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Words… Words… Words: My Writing Process Bloghop
Last week, Vidya Sury tagged me in her post On Writing
For me, the entire writing experience begins with words!
I LOVE words ! Words have feel… sound… nuance… texture…
I love the many facets of words ~ the push and pull… the ebb and flow… the literal and figurative… the foreign and familiar… clarity and ambiguity… evocative and provocative… I could go on and on…
Words have a life of their own! They can elevate you to dizzy heights and then drop you to the depths of despair. Such is the power of words!
Reading is also important. I was a reader LONG before I became a writer… and the two belong together. I've been wondering about the inextricable link between writing and reading. In the art of reading and writing is it:
a) the only people who read anymore, are writers… OR
b) today's readers are the undiscovered writers of tomorrow…
What are your thoughts on this?
Now on to the questions:
What am I working on/writing?
For this blog hop, I'm going to focus on flash fiction. I'm busy with a collection of 26 musically-inspired pieces/vignettes, posted during the 2012 A to Z Challenge
How does my work/writing differ from others in its genre?
I'm not too sure. Maybe those of you who are familiar with my writing can tell me in the comments below…?
Why do I write what I do?
For those of you who have NEVER attenpted flash fiction before – you have NO IDEA what you're missing out on!! It's loads of fun!
It's literary bungee jumping/ scuba diving (not that I've bungeed or had the deep sea diving experience…)
…an exciting, heady, addictive, adrenalin-pumping experience… for sure!
I think the perfect answer is best expressed in flash fiction form… And if you are a COFFEE LOVER (most writers are), then you'll identify even more…
"Imagine you are sipping coffee. Not cappucino. None of that froth. No flat whites: flatness is banned. This is espresso. In a teeny cup. All that flavour, darkness, fizz and jolt, smooth notes, a full scale — no less, no more. Finish your coffee. Put down the teeny cup. Sit and wonder. Sit and feel how you are altered. Stand up. Go on with your day."
–Tania Hershman (UK)
How does my writing process work?
These flash pieces have been inspired by prompts. I enjoy word prompts/photo prompts. First – read the prompt for the day. Second – allow my imagination to go off at a tangent, as I consider the connotation of the word.
I'll use an example from the five-sentence fiction pieces. The word prompt – ORANGE. Sometimes a picture accompanies the word… sometimes not. If there's no picture/photo, I sometimes look around for one, to enhance the entire process.
I toy around with all the words that come to mind – fruit, sunset colour, squishy and juicy, citrus family, sweetish acidic juice, lying between red and yellow on the colour spectrum, orange plants, aloes, etc. By the process of elimination, I try to get rid of all the ones that sound boring. This initial exercise gets the creative juices going. Then I turn to the unusual ways of using the word. Instead of using the normal 'fruit association', what about orange-ish coloured hair? I let that idea simmer for a day or so. I imagine that a person with orange hair would be bold/daring/risk-taker… more of an extrovert. I let the ideas simmer further…
As the piece begins to develop/unfold in my mind I try to make it exciting by incorporating the following:
~~ Sentence variation. This depends on the prompt and is also guided by my mood. It can range from five long sentences in one piece; or five short sentences in one piece; or long and short combined. I've discovered that use of a one-word, two-word or three-word sentence really packs a punch.
~~ I strive for a specific mood/tone in the piece.
~~ Use of the senses. At least one or two…
The topic of varying your sentence lengths was discussed in my previous post HERE
So the final product would look something like this:
The six-month-long search reached its finale.
Ten candidates strutted their stuff, with styles that ranged from downright ridiculous to over-the-top.
A little-known, pixie-faced girl, sported a riotous bulk of waist-length wild hair, interlaced with frizzy orange curls, like aloes in desperate need of attention.
Au naturelle.
Unknowingly, a trendsetter-in-the-making, blazing a new trail, leading the way on an unknown journey, taking the fledgling company to greater heights.
So there you have it! That's my writing process. What's yours? Have you written any flash fiction? Did you enjoy writing it?
* * * * *
I now have the privilege of tagging three lovely ladies, inviting them to talk about why they write and their writing process. Here's what they will do:
Answer the four questions on their blog next week June 17/18 and then tag three writers/bloggers of their choice, to answer these questions on writing, on their own blogs the following week. (Because this bloghop has been doing the rounds for a long, long time, there may not be any bloggers left to tag)
The questions are:
1. What am I working on/writing?
2. How does my work/writing differ from others in its genre?
3. Why do I write what I do?
4. How does my writing process work?
The blogger buddies I am tagging are –
Trisha Farnan of Word + Stuff
Trisha was born in 1980 (everyone can do their own maths there) in England, not far from the border with Scotland. Her parents are New Zealanders but they all live in Western Australia now. She has a brother who lives in Canada.
Trisha loves to travel to far-away exotic climes, but she always loves to come home. Cats are her favourite animal, and she tends to talk to them a lot. She also talks to inanimate objects. She is an animal lover in general, and greatly admires ants' ability to carry burdens MUCH heavier than themselves. Other loves of Trisha's include the Smashing Pumpkins, wine and Bailey's, Asian food, and thunderstorms.
Trisha writes songs, plays guitar, and sings. Her band's debut album was released in 2013.
Jen Chandler of The Cup And Page
Jen Chandler has been writing since she was first able to clasp her grubby little hands around a pencil. Jen chisels words out of thin air on a dialy basis, hoping soon they will fall into some semblance of pattern and produce a novel. She's tried her hand at many different genres but calls fantasy and magical realism home.
When she's not writing, Jen can be found reading, gardening, drinking copious amounts of tea, sewing, drawing and singing ridiculous songs to her cat. Her favorite sport is falling down rabbit holes and she has been known to see dragons in the vegetable garden. Jen blogs at The Cup and Page and ensures that there are gluten-free brownies for all who wander by for a visit.
Marie Abanga of Marie Abanga
l have 3 boys for now and they are my musketeers. I have a gentleman in my life right now but it was not always the case. I have a good job now but I do not intend to stop at that no, I am working myself towards leaving a mark on this earth when I depart. My principles are D, D and D- Determination, Discipline and Dedication! Life has not always been easy, it still is not. The truth now is, I am much more tougher than I was years ago and so all the more ready to live courageously and fiercely till the end. I am hungry, I am brave, I am not giving up and it ain´t over until I win or die.
* * * * *
By the end of a long evening working as a special set of eyes for the presidential security detail, all Kat Marengo wants is to kick off her shoes and stash two not-really-stolen rings in a secure spot. Plus, maybe sleep with Dave Krizak. No, make that definitely sleep with Dave Krizak. The next morning, she wishes her new top priorities were so simple.
98 Comments
Lovely Flash Fiction, Michelle! I like to write it in my journal. It's always great to find the gems and not so gems later and wonder what prompted them.
Wow! Love the sizzle in your words . . . all just from orange. Thank you. I like to write some flash, and I've even been experimenting with "hint" fiction lately. Hint fiction is 25 words or less. It's like just taking a tiny sip of explosive fiction. (sorry, I don't think I can match that awesome quote you have up there, but I thought I would try). I'm going to bookmark this post – love it and your thoughts on flash!
Aw Michelle,
I waited up late to read your post and be sure l will be up to the task. Sure l will just do it my Way and l think l will survive just like l did the A to Z challenge. Thanks for tagging me and let's see what l put up come next week. l may go out of line but heck, that's my own writer's journey right?
Kind regards, Marie author of My Unconventional Loves
Even though I write sparsely, I've never been happy with the flash fiction I've tried. It's very impressive 🙂
I think your flash fiction is amazing. Mine is truly pathetic. I love your writing proces and the amount of thought you put in constructing your work. You make everday things into art.
I did some flash fiction for Siv Ottem's book release, The Ash Tree. I wrote a quick story on Freya. It was super fun. What I liked best about it was just writing and not editing much. Just get the story out. Set up the problem, add a little drama/action, and wrap it up. I like the espresso coffee description of flash fiction.
My writing process – I practice "Thinkology" like in "The Music Man." In other words, I daydream a lot!!
Great post.
I am in awe of your process for flash fiction. I don't even think that hard for my novel work. (Maybe I should…?)
I love flash fiction, so I enjoyed your post. Thanks for participating in my cover reveal, too. Much appreciated!
I love coffee., too. Have a cup for me since it's on my no-no list.
Hey Michelle,
Good grief, what a long post! Yikes 🙂 You know, I agree with you on the varying length of sentences. Mix it up a bit. It's good. Of course, a really long sentence can also have the impact of making you wonder if the sentence will ever end and you start to realise you've been holding your breath reading the overly long sentence.
Thanks for your FLASH fiction. Now that I know it's not something rude. LOL
A worthy notation of links. Thanks for that.
Gary 🙂
Impressive. I need to try more prompt writing. Maybe if I do it the way you do, it will be easier.
Cute graphic! I have to say though, I know a lot of people who read and don't write. The two are definitely not entangled.
