Maintenance Complex Research Articles

Establishing an Extracorporeal Cardiopulmonary Resuscitation Program

Extracorporeal cardiopulmonary resuscitation (ECPR) is a complex, life-saving procedure that uses mechanical support for patients with refractory cardiac arrest, representing the pinnacle of extracorporeal membrane oxygenation (ECMO) applications. Effective ECPR requires precise patient selection, rapid mobilization of a multidisciplinary team, and skilled cannulation techniques. Establishing a program necessitates a cohesive ECMO system that promotes interdisciplinary collaboration, which is essential for managing acute cardiogenic shock and severe pulmonary failure. ECPR is suited for selected patients, emphasizing the need to optimize every step of cardiac arrest management—from public education to advanced post-resuscitation care. The flexibility of ECMO teams allows them to manage various emergencies such as cardiogenic shock, massive pulmonary embolism, and severe asthma, showcasing the program’s adaptability. Launching an ECPR program involves addressing logistical, financial, and organizational challenges. This includes gaining administrative approval, assembling a diverse team, and crafting detailed protocols and training regimens. The development process entails organizing teams, refining protocols, and training extensively to ensure operational readiness. A systematic approach to building an ECPR program involves establishing a team, defining patient selection criteria, and evaluating caseloads. Critical elements like patient transport protocols and anticoagulation management are vital for the program’s success. In conclusion, initiating an ECPR program demands thorough planning, collaborative effort across specialties, and ongoing evaluation to improve outcomes in critical cardiac emergencies. This guide offers practical insights to support institutions in navigating the complexities of ECPR program development and maintenance.

Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM

The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.

Assessing Evolution of Microservices Using Static Analysis

Microservices have gained widespread adoption in enterprise software systems because they encapsulate the expertise of specific organizational subunits. This approach offers valuable insights into internal processes and communication channels. The advantage of microservices lies in their self-contained nature, streamlining management and deployment. However, this decentralized approach scatters knowledge across microservices, making it challenging to grasp the holistic system. As these systems continually evolve, substantial changes may affect not only individual microservices but the entire system. This dynamic environment increases the complexity of system maintenance, emphasizing the need for centralized assessment methods to analyze these changes. This paper derives and introduces quantification metrics to serve as indicators for investigating system architecture evolution across different system versions. It focuses on two holistic viewpoints of inter-service interaction and data perspectives derived through static analysis of the system’s source code. The approach is demonstrated with a case study using established microservice system benchmarks.

Identification of Goat Supernumerary Teat Phenotype Using Wide-Genomic Copy Number Variants.

Supernumerary teats (SNTs) or nipples often emerge around the mammary line. This study performed a genome-wide selective sweep analysis (GWS) at the copy number variant (CNV) level using two selected signal calculation methods (VST and FST) to identify candidate genes associated with SNTs in goats. A total of 12,310 CNVs were identified from 37 animals and 123 CNVs, with the top 1% VST values including 84 candidate genes (CDGs). Of these CDGs, minichromosome maintenance complex component 3, ectodysplasin A receptor associated via death domain, and cullin 5 demonstrated functions closely related to mammary gland development. In addition, 123 CNVs with the top 1% FST values were annotated to 97 CDGs. 5-Hydroxytryptamine receptor 2A, CCAAT/enhancer-binding protein alpha, and the polymeric immunoglobulin receptor affect colostrum secretion through multiple signaling pathways. Two genes, namely, RNA-binding motif protein 46 and β-1,3-galactosyltransferase 5, showed a close relation to mammary gland development. Six CNVs were identified and annotated to five genes by intersecting the top 1% of candidate CNVs with both parameters. These genes include LOC102185621, LOC102190481, and UDP-glucose pyrophosphorylase 2, which potentially affect the occurrence of BC through multiple biological processes, such as cell detoxification, glycogen synthesis, and phospholipid metabolism. In conclusion, we discovered numerous genes related to mammary development and breast cancer (BC) through a GWS, which suggests the mechanism of SNTs in goats and a certain association between mammary cancer and SNTs.

