Structure and Biomechanics during Xylem Vessel Transdifferentiation in Arabidopsis thaliana

Individual plant cells are the building blocks for all plantae and artificially constructed plant biomaterials, like biocomposites. Secondary cell walls (SCWs) are a key component for mediating mechanical strength and stiffness in both living vascular plants and biocomposite materials. In this paper, we study the structure and biomechanics of cultured plant cells during the cellular developmental stages associated with SCW formation. We use a model culture system that induces transdifferentiation of Arabidopsis thaliana cells to xylem vessel elements, upon treatment with dexamethasone (DEX). We group the transdifferentiation process into three distinct stages, based on morphological observations of the cell walls. The first stage includes cells with only a primary cell wall (PCW), the second covers cells that have formed a SCW, and the third stage includes cells with a ruptured tonoplast and partially or fully degraded PCW. We adopt a multi-scale approach to study the mechanical properties of cells in these three stages. We perform large-scale indentations with a micro-compression system in three different osmotic conditions. Atomic force microscopy (AFM) nanoscale indentations in water allow us to isolate the cell wall response. We propose a spring-based model to deconvolve the competing stiffness contributions from turgor pressure, PCW, SCW and cytoplasm in the stiffness of differentiating cells. Prior to triggering differentiation, cells in hypotonic pressure conditions are significantly stiffer than cells in isotonic or hypertonic conditions, highlighting the dominant role of turgor pressure. Plasmolyzed cells with a SCW reach similar levels of stiffness as cells with maximum turgor pressure. The stiffness of the PCW in all of these conditions is lower than the stiffness of the fully-formed SCW. Our results provide the first experimental characterization of the mechanics of SCW formation at single cell level

Structure and Biomechanics during Xylem Vessel Transdifferentiation in Arabidopsis thaliana

Individual plant cells are the building blocks for all plantae and artificially constructed plant biomaterials, like biocomposites. Secondary cell walls (SCWs) are a key component for mediating mechanical strength and stiffness in both living vascular plants and biocomposite materials. In this paper, we study the structure and biomechanics of cultured plant cells during the cellular developmental stages associated with SCW formation. We use a model culture system that induces transdifferentiation of Arabidopsis thaliana cells to xylem vessel elements, upon treatment with dexamethasone (DEX). We group the transdifferentiation process into three distinct stages, based on morphological observations of the cell walls. The first stage includes cells with only a primary cell wall (PCW), the second covers cells that have formed a SCW, and the third stage includes cells with a ruptured tonoplast and partially or fully degraded PCW. We adopt a multi-scale approach to study the mechanical properties of cells in these three stages. We perform large-scale indentations with a micro-compression system in three different osmotic conditions. Atomic force microscopy (AFM) nanoscale indentations in water allow us to isolate the cell wall response. We propose a spring-based model to deconvolve the competing stiffness contributions from turgor pressure, PCW, SCW and cytoplasm in the stiffness of differentiating cells. Prior to triggering differentiation, cells in hypotonic pressure conditions are significantly stiffer than cells in isotonic or hypertonic conditions, highlighting the dominant role of turgor pressure. Plasmolyzed cells with a SCW reach similar levels of stiffness as cells with maximum turgor pressure. The stiffness of the PCW in all of these conditions is lower than the stiffness of the fully-formed SCW. Our results provide the first experimental characterization of the mechanics of SCW formation at single cell level

Multiscale Mechanical Characterization of Subcellular Structures in Living Walled Cells

