Structural and surface property characterization of titanium dioxide nanotubes for orthopedic implants

This research focused on the to modification of the surface structure of titanium implants with nanostructured morphology of TiO2 nanotubes and studied the interaction of nanotubes with osteoblast cells to understand the parameters that affect the cell growth. The electrical, mechanical, and structural properties of TiO2 nanotubes were characterized to establish a better understanding on the properties of such nanoscale morphological structures. To achieve the objectives of this research work I transformed the titanium and its alloys, either in bulk sheet form, bulk machined form, or thin film deposited on another substrate into a surface of titania nanotubes using a low cost and environmentally friendly process. The process requires only a simple electrolyte, low cost electrode, and a DC power supply. With this simple approach of scalable nanofabrication, a typical result is nanotubes that are each approximately 100nm in diameter and have a wall thickness of about 20nm. By changing the fabrication parameters, independent nanotubes can be fabricated with open volume between them. Titanium in this form is termed onedimensional since electron transport is narrowly confined along the length of the nanotube. My Ph.D. accomplishments have successfully shown that osteoblast cells, the cells that are the precursors to bone, have a strong tendency to attach to the inside and outside of the titanium nanotubes onto which they are grown using their filopodia – cell’s foot used for locomotion – anchored to titanium nanotubes. In fact it was shown that the cell prefers to find many anchoring sites. These sites are critical for cell locomotion during the first several weeks of maturity and upon calcification as a strongly anchored bone cell. In addition I have shown that such a surface has a greater cell density than a smooth titanium surface. My work also developed a process that uses a focused and controllably rastered ion beam as a nano-scalpel to cut away sections of the osteoblast cells to probe the attachment beneath the main cell body. Ultimately the more rapid growth of osteoblasts, coupled with a stronger cell-surface interface, could provide cost reduction, shorter rehabilitation, and fewer follow-on surgeries due to implant loosening.

Regional myocardial wall thinning at multidetector computed tomography correlates to arrhythmogenic substrate in postinfarction ventricular tachycardia: assessment of structural and electrical substrate

BACKGROUND A majority of patients undergoing ablation of ventricular tachycardia have implanted devices precluding substrate imaging with delayed-enhancement MRI. Contrast-enhanced multidetector computed tomography (MDCT) can depict myocardial wall thickness with submillimetric resolution. We evaluated the relationship between regional myocardial wall thinning (WT) imaged by MDCT and arrhythmogenic substrate in postinfarction ventricular tachycardia. METHODS AND RESULTS We studied 13 consecutive postinfarction patients undergoing MDCT before ablation. MDCT data were integrated with high-density 3-dimensional electroanatomic maps acquired during sinus rhythm (endocardium, 509±291 points/map; epicardium, 716±323 points/map). Low-voltage areas (<1.5 mV) and local abnormal ventricular activities (LAVA) during sinus rhythm were assessed with regard to the WT. A significant correlation was found between the areas of WT <5 mm and endocardial low voltage (correlation-R=0.82; P=0.001), but no such correlation was found in the epicardium. The WT <5 mm area was smaller than the endocardial low-voltage area (54 cm(2) [Q1-Q3, 46-92] versus 71 cm(2) [Q1-Q3, 59-124]; P=0.001). Among a total of 13 060 electrograms reviewed in the whole study population, 538 LAVA were detected and analyzed. LAVA were located within the WT <5 mm (469/538 [87%]) or at its border (100% within 23 mm). Very late LAVA (>100 ms after QRS complex) were almost exclusively detected within the thinnest area (93% in the WT<3 mm). CONCLUSIONS Regional myocardial WT correlates to low-voltage regions and distribution of LAVA critical for the generation and maintenance of postinfarction ventricular tachycardia. The integration of MDCT WT with 3-dimensional electroanatomic maps can help focus mapping and ablation on the culprit regions, even when MRI is precluded by the presence of implanted devices.

Seawater carbonate chemistry, processes and elements during experiments with planktonic foraminifera Orbulina universa, 2004

We investigate the sensitivity of U/Ca, Mg/Ca, and Sr/Ca to changes in seawater [CO3[2-]] and temperature in calcite produced by the two planktonic foraminifera species, Orbulina universa and Globigerina bulloides, in laboratory culture experiments. Our results demonstrate that at constant temperature, U/Ca in O. universa decreases by 25 +/- 7% per 100 µmol [CO3[2-]] kg**-1, as seawater [CO3[2-]] increases from 110 to 470 µmol kg**-1. Results from G. bulloides suggest a similar relationship, but U/Ca is consistently offset by ~+40% at the same environmental [CO3[2-]]. In O. universa, U/Ca is insensitive to temperature between 15°C and 25°C. Applying the O. universa relationship to three U/Ca records from a related species, Globigerinoides sacculifer, we estimate that Caribbean and tropical Atlantic [CO3[2-]] was 110 +/- 70 µmol kg**-1 and 80 +/- 40 µmol kg**-1 higher, respectively, during the last glacial period relative to the Holocene. This result is consistent with estimates of the glacial-interglacial change in surface water [CO3[2-]] based on both modeling and on boron isotope pH estimates. In settings where the addition of U by diagenetic processes is not a factor, down-core records of foraminiferal U/Ca have potential to provide information about changes in the ocean's carbonate concentration.

