Mitochondrial Network Size Scaling in Budding Yeast

Mitochondria must grow with the growing cell to ensure proper cellular physiology and inheritance upon division. We measured the physical size of mitochondrial networks in budding yeast and found that mitochondrial network size increased with increasing cell size and that this scaling relation occurred primarily in the bud. The mitochondria-to-cell size ratio continually decreased in aging mothers over successive generations. However, regardless of the mother's age or mitochondrial content, all buds attained the same average ratio. Thus, yeast populations achieve a stable scaling relation between mitochondrial content and cell size despite asymmetry in inheritance.

Variability in light absorption and scattering of phytoplankton in Patagonian waters: Role of community size structure and pigment composition

Intense phytoplankton blooms were observed along the Patagonian shelf-break with satellite ocean color data, but few in situ optical observations were made in that region. We examine the variability of phytoplankton absorption and particulate scattering coefficients during such blooms on the basis of field data. The chlorophyll-a concentration, [Chla], ranged from 0.1 to 22.3 mg m−3 in surface waters. The size fractionation of [Chla] showed that 80% of samples were dominated by nanophytoplankton (N-group) and 20% by microphytoplankton (M-group). Chlorophyll-specific phytoplankton absorption coefficients at 440 and 676 nm, a*ph(440) and a*ph(676), and particulate scattering coefficient at 660 nm, b*p(660), ranged from 0.018 to 0.173, 0.009 to 0.046, and 0.031 to 2.37 m2 (mg Chla)−1, respectively. Both a*ph(440) and a*ph(676) were statistically higher for the N-group than M-group and also considerably higher than expected from global trends as a function of [Chla]. This result suggests that size of phytoplankton cells in Patagonian waters tends to be smaller than in other regions at similar [Chla]. The phytoplankton cell size parameter, Sf, derived from phytoplankton absorption spectra, proved to be useful for interpreting the variability in the data around the general inverse dependence of a*ph(440), a*ph(676), and b*p(660) on [Chla]. Sf also showed a pattern along the increasing trend of a*ph(440) and a*ph(676) as a function of the ratios of some accessory pigments to [Chla]. Our results suggest that the variability in phytoplankton absorption and scattering coefficients in Patagonian waters is caused primarily by changes in the dominant phytoplankton cell size accompanied by covariation in the concentrations of accessory pigments.

Central nervous system of Rhipicephalus sanguineus ticks (Acari: Ixodidae): an ultrastructural study

This study performed the ultrastructural description of the synganglion of Rhipicephalus sanguineus males and females, aiming to contribute to the understanding of the cellular organization of this organ. The results show that the central nervous system of these individuals consists of a mass of fused nerves, named synganglion, from where nerves emerge towards several parts of the body. It is surrounded by the neural lamella, a uniform and acellular layer, constituted by repeated layers of homogeneous and finely granular material. The perineurium is just below, composed of glial cells, which extensions invaginate throughout the nervous tissue. The synganglion is internally divided into an outer cortex, which contains the cellular bodies of the neural cells and an inner neuropile. The neural cells can be classified into two types according to cell size, cytoplasm-nucleus relation, and neurosecretory activity. Type I cells are oval or spherical and present a large nucleus occupying most part of the cytoplasm, which contains few organelles. Type 2 cells are polygonal, present a great cytoplasm volume, and their nuclei are located in the cell periphery. The cytoplasm of these cells contains a well-developed rough endoplasmic reticulum, Golgi regions, mitochondria, and several neurosecretory granules. The subperineurium and the tracheal ramifications are found between the cortex and the neuropile. The latter is formed mainly by neural fibers, tracheal elements, and glial cells. The results obtained show that R. sanguineus males' and females' nervous tissue present an ultrastructural organization similar to the one described in the literature for other tick species.

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.

