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With Christmas activities taking place every day, it's easy to forget "the reason for the season."
I find three ways to connect, learn and grow closer to Jesus, for whom the holiday was named.
All three are crucial.
Year round. Including in December.
Personal quiet time
I'm a morning person, the first one up in my household. Always have been. When our kids were young, my job started before they got up for school. Even today, I'm up before 6:30 a.m. – without an alarm.
I start the coffee. I feed the cats. I pour a cup of the morning brew. I sit down in my living room chair, the cup in one hand, the Bible in the other, and often a cat on my lap.
That's the best part of my entire day. It's dark. It's quiet. It's warm (thanks to the cat).
God often speaks to me there.
Today I read the first three chapters of 1 Peter. "Finally, all of you, have unity of spirit …" (1 Peter 3:8) In church yesterday our pastor talked about the deepest longings of our hearts. A magazine did a survey on that recently, he said, and the most common longings were happiness, money, a relationship, peace and joy.
My deepest longing, however, didn't appear on the magazine's list: unity. I wish with all my heart that we as Americans and as citizens of the world would learn to get along with each other. I've written about this many times.
Most of our deepest longings are selfish. Mine is for unity among all people. That's selfish too, I suppose; I wish to be understood as well as I wish to understand you.
These thoughts ran through my mind in my quiet time this morning.
This happens frequently. A verse I read resonates, and my mind probes into it. What does it mean? What would it look like if we (I) truly lived this out?
Unity among believers is the last thing Jesus prayed for in the Garden of Gethsemane before He was crucified (John 17:20-24). Unity mattered to Jesus, too.
Many of you discredit the Christian faith because we Christians can't get along with each other, much less with you. Our message to you is fragmented. Some so-called Christians mis-lead you.
This is why we must read the Bible for ourselves. What's in there? I'm a journalist; I'm a cynic by nature. I'm not going to take your word for it. I will double-check you.
I will read it for myself.
Small group
Having said that, I can learn from you, and you from me. The best churches are organized with small groups of a dozen or so people who get together on a regular basis. My wife and I also attended Sunday School classes for many years. And we participate in Wednesday night men's and women's groups to study the Bible and issues of the day, with a Biblical perspective.
I have plenty to learn from you. You have insights into life that I don't have. I've experienced things that I can share with you as well. As we get to know each other better, we discover that we aren't as different as we thought we were.
We also can support each other through our struggles and trials of life. When someone in our group has a dying relative, others understand because they've been there. When someone new joins the group because he and his family just moved here, I can relate right away, because that's my experience too. When someone talks Browns-Steelers … well, I have ties to both cities, actually.
All these issues can unify us. We connect at this level in a group setting.
The best groups challenge me to learn something new about myself, to step out in faith and do something, to help me understand a truth about God in a different or deeper way. It's a safe place to be vulnerable. For guys, that's not normal. And even in a couple of men's groups I'm in, that doesn't come easy.
But it's necessary. I can learn things from you that I cannot learn on my own.
Uh oh, here it comes. Yes, there's a reason to attend a Sunday morning service every week.
The pastor, if he (or she) is inspired by the living God, has done his homework on his message for the day. As a general rule, the pastor dives deep into a verse or small section of the Bible. He offers insights and background that his training and study have taught him. And then he gives practical applications on how we can live out the truths that he is teaching us.
If this is the only Bible learning we do each week, we miss so much. Even if you hear one sermon a week for your entire life, you'll miss reading most of the Bible. That's why we must read the Bible on our own, and study it in small groups. We learn truths in different ways, and at different levels.
Real-world application
Does God speak to you when you are alone? Does He speak in your small groups? Is He speaking through your pastor?
I daresay that most of you who are critical of God or the church aren't participating in it at all, but are criticizing as outsiders. We in America are good at that. We Christians are good at criticizing you too.
All of us would do so much better if we got to know each other better. Find out what makes each of us tick. How each of us thinks.