Skeletal trade-offs in coralline algae in response to ocean acidification

Ocean acidification is changing the marine environment, with potentially serious consequences for many organisms. Much of our understanding of ocean acidification effects comes from laboratory experiments, which demonstrate physiological responses over relatively short timescales. Observational studies and, more recently, experimental studies in natural systems suggest that ocean acidification will alter the structure of seaweed communities. Here, we provide a mechanistic understanding of altered competitive dynamics among a group of seaweeds, the crustose coralline algae (CCA). We compare CCA from historical experiments (1981-1997) with specimens from recent, identical experiments (2012) to describe morphological changes over this time period, which coincides with acidification of seawater in the Northeastern Pacific. Traditionally thick species decreased in thickness by a factor of 2.0-2.3, but did not experience a change in internal skeletal metrics. In contrast, traditionally thin species remained approximately the same thickness but reduced their total carbonate tissue by making thinner inter-filament cell walls. These changes represent alternative mechanisms for the reduction of calcium carbonate production in CCA and suggest energetic trade-offs related to the cost of building and maintaining a calcium carbonate skeleton as pH declines. Our classification of stress response by morphological type may be generalizable to CCA at other sites, as well as to other calcifying organisms with species-specific differences in morphological types.

Phenotypic plasticity of coralline algae in a High CO2 world

It is important to understand how marine calcifying organisms may acclimatize to ocean acidification to assess their survival over the coming century. We cultured the cold water coralline algae, Lithothamnion glaciale, under elevated pCO2 (408, 566, 770, and 1024 µatm) for 10 months. The results show that the cell (inter and intra) wall thickness is maintained, but there is a reduction in growth rate (linear extension) at all elevated pCO2. Furthermore a decrease in Mg content at the two highest CO2 treatments was observed. Comparison between our data and that at 3 months from the same long-term experiment shows that the acclimation differs over time since at 3 months, the samples cultured under high pCO2 showed a reduction in the cell (inter and intra) wall thickness but a maintained growth rate. This suggests a reallocation of the energy budget between 3 and 10 months and highlights the high degree plasticity that is present. This might provide a selective advantage in future high CO2 world.

The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco

Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing.

Softening-up mannan-rich cell walls

The softening and degradation of the cell wall (CW), often mannan enriched, is involved in several processes during development of higher plants, such as meristematic growth, fruit ripening, programmed cell death, and endosperm rupture upon germination. Mannans are also the predominant hemicellulosic CW polymers in many genera of green algae. The endosperm CWs of dry seeds often contain mannan polymers, sometimes in the form of galactomannans (Gal-mannans). The endo-beta-mannanases (MANs) that catalyse the random hydrolysis of the beta-linkage in the mannan backbone are one of the main hydrolytic enzymes involved in the loosening and remodelling of CWs. In germinating seeds, the softening of the endosperm seed CWs facilitates the emergence of the elongating radicle. Hydrolysis and mobilization of endosperm Gal-mannans by MANs also provides a source of nutrients for early seedling growth, since Gal-mannan, besides its structural role, serves as a storage polysaccharide. Therefore, the role of mannans and of their hydrolytic enzymes is decisive in the life cycle of seeds. This review updates and discusses the significance of mannans and MANs in seeds and explores the increasing biotechnological potential of MAN enzymes.

Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium. We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Σ1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

Distinct Morphological Phenotypes of Cell Fusion Mutants

Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Δ fus2Δ, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus− zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.

Fission Yeast Pob1p, Which Is Homologous to Budding Yeast Boi Proteins and Exhibits Subcellular Localization Close to Actin Patches, Is Essential for Cell Elongation and Separation

The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain. Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth. Loss of its function leads to quick cessation of cellular elongation. Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation. Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen. In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis. Thus, this protein exhibits intracellular dynamics similar to F-actin patches. However, Pob1p constitutes a layer, rather than patches, at growing cell tips. It generates two split discs flanking the septum at cytokinesis. The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology. Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation. We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.

The mechanical properties of Saccharomyces cerevisiae

Cell-wall mechanical properties play an integral part in the growth and form of Saccharomyces cerevisiae. In contrast to the tremendous knowledge on the genetics of S. cerevisiae, almost nothing is known about its mechanical properties. We have developed a micromanipulation technique to measure the force required to burst single cells and have recently established a mathematical model to extract the mechanical properties of the cell wall from such data. Here we determine the average surface modulus of the S. cerevisiae cell wall to be 11.1 ± 0.6 N/m and 12.9 ± 0.7 N/m in exponential and stationary phases, respectively, giving corresponding Young's moduli of 112 ± 6 MPa and 107 ± 6 MPa. This result demonstrates that yeast cell populations strengthen as they enter stationary phase by increasing wall thickness and hence the surface modulus, without altering the average elastic properties of the cell-wall material. We also determined the average breaking strain of the cell wall to be 82% ± 3% in exponential phase and 80% ± 3% in stationary phase. This finding provides a failure criterion that can be used to predict when applied stresses (e.g., because of fluid flow) will lead to wall rupture. This work analyzes yeast compression experiments in different growth phases by using engineering methodology.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Hypersensitive Cell Death and Papilla Formation in Barley Attacked by the Powdery Mildew Fungus Are Associated with Hydrogen Peroxide but Not with Salicylic Acid Accumulation1

We analyzed the pathogenesis-related generation of H2O2 using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H2O2 accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H2O2 accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H2O2 was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H2O2 may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.

Nucellain, a Barley Homolog of the Dicot Vacuolar-Processing Protease, Is Localized in Nucellar Cell Walls1

The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.