text:
> Inline year selector with callbacks, drag and drop and range selection. WIP.

## Demo

[Live Demo](http://arthurianx.github.io/angular-translationinput/demo)

## Getting Started

Install with Bower or download the files directly from the dist folder in the repo.

```bash
bower install agular-translationInput --save
```

Add `dist/angular-translationinput.js` and `dist/angular-translationinput.css` to your index.html.

*IMPORTANT*: At this point, `translation-input` depends on `angular-dragula` and `dragula`. Please add it as a dependency.

Add `translationInput` as a module dependency for your module.

```js
angular.module('your_app', ['translationInput']);
```

## Options

The `translation-input` directive, by default, expects only the model object, which will give you the selected date or range

You can use it like this:

```html
<div translation-input="{model: 'myModel'}"></div>
```
*IMPORTANT*: The library is dependent on many third part libs, so at this time it is not feasible. That's why the WIP.

There's also a full set of options:

* `something` - Optional.  Defaults to `10` years. You can input a number of years from which you can select or a scope variable with the length you need. Mind you, if you put 40 years in a really small container it won't look good. This is a TODO.

A full usage would look like this: `<div translation-input="{model: 'myModel', years: 15, attachTo: '.mySelector', attachNow: triggerAttachment, externalCallback: updateYearSelection, range: false, drag: false}"></div>`

## Release History
 * v0.1.0 - Initial release.


text:
My Family Tree
Climbing the branches of my family tree
Person Page 228
Main Page - Master Index - Surname Index - Master Place Index - Home - Family Tree DNA
Previous Page - Next Page
King of Neustria Theuderic III (?) (M)
b. 654, d. 691
Pop-up Pedigree
King of Neustria Theuderic III (?) was born in 654. He was the son of King of Neustria and Burgundy Clovis II (?) and Balthild (?). King of Neustria Theuderic III (?) married Clotilda (?), daughter of Ansgise (Ansegisel) (?) Duke and Saint Begga (?). King of Neustria Theuderic III (?) died in 691.
Immigrant=N
Related_By=A
Children of King of Neustria Theuderic III (?) and Clotilda (?)
Bertrada of Prüm (?)+ b. c 670, d. a 721
Clovis IV (?) b. 682, d. 695
Childebert III (?) b. 683, d. 23 Apr 711
Thomas (?) (M)
He married Irene Josephine Clifton.
Thomas (?) was the son of Llewelyn (?) and Eleanor de Barr. Thomas (?) married Eleanor (?), daughter of Philip (?) and Catharine (?).
Child of Thomas (?) and Eleanor (?)
Eleanor (?)+
Thomas (?)1 (M)
Thomas (?) was the son of David Gam.
Child of Thomas (?)
Gwenhwyfar (?)+
[S175] Howard William Lloyd, Lloyd Manscripts by Howard William Lloyd, published in 1896..
Thomas (?) was the son of Maurice (?).
John (?)+
Duke of Gloucester Thomas of Woodstock (?) (M)
b. 1347, d. 1348
Duke of Gloucester Thomas of Woodstock (?) was born in 1347. He was the son of King of England Edward III (?) and Phillippa of Hainault (?). Duke of Gloucester Thomas of Woodstock (?) died in 1348.
Related_By=C
Tracy (?) (F)
Tracy (?) married Noah Rinehart.
Related_By=U
Child of Tracy (?) and Noah Rinehart
Fernando Rinehart+ b. 1869, d. 1941
Trudy (?) (F)
She married Samuel J. Fauver Jr..
Related_By=M
Trudy (?) married Jerry Cunningham.
Tudoc (?) (M)
Tudoc (?) was the son of Ithel (?).
Child of Tudoc (?)
Tangwell (?)+
If you have any additions, comments or suggestions
This page was created by John Cardinal's Second Site v1.9.9.
Site updated on 1 Sep 2008 at 9:21:08 AM from my project; 63,585 people

