The effect of weak inertia in rotating high-aspect-ratio vessel bioreactors

One method to grow artificial body tissue is to place a porous scaffold seeded with cells, known as a tissue construct, into a rotating bioreactor filled with a nutrient-rich fluid. The flow within the bioreactor is affected by the movement of the construct relative to the bioreactor which, in turn, is affected by the hydrodynamical and gravitational forces the construct experiences. The construct motion is thus coupled to the flow within the bioreactor. Over the timescale of a few hours, the construct appears to move in a periodic orbit but, over tens of hours, the construct drifts from periodicity. In the biological literature, this effect is often attributed to the change in density of the construct that occurs via tissue growth. In this paper, we show that weak inertia can cause the construct to drift from its periodic orbit over the same timescale as tissue growth. We consider the coupled flow and construct motion problem within a rotating high-aspect- ratio vessel bioreactor. Using an asymptotic analysis, we investigate the case where the Reynolds number is large but the geometry of the bioreactor yields a small reduced Reynolds number, resulting in a weak inertial effect. In particular, to accurately couple the bioreactor and porous flow regions, we extend the nested boundary layer analysis of Dalwadi et al. (J. Fluid Mech. vol. 798, pp. 88–139, 2016) to include moving walls and the thin region between the porous construct and the bioreactor wall. This allows us to derive a closed system of nonlinear ordinary differential equations for the construct trajectory, from which we show that neglecting inertia results in periodic orbits; we solve the inertia-free problem analytically, calculating the periodic orbits in terms of the system parameters. Using a multiple-scale analysis, we then systematically derive a simpler system of nonlinear ordinary differential equations that describe the long-time drift of the construct due to the effect of weak inertia. We investigate the bifurcations of the construct trajectory behaviour, and the limit cycles that appear when the construct is less dense than the surrounding fluid and the rotation rate is large enough. Thus, we are able to predict when the tissue construct will drift towards a stable limit cycle within the bioreactor and when it will drift out until it hits the bioreactor edg

G-overhang dynamics at Tetrahymena telomeres

To learn more about the structure of the DNA terminus at Tetrahymena thermophila telomeres, we have devised a ligation-mediated primer extension protocol to accurately measure the length of the G-strand overhang. We show that overhang length and the identity of the 3′-terminal nucleotide are tightly regulated. The majority of overhangs terminate in the sequence 5′-TTGGGGT and >80% are either 14–15 or 20–21 nucleotides in length. No significant changes in overhang length were detected as cells traversed the cell cycle. However, changes in length distribution were observed when cells exited the cell cycle, indicating an altered balance between DNA synthesis and degradation or end protection. We also provide evidence that rDNA molecules have overhangs on both telomeres. Full-length rDNA could be cloned by a strategy that depends on overhangs being present at both ends. Moreover, analysis of leading strand telomeres revealed that a significant fraction have overhangs ≥5 nucleotides. Our results indicate that generation of the terminal telomeric DNA structure is highly regulated and requires several distinct DNA-processing events

Modulation of Telomere Length Dynamics by the Subtelomeric Region of Tetrahymena Telomeres

Tetrahymena telomeres usually consist of ∼250 base pairs of T(2)G(4) repeats, but they can grow to reach a new length set point of up to 900 base pairs when kept in log culture at 30°C. We have examined the growth profile of individual macronuclear telomeres and have found that the rate and extent of telomere growth are affected by the subtelomeric region. When the sequence of the rDNA subtelomeric region was altered, we observed a decrease in telomere growth regardless of whether the GC content was increased or decreased. In both cases, the ordered structure of the subtelomeric chromatin was disrupted, but the effect on the telomeric complex was relatively minor. Examination of the telomeres from non-rDNA chromosomes showed that each telomere exhibited a unique and characteristic growth profile. The subtelomeric regions from individual chromosome ends did not share common sequence elements, and they each had a different chromatin structure. Thus, telomere growth is likely to be regulated by the organization of the subtelomeric chromatin rather than by a specific DNA element. Our findings suggest that at each telomere the telomeric complex and subtelomeric chromatin cooperate to form a unique higher order chromatin structure that controls telomere length

Comparative studies of two generations of NanoString nCounter System.

The NanoString nCounter System has been widely used in basic science and translational science research for the past decade. The System consists of two instruments: the PrepStation and the Digital Analyzer, and both have been continuously improved with evolving technologies. A great amount of research data have been generated at multiple research laboratories with the employment of different generations of the System. With the need of integrating multiple datasets, researchers are interested to know whether signals are comparable between different generations of the System. Toward this end, we designed a profiling study to compare performance of two generations of the NanoString nCounter System using a common set of biological samples. Using graphical tools and statistical models, we found that two different generations of NanoString nCounter System produced equivalent signals and signal deviations are in the range of random background noises for the medium-high expression levels

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell Cycle Checkpoint

The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3′ strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang

Regulation of actin function by protein kinase A-mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65+/+ cells. The ability of RelA/p65−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability