After being without their best guard for the past seven games, the Philadelphia 76ers received a piece of good news prior to their Friday night game against the Brooklyn Nets.
#SixersTalk MCW is listed officially as a game time decision however he said he was looking forward to getting back out there
— Dei Lynam (@dlynamCSN) December 20, 2013
Rookie point guard Michael Carter-Williams is expected to return to the lineup on Friday, and even with his extended absence, MCW ranks near the top of many Rookie of the Year ballots at this early stage. Carter-Williams has missed those 7 games as a result of a knee infection, and while Dei Lynam reports (see above) that he is “questionable” for tonight, all indications are that he’ll be back on the court.
His return sets the stage for what should be an incredibly interesting battle between the rookie and a healthy Deron Williams, and that gives a bit more intrigue to what could have been an ugly game. In fact, Carter-Williams makes any game more entertaining, as his flashy, unique style has also resulted in big-time numbers (17.7 points, 7.3 assists, 5.8 rebounds per game) so far in his short career.
In his absence, Philly has been unable to garner a single win, and that is a certain indication of just how valuable the young man is. He may not be at his peak due to their injury and some rust, but it’ll be great to see him on the floor.


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Congratulations to Maureen Scholl, CEO of Exchanges for
Aon Hewitt, for being named as one of Consulting Magazine’s
Top 25 Consultants for 2013.
Through her leadership, Aon Hewitt has been a pioneer in the health exchange industry,
offering a comprehensive portfolio of solutions including a first-of-its-kind multi-carrier health
exchange for large corporations and an industry leading retiree health exchange.
Together with her team, Maureen has helped to redefine health solutions for greater choice,
affordability and wellness, thereby securing a better future for organizations and individuals alike.
Now that’s empowering results!
To learn more about Aon Hewitt, visit us online at

domain: news


text:
Made with zesty lemon juice and fresh cilantro, these hearty and flavorful Chickpea Veggie Burgers are perfect for vegans, vegetarians and meat eaters!
🍔 My favorite food? Sandwiches!
When asked what my favorite kind of food is, after lots of internal debate I often answer "sandwiches." While my favorites are always changing, I never get sick of delicious food sandwiched between two slices of bread. Anything I can hold in my hands and take a big bite out of is right up my alley.
I love all kinds of sandwiches and thankfully there are so many plant-based ways to create a delicious sandwich. From simple sandwiches like peanut butter and jelly to more exciting combinations like lentil sloppy joes or buffalo chickpea salad, the possibilities are numerous!
But one of my favorite types of sandwiches are burgers... veggie burgers! (and we ate TONS of the best veggie burgers in Budapest!)
They're so hearty and filling and depending on what condiments you use, they can take a number of different forms. While I've always loved ketchup and mustard since I was little, I've grown to love veggies and hummus on my burgers, especially this Cilantro Chickpea Veggie Burger!
One thing Brett really misses about the States is being able to eat a burger. While I know I could buy meat here in China, the way its sold (hanging on hooks in the open air) still grosses me out so I've still yet to buy and prepare meat for Brett. Thankfully the produce is abundant here in China so I've been able to whip up some veggie burgers that both Brett and I to enjoy. We can dress them up however we please, giving us completely different flavor profiles, yet we still feel like we're eating the same meal.
These Cilantro Chickpea Veggie Burgers pair beautifully with my Pumpkin Hummus and are even better on my 40-minute homemade burger buns!
I hope you give them a try! If you do, let me know by leaving a comment or taking a picture and tagging #theconscientiouseater on Instagram!
Cilantro Chickpea Veggie Burgers
- 15 ounces chickpeas drained and rinsed
- 1 small bunch cilantro coarsely chopped
- 1 lemon all of the zest, half of the juice
- 2 cloves garlic coarsely chopped
- 1 teaspoon cumin
- ¼ teaspoon salt
- freshly ground black pepper to taste
- crushed red pepper flakes to taste
- 1 flax egg 3 tablespoons of water + 1 tablespoon [ground flaxseed]
- ¾ cups oat flour
- ½ red onion diced
- 2 tablespoons sunflower seeds coarsely chopped
- Preheat your oven to 375 °F and line a baking sheet with a silicone baking mat or non-stick baking paper. Set aside.
- Prepare your flax egg by whisking 1 tablespoon of ground flaxseed with 3 tablespoons of water in a small bowl. Set aside to thicken.
- In a food processor or high speed blender, combine the chickpeas, cilantro, lemon zest and juice, garlic and spices. Process until almost paste-like, but with a little texture.
- Pour the chickpea mixture into a large bowl and add in the flax egg, oat flour, red onion and sunflower seeds and stir until well combined.
- Divide the batter into 4-6 sections, depending on your preference, and use your hands to form the sections of dough into patties about ½ inch thick.
- Place the patties on your prepared baking tray and bake them in the oven for about 30 minutes, flipping half way through, or until the patties reach your desired dryness. Bake the patties longer for dryer, firmer patties or take them out sooner for softer, doughier patties.
- Serve on buns or a bed of lettuce with the toppings of your choice.
Would hemp seeds work instead of sunflower?
Hi Crystal! I haven't tried that, but I don't see why it wouldn't work. Go for it and let me know how they turn out!
Bethany @ Athletic Avocado says
Im always in the mood for a nice thick, filling and flavorful veggie burger like this one!! I love using chickpeas as a binder, and the cilantro adds tons of flavor!
Thick veggie burgers are the best!!
Rebecca @ Strength and Sunshine says
These look like they may be my new favorite chickpea burger! I never put flour in mine except chickpea flour or quinoa flakes once, but I do think it helps hold them together and bulk them up! These look fantastic!
Thanks Rebecca! Chickpea flour would have been perfect for these!

domain: food


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عنوان مقاله [English]
نویسندگان [English]چکیده [English]
According to the importance of careful review of crude oil market fluctuations on the Iranian economy, in this paper a multivariate model of Markov switching vector error correction model (have been used). Variables such as real gross domestic product in industrial sector, real effective exchange rate, real governmental expenditure, real import, inflation rate and real crude oil price is used to detect the asymmetric effects of crude oil market shocks in the expansion and recession phase of the Iran industrial sector by using the seasonal data from years 1368 to 1386. Results show asymmetric behavior of the model variables on the oil price fluctuations in different regimes of the Iran industrial sector, so that with moving from the recession phase to expansion phase of the Iran industrial sector, the negative effect of increase in oil price on the Iranian economy has been increased.
JEL Classifications: E31, E32, G14, Q41, Q43

domain: finance


text:
Fashion inspired Wedding
Photography & Cinematography Services
Wedding photos inspired from fairy tales and Fashion Magazines. Direction and lighting inspired from movies
Piece of art, for your house and an unforgettable experience!
It's time to have a professional photography experience!
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We love people so it helps us to capture their feelings.
Exciting and crazy moments that you will want to remember for a lifetime!
"A love story, with us as heroes"
Your wedding video should be as special as you are!
Articles & News
The art of telling a story is a separate art but is also contained in...
Photography in preparation of the bride, is one of the most difficult parts in a...
Yes the possibilities of Photoshop are endless! As well as the ways you can apply...
The existence of so many recorded emotions and tenderness is the proof that there is...
Contact us to receive our suggestions or make an appointment to meet you and learn more about you and your needs.

domain: artistic


text:
Licensed since 1986
|Association Name||Position Name||Duration|
|Washington State Trial Lawyers Association||N/A||N/A|
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Showcase your speaking engagements, awards, case histories, and more.

domain: law


CONVEY SYSTEM ROLLERS / CHAINS
To complement its wide range of wrapping machines, Pieri is also able to offer conveying systems for palletized loads with roller and chain conveyors. Pieri designs and manufactures motorized roller and chain conveyors, of all dimensions, personalised upon customers request. Depending on the type of the product, PIERI, thanks to its long running experience, is able to propose functional and complete solutions for every requirements. The project is developed according to definite and optimized criteria for a perfect line efficiency: in changing for example the diameter and pitch of the rollers or the height and width of the conveyor. PIERI can offer from floor level conveyors to chain conveyors with 2, 3, 4 or 5 chains. The manufacture of conveying systems uses only high quality materials and components, its steel frame and adjustable feet ensure stability and robustness even during the transportation.