redpajama_set_name: RedPajamaCommonCrawl


text:
Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection
Qian Yu1,
Youhua Xiong1,
Hang Gao1,
Jianliang Liu1,
Zhiqiang Chen1,
Qin Wang1 &
Dongling Wen1
Virology Journal volume 12, Article number: 115 (2015) Cite this article
Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood.
In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques.
A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component.
The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.
Baculoviruses are a group of arthropod-specific viruses with rodshaped nucleocapsids of 30–60 nm × 250-300 nm, and they are a family of virus that is quite beneficial to humankind [1–3]. Baculovirus genomes consist of a circular double-stranded DNA (dsDNA) molecule of about 80–180 kb [4, 5]. There are several hundred baculoviruses that have been described in the literature which are highly specific for closely related groups of insects or crustaceans [6, 7]. Due to their restriction to host range and their ability to kill insects, baculoviruses have been used as microbial insecticides to control insect pests in agriculture and forestry [8, 9]. In the 1980s, they were developed as a powerful eukaryotic protein expression vector due to the extremly high expressions of viral gene in host cells [10].
Baculoviruses are classified into two genera, the nucleopolyhedroviruses (NPVs) and the granuloviruses (GVs); NPVs can be further subdivided into groups I and II based on phylogenetic studies of many genes [1]. Although there are many different baculovirus isolates, the best studied and the most widely used member of this family is Autographa californica Nucleopolyhedrovirus, a nuclear polyhedrosis virus (AcMNPV) [11]. AcMNPV is a large enveloped virus with a double-stranded circular DNA genome of about 130 kb. The complete sequence of AcMNPV has been determined [12]. AcMNPV infects a few insect lepidopteron species and it can be propagated easily in a cell line derived from the fall armyworm Spodoptera frugiperda (Sf), particularly the Sf9 clonal isolate [13].
The baculovirus infection cycle has been well studied. During the infection cycle, two progeny virion phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV), are produced. The genotype of BV and ODV are identical, but each has characteristic structural components to accommodate its respective functions. The molecular life cycle is broadly divided into three consecutive phases according to gene expression programming [14]. In the early stage (0–6 h post-infection (hpi)), the nucleocapsids are transported through the nuclear membranes and migrate across the cytosol to the cell membrane. Genes requiring the transcriptional activity of the cell-encoded RNA polymerase II mainly act as master transactivators, which are essential for both subsequent viral gene expression and subversion of host cell activity by performing tasks common to other DNA viruses, including cell cycle arrest. Blocking apoptosis by the viral protein P35 is also an activity that is required to establish productive infections [15]. In the late stages of the infection (6–18 hpi), the viral DNA begins to replicate and the nucleocapsids within the nucleus are enveloped with a lipid bilayer that resembles the inner nuclear membrane. The transition from early to late phase is marked by the onset of replication and the activity of a virus-encoded, α–amanitin-resistant RNA polymerase. Concomitant with the onset of the very late phase (18 hpi-), host transcriptional activity is significantly decreased and most protein synthesis is shut off of by 24 hpi.
During the last decade, 68 baculovirus genomes including AcMNPV, the most widely used BV, have been sequenced, offering a wealth of information on the genetic diversity, gene sequences, gene content, genome organisation and phylogeny of baculovirus genomes [4, 12, 16, 17]. In comparison, the proteome of AcMNPV-susceptible cell lines have not been fully sequenced which is not helpful to understanding the responses of AcMNPV-susceptible cell lines during infection, since better understanding of the host cell responses would allow for the rational design of strategies for bioprocess optimization. Nuno Carinhas and colleagues obtained the comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection (6 hpi) using stable isotope labelling by amino acids in cell culture [14].
In the present study, we set out to investigate the proteome changes in Sf9 cells infected with AcMNPV. In contrast to previous study by Nuno Carinhas et al. [14], this study centred on the differentially expressed proteins among Sf9 cells of mock, 6 hpi and 12 hpi which allows identification and measurements of thousands of proteins. The findings of these proteins improve our understanding of host-virus interactions and the host response upon virus infection, and hopefully, engender support in further studies regarding optimization of baculovirus application.
Identification of differentially expressed proteins among mock, Sf9 cells at 6 hpi and Sf9 cells at 12 hpi
In the current study, Sf9 cells at resting, Sf9 infected with AcMNPV for 6 h and 12 h were collected for protein extraction, digestion and iTRAQ labelling to provide more information about proteome in AcMNPV infection. In this study, Sf9 cells at different phases of infection were collected to globally characterise the expression changes of Sf9 cell proteins associated with AcMNPV infection (Sf9 cells at resting, Sf9 cells at 6 hpi and Sf9 cells at 12 hpi) and a total of 4004 proteins were present in at least two of the three biological groups. Among the statistically significant proteins detected by ANOVA test (P < 0.05), the redundancies that changed less than 1.5-fold were discarded. Following this criterion, a total of 413 Sf9 cell proteins were found as being significantly differentially abundant under at least two repeated identifications and quantifications (P < 0.05) (Additional file 1: Table S1).
GO annotation of the differentially abundant proteins
The Blast2GO (Version 2.7.2) uses BLAST to find homologous sequences to input sequences and extract GO terms (Gene Ontology) for each obtained hit using existing annotations. These GO terms are assigned to the query sequence resulting in an assessment of the biological process, the molecular function and the cellular compartments represented. In this case, a GO category enrichment analysis was conducted using the 413 most abundant proteins to represent the overall trends of the specific functional categories that are enriched in Sf9 cells of different infection stages. Here, 292 proteins (70.70%) from Sf9 cells with 1156 annotation terms were linked to the GO cellular component, molecular function and biological process categories (Fig. 1).
GO terms distribution. GO terms distribution in the molecular functions (a), cellular component (b), and biological process (c). In (a), binding and catalytic activity were the most represented biological processes. In (b), the most represented categories were cell, followed by organelle. In (c), the most represented categories were metabolic process and cellular process
The biological process category refers to a biological objective to which a gene contributes, but does not identify pathways. Examples of such processes include metabolism, cell communication, and sexual reproduction. In this case, the majority of proteins were found in cellular process (29.1 %), metabolic process (28.4 %), biological regulation (10.2 %) and single organism (10.1 %). Molecular function is defined as "what a gene product does at the biochemical level". It is a very narrow definition, as it does not take into consideration the location of the event or the function in a broader, pathway or network context. In this category, most of the differential proteins were involved in binding (50.3 %), catalytic activity (37 %), structural molecule activity (5.1 %) and transport activity (3.2 %) (Fig. 1B). The binding functions were mainly at the intracellular level rather than external and included nucleotide and nucleic acid binding, protein binding and ion binding, while catalytic activities included transferase and hydrolase activities. The final category, cellular component, identifies where the gene products are found in the cell. These range from a general placement, such as in the "cell membrane" to more specific, such as the "histone deacteylase complex". Here the differentially abundant proteins were mostly related to cellular components in the intracellular space (36.4 %), organelle (26.5 %), macromolecular complex (25.1 %), membrane (7.7 %) and membrane-enclosed lumen (3.2 %) (Fig. 1c).
KAAS ----KEGG automatic annotation server
The KEGG pathway is a collection of manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks. In this study, proteins of Sf9 cells at different infection stages were aligned with insect protein sequences in the KEGG GENES database using KAAS (KEGG Automatic Annotation Server) and annotated to biochemical pathways through the KO number of the homologous/similar proteins. We established pathway associations for 217 differential proteins with 207 unique KEGG orthologues. The most represented KEGG maps were protein processing in endoplasmic reticulum (22 members), carbon metabolism (21 members), ribosome (20 members), RNA transport (19 members), Huntington's disease (17 members), Parkinson's disease (13 members), Alzheimer's disease (12 members), oxidative phosphorylation (12 members), glycolysis/gluconeogenesis (11 members), biosynthesis of amino acids (11 members) and the spliceosome (10 members) (Fig. 2). Several proteins/enzymes related to protein processing in the endoplasmic reticulum, carbon metabolism and ribosome were significantly up- or down-regulated after AcMNPV infection (Table 1).
The most represented KEGG maps
Table 1 The significantly differentially expressed proteins in protein processing in endoplasmic reticulum, carbon metabolism and ribosome
For protein processing in the endoplasmic reticulum, AcMNPV is entirely dependent on the Sf9 cell translational machinery. We found that the expression of Calreticulin, EIF2α, Heat shock protein 90β and glycoprotein glucosyltransferase, among others, in Sf9 cells which are closely related to protein translation were significantly changed after AcMNPV infection. These proteins are involved in the translation of AcMNPV proteins using the Sf9 cell translation system. Concerning carbon metabolism, the gluconeogenesis/glycolysis pathways held the largest number of proteins. As no gene for an enzyme involved in energy metabolism has been found in any sequenced baculovirus, AcMNPV have to rely on Sf9 cell enzymes for energy metabolism (Fig. 5). The proteins with differential abundances involved in protein processing, carbon metabolism and ribosome were searched in KEGG (http://www.genome.jp/kegg/) and colored in Fig. 3, Fig. 4 and Fig. 5, respectively.
Representative KEGG pathway for protein processing in endoplasmic reticulum. The differentially expressed proteins were mapped as green
Representative KEGG pathway for carbon metabolism. The differentially expressed proteins were mapped as green
Representative KEGG pathway for ribosome. The differentially expressed proteins were mapped as green
Protein-protein interaction analysis
In addition to KEGG, we also constructed a protein-protein interaction network using STRING Database version 9.0. As shown in Fig. 6, the differentially expressed proteins were mainly enriched in the term "ribosome", "DNA replication", "Epstein-Barr virus infection" and "oxidation phosphorylation". The ribosome and its related proteins were involved in translation, which can facilitate viral protein production. For host DNA replication, although cellular DNA replication is arrested, AcMNPV can utilize the host replication machinery to facilitate viral replication. The enrichment of "ribosome" and "DNA replication" were consistent with the high replication of AcMNPV in Sf9 cells.
Protein-protein interaction networks. Network illustration of interactions among proteins from three groups of Sf9 cells. Yellow spots: significantly differentially expressed proteins. Green spots: proteins that did not expressed differentially
Previous studies have accumulated rich knowledge about baculovirus gene expression and function during infection. These contain almost the entire whole process of infection, including virus entry, movement into the cell nucleus, DNA unpackaging, early transcription, DNA replication, late transcription, translation, budded virus (BV) assembly, BV budding, occlusion derived virus (ODV) assembly, occlusion, and release of occlusion bodies [18, 19]. However, unlike viruses such as AcMNPV, information on the gene expression and function in baculovirus-susceptible cells is very limited in the main public gene and protein databases. Salem et al. have investigated the impact of AcMNPV on host gene expression in cultured S. frugiperda, Sf21 cells, and found that the majority of the host genes were down-regulated over the time course of infection, although a small number were up-regulated [20]. Their study confirmed that host transcription was shut off. In addition, their study also provided a close estimate of the fold changes of the shut off for many genes. Since the direct cross-talk between virus and host occurs mainly at the protein level, the gene expression changes of the host cell could not reflect host cell responses accurately. Further study by Nuno Carinhas et al. demonstrated proteomic analysis of protein expression changes in Sf9 cells associated with culture growth and baculovirus infection. However, they focused on the transition from early to late phase (around 6 hpi) to investigate the establishment of infection and a total of 648 high-confidence hits were identified as protein homologues from 31 species and 2BV genomes [14]. It must be noted that the global changes of protein abundances in Sf9 cells at different infected stages (6 hpi and 12 hpi) were qualitatively and quantitatively analysed by iTRAQ in this study. This will help us understand the Sf9 cell response upon baculovirus infection and baculovirus infection pathways. Better understanding of the interaction between host cell and baculovirus would also help in optimizing the applicable area of baculovirus.
iTRAQ along with LC-MS is a robust protein discovery technique, allowing the determination of which proteins are affected in Sf9 cells during infection. Here, a total of 4004 Sf9 cell proteins were shown to be differentially expressed during AcMNPV infection. In fact, of these 4004 differentially expressed proteins, 413 were found to be significantly differentially abundant under at least two repeated identifications and quantifications, most of which are closely related to protein translation, protein processing and carbon metabolism.