Identification of Crucial Cancer Stem Cell Genes Linked to Immune Cell Infiltration and Survival in Hepatocellular Carcinoma.

Hepatocellular carcinoma is characterized by high recurrence rates and poor prognosis. Cancer stem cells contribute to tumor heterogeneity, treatment resistance, and recurrence. This study aims to identify key genes associated with stemness and immune cell infiltration in HCC. We analyzed RNA sequencing data from The Cancer Genome Atlas to calculate mRNA expression-based stemness index in HCC. A weighted gene co-expression network analysis was performed to identify stemness-related gene modules. A single-sample gene set enrichment analysis was used to evaluate immune cell infiltration. Key genes were validated using RT-qPCR. The mRNAsi was significantly higher in HCC tissues compared to adjacent normal tissues and correlated with poor overall survival. WGCNA and subsequent analyses identified 10 key genes, including minichromosome maintenance complex component 2, cell division cycle 6, forkhead box M1, NIMA-related kinase 2, Holliday junction recognition protein, DNA topoisomerase II alpha, denticleless E3 ubiquitin protein ligase homolog, maternal embryonic leucine zipper kinase, protein regulator of cytokinesis 1, and kinesin family member C1, associated with stemness and low immune cell infiltration. These genes were significantly upregulated in HCC tissues. A functional enrichment analysis revealed their involvement in cell cycle regulation. This study identified 10 key genes related to stemness and immune cell infiltration in HCC. These genes, primarily involved in cell cycle regulation, may serve as potential targets for developing more effective treatments to reduce HCC recurrence and improve patient outcomes.

ANXA4 restricts HBV replication by inhibiting autophagic degradation of MCM2 in chronic hepatitis B

BackgroundHepatitis B virus (HBV) is an enveloped DNA virus that causes chronic hepatitis B (CHB) infection. Annexin, a Ca2+-activated protein, is widely expressed in various organs and tissues and has potential utility in disease diagnosis and treatment. However, the relationship between the annexin family and CHB remains unclear.MethodsClinical samples from hepatitis patients and donors or healthy individuals were collected. Transcriptome sequencing in CHB liver tissues and HBV-infected cells were performed. HepG2.2.15 cells with the full-length HBV genome and HBV-infected HepG2-NTCP cell models were established. HBV-infected mouse model was constructed and adeno-associated virus was utilized.ResultsANXA4 expression was elevated during CHB infection. ANXA4 knockdown promoted HBV replication and aggravated liver injury, while ANXA4 overexpression alleviated that. Mechanistically, autophagy pathway was activated by ANXA4 deficiency, promoting autophagic degradation of minichromosome maintenance complex component 2 (MCM2). MCM2 inhibition activated HBV replication, while MCM2 overexpression attenuated ANXA4 deficiency-induced HBV replication and liver injury. Clinically, the expression of hepatitis B viral protein was negatively correlated with the ANXA4 levels, and CHB patients with high ANXA4 levels (> 8 ng/ml) showed higher sensitivity to interferon therapy.ConclusionsANXA4 functions as a protective factor during HBV infection. ANXA4 expression is elevated under HBV attack to restrict HBV replication by inhibiting autophagic degradation of MCM2, thereby alleviating liver injury and suppressing the CHB infection process. ANXA4 also enhances the sensitivity of CHB patients to interferon therapy. Therefore, ANXA4 is expected to be a new target for CHB treatment and prognostic evaluation.Graphical

Effect of ethanol addition on enhancing the cooling performance improvement of spray system