text:
Content with label jpeg2000+jpx+library in The Registry (See content from all spaces)
validation, video, pdf, characterisation, convert, too, jp2k, tool, formatissue, untagged, extraction, characterise, tiff, jp2, java, cli, tools, audio, migration
( - jpeg2000, - jpx, - library )
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text:
Demi Lovato celebrated her 19th birthday this weekend, and she did so with a glam rock-themed b-day bash. The "Skyscraper" singer, seen here with friend Jenna Schubart, rocks a part New York Dolls, part "Sex And The City" studded ensemble. (We're guessing that's the first time those two references ever happened in the same sentence.) According to sources, Demi provided guests with an open Red Bull bar (no alcohol), and party goers who weren't dressed the glam rock part were not admitted. Energy drinks and mandatory glitter? We thought Ke$ha patented that combo.
Also this weekend, Demi tweeted the cover art for her upcoming album Unbroken (below), and we are proud to report that homegirl looks STUNNING. In perfectly touseled hair, a fur vest, and burgundy lips, Demi looks off into the distance in front of a beautiful sunset as if to say, "There are so many No. 1s on this album, you guys don't even know." Or possibly, "Dontcha wish your girlfriend was hot like me?"

domain: celebrity


text:
The world is becoming a shelter for depression, anxiety is increasing. Youth are avoiding gatherings, sports, and outdoor activities to be secluded. Many are withdrawing from active personal, schooling and family life. These are symptoms of psychological bruises or conflicts. Many a time youth are facing a shock in their life, witnessing the corruption and conflicts in and around them. They were having nascent feelings and innocent perceptions about the world, which were challenged by the harsh reality. In the European Union, more than 11 million youth are in depression.
How to cope with depression?
Coping with Anxiety:
Active participation in academic and social life. Keeping the self academically and in social services busy helps to release tension and develop patience.
- Spiritual path gives us an understanding of the world and uplifts us beyond the material world.
- There is a divine world, the world of truth and true happiness and realization of the life beyond the mundane day-to-day activities helps in developing patience.
- Rationalize your expectations, if one keeps the self busy with low expectations then one can enjoy life.
- High expectations make one depressed. Meditation is the process to keep the self engaged positively at the level of thought.
- Forgetting the painful, sinful or abusive moments of the past and to start life afresh with a new beginning and determination.
- Let us kick start life without worries and hurry. Slow and steady wins the race.

domain: sociology


V-NECK PEASANT TOP
This semi-sheer blouse is finished with a touch of sparkle.
- Metallic Dot Ikat Georgette: 99% viscose, 1% metallic
- Made in USA of Italian fabric
- Dry clean
- Length: 25 inches (size S)
- Long blouson sleeves
- Split neck with ties
- Elastic cuffs
- Fifteen Twenty "V-Neck Peasant Top"


text:
Frances Mayes - widely published poet, gourmet cook, and travel writer - opens the door to a wondrous new world when she buys and restores an abandoned villa in the spectacular Tuscan countryside. In sensuous and evocative language, she brings the reader along as she discovers the beauty and simplicity of life in Italy.
This book is one of my favourites. I love the way Frances Mayes writes. She has very lyrical, poetic way with words. So I was quite excited of adding this book to my Audio list.
I lasted less than one paragraph. Believe me I really really tried to like it. The reason I disliked it so much was her voice, it really grates. Her heavy southern American drawl really grates. It is not the sort of voice that goes with such a well wrtten book.
A real shame.
7 of 9 people found this review helpful


text:
Sci fi/Fantasy image byCarolin Roske
I love mandalas. So I made a picture with a mandala. The woman is from Harper's Bazaar. It took me quite some time to colour my drawing, because I used so many shades of colours.
Published More than a year ago
Be the first to favourite this image.
This image is flagged as NSFW

domain: hobby


text:
Celebrating Art & Industry in Melbourne’s west
Be engrossed by the industrial history and creative grunge of Melbourne’s west.
Presented by Hobsons Bay City Council in partnership with Donna Jackson of Hubcap Productions, the 2018 Art & Industry Festival will run from 16 – 25th November.
The Festival has been developed over two years by artists, trades people and industries and will offer ten days of unique experiences that could only take place in Melbourne’s West.
See industrial-inspired fashion by local designers, new suburban hideaways, bars and gallery spaces. Join the Open Day program and take a behind-the-scenes pee at glass making, boat building. Or, go on an art walk, industrial heritage tour, or walk look through an explosives reserve.
The plethora of creative experiences during the Festival includes theatre, visual arts, photography, walking tours, pop-up container cinemas, large-scale projections, talks and live music.
“We live in the industrial heartland of Australia, and it’s a complex environment,” Festival Director Donna Jackson said. “Industry is neither good nor bad, it’s about where we live and it’s the truth of where we live.”
The Festival launch was held at a currently disused glass warehouse in Spotswood, now owned by Cadence Property group, where a select group of people were privy to a sneak peek at the fashion and performances to feature throughout the Festival.
Director of Cadence Property Group, Charlie Buxton, received a round of applause from those attending the launch when he offered a glimpse into the future plans for the glass factory at 1 Hudsons Road, Spotswood. “This building, and others on the site, will be retained and rejuvenated to accommodate the industries of the future, offices, light industries, some retail, some accommodation, hospitality as well as the creative industries,” he said.
The inaugural Art & Industry Festival was held in November 2016 and provided a catalyst for a unique group of partnerships between community groups, local government, industries, unions and small businesses.
The Art & Industry Festival 2018 program is available online and can be downloaded from www.artandindustryfestival.com.au
Ray Horsburgh AM with Donna Jackson and Cr Peter Hemphill; Hobsons Bay Mayor Cr Angela Adair
Costume designer & art teacher Anna Kolusniewski (Aa is for Art): Musician and Art & Industry Festival artist-in-residence James Henry


