The Influence of Long and Short Cycle Schemes of Self-Cycling Fermentation on the Growth of E. Coli and S. Cerevisiae

Self-cycling fermentation (SCF), a cyclic process in which cells divide once per cycle, has been shown to lead to improvements in productivity during bioconversion and, often, whole-culture synchronization. Previous studies have found that in some cases, the completion of synchronized cell replication occurred simultaneously with depletion of a limiting nutrient. However, exceptions were also observed when the end of cell doubling occurred before the exhaustion of the limiting nutrient. In order to better understand the underlying mechanisms and impacts of these growth patterns on bioprocessing, we investigated the growth of Escherichia coli and Saccharomyces cerevisiae in long- and short-cycle SCF strategies. Three characteristic events linked to SCF cycles were identified: (1) the completion of synchronized cell replication, (2) the depletion or a plateau of the limiting nutrient, and (3) characteristic points of control parameters (e.g., the minimum of dissolved oxygen and the maximum of carbon dioxide evolution rate). Three major trends stemming from this study and SCF literature were observed: (A) co-occurrence of the three key events in SCF cycles, (B) cycles for which cell replication ended prior to the co-occurrence of the other two events, and (C) cycles for which the time of depletion or a plateau of the limiting nutrient occurred later than the concurrence of the other two events. Based on these observations, a novel definition for SCF is proposed.

Computational model of 3D cell migration based on the molecular clutch mechanism

The external environment is a regulator of cell activity. Its stiffness and microstructure can either facilitate or prevent 3D cell migration in both physiology and disease. 3D cell migration results from force feedbacks between the cell and the extracellular matrix (ECM). Adhesions regulate these force feedbacks by working as molecular clutches that dynamically bind and unbind the ECM. Because of the interdependency between ECM properties, adhesion dynamics, and cell contractility, how exactly 3D cell migration occurs in different environments is not fully understood. In order to elucidate the effect of ECM on 3D cell migration through force-sensitive molecular clutches, we developed a computational model based on a lattice point approach. Results from the model show that increases in ECM pore size reduce cell migration speed. In contrast, matrix porosity increases it, given a sufficient number of ligands for cell adhesions and limited crowding of the matrix from cell replication. Importantly, these effects are maintained across a range of ECM stiffnesses’, demonstrating that mechanical factors are not responsible for how matrix microstructure regulates cell motility.

Kronos scRT: a uniform framework for single-cell replication timing analysis

Mammalian genomes are replicated in a cell-type specific order and in coordination with transcription and chromatin organization. Although the field of replication is also entering the single-cell era, current studies require cell sorting, individual cell processing and have yielded a limited number (<100) of cells. Here, we have developed Kronos scRT (https://github.com/CL-CHEN-Lab/Kronos scRT), a software for single-cell Replication Timing (scRT) analysis. Kronos scRT does not require a specific platform nor cell sorting, allowing the investigation of large datasets obtained from asynchronous cells. Analysis of published available data and droplet-based scWGS data generated in the current study, allows exploitation of scRT data from thousands of cells for different mouse and human cell lines. Our results demonstrate that, although most cells replicate within a close timing range for a given genomic region, replication can also occur stochastically throughout S phase. Altogether, Kronos scRT allows investigating the RT program at a single-cell resolution for both homogeneous and heterogeneous cell populations in a fast and comprehensive manner.

Reaction Network Modeling of Complex Ecological Interactions: Endosymbiosis and Multilevel Regulation

Endosymbiosis is a type of symbiosis where one species of microscopic scale inhabits the cell of another species of a larger scale, such that the exchange of metabolic byproducts produces mutual benefit. These benefits can occur at different biological levels. For example, endosymbiosis promotes efficiency of the cell metabolism, cell replication, and the generation of a macroscopic layer that protects the organism from its predators. Therefore, modeling endosymbiosis requires a complex-systems and multilevel approach. We propose a model of endosymbiosis based on reaction networks, where species of the reaction network represent either ecological species, resources, or conditions for the ecological interactions to happen, and the endosymbiotic interaction mechanisms are represented by different sequences of reactions (processes) in the reaction network. As an example, we develop a toy model of the coral endosymbiotic interaction. The model considers two reaction networks, representing biochemical traffic and cellular proliferation levels, respectively. In addition, the model incorporates top-down and bottom-up regulation mechanisms that stabilizes the endosymbiotic interaction.