On Facebook I follow a prominent expert on poverty, who travels the country seeking to alleviate or even end poverty. She commented recently about a library forgiving fines.
Are library fines a poverty issue? Yes. She explained how she grew up in a home with more than 20 relatives. She avoided libraries because if she took a book home, it would get lost in such a crowded place. She missed out on all a library offers – chances to discover new ideas, improve reading skills, learn history and other subjects from those who have lived it – because she was afraid of getting punished for using a library.
I had no idea.
The point of a personal quiet time, small groups and weekly preaching is to learn and grow closer to God and to each other. Is poverty a God issue? Absolutely, yes. It's easy to judge people who don't pay their library fines as lazy or thieves – until we understand why.
Unity. My deepest longing. Which I realized in a sermon. Then meditated on in a personal quiet time. And read about online.
I'm trying to live it out. It's a worthy goal.
This is how God works. Alone, in groups and in church. With real-world applications. It's all good.
And it's all necessary.


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- The expectations for American distance runner Mary Decker were sky high as she made her Olympic debut in the 3,000 meters at the 1984 Los Angeles Games. Following numerous setbacks that prevented her from the 1976 and 1980 Olympics, she was ready to validate her greatness. But as fate would have it, a major collision would define her career.
- First Aired: August 13, 2013 - 51 Minutes Runtime
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Last year's boating accident in Anstruther has led to new safety guideline recommendations. The accident, which occurred on the Firth of Forth last summer involved the collision of two inflatable boats. A 45-year-old woman and mother of two children suffered permanent damage to her eyesight and a punctured lung in the accident. The woman's injuries were the result of being crushed. Osprey II, the boat involved in the accident, was carrying 11 passengers instead of the boat's normal 8 passenger occupancy. The boat had seven adults and four children when the accident occurred. The boat often sold two additional tickets when the weather was good. The two additional passengers were seated in inflatable tubes rather the boat's interior bench.
The woman involved in the accident was sitting on an inflatable tube at the time the boating accident occurred.
Captains of each ship, both the Osprey and the Osprey II increased speed and then attempted to power turn away from each other. The captains both reacted by reducing their speed and tried to avoid an accident knowing that the boats were about to collide. The quick actions by the two boats did little to prevent the accident.
The mother of two was on the ship with her husband and two children. When the boats collided, she was taken to the hospital with a punctured lung, lacerations, bruising, broken collar bones and five broken ribs. Internal injuries resulted in damage to both of her eyes.
Incident reports indicate that passengers not on the inboard seating are at a much higher risk of injuries and falling overboard if a collision occurs.
The boats, considered rigid inflatable boats (RIB) did not break regulations during the accident. No regulations are in place that prohibit passengers from sitting on the inflatable tubes despite the heightened risk of injuries.
The Marine Accident Investigation Branch (MAIB) has advised the Maritime and Coastguard Agency that regulations be put in place that limit the allowable numbers of passengers to the amount of suitable seats for passengers.
Owners of the Osprey and Osprey II, Isle of May Boat Trips Ltd, have banned passengers from sitting on the inflatable tubes following the accident. The owners have also banned crew members from sitting on the tubes and have limited the number of passengers to 12 on the Osprey and 8 on the Osprey II, the seating corresponds to the number of non-inflatable tube seats on each vessel.
Twin RIB operations have also been banned by the company expect in emergency situations.
Passenger limit recommendations have been made to the Maritime and Coastguard Agency. The ships' captains both acted promptly to reduce their speed to lessen the impact during collision. The captains were both found to be unable to prevent the accident.
The risk assessment of the maneuver had not been performed prior to the accident despite the move being performed with success in the past. The investigation states that there was no thought given to what to do in the event of a collision situation.
Osprey II's skipper tried to reduce the engine's speed, turn to port and engage the astern gear to prevent the accident. The skipper of the Osprey II assessed the passenger's injuries but couldn't relocate the woman to the bench seats due to the passenger's injuries.