The widespread application of spray technology has significantly improved outdoor environmental thermal comfort. However, the spray system is unable to fulfill special comfort requirements. Utilizing mechanical ventilation and control systems effectively enhances the cooling performance of spray systems; however, those approaches are associated with elevated costs and maintenance complexities. The combination of ethanol and water has been demonstrated to produce a lower surface tension and a higher latent heat of evaporation, a property that contributes to the heat transfer efficiency and evaporation rate of the droplets. Considering the convenience and cost-effectiveness of adding ethanol as an additive, it emerges as the optimal choice. To evaluate the effectiveness of ethanol on the evaporative cooling performance of the spray system, a comparative study was conducted, comparing the cooling performances of different concentrations of ethanol with a spray system without ethanol addition. Five operating conditions were designed, including a no-spray case, a non-additive case, a 0.5 % ethanol concentration case, a 1.0 % ethanol concentration case, and a 1.5 % ethanol concentration case. The results demonstrated that adding ethanol effectively enhanced the cooling performance of the spray system. At 1.7m above ground level (0.4m away from the nozzle), compared to the non-additive case without ethanol, the 0.5 % ethanol concentration case, 1.0 % ethanol concentration case, and 1.5 % ethanol concentration case further reduced the temperature by 0.95 °C, 1.64 °C, and 2.27 °C. Moreover, the relative humidity grew by 3.57 %, 9.25 %, and 13.5 %, respectively. Adding ethanol significantly enhanced the evaporative cooling effect of the spray system, with the optimal concentration observed at 1.5 %. The results demonstrate that enhancing the evaporative cooling efficacy of a spray system by introducing ethanol into the spray medium represents a viable strategy and improve urban thermal environment in summer. Furthermore, this study aims to examine the impact of droplet evaporation on relative humidity, establishing a foundation for future enhancements aimed at addressing the issue of over-humidification in spray systems.

SMC5/6-Mediated Transcriptional Regulation of Hepatitis B Virus and Its Therapeutic Potential.

Cells have developed various mechanisms to counteract viral infections. In an evolutionary arms race, cells mobilize cellular restriction factors to fight off viruses, targeted by viral factors to facilitate their own replication. The hepatitis B virus (HBV) is a small dsDNA virus that causes acute and chronic infections of the liver. Its genome persists in the nuclei of infected hepatocytes as a covalently closed circular DNA (cccDNA) minichromosome, thus building up an episomal persistence reservoir. The chromosomal maintenance complex SMC5/6 acts as a restriction factor hindering cccDNA transcription, whereas the viral regulatory protein HBx targets SMC5/6 for proteasomal degradation, thus relieving transcriptional suppression of the HBV minichromosome. To date, no curative therapies are available for chronic HBV carriers. Knowledge of the factors regulating the cccDNA and the development of therapies involving silencing the minichromosome or specifically interfering with the HBx-SMC5/6 axis holds promise in achieving sustained viral control. Here, we summarize the current knowledge of the mechanism of SMC5/6-mediated HBV restriction. We also give an overview of SMC5/6 cellular functions and how this compares to the restriction of other DNA viruses. We further discuss the therapeutic potential of available and investigational drugs interfering with the HBx-SMC5/6 axis.

SETD3-mediated histidine methylation of MCM7 regulates DNA replication by facilitating chromatin loading of MCM.

The minichromosome maintenance complex (MCM) DNA helicase is an important replicative factor during DNA replication. The proper chromatin loading of MCM is a key step to ensure replication initiation during S phase. Because replication initiation is regulated by multiple biological cues, additional changes to MCM may provide better understanding towards this event. Here, we report that histidine methyltransferase SETD3 promotes DNA replication in a manner dependent on enzymatic activity. Nascent-strand sequencing (NS-seq) shows that SETD3 regulates replication initiation, as depletion of SETD3 attenuates early replication origins firing. Biochemical studies reveal that SETD3 binds MCM mainly during S phase, which is required for the CDT1-mediated chromatin loading of MCM. This MCM loading relies on histidine-459 methylation (H459me) on MCM7 which is catalyzed by SETD3. Impairment of H459 methylation attenuates DNA synthesis and chromatin loading of MCM. Furthermore, we show that CDK2 phosphorylates SETD3 at Serine-21 during the G1/S phase, which is required for DNA replication and cell cycle progression. These findings demonstrate a novel mechanism by which SETD3 methylates MCM to regulate replication initiation.