text:
Original Research ARTICLE
A new mouse avatar model of non-small cell lung cancer
- 1Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
- 2Department of Pathophysiology and Transplantation, Doctorate School in Molecular and Translational Medicine, University of Milan, Milan, Italy
- 3Center for Preclinical Surgical Research, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
- 4Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy
- 5Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy
- 6Division of Thoracic Surgery and Lung Transplantation, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
- 7Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi” (INGM), Milan, Italy
Introduction: Lung cancer remains the leading cause of tumor-related deaths, despite advances in the understanding of the disease pathogenesis and in its clinical treatment. It is crucial to develop novel technologies to discover disease biomarkers and predict individual therapy response.
Materials and methods: We established 48 patients-derived tumor xenografts (PDTXs) implanted in the subrenal capsule of immunodeficient mice using thin, precision-cut tumor tissue slices, derived from five patients affected by non-small cell lung cancer. Twenty-six tissue slices were immediately processed and implanted at sample recovery [patients-derived tumor xenografts derived from fresh tissue (dPDTX)], whereas the remaining sections were cultured on specific organotypic supports at 37°C and 5% CO2 for 24 h before grafting [patients-derived tumor xenografts derived from cultured tissue (cPDTX)]. At sacrifice, xenografts tissue morphology, proliferation (Ki67), and histotype markers were analyzed. Oncogenic miRNAs profiles were assessed in PDTXs, human tumors, and serum from one patient.
Results: Xenografts retained the original cancer features and there were no differences between dPDTXs and cPDTXs. Squamous cell carcinoma (SCC) xenografts showed a higher engraftment rate than adenocarcinoma (AC)-derived tumors. At basal time, Ki67 levels were higher in SCCs than in ACs, and the expression levels of genes associated to a stem cell-like phenotype were also more expressed in SCC samples. The analysis of oncogenic miRNAs showed that circulating miR-19b, -21, and -210 levels were correlated with higher Ki67 expression in xenografts.
Conclusion: Our study implemented the PDTX model with thin, precision-cut tumor slices from small tumors, which could be useful for clinical applications and predictive purposes. The different engraftment success is likely determined by tumor histotype, high proliferation index, and the expression of genes essential for cancer stem cells maintenance. Our PDTXs model could be a valid tool to expand primary tumors for the discovery of new biomarkers and explore therapeutic options.
Lung cancer is the principal cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is the major form, accounting for about 85% of all lung tumors, with three histological subtypes: adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma (1).
Although much effort is focused in early disease detection, NSCLC is often diagnosed at advanced stages, not suitable for surgical resection and characterized by a dismal prognosis. Moreover, despite several advances in our insights of molecular mechanisms of lung tumorigenesis and therapeutic response, the current therapies offer only modest survival benefits and drug resistance commonly arise (2). This failure may at least in part be due to the pathological and molecular heterogeneity characteristic of this disease (3).
It is crucial to develop novel technologies in order to discover new biomarkers and predict individual therapy response. In vivo models able to mimic patients’ malignancies are useful tools to target this need.
The patients-derived tumor xenografts (PDTXs), also called mice avatar, are in vivo models which are based on the graft of human tumor fragments in immunocompromised mice (4). Xenografts retain the histopathological and genetic features of the original cancers making the PDTX a valid method to expand primary tumors, predict cancer response to therapy, and to determine new therapeutic markers (5). However, different factors including tumor histotype, grade, and the site of the transplant can influence the success of the graft. Moreover, tissue fragments must be rapidly processed and the original material is often insufficient (6, 7).
Recently, we perfected an ex vivo platform suitable to culture thin, precise-cut tissue slices from a variety of pathological and normal tissues in a biological context that strictly resemble the original microenvironment (8, 9).
In order to overcome PDTXs limits, we combined PDTX method with our ex vivo tissue culture platform. We established 48 subrenal capsule grafts using thin, precise-cut, 300 μm-thick tumor tissue slices derived from five NSCLC patients. Tissue slices were immediately processed and implanted at the sample recovery [patients-derived tumor xenografts derived from fresh tissue (dPDTX)] or cultured for 24 h and subsequently grafted with the same technique [patients-derived tumor xenografts derived from cultured tissue (cPDTX)].
Materials and Methods
Lung Cancer Specimens
Primary human lung tumors were obtained from five patients who underwent surgery for therapeutic purposes at Fondazione IRCCS Ca’ Granda, Policlinico Hospital (Milan, Italy). The patients’ clinicopathologic features are shown in Table 1. Patients did not receive neo-adjuvant chemotherapy and/or radiotherapy before surgery. Blood samples were collected from two patients. Informed consent was obtained from all patients and the study was approved by the Institutional Review Board of the Fondazione IRCCS Ca’ Granda.
Tissue Slices Preparation
Lung cancer tissue slices (300 μm-thick) were obtained through serial cutting of the individual samples using a vibratome VT1200 (Leica Microsystems, Wetzlar, Germany), as previously described (8). Tissue processing was performed within 20 min after surgical resection. For all specimens, the first tissue slice was collected (baseline sample, T0), formalin-fixed and paraffin-embedded (FFPE) for morphological and immunohistochemical analyses.
Organotypic Tissue Cultures
Tissue slices were cultured as already described (8). Briefly, NSCLC sections were cultured on specific organotypic inserts (Millipore, Darmstadt, Germany) for 24 h at 37°C in a humidified incubator with 5% CO2. Culturing media consisted of Ham F-12 media supplemented with 20% inactivated FBS (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin (Life Technologies), 100 μg/mL streptomycin (Life Technologies), 2.5 μg/mL amphotericin B, and 100 μg/mL of kanamycin (Sigma Aldrich, St. Louis, MO, USA). For each tumor, a tissue slice was harvested after 24 h of culture, FFPE, and used as control [24 h culture control (NTC24)] for morphological and immunohistochemical analyses.
Three hundred micrometer-thick tissue slices were cut into multiple 3 mm × 3 mm pieces and submerged in sterile PBS supplemented with antibiotics (10% Pen Strep; Life Technologies). Forty-eight tissue fragments were grafted under the renal capsule of 6–8 weeks old CD1 athymic mice (Charles River Laboratories, Calco, Italy; one graft/kidney) within 1 h, as previously described (10). Twenty-six tissue slices were immediately processed and implanted at sample recovery (dPDTX) whereas the others were cultured for 24 h and subsequently grafted (cPDTX). At 1.5, 3, and 6 months from the graft, mice were sacrificed and xenografts were collected. Individual tumor volumes were calculated as V = (LWS)/2 where L and W are longer and shorter axis, respectively, and S is the xenograft thickness. L and W were measured using a caliper, whereas S was determined by DMD108 microscope (Leica Microsystems). The variation of tumor size was expressed as the final volume (Vf) and initial volume (Vi = 1.35 mm3) ratio. Tumors were considered engrafted when they retained viable cancer tissue (engraftment rate). Lungs, liver, the contralateral normal kidney, and spleen were harvested and FFPE for evaluation of metastatic foci. For 42 mice, blood sample was collected at sacrifice by cardiac puncture.
Animal care and experiments were performed in accordance with the Principles of Laboratory Animal Care (NIH Publication No. 86-23, revised 1985) and approved by the Local Committee for Experimental Animal Research. Mice were anesthetized by i.p. injection with fresh 2,2,2-tribromoethanol (Avertin, Sigma Aldrich) and sterile practices were followed during the surgical procedure. SCC4 mice were sacrificed after heavy sedation with a dose of 500 mg/kg of avertin by i.p (Sigma Aldrich) at 1.5 and 3 months because they showed pain symptoms with a progressive degeneration of their physical condition starting from the first month after grafting.
Morphological and Immunohistochemical Analyses
In order to assess tissue morphology, hematoxylin and eosin (H&E) staining was performed. Tumor tissues immunoreactivity for Ki67, CK5/6, P63, TTF1, NAP-A, CD31, vimentin (antigen retrieval by EDTA at 95°C for 36 min), or SOX2 (antigen retrieval by citrate buffer at 95°C for 44 min) was analyzed. All primary antibodies except SOX2 (Cell Signaling, Danvers, MA, USA) were from Ventana Medical Systems (Roche Group, Tucson, AZ, USA). Individual primary antibodies were detected using specific secondary antibodies and visualized by 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. Negative controls were prepared in the absence of primary antibody and included in each reaction. Three investigators (Silvano Bosari, Alessandro Del Gobbo, and Stefano Ferrero) independently examined and scored the slides. When differences arose, the cases were reviewed until a consensus was reached. For quantification of proliferative activity, a Ki67 score was determined as the percentage of positive tumor cells. Immunoreactivity for CK5/6, P63, TTF1, NAP-A was quantified as the percentage of positive tumor cells. SOX2 levels were determined using a two-score system for percentage of positive cells and intensity of staining. The staining intensity was expressed in a scale of 0 (absent staining) to 3 (strong staining). Representative images were acquired using a DMD108 system (Leica Microsystems) and contrast/brightness was adjusted using identical settings with Photoshop (Adobe Systems Inc., San Jose, CA, USA).
RNA Purification and Reverse Transcription
Total RNA was purified from FFPE samples of NSCLC tissues and corresponding non-neoplastic lung parenchyma using the MasterPure RNA purification kit (Epicentre, Madison, WI, USA) following the manufacturer’s instructions. RNA from serological specimens of human or murine origin was isolated using miRNeasy serum/plasma kit (Qiagen, Limburg, Netherlands) according to the supplier’s protocol. Before RNA purification, an exogenous small RNA (cel-miR-39; Qiagen) was added to each serum samples for normalization of extraction procedures. RNA was quantified spectrophotometrically. For gene expression quantification, 300 ng of total RNA were reverse-transcribed using the high-capacity cDNA reverse transcription kit (Life Technologies) in a Vf of 20 μL.
Conversely, for miRNAs profiling, 33 ng of total RNA were reverse-transcribed using the TaqMan MicroRNA reverse transcription kit with the Megaplex RT Primers Human Pool A v.2.1 and then preamplified using the TaqMan PreAmp Master Mix with the Megaplex PreAmp Primers Human Pool A v.2.1, according to the manufacturer’ specifications (Life Technologies). For serum miRNAs analysis, cel-miR-39-specific RT primers and probe (192 and 32 nM, respectively; from Life Technologies) were added to the reverse transcription and pre-amplification reactions, respectively.
Real Time RT-PCR (qPCR)
Expression levels of genes and miRNAs were analyzed in duplicate using gene-specific primers and TaqMan probes (Table S1 in Supplementary Material) and the ABI Prism 7900HT sequence detection system (Life Technologies). Targets raw data (Ct values) were converted into relative quantities using GeNorm software and then median-normalized and log2-transformed for statistical analysis (11). β-2 microglobulin (β2M) and actinβ (ACTβ) were used as reference genes for target relative quantification. MammaryU6 and RNU48, or miR-16 and miR-39 were used as reference genes for relative quantification of tissue or serum target miRNAs, respectively. The serum of a 3 and 6 months old mouse without grafts was used as control in order to normalize serum miRNAs contents with respect to the animals’ age.
The association between Ki67 levels and PDTXs volume increase was evaluated by Fisher’s exact test. Data were analyzed using Prism 4.0 (GraphPad Inc., La Jolla, CA, USA). The expression profiles of genes and miRNAs in tissue and serum samples were analyzed using the DNA-Chip Analyser Software (http://www.dchip.org) as previously described (12). The variance of miRNAs levels according to xenografts features, such as volume variation, Ki67 score, duration of grafts, and engraftment success was analyzed by ANOVA test provided within DNA-Chip Analyser Software. Differences among samples group were analyzed using the Mann–Whitney U test (GraphPad Inc.). Volume variation was considered high (H) when it was >1 and low (L) when it was ≤1. Ki67 levels were considered high (H) when Ki67 immunoreactivity was >50% and low (L) when it was ≤50%.
Statistical significance was assumed if the probability value (p) was <0.05.
Establishment of PDTXs
Tumor tissue slices of 300 μm thickness were obtained from five patients affected by NSCLC and used to establish 48 first generation PDTXs, as outlined in Figure 1. At mice sacrifice (1.5, 3, and 6 months after implantation) xenografts, and specifically SCC-derived PDTXs displayed evident vascularization (asterisks in Figure 2A). Morphologic analysis of the primary tumors and the corresponding xenografts revealed that 29 PDTXs closely resembled the original cancer (H&E staining; Figure 2B). Conversely, the remaining 19 xenografts (39.6%) did not retain tumor epithelial compartment resulting in a total engraftment rate of 60.4% (Figure 2C). In particular, PDTXs derived from SCC tumors (SCC-PDTXs) showed a higher engraftment rate than xenografts derived from AC tissues (AC-PDTXs, 95.5 vs. 30.8%). AC-PDTXs retained the tumor epithelial component only at 1.5 months from the graft (Figure 2B), whereas these xenografts showed only stromal tissue at 3 and 6 months from the graft as evidenced by microscopic (H&E staining, Figure 2B) and immunohistochemistry (vimentin staining, Figure S1 in Supplementary Material) analyses.
Figure 2. PDTX establishment from vibratome-generated NSCLC tissue sections. First generation PDTXs were established by implanting 300 μm-thick NSCLC tissue slices under mice renal capsule. (A) Representative macroscopic images of three xenografts at harvesting. Asterisks indicate xenograft superficial vascularization. (B) Tissue morphology was determined by H&E and representative images of SCC and AC-PDTXs are shown. AC-PDTXs exhibited the epithelial compartment only at 1.5 months from the graft. The absence of epithelial cancer cells is evident in AC-PTDTXs harvested at 3 and 6 months. Black arrow heads indicate stromal cells. Original magnification is 200×. (C) Schematic representation of PDTXs engraftment rates. (D,E) Quantification of AC (average of AC2, 3, and 5 samples); (D) SCC1 and SCC4-PDTXs. (E) Volume variation compared to initial tumor volume (Vi) at different time points, respectively. For all experiments, bars represent mean ± SEM.
In regards to the ex vivo cultures for 24 h of NSCLC tissue slices before grafting, no significant difference in engrafting rate or retention of epithelial compartment was evidenced in cPDTXs compared to dPDTXs. Moreover, dPDTXs and cPDTXs exhibited comparable engraftment rates in SCC (91.7 vs. 100%) or AC (28.6 vs. 33.3%) derived grafts, as shown in Figure 2C.
When we analyzed the xenografts volume variation, we observed that tumors growth was higher in SCC-PDTXs (volume variation range: 0.5–363.8) than AC-PDTXs (volume variation range: 0.17–1.36). Indeed, most of the xenografts derived from AC samples (24/26, 92.3%) did not increase their volume compared to their initial mass (Figure 2D). In contrast, 19 (86.3%) xenografts derived from SCC slices increased their mass compared to their original volume (Figure 2E). Interestingly, SCC-PDTXs showed heterogeneity in tumor growth rate, and two patterns characterized by a slow or fast volume increase were identified. Morphological examination of PDTXs revealed that SCC4-derived xenografts presented large necrotic areas, whereas necrosis was absent in SCC1-PDTXs (not shown). Nevertheless, the difference in cell death did not prevent SCC4-PDTXs to have a higher and faster growth rate than SCC1-derived xenografts (Figure 2E).
Finally, we analyzed liver, lung, contralateral kidney, and spleen for presence of distant metastases at animals’ sacrifice. No metastatic foci were detected by macroscopic or microscopic (H&E) analyses (Figure S2 in Supplementary Material).
Retention by Xenografts of the Original Cancer Biological Features
In order to compare primary tumors and corresponding PDTXs, xenografts were investigated for the presence of distinctive histological markers of their original cancer. Therefore, P63 and CK5/6 immunoreactivity was analyzed in xenografts derived from SCC samples (Figures 3A–C), whereas NAP-A and TTF1 stains were investigated in AC-PDTXs (Figures 3D–F). As previously described (8), the levels of distinctive histological markers were preserved after 24 h of ex vivo culture (Figure S3 in Supplementary Material).
Figure 3. Morphological and histological analysis of PDTXs. NSCLC histotypes-specific markers and morphology were analyzed in xenografts. (A–C) SCC-derived PDTXs were analyzed for P63 and CK5/6 immunoreactivity at the indicated harvesting schedules and compared to the corresponding human tissue. Representative images of P63 and CK5/6 staining SCC cases are shown in (A), and are quantified in (B,C). Original magnification 200×. (D–F) Immunoreactivity of AC-PDTXs and corresponding human tissues for NAP-A and TTF1 markers was analyzed, and a representative AC case is shown. (D) Quantification of NAP-A (E) and TTF1 (F) proteins levels in T0, NTC24 samples, and relative xenografts at 1.5 months after the graft. Original magnification 200×. For all experiments, bars represent mean ± SEM.
In xenografts cell type-specific markers, levels were preserved at all harvesting points compared to the original cancer and irrespective of the NSCLC histotype. Moreover, despite a marginal reduction in CK5/6 observed in SCC-cPDTXs at 1.5 months, no significant difference of these proteins levels was observed between dPDTXs and cPDTXs at later harvesting schedules, i.e., 3 or 6 months. TTF1 immunoreactivity was preserved in AC-PDTXs at 1.5 months, whereas NAP-A levels showed a slight reduction in PDTXs tissues that was not statistically significant.
Ki67 levels were analyzed in xenografts, in NTC24 cultures, and in the corresponding human cancer. No loss of tissue proliferative potential was evidenced between baseline (T0) and NTC24 samples. Regarding xenografts, SCC-PDTXs maintained high Ki67 levels compared to their primitive cancer and NTC24 at all time points (Figures 4A,B). No significant difference in Ki67 immunoreactivity was shown between dPDTX and cPDTX derived from SCC samples. Conversely, AC-PDTXs exhibited lower Ki67 levels than their original cancer (Figures 4A,C). Nevertheless, no significant difference was highlighted at 1.5 months after the graft between dPDTX and cPDTX derived from AC samples.
Figure 4. Evaluation of NSCLC tissues proliferative activity. Ki67 immunohistochemical levels were analyzed in PDTXs and in their corresponding human cancer tissues. (A) Ki67 immunoreactivity is shown for SCC- and AC-PDTXs compared to their relative controls and at the indicated time points. (B,C) Representative images of Ki67 staining for SCC- (B) or AC5-NSCLC cases (C) and matched PDTXs. Original magnification 200×. (D) Analysis of Ki67 levels in SCC- or AC-NSCLCs at baseline and after 24 h of ex vivo culture. (E) The association of high proliferative activity (Ki67 >50%) with PDTXs volume variation was analyzed (high volume variation = volume variation >1). ***p < 0.0001 (Fisher exact test). For all experiments, bars represent mean ± SEM.
We therefore looked for differences in proliferative potential in human NSCLC samples that could account for the distinct behavior of SCC-PDTXs respect to AC-PDTXs. SCC tissues showed higher proliferative index than AC samples at baseline (Figure S4 in Supplementary Material) or after 24 h of ex vivo culture (Ki67 score range: 55–80 or 5–20%, respectively; Figure 4D). In particular, SCC4 tumor showed higher Ki67 levels than SCC1 (80 vs. 55% of Ki67-positive cells; Figure S4 in Supplementary Material). Moreover, PDTXs generated from tissues exhibiting an elevated proliferative index (Ki67-positive cells >50%) significantly displayed a higher growth in vivo (volume variation >1, p < 0.0001; Figure 4E).
Expression Analysis of Genes Related to a Stem Cell Phenotype
To gain insights on the different SCC and AC engraftment rates, the expression levels of four stem cell-related genes, namely ALDH1A1, c-MYC, NANOG, and SOX2 (Table S1 in Supplementary Material), were analyzed in T0 and in NTC24 samples. The gene expression analysis identified SOX2 and ALDH1A1 as the most deregulated transcripts in SCC compared to AC samples at baseline and after 24 h of ex vivo culture, with an increased expression of more than 100- or 16-folds in SCC tumors respect to AC. Moreover, SOX2 and ALDH1A1 expression levels were significantly higher in SCC tissues compared to AC and correlated with the engraftment success (p < 0.001 by ANOVA; Figure 5A). c-MYC was also more expressed in SCC than in AC tissues (7- and 36.4-fold differences for T0 and NTC24 samples, respectively), whereas NANOG showed a mild overexpression in AC-NSCLC tissues at baseline (0.5-fold difference) that was not confirmed in NTC24 samples (1.6-fold difference). Finally, c-MYC and NANOG expression levels were not correlated to the engraftment rates.
Figure 5. Analysis of stem cell-related genes in PDTXs and corresponding human tumors. Expression profiles of stem cell-related genes were investigated in SCC or AC samples at baseline. (A) Heatmap of NANOG, c-MYC, SOX2, and ALDH1A1 expression in NSCLC tissues at T0 and after 24 h of ex vivo culture. SOX2 and ALDH1A1 were significantly more expressed in SCC cases, and in PDTXs with successful engraftment (**p < 0.001, by ANOVA). Diagnosis: S, SCC; A, AC. Engraftment: H, high; L, low. (B) Representative SCC and AC cases stained with an antibody directed against SOX2 at T0 and after 24 h of ex vivo cultures (NTC24). Original magnification 200×. (C) SOX2 immunoreactivity is shown for SCC- and AC-PDTXs and their relative controls. For all experiments, bars represent mean ± SEM.
SOX2 protein levels were investigated by immunohistochemistry in control tissues (T0 and NTC24 samples), and in their corresponding xenografts. Confirming gene expression data, SOX2 protein levels were more elevated in SCC than AC samples (Figure 5B). SOX2 expression was maintained after 24 h of ex vivo culture and in xenografts derived from SCC tissue slices (Figure 5C). In contrast, SOX2 was poorly expressed by AC samples, either in T0, NTC24 samples or in their corresponding PDTXs at all harvesting points (Figures 5B,C). No significant differences were demonstrated in SOX2 staining between dPDTX and cPDTX derived from either SCC or AC lung cancers (Figure 5C).
Tissue and Serum miRNAs Expression Analysis
Lastly, to investigate whether our model supported the discovery of new biomarkers, we analyzed in SCC tissues or serum the expression of seven miRNAs (miR-19a, miR-19b, miR-20a, miR-21, miR-31, miR-150, and miR-210) known to be involved in lung cancer (13, 14).
Variation of miRNAs expression levels in SCC-PDTXs tissues (Figure 6A) or serum (Figure 6B) was investigated relative to xenografts proliferation, volume variation, and type and duration of the graft. MiR-19a levels in PDTX tissues were inversely correlated with Ki67 immunoreactivity (p = 0.03 by ANOVA; Figure 6A). Whereas tissue levels of miR-19b, -20a, -21, and -31 significantly decreased at 6 months compared to the earlier PDTX harvesting points (p = 0.05, p = 0.004, p = 0.01, and p = 0.03 by ANOVA, respectively; Figure 6A). Globally, tissue miRNAs expression levels showed a general downregulation in PDTXs compared to baseline T0 and NTC24 samples.
Figure 6. Analysis of NSCLC-related miRNAs in PDTXs, corresponding human tumors, and serum. Expression levels of miRNAs involved in lung cancer progression were analyzed in PDTXs or human tumor tissues and serum derived from SCC patients. (A) Heatmap of SCC1, SCC4, and their relative PDTXs tissues analyzed for miRNAs expression levels. miR-19a levels were inversely correlated with Ki67 immunoreactivity (*p = 0.03 by ANOVA). Tissue levels of miR-19b, -20a, -21, and -31 significantly decreased at 6 months compared to the PDTXs harvested at 1.5 and 3 months (respectively *p = 0.05, **p = 0.004, *p = 0.01, and *p = 0.03 by ANOVA). (B) Heatmap of SCC1- and SCC4-PDTXs serum analyzed for miRNAs expression levels. miR-19b, miR-20a, and miR-31 were significantly higher in 1.5 months old PDTXs (respectively *p = 0.02, **p = 0.007, and **p = 0.003, by ANOVA), and miR-21 and -210 levels were correlated with PDTXs proliferative activity (respectively *p = 0.03, *p = 0.02 by ANOVA). (C) PDTXs with high Ki67 levels (>50%) exhibited significantly high circulating miR-19b and -21 levels (*p = 0.04 and *p = 0.03 by Mann–Whitney U test, respectively). For all experiments, bars represent mean ± SEM. V. Variation, volume variation; H, high; L, low; C, graft after 24 h culture; D, direct graft.
Serological analysis of the aforementioned miRNAs documented that circulating miR-21 and miR-210 levels were differentially modulated according to PDTXs proliferative activity (p = 0.03, p = 0.02 by ANOVA, respectively; Figure 6B). Conversely, serological levels of miR-19b, miR-20a, and miR-31 significantly decreased at the latter PDTX harvesting point compared to the 1.5 and 3 months schedule (p = 0.02, p = 0.007, and p = 0.003 by ANOVA, respectively; Figure 6B), mimicking what observed for these small regulatory RNAs in PDTXs tissues (Figure 6A).
In particular, PDTXs with a Ki67 immunoreactivity >50% showed significantly higher expression of circulating miR-19b and miR-21 levels (p = 0.04 and p = 0.03 by Mann–Whitney U test, respectively; Figure 6C).
No other correlations could be evidenced between miRNAs and PDTX features.
The improvement of in vivo models that closely mimic primary NSCLC and that recapitulate tumor heterogeneity at molecular and morphological level is a crucial element to pursue a personalized therapeutic approach and to discover new targets for innovative treatments. In this context, the PDTXs experimental system represents a noteworthy opportunity. Furthermore, in vivo models of human tumors could provide novel prognostic biomarkers besides contributing to innovative therapeutic targets discovery and testing.
To address these ambitious goals, technical procedures must be optimized and standardized to ensure feasibility and reproducibility of PDTX generation from small tumors, the majority of currently resected NSCLCs. In addition, the possibility to use samples from primary tumors in a deferred setting, and not immediately after surgery, could strongly expand the usefulness of tumor xenografts.
In this scenario, our study implemented the PDTX model by implanting 300 μm-thick, precision-cut NSCLC tissue slices under the renal capsule of CD1 athymic mice and by ex vivo culturing procedures of tumor sections before grafting. PDTX models are entering the research laboratories to study tumor biology (10, 15, 16) and anticancer therapy (4, 17), for different malignancies. To date, scarce data are available regarding the establishment of PDTX from precision-cut tissue slices (18, 19) and the majority of previously reported NSCLC xenograft models were established from minced tissues (10, 20–22). Our model has the potential to provide several improvements compared to PDTX derived from minced tumor tissues. Reduced tissue thickness might improve the proper exchange of nutrients and drugs. Furthermore, tissue slices obtained with vibratome sectioning reduces tissue injury compared to mincing and allows the achievement of implantable tissues with accurate and uniform dimensions (18). With the precision-cut technique presented here, more tumor sections from the same samples can be obtained, compared to the tissue mincing technique. Lastly, tissue sectioning by vibratome ensures a much greater reproducibility of the implanted xenografts in different animals and allows greater consistency of the tumor cell population present in the serial sections.
The possibility to culture ex vivo tumor sections for 24 h before engrafting allows to delay graft without causing tissues damage, loss of tumor viability, or alterations of morphological features.
Patients-derived tumor xenograft models show three principal applications. First, they are useful to test new anticancer drugs in a context that considers both a patient-derived tumor tissue and the in vivo situation. Second, PDTX method is appropriate to identify biomarkers of response to therapies and, third, it could support the delineation of a personalized therapeutic algorithm. Indeed PDTXs may allow the preclinical evaluation of patients’ tumor sensitivity to specific therapies in vivo.
One limitation of the previously described techniques is the use of minced tumor tissues or of tumor pieces. These procedures require a larger amount of tumors than precise-cut tissue slices and, moreover, they did not grant a controlled or homogeneous tissue grafting among the different receiving mice per human tumor. The availability of large tumors is expected to decrease due to early prevention and the use of neo-adjuvant regimens before surgery. Furthermore, this topic is particularly relevant in lung cancer since advanced disease is not surgically treatable.
Our study improves the use of PDTXs models because it allows the generation of serial xenografts starting from small tumor masses. The establishment of PDTXs from small tumors is useful for clinical applications where tissues availability is limited. Moreover, PDTX obtained from thin organ slices are more homogeneous and this may facilitate their implantation compared to minced specimens.
Lastly, the possibility to culture tumor sections for 24 h grants a time-frame that can be exploited to modulate important signaling pathways or to delay grafting to a more convenient moment.
The data obtained from the established 48 NSCLC-PDTXs show that PDTX tumors reproduce the histological features and the histotype distinctive markers of patients’ primary cancers up to 6 months after the graft. Moreover, tissue culture does not affect PDTX ability to preserve original tumors’ characteristics nor does it affect engrafting rate, PDTX growth, or xenografts’ proliferative potential.
Pertaining the implantation site of PDTXs under the renal capsule, our data confirm that this is suitable to obtain vascularization of the xenograft. As described in the literature, the renal capsule improves tumor engraftment due to the development of rich vascularity, which is a key feature for graft survival especially during the initial phase after implantation (16). The use of thin tumor tissue slices has improved this procedure, and the presence of vascularization was noted initially in all SCC- and AC-PDTXs, irrespective of the final engraftment success.
However, our data suggest that host-mediated vascularization of the PDTX is not sufficient for graft survival and expansion. In fact our results show that PDTX growth is more dependent on the proliferative ability of the primary cancer. Moreover, other core elements such as stem-related factors may be involved in adaptation of the xenograft in the host microenvironment, determining a successful engraftment.
We also show that the tumor histotype is importantly involved in PDTX survival and expansion. As previously reported in literature in different settings (23, 24), our model highlighted that SCC samples show a higher engraftment ability than AC specimens. Indeed, SCC samples that showed elevated Ki67 levels generated SCC-PDTXs that retained a high proliferative index and increased tumor volume.
Albeit limited by the small number of primary tumors used in this study, no correlation was observed between the engraftment rate and other clinical features, such as lymph node status, tumor grade, and size. These data indicate that tumor ability to engraft and survive is more often associated with elevated proliferative potential rather than aggressive features such as presence of nodal metastases. Future studies with larger clinical series are needed to confirm these conclusions.
However, to date, the main determinants affecting PDTX success rate are still unknown. In order to understand other factors involved in tumor engraftment, we analyzed the expression levels of four stem cell-related genes at baseline levels in this set of human NSCLCs. SOX2 and ALDH1A1 expression were more elevated in SCC specimens than in AC tissues. In particular, SOX2 levels were retained by SCC-PDTXs and lost by AC-PDTXs compared to their controls. SOX2 is a stem cell transcription factor that plays a crucial role in the regulation of embryonic development and it is one of the genes involved in reprograming human somatic cells to pluripotent stem cells (25). Overexpression of SOX2 has been described in lung cancer, in particular in SCC, where it is frequently amplified and promotes cancer progression (26). Our data show that this stem-factor is overexpressed at either the gene or protein level in SCC-PDTXs, therefore suggesting that SOX2 favors tumor cell survival and adaptation to different microenvironments, eventually sustaining tumor growth.
Lastly, in order to investigate the suitability of our model for novel molecular biomarkers discovery, we analyzed tissue and serum miRNAs expression in PDTXs and corresponding human SCC tissue. As a proof of concept, we analyzed miRNAs involved in lung cancer.
Human miRNAs were preserved in murine hosts and could be detected in serum and tissue of our PDTXs model. In particular, circulating miR-19b, -21, and miR-210 levels were directly correlated with PDTXs proliferation. As a member of the miR-17-92 cluster, miR-19b is an oncogenic key factor present in different types of cancer (27, 28). Recently, miR-19b has been shown to induce tumor growth and metastasis in vivo (29). Moreover, circulating miR-19b levels have been associated with a worse disease progression in patients affected by NSCLC (30). miR-21 acts as an oncogenic miRNA and is involved in the regulation of cell cycle, apoptosis, invasion, and metastasis. Recent studies demonstrated that miR-21 promoted cell proliferation (31, 32) and its inhibition induced a cell cycle arrest at G2/M phase (31). MiR-210 is a hypoxia-inducible miRNA in lung cancer and melanoma (33) and it has been detected in exosome in vitro and in vivo (34). Moreover, serological miR-210 levels were significantly upregulated in NSCLC patients compared to healthy controls and were correlated with an advanced disease (35). In line with the literature, we found increased circulating miR-210 levels in PDTX characterized by elevated tumor proliferation.
These results suggest that our model could be a useful tool for biomarker discovery, allowing time-course investigations over several months of tumor engraftment and growth.
Overall, our study indicates that PDTXs established from precise-thick tissue slices represent a valid tool to investigate cancer biology and to discover novel biomarkers. Our results highlight the importance of PDTX models to achieve a personalized therapeutic approach with in vivo analysis and the monitoring of tumors response to anticancer agents.
Author contributions included: work concept and design (MR, AF, SG, VV, SB), data acquisition (MR, AF, DR, AG, AP, SF), analysis (MR, AF, SG, ADG, SF, AP), and interpretation (MR, AF, SG, AG, SF, VV, SB), drafting of the manuscript (MR, AF, SG, VV), editing of the manuscript (AF, VV, SB), approval of final content for journal submission and publication (MR, AF, SG, AG, SF, DR, AP, VV, and SB).
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank Fabio Ambrosetti for outstanding technical assistance. This work was supported by the Italian Minister of Health (GR2011-02351626 to VV), by Fondazione Cariplo (2010-0846 to SB), and by Italian Minister of University and Research (Grant CTN01_00177_817708 “DNA on Disk” to SB).
The Supplementary Material for this article can be found online at http://www.frontiersin.org/Journal/10.3389/fonc.2015.00052/abstract
AC, adenocarcinoma; DAB, 3,3′-diaminobenzidine; FFPE, formalin-fixed and paraffin-embedded; H&E, hematoxylin and eosin; NSCLC, non-small cell lung cancer; NTC24, 24 h culture control; PDTXs, patients-derived tumor xenografts; cPDTXs, patients-derived tumor xenografts derived from cultured tissues; dPDTXs, patients-derived tumor xenografts derived from fresh tissues; RQ, relative quantity; SCC, squamous cell carcinoma; T0, baseline time; Vf, final volume; Vi, initial volume.
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Keywords: NSCLC, patients-derived tumor xenografts, tissue slices, organotypic culture, miRNA
Citation: Russo MV, Faversani A, Gatti S, Ricca D, Del Gobbo A, Ferrero S, Palleschi A, Vaira V and Bosari S (2015) A new mouse avatar model of non-small cell lung cancer. Front. Oncol. 5:52. doi: 10.3389/fonc.2015.00052
Received: 08 December 2014; Accepted: 13 February 2015;
Published online: 03 March 2015.
Edited by:Victoria Virador, Virador and Associates, USA
Reviewed by:Vilma A. Sardao, Center for Neuroscience and Cell Biology, Portugal
Lori D. Dwyer-Nield, University of Colorado, USA
Copyright: © 2015 Russo, Faversani, Gatti, Ricca, Del Gobbo, Ferrero, Palleschi, Vaira and Bosari. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Valentina Vaira, Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, via F. Sforza 35, Milan 20122, Italy e-mail: email@example.com;
Silvano Bosari, Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, via F. Sforza 35, Milan 20122, Italy e-mail: firstname.lastname@example.org
†Maria Veronica Russo and Alice Faversani are co-first authors and have equally contributed to this work.
‡Valentina Vaira and Silvano Bosari are co-last authors and have equally contributed to this work.