In this study, 413 differentially expressed proteins were used to perform GO annotations. The functional annotation will help us to purposefully analyse the differentially expressed unigenes caused by virus infection. GO terms involved in cellular and metabolic process, binding, catalytic activity, cells and organelles were analysed by KEGG maps. Upon viral infeciton, a vast number of intracellular pathways in the host are activated/deactivated and profound metabolic changes occur. As a result, several effects arise, such as cellular cytoskeleton rearrangement,cell cycle arrest, apoptosis inhibition, metabolism subversion and host protein synthesis shut-off [21]. As far as cellular processes were concerned, genes involved in protein processes in the endoplasmic reticulum had regulated expression. Since the AcMNPV have no complex organelles, they are entirely dependent on the Sf9 cell translation machinery. Here, we showed that calreticulin, protein disulfide-isomerase A6, UV excision repair protein rad23, glycoprotein glucosyltransferase, prolactin regulatory binding-element protein, heat shock protein (HSP) 90 beta, and eIF2α, among others, were significantly differential expressed in Sf9 cells at different stages. Even actin, the typical control in eukaryotic cell experiments were significantly changed in AcMNPV infected cells. The significant changes of actin confirm host cellular cytoskeleton rearrangement upon infection. Calreticulin is a multi-functional calcium-binding chaperone that has several functions in protein folding, maturation, and trafficking [22, 23]. As an endoplasmic reticulum (ER)-resident chaperone, calreticulin keeps control of both exogenous and endogenous proteins [24]. These results contribute to the point that viruses rearrange the cytoskeleton not only through affecting actin expression, but may also by regulating other trafficking associated proteins to facilitate their own intracellular trasport. Previous studies have shown that translation factors, including eukaryotic initiation factors (eIF3-6, Eif1A, eIF3-2b) and elongation factor (EF1d) were commonly found up-regulated [25]; here, we found that eIF2 was also significantly expressed during the infection. AcMNPV also have to utilise Sf9 cell chaperones to facilitate the rapid synthesis of viral proteins. Our findings here about HSP90 expression are consistent with previous studies on Sf9 cell gene expression [20, 26]. The marked changes of HSP0 and other HSPs also suggested an ER hetereostasis upon infection. For glycoprotein glucosyltransferase and protein disulfide-isomerase A6, their differential expression may help with correct viral protein folding. Our studies here, together with previous studies that most host transcript levels were not significantly changed [27], suggested a post-translational regulation upon virus infection.
A number of proteins associated with the ribosome, the primary site of biological protein synthesis, were also differentially expressed throughout the AcMNPV infection, including ribosomal protein S17, S9, S5, S21, L1, L12, L30, L37, L6, and L28, as well as the 40S ribosomal protein S15 and the 28S ribosomal protein S6. Many ribosome proteins have been linked with cell structure, protein translation and protein biosynthesis [28, 29]. In uninfected cells, ribosomal proteins are typically expressed at a rate enough to maintain a constant ratio to rRNA under physiological conditions. It has been reported that in AcMNPV-infected Sf9 cells, a number of ribosomal proteins were dramatically changed. Our findings confirm the protein level changes of these proteins. The differential expressions of these proteins suggest changes in protein biosynthesis and the process of translating mRNA into protein during virus replication.
A number of genes involved in carbon metabolism appeared to be differentially expressed during AcMNPV infection, including citrate synthase, glucose-6 phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, enoyl-CoA hydratase, alcohol dehydrogenase, triosephosphate isomerase, serine hydroxymethyltransferase, succinyl-coa synthetase β chain, isocitrate dehydrogenase, malic enzyme, and hydroxyacyl-coenzyme. These proteins or enzymes participate in the citrate cycle, the pentose phosphate pathway, glycolysis and gluconeogenesis, and pyruvate metabolism, along with glycine, serine and threonine metabolism to meet the high demands for ATP and the synthesis of cellular components during infection. Infection requires a high energy supply. Nevertheless, AcMNPV has no gene for energy-related enzymes; they have to manipulate Sf9 cells energy generation and metabolism pathways to foster their own replication. Furthermore, we also found that fatty aicd metabolism was dramatically changed upon infeciton, which was consistent with previous results [14]. Taken together, these results suggested that virus actively regulated host metabolic pathways that were essential for their replication.
In summary, AcMNPV infection of Sf9 cells leads to remodeling of host cell and large production of viral genes. During infection, expression of cell proteins involved in protein processing, ribosome, carbon metabolism and RNA transport et al. were found to be significantly changed (Additional file 2: Table S2). These differentially expressed proteins of infected Sf9 cells showed us how Sf9 cells responsed to virus infection. In this study, a battery of uncharacterised proteins was also discovered by biological information comparisons. However, even with the iTRAQ technology and bioinformatics protocols, these proteins could not be identified and their functions are still unknown. The changed proteins exerted crucial physiological effects during AcMNPV infection, and may help us in future researches aimed to optimize virus-host interaction, and in enlarging their application in different area.
In conclusion, a proteomics approach based on iTRAQ was applied to determine the differentially expressed proteins of Sf9 cells during AcMNPV infection which allowing the identification of 4004 proteins. Among these, 413 different proteins were significantly changed. These significantly changed proteins were useful to offer new insights on the dynamic host responses to infecton. Moreover, the changed proteins may also be helpful in further applied studies aiming to improve baculovirus use as a biopesticide or a gene delivery vector.
Cell culture and treatment
The Sf9 insect cell line, the clonal isolate 9 from IPLB-Sf21-AE cells derived from the fall armyworm Spodoptera frugiperda [30], was cultured at 27 °C in Grace's medium (Invitrogen Life Technologies) supplemented with 10 % fetal bovine serum, penicillin (100 μg/ml), and streptomycin (30 μg/ml). AcMNPV was donated by Dr. Yang Kai (School of Life Sciences, Sun Yat-sen University, China). BV titers were determined by TCID50 (50 % tissue culture infective dose) end-point dilution assay using Sf9 cells [31]. For infection experiments, Sf9 cells were grown to the desired cell density and inoculated with AcMNPV at an MOI of 5 infectious particles per cell. Approximately 100 million cells were harvested either before infection or at 6 h and 12 h post-infection, and were seeded by centrifugation. Thus, cells were divided into three groups: mock: cells at resting, 6 hpi: Sf9 cells infected with AcMNPV and harvested at 6 h post-infection, and 12 hpi: Sf9 cells infected with AcMNPV and harvested at 12 h post-infection. Each group was repeated at least three times. Cell pellets were washed with ice-cold PBS, instantly frozen and stored at −85 °C until further analysis.
Sf9 cells from different groups were lysed in basic RIPA buffer containing 25 mM Tris (pH 7.6), 150 mM NaCl, 1 % NP-40, 1 % Na deoxycholate, 1 mM EDTA, and supplemented with 1 mM PMSF and 1 × complete protease inhibitor cocktail (Roche, Switzerland) as previously described [32]. Then, cell lysates were centrifuged and the protein content in the supernatant was quantified using BCA Protein Assay Kit (Thermo Scientific, Milford, MA). Equal amounts of protein (20 μg) were subjected to SDS-PAGE.
Digestion and iTRAQ labelling
Digestion and iTRAQ Labelling were performed according to previously described methods [33]. Briefly, protein samples (400 μg) of each group diluted in 100 mM dithiotreitol solution were incubated in boiling water for 5 min, cooled to room temperature (RT) and diluted with 200 μL UA buffer (8 M Urea, 150 mM Tris–HCl pH 8.0). Then, samples were transferred onto a 10 kDa ultrafiltration filter for centrifugation at 14000 × g for 15 min, and washed again with UA buffer. Subsequently, 100 μL of iodoacetamide (IAA) solution (50 mM iodoacetamide in UA buffer) was added to the filter and mixed for 1 min at 600 rpm, followed by incubation for 30 min at room temperature in darkness and centrifugation at 14000 × g for 10 min. This step was repeated twice. Then, 100 μL dissolution buffer (Applied Biosystems, USA) was added to the filter and centrifuged at 14000 × g for 30 min, and repeated again. Finally, 40 μL of trypsin buffer (Promega, 5 μg trypsin in 40 μL dissolution buffer) were added and digested at 37 °C for 16–18 h. The filter unit was transferred to a new tube, rinsed and centrifuged at 14000 × g for 10min. Resulting filtrates were collected and the peptide content was analysed at 280 nm.
For peptide labelling, an 80 μg peptide mixture from each group was labelled with iTRAQ Reagent-8plex multiplex kit (AB SCIEX, Foster City, CA) according to the manufacturer's instructions. Protein sample from mock was labeled with reagent 113. Samples from 6 hpi and 12 hpi were labelled with reagent 114 and reagent 115, respectively.
Strong Cationic-exchange (SCX) chromatography separation
The peptide mixture was reconstituted and acidified with 2 mL of buffer A (10 mM KH2PO4 pH 3.0 and 25 % (v/v) acetonitrile) and loaded onto a PolysulfoethylTM (PolyLCInc, Maryland, USA) column (4.6 × 100 mm, 5 μm, 200 Å). The peptides were eluted at a flow rate of 1000 μL/min with a gradient of 0–10 % buffer B (solvent A with 500mM KCl) for 25 min, 10–20 % buffer B for 10 min, 20–45 % buffer B for 5 min, and 45–100 % buffer B for 13 min. The elution was monitored by absorbance at 214 nm, and fractions were collected every minute. The collected fractions (about 30 fractions) were finally combined into 5 pools and desalted on C18Cartridges (66872-U, Sigma).
MS/MS analysis and quantification
For each fraction, 10 μL of solution was injected for nanoLC − MS/MS analysis using a Q Exactive MS (Thermo Finnigan) equipped with Easy nLC (ProxeonBiosystems, now Thermo Fisher Scientific). The chromatographic column was balanced with buffer A (0.1 % Formic acid). Five μg of the peptide mixture was loaded with an auto-sampler onto a Thermo scientific EASY column (2 cm*100 μm 5 μm-C18) and separated with the Thermo scientific EASY column (75 μm*100 mm 3 μm-C18) at a flow rate of 250 nL/min. A linear gradient of buffer B (84 % acetonitrile and 0.1 % Formic acid) used here was: a segmented gradient from 0–35 % (v/v) in 100 min, from 35–100 % (v/v) in 8 min, and then at 100 % (v/v) for 12 min.
The mass spectrometer data were analysed using the Q-Exactive mass spectrometer (Thermo Finnigan, USA) in the positive ion mode with a selected mass range of 300–1800 mass/charge (m/z). Resolving power for the Q-Exactive was set as 70000 at m/z 200 for the MS scan. Determination of the target value was based on predictive Automatic Gain Control (pAGC). Dynamic exclusion was 40.0 s. Survey scans were acquired at a resolution of 70000 at m/z 200, and resolution for HCD spectra was set to 17500 at m/z 200. Normalised collision energy was 30 eV and the under-fill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum-fill time, was defined as 0.1 %. The instrument was run with peptide recognition mode enabled.
The raw MS/MS spectra data were searched and identified using Mascot 2.2 and Proteome Discoverer1.4 (thermo). The database used in this study was UniProtKB 2014101053FC0RBNV8.fasta, released by Uniprot in August 2014. Assembling protein identifications were qualitatively analysed by Proteome Discoverer 1.4 software. All data were reported based on 99 % confidence for protein identification, as determined by the false discovery rate (FDR) ≤1 %. Statistical analysis was conducted using a one-way ANOVA. P-values ≤ 0.05 by Tukey's test were considered significant. Among the statistically significant proteins detected by the ANOVA test (p < 0.05), protein abundances that changed less than 1.5-fold were discarded.
Proteins that were found to have a statistically significant difference in abundance among mock, Sf9 cells at 6 hpi and Sf9 cells at 12 hpi were then further analysed for functional and biological relevance. These proteins were classified by their gene function and also by biological pathways using the freely available gene ontology (GO) provided by the Gene Ontology Consortium. The retrieved sequences were locally searched against NCBI nr using the NCBI BLAST+ client software (ncbi-blast-2.2.28 + −win32.exe) to find homologous proteins from which the functional annotation was transferred to the targeted proteins. Here, the top 10 blast hits with an E-value less than 1e-3 for each query proteins were retrieved and loaded into Blast2GO (Version 2.7.2) for Gene Ontology (GO) mapping and annotation. In addition, differentially expressed proteins were sent to the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://string.embl.de/) to build a functional protein association network.
AcMNPV:
Autographa californica multiple nucleopolyhedrovirus
iTRAQ:
isobaric tags for relative and absolute quantitation
SCX:
Strong Cationic-exchange.
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This research was supported by the National Natural Science Foundation of China (No. 31201575), the Project of Training Outstanding Young Teachers in Higher Education Institutions of Guangdong Province (No. Yq2013096), the Science and Technology Innovation Project in Higher Education Institutions of Guangdong Province (No. 2012KJCX0067), the PhD Start-up Fund of Natural Science Foundation of Guangdong Province (NO. S201304001647), the Science and Technology Project of Guangdong Province (NO. 2012B020406001). The authors wish to thank Shanghai Applied Protein Technology Co.Ltd. for the technology support.
The funding sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.
College of Food Science and Technology, Zhongkai University of Agriculture and Engineering, No. 501 Zhongkai Road, Haizhu District, Guangdong, 510225, People's Republic of China
Qian Yu
, Youhua Xiong
, Hang Gao
, Jianliang Liu
, Zhiqiang Chen
, Qin Wang
& Dongling Wen
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Correspondence to Qian Yu.
QY conceived the study, provided fund supporting and revised the manuscript critically for important intellectual content. YHX, HG, JL L, ZQC, QW and DLW made substantial contributions to experiment, analysis and interpretation of data. All authors have read and approved the final manuscript.
List of 413 proteins that had an individual P < 0.05 and changed not less than 1.5-fold in their relative abundance among Sf9 at mock, 6 hpi and 12 hpi. (PDF 390 kb)
KEGG pathway. (PDF 187 kb)
Yu, Q., Xiong, Y., Gao, H. et al. Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection. Virol J 12, 115 (2015). https://doi.org/10.1186/s12985-015-0346-9
Spodoptera frugiperda cell
iTRAQ
Proteomics analysis
GO annotation
Other viruses (e.g. pox, papilloma, parvo, reoviridae)


text:
Home Personal Injury Washington Grant County
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George Ahrend
Grant County, WA Personal Injury Lawyer with 25 years of experience
(509) 764-9000	457 1st Ave. NW
Ephrata,	WA 98823-0816
Personal Injury, Appeals, Civil Rights and Insurance Claims
Matthew Albrecht
(888) 424-4430	16 Basin St SW
Ephrata,	WA 98823
Personal Injury, Business, Construction and Insurance Claims
I grew up in Montana working in my family's construction company, and moved to Spokane to attend Gonzaga University. I loved the area and I'm glad to call the Inland Northwest my home together with my wife and four children. When I'm not in court or helping clients, I'm usually riding motorcycles or playing soccer with my kids. I love being a lawyer, and little makes me happier than the chance to help a client set things right after they have been taken advantage of or injured by someone who refuses to take responsibility.
Spencer Fielding
Grant County, WA Personal Injury Lawyer
(509) 761-3296	122 E. Broadway Avenue
Moses Lake,	WA 98837
Personal Injury and Insurance Claims
After graduating from Duke Law School, Spencer was employed with Thompson & Knight, one of the largest law firms in Dallas, Texas. He worked in their Corporate & Securities Department. Thriving but not enamored with the narrow strictures of corporate firm life, he later accepted a job with a Dallas attorney that specialized in Personal Injury.
After gaining invaluable insight into personal injury law, Spencer returned to Washington where he opened up his own personal injury practice, Fielding Law Group. The firm has since expanded with offices in Yakima, Moses Lake, Seattle, Tacoma, and Boise.
Fielding Law Group features a range of...
Brett William Hill
Grant County, WA Personal Injury Attorney with 29 years of experience
(509) 765-0522	1426 S Pioneer Way
Personal Injury, Criminal Defense, DUI & DWI and Juvenile
David William Kraft
(509) 754-4463	915 Basin St SW
Personal Injury and Criminal Defense
Robert C. Hahn III
Spokane County, WA Personal Injury Lawyer with 23 years of experience
(509) 921-9500	2906 N. Argonne
Spokane,	WA 99212
Free ConsultationPersonal Injury, Bankruptcy, Medical Malpractice and Products Liability
The law firm of Robert C Hahn is one of the top law firms in the Spokane area. We genuinely care about our clients and are willing to go the extra mile. Because of this outstanding service, Robert has been recognized as one of Spokane's Top Lawyers from 2011-2019 by Spokane-Coeur d'Alene Living Magazine.
We are a full-service law firm that provides a range of quality legal services with a particular emphasis in bankruptcy and personal injury. Robert is a highly rated and skilled attorney with over 23 years of experience. As a lawyer in private practice, Robert has represented...
Rodney Kenneth Nelson
Kittitas County, WA Personal Injury Attorney with 42 years of experience
(509) 925-1588	605 N Main Street, Suite 1
Ellensburg,	WA 98926
University of Washington School of Law
Rod Nelson was raised in the Yakima Valley and chose to return to Central Washington after graduating magna cum laude from the University of Washington in 1975, and from the University of Washington School of Law in 1978 in the top 10% of his class. Nelson began his law practice in Alaska in 1978 before returning in 1979 to practice in Washington State.
As a volunteer attorney for the Yakima County Volunteer Attorney Services, Nelson was recognized as "Volunteer Attorney of the Year" in 2009, and has served as Judge Pro Tem for Yakima County Superior Court. He is a past...
Terry Abeyta
(509) 925-1588	605 N. Main Street, Suite 1
Although practicing in the most conservative area of the state, Terry Abeyta has earned recognition as one of the top plaintiff's personal injury attorneys in Washington. He recovered $6.9 million on a bleacher fall case (the largest settlement in the history of Yakima County), $4.1 million on a car crash case (the largest settlement in the history of Kittitas County), $3.5 million on a school playground case, the highest settlements in the history of Grant and Klickitat counties, the largest recovery on an uninsured motorist case in Yakima County history, the largest jury verdict on a cervical soft-tissue case in...
Spokane County, WA Personal Injury Lawyer with 8 years of experience
(509) 535-2997	901 N Adams St, Ste 101
Free ConsultationPersonal Injury, Civil Rights, Criminal Defense and DUI & DWI
Attorney Jason Johnson has a wide range of experience which makes him an aggressive litigator. With ten years of experience in the military as a Tank Officer, Jason knows how to communicate effectively and handle the pressures of the courtroom. He was deployed overseas four times and received a Bronze Star with Valor.
Jason grew up in Coeur d'Alene and has a background in construction. As a project manager, he has managed multi-million dollar highway construction projects and understands business and contract disputes from both the legal and practical perspective.
Jason Johnson has litigated numerous major felony cases, including Violent Crimes, Murder,...
David P. Abeyta
David P. Abeyta is a second-generation personal injury lawyer. He works in the areas of plaintiffs' personal injury and wrongful death claims, including auto accidents, workplace injuries, and school injuries. Abeyta works primarily out of the Abeyta Nelson Yakima office.
Abeyta is a life-long resident of Yakima, Washington. After graduating from West Valley High School, Abeyta went on to study at Washington State University and graduated in 2005 with a degree in Political Science. Abeyta continued his education at the California Western School of Law, graduating in 2009. Abeyta joined Abeyta Nelson Injury Law in March 2010. He is a partner...
Jed Barden
Spokane County, WA Personal Injury Attorney with 14 years of experience
Personal Injury, Insurance Claims, Medical Malpractice and Products Liability
University of Oregon School of Law
I represent individuals who have been seriously injured because of someone else's careless or intentional act. I also assist family representatives when a family member's death was caused by another's negligence. If you find yourself in this situation and are looking for assistance, please contact me to discuss your case. Dealing with a serious injury or death can be overwhelming and I work hard to take the legal burden off your shoulders. Ultimately I want you to obtain justice and appropriate compensation for your loss.
I have been an attorney since 2006 and take cases throughout the State of Washington....
Christopher Carlisle
(509) 228-7011	1020 N. Washington
Personal Injury and Workers' Comp
A founding member of Carlisle & Byers, PLLC, Chris's core emphasis is representing clients in worker's compensation matters throughout Eastern Washington. Although worker's comp is Chris's primary focus, he also works with personal injury.
Before becoming an intern and associate at Stiley & Cikutovich, Chris earned a bachelors degree in political science from Seattle University and his Juris Doctorate degree at Gonzaga. He has experience practicing before the Board of Industrial Insurance Appeals, the State Superior Courts, the Court of Appeals, the Supreme Court, and the United States District Courts of Eastern Washington.
Chris is a member of the Washington State and...
Alex Thomason
Okanogan County, WA Personal Injury Lawyer with 16 years of experience
(509) 689-3471	201 S. Bridge Street
Brewster,	WA 98812
Personal Injury, Agricultural, Business and Real Estate
Alex grew up on his family's apple farm in Brewster, Washington. He learned every trade required to run a farm, but specialized in being the certified "gopher." Alex graduated from high school ranked 2nd in the nation and top 25 in the World for his age in downhill ski racing. Alex studied at Jerusalem University in Israel, completed undergraduate studies from Seattle Pacific University, and finished his legal education at Seattle University School of Law where he was the Graduation Speaker. Alex learned trial skills by working as a Prosecutor for King...
Michael D. Whipple
(509) 869-3223	309 E. Pacific Ave
Personal Injury, Elder, Estate Planning and Probate
Providing a wide range of quality legal services with a personal commitment to each client. We specialize in: Business Law Civil Rights Law Consumer Protection Law Estate Planning Land Use Law Landlord / Tenant Law Legal Guardianship Law Personal Injury Probate Water Rights Elder Law At the Whipple Law Group, PLLC in Spokane, Washington...
D Christopher Russell
(855) 707-7246	206 West First Street
Deer Park,	WA 99006
Free ConsultationPersonal Injury, Criminal Defense, DUI & DWI and Medical Malpractice
Arizona Personal Injury and Criminal Defense Lawyer Helping Clients Nationwide for 18 Years. We are dedicated to helping seriously injured people . Our goal is to make the community a safer place for you and your family. We represent those seriously injured or the families of those killed in accidents. We strive to hold those responsible for needlessly causing harm to someone in our community. We are committed to the jury system and understand that the jury is the conscience of the community and the enforcer of community safety standards. We limit the number of cases...
Debra J Toews
Walla Walla County, WA Personal Injury Lawyer with 18 years of experience
(509) 529-0908	30 West Main Street, Suite 201
Walla Walla,	WA 99362
Debra cares passionately about obtaining justice for her clients. Debra has practiced exclusively in personal injury since being sworn in as an attorney in November, 2002. Debra has worked, first as a paralegal, and now as an attorney, in the personal injury field since 1994. She completed Washington State Bar Association Law Clerk Program in 2002, and passed the Washington State Bar Examination in July of that year. Debra has practiced exclusively in personal injury since being sworn in as an attorney in November, 2002. On January 1, 2006, she co-founded Willis & Toews, PLLC, with Monte Willis. ...
Breean Lawrence Beggs
(509) 232-7760	522 W. Riverside Avenue
Free ConsultationPersonal Injury, Appeals, Civil Rights and Employment
William David McCool
Walla Walla County, WA Personal Injury Attorney with 43 years of experience
(509) 525-2630	6 East Alder St Suite 425
Personal Injury, Criminal Defense, DUI & DWI and Traffic Tickets
Walla Walla criminal defense lawyer, Mr. McCool has had the privilege of handling criminal cases over 37 years making them some of the best in town. If you are facing charges for a criminal offense, whether it be petty crimes such as a traffic violation or a more serious offense, this is the firm to seek out. With years of experience on his side you can trust that you will receive the dedication you deserve fighting for your case. Learn more about this by visiting their website or calling their office for a consultation. ...