An Integrated Approach to the Regional Estimation of Soil Moisture

Automatic or smart irrigation systems benefit irrigation water management. However, measurement sensor networks in automatic irrigation systems are complex, and maintenance is essential. Regional soil moisture estimation avoids the multiple measurements necessary when deploying an irrigation system. In this sense, a fuzzy estimation approach based on decision-making (FEADM) has been used to obtain soil moisture point estimates. However, FEADM requires intelligent weather adjustment based on spatial features (IWeCASF) to perform regional soil moisture estimation. The IWeCASF-FEADM integrated approach for regional soil moisture estimation is developed in this work. IWeCASF provides the inputs for FEADM. FEADM is performed R times; R is the number of checkpoints at which a point estimate is obtained. In this way, regional estimation is achieved when the set of R soil moisture point estimates is completed. Additionally, IWeCASF-FEADM considers the irrigation water records, which are not included in either method individually. This method can detect when the soil moisture is deficient in a region, allowing actions to prevent water stress. This regional estimation reduces an irrigation system’s operational and maintenance complexity. This integrated approach has been tested over several years by comparing the results of regional soil moisture estimation with measurements obtained at many points in the study region.

HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.

Postnatal mammalian cardiomyocytes (CMs) rapidly lose proliferative capacity and exit the cell cycle and undergo further differentiation and maturation. Cell cycle activation has been a major strategy to stimulate postnatal CM proliferation, albeit achieving modest effects. One impediment is that postnatal CMs may need to undergo dedifferentiation before proliferation, if not simultaneously. Here, we report that overexpression of Hdac7 in neonatal mouse CMs results in significant CM dedifferentiation and proliferation. Mechanistically, we show that HDAC7-mediated CM proliferation is contingent on dedifferentiation, which is accomplished through suppressing MEF2. Hdac7 overexpression in CM shifts the chromatin state from binding MEF2, which favors the differentiation transcriptional program to AP-1, which favors the proliferative transcriptional program. Further, we found that HDAC7 interacts with minichromosome maintenance complex (MCM) components to initiate cell cycle progression. Our findings reveal that HDAC7 promotes CM proliferation by its dual action on CM dedifferentiation and proliferation, uncovering a potential new strategy for heart regeneration/repair.

A Novel Ultrasonic Leak Detection System in Nuclear Power Plants Using Rigid Guide Tubes with FCOG and SNR.

Leak detection in nuclear reactor coolant systems is crucial for maintaining the safety and operational integrity of nuclear power plants. Traditional leak detection methods, such as acoustic emission sensors and spectroscopy, face challenges in sensitivity, response time, and accurate leak localization, particularly in complex piping systems. In this study, we propose a novel leak detection approach that incorporates a rigid guide tube into the insulation layer surrounding reactor coolant pipes and combines this with an advanced detection criterion based on Frequency Center of Gravity shifts and Signal-to-Noise Ratio analysis. This dual-method strategy significantly improves the sensitivity and accuracy of leak detection by providing a stable transmission path for ultrasonic signals and enabling robust signal analysis. The rigid guide tube-based system, along with the integrated criteria, addresses several limitations of existing technologies, including the detection of minor leaks and the complexity of installation and maintenance. By enhancing the early detection of leaks and enabling precise localization, this approach contributes to increased reactor safety, reduced downtime, and lower operational costs. Experimental evaluations demonstrate the system's effectiveness, focusing on its potential as a valuable addition to the current array of nuclear power plant maintenance technologies. Future research will focus on optimizing key parameters, such as the threshold frequency shift (Δf) and the number of randomly selected frequencies (N), using machine learning techniques to further enhance the system's accuracy and reliability in various reactor environments.