I love your flash fiction pieces! You make it sound so exciting. I should do this more, just for fun. No wonder yours are so good though. Makes sense now. ^_^
While I was blissfully asleep, you were busy, Michelle! Love your post. And I love flash fiction – my English teacher would be so proud! Similar to 55, right? Mmm.
What I love about your writing is is instant connection you make with your reader and leave them smiling!
So glad I tagged you on this blog hop. Such a great topic, right? Hugs! Love the "badge" for your writing process. I enjoy picture prompts too because they leave so much to our interpretation. I also like the idea of looking for a pic to match a word prompt to get the creativity flowing.
Thank you! I am now looking forward to reading the posts of those you tagged!
Hi Michelle .. love your Orange take … I can see that a new frothy beginning … while the process can mull around in the head can't it … interesting three you've tagged – I know Trisha .. but the others are new .. I must get over to meet them ..
Carol's book – shines out so brightly … like her photo … and I sure hope does well … cheers Hilary
I love the way you ponder a word before you write.
Congrats to Carol.
Michelle – I'm in awe of your disciplined and obviously effective process. I learn so much from you, and you connect us with so many others in the blogging community. This is truly an inspiring post!
I've written my share of flash fiction and it was an exciting challenge. Writing it is so different from writing novels. The thrill of actually finishing and revising in a day or two is almost addictive versus the loooong plooood of novel writing. I enjoyed your post – have a great week!
I've never tried writing flash fiction, but it sounds like fun!
Flash fiction. In one of my writing classes in college we worked on it and we read books full of it. I had never paid attention to it befire, but I really liked it, and it encouraged me to write some. It was such fun. I might start up again 🙂
My picture didn't work 🙁 Boooo.
Thanks for tagging me 🙂 I'm trying to find people who I can tag now in turn. 😛
I find flash fiction very intimidating. I like how you explain your process. It gives me hope I can try it some day.
Wow, what an inspiring legacy. I think your approach to writing is very impressive. About reading, my husband reads on average 10 books a month. His love of reading inspires me on those not so productive days.
Great post! Flash fiction, eh?
You know, I used to love FF… I did a lot before. Several a week for challenges on a writing forum. But I've not done any for a couple of years, if not longer. That's shocking of me. I've book marked this. Great post, and scintillating FF. I love how you highlighted sentence length, too. Never thought about it consciously, but I do it instinctually I think… kind of move stuff around until it hits the right beat in my head. Would be better to do that consciously though, come to think of it. 🙂
Wonderful flash fiction! Ive tried shorts before but nothing like that! You paint such lovely word pictures and it is obvious you love each word you choose :). Thanks for sharing your process with us and thanks for the tag! My turn? Yikes!
I love your flash fiction. I would like to write more of it. I usually do them for blog hops. They're fun and challenging.
Flash fiction is a lot of fun and a great way to practice writing as well. I know I wouldn't be the writer I am today without writing so much flash fiction and other shorter works.
Wahoo! I love Carol's cover. Seriously. And flash fiction is a pet love. I haven't written any for a while, but I do adore it. If I put my mind to it, I could be good at it. In the meantime I'll stick to my novels. =)
Hey, MIchelle,
I like the idea of toying with words and trying to do something different with them. No doubt, that's why you create such interesting short fiction.
I know plenty of readers who don't write.
Words can be so powerful and it's so irritating when I can't think of the right one. Quit hiding, I tell it. Does it listen? No. So I'm grateful for the online thesaurus
What a great flash piece prompted by orange. As you know, i love prompts, but I haven't tried them for flash fiction yet. Maybe someday. 🙂
I haven't done much flash fiction – even less now that I'm out of the classroom and working in Spec Ed. I find my stories tend to take over and soon they're not flash fiction any more 🙂
I do enjoy writing flash fiction. It stretches your writing ability to be able to tell a full story in brief. Fun example you posted!
Great answers! I know plenty of people who read with no intention of ever writing. Maybe secretly they dream of it? I don't know…they just seem content reading, and some are near retirement with still no mention of wanting to write.
I just love posts like this where I can learn a writer's process – I figure the more of them I know, the more mine will gel. Also, I get the chance to meet some of your friends!
Also, it's so good to see Carole's book everywhere! I wish here the best of luck.
Tina @ Life is Good
On the Open Road! @ Join us for the 4th Annual Post-Challenge Road Trip!
A huge part of me wishes I hadn't become a writer, LOL… because it's ruined reading for me!!!! I wish I could just get swallowed up in a book like I used to…
And LOVE the Carol highlight and the other three gals. So great, Michelle. And also, thank you for your kind words on my blog today. They certainly help. 🙂
Very inspirational post! This is why I came back 🙂
I like your writing process. It's nice to read how others work, because it helps me with my own stuff. Great post, Michelle. 🙂
Hi Michelle, I simply LOVED the flash fiction. It was super. And I also enjoyed reading about your writing process. I fell in love with words as a kid and the love for words made me want to write my own stories.
I struggle with flash fiction. Sometimes I think my mind just isn't geared for stories that short. 🙂
Have I mentioned lately that you're the Queen of flash fiction?! It takes a lot of patience, and skill to make each sentence sail smoothly onto the next.
Flash fiction is a lot of fun. Thanks for the reminder that as writers we can give ourselves permission to play with words. I am encouraging folks to write about their life stories on my blog for the next few weeks and one of the exercises is to write about your life using only six words. There's a prompt for ya'. Thanks for explaining how you go about writing your flash fiction. Great info here.
That's a lovely piece of Flash, Michelle. I've been asked to be tagged on this bloghop too, but with everything I need to hold down, I just haven't said yes, yet. Great job with yours, and I know all the authors you tagged– would enjoy getting to know them even better 🙂
Interesting post.
I write flash fiction, specifically drabbles, every Tuesday for the FB3X Drabble Cascades. I know what you mean about words, and short fiction does really put them into perspective, because every single one counts. I feel a lot freer in drabbles to experiment as well, play with grammar (and in some cases ignore it).
I thought your approach to developing your fic was interesting, letting it simmer, a considered approach. I find with drabbles I'm a lot less considered than I am with longer work – I tend to jump in where angels fear to tread ;P. I think it's because I know I have time to write another one if another idea occurs to me and I'm like a kid with a new toy, I just wanna try the idea out. That has led me to write a whole drabble and then practically change or rearrange every word in it before I was done with it though :).
wow, mish! jam packed and seal wrapped!
i enjoyed it all! and you are the queen of flash, able to write about anything and make us feel it in so few words!
Flash fiction is fun! For years I was unable to write short stories, and I thought I would never be able to, but then I got the idea for one, wrote it, and since then several ideas have come to me. I'm glad they have, too. 🙂
I really enjoy flash fiction (reading and writing) although I haven't done any for too long! The challenge of working with prompts is fun because there are so many ways it can go. You really did a great job of creating an intriguing character in just a few lines with this story.
I love that your process starts with straight words. Each one packs a different emotion, and when you've got the right combo it makes for a powerful piece – like the one you posted here!

subset: commoncrawl


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Get count of lines in scripts (shell)
If you have tried to get the count of lines in file, the you would know about "nl" or "wc -l". But as you are aware these give you number of lines with other details as well and you need to post process the number to make sure that you have only number and nothing else. In such cases, it is useful to use the count feature of grep and here is a shorthand to get the count of lines in any shell script:
lines=$(grep -c . <filename>)


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Apparently the default settings for .htaccess files and URL rewrites in Ubuntu (8.04 anyway) is kinda jacked. Apache wasn't seeing my .htaccess files, and even after setting up the configuration correctly, I was getting Internal Server (500) errors from .htaccess files.
The default setting for Apache2 on Ubuntu for .htaccess is "ignore it". You need to enable it by going to /etc/apache2/sites-available/default and changing the AllowOverride directive, which you can find inside the
AllowOverride ALL
If you want to enable URL rewrites, you need to enable the mod_rewrite module as it is not enabled by default:
[11:31:14] vivin@dauntless ~/Projects/www/skyblue
$ sudo a2enmod rewrite
Module rewrite installed; run /etc/init.d/apache2 force-reload to enable.
[11:31:20] vivin@dauntless ~/Projects/www/skyblue
$ sudo /etc/init.d/apache2 force-reload
* Reloading web server config apache2


text:
In this issue of the JCI, Horng et al. observed a protective response to inflammation in the glia limitans of mice and in cultured human astrocytes. Increases in blood-brain barrier permeability provoked tight junction formation in glia limitans astrocytes, creating an inducible barrier to leukocyte and humoral infiltration. On the cover, primary human astrocytes (red) corral CD3+ T lymphocytes (blue) within a network of tight junctions after exposure to proinflammatory IL-1β. Image credit: Candice Chapouly.