The small molecule kobusone can stimulate islet β-cell replication in vivo

Objective To investigate the ability of kobusone to reduce high glucose levels and promote β-cell proliferation. Methods Four-week-old female db/db mice were assigned to the kobusone (25 mg/kg body weight, intraperitoneally twice a day) or control group (same volume of PBS). Glucose levels and body weight were measured twice a week. After 6 weeks, intraperitoneal glucose tolerance tests and immunohistochemical studies were performed, and insulin levels were determined. The expression of mRNAs involved in cell proliferation, such as PI3K, Akt, cyclin D3 and p57Kip 2 , was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results Kobusone reduced blood glucose levels after 3 weeks and more strongly increased serum insulin levels than the vehicle. Immunohistochemistry illustrated that kobusone increased 5-bromo-2′-deoxyuridine incorporation into islet β-cells, suggesting that it can stimulate islet β-cell replication in vivo. RT-qPCR indicated that kobusone upregulated the mRNA expression of PI3K, Akt, and cyclin D3 and downregulated that of p57Kip2. Conclusion Our findings suggest that kobusone is a potent pancreatic islet β-cell inducer that has the potential to be developed as an anti-diabetic agent.

Epigenetic Programming during thymic development sets the stage for optimal function in effector T cells via DNA demethylation

The potential for early thymic developmental events to program epigenetic states that influence adult T cell physiology remains an important question in health. Herein using the Cd4 locus as a paradigm for early developmental programming, we demonstrate that DNA demethylation during thymic development is critical for the licensing of a novel stimulus-responsive element that serves to maintain CD4 gene expression in effector T cells. We document the importance of maintaining high CD4 expression during parasitic infection and show that by driving transcription, this stimulus-responsive element allows for the maintenance of H3K4me3 levels during T cell replication, which is critical for repelling de novo DNA methylation at the Cd4 promoter. A failure to undergo epigenetic programming during development leads to gene silencing during effector T cell replication, thus providing evidence that early development can program stimulus-responsive elements to propagate a stable epigenetic state in effector T cells, with important biological consequences.

Weak Electromagnetic Fields Accelerate Fusion of Myoblasts

Weak electromagnetic fields (WEF) alter Ca2+ handling in skeletal muscle myotubes. Owing to the involvement of Ca2+ in muscle development, we investigated whether WEF affects fusion of myoblasts in culture. Rat primary myoblast cultures were exposed to WEF (1.75 µT, 16 Hz) for up to six days. Under control conditions, cell fusion and creatine kinase (CK) activity increased in parallel and peaked at 4–6 days. WEF enhanced the extent of fusion after one and two days (by ~40%) vs. control, but not thereafter. Exposure to WEF also enhanced CK activity after two days (almost four-fold), but not afterwards. Incorporation of 3H-thymidine into DNA was enhanced by one-day exposure to WEF (~40%), indicating increased cell replication. Using the potentiometric fluorescent dye di-8-ANEPPS, we found that exposure of cells to 150 mM KCl resulted in depolarization of the cell membrane. However, prior exposure of cells to WEF for one day followed by addition of KCl resulted in hyperpolarization of the cell membrane. Acute exposure of cells to WEF also resulted in hyperpolarization of the cell membrane. Twenty-four hour incubation of myoblasts with gambogic acid, an inhibitor of the inward rectifying K+ channel 2.1 (Kir2.1), did not affect cell fusion, WEF-mediated acceleration of fusion or hyperpolarization. These data demonstrate that WEF accelerates fusion of myoblasts, resulting in myotube formation. The WEF effect is associated with hyperpolarization but WEF does not appear to mediate its effects on fusion by activating Kir2.1 channels.

