Identification of male- and female-specific olfaction genes in antennae of the oriental fruit fly (Bactrocera dorsalis)

The oriental fruit fly (Bactrocera dorsalis) is a species of tephritid fruit fly, endemic to Southeast Asia but also introduced to many regions of the US, and it is one of the major pest species with a broad host range of cultivated and wild fruits. Although males of B. dorsalis respond strongly to methyl eugenol and this is used for monitoring and estimating populations, the molecular mechanism of the oriental fruit fly olfaction has not been elucidated yet. Therefore, in this project, using next generation sequencing technologies, we sequenced the transcriptome of the antennae of male and female adults of B. dorsalis. We identified a total of 20 candidate odorant binding proteins (OBPs), 5 candidate chemosensory proteins (CSPs), 35 candidate odorant receptors (ORs), 12 candidate ionotropic receptors (IRs) and 4 candidate sensory neuron membrane proteins (SNMPs). The sex-specific expression of these genes was determined and a subset of 9 OR genes was further characterized by qPCR with male and female antenna, head, thorax, abdomen, leg and wing samples. In the male antennae, 595 genes showed a higher expression, while 128 genes demonstrated a higher expression in the female antennae. Interestingly, 2 ORs (BdorOR13 and BdorOR14) were highly and specifically expressed in the antennae of males, and 4 ORs (BdorOR13, BdorOR16, BdorOR18 and BdorOR35) clustered with DmOR677, suggesting pheromone reception. We believe this study with these antennae-enriched OBPs, CSPs, ORs, IRs and SNMPs can play an important role in the detection of pheromones and general odorants, and so in turn our data improve our current understanding of insect olfaction at the molecular level and provide important information for disrupting the behavior of the oriental fruit fly using chemical communication methods

Functional impairment of human resident cardiac stem cells by the cardiotoxic antineoplastic agent trastuzumab

Trastuzumab (TZM), a monoclonal antibody against the ERBB2 protein, increases survival in ERBB2-positive breast cancer patients. Its clinical use, however, is limited by cardiotoxicity. We sought to evaluate whether TZM cardiotoxicity involves inhibition of human adult cardiac-derived stem cells, in addition to previously reported direct adverse effects on cardiomyocytes. To test this idea, we exposed human cardiosphere-derived cells (hCDCs), a natural mixture of cardiac stem cells and supporting cells that has been shown to exert potent regenerative effects, to TZM and tested the effects in vitro and in vivo. We found that ERBB2 mRNA and protein are expressed in hCDCs at levels comparable to those in human myocardium. Although clinically relevant concentrations of TZM had no effect on proliferation, apoptosis, or size of the c-kit-positive hCDC subpopulation, in vitro assays demonstrated diminished potential for cardiogenic differentiation and impaired ability to form microvascular networks in TZM-treated cells. The functional benefit of hCDCs injected into the border zone of acutely infarcted mouse hearts was abrogated by TZM: infarcted animals treated with TZM + hCDCs had a lower ejection fraction, thinner infarct scar, and reduced capillary density in the infarct border zone compared with animals that received hCDCs alone (n = 12 per group). Collectively, these results indicate that TZM inhibits the cardiomyogenic and angiogenic capacities of hCDCs in vitro and abrogates the morphological and functional benefits of hCDC transplantation in vivo. Thus, TZM impairs the function of human resident cardiac stem cells, potentially contributing to TZM cardiotoxicity

Characterization of cold shock domain proteins and SUMOylation system from Oryza sativa