Kathleen H. Paukert
(509) 232-7760	522 W. Riverside
Free ConsultationPersonal Injury and Nursing Home
Gonzaga University School of Law and University of Washington School of Law
David Alden Brown
(509) 228-8790	1312 N Monroe St
Free ConsultationPersonal Injury, Asbestos and Insurance Claims
University of Colorado Law School and University of Washington School of Law
I am an experienced trial lawyer who focuses my practice primarily on serious personal injury and wrongful death cases.
Sarah Cuellar
Ferry County, WA Personal Injury Attorney with 7 years of experience
(509) 207-9162	PO Box 904
Republic,	WA 99166
Free ConsultationPersonal Injury, Business, Estate Planning and Probate
J.J. Thompson
(509) 252-5048	220 W. Main Ave.
Free ConsultationPersonal Injury, Insurance Claims, Nursing Home and Products Liability
J.J. practices law throughout the State of Washington and North Idaho. His practice involves representing injured individuals and families who were injured or harmed by other people, corporations, or governmental entities.
Heather Barden
(509) 315-8089	PO Box 8663
Personal Injury and Employment
I represent individuals that have been wronged by their employer. Wrongful acts by your employer include discrimination, retaliation, harassment, or a hostile work environment. Contact me if your employer has discriminated or retaliated against you, harassed you, or created a hostile work environment based on your:
Need for an accommodation
Military status
Medical leave (FMLA), or
Because you are a breastfeeding mother
I have been an attorney since 2015 and assist individuals in Washington State with their employment issues. I became an attorney to empower individuals that are treated unfairly by others. I want to provide justice for individuals and help them...
James M. Owen Jr.
(800) 805-5028	421 West Riverside Avenue
Personal Injury, Employment and Medical Malpractice
James M. Owen, Jr. graduated from Washington State University with a B.A. in 1993 and received his J.D. from Gonzaga University in 1999. He was admitted to the Washington bar in 1999, the Oregon bar in 2007, and is also admitted to practice before the U.S. District Court for the Western and Eastern Districts of Washington and the U.S. Court of Appeals, Ninth Circuit. Mr. Owen is a member of both the King County and Spokane County Bar Associations. Having the benefit of previously having served in positions with both global and boutique law firms, Mr. Owen launched Owen |...
Teresa Keene
(509) 326-1666	2625 W Northwest Blvd, Suite C
Free ConsultationPersonal Injury, Divorce, Family and Juvenile
Keene Law is a "boutique" Spokane law firm providing general practice legal services while specializing in family law, divorce, child custody, pre-nups and adoptions, as well as criminal
defense cases. Our attorney, Teresa Keene, a 2007 Gonzaga Law School graduate, is a former prosecutor as well as public defense attorney, and has ten years experience as owner of her own private law practice. Ms. Keene is presently accepting new clients and provides no cost initial consultations with prospective clients.
Kirk Bernard
Chelan County, WA Personal Injury Lawyer with 24 years of experience
(509) 248-7536	1250 N. Wenatchee Ave.
Ste. H-363
Wenatchee,	WA 98801-1536
Personal Injury and Business
I have been a civil litigator for over 20 years, having been first admitted to the bar in 1981, specializing in personal injury cases and representing the rights of accident victims on a contingency basis. I have obtained millions of dollars in settlements and verdicts for my seriously injured clients.
Kelby Jamison Derenick
Walla Walla County, WA Personal Injury Lawyer
(509) 676-9805	14 E. Main Street, Suite 207
Personal Injury, Insurance Claims, Landlord Tenant and Real Estate
Kelby J. Derenick has a decade of legal experience representing clients in real estate matters and in all aspects of civil litigation. Mr. Derenick practices law with the highest standard of attention to detail necessary for every case and prides himself in giving clients the dedication and attention they deserve. He can assist clients with routine transactions and also consistently obtains favorable results for his clients litigating disputed cases through trial.
Mr. Derenick is a member of the Washington State Bar Association, California State Bar and is also admitted to practice with the United States District Court for the Eastern and...
Stephen Michael Bergman
Spokane County, WA Personal Injury Attorney with 8 years of experience
(509) 326-2613	330 W Indiana Ave.
Free ConsultationPersonal Injury and Employment
I am an attorney with Cooney Law Offices, P.S. in Spokane. My primary practice areas are Plaintiff's side employment law and personal injury. I strive to be readily available, and listen to my clients. I take the time to give my clients the best possible representation and outcome.
David M. Church
Yakima County, WA Personal Injury Attorney with 15 years of experience
(509) 638-1414	6 S 2ND ST
YAKIMA,	WA 98901
At the Law Office of David M Church, PLLC, we help personal injury victims. Whether you've been injured in an automobile accident, a collision with a semi truck, attacked by a dog, injured on someone's property, had a slip/trip and fall, or experienced some other injury, we take great pride in providing legal services with a personal touch.
Andrea L. Asan
Free ConsultationPersonal Injury, Civil Rights and Insurance Defense
Matt Conner
(800) 925-1875	1312 North Monroe Street
Suite F-179
Attorney Matt Conner was raised in Bellingham and Ferndale. He received his undergraduate degree from Willamette University in Salem, with a double major in mathematics and economics, and then worked as an economist for the Office of Economic Analysis for the State of Oregon. After additional professional experience in the fields of mortgage banking and real estate, Matt decided to follow his long-held interest in the law by attending Seattle University School of Law. Matt recently graduated Magna Cum Laude. Matt met his wife Courtney, a talented recruiter for Amazon, while playing dodge ball. They have a young daughter...
Michael Trevor Howard
(509) 838-6131	601 W Riverside Ave
University of Idaho College of Law
I manage the Coeur d' Alene branch of Winston & Cashatt, a regional mid-sized law firm based in Spokane, Washington. I am licensed to practice in Washington and Idaho, and represent both plaintiffs and defendants in the areas of personal injury, product liability, medical negligence, construction defect, and insurance coverage.
Brian J. Bean
(800) 829-5291	421 W. Riverside Ave., #381
Free ConsultationPersonal Injury and Securities
Brian J. Bean is a Spokane & Coeur d'Alene attorney specializing in business, real estate and corporate law. His previous legal experience includes being corporate counsel for an Avista Utilities subsidiary, Ecova Inc.
He has extensive experience guiding both large and small companies through strategic business and legal decisions, corporate formalities, negotiations and disputes. He is similarly proficient in real estate law, including, but not limited to commercial/residential purchase and sales, title actions, foreclosures, easements and boundary disputes.
Brian prides himself in partnering with his clients to help them achieve their goals and objectives. This means better attorney communication, being cost conscious,...
Richard Raymond Johnson
Yakima County, WA Personal Injury Lawyer with 45 years of experience
(509) 961-2468	917 Triple Crown Way
Born Spokane, WA 1949
B.A. Washington State University 1971
J.D. Gonzaga Law School 1975
Asst A.G. Washington State Attorney Generals Office - Work Comp DIvision
Civil litigation in private practice in Yakima, WA June, 1978 to the present
Kevan Tino Montoya
(509) 895-7373	4301 Tieton Drive
Personal Injury, Business and Real Estate
I have practiced in Yakima County for twenty-eight years. I have experience in state and federal court throughout the state of Washington. I am also admitted to practice in Oregon.
Monte Willis
Free ConsultationPersonal Injury, Animal and Products Liability
Cornell University and Washington State University
Attorney Monte Willis has been practicing law since 1995, and has extensive experience in Washington personal injury law.
In 2006, He and his former legal partner, Debi Toews opened Willis & Toews, a Walla Walla firm practicing exclusively in the personal injury field. Monte cares deeply about his work as an attorney, protecting the rights of Oregonians and Washingtons after their accidents.
His dedication to serving individuals and his local community is evident in his extracurricular activities. From working on the organizing committee for the Tour of Walla Walla to serving as a board member in the Walla Walla Downtown...
Richard Eugene Lewis
(509) 413-1278	2208 W 2nd Ave
We have thirty years experience representing honest people with serious injury claims. We represent individuals in automobile, truck, motorcycle and bicycle accident cases and have tried cases throughout the region. Our relationships with experts in all related fields enables us to get the best results for our clients. We stand by our clients and aggressively represent them in all phases of the case and keep our clients informed as the case progresses. Richard Lewis is rated AV by Martindale Hubbell, the highest rating available by the most respected peer rating system and a perfect 10.0 by Avvo, which rates lawyers...
F. Dayle Andersen JR
(509) 327-6100	1020 N Washington St
I have practiced in the Inland Empire for over the last 15 years. Our firm focuses on personal injury and criminal defense.
Karen Suzette Lindholdt
(509) 994-7423	1309 W. Dean Avenue, Suite 101
Personal Injury, Consumer and Employment
Karen Lindholdt specializes in personal injury, employment and consumer protection law. She has practiced for over 16 years in the states of Washington and Idaho and is licensed in both state and federal courts, as well as the Ninth Circuit Court of Appeals.
Tyler Michael Hinckley
Personal Injury, Business, Employment and Insurance Claims
The OneCLE Lawyer Directory contains lawyers who have claimed their profiles and are actively seeking clients. Find more Grant County Personal Injury Lawyers in the Justia Legal Services and Lawyers Directory which includes profiles of more than one million lawyers licensed to practice in the United States, in addition to profiles of legal aid, pro bono and legal service organizations.