PFKFB3 DEPLETION ACTIVATES β–CELL REPLICATION BY CELL COMPETITIVE CULLING OF COMPROMISED β–CELLS UNDER STRESS

Highly conserved hypoxia–inducible factor 1 alpha (HIF1α) and its target 6–phosphofructo–2–kinase/fructose–2,6–biphosphatase 3 (PFKFB3) play a critical role in the survival of damaged β–cells in type 2 diabetes (T2D) while rendering β–cells non–responsive to glucose stimulation by mitochondrial suppression. HIF1α –PFKFB3 is activated in 30–50% of all β–cells in diabetic islets, leaving an open question of whether targeting this pathway may adjust β–cell mass and function to the specific metabolic demands during diabetogenic stress. Our previous studies of β–cells under amyloidogenic stress by human islet amyloid polypeptide (hIAPP) revealed that PFKFB3 is a metabolic execution arm of the HIF1α pathway with potent implications on Ca2+ homeostasis, metabolome, and mitochondrial form and function. To discriminate the role of PFKFB3 from HIF1α in vivo, we generated mice with conditional β–cell specific disruption of the Pfkfb3 gene on a heterozygous hIAPP background and a high–fat diet (HFD) [PFKFB3βKO + diabetogenic stress (DS)]. PFKFB3 disruption in β–cells under diabetogenic stress led to selective purging of hIAPP–damaged β–cells and the disappearance of bihormonal insulin– and glucagon–positive cells, thus compromised β–cells. At the same time, PFKFB3 disruption led to a three–fold increase in β–cell replication resembling control levels as measured with minichromosome maintenance 2 protein (MCM2). PFKFB3 disruption depleted bihormonal cells while increased β–cell replication that was reflected in the increased β–/α–cell ratio and maintained β–cell mass. Analysis of metabolic performance indicated comparable glucose intolerance and reduced plasma insulin levels in PFKFB3βKO DS relative to PFKFB3WT DS mice. In the PFKFB3βKO DS group, plasma glucagon levels were reduced compared to PFKFB3WT DS mice and were in line with increased insulin sensitivity. Glucose intolerance in PFKFB3βKO DS mice could be explained by the compensatory expression of HIF1α after disruption of PFKFB3. Our data strongly suggest that the replication and functional recovery of β–cells under diabetogenic stress depend on selective purification of HIF1α and PFKFB3–positive β–cells. Thus, HIF1α–PFKFB3–dependent activation of cell competition and purging of compromised β–cells may yield functional competent β–cell mass in diabetes.

9-(2-Phosphonyl-methoxyethyl)-adenine promotes erythrocytic differentiation and disrupts cell replication in chronic myelogenous leukemia K562 cells

Disruption during cellular differentiation can cause hematopoietic stem cells to proliferate uncontrollably, resulting in the development of cancer. Differentiation therapies are being investigated as a type of cancer treatment which involve inducing agents that promote the differentiation of cancer cells into those with similar properties to normal blood cells. These cells can then undergo apoptosis at an accelerated and controlled rate compared to cancer cells, making this a potential therapeutic technique. In this study, the ability of human chronic myelogenous leukemia K562 cells to undergo cellular differentiation in response to the inducing agent 9-(2-Phosphonyl-methoxy ethyl)-adenine (PMEA) is investigated. PMEA has previously been shown to disrupt cell replication, and promote erythrocytic differentiation in K562 cells. In order to further test the effectiveness of this inducer, cell proliferation was measured with a cell growth curve, hemoglobin presence was measured with benzidine staining, and gamma-globin expression (a protein subunit of fetal hemoglobin) was measured in both induced and uninduced K562 cell cultures via RT-qPCR and western blotting. The results indicate that PMEA slows cell replication, and promotes hemoglobin (and subsequently gamma-globin) expression in treated cells. In summary, the findings support the conclusion that PMEA is able to promote erythrocytic differentiation in K562 cells, and provides information that supports differentiation therapies as a method for cancer treatment.

Review of methods for the detection of Lawsonia intracellularis infection in pigs

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis–specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide.