Replicative Potential Research Articles

Telomerase and hallmarks of cancer: An intricate interplay governing cancer cell evolution

Transformed cells must acquire specific characteristics to be malignant. Weinberg and Hanahan characterize these characteristics as cancer hallmarks. Though these features are independently driven, substantial signaling crosstalk in transformed cells efficiently promotes these feature acquisitions. Telomerase is an enzyme complex that maintains telomere length. However, its main component, Telomere reverse transcriptase (TERT), has been found to interact with various signaling molecules like cMYC, NF-kB, BRG1 and cooperate in transcription and metabolic reprogramming, acting as a strong proponent of malignant features such as cell death resistance, sustained proliferation, angiogenesis activation, and metastasis, among others. It allows cells to avoid replicative senescence and achieve endless replicative potential. This review summarizes both the canonical and noncanonical functions of TERT and discusses how they promote cancer hallmarks. Understanding the role of Telomerase in promoting cancer hallmarks provides vital insight into the underlying mechanism of cancer genesis and progression and telomerase intervention as a possible therapeutic target for cancer treatment. More investigation into the precise molecular mechanisms of telomerase-mediated impacts on cancer hallmarks will contribute to developing more focused and customized cancer treatment methods.

2283P Assessing the replicative potential of the oncolytic virus VSV-GP in patient-derived tumor biopsies ex vivo

2283P Assessing the replicative potential of the oncolytic virus VSV-GP in patient-derived tumor biopsies ex vivo

Soft Sensing of LPG Processes Using Deep Learning.

This study investigates the integration of soft sensors and deep learning in the oil-refinery industry to improve monitoring efficiency and predictive accuracy in complex industrial processes, particularly de-ethanization and debutanization. Soft sensor models were developed to estimate critical variables such as the C2 and C5 contents in liquefied petroleum gas (LPG) after distillation and the energy consumption of distillation columns. The refinery's LPG purification process relies on periodic sampling and laboratory analysis to maintain product specifications. The models were tested using data from actual refinery operations, addressing challenges such as scalability and handling dirty data. Two deep learning models, an artificial neural network (ANN) soft sensor model and an ensemble random forest regressor (RFR) model, were developed. This study emphasizes model interpretability and the potential for real-time updating or online learning. The study also proposes a comprehensive, iterative solution for predicting and optimizing component concentrations within a dual-column distillation system, highlighting its high applicability and potential for replication in similar industrial scenarios.

S10-1 Research on sub-national initiatives in HEPA policy making from Europe, Japan and the USA

Abstract The importance of local governments' engagement in physical activity promotion at local level has been acknowledged and advocated by the World Health organization in documents such as: “Promoting physical activity and active living in urban environments: the role of local governments” or “Global action plan on physical activity 2018–2030: more active people for a healthier world”. However, given the relative “youngness” of the physical activity policy research domain, the local level initiatives have been underrepresented in the scientific literature. The current symposium aims to present a number of diverse initiatives to promote health enhancing physical activity at sub-national level, each having a very unique context and approach. The paper from Eastern Michigan University presents the actions taken to implement Active People, Healthy Nation (APHN) program in geographic areas in Michigan where the priority population (Asian Americans) lives and the outcomes of this initiative. The contribution from Japan describes the process of developing and applying the L-PAT (local policy audit tool), an instrument comprising 11 items based on WHO’s HEPA Policy Audit Tool (HEPA PAT) and the results of this survey within 47 Japanese prefectures and the C-PAT (city policy audit tool), a 6 items tool, in a selection of 272 Japanese municipalities. The EU study presented in this symposium comprises the results of evaluation, using the CAPLA- Santé, a validated instrument, of local HEPA policies in 4 selected municipalities, one from each of the following countries: Finland, France, Germany, Romania and from one municipality in Japan. The responsibilities of local authorities in health promotion and protection are undeniable. The way the authorities manage to fulfil these responsibilities is gaining more and more interest from various stakeholders from local level activists to researchers and policymakers at national and international level. Local communities are increasingly stimulated to contribute to their own wellbeing, so data on the status quo and progress is needed more than ever. The initial discussion points of the symposium will focus on strengths and weaknesses of the initiatives presented, as well as the potential for knowledge exchange and replication & scaling-up.