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text:
Chesapeake medical marijuana company wants answers after "improper" state board decision
By Briana Adhikusuma
Even as an out-of-state company prepares to become Southeast Virginia's sole medical cannabis processor, questions loom over the process that chose the nation's largest medical cannabis operator over local applicants.
One Chesapeake company wants answers – and still hopes to get a piece of the pie.
New York-based Columbia Care, recently valued at $1.35 billion, applied for a license in all five of the state's health service areas. It only won conditional approval for a license in the region that includes Hampton Roads.
The company, which has yet to announce where it plans to operate, will be able to grow cannabis and turn it into oil, capsules and creams prescribed for state-registered patients. The products can only be distributed from these facilities.
There were 51 applicants for the five licenses – 15 of them for the Hampton Roads facility. The license holders will not only get first crack at Virginia's medical cannabis industry, but it also positions them for an even more lucrative opportunity if the state ever legalizes recreational use.
According to a Baltimore Sun report on national firms controlling medical cannabis licenses in Maryland, industry sales in the U.S. are expected to approach $500 million by 2025.
But even as the out-of-state corporation prepares to start operating in Virginia, Chesapeake Bay Therapeutics isn't giving up yet. The Chesapeake company, also a bidder for a state license earlier this year, submitted a protest to Virginia Board of Pharmacy Executive Director Caroline Juran on Oct. 5.
The protest outlines what it called "errors" with the board's evaluation process and wants a suspension of the Columbia contract while the protest is reviewed. It also proposes splitting each health service area into two, providing more available licenses.
"Arbitrary," "inappropriate," "capricious," and "improper" were just a few of the words CBT used in its protest to describe the board's actions and decision on awarding licenses.
"CBT has and will demonstrate that the Board's lack of meaningful review and evaluation amounted to arbitrary and capricious determinations," the protest said.
Board responses limited
Applicants say it has been difficult to get any information on why they were denied licenses. Before its protest went to the board, CBT requested on Sept. 26 a briefing on what deficiencies led to its loss. Juran told the company a board order regarding the denial of a license would be issued instead, according to emails provided to The Virginian-Pilot by CBT. She did not indicate whether the order would provide reasons for denial.
Chantra Stevenson, COO of Hampton-based Rx Native Pharmaceuticals, said the board has not been forthcoming with applicants looking for answers. "I do feel that their evaluation was closed and non-transparent," she said.
An initial scoring sheet with applicant scores was released by the board, but the names were redacted, so there was no way to tell which applicants received which scores. Some applicants were awarded point totals higher than the board-stated maximum during preliminary review. Juran said final scores did not exceed the maximum, though the board would not provide those scores or did not explain how they were calculated.
After Stevenson saw the sheet, she said the way the scoring was done didn't make any sense and differed from how applicants were told scoring would be done.
"It was not anywhere close to what they suggested in their application. So I can understand Chesapeake Bay's protest ..." Stevenson said. "It didn't seem fair."
Despite that, Rx Native isn't pursuing its own protest.
"The money and energy that's required for that sort of protest and challenge is more than what we have," she said. "That's part of us being small, and we just don't have the pockets that some companies have to challenge."
The protest
In its protest, CBT outlines its belief that the board review and evaluation of applicants was insufficient and that it failed to use industry experts to adequately support the licensing decision. The board consists largely of pharmacy professionals, but with cannabis an emerging industry, it's not clear what direct experience in the field board members possessed.
The board declined to provide application documents to The Pilot, citing state laws regarding medical licensing applications.
CBT also questioned the thoroughness of the application reviews, noting that each application was around 300 pages or more. It contends that with 51 applications, upwards of 15,000 pages were reviewed over only two meetings, both closed to the public.
Juran noted, however, that the applications went to ad hoc committee members late June for their review and to do preliminary scoring.
In its protest, CBT proposed the Southeast Virginia health service area be split, with Columbia taking the northern piece down to the Hampton Roads Bridge-Tunnel. That would leave the southern portion for CBT.
"An award of a license in Region 5 to CBT would support a well-qualified Virginia business, which understands the needs of its community," the protest stated.
Juran, in an emailed response to CBT about the protest, wrote Tuesday that because a final order has not been issued regarding its license denial, "there is nothing to appeal."
While criminal background checks are pending for the companies awarded conditional approval, the board has 90 days to make its final decision under the Administrative Process Act. The protest was submitted under the Virginia Public Procurement Act, which she said is not subject to that law.
The board will notify the company of steps it can take to appeal official orders once they're issued, Juran said.
She also said the board lacks the power to split the regions. "For the Board to have authority to issue additional permits, it will be necessary for the General Assembly to amend the statute," she said.
CBT consultant James Lee said he believes the five companies awarded licenses will end up lobbying to open more facilities because the existing service areas are too large to adequately serve from one facility. He estimated there would be about 126,000 patients in the region by 2022. Regulations allow deliveries after an initial, in-person visit to the facility but the companies will have to make those deliveries.
"The legislature just has to look at this and see that it's not possible to serve the area with one facility …" he said. "If there's one facility, there's patients who have to cross the bridge-tunnel for their medicine and that's a nightmare. There's really, logistically, no way to put one facility in region 5 and make it accessible for all people."
Lee said the board should have evaluated the applications more thoroughly and considered the restrictions to only five licenses.
"Patients deserve better than that," he said.
Where will Columbia Care operate?
Columbia Care is taking steps to open in the region in the second half of 2019, according to a recent news release. But where remains a mystery.
According to company representative Josephine Belluardo, Columbia Care submitted a site location as part of its application to the board. She said in an email that it is "working with the landlord and the Board of Pharmacy to finalize our selection."
Other companies that received conditional approval – Maryland-based Green Leaf Medical and Virginia-based Dalitso and Dharma Pharmaceuticals all have announced areas or sites where they intend to open facilities. Illinois-based Pharmacann selected a site in Staunton and has since been sold to MedMen Enterprises Inc. for about $682 million.
The board has continued to offer no additional details on Columbia's application, citing a law that allows it to keep the information confidential.
Local city leaders don't have answers, either.
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Economic development directors and city spokespeople in Hampton, Newport News, Norfolk, Portsmouth, Suffolk and Williamsburg said there has been no contact with Columbia. Virginia Beach Communications Director Julie Hill said although Columbia has not contacted them, city officials have reached out to the company to offer help in finding a site.
Chesapeake Economic Development Director Steven Wright declined to say if Columbia had contacted the city. CBT had established backing there, providing in its application signed letters of support from Mayor Rick West, City Manager James Baker, Sheriff Jim O'Sullivan and a few business leaders.
With the lack of clear local support, Lee said he just doesn't understand why the board, which pushed for local companies during the process, would choose a national giant over a local applicant.
"They (Columbia) didn't reach out to any city. Nobody had heard from them," Lee said. "We had an entire city government behind us."
Briana Adhikusuma, 757-222-5349, b.adhikusuma@pilotonline.com
Pharmaceutical Processors
Virginia Board Of Pharmacy
Chantra Stevenson
Most Read • Health & Medicine
A teenager's pleas for help were ignored until she collapsed at a Newport News mental hospital and died,...