The physiology of walled cells is dramatically different from that of human cells, but the biomechanics of walled cells are far less studied. Most bacterial, fungal, and plant cells have a strong cell wall (CW), which allows them to withstand large hydrostatic pressures in the cytoplasm, called turgor. Turgor pressure conflates the mechanics of subcellular components and complicates the characterization of the cell. In this dissertation, new models are introduced and explored for single cells to investigate the multiscale mechanics of plant and bacterial cells using micro- and nano-indentation experiments. A multi-scale biomechanical assay is used to study the mechanical properties of plant cells. The plant CW is typically around 5% of the width of the entire cell, and is thought to carry most of the mechanical load. Large-scale indentations using a micro-indentation system probe the behavior of the overall cell structure, and atomic-force microscopy (AFM) nano-scale indentations are used to isolate the CW response. To determine the effect of external osmotic pressure, indentations are performed on cells in different osmotic conditions: hypotonic, isotonic, and hypertonic. The cell is idealized as two springs acting in series, one to represent the CW and one to represent the cytoplasm. The model uses the experimentally determined initial stiffnesses as input to the model to determine the relative stiffness contributions of the CW and the cytoplasm. The first type of walled cells investigated is the xylem vessel element of Arabidopsis thaliana. The xylem is responsible for transporting water through the stem of any vascular plant (more commonly known as a land plant), and hence it must maintain structural integrity against high internal pressures while transporting water from the roots to the leaves. For extra structural support, xylem vessel elements develop secondary cell walls (SCWs), which are known to be a key component for mediating mechanical strength and stiffness in vascular plants. The structure and biomechanics of cultured plant cells are investigated during the cellular developmental stages associated with SCW formation using the multi-scale biomechanical assay described above. To determine the effect of morphological changes during differentiation, micro- and nano-indentations are performed on cells in different observed stages of the differentiation process.Prior to triggering differentiation, cells in hypotonic pressure conditions are significantly stiffer than cells in isotonic or hypertonic conditions, highlighting the dominant role of turgor pressure. Plasmolyzed cells with a SCW reach similar levels of stiffness as cells with maximum turgor pressure. Analysis using the two-spring model shows that the stiffness of the primary CW in all of these conditions is lower than the stiffness of the fully-formed SCW. These results provide the first experimental characterization of the mechanics of SCW formation at the single-cell level in plant cells. Next, the mechanical response of individual Nicotiana tabacum cells from a suspension culture is studied using the same multi-scale biomechanical assay. The role played by the microtubules (MTs) and actin filaments (AFs) is determined through the use of drug treatments which selectively remove MTs and AFs. A generative statistical model is added to the two-spring model to quantify the stiffnesses of the CW, cytoplasm, turgor pressure, MTs, and AFs. Analysis of the initial stiffness and energy dissipation calculated from micro-indentation experiments indicates that the MTs and AFs contribute significantly to the mechanical response of a cell under compression. Micro- and nano-indentation tests confirm that turgor pressure is the most significant contributor to the stiffness response of turgid cells in compression. Finally, the results reveal that turgor pressure exerts stress on the CW, which leads to a measurable stiffening of the CW. The studies described above focused on developing a discrete model to describe the mechanics of a cell in indentation experiments. However, the most common type of model used to evaluate the mechanics of a cell are continuum models. Continuum models are also necessary to decouple the material properties of subcellular components from their structure. In the final section, AFM indentations are simulated on a gram-negative bacterium, Escherichia coli, and a sensitivity study and inverse analysis are performed to solve for the CW elastic modulus and turgor pressure simultaneously. Sensitivity study results reveal that uncertainty in turgor pressure and CW elasticity indeed contribute the most to variability in force spectra from AFM measurements. The parameter space of possible values for CW elastic modulus and turgor pressure is discretized using triangular elements. "Simulated experiments" are tested throughout the parameter space, and correlations between the CW elastic modulus and turgor pressure, which depend on the type of objective function, are investigated. Two unique objective functions are tested in the inverse analysis, and a third objective function, which is a weighted sum of the first two, is found to reduce errors in estimated CW elastic modulus and turgor pressure by 20% and 11%, respectively. The use of this type of inverse analysis has the potential to elucidate the material properties of CWs using a single indentation measurement and reliably decouple these properties from the high turgor pressures inside walled cells.</p

Reducing the cesarean delivery rate

BACKGROUND: The cesarean delivery rate has been rising in recent years, having associated maternal morbidities. Elective induction of labor has also been seen to rise during this same time period. OBJECTIVE: This current study investigated the difference in the cesarean delivery rate between induction of labor and spontaneous labor among nulliparous, term, singleton, and vertex-presenting women. STUDY DESIGN: A retrospective cohort in a single institution over a seven-year period was used for this analysis, observing the difference in cesarean delivery rate at different term gestational ages and neonatal morbidity using the 5-minute Apgar score \u3c 5. RESULTS: A statistically significant difference was found in cesarean delivery rate between those women whose labor was induced and those whose labor began spontaneously, at each term gestational age of labor initiation (P \u3c 0.001). The proportion of indications for induction was described (i.e. elective vs. medically-indicated), and no difference was found for neonatal morbidity between the groups analyzed, using the 5-minute Apgar score as the perinatal outcome measure. CONCLUSION: A comparison was made between spontaneous and induced labor regarding the resultant cesarean delivery rate, and a significant difference was found favoring spontaneous labor. This should be considered when electing to deliver using an induction methodology for nulliparous women, especially when there are no medical indications for it

Aptamer-based proteomic signature of intensive phase treatment response in pulmonary tuberculosis

BACKGROUND: New drug regimens of greater efficacy and shorter duration are needed for tuberculosis (TB) treatment. The identification of accurate, quantitative, non-culture based markers of treatment response would improve the efficiency of Phase 2 TB drug testing. METHODS: In an unbiased biomarker discovery approach, we applied a highly multiplexed, aptamer-based, proteomic technology to analyze serum samples collected at baseline and after 8 weeks of treatment from 39 patients with pulmonary TB from Kampala, Uganda enrolled in a Centers for Disease Control and Prevention (CDC) TB Trials Consortium Phase 2B treatment trial. RESULTS: We identified protein expression differences associated with 8-week culture status, including Coagulation Factor V, SAA, XPNPEP1, PSME1, IL-11 Rα, HSP70, Galectin-8, α2-Antiplasmin, ECM1, YES, IGFBP-1, CATZ, BGN, LYNB, and IL-7. Markers noted to have differential changes between responders and slow-responders included nectin-like protein 2, EphA1 (Ephrin type-A receptor 1), gp130, CNDP1, TGF-b RIII, MRC2, ADAM9, and CDON. A logistic regression model combining markers associated with 8-week culture status revealed an ROC curve with AUC=0.96, sensitivity=0.95 and specificity=0.90. Additional markers showed differential changes between responders and slow-responders (nectin-like protein), or correlated with time-to-culture-conversion (KLRK1). CONCLUSIONS: Serum proteins involved in the coagulation cascade, neutrophil activity, immunity, inflammation, and tissue remodeling were found to be associated with TB treatment response. A quantitative, non-culture based, five-marker signature predictive of 8-week culture status was identified in this pilot study