In the first part of this study, two novel c&barbelow;old s&barbelow;hock domain p&barbelow;roteins from rice (OsCSP) were cloned and subsequently characterized their roles during stress conditions and development. OsCSP1 and OsCSP2 ( Oryza sativa CSD protein) encode putative proteins consisting of an N-terminal CSD and glycine-rich regions that are interspersed by 4 and 2 CX2CX4HX4C (CCHC) retroviral-like zinc fingers, respectively. Using an in vitro DNA binding assay, I demonstrate that OsCSPs exhibit conserved ssDNA binding activity. In vivo functional complementation in a cold-sensitive bacterial strain, that lacks four cold inducible cold shock domain proteins revealed that OsCSPs function as RNA chaperones, similar to their bacterial and winter wheat counterparts. To understand the functions of these genes in rice, I studied the transcriptional regulation in response to abiotic stress conditions. Under cold stress, OsCSP transcript levels are only transiently and marginally increased and the encoded proteins did not accumulate. These transcript and protein data are in sharp contrast with the observed data for winter wheat and Arabidopsis cold shock domain proteins under cold stress. In these species, both transcripts and protein levels of CSPs are increased upon cold stress. Based on these data, it can be hypothesized that the accumulation of cold shock domain proteins may play an important role in determining the cold acclimation capability of the plants. Expression analysis at the protein and RNA levels during development revealed that OsCSPs are highly expressed in the reproductive and meristematic tissues. These results indicate a potential role for rice cold shock domain proteins in plant growth and reproductive development.;In this study, I also characterized the post-translational modification of plant cold shock domain proteins by SUMOylation. Post-translational modifications can impart rapid changes in protein function. SUMOylation involves the reversible attachment of a small protein called SUMO (small ubiquitin-like modifier) to target proteins. The SUMO protein has a similar three dimensional structure as that of ubiquitin and the process of SUMOylation is very similar to that of ubiquitination. However, unlike ubiquitination, SUMOylation is not implicated in protein degradation. SUMO modification can affect the target protein stability, sub-cellular localization protein-protein interactions. Using a computational approach on rice and Arabidopsis cold shock domain proteins, I identified canonical SUMOylation motifs in both rice CSPs and one of the Arabidopsis CSPs. Using in vitro assays, I demonstrated that both OsCSPs can undergo SUMOylation. Using mutational approaches, I identified an important lysine residue for SUMOylation in Arabidopsis AtCSP1. By employing GFP-tagged wild-type and variant AtCSP1 proteins, I demonstrate that SUMOylation appears to affect AtCSP1 protein localization.;In another study, I characterized the entire SUMO conjugation system in rice. The process of SUMOylation involves a cascade of enzymatic reactions involving activation (E1) enzymes, conjugation (E2) enzymes and ligation (E3) enzymes. I compared the protein sequences of all these genes from rice with those from Arabidopsis, yeast and human. This revealed a high amino acid sequence conservation of individual SUMOylation components from yeast to plants and animals. In Arabidopsis, the SUMOylation system has been implicated in plant development and in mediating abiotic stress responses. To understand the role of the rice SUMOylation system during development, I studied the SUMO conjugate profiles and the expression of individual SUMO component genes in various tissues at different stages of plant development. The highest levels of SUMOylated proteins were observed in panicles and meristematic tissues. Expression studies revealed that SUMO component genes are highly expressed in reproductive tissues like developing seeds and panicles. Together, these data implicate an important role for the rice SUMOylation system in plant growth and reproductive development. To understand the role of SUMOylation system in rice, I studied SUMO conjugate profiles and the transcriptional regulation of individual SUMO components during cold, salt and ABA stress conditions. Rice responds to these stresses by accumulating SUMO conjugated proteins, suggesting that protein SUMOylation helps to mediate plant stress responses. Studies on the transcriptional regulation of individual SUMO pathway genes during these stress conditions revealed that most are transcriptionally down-regulated. However, a particular SUMO E3 ligase gene (OsSIZ2) is transiently up-regulated upon exposure to all three stress conditions. Considering the importance of E3 ligases in improving the efficiency and specificity of the SUMO conjugation reactions, OsSIZ2 may mediate accumulation of SUMO conjugates during these stress conditions. Taken together, these data suggest a role for SUMOylation in rice development and stress responses. (Abstract shortened by UMI.)