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Nancy Water Transcript of Interview
Nancy Waters was born at Mill Green, Turvey in 1931. Her hard working parents ensured that Nancy, her brother and two sisters enjoyed a healthy upbringing. Nancy recalls her childhood in Turvey, helping her mother and father, her schooldays and her Saturday jobs for local shops. After leaving school Nancy went to work in Bedford, firstly for Clayton's and soon after for Igranic. Once her own children were all at school, Nancy found local part time work. Initially, this involved packing herbs and spices for a company based in the Old Chapel in Carlton Road and then at Bon Appetit in Bamfords Lane, Turvey.
CreatorAnjum Gray
Date of creation2019
Date of coverage1931 - 2019
ContributorAnjum Gray
CopyrightTurvey History Society
LicenseCreative Commons Attribution-NonCommercial-NoDerivs (CC-BY-NC-ND)
Reference numberTHS/VCS/006
Document (276)
Transcript (9)
Turvey People (86)
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Our Mission
The Pennsylvania Alliance to Control Tobacco (PACT), under the American Lung Association in Pennsylvania, is a statewide coalition that educates the public and elected officials about clean indoor air, tobacco taxes, and other critical tobacco control issues. PACT is primarily funded by a grant through the Pennsylvania Department of Health. Members of PACT include tobacco control advocates, healthcare professionals, government agencies, community leaders, business owners, and the general public.
PACT was founded in 2001 by the American Cancer Society, American Heart Association, and American Lung Association. For over 15 years, PACT has worked with local coalitions, regional contractors, and national health organizations to strengthen tobacco control laws across Pennsylvania that can prevent youth from initiating tobacco use, help people who use tobacco quit, and reduce exposure to secondhand smoke. PACT develops educational materials to share with legislators and community members, provides training for members on advocacy strategies and tobacco control issues, and supports the Tobacco Resistance Unit (TRU), Pennsylvania's youth-led tobacco control movement.
Our Staff
Sarah Lawver
Sarah Lawver is the Director of Advocacy for both Pennsylvania and West Virginia with American Lung Association for the Pennsylvania Alliance to Control Tobacco (PACT). Sarah leads policy, statewide advocacy strategy, program development and implementation, and continual management of all advocacy projects. Prior to joining PACT, Sarah excelled in chronic disease integration efforts and served as the Tobacco Control Program Manager for the North Central Region of Pennsylvania. Sarah is currently pursuing a Master's of Public Administration at Shippensburg University.
Molly Pisciottano
Molly Pisciottano is a Health Promotions Specialist with the American Lung Association in Pennsylvania and her primary role consists of managing the youth tobacco prevention program known as the Tobacco Resistance Unit (TRU). Molly received her Master's Degree in Healthcare Administration and Management from Point Park University. She presently oversees TRU recruitment/member engagement, while providing necessary trainings to TRU Advisors as well as youth advocacy trainings for TRU Youth. In addition, she assists with the organization of TRU Storms/Events, keeps up to date on TRU social media and the TRU website, and works on other tobacco control initiatives such as Tobacco 21, smoke-free housing and smoke-free college campuses.


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Attilio Fiandrotti <firstname.lastname@example.com> scrisse: > There is huge amount of already existing themes for GTK2 [1] and it > seems to me that the one actually most suitable for the g-i is > H20-gtk2-Ruby (see attached PNG). The page for the theme is http://art.gnome.org/themes/gtk2/409 However I have two notes about it: * There's no license at this page nor in the tarball. So it is currently not DFSG-free. Maybe we should ask the author for a free license. * There are some thumbnail in the .xvpics directory. (If it will be released under a free license) I think they can be removed. Greetings, Luca -- .''`. ** Debian GNU/Linux ** | Luca Bruno : :' : The Universal O.S. | luca.br(AT)uno.it `. `'` | GPG Key ID: 3BFB9FB3 `- http://www.debian.org | Proud Debian GNU/Linux User
Description: PGP signature


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Really looking forward to this! I'm good with writing if you need someone to write some dialogue!