P14.09.B IS BLOOD-DERIVED EXOSOMAL HTERT TRANSCRIPT A MARKER OF ONCOGENIC ACTIVITY IN PRIMARY BRAIN TUMORS? AN EXPLORATORY STUDY

Abstract BACKGROUND In order to proliferate indefinitely, all tumors require a telomere maintenance mechanism. The expression of human telomerase reverse transcriptase (hTERT) enables telomere maintenance and provides cancer cells with limitless replicative potential. As such, it may serve as an attractive biomarker for oncogenic activity. This study explored whether a liquid biopsy that analyses blood derived exosomal hTERT transcript (e-hTERT-trans) may serve as such a biomarker in gliomas and meningiomas when compared to healthy controls. MATERIAL AND METHODS Exosomes were isolated from the pre-operative sera of patients’ samples stored in the biobank of both Rabin and Sheba Medical Centers. The levels of e-hTERT-trans were measured in 81 healthy controls, 117 meningiomas, 17 low-grade gliomas, and 61 glioblastomas. Clinical parameters of the patients were collected retrospectively and compared to the levels of the e- hTERT-trans. RESULTS The Upper normal limit of controls e-hTERT-trans was 1.85 relative quantitation (RQ). The rate of detection increased with rising tumor grade and correlated with tumor recurrence in meningiomas: mean RQ without recurrence (2.17+11.7) vs. with recurrence (3.59+4.42; p=0.002). In glioblastomas, pre-operative measurements correlated with tumor volume and with the disease course on serial sampling. CONCLUSION We demonstrated for the first time that the expression of e-hTERT-trans transcript can be measured in the serum of primary brain tumors. This exosomal marker carries the potential to serve as a biomarker once used in conjunction with other clinical and radiological parameters. Future studies are required to investigate whether the sensitivity could be augmented and whether it can be implemented into routine patients care.

Strengthening regional governance and the health care network in small municipalities in BrazilFortalecimiento de la gobernanza regional y de la red de atención de salud en municipios pequeños de Brasil

In Brazil, 67.7% of the municipalities are characterized as small, with a population of less than 20 thousand. The objective of this article is to systematize the experience and identify the challenges and lessons learned in the implementation of the model for strengthening regional governance and organizing the health care network (HCN) in a region of Brazil composed of these municipalities, which present low technical and budgetary capacity, in addition to various fragilities related to the organization of the HCN. With a view to strengthening regional governance and the organization of the primary health care (PHC) work process and, consequently, the workflow with other levels of care, a strategy entitled More Care More Health (Mais Cuidado Mais Saúde) was proposed, developed in partnership with the Pan American Health Organization/World Health Organization (PAHO/WHO) and the Ministry of Health. The project was developed through permanent health education and institutional capacity building in the region of Ivaiporã, state of Paraná, based on workshops and addressing three priority axes: HCN integration, capacity building and information management. The target audience were the extended PHC teams. The project made it possible to develop local and governance capacities through joint reflections on the health care model, its components and the necessary changes in work processes for health promotion with a focus on the quality of life of users. Because it is an experience that considers the weaknesses, needs and autonomy of local actors, the project has a high potential for replication and customization for other regions with similar characteristics inside and outside Brazil.

A social emotional learning intervention to reduce psychosocial difficulties among rural children in central China.

Programmes based on social emotional learning (SEL) have been effective in reducing psychosocial difficulties in a number of countries. In Mainland China, there has been no empirical research on the prevention of children's psychosocial difficulties using the SEL approach. This study aimed to assess whether an adapted version of the SEL programme can reduce psychosocial difficulties of primary school children in rural China. The intervention consisted of 16 weekly 90-min class sessions, conducted among 206 children aged 8-12 years (with 290 controls) in a poor rural area of Central China. Self-report questionnaires were administered at baseline, post-intervention and 5-month follow-up. The results suggested that the programme (1) can reduce children's total difficulties (measured using the Strength and Difficulties Questionnaire) at post-intervention (d= -0.18) and 5-month follow-up (d= -0.19), (2) was more effective among children experiencing verbal abuse (d= -0.21) or physical abuse (d= -0.24) from caregivers and (3) was popular among more than 90% of the participants. The programme is cheap, easy to implement and can be delivered in school hours. Therefore, it has clear potential for replicability and sustainability.