PRINCIPLES OF COMPLEX POLYUBIQUITIN SIGNALING, THE STRUCTURAL BASIS FOR UBIQUITIN-UBISTATIN INTERACTIONS, AND NOVEL ASSAYS FOR THE CHARACTERIZATION OF DEUBIQUITINASES

Ubiquitination is the most versatile and is certainly one of the most difficult post-translation modifications to understand in eukaryotic life. In the process of ubiquitination the C-terminus of ubiquitin (Ub), a small 8.65 kDa protein is covalently attached to εNH2 groups of lysine side chains on target proteins. Once attached, additional Ubs can be added to the original Ub at eight unique linkage sites (M1, K6, K11, K27, K29, K33, K48, or K63) to form polyUb chains. This internal Ub-Ub linkage dictates the structural conformation of the polyUb chain, which in turn governs the receptors that can recognize a given chain. PolyUb chains were thought to be homogeneously linked until very recently when mixed linkage polyUb chains were detected on several cellular pathways. This observation implied that instead of having just eight distinct polyUb signals, there were now potentially quadrillions of unique chains. The results presented within represent the first in depth studies of mixed linkage polyUb chains, focusing on the structural impact of linkage mixing. For mixed K48 and K63 linked chains the findings support that their individual linkage properties are preserved regardless of linkage mixing. However, simulations for mixed linkage chains containing different linkages imply that many novel polyUb signals are possible. The ubiquitin-proteasome pathway is the primary mechanism to degrade short lived proteins in the cell and has also emerged as a top therapeutic target. Ubistatins, a class of small molecules bring about the same effects as existing proteasome inhibitor drugs by directly binding the polyUb chain. However, virtually nothing is known about the structural properties for any ubistatin/Ub complex. Here is provided the first structure of a ubistatin/Ub complex along with data that overwhelmingly validates the structure. Other important factors regarding the ubistatin/Ub interaction including the stoichiometry and dual hydrophobic / electrostatic binding mechanism are also uncovered. Proteomic analysis of polyUb conjugates has been limited to determining which linkage types are present. A novel method for K63 linked polyUb conjugates, which can measure consecutive K63 linkages is described here. This method allows the proteomics community to gain unprecedented information on cellular pathways utilizing K63 linkages

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme

Metrics for Broadband Networks in the Context of the Digital Economies

In a transition to automated digital management of broadband networks, communication service providers must look for new metrics to monitor these networks. Complete metrics frameworks are already emerging whereas majority of the new metrics are being proposed in technical papers. Considering common metrics for broadband networks and related technologies, this chapter offers insights into what metrics are available, and also suggests active areas of research. The broadband networks being a key component of the digital ecosystems are also an enabler to many other digital technologies and services. Reviewing first the metrics for computing systems, websites and digital platforms, the chapter focus then shifts to the most important technical and business metrics which are used for broadband networks. The demand-side and supply-side metrics including the key metrics of broadband speed and broadband availability are touched on. After outlining the broadband metrics which have been standardized and the metrics for measuring Internet traffic, the most commonly used metrics for broadband networks are surveyed in five categories: energy and power metrics, quality of service, quality of experience, security metrics, and robustness and resilience metrics. The chapter concludes with a discussion on machine learning, big data and the associated metrics

Genomic and transcriptomic analyses reveal distinct biological functions for cold shock proteins (<i>Vpa</i>CspA and <i>Vpa</i>CspD) in <i>Vibrio parahaemolyticus</i> CHN25 during low-temperature survival