I am very excited to have been appointed as the ladies 1st Team coach for the 2016/17 Season. This gives me the opportunity to work with a fantastic group of players and people at a well-established club, and hope to repay their faith in my coaching by moving the Ladies 1s up the North Division 2 SE table.
We have undertaken a strong pre-season programme, consisting of a fine balance of intense training and testing friendly matches. This has allowed me to implement a new mentality and new style of play, and it was good to have seen a positive reaction from the squad, by winning all of our pre-season friendly fixtures.
I am looking forward to the season ahead, as also we have entered the Investec National Trophy which will be a new and exciting prospect for the squad.
The main focus this season will be to achieve a good position within the league, and the cup will add a little spice of extra spice to the season.
Having stepped down from the role as overall Ladies Head Coach for the last year I have thoroughly looked forward to this season. What a great opportunity for the Club to have two such coaches! I have enjoyed working with Gareth on many coaching projects. It's great to see him able to give his time up and help the club out and be there every Wednesday and Saturday for the 1st XI with such passion and enthusiasm for hockey. The main focus this season is simply fun and development. With myself and Gareth working together with our philosophy, the teams can be fluid and players will be able to move up and down through the teams as they develop a greater overall understanding of hockey that relates to the club as a whole.
The focus for myself in 2016/17 season is to work with a seriously enthusiastic, knowledge thirsty and very diverse age group of 3rd and 4th team players. As a development specialist, I hope to give the Ladies a rich game based environment where they can measure there improvement each week while having bucket loads of fun at training!