Producción de tomate tipo italiano en función del volumen de la celda y de la edad de las mudas

Con el objetivo de evaluar la influencia de las bandejas de celdas sobre la producción de tomate tipo italiano en el campo, se realizó este trabajo en Piracicaba, SP, Brasil, de mayo a agosto del 2005. Los tratamientos consistieron en cuatro volúmenes de recipiente, tres bandejas de poliestireno expandido de 121,2; 34,6 y 12,0 cm³ y de una bandeja de plástico rígido de 14,0 cm³ (72, 128, 288 y 450 celdas, respectivamente) combinadas con cuatro edades para el trasplante (19, 24, 29 y 34 días después de la siembra). El delineamiento para la producción de mudas fue completamente al azar, con cinco plantas por parcela y tres repeticiones. Se analizaron área foliar, altura, masa verde y seca de la parte aérea y raíz y la calidad de las mudas. En la producción a campo, el delineamiento fue en bloques al azar con diez plantas por parcela y tres repeticiones. Fueron evaluadas la precocidad para inicio de cosecha, producción comercial y total por planta. Volúmenes mayores de recipiente (121,2 y 34,6 cm³) presentaron mejor calidad de mudas. En la producción de frutos, el número comercial y total de frutos por planta fue superior en la muda de 24 días de edad, sin embargo, en la producción total de frutos, no hubo diferencia entre los tratamientos. Por otro lado, también se obtuvo precocidad para la cosecha en los tratamientos realizados en los volúmenes de 121,2 y 34,6 cm³. El volumen de recipiente de 14,0 cm³ (450 celdas) resultó en mudas de calidad muy inferior, alongadas y raquíticas.

Effect of the relationship between particle size, inter-particle distance, and metal loading of carbon supported fuel cell catalysts on their catalytic activity

The effect of the relationship between particle size (d), inter-particle distance (x(i)), and metal loading (y) of carbon supported fuel cell Pt or PtRu catalysts on their catalytic activity, based on the optimum d (2.5-3 nm) and x(i)/d (>5) values, was evaluated. It was found that for y < 30 wt%, the optimum values of both d and x(i)/d can be always obtained. For y >= 30 wt%, instead, the positive effect of a thinner catalyst layer of the fuel cell electrode than that using catalysts with y < 30 wt% is concomitant to a decrease of the effective catalyst surface area due to an increase of d and/or a decrease of x(i)/d compared to their optimum values, with in turns gives rise to a decrease in the catalytic activity. The effect of the x(i)/d ratio has been successfully verified by experimental results on ethanol oxidation on PtRu/C catalysts with same particle size and same degree of alloying but different metal loading. Tests in direct ethanol fuel cells showed that, compared to 20 wt% PtRu/C, the negative effect of the lower x(i)/d on the catalytic activity of 30 and 40 wt% PtRu/C catalysts was superior to the positive effect of the thinner catalyst layer.

Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)calcium phosphate (PLGACaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470590, 590850 and 8501200 mu m. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470590 mu m. These results show that PLGACaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (similar to 1000 mu m) and smaller (similar to 500 mu m) pores in a single scaffold would optimize cellular and tissue responses during bone healing. Copyright (C) 2011 John Wiley & Sons, Ltd.

The Endothelial Sample Size Analysis in Corneal Specular Microscopy Clinical Examinations

Purpose: To evaluate endothelial cell sample size and statistical error in corneal specular microscopy (CSM) examinations. Methods: One hundred twenty examinations were conducted with 4 types of corneal specular microscopes: 30 with each BioOptics, CSO, Konan, and Topcon corneal specular microscopes. All endothelial image data were analyzed by respective instrument software and also by the Cells Analyzer software with a method developed in our lab(US Patent). A reliability degree (RD) of 95% and a relative error (RE) of 0.05 were used as cut-off values to analyze images of the counted endothelial cells called samples. The sample size mean was the number of cells evaluated on the images obtained with each device. Only examinations with RE<0.05 were considered statistically correct and suitable for comparisons with future examinations. The Cells Analyzer software was used to calculate the RE and customized sample size for all examinations. Results: Bio-Optics: sample size, 97 +/- 22 cells; RE, 6.52 +/- 0.86; only 10% of the examinations had sufficient endothelial cell quantity (RE<0.05); customized sample size, 162 +/- 34 cells. CSO: sample size, 110 +/- 20 cells; RE, 5.98 +/- 0.98; only 16.6% of the examinations had sufficient endothelial cell quantity (RE<0.05); customized sample size, 157 +/- 45 cells. Konan: sample size, 80 +/- 27 cells; RE, 10.6 +/- 3.67; none of the examinations had sufficient endothelial cell quantity (RE>0.05); customized sample size, 336 +/- 131 cells. Topcon: sample size, 87 +/- 17 cells; RE, 10.1 +/- 2.52; none of the examinations had sufficient endothelial cell quantity (RE>0.05); customized sample size, 382 +/- 159 cells. Conclusions: A very high number of CSM examinations had sample errors based on Cells Analyzer software. The endothelial sample size (examinations) needs to include more cells to be reliable and reproducible. The Cells Analyzer tutorial routine will be useful for CSM examination reliability and reproducibility.