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This year, the Novemberfest Talent show will take place after school during Family Day. The hosts of the show will be NCS parents, Melanie Beiter, Laura Bray and Kristen Leniz. The show will take place on Wednesday, November 14th from 12:30 - 2:30pm under the tent on the main stage!
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OFFICE ACTG. ASST. PROVOST-MARSHAL-GENERAL,
Montpelier, Vt., June 18, 1863.
Respectfully forwarded, and recommend that a military force may be sent to Rutland.
T. G. PITCHER,
Brigadier-General and Actg. Asst. Provost-Marshal-General.
[JUNE 19, 1863.- For correspondence relating to raising troops in Delaware, see Schenck to Cannon, Series I, Vol. XXVII, Part III, p. 221.]
HEADQUARTERS OF THE ARMY,
Washington City, D. C., June 19, 1863.
Major General A. E. BURNSIDE,
GENERAL: I have recently conversed with several prominent citizens of Ohio and Indiana, including Governor Morton, and they are unanimously of the opinion that the policy of appointing district commanders in those States is injurious. They say that it conveys to the people an idea that they are being subjected to military constraint; that difficulties are multiplied between the civil and military authorities, the former taking offense at the assumption of the latter; that the public feeling is becoming strong that these military commanders, with their numerous staffs, had much better be in the field fighting the enemy than in exercising unnecessary military authority in the loyal States.
I know that representations to the same effect have for some time been made to the War Department, and I think it would be well for you to consider this matter and the propriety of withdrawing these district commanders and leaving the control, as much as possible, in the hands of the Governors and civil authorities. For example, why not leave Governor Morton, with the commanding officer at Indianapolis, to manage affairs in that State and put General Willcox and his staff in the field? I know that Willcox is an excellent officer, but he is a stranger in Indiana and is not likely to understand their political squabbles. The Governor has made no complaints of him or of any one else, but he does not see any necessity for his being there at all. Moreover, there are daily applications for more generals in the field, with recommendations innumerable to make new ones, for which there are no vacancies. Now, if generals can be spared for the unimportant duties of commanding districts in the loyal States, where there are only a handful of troops, they should be sent to other armies in y wanted.
I write this simply as a suggestion for your consideration.
Very respectfully, your obedient servant,
H. W. HALLECK,
WASHINGTON, June 19, 1863.
MY DEAR SCHOFIELD: The only force I will have in many places to enforce the enrollment and draft will be the Invalid Corps.
25 R R-SERIES III, VOL III


