Differentiation of Solenopsis invicta social forms using high resolution melt PCR.

Solenopsis invicta Buren (red imported fire ant) are invasive pests that have the capability of major destructive impacts on lifestyle, ecology and economy. Control of this species is dependent, in part, upon ability to estimate the potential spread from newly discovered nests. The potential for spread and the spread characteristics differ between monogyne and polygyne social forms. Prior to this study, differentiation of the two social forms in laboratory test samples commonly used a method involving restriction endonuclease digestion of an amplified Gp-9 fragment. Success of this assay is limited by the quality of DNA, which in the field-collected insects may be affected by temporary storage in unfavourable conditions. Here, we describe an alternative and highly objective assay based upon a high resolution melt technique following preamplification of a significantly shorter Gp-9 fragment than that required for restriction endonuclease digestion. We demonstrate the application of this assay to a S. invicta incursion in Queensland, Australia, using field samples from which DNA may be partially degraded. The reductions in hands-on requirements and overall duration of the assay underpin its suitability for high-throughput testing.

Stanniocalcin-1 in cell stress and differentiation

Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.

Proinflammatory cytokines regulate cementogenic differentiation of periodontal ligament cells by Wnt/Ca2+ signaling pathway

Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca2+ signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca2+ signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca2+ signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca2+ pathway was blocked by Ca2+/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca2+ pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca2+ pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases.

Differentiation of Indian, East Timorese, Papuan and Floridian Candidatus Liberibacter asiaticus' isolates on the basis of simple sequence repeat and single nucleotide polymorphism profiles at 25 loci

Japanese isolates of Candidatus Liberibacter asiaticus have been shown to be clearly differentiated by simple sequence repeat (SSR) profiles at four loci. In this study, 25 SSR loci, including these four loci, were selected from the whole-genome sequence and were used to differentiate non-Japanese samples of Ca. Liberibacter asiaticus (13 Indian, 3 East Timorese, 1 Papuan and 8 Floridian samples). Out of the 25 SSR loci, 13 were polymorphic. Dendrogram analysis using SSR loci showed that the clusters were mostly consistent with the geographical origins of the isolates. When single nucleotide polymorphisms (SNPs) were searched around these 25 loci, only the upstream region of locus 091 exhibited polymorphism. Phylogenetic tree analysis of the SNPs in the upstream region of locus 091 showed that Floridian samples were clustered into one group as shown by dendrogram analysis using SSR loci. The differences in nucleotide sequences were not associated with differences in the citrus hosts (lime, mandarin, lemon and sour orange) from which the isolates were originally derived.

Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc), causal agent of fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. The development of a molecular diagnostic capable of reliably distinguishing between the various races of the pathogen is of key importance to disease management. However, attempts to distinguish isolates using the standard molecular loci typically used for fungal phylogenetics have been complicated by a poor correlation between phylogeny and pathogenicity. Among the available alternative loci are several putative effector genes, known as SIX genes, which have been successfully used to differentiate the three races of F. oxysporum f. sp. lycopersici. In this study, an international collection of Foc isolates was screened for the presence of the putative effector SIX8. Using a PCR and sequencing approach, variation in Foc-SIX8 was identified which allowed race 4 to be differentiated from race 1 and 2 isolates, and tropical and subtropical race 4 isolates to be distinguished from one another.

Natural Abundant Solid State NMR Studies in Designed Tripeptides for Differentiation of Multiple Conformers

Solid state NMR (SSNMR) experiments on heteronuclei in natural abundance are described for three synthetically designed tripeptides Piv-(L)Pro_(L)Pro-(L)Phe-OMe (1), Piv-(D)Pro_(L)Pro_(L)Phe-OMe (2), and Piv-(D)Pro_(L)Pro_(L)Phe-NHMe (3). These peptides exist in different conformation as shown by solution state NMR and single crystal X-ray analysis (Chatterjee et al., Chem Eur J 2008, 14, 6192). In this study, SSNMR has been used to probe the conformations of these peptides in their powder form. The C-13 spectrum of peptide (1) showed doubling of resonances corresponding to cis/cis form, unlike in solution where the similar doubling is attributed to cis/trans form. This has been confirmed by the chemical shift differences of C-beta and C-gamma carbon of Proline in peptide (1) both in solution and SSNMR. Peptide (2) and (3) provided single set of resonances which represented all transform across the di-Proline segment. The results are In agreement with the X-ray analysis. Solid state N-15 resonances, especially from Proline residues provided additional information, which is normally not observable in solution state NMR. H-1 chemical shifts are also obtained from a two-dimensional heteronuclear correlation experiment between H-1-C-13. The results confirm the utility of NMR as a useful tool for identifying different conformers in peptides in the solid state. (C) 2009 Wiley Periodicals, Inc. Biopolymers 91: 851-860, 2009.

Solutions of Boundary Layer Equations Governing Free Convective and Magnetohydrodynamic Flows through Parametric Differentiation

In der vorliegenden Arbeit wird die Methode der parametrischen Differentiation angewendet, um ein System nichtlinearer Gleichungen zu lösen, das zwei- und dreidimensionale freie, konvektive Grenzschichströmungen bzw. eine zweidimensionale magnetohydrodynamische Grenzschichtströmung beherrscht. Der Hauptvorteil dieser Methode besteht darin, daß die nichlinearen Gleichungen auf lineare reduziert werden und die Nichtlinearität auf ein System von Gleichungen erster Ordnung beschränkt wird, das, verglichen mit den ursprünglichen Nichtlinearen Gleichungen, viel leichter gelöst werden kann. Ein anderer Vorzug der Methode ist, daß sie es ermöglicht, die Lösung von einer bekannten, zu einem bestimmten Parameterwert gehörigen Lösung aus durch schrittweises Vorgehen die Lösung für den gesamten Parameterbereich zu erhalten. Die mit dieser Methode gewonnenen Ergebnisse stimmen gut mit den entsprechenden, mit anderen numerischen Verfahren erzielten überein.

