Unique Regulatory Mechanisms For Dna Replication And Genome Maintenance In Mammals

DNA replication is a fundamental process in all organisms. Using single stranded DNA (ssDNA) as a template, DNA polymerase synthesizes new DNA to produce an exact copy of the genome. However, most DNA polymerases lack the ability to unwind the double stranded DNA (dsDNA) necessary to expose ssDNA [1]. Therefore, an additional DNA helicase is required to initiate DNA replication by unwinding dsDNA and exposure of ssDNA as a template. In eukaryotes, genetic and biochemical assays have shown the MCM (minichromosome maintenance) family of proteins functions in a hexameric complex as the genomic DNA replication helicase [2]. MCMs have been well studied through in vitro biochemistry, cells in culture, and simple model organisms including S. cerevisiea and Xenopus laevis. These experimentally tractable systems have shown that misrelated or dysfunctional MCMs have deleterious consequences, especially genomic instability (GIN). However, there are still several major unresolved issues that need to be addressed. (1) The "MCM paradox" described that the MCMs are in excess of the number of origins. What is the function of these excess MCMs, and how does it relate to cells and animals? (2) The "MCM puzzle" indicates that mini-MCM complexes exist, but their functions are still unclear (3) Very little is known about the function of the MCM helicase with respect to the health of whole animals. To accomplish these issues and explore the relationship between MCMs and disease, I generated mice deficient in MCMs as a model of in vivo disease in this thesis. The mice which carry 50% reduction of Mcm2, 3, 4, 6, and 7 is phenotypically identical to wild-type at least through 1 year of age. Further reduction of Mcms to 70% causes several detrimental phenotypes, including embryonic lethality, growth retardation, genomic instability, and cancer susceptibility. Most importantly, the reduction of MCM3 rescues most of the detrimental phenotypes in other MCM deficient mice, suggesting a unique function of MCM3. Highly similar to in vitro results, I showed that the MCM3/5 dimer inhibits the MCM2-7 complex from binding chromatin and hinders cell cycle. I also discovered that the Mcm4Chaos3 mutation induces a pan-downregulation of Mcm2-7 post-transcriptionally. The pan-down regulation of Mcm2-7 is a self-preservation mechanism because it reduces MCM3 levels that block the recruitment of chromatin bound MCM2-7. Finally, I identified that Mcm hypomorphic mice possess a unique gender bias phenotype. The male animals are more resistant to MCM insufficiency due to a testosterone protective effect. In summary, this dissertation explores the function of the excess MCMs in aspects of cell cycle and in whole animals. It builds understanding about the regulation of MCMs with emphasis upon cancer formation as a result of MCM deficiency. The MCM hypomorphic mice also reveal a post-transcriptional regulation of Mcms that responded to helicase complex instability or insufficiency. The unique negative function of the MCM3/5 dimer overturns the current theory that the MCM2-7 heterohexamer is the only type of replication helicase that forms

Variations of Physiological Parameters and HSP70 and HSP90 Polymorphisms in Water Buffaloes in Taiwan During Cool and Warm Season

Background: This study examined the physiological parameters of water buffaloes in Taiwan in the cool (February) and warm (August) seasons of 2020 and 2021. Methods: Data was collected for a study in February, August 2020, and 2021. The ambitious temperature, humidity, water buffaloes’ rectal temperature (RT), and respiratory rate (RR) were recorded. The plasma expression levels of heat-shock protein (HSP)70 and HSP90 were examined using an ELISA kit. Furthermore, the HSP70 and HSP90 fragment genetic sequence variations were analyzed using the PCR method and MEGA6 software. Results: The results revealed that in the warm season, the rectal temperature (RT), respiratory rate (RR), and heat tolerance coefficient (HTC) were significantly higher compared to the cool season (all P < 0.05). Additionally, the temperature-humidity index (THI) had moderate to high correlations with RT (0.518), RR (0.744), and HTC (0.757). Plasma HSP70 expression levels were higher in the warm season than in the cool season (P < 0.05). The genetic sequences of HSP70 and HSP90 fragments were compared, and five single-nucleotide variation (SNV) sites were identified. However, each genotype showed no significant physiological difference between the cool and warm seasons. Conclusion: Temperature and humidity changes in Taiwan had a significant correlation with the physical condition of water buffaloes. This information can be valuable in improving the living conditions of these animals, leading to better animal welfare. Additionally, the HSP70 and HSP90 gene variations in water buffaloes in Taiwan could be used as a reference for future research on breeding and identifying molecular markers

Technetium-99m-Labeled Autologous Serum Albumin: A Personal-Exclusive Source of Serum Component

Technetium-99m human serum albumin (99mTc-HSA) is an important radiopharmaceutical required in nuclear medicine studies. However, the risk of transfusion-transmitted infection remains a major safety concern. Autopreparation of serum component acquired from patient provides a “personal-exclusive” source for radiolabeling. This paper is to evaluate the practicality of on-site elusion and subsequent radiolabeling efficacy for serum albumin. Results showed that the autologous elute contained more albumin fraction than serum without extraction procedure. Good radiochemical purity and stability were demonstrated after radiolabeling. Biodistribution study showed that labeled albumin accumulated immediately in the lung, liver, and kidney. It was cleared steadily and excreted in the urine. The biologic half-life was defined, and all samples passed the pyrogenicity and sterility tests. In conclusion, autoalbumin could be extracted and radiolabeled properly in a nuclear medicine setting. Moreover, the risk of transfusion-transmitted infection associated with nonautologous, multisource 99mTc-HSA agents can be reduced

An Expanded Conformation of Single-Ring GroEL-GroES Complex Encapsulates an 86 kDa Substrate

SummaryElectron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with ∼80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)7 complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (αβ) assembly intermediate of mitochondrial branched-chain α-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both α and β subunits of the substrate from tryptic digestion