Analysis of Plant Watering Efficiency Using IoT Technology Controlled Through Google Assistant

This paper presents a systematic literature review (SLR) of 120 academic documents sourced from the Scopus database, which analyses the efficiency of crop watering systems utilizing Internet of Things (IoT) technology controlled via Google Assistant. The review explores key advances in IoT-based irrigation systems, highlighting how real-time data from sensors and voice-controlled automation improve water efficiency and user experience. The findings reveal that IoT systems can reduce water wastage by 25-30%, optimise plant health, and offer convenience through voice commands. However, challenges such as connectivity issues, high implementation costs, and system maintenance complexity also need to be addressed. This paper discusses potential future research directions, including scalability, AI integration, and cost-effective solutions to expand the adoption of these technologies in agriculture and horticulture.

Development of Detection and Mitigation of Advanced Persistent Threats Using Artificial Intelligence and Multi-Layer Security on Cloud Computing Infrastructure

This research proposes a novel approach for detecting and mitigating Advanced Persistent Threats (APTs) in cloud computing infrastruc ture, offering more comprehensive protection compared to previous methods. By integrating detection and mitigation, this study addresses the shortcomings of prior research that focused solely on detection. Based on the conducted research, Artificial Intelligence (AI) detected Cross-Site Scripting (XSS) attacks with an accuracy of 0.9951, SQL Injection (SQLI) at 0.9964, and Remote Code Execution (RCE) at 0.9876. In trials against new attacks, the detection success rates reached 70% for XSS, 98% for SQLI, and 100% for RCE. During the deployment phase, the system successfully identified 23.040 out of 108.394 requests as XSS attacks, 2.684 out of 128.750 as SQLI attacks, and 1.135 out of 46.450 as RCE attacks. The detection and mitigation methods were directly tested on cloud server experiencing APT attacks. The daily attacks on the server reached 1.980, with 663.000 requests. Additionally, the number of attacks directed at authentication or sensitive pages reached 17.913.701. Attack mitigation was tested through seven layers of security, including DNS Protection, Config Server Firewall (CSF), OWASP ModSecurity, HTTP middleware, data filter or sanitizer, template engine, and manual mitigation successfully blocking million of persistent attacks. The DNS protection layer successfully mitigated 59,000 out of a total of 19 million requests. The CSF layer mitigated 173 sources IP of DDoS attacks. The ModSecurity layer mitigated 17,916,204 attacks. All attacks were successfully mitigated before reaching the HTTP Middleware stage or next layer. The use of NIST 2.0 standards helps manage security risks through identification, protection, detection, response, and recovery. Test results indicate that this multi-layered system is more efficient and effective in detecting and mitigating attacks compared to traditional methods. However, the complexity of implementation and maintenance poses challenges that must be addressed. This research significantly contributes to a more adaptive and sustainable cybersecurity strategy.

Expression, potential biological behaviour and clinical significance of MCM3 in pancreatic adenocarcinoma: a comprehensive study integrating high throughput sequencing, CRISPR screening and in-house immunohistochemistry