Single-Cell Proliferation Microfluidic Device for High Throughput Investigation of Replicative Potential and Drug Resistance of Cancer Cells.

Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes. We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin. The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01µM, 0.001µM, 0.0001µM), we observed that 50-80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension. The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells. The online version contains supplementary material available at 10.1007/s12195-023-00773-z.

Humanized mice generated by intra-bone marrow injection of CD133-positive hematopoietic stem cells: application to HIV-1 research

Animal models are essential for basic and clinical research on virus diseases. Humanized mice (mice reconstituted with human hematopoietic cells) have been effectively used for various virus studies as small animal models. Studies on human-tropic HIV-1 have also been performed using different humanized mouse models. Various humanized mice have been generated using distinct mouse strains and engraftment methods. These different techniques affect the reconstitution of human hematopoietic cells in individual mice, and in turn the HIV-1 replication in vivo. In this report, we describe the details of the generation method of humanized mice, i.e., severely immunodeficient mice (NSG mice) transplanted with human CD133-positive cells via intra-bone marrow injection (IBMI). It has been shown that the CD133-positive cells are highly capable to generate CD34-positive cells in vivo and IBMI is an excellent methodology for lymphoid and myeloid cell repopulation. In humanized mice transplanted with CD133-positive cells into the bone marrow, human lymphocytes were increased 3 months after the transplantation and a steady increase in CD4-positive cells was observed until 6–8 months after the transplantation. In order to test the utility of our system, CXCR4-tropic and CCR5-tropic HIV-1 clones were intraperitoneally inoculated into the resultant humanized mice 6–8 months after the transplantation. Upon inoculation at the same dose of viruses, the plasma viral load in CCR5-tropic HIV-1-inoculated mice peaked earlier than that in CXCR4-tropic HIV-1-inoculated mice (2–3 weeks vs 5–10 weeks post-inoculation). While a rapid decrease in CD4-positive cells was observed at the peak or prior to the peak of viremia for CXCR4-tropic HIV-1-inoculated mice, CD4-positive cells were gradually decreased in CCR5-tropic HIV-1-inoculated mice. Upon inoculation at the same dose of viruses, a Nef-deleted R5-tropic HIV-1 exhibited retarded growth kinetics in the inoculated mice compared to the parental virus (around 8 weeks vs 2–3 weeks post-inoculation), which appears to reflect the decrease in replication potential in primary cells. Taken all together, in addition to the humanized mice reported so far, our humanized mice generated by transplanting CD133-positive cells with the IBMI method would be an appropriate prototype model for understanding HIV-1 biology in vivo.

Preventing occludin tight-junction disruption via inhibition of microRNA-193b-5p attenuates viral load and influenza-induced lung injury

Virus-induced lung injury is associated with loss of pulmonary epithelial-endothelial tight junction integrity. While the alveolar-capillary membrane may be an indirect target of injury, viruses may interact directly and/or indirectly with miRs to augment their replication potential and evade the host antiviral defense system. Here, we expose how the influenza virus (H1N1) capitalizes on host-derived interferon-induced, microRNA (miR)-193b-5p to target occludin and compromise antiviral defenses. Lung biopsies from patients infected with H1N1 revealed increased miR-193b-5p levels, marked reduction in occludin protein, and disruption of the alveolar-capillary barrier. In C57BL/6 mice, the expression of miR-193b-5p increased, and occludin decreased, 5-6days post-infection with influenza (PR8). Inhibition of miR-193b-5p in primary human bronchial, pulmonary microvascular, and nasal epithelial cells enhanced antiviral responses. miR-193b-deficient mice were resistant to PR8. Knockdown of occludin, both invitro and invivo, and overexpression of miR-193b-5p reconstituted susceptibility to viral infection. miR-193b-5p inhibitor mitigated loss of occludin, improved viral clearance, reduced lung edema, and augmented survival in infected mice. Our results elucidate how the innate immune system may be exploited by the influenza virus and how strategies that prevent loss of occludin and preserve tight junction function may limit susceptibility to virus-induced lung injury.