Type 1 diabetes (T1D) is a chronic autoimmune disease that causes severe loss of pancreatic β cells. Autoreactive T cells are key mediators of β cell destruction. Studies of organ donors with T1D that have examined T cells in pancreas, the diabetogenic insulitis lesion, and lymphoid tissues have revealed a broad repertoire of target antigens and T cell receptor (TCR) usage, with initial evidence of public TCR sequences that are shared by individuals with T1D. Neoepitopes derived from post-translational modifications of native antigens are emerging as novel targets that are more likely to evade self-tolerance. Further studies will determine whether T cell responses to neoepitopes are major disease drivers that could impact prediction, prevention, and therapy. This Review provides an overview of recent progress in our knowledge of autoreactive T cells that has emerged from experimental and clinical research as well as pathology investigations.
Alberto Pugliese
Coronary revascularization is an effective means of treating ischemic heart disease; however, current therapeutic revascularization strategies are limited to large caliber vessels. Because the mammalian heart scars following cardiac injury, recent work showing that cardiac fibroblasts can transdifferentiate into new coronary endothelium raises a new and exciting approach to promoting endogenous revascularization following cardiac injury. In this issue of the JCI, He et al. report on their employment of a battery of lineage-tracing tools to address the developmental origins of fibroblasts that give rise to new endothelial cells. Surprisingly, cardiac fibroblasts did not appear to contribute appreciably to regeneration of cardiac endothelium. Instead, cardiac endothelial cells were likely to proliferate and generate new endothelium following injury. As these conclusions diverge from prior findings, additional work will be required to understand the sources that generate cardiac endothelium in new blood vessels after injury. Clarification of the origins of coronary endothelial cells during cardiac repair is essential for identifying improved approaches to revascularizing damaged myocardium in patients with ischemic heart disease.
Ravi Karra, Agoston O. Walter, Sean M. Wu
Leukotrienes are proinflammatory lipid mediators that have been shown to be upregulated in several diseases, including asthma, aspirin-exacerbated respiratory disease (AERD), inflammatory bowel disease, and acute respiratory distress syndrome. Leukotrienes have been explored as therapeutic targets for these diseases and others; however, leukotriene inhibitors have had limited success in the clinic. There are noted differences in the incidence of leukotriene-mediated diseases in males and females, but sex as a factor in the response to leukotriene inhibitors has not been fully explored. In this issue of the JCI, Pace and colleagues present evidence that there are sex-specific differences in the effectiveness of certain leukotriene inhibitors and link the differences in response to the presence of androgens. The results of this study indicate that sex needs to be taken into consideration in the future evaluation of leukotriene inhibitors to treat disease.
Lewis J. Smith
The blood-brain barrier (BBB) and the glia limitans serve to prevent the migration of cells and other large molecules from the blood into the CNS. Neuroinflammatory diseases are characterized by disruption of the BBB and increased leukocyte infiltration into the CNS. In this issue of the JCI, Horng and colleagues demonstrate that astrocytes of the glia limitans induce tight junction formation in response to inflammatory cues, thereby tightening the border to limit the number of activated T cells infiltrating the CNS. Moreover, preventing the formation of this inducible barrier in mice increased disease severity in models of neuroinflammation. Together, the results of this study indicate that the inducible barrier of the glia limitans should be further explored as a therapeutic target.
Francisco J. Quintana
Proteinopathies are characterized by the accumulation of misfolded proteins, which ultimately interfere with normal cell function. While neurological diseases, such as Huntington disease and Alzheimer disease, are well-characterized proteinopathies, cardiac diseases have recently been associated with alterations in proteostasis. In this issue of the JCI, Fang and colleagues demonstrate that mice with cardiac-specific deficiency of the co-chaperone protein BCL2-associated athanogene 3 (BAG3) develop dilated cardiomyopathy that is associated with a destabilization of small HSPs as the result of a disrupted interaction between BAG3 and HSP70. Together, the results of this study suggest that strategies to upregulate BAG3 during cardiac dysfunction may be beneficial.
Wataru Mizushima, Junichi Sadoshima
Atherosclerosis is a chronic inflammatory disease, and developing therapies to promote its regression is an important clinical goal. We previously established that atherosclerosis regression is characterized by an overall decrease in plaque macrophages and enrichment in markers of alternatively activated M2 macrophages. We have now investigated the origin and functional requirement for M2 macrophages in regression in normolipidemic mice that received transplants of atherosclerotic aortic segments. We compared plaque regression in WT normolipidemic recipients and those deficient in chemokine receptors necessary to recruit inflammatory Ly6Chi (Ccr2–/– or Cx3cr1–/–) or patrolling Ly6Clo (Ccr5–/–) monocytes. Atherosclerotic plaques transplanted into WT or Ccr5–/– recipients showed reduced macrophage content and increased M2 markers consistent with plaque regression, whereas plaques transplanted into Ccr2–/– or Cx3cr1–/– recipients lacked this regression signature. The requirement of recipient Ly6Chi monocyte recruitment was confirmed in cell trafficking studies. Fate-mapping and single-cell RNA sequencing studies also showed that M2-like macrophages were derived from newly recruited monocytes. Furthermore, we used recipient mice deficient in STAT6 to demonstrate a requirement for this critical component of M2 polarization in atherosclerosis regression. Collectively, these results suggest that continued recruitment of Ly6Chi inflammatory monocytes and their STAT6-dependent polarization to the M2 state are required for resolution of atherosclerotic inflammation and plaque regression.
Karishma Rahman, Yuliya Vengrenyuk, Stephen A. Ramsey, Noemi Rotllan Vila, Natasha M. Girgis, Jianhua Liu, Viktoria Gusarova, Jesper Gromada, Ada Weinstock, Kathryn J. Moore, P'ng Loke, Edward A. Fisher
Non-muscle–invasive bladder cancer (NMIBC) is a highly recurrent tumor despite intravesical immunotherapy instillation with the bacillus Calmette-Guérin (BCG) vaccine. In a prospective longitudinal study, we took advantage of BCG instillations, which increase local immune infiltration, to characterize immune cell populations in the urine of patients with NMIBC as a surrogate for the bladder tumor microenvironment. We observed an infiltration of neutrophils, T cells, monocytic myeloid-derived suppressor cells (M-MDSCs), and group 2 innate lymphoid cells (ILC2). Notably, patients with a T cell–to-MDSC ratio of less than 1 showed dramatically lower recurrence-free survival than did patients with a ratio of greater than 1. Analysis of early and later time points indicated that this patient dichotomy existed prior to BCG treatment. ILC2 frequency was associated with detectable IL-13 in the urine and correlated with the level of recruited M-MDSCs, which highly expressed IL-13 receptor α1. In vitro, ILC2 were increased and potently expressed IL-13 in the presence of BCG or tumor cells. IL-13 induced the preferential recruitment and suppressive function of monocytes. Thus, the T cell–to-MDSC balance, associated with a skewing toward type 2 immunity, may predict bladder tumor recurrence and influence the mortality of patients with muscle-invasive cancer. Moreover, these results underline the ILC2/IL-13 axis as a targetable pathway to curtail the M-MDSC compartment and improve bladder cancer treatment.
Mathieu F. Chevalier, Sara Trabanelli, Julien Racle, Bérengère Salomé, Valérie Cesson, Dalila Gharbi, Perrine Bohner, Sonia Domingos-Pereira, Florence Dartiguenave, Anne-Sophie Fritschi, Daniel E. Speiser, Cyrill A. Rentsch, David Gfeller, Patrice Jichlinski, Denise Nardelli-Haefliger, Camilla Jandus, Laurent Derré
Programmed death-1–directed (PD-1–directed) immune checkpoint blockade results in durable antitumor activity in many advanced malignancies. Recent studies suggest that IFN-γ is a critical driver of programmed death ligand-1 (PD-L1) expression in cancer and host cells, and baseline intratumoral T cell infiltration may improve response likelihood to anti–PD-1 therapies, including pembrolizumab. However, whether quantifying T cell–inflamed microenvironment is a useful pan-tumor determinant of PD-1–directed therapy response has not been rigorously evaluated. Here, we analyzed gene expression profiles (GEPs) using RNA from baseline tumor samples of pembrolizumab-treated patients. We identified immune-related signatures correlating with clinical benefit using a learn-and-confirm paradigm based on data from different clinical studies of pembrolizumab, starting with a small pilot of 19 melanoma patients and eventually defining a pan-tumor T cell–inflamed GEP in 220 patients with 9 cancers. Predictive value was independently confirmed and compared with that of PD-L1 immunohistochemistry in 96 patients with head and neck squamous cell carcinoma. The T cell–inflamed GEP contained IFN-γ–responsive genes related to antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance, and these features were necessary, but not always sufficient, for clinical benefit. The T cell–inflamed GEP has been developed into a clinical-grade assay that is currently being evaluated in ongoing pembrolizumab trials.
Mark Ayers, Jared Lunceford, Michael Nebozhyn, Erin Murphy, Andrey Loboda, David R. Kaufman, Andrew Albright, Jonathan D. Cheng, S. Peter Kang, Veena Shankaran, Sarina A. Piha-Paul, Jennifer Yearley, Tanguy Y. Seiwert, Antoni Ribas, Terrill K. McClanahan
An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein β-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific β-arrestin 2 deficiency did not affect hepatic insulin sensitivity or β-adrenergic signaling. Adult mice lacking β-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of β-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic β-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes.