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Any opinion on changing all the traits to their corresponding tag?
e.g. instead of "Cursed" -> Cursed
There was a section on Plants at the bottom, but when bringing up the edit page that section was not visible. After editing the page to show the Advanced monsters that section disappeared. Since the monsters are more important I decided to do it anyway.


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Onshore flow is keeping the coast foggy Thursday morning, which means another cool day for the beaches. On Wednesday, the marine layer mixed out by late morning for Avila Beach And Shell Beach but stayed foggy over the Five Cities. It's possible there could be a similar trend Thursday due to the onshore flow remaining under a ridge of high pressure.
Daytime highs at the coast will range from the mid-50s to mid-60s with cooler conditions toward the north coast. For the coastal valleys, peak temperatures will stay between the mid-60s and mid-70s as the inland valleys could heat up to the 80s and 90s. The microclimates will be very apparent Thursday as a wide range continues to separate the peak temperatures of coastal and inland valley communities.
Happy #FridayJunior! I'm checking on our peak temperatures from Wednesday and found that San Miguel reached 101 degrees! Meanwhile, Morro Bay reached 56 degrees. A 45-degree difference separated by 37 miles... wild! @KSBY— Brooke Martell (@BrookeMartell) June 3, 2021
High pressure aloft remains which will allow for the warmer temperatures to play out across the interiors. Over the weekend, the ridge will weaken and a trough from the Pacific Northwest will bring some influence to the region in the form of cooler daytime highs. As for the coastal areas, not much will change as June Gloom persists.