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma

Background. Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. Material and methods. We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. Results. Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN+) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN+ with an accuracy of 80.0% (p = 0.012). Conclusions. The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our "Nodal Index" model are predictive of lymph node metastasis in OSCC.

Adipose Tissue-Derived Stem Cell Treatment Prevents Renal Disease Progression

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 x 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 x 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4. IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-alpha, KC, RANTES, and IL-1 alpha. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.

The Influence of Osteoblast Differentiation Stage on Bone Formation in Autogenously Implanted Cell-Based Poly(Lactide-Co-Glycolide) and Calcium Phosphate Constructs

We tested the hypothesis that the osteoblast differentiation status of bone marrow stem cells (BMSCs) combined with a three-dimensional (3D) structure modulates bone formation when autogenously implanted. Rat BMSCs were aspirated, expanded, and seeded into a 3D composite of poly(lactide-co-glycolide) and calcium phosphate (PLGA/CaP) to produce a hybrid biomaterial. Calvarial defects were implanted with (1) scaffold without cells (SC/NC), (2) scaffold and BMSCs (SC + BMSC), (3) scaffold and osteoblasts differentiated for 7 days (SC + OB7), and (4) for 14 days (SC + OB14). After 4 weeks, there was more bone formation in groups combining scaffold and cells, SC + BMSC and SC + OB7. A nonsignificant higher amount of bone formation was observed on SC + OB14 compared with SC/NC. Additionally, more blood vessels were counted within all hybrid biomaterials, without differences among them, than into SC/NC. These findings provide evidences that the cell differentiation status affects in vivo bone formation in autogenously implanted cell-based constructs. Undifferentiated BMSCs or osteoblasts in early stage of differentiation combined with PLGA/CaP scaffold favored bone formation compared with plain scaffold and that one associated with more mature osteoblasts.

The influence of pore size on osteoblast phenotype expression in cultures grown on porous titanium

This study investigated the effect of pore size on osteoblastic phenotype development in cultures grown on porous titanium (Ti). Porous Ti discs with three different pore sizes, 312 mu m (Ti 312), 130 mu m (Ti 130) and 62 mu m (Ti 62) were fabricated using a powder metallurgy process. Osteoblastic cells obtained from human alveolar bone were cultured on porous Ti samples for periods of up to 14 days. Cell proliferation was affected by pore size at day 3 (p = 0.0010), day 7 (p = 0.0005) and day 10 (p = 0.0090) in the following way: Ti 62 < Ti 130 < Ti 312. Gene expression of bone markers evaluated at 14 days was affected, RUNX2 (p = 0.0153), ALP (p = 0.0153), BSP (p = 0.0156), COL (p = 0.0156), and OPN (p = 0.0156) by pore size as follows: Ti 312 < Ti 130 < Ti 62. Based on these results, the authors suggest that porous Ti surfaces with pore sizes near 62 mu m, compared with those of 312 mu m and 130 mu m, yield the highest expression of osteoblast phenotype as indicated by the lower cell proliferation rate and higher gene expression of bone markers.

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract Introduction Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway. Methods During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay. Results Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α. Conclusion We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.