Weight Date - A RPG maker dating sim
would love to play it but it isn't Mac compatible
I'm glad your looking forward to it.
Dialogue isent really the trouble since I sort of fast tracked it to get the concept ready it's more so going back and proof reading. Thanks for the offer
i love the game, i look foward to the next updates
A really nice little concept, I would love to give suggestions but I want to wait for you to have the chance to build out your systems a little more first. I enjoyed what I played!
in terms of the system i kinda have it all planned out. the only things that are not final are roster and what events take place in what days as I do plan of having those on specific days.
I like it. Reminds me of "Codename : D.E.B.U."
This could be it's spiritual successor.
Using the same program for the characters yea it pretty much is
sad to see that project canceled
I liked your game a lot! I hope you get the time and will to finish it.
The "shy" cowgirl is very cute :3
Thank you for the demo!
Oh, this sounds fun!
I like it its cute You will still work in this??
I've gotta say, I'm super hyped about this! As someone mentioned before, it's like Project D.E.B.U. is back from the dead, which makes me happy lol. I'll definitely be checking out all the new builds
Due to Uni stuff I've only been working on story paths and events listing but it's very slow. things should pick up near christmas I just have so much on my plate and the only one working on it. as I said this will take a long time to make
I have high hopes for this dating sim.
I like this demo game I wait more things.
Hey yall
first of all Merry Christmas as of the time of this post
and second of all I wish to thank all of you that have shown support and interest towards weight date. I had some axiety over this project for a need to finish this wile at uni but I've learned that I have to pace myself as the last 3 months have been a little crazy. As such there has been little development towards the game mostly flowcharting the story and going back to the build to fix the oh so many spelling mistakes (this was one of my many concerns as my english is well shit). As such I think the next way forward for the game is to work on a grammer/spelling fix update to start in january and before the end of that month maybe have something new. Maybe have a discord or something to give more motivation and give you guys more insight to where it may go.
Once again I thank each and every one of you for downloading and playing the game. I hope the finish product will satisfy all of you
I played the demo. I love this! Keep working on it!
Update 26th january 2020
The month is almost over and I havent said anything since before christmas. I apologise as i've been busy with other matters and only developing the game in small bursts. I thought about the project long and hard and ofcourse about me not being as acitive here as I should be. Thus I am going to pull a no man sky approch and kinda keep quiet about certine things. this being if I do upload a new file or update the demo it will be the full game. unfortunantly uni starts up again so I will have 3 months to focus on that but after I get a long summer and that is when I get back to implementing all the stuff I need. as for progress the roomba and terra chapters have been done up to 70% but the new text needs proof read.
Why the change from last post? I'm achually not very confident and the peer pressure to get a good game for everyone wanting this is making me a little stressed. that's why i'm trying to pace myself and just work on it when I can even during Uni. I'm still happy to talk or be asked questions and such but as for when the game will be finished I cannot say
Don't worry too much. After all, this is a side project, so do what you need to do. I'm pretty sure most of us can sympathize with university being a pain, so don't be stressed out on our behalf. It'll be done when it's done, and we can live with that.
Just played through the demo and this looks great so far. I wish you luck on you're project in the future!


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Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine β-defensins (mBD) 3 and 4, the murine homologs of human β-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-γ, MIP-2, IL-1β, TNF-α, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-κB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.
Clinically, Pseudomonas aeruginosa-induced keratitis is a rapidly progressing corneal disease that results in inflammatory epithelial edema, stromal infiltration, corneal ulceration, stromal tissue destruction, and, frequently, vision loss (1). Experimentally, P. aeruginosa challenge leads to corneal perforation in susceptible C57BL/6 (B6) mice and less severe disease in resistant BALB/c mice (2). In this regard, the antimicrobial properties of the cornea are attributed to several dynamic components, such as defensins, which can kill invading pathogens, regulate inflammation and TLR activation, as well as chemoattract a variety of immune cells (3, 4, 5).