Lu Zhu, Mario Rossi, Yinghong Cui, Regina J. Lee, Wataru Sakamoto, Nicole A. Perry, Nikhil M. Urs, Marc G. Caron, Vsevolod V. Gurevich, Grzegorz Godlewski, George Kunos, Minyong Chen, Wei Chen, Jürgen Wess
In rheumatoid arthritis (RA), immunological triggers at mucosal sites, such as the gut microbiota, may promote autoimmunity that affects joints. Here, we used discovery-based proteomics to detect HLA-DR–presented peptides in synovia or peripheral blood mononuclear cells and identified 2 autoantigens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), as targets of T and B cell responses in 52% and 56% of RA patients, respectively. Both GNS and FLNA were highly expressed in synovia. GNS appeared to be citrullinated, and GNS antibody values correlated with anti–citrullinated protein antibody (ACPA) levels. FLNA did not show the same results. The HLA-DR–presented GNS peptide has marked sequence homology with epitopes from sulfatase proteins of the Prevotella sp. and Parabacteroides sp., whereas the HLA-DR–presented FLNA peptide has homology with epitopes from proteins of the Prevotella sp. and Butyricimonas sp., another gut commensal. Patients with T cell reactivity with each self-peptide also had responses to the corresponding microbial peptides, and the levels were directly correlated. Furthermore, HLA-DR molecules encoded by shared-epitope (SE) alleles were predicted to bind these self- and microbial peptides strongly, and these responses were more common in RA patients with SE alleles. Thus, sequence homology between T cell epitopes of 2 self-proteins and a related order of gut microbes may provide a link between mucosal and joint immunity in patients with RA.
Annalisa Pianta, Sheila L. Arvikar, Klemen Strle, Elise E. Drouin, Qi Wang, Catherine E. Costello, Allen C. Steere
Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. It has shown promise as a cancer therapeutic in preclinical studies and early-phase clinical trials. Here, we have identified anthrax toxin receptor 1 (ANTXR1) as the receptor for SVV using genome-wide loss-of-function screens. ANTXR1 is necessary for permissivity in vitro and in vivo. However, robust SVV replication requires an additional innate immune defect. We found that SVV interacts directly and specifically with ANTXR1, that this interaction is required for SVV binding to permissive cells, and that ANTXR1 expression is necessary and sufficient for infection in cell lines with decreased expression of antiviral IFN genes at baseline. Finally, we identified the region of the SVV capsid that is responsible for receptor recognition using cryoelectron microscopy of the SVV-ANTXR1-Fc complex. These studies identify ANTXR1, a class of receptor that is shared by a mammalian virus and a bacterial toxin, as the cellular receptor for SVV.
Linde A. Miles, Laura N. Burga, Eric E. Gardner, Mihnea Bostina, John T. Poirier, Charles M. Rudin
The mechanisms that promote the generation of new coronary vasculature during cardiac homeostasis and after injury remain a fundamental and clinically important area of study in the cardiovascular field. Recently, it was reported that mesenchymal-to-endothelial transition (MEndoT) contributes to substantial numbers of coronary endothelial cells after myocardial infarction. Therefore, the MEndoT has been proposed as a paradigm mediating neovascularization and is considered a promising therapeutic target in cardiac regeneration. Here, we show that preexisting endothelial cells mainly beget new coronary vessels in the adult mouse heart, with essentially no contribution from other cell sources through cell-lineage transdifferentiation. Genetic-lineage tracing revealed that cardiac fibroblasts expand substantially after injury, but do not contribute to the formation of new coronary blood vessels, indicating no contribution of MEndoT to neovascularization. Moreover, genetic-lineage tracing with a pulse-chase labeling strategy also showed that essentially all new coronary vessels in the injured heart are derived from preexisting endothelial cells, but not from other cell lineages. These data indicate that therapeutic strategies for inducing neovascularization should not be based on targeting presumptive lineage transdifferentiation such as MEndoT. Instead, preexisting endothelial cells appear more likely to be the therapeutic target for promoting neovascularization and driving heart regeneration after injury.
Lingjuan He, Xiuzhen Huang, Onur Kanisicak, Yi Li, Yue Wang, Yan Li, Wenjuan Pu, Qiaozhen Liu, Hui Zhang, Xueying Tian, Huan Zhao, Xiuxiu Liu, Shaohua Zhang, Yu Nie, Shengshou Hu, Xiang Miao, Qing-Dong Wang, Fengchao Wang, Ting Chen, Qingbo Xu, Kathy O. Lui, Jeffery D. Molkentin, Bin Zhou
The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4–containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.
Ya-Ting Wang, Jocelyn Chen, Chou-Wei Chang, Jayu Jen, Tzu-Yu Huang, Chun-Ming Chen, Roger Shen, Suh-Yuen Liang, I-Cheng Cheng, Shuenn-Chen Yang, Wu-Wei Lai, Kuang-Hung Cheng, Tao-Shih Hsieh, Ming-Zong Lai, Hung-Chi Cheng, Yi-Ching Wang, Ruey-Hwa Chen
Angioimmunoblastic T cell lymphoma (AITL) represents a distinct, aggressive form of peripheral T cell lymphoma with a dismal prognosis. Recent exome sequencing in patients with AITL has revealed the frequent coexistence of somatic mutations in the Rho GTPase RhoA (RhoAG17V) and loss-of-function mutations in the 5-methylcytosine oxidase TET2. Here, we have demonstrated that TET2 loss and RhoAG17V expression in mature murine T cells cooperatively cause abnormal CD4+ T cell proliferation and differentiation by perturbing FoxO1 gene expression, phosphorylation, and subcellular localization, an abnormality that is also detected in human primary AITL tumor samples. Reexpression of FoxO1 attenuated aberrant immune responses induced in mouse models adoptively transferred with T cells and bearing genetic lesions in both TET2 and RhoA. Our findings suggest a mutational cooperativity between epigenetic factors and GTPases in adult CD4+ T cells that may account for immunoinflammatory responses associated with AITL patients.
Shengbing Zang, Jia Li, Haiyan Yang, Hongxiang Zeng, Wei Han, Jixiang Zhang, Minjung Lee, Margie Moczygemba, Sevinj Isgandarova, Yaling Yang, Yubin Zhou, Anjana Rao, M. James You, Deqiang Sun, Yun Huang
The WD40-containing E3 ubiquitin ligase RFWD3 has been recently linked to the repair of DNA damage by homologous recombination (HR). Here we have shown that an RFWD3 mutation within the WD40 domain is connected to the genetic disease Fanconi anemia (FA). An individual presented with congenital abnormalities characteristic of FA. Cells from the patient carrying the compound heterozygous mutations c.205_206dupCC and c.1916T>A in RFWD3 showed increased sensitivity to DNA interstrand cross-linking agents in terms of increased chromosomal breakage, reduced survival, and cell cycle arrest in G2 phase. The cellular phenotype was mirrored in genetically engineered human and avian cells by inactivation of RFWD3 or introduction of the patient-derived missense mutation, and the phenotype was rescued by expression of wild-type RFWD3 protein. HR was disrupted in RFWD3-mutant cells as a result of impaired relocation of mutant RFWD3 to chromatin and defective physical interaction with replication protein A. Rfwd3 knockout mice appear to have increased embryonic lethality, are subfertile, show ovarian and testicular atrophy, and have a reduced lifespan resembling that of other FA mouse models. Although RFWD3 mutations have thus far been detected in a single child with FA, we propose RFWD3 as an FA gene, FANCW, supported by cellular paradigm systems and an animal model.
Kerstin Knies, Shojiro Inano, María J. Ramírez, Masamichi Ishiai, Jordi Surrallés, Minoru Takata, Detlev Schindler
BACKGROUND. Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients. METHODS. A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions. RESULTS. Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti–type VII collagen autoantibodies in patients' blood or skin. CONCLUSION. Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations. TRIAL REGISTRATION. ClinicalTrials.gov NCT02698735. FUNDING. Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.
David T. Woodley, Jon Cogan, Yingping Hou, Chao Lyu, M. Peter Marinkovich, Douglas Keene, Mei Chen
Current anti-VEGF therapies for colorectal cancer (CRC) provide limited survival benefit, as tumors rapidly develop resistance to these agents. Here, we have uncovered an immunosuppressive role for nonclassical Ly6Clo monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following VEGF signaling blockade, resulting in recruitment of CX3CR1+Ly6Clo monocytes into the tumor. We also found that treatment with VEGFA reduced expression of CX3CL1 in endothelial cells in vitro. Intravital microscopy revealed that CX3CR1 is critical for Ly6Clo monocyte transmigration across the endothelium in murine CRC tumors. Moreover, Ly6Clo monocytes recruit Ly6G+ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6Clo monocyte or Ly6G+ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, a gene therapy using a nanoparticle formulated with an siRNA against CX3CL1 reduced Ly6Clo monocyte recruitment and improved outcome of anti-VEGFR2 therapy in mouse CRCs. Our study unveils an immunosuppressive function of Ly6Clo monocytes that, to our knowledge, has yet to be reported in any context. We also reveal molecular mechanisms underlying antiangiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies.