domain: geography


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We usually bomb the tag a couple of times a month. But selfies are welcome wherever!
Feel free to send us as many as you want :)
I am a child of the Americas,
a light-skinned mestiza of the Caribbean,
a child of many diaspora, born into this continent at a crossroads.
I am a U.S. Puerto Rican Jew,
a product of the ghettos of New York I have never known.
An immigrant and the daughter and granddaughter of immigrants.
I speak English with passion: it’s the tongue of my consciousness,
a flashing knife blade of crystal, my tool, my craft.
I am Caribeña, island grown. Spanish is my flesh,
Ripples from my tongue, lodges in my hips:
the language of garlic and mangoes,
the singing of poetry, the flying gestures of my hands.
I am of Latinoamerica, rooted in the history of my continent:
I speak from that body.
I am not African. Africa is in me, but I cannot return.
I am not taína. Taíno is in me, but there is no way back.
I am not European. Europe lives in me, but I have no home there.
I am new. History made me. My first language was spanglish.
I was born at the crossroads and I am whole.
did you know that Mexico is
So you’re basically saying only Mexican women who have white features are worthy of being called beautiful and of representation.
RIGHT THO. RIIIIGHHTTT. BLESS THIS POST.
Greetings everyone! My name is Alex and I am a member of Girl Fry, a latin@, queer punk band based out of Los Angeles.
I am here to ask for your help.
We need a drummer.
Ours informed us the other night that she can no longer devote as much time as before to the band and that she needs to focus on her own personal goals. We respect that and wish her the best of luck in her endeavors.
That being said, she has agreed to finish up the album with us and play a show in May. After May we will need a new drummer, especially for the summer tour in August.
The requirements are the following:
(Girls and feminine persons strongly encouraged to audition!)
If you are interested, please email us at firstname.lastname@example.org or via our Facebook.
Thank you for your time.


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The Meroe Pyramids mark the southern extreme of Egyptian influence as you follow the course of the Nile river south towards its source. After the glories of the New Kingdom had faded, the great Egyptian empire fell into an era of in-fighting which culminated in the Balkanisation of the realm into smaller (usually warring) entities – one of which was the great Meroitic dynasty that ruled this area of the Nubian Desert from 592 BC to 350 AD. Although the pyramid tombs they left behind are nowhere near the gargantuan majesty of the Great Pyramids of Saqqara, Dahshur and Giza (Cheops’ Pyramid is 146.5 metres tall, the Meroe Pyramids barely make double figures), they are still definitely worth a day trip, situated four hours north of Khartoum.
However, this being Sudan, getting there is only half the problem. To travel anywhere or take photos of anything (you can forget about video) you need written permission from the Ministry of Tourism, Antiquities and Wildlife. The reason for this (and the reason I wasn’t allowed to make the trip from Gallabat to Khartoum under my own steam) is that the leader of Sudan is a wanted war criminal. That is all I have to say on the matter, although in general I will use this opportunity to reiterate my belief that if you’re going to have a revolution, revolt, war, insurrection, coup d’etat whatever, your best bet for a freer, wealthy, more egalitarian state would be to rid your country of not just the President, but the whole godforsaken institution of the Presidency entire. Three words for you my friend: Parliament, Parliament, Parliament. All else will follow.
There has been a time in pretty much every republic of Planet Earth (including the US, fact fans!) has had a known criminal in charge of the government, military and country. This is not the best of all possible worlds, believe me.
After dropping his daughters at school, Mr. Mohammed took me to the Ministry of Tourism, Antiquities and Wildlife and we got my application processed, culminating in me having ten copies (for any police checkpoint that wanted them) of my permission to travel to and take pictures of the Meroe Pyramids. Overkill? Well, I guess it depends where you’re coming from. For a North Korean, I’m sure it would feel like a breath of fresh air. For me, I felt like I was in North Korea.
Mr. Mohammed was good enough to drive me over to the Atbara bus station, and from there I jumped the next bus headed north. It took a good four hours to get to the Pyramids, and I arrived at around 2.45pm, the only person getting off the bus at this point along the lonely highway. It was then a 700m hike across the stony desert to the Pyramids themselves.
“Had the place to myself. Felt very Indiana Hughes.”
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It’s a testament to Sudan’s burgeoning tourist industry that I was the only person there. Maybe if they didn’t make it nigh on impossible to get a visa…
I left the site at around 5.30, the sun hanging low in the sky. I stood at the side of the road for an age with my thumb out waiting for somebody to stop, which thankfully they did before the sun actually set. Only they didn’t take me to Khartoum, or even to Shendi, the nearest outpost of civilisation. No, they dropped me at a deserted desert rest stop. The people there (predictably) wanted me to stay the night, but that wasn’t going to happen: I had to be on the bus to Wadi Halfa in the morning – I needed to get back to Khartoum.
So I started walking along the highway, firm in the belief that somebody – anybody – would stop and pick me up. They didn’t. It grew dark. I walked for over an hour. Eventually I came to another rest stop and started asking around. One guy wanted 100 Sudanese pounds to get back to Khartoum. Although this equated to about $20, I thought it fairly reasonable, I was tired and I wanted to get back as soon as possible. But then he pulled the old ‘Oh no, I meant 100 dollars’ shite that I’ve had to put up with so many times it has definitely ceased being funny anymore, not that it ever was in the first place.
Eventually I found a trucker willing to take me to the next town, Shendi. He wanted 100 Sudanese pounds too, but considering Shendi is not even halfway back to Khartoum, I flat refused to pay anymore than £50. That didn’t stop him attempting to dive his hand into my shirt pocket every ten minutes in a crappy attempt to grab another fifty. I arrived at Shendi around 9pm and waited at the police checkpoint for the next bus to Khartoum. Incredible: my last night in Sub-Saharan Africa and I finally find a use for all these damn police checkpoints – the buses have to stop!
It was 11.10pm before I got back to Khartoum. Mr. Mohammed, being a good soul, came to pick me up from the drop-off point, bringing his son Hamed with him. That night I grabbed a couple of hours sleep on the roof of Mr. Mohammed’s apartment block. There was a cool winter’s breeze, a portent of what was to come as I climb the latitudes north-north-west.