Expression of anti-chikungunya single-domain antibodies in transgenic Aedes aegypti reduces vector competence for chikungunya virus and Mayaro virus.

Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.

Survival Strategies of Duckweeds, the World's Smallest Angiosperms.

Duckweeds (Lemnaceae) are small, simply constructed aquatic higher plants that grow on or just below the surface of quiet waters. They consist primarily of leaf-like assimilatory organs, or fronds, that reproduce mainly by vegetative replication. Despite their diminutive size and inornate habit, duckweeds have been able to colonize and maintain themselves in almost all of the world's climate zones. They are thereby subject to multiple adverse influences during the growing season, such as high temperatures, extremes of light intensity and pH, nutrient shortage, damage by microorganisms and herbivores, the presence of harmful substances in the water, and competition from other aquatic plants, and they must also be able to withstand winter cold and drought that can be lethal to the fronds. This review discusses the means by which duckweeds come to grips with these adverse influences to ensure their survival. Important duckweed attributes in this regard are a pronounced potential for rapid growth and frond replication, a juvenile developmental status facilitating adventitious organ formation, and clonal diversity. Duckweeds have specific features at their disposal for coping with particular environmental difficulties and can also cooperate with other organisms of their surroundings to improve their survival chances.

Identification and analysis of a clinically isolated strain of Halomonas based on whole-genome sequencing and comparative genomics.

The aim of this study was to identify the species of a Halomonas strain isolated from a neonatal blood sample and to understand the potential pathogenicity and characteristic genes of the strain. The genomic DNA of strain 18071143 (identified as Halomonas by matrix-assisted laser desorption-ionization time of flight-mass spectrometry and the 16S ribosomal RNA (rRNA) gene sequence) was sequenced using Nanopore PromethION platforms. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated using the complete genome sequences of the strain. Comparative genomic analyses were performed on strain 18071143 and 3 strains of Halomonas (Halomonas stevensii S18214, Halomonas hamiltonii KCTC 22154, and Halomonas johnsoniae KCTC 22157) that were associated with human infections and had high genomic similarity to strain 18071143. Phylogenetic, ANI, and dDDH similarity analyses based on genome sequence indicated that strain 18071143 belonged to the species H stevensii. Similarities exist between strain 18071143 and the other 3 Halomonas strains in terms of gene structure and protein function. Nonetheless, strain 18071143 has greater potential for DNA replication, recombination, repair, and horizontal transfer. Whole-genome sequencing holds great promise for accurate strain identification in clinical microbiology. In addition, the results of this study provide data for understanding Halomonas from the perspective of pathogenic bacteria.

Immunometabolic reprogramming, another cancer hallmark.

Molecular carcinogenesis is a multistep process that involves acquired abnormalities in key biological processes. The complexity of cancer pathogenesis is best illustrated in the six hallmarks of the cancer: (1) the development of self-sufficient growth signals, (2) the emergence of clones that are resistant to apoptosis, (3) resistance to the antigrowth signals, (4) neo-angiogenesis, (5) the invasion of normal tissue or spread to the distant organs, and (6) limitless replicative potential. It also appears that non-resolving inflammation leads to the dysregulation of immune cell metabolism and subsequent cancer progression. The present article delineates immunometabolic reprogramming as a critical hallmark of cancer by linking chronic inflammation and immunosuppression to cancer growth and metastasis. We propose that targeting tumor immunometabolic reprogramming will lead to the design of novel immunotherapeutic approaches to cancer.

Infectious SARS-CoV-2 Particles from Rectal Swab Samples from COVID-19 Patients in Brazil.