Keehoon Jung, Takahiro Heishi, Omar F. Khan, Piotr S. Kowalski, Joao Incio, Nuh N. Rahbari, Euiheon Chung, Jeffrey W. Clark, Christopher G. Willett, Andrew D. Luster, Seok Hyun Yun, Robert Langer, Daniel G. Anderson, Timothy P. Padera, Rakesh K. Jain, Dai Fukumura
BACKGROUND. Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2–inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. FUNDING. The National Cancer Institute.
Meixiao Long, Kyle Beckwith, Priscilla Do, Bethany L. Mundy, Amber Gordon, Amy M. Lehman, Kami J. Maddocks, Carolyn Cheney, Jeffrey A. Jones, Joseph M. Flynn, Leslie A. Andritsos, Farrukh Awan, Joseph A. Fraietta, Carl H. June, Marcela V. Maus, Jennifer A. Woyach, Michael A. Caligiuri, Amy J. Johnson, Natarajan Muthusamy, John C. Byrd
The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4–/– mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.
Samuel Lessard, Emily Stern Gatof, Mélissa Beaudoin, Patrick G. Schupp, Falak Sher, Adnan Ali, Sukhpal Prehar, Ryo Kurita, Yukio Nakamura, Esther Baena, Jonathan Ledoux, Delvac Oceandy, Daniel E. Bauer, Guillaume Lettre
Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity–dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.
Jia Wang, Peng Cheng, Marat S. Pavlyukov, Hai Yu, Zhuo Zhang, Sung-Hak Kim, Mutsuko Minata, Ahmed Mohyeldin, Wanfu Xie, Dongquan Chen, Violaine Goidts, Brendan Frett, Wenhao Hu, Hongyu Li, Yong Jae Shin, Yeri Lee, Do-Hyun Nam, Harley I. Kornblum, Maode Wang, Ichiro Nakano
BACKGROUND. The tumor immune response is increasingly associated with better clinical outcomes in breast and other cancers. However, the evaluation of tumor-infiltrating lymphocytes (TILs) relies on histopathological measurements with limited accuracy and reproducibility. Here, we profiled DNA methylation markers to identify a methylation of TIL (MeTIL) signature that recapitulates TIL evaluations and their prognostic value for long-term outcomes in breast cancer (BC). METHODS. MeTIL signature scores were correlated with clinical endpoints reflecting overall or disease-free survival and a pathologic complete response to preoperative anthracycline therapy in 3 BC cohorts from the Jules Bordet Institute in Brussels and in other cancer types from The Cancer Genome Atlas. RESULTS. The MeTIL signature measured TIL distributions in a sensitive manner and predicted survival and response to chemotherapy in BC better than did histopathological assessment of TILs or gene expression–based immune markers, respectively. The MeTIL signature also improved the prediction of survival in other malignancies, including melanoma and lung cancer. Furthermore, the MeTIL signature predicted differences in survival for malignancies in which TILs were not known to have a prognostic value. Finally, we showed that MeTIL markers can be determined by bisulfite pyrosequencing of small amounts of DNA from formalin-fixed, paraffin-embedded tumor tissue, supporting clinical applications for this methodology. CONCLUSIONS. This study highlights the power of DNA methylation to evaluate tumor immune responses and the potential of this approach to improve the diagnosis and treatment of breast and other cancers. FUNDING. This work was funded by the Fonds National de la Recherche Scientifique (FNRS) and Télévie, the INNOVIRIS Brussels Region BRUBREAST Project, the IUAP P7/03 program, the Belgian "Foundation against Cancer," the Breast Cancer Research Foundation (BCRF), and the Fonds Gaston Ithier.
Jana Jeschke, Martin Bizet, Christine Desmedt, Emilie Calonne, Sarah Dedeurwaerder, Soizic Garaud, Alexander Koch, Denis Larsimont, Roberto Salgado, Gert Van den Eynden, Karen Willard Gallo, Gianluca Bontempi, Matthieu Defrance, Christos Sotiriou, François Fuks
Adipocytes secrete the hormone leptin to signal the sufficiency of energy stores. Reductions in circulating leptin concentrations reflect a negative energy balance, which augments sympathetic nervous system (SNS) activation in response to metabolically demanding emergencies. This process ensures adequate glucose mobilization despite low energy stores. We report that leptin receptor–expressing neurons (LepRb neurons) in the periaqueductal gray (PAG), the largest population of LepRb neurons in the brain stem, mediate this process. Application of noxious stimuli, which often signal the need to mobilize glucose to support an appropriate response, activated PAG LepRb neurons, which project to and activate parabrachial nucleus (PBN) neurons that control SNS activation and glucose mobilization. Furthermore, activating PAG LepRb neurons increased SNS activity and blood glucose concentrations, while ablating LepRb in PAG neurons augmented glucose mobilization in response to noxious stimuli. Thus, decreased leptin action on PAG LepRb neurons augments the autonomic response to noxious stimuli, ensuring sufficient glucose mobilization during periods of acute demand in the face of diminished energy stores.
Jonathan N. Flak, Deanna Arble, Warren Pan, Christa Patterson, Thomas Lanigan, Paulette B. Goforth, Jamie Sacksner, Maja Joosten, Donald A. Morgan, Margaret B. Allison, John Hayes, Eva Feldman, Randy J. Seeley, David P. Olson, Kamal Rahmouni, Martin G. Myers Jr.
After traumatic brain injury (TBI), glial cells have both beneficial and deleterious roles in injury progression and recovery. However, few studies have examined the influence of reactive astrocytes in the tripartite synapse following TBI. Here, we have demonstrated that hippocampal synaptic damage caused by controlled cortical impact (CCI) injury in mice results in a switch from neuronal to astrocytic d-serine release. Under nonpathological conditions, d-serine functions as a neurotransmitter and coagonist for NMDA receptors and is involved in mediating synaptic plasticity. The phasic release of neuronal d-serine is important in maintaining synaptic function, and deficiencies lead to reductions in synaptic function and plasticity. Following CCI injury, hippocampal neurons downregulated d-serine levels, while astrocytes enhanced production and release of d-serine. We further determined that this switch in the cellular source of d-serine, together with the release of basal levels of glutamate, contributes to synaptic damage and dysfunction. Astrocyte-specific elimination of the astrocytic d-serine–synthesizing enzyme serine racemase after CCI injury improved synaptic plasticity, brain oscillations, and learning behavior. We conclude that the enhanced tonic release of d-serine from astrocytes after TBI underlies much of the synaptic damage associated with brain injury.
Enmanuel J. Perez, Stephen A. Tapanes, Zachary B. Loris, Darrick T. Balu, Thomas J. Sick, Joseph T. Coyle, Daniel J. Liebl
BACKGROUND. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) can increase HIV RNA expression in vivo within resting CD4+ T cells of aviremic HIV+ individuals. However, while studies of VOR or other HDAC inhibitors have reported reversal of latency, none has demonstrated clearance of latent infection. We sought to identify the optimal dosing of VOR for effective serial reversal of HIV latency. METHODS. In a study of 16 HIV-infected, aviremic individuals, we measured resting CD4+ T cell–associated HIV RNA ex vivo and in vivo following a single exposure to VOR, and then in vivo after a pair of doses separated by 48 or 72 hours, and finally following a series of 10 doses given at 72-hour intervals. RESULTS. Serial VOR exposures separated by 72 hours most often resulted in an increase in cell-associated HIV RNA within circulating resting CD4+ T cells. VOR was well tolerated by all participants. However, despite serial reversal of latency over 1 month of VOR dosing, we did not observe a measurable decrease (>0.3 log10) in the frequency of latent infection within resting CD4+ T cells. CONCLUSIONS. These findings outline parameters for the experimental use of VOR to clear latent infection. Latency reversal can be achieved by VOR safely and repeatedly, but effective depletion of persistent HIV infection will require additional advances. In addition to improvements in latency reversal, these advances may include the sustained induction of potent antiviral immune responses capable of recognizing and clearing the rare cells in which HIV latency has been reversed. TRIAL REGISTRATION. Clinicaltrials.gov NCT01319383. FUNDING. NIH grants U01 AI095052, AI50410, and P30 CA016086 and National Center for Advancing Translational Sciences grant KL2 TR001109.
Nancie M. Archin, Jennifer L. Kirchherr, Julia A.M. Sung, Genevieve Clutton, Katherine Sholtis, Yinyan Xu, Brigitte Allard, Erin Stuelke, Angela D. Kashuba, Joann D. Kuruc, Joseph Eron, Cynthia L. Gay, Nilu Goonetilleke, David M. Margolis
Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.