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It’s officially been a year since the first Covid-19 cases were reported in the United States, and most of us are ready to see any semblance of the “normal” we knew before this pandemic. Of course, the most essential way to get through this pandemic is by getting vaccinated as soon as possible. Since we last reported, (check out our previous article here about Covid-19 and vaccines) many advancements have been made in the vaccine rollout, however much delayed. Following the election and President Biden’s inauguration, the new administration is pushing forward to get as many Americans vaccinated as possible.
On Tuesday, January 26th, President Biden announced that the government will be buying 200 million more vaccine doses, a move to fully inoculate 300 million Americans by the end of the summer. The government is in the process of getting 100 million doses from Pfizer/BioNTech and 100 million from Moderna. This would be in addition to the 400 million doses the companies have already committed to providing for Americans. Tuesday marked the grim milestone of over 100,000,000 cases worldwide, with the United States reporting 25% of them. These numbers are a stark reminder of the reality of the pandemic and the Biden administration is responding with substantial measures during this “wartime” as Biden has called it. Employing the Defense Protection Act, the administration has begun buying more of a “special syringe that can extract more doses per vaccine vial, and it plans to use the wartime law for other raw materials, like lipid nanoparticles and bioreactor bags, if necessary,” according to a senior administration official. However, these materials are rare and there is a risk of disrupting the production of other healthcare products and international competition.
The U.S. Food and Drug Administration began distributing the first supply of Covid-19 vaccines in the U.S. on December 14th, 2020. During the initial period of distribution, Phase 1a, the vaccine was allocated to health care personnel and long term care residents and staff. Illinois entered Phase 1b on Monday, January 25th after the substantial completion of the first phase. This new phase now includes people aged 65 and older, as well as frontline workers. According to the state, 3.2 million residents fall under Phase 1b. For a more detailed outline of exactly who can get vaccinated in what phase, refer to the chart below.
Chicago Department of Public Health Commissioner Dr. Allison Arwady announced potential dates for the remaining phases. According to her, these dates include:
Phase 1C (essential workers and residents aged 16 and older with underlying health conditions): March 29
Phase 2 (All Chicagoans aged 16 and older): May 31
Dr. Allison Arwady also added “I want to make very clear that date is totally dependent on how many vaccines we get, what changes we may see with the federal government, but broadly, we’re thinking Phase 1B, we’ll be working through these groups over about two months,” Arwady said. “And then opening beyond that, and then looking ahead to Phase 2, which is really when a vaccine is available to all Chicagoans.”
Despite these pushes to distribute and administer vaccines as soon as possible, state and local officials have been complaining that they lack the adequate supply of vaccines to fuel the high demand from the public for shots. On Tuesday, New York Mayor Bill de Blasio said on MSNBC that the city has the capacity to administer 500,000 doses a week but hasn’t been able to do so because it is waiting on more vaccine supplies from the federal government. Federal officials say that they do not fully understand the reason for these delays but several factors can be attributed. Primarily, states have been holding back doses to be given out to nursing homes and other long-term care facilities. Only 8% of the vaccines designated to these centers have been distributed.
The demand for vaccines dipped over Christmas and New Year’s Day since many people took days off work and clinics also had reduced hours. Another critical factor for the rollout delay, according to public health experts, is that federal officials have left details of the final stage of vaccine distribution (scheduling and staffing) to local health officials and hospitals which are already overburdened by responding to the pandemic. All states are administering vaccines at different rates; for example, West Virginia finished giving the first round of doses to residents and workers at all of the state’s 214 long-term care facilities on Wednesday. Operation Warp Speed officials are expecting the rollout rate to increase once pharmacies including Costco, Walmart, and CVS begin to administer vaccines.
Additionally, an email was sent out to the staff and student body last week that WTHS has now been approved to conduct Covid-19 rapid tests. These tests are available for students or staff members who report or display symptoms while on campus, assuming consent is provided by a parent or guardian. For steps on how to give consent, see here. If the person tests negative, they will be directed to get a PCR Molecular test to confirm the result, or need to get an alternative diagnosis from their doctor. These PCR tests are available at most doctor offices, and for more information refer here. If the test is positive, however, then the person will have to isolate and follow protocol to exit the building and seek medical treatment safely.
With the new and potentially more contagious variants from the UK, South Africa, and Brazil, it is more important than before to wear double or upgraded masks, socially distance, and wash your hands. If you are an eligible individual to receive a vaccine, as outlined in Phases 1a and 1b, you can find the nearest vaccination site here. Additionally, for more detailed information about the Illinois Covid-19 vaccination plan, please reference here. And finally, for an interactive map of vaccination tracker in each Illinois county, click here.


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Reflection Art Competition is a WA state PTA and National PTA program that starts at school level and is one of the key programs sponsored by SES PTSA. Each year students are invited to create original works of art around a theme. Each piece is evaluated by a local artist and/or art educators. Top scoring works advance to the district level with the opportunity to advance to the state and even national level.
The theme for the 2022-23 is Show your voice! All students are invited to submit work on this theme within 6 art categories. Entries must be original work of one student and it must be created for this year’s competition. Please look over the rules for the requirements, it will not advance to the next level. Submissions with completed entry forms can be turned in online by November 18th 2022.
Remember: The theme matters! Judges will use the artist statement to take into account how the artist has interpreted the theme.
Special Artist Division: Students with disabilities may receive special accommodations by entering the special artist division and are encouraged to enter any category of their choice.
While the primary focus of Reflections is a celebration of creativity through positive recognition, finalists from local PTAs will move on to their council PTA (if applicable), then finalists from councils will move on to the state round. Washington State PTA finalists who receive an “Outstanding Interpretation” award will advance to the National PTA round. National PTA Reflections awards can be found here.
Reflections entries will be submitted digitally again this year. If an entry moves on to the National level a physical entry may be needed, so please keep them!
Entries will also be displayed on the bulletin board in school.
ENTRY SUBMISSION DEADLINE: November 18, 2022.
ENTRY SUBMISSION OPEN!
To submit an entry for the 2022-2023 Reflections contest, please complete the google form at the following link. Entries can be directly uploaded in the Google form.
– OR –
Complete the student entry form below and email it along with your file to firstname.lastname@example.org.
Art Education Program
Did you know that the art program at Snoqualmie Elementary is primarily funded by the PTSA? Thank you to all the families who participate in our fundraising efforts and allow students to receive 5 art lessons a year!
Smart With Art, in partnership with the SES PTSA, will work together for the 2022-2023 school year to bring visual arts education to the young artists at Snoqualmie Elementary. Sponsoring art education is one of the regular programs the PTSA supports each year with donations and fundraising from its members, parents, and the community.
Students are encouraged to express their own unique styles as they explore a variety of art mediums and learn about historical art movements. Each project focuses heavily on the CREATIVE PROCESS with emphasis on developing skills and understanding applicable art elements and design principles while working toward a specific objective.
Each grade level will receive 5 sessions of art covering 5 art mediums: drawing, painting, printmaking, fiber arts and metal working.
Family Fun Events
The SES PTSA plans, organizes, and runs numerous family events throughout the year! These fun events are designed to promote family and community involvement and provide a fun environment for our children to learn, grow, and develop relationships with other students. Some of these events include:
- Cougar Cub Club (pre-kindergarten meetup)
- Boo-Hoo Coffee
- Back-to-School Ice Cream Social
- Book Fair
- Family STEM Night
- Bingo Night
- Field Day
For more information on upcoming Family Fun Events, please see our calendar or follow our Facebook Page!
Room Parents play a key role in connecting families with their class and the SES community. They help the teacher coordinate classroom events and parties and help support the teacher. Room Parents are also periodically asked to assist the PTSA with event communications and teacher appreciation activities.