The main objective of this study was to investigate the dynamic of SARS-CoV-2 viral excretion in rectal swab (RS), saliva, and nasopharyngeal swab (NS) samples from symptomatic patients and asymptomatic contacts. In addition, in order to evaluate the replication potential of SARS-CoV-2 in the gastrointestinal (GI) tract and the excretion of infectious SARS-CoV-2 from feces, we investigated the presence of subgenomic nucleoprotein gene (N) mRNA (sgN) in RS samples and cytopathic effects in Vero cell culture. A prospective cohort study was performed to collect samples from symptomatic patients and contacts in Rio de Janeiro, Brazil, from May to October 2020. One hundred and seventy-six patients had samples collected at home visits and/or during the follow up, resulting in a total of 1633 RS, saliva, or NS samples. SARS-CoV-2 RNA was detected in 130 (73.9%) patients who had at least one sample that tested positive for SARS-CoV-2. The presence of replicating SARS-CoV-2 in RS samples, measured by the detection of sgN mRNA, was successfully achieved in 19.4% (6/31) of samples, whilst infectious SARS-CoV-2, measured by the generation of cytopathic effects in cell culture, was identified in only one RS sample. Although rare, our results demonstrated the replication capacity of SARS-CoV-2 in the GI tract, and infectious viruses in one RS sample. There is still a gap in the knowledge regarding SARS-CoV-2 fecal-oral transmission. Additional studies are warranted to investigate fecal or wastewater exposure as a risk factor for transmission in human populations.

The spontaneous immortalization probability of mammalian cell culture strains, as their proliferative capacity, correlates with species body mass, not longevity

BackgroundThe Peto's paradox consists in the observation that individuals from long-lived and large animal species do not experience a higher cancer incidence, despite being exposed for longer time to the possibility of accumulating mutations and having more target cells exposed to the phenomenon. The existence of this paradox has been recently confirmed (Vincze et al., 2022). Concurrently, robust evidence has been published that longevity involves a convergent evolution of cellular mechanisms that prevent the accumulation of mutations (Cagan et al., 2022). It remains unclear which cellular mechanisms are critical to allow the evolution of a large body mass while keeping cancer at bay. MethodsAdding to existing data linking cellular replicative potential and species body mass (Lorenzini et al., 2005), we have grown a total of 84 skin fibroblast cell strains from 40 donors of 17 mammalian species and analyzed their Hayflick's limit, i.e., their senescent plateau, and eventual spontaneous immortalization escape. The correlation of immortalization and replicative capacity of the species with their longevity, body mass and metabolism has been assessed through phylogenetic multiple linear regression (MLR). ResultsThe immortalization probability is negatively related to species body mass. The new evaluation and additional data about replicative potential strengthen our previous observation, confirming that stable and extended proliferation is strongly correlated with the evolution of a large body mass rather than lifespan. ConclusionThe relation between immortalization and body mass suggests a need to evolve stringent mechanisms that control genetic stability during the evolution of a large body mass.

Higher replication potential of West Nile virus governs apoptosis induction in human neuroblastoma cells.

Theextent of neuronal cell damage caused by West Nile virus (WNV) infection governs the disease severity ranging from mild, febrile illness to fatal encephalitis. Availability of naturally occurring genetic variants is helpful to study viral factors governing differential pathogenesis. During WNV infection, apoptosis serves as a virulence determinant positively contributing to viral pathogenesis. We investigated the levels of apoptosis induced by a low neurovirulent WNV lineage 5 strain 804994 and a high neurovirulent lineage 1 strain 68856 in human neuroblastoma cells, IMR-32. Our investigations clearly show the correlation between higher multiplication capacities of 68856 with higher levels of cytopathology induced by apoptosis. We observed activation of both the extrinsic and intrinsic apoptotic pathways during WNV infection. Infection with higher neurovirulent strain resulted in higher upregulation of pro-apoptotic proteins including death receptors (DR), adaptor protein, BH3-only regulatory proteins and higher cleavage of initiator caspases of both pathways. These results suggest that the virulence of a WNV strain may correlate with its higher replication fitness and ability to cause more cellular damage.

Preliminary Study: Micropropagation Using Five Types of Chelated Iron and the Subsequent Acclimation of Blue Honeysuckle (Lonicera caerulea var. kamtschatica Sevast.)