Sam Horng, Anthony Therattil, Sarah Moyon, Alexandra Gordon, Karla Kim, Azeb Tadesse Argaw, Yuko Hara, John N. Mariani, Setsu Sawai, Per Flodby, Edward D. Crandall, Zea Borok, Michael V. Sofroniew, Candice Chapouly, Gareth R. John
Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fibγ390–396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMβ2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fibγ390–396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.
Anna K. Kopec, Sara R. Abrahams, Sherry Thornton, Joseph S. Palumbo, Eric S. Mullins, Senad Divanovic, Hartmut Weiler, A. Phillip Owens III, Nigel Mackman, Ashley Goss, Joanne van Ryn, James P. Luyendyk, Matthew J. Flick
Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO–activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor–induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.
Simona Pace, Carlo Pergola, Friederike Dehm, Antonietta Rossi, Jana Gerstmeier, Fabiana Troisi, Helmut Pein, Anja M. Schaible, Christina Weinigel, Silke Rummler, Hinnak Northoff, Stefan Laufer, Thorsten J. Maier, Olof Rådmark, Bengt Samuelsson, Andreas Koeberle, Lidia Sautebin, Oliver Werz
Adoptive transfer of T cells engineered to express a hepatitis B virus–specific (HBV-specific) T cell receptor (TCR) may supplement HBV-specific immune responses in chronic HBV patients and facilitate HBV control. However, the risk of triggering unrestrained proliferation of permanently engineered T cells raises safety concerns that have hampered testing of this approach in patients. The aim of the present study was to generate T cells that transiently express HBV-specific TCRs using mRNA electroporation and to assess their antiviral and pathogenetic activity in vitro and in HBV-infected human liver chimeric mice. We assessed virological and gene-expression changes using quantitative reverse-transcriptase PCR (qRT-PCR), immunofluorescence, and Luminex technology. HBV-specific T cells lysed HBV-producing hepatoma cells in vitro. In vivo, 3 injections of HBV-specific T cells caused progressive viremia reduction within 12 days of treatment in animals reconstituted with haplotype-matched hepatocytes, whereas viremia remained stable in mice receiving irrelevant T cells redirected toward hepatitis C virus–specific TCRs. Notably, increases in alanine aminotransferase levels, apoptotic markers, and human inflammatory cytokines returned to pretreatment levels within 9 days after the last injection. T cell transfer did not trigger inflammation in uninfected mice. These data support the feasibility of using mRNA electroporation to engineer HBV TCR–redirected T cells in patients with chronic HBV infection.
Janine Kah, Sarene Koh, Tassilo Volz, Erica Ceccarello, Lena Allweiss, Marc Lütgehetmann, Antonio Bertoletti, Maura Dandri
Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.
Xi Fang, Julius Bogomolovas, Tongbin Wu, Wei Zhang, Canzhao Liu, Jennifer Veevers, Matthew J. Stroud, Zhiyuan Zhang, Xiaolong Ma, Yongxin Mu, Dieu-Hung Lao, Nancy D. Dalton, Yusu Gu, Celine Wang, Michael Wang, Yan Liang, Stephan Lange, Kunfu Ouyang, Kirk L. Peterson, Sylvia M. Evans, Ju Chen


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April Field Visit – Heart of the Forest Wildlife Recorders Group
Would you like to help us record and understand the wide range of wonderful wildlife living in the Heart of the National Forest? If so then this volunteer group is for you!
Open to novices and experts alike, this group meets every second Thursday of the month between February – November, from 10am to approx 2pm.
Suitable outdoor clothing required including sturdy footwear. Please also bring your own lunch and drinks.
For each field visit (other than in May) the group will meet at the car park of Conkers Waterside and then head off to a nearby location within the Heart of the Forest.
Please email firstname.lastname@example.com or call 01283 551211 to book and for more information.
Event Information
Date:12 Apr 2018
Time:10:00 am - 2:00 pm
Address:Conkers Waterside
Bath Yard
Get directions
Contact Us
To find out more information about our Black to Green project or any upcoming events and opportunities then get in touch...
Call: (01283) 551211
Email: firstname.lastname@example.com


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Los Angeles LED Screens
Los Angeles' #1 Audio Visual Rental & Installation Company
LED screens and audiovisual make events come alive - concerts, festivals, tradeshows and presentations, annual parties and other retail industry shows. These advanced multimedia display technology open up a whole new world of possibilities that traditional projector displays cannot provide.
Los Angeles (US: (listen) lawss AN-jəl-əs; Spanish: Los Ángeles [los ˈaŋxeles], lit. 'The Angels'), often referred to by its initials L.A., officially the City of Los Angeles, is the most populous city in the U.S. state of California. With roughly 3.9 million residents within the city limits as of 2020, Los Angeles is the second-most populous city in the United States, behind only New York City; it is the commercial, financial and cultural center of the Southern California region. Los Angeles has a Mediterranean climate, an ethnically and culturally diverse population, in addition to a sprawling metropolitan area. The majority of the city proper lies in a basin in Southern California adjacent to the Pacific Ocean in the west and extending partly through the Santa Monica Mountains and north into the San Fernando Valley, with the city bordering the San Gabriel Valley to its east. It covers about 469 square miles (1,210 km2), and is the county seat of Los Angeles County, which is the most populous county in the United States with an estimated 9.86 million residents as of 2022.
Arlington Heights, Arts District, Baird Park, Baldwin Village, Baldwin Vista, Beverly Grove, Boston Heights, Boyle Heights, Brentwood, Brentwood Glen, Brookside, Castle Heights, Central Alameda, Central LA, City Terrace


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Welcome to the November 13, 2018 edition of Around the BCHL.
Hair controversy in the BCHL?
It's true, according to an article by Penticton Western News editor Kristi Patton. Patton points out a poll that was put on the league's @goBCHL Twitter account last week, asking fans to identify the player with the best hair.
New poll question - who has the best hair in the BCHL?
Ty Westgard, @SurreyEagles
Alex Newhook, @BCHLGrizzlies
Diarmad DiMurro, @BCHLSmokeEaters
Regan Kimens, @BCHLExpressOr someone else? Tweet us your replies and photos pic.twitter.com/tIV9vOknGX
— BC Hockey League (@GoBCHL) November 9, 2018
Ty Westgard (Surrey), Alex Newhook (Victoria), Regan Kimens (Coquitlam) and Diarmad DiMurro (Trail) were the candidates put forth, with Penticton's Cassidy Bowes the most notable snub.
Bowes has left his hair uncut since the start of the season as he raises money for the Children's Cancer Research Fund, pledging to not get a trim until the fundraising target of $9,292 is met.
He's up to $3,224 so far and his flow is spectacular.
But, according to Patton's article, the league excluded Bowes from the poll because he's growing his hair out for a cause.
The Vees Twitter account immediately went to bat for Bowes and teammate Drew Elser, who is also rocking some great hockey hair.
Those wanting to donate to @Bowesie92's GoFundMe, but aren't comfortable donating online, there will be a cash donation box set up at the @SOEC during Saturday's game against the @BCHLSmokeEaters!
GoFundMe: https://t.co/5X26WfAOZg pic.twitter.com/l6weemLYLp
— Penticton Vees (@PentictonVees) November 8, 2018
No matter what happens the rest of the season, this is undoubtedly the story of 2018-19.
The Chilliwack Chiefs put a 10 on the scoreboard in a Monday afternoon road rout of the Surrey Eagles.
Cayden Bailey got the start in goal for Surrey, lasting three minutes and 58 seconds.
He faced three shots. All three got by him with Brett Willits scoring twice and Ethan Bowen adding a single. Willits and Bowen ended the game with hat-tricks. Cole Donhauser had a pair with Marcus Tesink and Will Dow-Kenny also scoring as the Chiefs improved their league-leading record to 18-7-0-0.
The Eagles fell to 6-19-0-1. Points-wise they're tied with Cowichan for last overall, but their .250 win percentage is worst in the 17 team league. What's most amazing about Surrey is their goals-against. Take away Monday's game and the Eagles are still the only team to have coughed up more than 100 goals this season (111 without Chiliwack game, 121 with). Only one other team has given up more than 90 (Cowichan with 91).
If you're looking for silver linings, Ty Westgard is fifth in BCHL scoring with nine goals and 33 points in 23 games.
But that's about it, and Surrey's situation could get a lot worse before it gets better.
One other note on the Eagles before moving on.
Surrey has played 11 games at home this season and has just one win. Something about the South Surrey Arena isn't sitting right with the team this year. Away from home they are much better (only by comparison), with five wins in 15 games.
Normally a team would look at their schedule, see a home-heavy back half and be happy.
The Eagles might be the exception.
On the plus side, here's Teddy Prior's first BCHL goal, a small bright spot for Surrey in Monday's Chilliwack romp.
Updating the Vernon Viper heartbreaking loss index with another defeat.