A parametric differentiation version with finite-difference scheme applicable to a class of problems in boundary layer flow with massive blowing

A numerical procedure, based on the parametric differentiation and implicit finite difference scheme, has been developed for a class of problems in the boundary-layer theory for saddle-point regions. Here, the results are presented for the case of a three-dimensional stagnation-point flow with massive blowing. The method compares very well with other methods for particular cases (zero or small mass blowing). Results emphasize that the present numerical procedure is well suited for the solution of saddle-point flows with massive blowing, which could not be solved by other methods.

Genetic differentiation analysis for the identification of complementary parental pools for sorghum hybrid breeding in Ethiopia

Key message The potential for exploiting heterosis for sorghum hybrid production in Ethiopia with improved local adaptation and farmers preferences has been investigated and populations suitable for initial hybrid development have been identified. Abstract Hybrids in sorghum have demonstrated increased productivity and stability of performance in the developed world. In Ethiopia, the uptake of hybrid sorghum has been limited to date, primarily due to poor adaptation and absence of farmer’s preferred traits in existing hybrids. This study aimed to identify complementary parental pools to develop locally adapted hybrids, through an analysis of whole genome variability of 184 locally adapted genotypes and introduced hybrid parents (R and B). Genetic variability was assessed using genetic distance, model-based STRUCTURE analysis and pair-wise comparison of groups. We observed a high degree of genetic similarity between the Ethiopian improved inbred genotypes and a subset of landraces adapted to lowland agro-ecology with the introduced R lines. This coupled with the genetic differentiation from existing B lines, indicated that these locally adapted genotype groups are expected to have similar patterns of heterotic expression as observed between introduced R and B line pools. Additionally, the hybrids derived from these locally adapted genotypes will have the benefit of containing farmers preferred traits. The groups most divergent from introduced B lines were the Ethiopian landraces adapted to highland and intermediate agro-ecologies and a subset of lowland-adapted genotypes, indicating the potential for increased heterotic response of their hybrids. However, these groups were also differentiated from the R lines, and hence are different from the existing complementary heterotic pools. This suggests that although these groups could provide highly divergent parental pools, further research is required to investigate the extent of heterosis and their hybrid performance.

RNA polymerase activity in isolated nuclei of Nicotiana sanderae callus: Characteristics and modulation during differentiation

Isolated nuclei from differentiating cultures of Nicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg2+ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH4)2 SO4 and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.

Otolith morphology of four mackerel species (Scomberomorus spp.) in Australia: Species differentiation and prediction for fisheries monitoring and assessment

Four species of large mackerels (Scomberomorus spp.) co-occur in the waters off northern Australia and are important to fisheries in the region. State fisheries agencies monitor these species for fisheries assessment; however, data inaccuracies may exist due to difficulties with identification of these closely related species, particularly when specimens are incomplete from fish processing. This study examined the efficacy of using otolith morphometrics to differentiate and predict among the four mackerel species off northeastern Australia. Seven otolith measurements and five shape indices were recorded from 555 mackerel specimens. Multivariate modelling including linear discriminant analysis (LDA) and support vector machines, successfully differentiated among the four species based on otolith morphometrics. Cross validation determined a predictive accuracy of at least 96% for both models. An optimum predictive model for the four mackerel species was an LDA model that included fork length, feret length, feret width, perimeter, area, roundness, form factor and rectangularity as explanatory variables. This analysis may improve the accuracy of fisheries monitoring, the estimates based on this monitoring (i.e. mortality rate) and the overall management of mackerel species in Australia.

A deadline-constrained 802.11 MAC Protocol with QoS differentiation for soft real-time control

As one of the most widely used wireless network technologies, IEEE 802.11 wireless local area networks (WLANs) have found a dramatically increasing number of applications in soft real-time networked control systems (NCSs). To fulfill the real-time requirements in such NCSs, most of the bandwidth of the wireless networks need to be allocated to high-priority data for periodic measurements and control with deadline requirements. However, existing QoS-enabled 802.11 medium access control (MAC) protocols do not consider the deadline requirements explicitly, leading to unpredictable deadline performance of NCS networks. Consequentially, the soft real-time requirements of the periodic traffic may not be satisfied, particularly under congested network conditions. This paper makes two main contributions to address this problem in wireless NCSs. Firstly, a deadline-constrained MAC protocol with QoS differentiation is presented for IEEE 802.11 soft real-time NCSs. It handles periodic traffic by developing two specific mechanisms: a contention-sensitive backoff mechanism, and an intra-traffic-class QoS differentiation mechanism. Secondly, a theoretical model is established to describe the deadline-constrained MAC protocol and evaluate its performance of throughput, delay and packet-loss ratio in wireless NCSs. Numerical studies are conducted to validate the accuracy of the theoretical model and to demonstrate the effectiveness of the new MAC protocol.

N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.