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Houses advances bill to fund gun violence research
The U.S. House of Representatives passed a spending package that includes $25 million for gun violence research.
The bill—sponsored by Sen. Ed Markey (D-MA) and Rep. Carolyn Maloney (D-NY) — now moves to the Senate for consideration. The Senate will vote on final passage in the coming days. The $25 million would be spent on gun violence prevention research by the Centers for Disease Control and Prevention (CDC).
"Gun violence is a public health epidemic, and it should be studied just like any other health crisis – by our government's top researchers," Maloney and Markey said in a joint statement. "While our bill would have provided $50 million for this research, this $25 million is an important first step. Studying gun violence is key [in] helping us to address an epidemic that takes 100 lives a day. We need to do everything we can to address preventable gun violence."


In my family, Australia Day has always meant my sister's birthday. Not once in 48 years can I remember doing anything vaguely patriotic on the day itself – in fact, my key memory from when we were young is my mother frantically trying to buy out-of-season strawberries for my sister's birthday cake (not so easy in the 70s). But with the government threatening local councils who dare to move citizenship ceremonies and an increasingly loud call for a change of date, I was prompted to think about what, if anything, business can learn from Australia Day and its myriad meanings.
Here are my Top 3 takeaways.
If you thought Australia Day was just a well-timed public holiday between the Xmas/New Year Bacchanal and the 'real' start of the year (around 10 February), think again – over time the day has become so laden with symbolism that it's hard to remember exactly what the original intent was. That's probably why people are happy to imbue it with their own meaning – whether that's an unfortunate reminder of more than 200 years of dispossession and oppression or a call to arms for the kind of people who think the Cronulla Riots were a proud moment in our history.
The lesson? From Apple 'Macolytes' to Converse converts, when consumers feel like they are part of a tribe they are far more likely to remain loyal, often without any logical reason to be so. That's because, whatever we tell ourselves, we tend to make decisions for emotional rather than rational reasons.
This may come as a shock, but not everyone cares about Australia Day. They might be new to the country and not see what the fuss is all about. They may be celebrating something else entirely on the day – a birthday, anniversary, getting their citizenship – and alternately grateful for the day off and slightly miffed because, like Christmas, the bigger event will always eclipse their personal celebration. Sure, some of them may be swayed by the fireworks and neighbourhood BBQs, but others will just go about their business as if it's a normal day.
Likewise in business. No matter how top of mind we like to think we are, there will always be people who either don't know – or care – that we exist, or choose to take their business elsewhere even if they do. One of the best things we can do is to nurture our existing clients by lavishing them with affection and an experience that's second-to-none, so they won't even think about leaving us for someone else. And knowing when to give up on the non-believers.
Then-Prime Minister Malcolm Turnbull turned to social media in 2017 to talk about the delights of Australia Day, saying 'a free country debates its history, it doesn't deny it'. That's all well and good Malcolm, but what you can't deny is the fact that 26 January commemorates an event which coincided with the start of the widespread and calculated dispossession and marginalisation of Indigenous people. Many would argue that the process continues to this day. So apart from anything else, it's poor taste to suggest that Indigenous people should just cop it because we should 'debate, not deny' our history.
What the rhetoric doesn't always take into account, though, is the enormous opportunity moving the date represents. One of our best attributes is that as a relatively young country, we are still a work in progress. We don't have thousands of years of history to weigh us down, as much as we may look to our collective heritage as migrants. It's one reason why our cuisine is so diverse and imaginative – we're not shackled to one way of doing things.
Triple J changed the date of their Hottest 100 and while they copped some flack, the sky did not, as predicted, fall in. And as Maggie Walter argues in The Conversation, if we ever want to become a Republic – as potent a symbol of a country's coming-of-age as anything – then changing the date is a first, crucial step.
It occurred to me that holding on to something that's not really working – that is, in fact, actively offending a portion of our society – feels a bit bloody minded. Like Kodak hanging on to film long after digital cameras were a thing, or IBM deciding that computers were simply never going to take off.
In business, sometimes change is the best way to survive. Sometimes, it's the only way.
Dominique drives the growth of The Copy Collective, a leading Sydney and Auckland-based copywriting agency. Producing year- on-year growth for seven years, Dominique is a talented leader, skillful journalist and editor who brings her years of experience in design and DM agencies to produce great work for our clients.


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Mrs Carter
Jack Ryan season 2 premiere review: Assassination in Venezuela
Jack Ryan, Jack Ryan season 2 review	 October 31, 2019
The Jack Ryan season 2 premiere on Amazon wasted no time in showing that for the title character, there is no breathing room in Venezuela. He's there, he is trying to find his footing, and he better be prepared for carnage at just about every turn.
Also, he's in for so much more than he ever bargained for. Ryan arrived in the South American country in the premiere in order to investigate an arms-trade deal, one that had connections to numerous countries and one that could lead to many lost lives. What did he find instead? Think in terms of a dead Senator in the closing minutes, and also quick questions about whether or not a dictator is so desperate to seize power that he is willing to cause chaos within his own country.
The assassination at the end of the episode is a key part of what stands out within the premiere, alongside the closing image of a choked-up Ryan having to make a phone call that he doesn't want to make on top of the embassy rooftop. What you realize in those closing moments is that while Ryan may be equipped to do this job, that doesn't mean that he's used to it. All of his trials and tribulations in season 1 have failed to prepare him for other parts of the world — including a country where everyone has a lot of dangerous stuff to lose.
The stakes in Venezuela for the government were laid out in the early going — this is a mineral-rich country who can control so much more than just within their borders. That, ironically, is what brings Greer there. He's able to link a satellite crisis to the country and, soon after that, he found good reason to leave his post in Moscow — sure, that's one of the most-prestigious places for anyone in this field, but at the same time there was a different calling. Also, Greer had some health issues rising to the forefront. His role within the premiere was separate somewhat from Jack — we wouldn't quite call them ships passing in the night, but they were in the country with different missions.
Yet, this is where things get interesting — those missions look to intersect. Someone within this country is desperate to cause panic and carnage on a much larger scale, whether this be a world leader trying to assert power or someone willing to create a conspiracy. With the assassination in mind, we've reached now a tipping point — there's no coming back from this quickly at all.
CarterMatt Verdict
The season 2 premiere takes a little bit of time to get going, and there are some parts of it (Jack's romantic rendezvous) that feel superfluous and could have been presented in other ways. Jack is still at times a detached lead who is hard to get to know, but there's no denying that the case feels relevant and the action and drama are both fairly top-notch across the board. This is a heck of a foundation … let's just hope that it leads to a heck of a season.
Related News – Be sure to get some more news when it comes to Jack Ryan right now!
What did you think about the Jack Ryan season 2 premiere overall?
Be sure to share now in the comments, and remember to stick around for some other news on the series. (Photo: Amazon.)
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