The Salmon Arm Silverbacks beat Vernon by a 4-3 count in overtime Saturday night, with Olson Werenka scoring the winner. That's six times the Vipers have fallen in OT this season. Add in two shootout losses and more than a third of their games in 2018-19 (23) have ended with a bad result in extra time. What makes the Salmon Arm loss even more heartbreaky is that Vernon rallied to tie the game with 1:08 to go in the final frame on a goal by Ben Helgeson.
Just when they thought a break had finally gone their way, they had the rug pulled out from under them, and you've got to think this might be getting into their heads.
I've pointed this out in a previous column, but had the Vipers picked up an extra point in half of their overtime and shootout losses, they'd be fourth in the division, three points out of first place. Instead they're sixth, seven points back of the front-running Merritt Centennials.
Sticking with the Interior division and the Centennials, who continue to boast the league's top offensive attack.
Merritt tagged the West Kelowna Warriors for eight goals in an 8-3 Remembrance Day win.
The Centennials have 99 goals on the season.
Checking the league leaders this morning, Merritt has three players inside the top seven. Bradley Cocca is third in the points race with 34. Nick Granowicz is fourth with 33 and Mathieu Gosselin is seventh with 30.
We're only in mid-November but the Centennials already have five players who've hit double digits in goals; Cocca (12), Granowicz (18), Gosselin (13), Matthew Kopperud (10) and Rylan Van Unen (10). Brendan Schneider (nine) will join that list with his next goal.
Merritt's big problem is the other end of the ice, where they continue to bleed goals against.
Only Surrey (121), Cowichan (91) and Trail (89) have given up more goals than the Centennials (87). They've been able to out-score those issues so far, but defensive play will tighten up league wide as teams shift into playoff-race mode in 2019, and offence-first clubs have a long history of fading down the stretch.
Merritt will need to fix their 200-foot game if they want to be anything more than a fun-to-watch also-ran next spring.
I was wondering when BCHL points and goals leader Kevin Wall would get an NCAA scholarship and it finally happened.
Wall has signed on with Penn State for the 2019-20 season, one of five BCHL players announcing college commitments over the last few days.
The dynamic Wenatchee duo of Christophe Fillion and Cristophe Tellier have both chosen Quinnipiac University and will join the Bobcats in 2020-21.
That's hard work paying off for Fillion.
Long-named Cowichan defenceman Dimitri Mikrogiannakis will become a problem for Rochester Institute of Technology radio and newspaper guys in 2020-21 and Powell River's Ryan Brushett is set to join the University of Nebraska-Omaha in 2019-20.
Eric Welsh is the sports editor at the Chilliwack Progress and has been covering junior A hockey in B.C. and Alberta since 2003.
Email email@example.com


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How to Know What Face Mask Is Right For You
Once you've mastered your skin care routine, the best way to keep your complexion glowing is to add in a mask treatment. Skin expert Kate Somerville recommends using a mask once or twice a week depending on your skin's needs. To find out what you should be using for your skin type, read on.
Sensitive: For sensitive skin types that experience redness, Somerville advises looking "for something with minerals to calm any inflammation." Try Avène's Soothing Moisture Mask ($26), a creamy option that features natural minerals from the thermal waters in the Cévennes mountains.
Acne-Prone: "If you've done a lot of extractions, a clay mask is a great choice, because it will pull any remaining infection out of the pores and close them down," Somerville explains. A clay mask is also a wonderful option for oily complexions, because it will also help to absorb any excess sebum. Try the Kate Somerville Clearing Mask ($45), which is formulated with kaolin clay to combat oily skin.
Dry: Add moisture back to a parched complexion with a hydrating treatment, like Caudalie's Moisturizing Cream Mask ($39). You can also use an exfoliating mask that will dissolve away dry, dead skin cells, such as Ren's Glycol Lactic Radiance Renewal Mask ($55), which contains glycolic acid to smooth roughness.
Combination: If your skin is truly combination — think: an oily T-zone matched with a dry complexion — then try one oil-absorbing mask where you need it and a moisture-rich option on the dry areas of your face.


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City Council Meetings and Workshops are held in the City Council Chambers at 101 NW A Street.
For more information, go to the City Council page.
Agendas are available at the Administration Office on Friday before each Council meeting and posted online in the Agenda Center.
For more information, contact Administration at 541-450-6000.
Show All Answers
Go to the City Council page or call Administration Office at 541-450-6000.
The City of Grants Pass has 8 Council members. There are 2 elected Council members for each Ward. Check the City Council page for the names of your specific Council members and their contact information.
The term of office for Council members is 4 years.
The City Council elections are held in November during the general election.
Please call Administration Office at 541-450-6000 for a copy of the City Resolution or Ordinance. Go to the Ordinance & Resolutions page for more information.
Open applications for City Committees and Commissions are advertised through our local newspaper and posted on the Committees & Commissions page, as well as the homepage. If you are interested in any city volunteer committee or commission, please contact the Administration Office at 541-450-6000.
You can submit your application online with the City Committee / Commission Appointment Application.


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- List your giveaways below.
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Tree Squad
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Tree Squad offers all types of tree services from technical removals to yearly trimming of all species of trees! We trim live oaks and shade trees, grind stumps, and clean up storm debris--from Palm Coast to Ponte Vedra. We also offer discounts to first responders, police officers, firefighters, Active Military, and Retired Military! For a free, no-obligation estimate, call us today! Tree Service, Tree Trimming and Tree Removal, Arborists, Tree Surgeons, Tree Cutting, Tree Pruning. Tree Felling, Tree Management. Tree Expert Tree Management Tree Surgeon Complete Tree Caring Certified Arborist
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- The latest SOFA, version 1.2.2, lets you import from Excel XLSX files (previously, Excel files had to be in the XLS format).
- Normality analyses can be included in reports, saved as output etc.
- And there is support for CUBRID databases. CUBRID is an open source relational database highly optimized for Web Applications. That brings to 6 the total number of SQL-type databases that SOFA can directly link to.
A few bugs were also fixed:
- Restored standard deviation option to row stats report tables.
- Fixed bug which meant row % was appended multiple times to config col dialog (until session closed).
- Restored PostgreSQL functionality by fixing faulty psycopg import statement.


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It is not often the world welcomes a brand-new nation into its fold. But that will be the case on Saturday, when South Sudan becomes the United Nations' 193rd member and the African Union's 54th.
Independence comes after a bitter and long-fought conflict against Sudan's north, which left the south marginalized, underdeveloped and facing huge challenges — and the specter of more fighting.
But in Juba, the new capital, South Sudan is preparing to celebrate.
Ahead of Saturday's independence festivities, traditional dancers, the national choir and South Sudan's military and other uniformed groups gathered together for a full dress rehearsal and flag-raising ceremony this week in the sweltering heat.
The practice run and the event itself are being held at the Dr. John Garang Mausoleum in Juba. Hailed as a national hero, the late Garang was the leader of Africa's longest civil war between mainly Arabic and Muslim northern Sudan and the largely black African and Christian south. The 20-year war ended in a 2005 peace deal that the U.S. helped broker.
Veteran southern Sudanese soldiers were among those proudly marching in the parade, like John Ngum Lang Ngum, who fought the first civil war in the bush and calls himself a freedom fighter.
"You know, I feel now happy, because we are finally liberated from the Arab rule. Myself, I am one of the founders. I'm one of the people who started the war," he says. "But I'm happy that the flag has been hoisted today while I'm alive. And I'm seeing our flag of freedom waving in the air."
Challenges Ahead
Schoolchildren were singing at the parade ground in a mood of excitement and expectation, despite the monumental hurdles an independent, oil-rich South Sudan still confronts.
It needs to build roads, railways, airports, homes, hospitals, schools and other major infrastructure; tap the new nation's agricultural and oil wealth; and maintain neighborly and peaceful relations with the northern half of the country after the formal divorce that came with an overwhelming "yes" vote in January's referendum for independence in the south.
"You can see we are sitting at the heart of this continent. This is the real heart, because it is the real area where the virgin resources of this continent are located. They have not been exploited. So, it needs peace," says Barnaba Marial Benjamin, South Sudan's information minister.
Benjamin says that after years of conflict, relations with northern Sudan must be cordial.
"Destabilizing each other is not in the interests of the two nations. That's why we believe having a viable state in the north and a viable state in the south, living side by side, cooperating in trade in business, discussing issues that may bring conflict is the best way forward," he says.
After blowing hot and cold about South Sudan's independence, the president of Sudan, Omar al-Bashir, has announced he will attend the ceremony in Juba on Saturday.
Most of Sudan's oil reserves are in the south, but the crude is exported via pipelines in the north. The two independent states must come to a wealth-sharing agreement and iron out other unresolved issues.
Meanwhile, there has been renewed fighting in recent weeks in the disputed oil-producing region of Abyei, which lies between the north-south border, and whose future as part of either the independent north or south has yet to be decided. Northern forces have pledged to pull out of Abyei and both sides have agreed to allow peacekeepers from neighboring Ethiopia to be deployed to Abyei to create a buffer zone.