Effects of insecticide-treated net access and use on infant mortality in Malawi: A pooled analysis of demographic health surveys

Malaria continues to be among the top causes of death in children and insecticide-treated nets (ITNs) are considered among the most effective malaria control methods. However, information on the association between universal ITN coverage and infant mortality is limited. A Cox proportional hazard model was applied to Malawi Demographic and Health Surveys to determine the association between ITN access and use and infant mortality between 2004 and 2015–2016. The overall infant mortality rate for the entire period was 47.9/1000 live births. Infants from the ITN-user households exhibited a lower risk of mortality [adjusted Hazard Ratio (aHR) = 0.61, 95% Confidence Interval (CI) = 0.44–0.85] than those from the ITN-nonuser households. Similarly, the infants from the high-access households exhibited a lower risk of death (aHR = 0.63, 95% CI = 0.46–0.86) than those from the no-access households. Infants from the ITN-user and high-access households exhibited a significantly lower risk of death (aHR = 0.57, 95% CI = 0.40–0.82) than those from the ITN-nonuser and no-access households. The relationship between ITN access and use and infant mortality was significant among female infants with a second or higher birth order and interval of ≥2 years. The findings of the present population-based study emphasized the importance of ITN access and use in providing optimal protection against malaria to infants in Malawi. Malaria control programs should ensure high ITN access and use in Malawi to reduce infant mortality

Multilevel analysis of factors associated with treatment-seeking behaviors among caregivers with febrile children in Malawi

Early diagnosis and treatment of childhood fever, an important sign of potentially serious infections such as malaria, is essential for controlling disease progression, and ultimately, preventing deaths. This study examined individual- and community-level factors associated with treatment-seeking behaviors and promptness in these behaviors among caregivers of febrile under-five children in Malawi. The 2015-2016 Malawi Demographic Health Survey was used to analyze a nationally representative sample of 4,133 under-five children who had fever within 2 weeks before the survey. A multilevel logistic regression model was used to examine the association between individual- and community-level factors and treatment-seeking behaviors. Approximately 67.3% of the caregivers reported seeking treatment for their febrile child, whereas only 46.3% reported promptly seeking treatment. Children from communities with moderate and high percentages of educated caregivers were more likely to be taken for treatment (adjusted odds ratio [aOR] = 1.26, 95% CI = 1.01-1.58 and aOR = 1.31, 95% CI = 1.02-1.70, respectively) than those from communities with a low percentage of educated caregivers. Children from communities with moderate and high percentages of caregivers complaining about the distance to a health facility were less likely to be taken for treatment (aOR = 0.74, 95% CI = 0.58-0.96 and aOR = 0.67, 95% CI = 0.51-0.88, respectively). At the individual level, having a cough in the last 2 weeks, region, religion, and having better health behaviors in other health dimensions were associated with fever treatment-seeking behaviors among Malawian caregivers. Programs aimed at improving treatment-seeking behaviors should consider these factors and the regional variations observed in this study

Influence of maternal exposure to malaria social and behavioral change messages and effectiveness of communication media on bed net use and malaria infection in Malawi.

Background: Malawi is a malaria-endemic country. A national malaria communication strategy was adopted to disseminate malaria messages with the aim of improving knowledge and adoption of malaria interventions. Objective: To examine the effect of maternal exposure to malaria messages and the medium through which such messages are delivered on insecticide-treated net (ITN) use and malaria infection among children under 5 years of age in Malawi. Methodology: Utilizing the data from the 2017 Malawi Malaria Indicator Survey, 2,055 children (aged under 5 years) and 1,886 children (aged 6–59 months) were analyzed for ITN use and malaria infection outcomes, respectively. Components of exposure to malaria messages were tested for association with ITN use and malaria infection outcomes using multiple logistic regression models. Results: Children whose mothers had reported hearing any malaria-related message in the past 6 months were more likely to sleep under an ITN and less likely to have malaria infection compared with those whose mothers had not heard any malaria-related message. Region and sex of the child were effect modifiers on the relationship between exposure to any malaria-related message and malaria infection. Knowledge regarding cause or protection methods partially mediated the relationship between exposure to any malaria message and malaria infection. Discussion and Conclusion: Health workers were an effective communication channel. Strengthening topic-specific malaria messages and building the capacity of health workers while alternately strengthening other message outlets may prove vital for effective malaria communication

Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation

<p>Abstract</p> <p>Background</p> <p>To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC.</p> <p>Methods</p> <p>Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens.</p> <p>Results</p> <p>Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage.</p> <p>Conclusions</p> <p>Coronin-1C could be a candidate biomarker to predict HCC invasive behavior.</p

Traffic agents for improving QoS in mixed infrastructure and ad hoc modes wireless LAN

As an important complement to infrastructured wireless networks, mobile ad hoc networks (MANET) are more flexible in providing wireless access services, but more difficult in meeting different quality of service (QoS) requirements for mobile customers. Both infrastructure and ad hoc network structures are supported in wireless local area networks (WLAN), which can offer high data-rate wireless multimedia services to the mobile stations (MSs) in a limited geographical area. For those out-of-coverage MSs, how to effectively connect them to the access point (AP) and provide QoS support is a challenging issue. By mixing the infrastructure and the ad hoc modes in WLAN, we propose in this paper a new coverage improvement scheme that can identify suitable idle MSs in good service zones as traffic agents (TAs) to relay traffic from those out-of-coverage MSs to the AP. The service coverage area of WLAN is then expanded. The QoS requirements (e.g., bandwidth) of those MSs are considered in the selection process of corresponding TAs. Mathematical analysis, verified by computer simulations, shows that the proposed TA scheme can effectively reduce blocking probability when traffic load is light