that this interaction influences DNA replication. ORF59's interactions with MCMs were confirmed in both endogenous and overexpression systems, which showed its association with MCM3, MCM4, MCM5, and MCM6. Interestingly, MCM6 interacted with both the N- and C-terminal domains of ORF59, and its depletion in BCBL-1 and BC3 cells led to an increase in viral genome copies, viral late gene transcripts, and virion production compared to the control cells following reactivation. MCMs perform their function by loading onto the replication competent DNA, and one means of regulating chromatin loading/unloading, in addition to enzymatic activity of the MCM complex, is by posttranslational modifications, including phosphorylation of these factors. Interestingly, a hypophosphorylated form of MCM3, which is associated with reduced loading onto the chromatin, was detected during lytic reactivation and correlated with its inability to associate with histones in reactivated cells. Additionally, chromatin immunoprecipitation showed lower levels of MCM3 and MCM4 association at cellular origins of replication and decreased levels of cellular DNA synthesis in cells undergoing reactivation. Taken together, these findings suggest a mechanism in which KSHV ORF59 disrupts the assembly and functions of MCM complex to stall cellular DNA replication and promote viral replication.IMPORTANCE KSHV is the causative agent of various lethal malignancies affecting immunocompromised individuals. Both lytic and latent phases of the viral life cycle contribute to the progression of these cancers. A better understanding of how viral proteins disrupt functions of a normal healthy cell to cause oncogenesis is warranted. One crucial lytic protein produced early during lytic reactivation is the multifunctional ORF59. In this report, we elucidated an important role of ORF59 in manipulating the cellular environment conducive for viral DNA replication by deregulating the normal functions of the host MCM proteins. ORF59 binds to specific MCMs and sequesters them away from replication origins in order to sabotage cellular DNA replication. Blocking cellular DNA replication ensures that cellular resources are utilized for transcription and replication of viral DNA.

MCM family in HCC: MCM6 indicates adverse tumor features and poor outcomes and promotes S/G2 cell cycle progression.

BACKGROUND: Minichromosome Maintenance family (MCMs), as replication licensing factors, is involved in the pathogenesis of tumors. Here, we investigated the expression of MCMs and their values in hepatocellular carcinoma (HCC). METHODS: MCMs were analyzed in 105 samples including normal livers (n = 15), cirrhotic livers (n = 40), HCC (n = 50) using quantitative polymerase chain reaction (qPCR) (Cohort 1). Significantly up-regulated MCMs were verified in 102 HCC and matched peritumoral livers using PCR (Cohort 2), and the correlations with clinical features and outcomes were determined. In addition, the focused MCMs were analyzed in parallel immunohistochemistry of 345 samples on spectrum of hepatocarcinogenesis (Cohort 3) and queried for the potential specific role in cell cycle. RESULTS: MCM2-7, MCM8 and MCM10 was significantly up-regulated in HCC in Cohort 1. In Cohort 2, overexpression of MCM2-7, MCM8 and MCM10 was verified and significantly correlated with each other. Elevated MCM2, MCM6 and MCM7 were associated with adverse tumor features and poorer outcomes. In Cohort 3, MCM6 exhibited superior HCC diagnostic performance compared with MCM2 and MCM7 (AUC: 0.896 vs. 0.675 and 0.771, P < 0.01). Additionally, MCM6 other than MCM2 and MCM7 independently predicted poorer survival in 175 HCC patients. Furthermore, knockdown of MCM6 caused a delay in S/G2-phase progression as evidenced by down-regulation of CDK2, CDK4, CyclinA, CyclinB1, CyclinD1, and CyclinE in HCC cells. CONCLUSIONS: We analyze MCMs mRNA and protein levels in tissue samples during hepatocarcinogenesis. MCM6 is identified as a driver of S/G2 cell cycle progression and a potential diagnostic and prognostic marker in HCC.

Expression and Prognostic Value of MCM Family Genes in Osteosarcoma.

We performed a detailed cancer VS normal analysis to explore the expression and prognostic value of minichromosome maintenance (MCM) proteinsin human sarcoma. The mRNA expression levels of the MCM family genes in sarcoma were analyzed using data from ONCOMINE, GEPIA and CCLE databases. KEGG database was used to analyze the function of MCM2-7 complex in DNA replication and cell cycle. QRT-PCR and western blot were used to confirm the differential expression of key MCMs in osteosarcoma cell lines. Cell Counting Kit-8 and flow cytometry method were used to detect the cell proliferation and apoptosis of hFOB1.19 cells. The results showed that MCM1-7 and MCM10 were all upregulated in sarcoma in ONCOMINE database. MCM2, and MCM4-7 were highly expressed in sarcoma in GEPIA database. Moreover, all these ten factors were highly expressed in sarcoma cell lines. Furthermore, we analyzed the prognostic value of MCMs for sarcoma in GEPIA and found that MCM2, MCM3, MCM4, and MCM10 are prognostic biomarkers for human sarcoma. Analysis results using KEGG datasets showed that MCM4 and MCM6-7 constituted a core structure of MCM2-7 hexamers. We found that AzadC treatment and overexpression of MCM4 significantly promoted hFOB1.19 cell proliferation and inhibited apoptosis. The present study implied that MCM2-4 and 10 are potential biomarkers for the prognosis of sarcoma. The prognostic role of MCM4 may be attributable to the change in its DNA methylation patterns.