There are also continuing deadly clashes in South Kordofan, northern Sudan's last remaining oil state. Although located in the north, Kordofan remains home to members of the Nuba community, who fought with the south during the civil war. The Nuba say independence in South Sudan will leave them isolated, persecuted and targeted by Sudanese government forces. Human-rights campaigners warn ethnic cleansing is being perpetrated in the Nuba Mountains.
Closer to home in the south, the Sudan People's Liberation Army is facing armed opposition from disenchanted militia groups that split from the once revolutionary movement that fought for and won southern liberation.
A Return Home, To A New Nation
Tens of thousands of southern Sudanese who have lived in exile in neighboring countries and overseas, or who were refugees in camps in Sudan's capital, Khartoum, and elsewhere, are returning to celebrate their hard-won independence — and to settle in their own newborn nation.
Hopes are high, says Agyedho Adwok Nyaba, sitting in an outdoor cafe. The tall, smiling and thoughtful 25-year-old development worker, who has spent almost her entire life outside South Sudan — first as a child refugee, then as a student — came back last November.
"When you are away from home — in this case, you know, I didn't know what home was," Nyaba says. "For me, if you ask me today what is home, I say home is where my heart is. There is something special about where you come from. So, for me, coming back home, it was just a pulling."
Across town at St. Theresa's Roman Catholic cathedral, children recite a prayer-poem for South Sudan: "Loving God, give us courage to reject resentment as well as ethnic conflicts. ... Help us to overcome hurt, hostility and bitterness in our hearts, so that we become reconciled citizens in our new nation ... in the spirit of service, unity and lasting peace.
"God bless the Republic of South Sudan. God bless the Republic of Sudan."
The prayer stresses that north and south Sudan should be partners, but there seems to be little trepidation in South Sudan about a future without the north, and simply relief that the time has come for the south to break away and savor independence. Southerners say they were second-class citizens in a united Sudan and are now desperate for freedom and lasting peace.
Copyright 2022 NPR. To see more, visit https://www.npr.org.


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A few days ago, I had a birthday – so I had to laugh out loud when I saw this Hampton Art/ Jillibean Soup stamp. I would much rather think that I'm vintage (instead of just old!)
This card was easy to make, but would be perfect to have on
hand for a last minute Birthday card.
I cut the fish-tailed paper banners from coordinating
patterned papers, and inked all of the edges.
The center banner was adhered with foam adhesive, to pop it up a
little. Next, the ribbon was added (over
the banners).
The sentiment was stamped on patterned paper, cut out, and
adhered with the foam adhesives.
Super easy and super cute!
Hampton Art/ Jillibean Soup Irish Farm House Soup jb0012
Clearsnap: Donna
Salazar Chox (Sage)
Other Supplies
Authentique Patterned Papers (Grace Collection)
Bazzill Cardstock
May Arts Ribbon – Twill/ Chevron Stripe 382-34-17
Xyron Mega Runner
Xyron Creatopia


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Launching a beauty business, let alone a successful one, is a delicate dance of elements: market research, product development and, let's not forget, good old-fashioned ingenuity. In fact, launching a beauty business requires keeping so many plates up in the air, it's easy for new brands entering this ring to become overwhelmed with the details before they've even hit the product development stage. But it doesn't need to be this way.
The Benchmarking Company (TBC) has worked with large and small brands, established brands and newbies, and has clearly established that, regardless of the stage of development of your company or product, for the best chance of success in the market, a game plan and a blueprint needs to be in place before too many plates are up in the air. These established plans will guide all the developmental aspects of a new business and new brand. Although the following five steps are not all the steps that must be taken, they are key building blocks that will help you kick-off your new venture with a solid foundation so you can hit the ground running.
Step #1: Have an Idea, But Not Just Any Idea
In order for your beauty business to succeed, you have to have a truly unique and different idea on which to lay the foundation of your entire operation. Have a new beauty business idea, but not just any idea. Are you fascinated with runway looks and want to bring them to women everywhere? Well, you'd better have a bigger idea than that because too many established brands already own that space. Let your ingenuity and entrepreneurial spirit help guide this step: What's new, unique or passionate about your plan? Why will women, and maybe even men, everywhere love and have to have it? Why should a consumer pay attention to your idea in-market? Why might she not pay attention? Take time and flesh out this idea, and perhaps even consider vetting it out with a consumer test or study before you start writing checks to the development houses and PR firms, and you will be way ahead of the pack.
Step #2: Know Your Audience/Know Your Market
Before you can even think about launching a new beauty business, you have to first know the marketplace inside and out. Who are the leaders? Who's entering the arena and who's leaving? What products do consumer publication beauty editors love—and, more importantly, what products are consumers buying and what keeps them clamoring for more?
Nearly as important as having a great idea, have a thorough understanding of the competitive market where your new beauty business will ultimately be living and breathing. What are the leaders in your category saying and doing? What's the latest innovation or hot new ingredient? How are brands interacting with their consumers online? What claims and results is your competition promising and can you match or exceed them?
Getting a handle on this vast pool of information may seem daunting or even impossible, but by starting with a clear and focused idea, you will be able to target exactly what intelligence will be most beneficial to your new beauty business and tailor all research efforts in support of this goal.
Step #3: Point Out the Difference
Having a point of difference is important from a product standpoint, but what about from a business standpoint? How will your beauty business be different from the Estées, Almays and Cover Girls of the world? When it comes to points of difference, how and where you decide to stake your claim will have a direct impact on every aspect of your business: profits, innovation, development, growth, creativity and even success. Think both creatively and strategically about how you will both craft this difference and how you will share it with your consumer—for which you have more options than ever: packaging copy, editorials and most notably, your online presence. Twitter, Facebook and YouTube are great vehicles to help carve out this difference and maintain it with an incredibly diverse and ever-expanding audience. Without a clearly defined point of difference, your new beauty business will never make the splash it needs to capture market share and to keep it.
Step #4: De-code Your DNA
To know your business's point of difference is to capture the very essence and DNA of your company. If you stand for technologically advanced beauty, then own that category and own it like no other brand taking up shelf space at Sephora or Ulta. Tailor all messaging, product development and packaging to reflect this aesthetic so that when consumers see anything connected to your brand, they instantly know it is you. Importantly, once you establish your point of difference and enter the market, you must maintain it.
Establish and leverage your point of difference to foster a positive association with this difference and your brand. A great example is Bare Escentuals' "Swirl, Tap, Buff" point of difference. It could not be co-opted because it is so well established that consumers will think of Bare Escentuals regardless of another company using a similar point of difference.
Also, there exists the very real potential that you will lose consumers if you are not true and continually change what you stand for in-market. The target consumers for your technologically advanced beauty brand, for example, will likely not find or connect to your brand if you don't fully establish or veer from a technology point of difference and, instead, tout 100% green, sustainable, vegan beauty. If you stray into messaging that doesn't support why you are different in the marketplace, and why they should both believe in and be loyal to your brand, they won't be.
Step #5: Innovate and Innovate Some More
The truth is, product and brand development don't always happen concurrently, yet it's the most successful brands that tackle these two steps in tandem. When developing your products, be choosy. Choose the most interesting, advanced ingredients you can afford; choose a lab that shares your vision and dedication; choose an SKU lineup that gives consumers both what they know and don't know they need; and make them want it all. Choose products that stand high above the competition, and bring your A-game. These days, new brands are flooding the market and consumers have more choices than ever before, which means you've got to enter the fray with the best you've got in order to even be granted the chance to introduce your next great idea to the market.
At the same time, while you are busy with all of this innovative development, begin creating the fun, strategic brand identity materials you will need to fit the emerging personality of your products.
While creating these branding documents, remember the other steps of the process: your audience and your market; your point of difference, and your DNA are key elements in the creation of brand name, tagline, positioning statements and brand stories. Ask yourself, "What does my brand stand for? What does the name and tagline say both about my brand and to the consumer? How will my final product fit into the marketplace and will it have the personality and pizzazz it must in order to capture both interest and loyalty?" Are product and brand development efforts synergistic or are they at odds? Keep the innovation flowing and your brand will always be relevant, viable and, more importantly, fresh in consumers' minds.
So, What Else Should a Start-up Beauty Entrepreneur Do?
Exhaustive due diligence. Before one dollar is spent on packaging, marketing or social media campaigns, thoroughly vet out your idea—both internally and with consumers. But not just any consumer—the loyal beauty brand consumer you want, and need, to dazzle. Test your business and product concepts, run consumer focus and trial groups, and really listen to what they have to say before you even think about gondola space or shelf talkers.
Consumer approval is the key to success in the beauty industry. But, for instance, with more than 158 million women living in the United States, how do you know if your new beauty business will capture the attention and dollars of so many? Don't miss your mark—or give your market share to anyone else—established brand or not. Instead, set your business up for immediate success by following these simple steps for proven launch success.
For additional information on consumer testing, read Alisa Marie Beyer's Testing, Testing ... 1, 2, 3 contribution in the March 2011 issue.