Abstract Background Vibrio parahaemolyticus causes serious seafood-borne gastroenteritis and death in humans. Raw seafood is often subjected to post-harvest processing and low-temperature storage. To date, very little information is available regarding the biological functions of cold shock proteins (CSPs) in the low-temperature survival of the bacterium. In this study, we determined the complete genome sequence of V. parahaemolyticus CHN25 (serotype: O5:KUT). The two main CSP-encoding genes (VpacspA and VpacspD) were deleted from the bacterial genome, and comparative transcriptomic analysis between the mutant and wild-type strains was performed to dissect the possible molecular mechanisms that underlie low-temperature adaptation by V. parahaemolyticus. Results The 5,443,401-bp V. parahaemolyticus CHN25 genome (45.2% G + C) consisted of two circular chromosomes and three plasmids with 4,724 predicted protein-encoding genes. One dual-gene and two single-gene deletion mutants were generated for VpacspA and VpacspD by homologous recombination. The growth of the ΔVpacspA mutant was strongly inhibited at 10 °C, whereas the VpacspD gene deletion strongly stimulated bacterial growth at this low temperature compared with the wild-type strain. The complementary phenotypes were observed in the reverse mutants (ΔVpacspA-com, and ΔVpacspD-com). The transcriptome data revealed that 12.4% of the expressed genes in V. parahaemolyticus CHN25 were significantly altered in the ΔVpacspA mutant when it was grown at 10 °C. These included genes that were involved in amino acid degradation, secretion systems, sulphur metabolism and glycerophospholipid metabolism along with ATP-binding cassette transporters. However, a low temperature elicited significant expression changes for 10.0% of the genes in the ΔVpacspD mutant, including those involved in the phosphotransferase system and in the metabolism of nitrogen and amino acids. The major metabolic pathways that were altered by the dual-gene deletion mutant (ΔVpacspAD) radically differed from those that were altered by single-gene mutants. Comparison of the transcriptome profiles further revealed numerous differentially expressed genes that were shared among the three mutants and regulators that were specifically, coordinately or antagonistically modulated by VpaCspA and VpaCspD. Our data also revealed several possible molecular coping strategies for low-temperature adaptation by the bacterium. Conclusions This study is the first to describe the complete genome sequence of V. parahaemolyticus (serotype: O5:KUT). The gene deletions, complementary insertions, and comparative transcriptomics demonstrate that VpaCspA is a primary CSP in the bacterium, while VpaCspD functions as a growth inhibitor at 10 °C. These results have improved our understanding of the genetic basis for low-temperature survival by the most common seafood-borne pathogen worldwide

Fundamental structural and biochemical features for the obestatin/GPR39 system mitogenic action

In 2005, a new peptide derived from the ghrelin peptide precursor, named obestatin, was discovered. One of the most important functions of obestatin is its mitogenic activity. Obestatin and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; this fact prompted us to investigate obestatin/GPR39 signalling and the relationship between the system and gastric cancer progression, and explored their potential functional roles. The main objective of this doctoral thesis is to establish the relationship between obestatin and GPR39 receptor in healthy and tumour-like surroundings, from structural to tissue level, to determinate the fundamental parameters of its mitogenic bioactivity. This issue will be accomplished with the following points: 1. To determine the structural features of obestatin required for the interaction with its receptor. 2. To elucidate the detailed activation/regulation mechanism of GPR39 receptor signalling triggered by obestatin. 3. To analyse the role of obestatin/GPR39 system in the development and malignity of tumours. Our results demonstrated that: At structural level, the amidation at the C-terminus is essential to adopt an α-helix structure and stabilize the GPR39 conformations necessary for the full range of receptor activities. Furthermore, human (11-23)-obestatin is able to induce selective coupling to the β-arrestin-dependent signalling, representing the first example of an endogenous biased ligand for GPR39. Meanwhile, mouse and human obestatin exhibit clear conformational differences beyond their primary structure. This evidence supports the species-specific activity of this peptide. Additionally, obestatin-GPR39 interaction might involve an E/Z isomerization of the peptide and the posibility that GPR39 could be acting as a prolyl cis-trans isomerase. Regarding the activation/regulation mechanism of GPR39 signalling triggered by obestatin, our results show that obestatin increases GPR39 phosphorylation and induces receptor endocytosis. In this signalling network, the transactivation process induced by obestatin GPR39-EGFR is a key mechanism, regulated by MMPs. The RTKs and proteases expression profiles confirm the implication of EGFR and MMPs in the obestatin signalling pathway, and introduce other proteases and RTKs in this cross-talk. In human tissues, we observe that the obestatin/GPR39 system regulates pepsinogen secretion. This result provide the first biological function for the obestatin/GPR39 system in healthy stomach. This system also regulates proliferation, motility, EMT, and invasion of gastric cancer cells. More importantly, the GPR39 expression levels found in human gastric adenocarcinomas provide the rationale for including GPR39 as a prognostic marker of these tumours. The ubiquitous expression of GPR39 and its cancer-associated overexpression, together with obestatin, provokes the proliferation and cell motillity of diverse human cancer cell lines. Moreover, these effects depend not only on GPR39, but also on the expression of key components of its signalling pathway, i.e. RTKs, proteases