Blue honeysuckle (Lonicera caerulea var. kamtschatica Sevast.) is a valuable berry crop with a unique biochemical composition. It is rich in vitamins, minerals, and biologically active substances. Different species and cultivars of honeysuckle require different cultivation conditions in the field of accelerated reproduction in vitro. Taking into account the high clonal replication potential of the plant, we conducted research on the chelated-iron form’s influence on the micropropagation productivity of the blue honeysuckle “Lulia” cultivar at the multiplication, rooting, and subsequent acclimatization stages of microplants. In a preliminary study, five types of iron chelates were tested with carboxyl- and phosphorus-containing ligands: Fe(III)-EDTA, Fe(III)-DTPA, Fe(III)-EDDHA, Fe(III)-HEDP, and Fe(II)-HEDP. Each type of iron chelate was applied at four concentrations: standard, decreased by 2 times, and increased by 1.5 times and 2 times in the basic Murashige and Skoog (MS) nutrient medium. It was found that the blue honeysuckle “Lulia” had a selectivity to the type of iron chelate that was used. The nutrient-medium modifications with iron chelates, which caused the plant response, contributed to a significant improvement in the plant’s physiological status and increased its survival rate during the microplant’s acclimation to the nonsterile conditions stage. At the rooting stage, an increase in the rooting rate of up to 100% (Fe (III)-EDDHA), an increase in the number of shoots by 1.5–2 times, and an increase in the number of roots by 1.4–1.9 times were observed. The positive effect of the iron’s chelated forms was also observed at the acclimation stage. According to the results of the research, the most suitable iron forms for clonal micropropagation of blue honeysuckle were carboxyl-containing Fe(III)-EDDHA and phosphonate-containing Fe(II)-HEDP. Moreover, the effectiveness of both complexonates was confirmed in a wide concentration range: Fe(III)-EDDHA from (×1.0) to (×2.0), and Fe(II)-HEDP from (×0.5) up to (×1.5).

Telomerase RNA plays a major role in the completion of the life cycle in Ustilago maydis and shares conserved domains with other Ustilaginales.

The RNA subunit of telomerase is an essential component whose primary sequence and length are poorly conserved among eukaryotic organisms. The phytopathogen Ustilago maydis is a dimorphic fungus of the order Ustilaginales. We analyzed several species of Ustilaginales to computationally identify the TElomere RNA (TER) gene ter1. To confirm the identity of the TER gene, we disrupted the gene and characterized telomerase-negative mutants. Similar to catalytic TERT mutants, ter1Δ mutants exhibit phenotypes of growth delay, telomere shortening and low replicative potential. ter1-disrupted mutants were unable to infect maize seedlings in heterozygous crosses and showed defects such as cell cycle arrest and segregation failure. We concluded that ter1, which encodes the TER subunit of the telomerase of U. maydis, have similar and perhaps more extensive functions than trt1.

A Novel Approach for Determining the Critical Quality Attributes of Mesenchymal Stem Cells by Specifying Cell Population With Replication Potential

We introduce a novel approach to determine the critical quality attributes (CQAs) of mesenchymal stem cells (MSCs) expected to exert immunosuppressive effects. MSCs retained homeostatic replication potentials, such as sustainable growth and consistent cell morphology as a population, in early passages, but lost them in late passages. Characteristic surface markers of MSCs (ie, CD73, CD90, and CD105) were no longer expressed at 2 weeks after subcutaneous transplantation into NOG mice when MSCs from late passages were transplanted, but not when MSCs from early passages were transplanted, suggesting that the biological effects of the MSCs differed according to the timing of cell harvesting and highlighting the importance of specifying MSCs that retained homeostatic features to define the CQAs. The homeostatic features of MSCs related to the balance of the redox system, nutrient requirements, and mitochondrial function were also observed until a certain passage. Therefore, we could define the CQAs of MSCs related to manufacturing by selecting process parameters (PPs) underlying the homeostatic features of MSCs and measuring these PPs quantitatively to specify the cell population with homeostatic features by limiting the passage number. The validity of the PPs stipulated in our pilot study was verified using an SKG murine arthritis model, and critical PPs (CPPs) were then selected among the PPs. Thus, CQAs related to manufacturing in the developmental phase could be defined by the CPPs in this manner, and the concept of CQAs could be refined continuously toward commercial manufacturing.