Comparative growth,cross stress resistance,transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity

Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5.     

Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis

Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.     

The Mitogen-activated Protein (MAP) Kinases p38 and Extracellular Signal-regulated Kinase (ERK) Are Involved in Hepatocyte-mediated Phenotypic Switching in Prostate Cancer Cells

The greatest challenge for the seeding of cancer in metastatic sites is integration into the ectopic microenvironment despite the lack of an orthotopic supportive environment and presence of pro-death signals concomitant with a localized “foreign-body” inflammatory response. In this metastatic location, many carcinoma cells display a reversion of the epithelial-to-mesenchymal transition that marks dissemination in the primary tumor mass. This mesenchymal to epithelial reverting transition (MErT) is thought to help seeding and colonization by protecting against cell death. We have previously shown that hepatocyte coculture induces the re-expression of E-cadherin via abrogation of autocrine EGFR signaling pathway in prostate cancer (PCa) cells and that this confers a survival advantage. Herein, we show that hepatocytes educate PCa to undergo MErT by modulating the activity of p38 and ERK1/2. Hepatocytes inhibited p38 and ERK1/2 activity in prostate cancer cells, which allowed E-cadherin re-expression. Introduction of constitutively active MEK6 and MEK1 to DU145 cells cocultured with hepatocytes abrogated E-cadherin re-expression. At least a partial phenotypic reversion can be achieved by suppression of p38 and ERK1/2 activation in DU145 cells even in the absence of hepatocytes. Interestingly, these mitogen-activated protein kinase activities were also triggered by re-expressed E-cadherin leading to p38 and ERK1/2 activity in PCa cells; these signals provide protection to PCa cells upon challenge with chemotherapy and cell death-inducing cytokines. We propose that distinct p38/ERK pathways are related to E-cadherin levels and function downstream of E-cadherin allowing, respectively, for hepatocyte-mediated MErT and tumor cell survival in the face of death signals.     

Evolving anisotropy and degree of elastolytic insult in abdominal aortic aneurysms: Potential clinical relevance?

Accurately estimating patient-specific rupture risk remains a primary challenge in timing interventions for abdominal aortic aneurysms (AAAs). By re-analyzing published biaxial mechanical testing data from surgically repaired human AAAs, material anisotropy emerged as a potentially important determinant of patient-specific lesion progression. That is, based on a new classification scheme, we discovered that anisotropic aneurysmal specimens correlated with increased patient age at surgery when compared with more isotropic specimens (79.7 vs. 70.9 years, p<0.002), despite no significant difference in maximum diameter. Furthermore, using an idealized axisymmetric, finite-element growth and remodeling model of AAA progression, we found that both the initial axial extent of elastin loss and ongoing damage to elastin in the shoulder region of the AAA directly affected the degree of anisotropy as the lesion evolved, with more extensive insults increasing the anisotropy. This effect appeared to be mediated by alterations in axial loading and subsequent differences in orientation of deposited collagen fibers. While the observed increased age before surgical intervention may suggest a potential benefit of anisotropic remodeling, future biaxial tests coupled with pre-surgical data on expansion rates and detailed theoretical analyses of the biostability of a lesion as a function of anisotropy will be required to verify its clinical relevance to patient-specific rupture risk.     

Modeling the formation of root apices

The formation of new root apices from small groups of cells with different cellular patterns has been simulated using an existing model based on growth tensors. To generate an apex, a steady growth field was used. The pattern of cells evolved to approach the steady state. Two extreme types of progressions have been obtained : one leading to an apex with a single or a few apical cells, and the other to an apex with a quiescent centre. The change of structure while applying a steady growth tensor indicates that development may involve a succession of discrete growth tensors.     

Hydroclimatic drivers of the growth of riparian cottonwoods at the prairie margin: River flows,river regulation and the Pacific Decadal Oscillation

Cottonwoods, riparian poplars, are facultative phreatophytes and can obtain water from shallow soil moisture originating from rainfall, or from the deeper capillary fringe above the alluvial water table that is recharged by river water infiltration. The correspondence between cottonwood growth and river flows should reveal the dependency upon alluvial groundwater and subsequently, the vulnerability to reduced river flows. To explore this association, we analyzed historic growth patterns of plains cottonwoods (Populus deltoides) along the Red Deer River (RDR), which is at the northwestern limit of the North American Great Plains. We developed chronologies of yearly radial increments (RI) and basal area increments (BAI) and explored correspondences with the environmental records from the past century. In this semi-arid region, the RI or BAI were not correlated with local precipitation while negative correlation with growth season temperature (T) (r = −0.37, p < 0.01) could reflect reduced growth with hot summers. There was correlation between growth and annual river discharge (Q, and particularly log Q that approximates river stage) and this increased with two year averaging (r = 0.51, p < 0.01), reflecting carry-over in the watershed hydrology and in the ecophysiological response. There was correspondence with the Pacific Decadal Oscillation index (PDO, r = −0.45, p < 0.01), which provides multi-decade transitions that influence Rocky Mountain headwater precipitation and other weather characteristics, and river flows. The combination of Q, PDO and T provided the strongest multiple regression model, accounting for 44% of the historic growth variation (52% correspondence for 1953–2013). The RDR was dammed in 1983, enabling winter flow augmentation, but summer flows were sustained and cottonwood growth and the streamflow correspondence persisted. This indicates that it is the pattern of dam operation and not damming per se that determines the fate of established riparian cottonwoods downstream. This study revealed that these cottonwoods are phreatophytic and dependent upon alluvial groundwater that is recharged from the river. This provides a research strategy to determine whether riparian woodlands along other regulated rivers are similarly groundwater-dependent and could be vulnerable to river flow reductions from excessive water withdrawal for irrigation or other uses, or with climate change.     

Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease: A NOVEL MECHANISM FOR GPCR-EGFR TRANSACTIVATION*

Agonist stimulation of G protein-coupled receptors (GPCRs) can transactivate epidermal growth factor receptors (EGFRs), but the precise mechanisms for this transactivation have not been defined. Key to this process is the protease-mediated “shedding” of membrane-tethered ligands, which then activate EGFRs. The specific proteases and the events involved in GPCR-EGFR transactivation are not fully understood. We have tested the hypothesis that transactivation can occur by a membrane-delimited process: direct increase in the activity of membrane type-1 matrix metalloprotease (MMP14, MT1-MMP) by heterotrimeric G proteins, and in turn, the generation of heparin-binding epidermal growth factor (HB-EGF) and activation of EGFR. Using membranes prepared from adult rat cardiac myocytes and fibroblasts, we found that MMP14 activity is increased by angiotensin II, phenylephrine, GTP, and guanosine 5′-O-[γ-thio]triphosphate (GTPγS). MMP14 activation by GTPγS occurs in a concentration- and time-dependent manner, does not occur in response to GMP or adenosine 5′-[γ-thio]triphosphate (ATPγS), and is not blunted by inhibitors of Src, PKC, phospholipase C (PLC), PI3K, or soluble MMPs. This activation is specific to MMP14 as it is inhibited by a specific MMP14 peptide inhibitor and siRNA knockdown. MMP14 activation by GTPγS is pertussis toxin-sensitive. A role for heterotrimeric G protein βγ subunits was shown by using the Gβγ inhibitor gallein and the direct activation of recombinant MMP14 by purified βγ subunits. GTPγS-stimulated activation of MMP14 also results in membrane release of HB-EGF and the activation of EGFR. These results define a previously unrecognized, membrane-delimited mechanism for EGFR transactivation via direct G protein activation of MMP14 and identify MMP14 as a heterotrimeric G protein-regulated effector.     

Tuning cellular responses to BMP-2 with material surfaces

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications.     

Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-based indicators for growth and condition the method of choice?

A laboratory calibration study was undertaken with juvenile Sepia officinalis (80-85 g initial wet weight) to investigate the effects of different food rations and different starving intervals on RNA/dry weight (DW) ratios and RNA/DNA ratios in cephalopod mantle muscle at two different temperatures. The digestive gland index was also used as an additional indicator of recent growth. High food rations and low temperature went along with high RNA/DW ratios and high RNA/DNA ratios. Starving resulted in a linear decline in growth performance and a concomitant decrease in RNA/DW and RNA/DNA ratio, with RNA/DNA ratios representing the growth data better. RNA/DNA ratios decreased faster at higher temperatures. A fluorimetric assay for nucleic acid analysis was optimized for cephalopod mantle tissues and yielded reproducible RNA/DNA ratios with a relative variance below 10%. Thus, it may be possible to use this estimator of recently encountered feeding regime for the evaluation of mortality rates of early teuthid paralarvae to eventually support stock management. Also, log relative digestive gland weight showed a strong relationship with starving time, but, surprisingly, not with temperature. Data from the two temperatures analyzed could be combined to form a common regression line of relative digestive gland index with starving time. This indicator for recent growth might be especially suitable for large specimens with a well-developed digestive gland.     

Yield stability of determinate and indeterminate dry bean cultivars

Summary Yield stability of determinate and indeterminate dry bean (Phaseolus vulgaris L.) cultivars was compared using regression of genotypic performance on environmental means. Yields of 28 dry bean cultivars differing in plant growth habit and commercial class designation were obtained from 42 Michigan performance nurseries over the 6 year period 1980 to 1985. The determinate type I large-seeded kidney and cranberry bean cultivars had below-average seed yield and large mean square deviations from regression. Lower yielding determinate small-seeded navy cultivars had low deviation mean square values, while higher yielding determinate navy cultivars had correspondingly higher mean square deviations from regression. Although seed yield of cultivars with an indeterminate growth habit was greater than determinate cultivars, prostrate type III indeterminate cultivars had deviation mean square values equivalent to those of large-seeded determinate cultivars. The erect, short vine type II indeterminate cultivars (architypes) had greater than average seed yields and minimum deviations from regression. Compared with other plant types, the architype group showed a greater yield response to more productive environments, with regression coefficient values significantly greater than unity. These results indicate that the type II growth habit offers the breeder the best opportunity of obtaining greater seed yield without incurring loss of yield stability as occurs with the type I and type III growth habits. Since the dry bean cultivars utilized in this study represent two distinct centers of domestication, the regression analysis suggests that cultivars from the predominant genetic center demonstrate more yield stability. A non-significant rank correlation coefficient between the combined and separate analyses for deviation mean square values of large-seeded cultivars implies that commercial dry bean classes should be compared separately based on center of domestication.Contributions from Michigan Bean Commission, Michigan Bean Shippers Assoc. and Michigan Agric. Exp. Sta., Michigan Agric. Exp. Sta. Journal Article No. 11986