Comprehensive Analysis of the Expression and Prognosis for MCMs in Human Gastric Cancer.

PURPOSES: Minichromosome maintenance (MCM) proteins play an important role in replication and cell cycle progression. Even so, their expression and prognostic roles in cancer remain controversial. METHODS: To address this issue, the study investigated the roles of MCMs in the prognosis of GC by using ONCOMINE, GEPIA2, UALCAN, Cancer Cell Line Encyclopedia (CCLE), the Human Protein Atlas, Kaplan-Meier Plotter, cBioPortal, GeneMANIA, and DAVID databases. RESULTS: Over expressions of mRNA and cell lines were found in all members of the MCM family, and MCMs were found to be significantly associated with pathological tumor grades in GC patients. Besides, higher mRNA expressions of MCM1/5/7 were found to be significantly associated with shorter overall survival (OS) and progression-free survival (FP) in GC patients, while higher mRNA expression of MCM4/6/9 were connected with favorable OS and FP. Moreover, a high mutation rate of MCMs (68%) was also observed in GC patients. CONCLUSIONS: The results indicated that MCM1/5/7 were potential targets of precision therapy for patients with GC. And MCM4/6/9 were new biomarkers for the prognosis of GC. The results of the study will contribute to supplement the existing knowledge, and help to explore therapeutic targets and enhance the accuracy of prognosis for patients with GC.

Size tunable mesoporous carbon microspheres using Pluronic F127 and gelatin as co-template for removal of ibuprofen.

Size tunable mesoporous carbon microspheres, MCMs were obtained using Pluronic F127 and gelatin in co-templating method via hydrothermal and pyrolysis treatments. The presence of gelatin increased the mechanical strength of Pluronic F127 which can sustain the uniform microspherical structure of carbon following pyrolysis at 950 °C. The diameter of MCMs were controlled by variation of weight ratios between Pluronic F127 to gelatin from 1:0.01 to 1:1. MCMs exhibited inter-particulate mesoporous structure with high thermal stability (<500 °C). The MCMs were used as adsorbent for removal of ibuprofen and the kinetic studies using linear regression analysis revealed the adsorption fits pseudo second-order kinetic. The rate of adsorption and the amount of adsorbed ibuprofen were correlated well with the surface area and the crystallite size of MCMs. The efficiencies of ibuprofen adsorption on MCMs were also investigated when ibuprofen was dissolved at different concentration of water and hexane mixtures, the effect temperature variation and the amount MCMs to the volume of ibuprofen solution.

Magnetic Carbon Microspheres as a Reusable Adsorbent for Sulfonamide Removal from Water.

Novel reusable magnetic carbon microspheres (MCMs) were prepared by hydrothermal method with glucose as carbon source and Fe3O4 nanoparticles as magnetic raw materials. And adsorption performance of MCMs for sulfonamide removal from water was investigated in detail. The results indicated that the calcination temperature and calcination time had significant effects on the surface area and its volume porous of MCMs. When MCMs were calcined in 600 °C for 1 h, the surface area and volume porous of MCMs were 1228 m2/g and 0.448 m3/g, respectively. The adsorption results showed that the adsorption data fitted well with the Langmuir isotherm model and followed pseudo-second-order kinetics. When the pH value was changed from 4.0 to 10.0, the adsorption capacity of MCMs for sulfonamide was decreased from 24.6 to 19.2 mg/g. The adsorption capacity of as-synthesized MCMs achieved 18.31 mg/g after it was reused four times, which exhibited a desirable adsorption capacity and reusability.

Yellow head-like viruses affecting the penaeid aquaculture industry: a review.

This review focuses on relevant scientific information regarding the current knowledge of the yellow head complex viruses, yellow head virus and gill-associated virus. The yellow head complex viruses have been problematic within the aquaculture industry for over 10 years and still retain their research topicality. Presently, there are numerous research papers from different journals covering the identification, disease expression and spread, pathogenesis, detection, morphology, genomic sequence and protein profiles of the yellow head complex viruses. Indeed, there has been no extensive review to compare these studies, and as a corollary, to assess flaws in contemporary research and knowledge. Additionally, the yellow head complex viruses rank within the top four prawn viruses with respect to disease impact and economic loss. This review collectively reports on all the findings and current methods of research and aims to identify weak areas of research where conclusions have been unjustifiably drawn and furthermore to elucidate areas that have a gap of knowledge.