Background Minichromosome maintenance complex component 3 (MCM3) plays a key role in various tumours. However, it remains largely unknown what the specific role and clinical significance of MCM3 in pancreatic adenocarcinoma (PAAD) are. Materials and Methods We integrated high-throughput data from PAAD worldwide to analyse the expression level of MCM3 mRNA. We used immunohistochemistry to analyse MCM3 protein expression levels in 145 cases in the PAAD group and 29 cases in the non-PAAD group. We also mainly analysed the necessity of MCM3 for PAAD growth based on CRISPR screen data. In addition, we used enrichment analysis and protein–protein interaction networks to explore the molecular mechanism of MCM3 in PAAD. We also analysed the correlation between MCM3 expression, components of the immune microenvironment in PAAD tissue and clinical prognosis. Results In PAAD, we observed for the first time that MCM3 was significantly highly expressed at both the mRNA (SMD = 0.67, 95% CI: 0.38 ∼ 0.96) and the protein level (p < 0.05). The mRNA (AUC = 0.78, 95% CI: 0.74 ∼ 0.81; sensitivity = 0.66, 95% CI: 0.55 ∼ 0.76; specificity = 0.76, 95% CI: 0.67 ∼ 0.84) and protein (AUC = 0.929) expression levels of MCM3 had a good ability to distinguish between PAAD and non-PAAD tissue. There was heterogeneity reflected by the differential expression of MCM3 protein in PAAD cells. MCM3 played an essential role in PAAD growth, through abnormal DNA replication, p53 signalling and cell cycle checkpoints. PAAD with high MCM3 expression was sensitive to c-75, brivanib, flavopiridol and VNLG/124 drugs, with stable molecular docking models. Conclusion MCM3 is likely to be a critical element in promoting the initiation and growth of PAAD. Flavopiridol may exert its anti-PAAD effect through the interaction between MCM3, classic CDK1 targets in the cell cycle checkpoint and p53 pathway as well as related molecules in other pathways.

Novel insights into the circadian modulation of lipid metabolism in chicken livers revealed by RNA sequencing and weighted gene co-expression network analysis

The circadian clock is crucial for maintaining lipid metabolism homeostasis in mammals. Despite the economic importance of fat content in poultry, research on the regulatory effects and molecular mechanisms of the circadian clock on avian hepatic lipid metabolism has been limited. In this study, we observed significant diurnal variations (P<0.05) in triglyceride (TG), free fatty acids (FFA), fatty acid synthase (FAS), and total cholesterol (TC) levels in the chicken embryonic liver under 12-h light/12-h dark incubation conditions, with TG, FFA, and TC concentrations showing significant cosine rhythmic oscillations (P<0.05). However, such rhythmic variations were not observed under complete darkness incubation conditions. Using transcriptome sequencing technology, we identified 157 genes significantly upregulated at night and 313 genes significantly upregulated during the 12-h light/12-h dark cycle. These circadian differential genes are involved in processes and pathways such as lipid catabolic process regulation, meiotic cell cycle, circadian rhythm regulation, positive regulation of the MAPK cascade, and glycerolipid metabolism. Weighted gene co-expression network analysis (WGCNA) revealed 3 modules-green, blue, and red-that significantly correlate with FFA, FAS, and TG, respectively. Genes within these modules were enriched in processes and pathways including the cell cycle, light stimulus response, circadian rhythm regulation, phosphorylation, positive regulation of the MAPK cascade, and lipid biosynthesis. Notably, we identified ten hub genes, including protein kinase C delta (PRKCD), polo like kinase 4 (PLK4), clock circadian regulator (CLOCK), steroid 5 alpha-reductase 3 (SRD5A3), BUB1 mitotic checkpoint serine/threonine kinase (BUB1B), shugoshin 1 (SGO1), NDC80 kinetochore complex component (NDC80), NIMA related kinase 2 (NEK2), minichromosome maintenance complex component 4 (MCM4), polo like kinase 1 (PLK1), potentially link circadian regulation with lipid metabolic homeostasis. These findings demonstrate the regulatory role of the circadian clock in chicken liver lipid metabolism homeostasis and provide a theoretical basis and molecular targets for optimizing the circadian clock to reduce excessive fat deposition in chickens, which is significant for the healthy development of the poultry industry.

Scalable Microservices Architecture: Leadership Approaches for High-Performance Retail Systems

The rapid evolution of retail systems in the digital age necessitates the adoption of scalable microservices architectures to meet the demands of high-performance and responsive platforms. As the retail sector increasingly relies on technology to drive customer engagement, operational efficiency, and competitive advantage, the role of leadership in designing and implementing scalable microservices architectures becomes critical. This paper explores leadership approaches that are essential for guiding the successful deployment of microservices in high-performance retail systems. The shift from monolithic to microservices architectures represents a fundamental change in how retail systems are built and maintained. Microservices, characterized by their modularity, independence, and scalability, offer significant advantages over traditional monolithic architectures, particularly in handling the dynamic demands of modern retail environments. However, the successful implementation of microservices requires more than just technical expertise; it demands strategic leadership that understands the complexities of system design, cross-functional collaboration, and long-term maintenance.

Prognostic and Clinical Significance of the Proliferation Marker MCM7 in Breast Cancer.

Minichromosome maintenance complex component 7 (MCM7) plays an essential role in proliferation and DNA replication of cancer cells. However, the expression and prognostic significance of MCM7 in breast cancer (BC) remain to be defined. In this study, we aimed to evaluate the role of MCM7 in BC. We conducted immunohistochemistry staining of MCM7 in 1,156 operable early-stage BC samples and assessed MCM7 at the transcriptomic levels using publicly available cohorts (n = 13,430). MCM7 expression was evaluated and correlated with clinicopathological parameters including Ki67 labelling index and patient outcome. At the transcriptomic level, there was a significant association between high MCM7 mRNA levels and shorter patient survival in the whole cohort and in luminal BC class but not in the basal-like molecular subtype. High MCM7 protein expression was detected in 43% of patients and was significantly associated with parameters characteristic of aggressive tumour behaviour. MCM7 was independently associated with shorter survival, particularly in oestrogen receptor-positive (luminal) BC. MCM7 stratified luminal tumours with aggressive clinicopathological features into distinct prognostic groups. In endocrine therapy-treated BC patients, high MCM7 was associated with poor outcome, but such association disappeared with administration of adjuvant chemotherapy. Patients with high expression of Ki67 and MCM7 showed worst survival, while patients with double low expression BC showed the best outcome compared with single expression groups. The current findings indicate that MCM7 expression has a prognostic value in BC and can be used to identify luminal BC patients who can benefit from adjuvant chemotherapy.

Identification of biallelic mutations in MCM3AP and comprehensive literature analysis.

Minichromosome maintenance complex component 3 associated protein (MCM3AP) is a gene in which mutations can result in autosomal recessive peripheral neuropathy with or without impaired intellectual development. The MCM3AP genotype-phenotype correlation and prognosis remain unclear. The aim of this study was to explore the genotype-phenotype correlations pertaining to MCM3AP. Whole-exome sequencing (WES) combined with copy number variation sequencing (CNV-seq) were performed on the genomic DNA isolated from a Chinese family, and Sanger sequencing, quantitative PCR and cDNA analyses were performed to examine the mutations. The retrospective study was conducted on 28 individuals with biallelic MCM3AP mutation-related diseases, including features such as mutations, motor development impairment, intellectual disability, weakness/atrophy, and cerebral magnetic resonance imaging abnormalities. Sequencing identified novel compound heterozygous mutations in MCM3AP, namely, a paternal variant c.1_5426del (loss of exons 1-25) and a maternal splicing variant c.1858 + 3A>G. Functional studies revealed that the variant c.1858 + 3A>G resulted in the heterozygous deletion of exon 5, thereby affecting splicing functionality. Furthermore, the compound heterozygous mutation may affect the functionality of the protein domain. Retrospective analysis revealed different genotype-phenotype correlations for the pathogenic variants in biallelic MCM3AP: all individuals (100%) with mutations outside the Sac3 domain exhibited early-onset symptoms, motor developmental delays, and cognitive abnormalities, conversely, the proportions of individuals carrying mutations within the domain were 26.7% (motor delays) and 46.7% (cognitive abnormalities). Our findings further expand the genetic mutation spectrum of biallelic MCM3AP and highlight the genotype-phenotype associations. Additionally, we elaborate on the importance of rehabilitation intervention.

Dynamics of Replication-Associated Protein Levels through the Cell Cycle.

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.