In mice, the most studied defensins are murine β-defensins (mBDs)3 1–4, which are chiefly expressed in a variety of epithelial cells (6, 7, 8, 9) and promote host resistance against infection in airway tissues (6, 7, 8, 9). mBD2 also can chemoattract and activate immature dendritic cells via TLR4 (10). Recently, studies using susceptible/resistant murine models have demonstrated that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection (11); however, little is known regarding the role of the other two murine defensins in the eye. In this regard, other studies have demonstrated that hBD2 and hBD3, the inducible human homologs of mBD3 and mBD4 (8, 9), play an important role in the ocular immune defense system. They do so by regulating a variety of immune events, including bacterial killing, cytokine release, mast cell histamine release, dendritic cell activation, immune cell chemotaxis, as well as epithelial cell migration and wound repair (3, 4, 5, 12, 13, 14, 15), suggesting that their homologs could be significant in murine ocular immunity. Moreover, in vitro studies have demonstrated synergistic activities of hBDs against Staphylococcus aureus and Escherichia coli (16), indicating a significant potential for mBDs to act together to protect the ocular surface from invading pathogens.
Thus, studies described herein investigated the expression and function of mBD3 and mBD4 in susceptible B6 vs resistant BALB/c mice and whether either defensin interacts with mBD2, previously shown to be of importance in this disease (11). Our data provide evidence that mBD3 and mBD4 are inducibly and disparately expressed in BALB/c vs B6 corneal epithelium and stroma after P. aeruginosa infection. However, of the two, only mBD3 is required for host resistance and interacts with mBD2 to protect against bacterial infection. Additionally, in vivo studies identified the cell sources of mBD2 and mBD3 in the corneal stroma after infection and demonstrated that despite their individual effects on disease outcome (e.g., inducible NO synthase (iNOS) expression), these two defensins act together to promote host resistance against corneal infection. This is achieved through modulation of bacterial load and regulation of both polymorphonuclear neutrophil (PMN) infiltration and production of proinflammatory cytokines and TLR signaling molecules.
Materials and Methods
Infection of mice
Eight-week-old female BALB/c (resistant) and B6 (susceptible) mice (The Jackson Laboratory) were anesthetized with ether and placed beneath a stereoscopic microscope at ×40 magnification. The cornea of the left eye was wounded with three 1-mm incisions using a sterile 25-gauge needle. A 5-μl aliquot containing 1 × 106 CFU of P. aeruginosa (American Type Culture Collection, strain 19660), prepared as described before (17), was topically applied to the corneal surface. Eyes were examined at 1 day postinfection (p.i.) and/or at times described below to ensure that mice were similarly infected and to monitor disease. Animals were treated humanely and in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Ocular response to infection
Corneal disease was graded using an established scale (18). A clinical score was recorded for each mouse after infection for statistical comparison of disease severity, and photography with a slit lamp was used to illustrate the disease response.
RNA interference
In vivo use of small interfering RNA (siRNA) has been described by others (19) as well as by studies from our laboratory (11, 20). For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 μl per mouse at a concentration of 8 μM) into the left eye of BALB/c mice (n = 5/group/time) 1 day before infection and then topically applied onto the infected corneas (5 μl/mouse/time at a concentration of 4 μM, once on the day of infection and twice on both 1 and 3 days p.i.). The efficacy and specificity of silencing of each defensin was tested by RT-PCR. All of the siRNAs used in the studies herein were shorter than 21 nucleotides in length to avoid nonspecific siRNA suppression effects via cell-surface TLR3 (21).
Separation of corneal epithelium and stroma
Corneas from normal, uninfected, or infected eyes of BALB/c and B6 mice were hemisected at 1 and 5 days p.i. and placed into 0.02 M EDTA-PBS buffer (pH 7.2) at 37°C for 15–30 min. Then, the corneal epithelium was gently separated from the underlying stroma using fine forceps and the tissue was processed for real-time RT-PCR.
Real-time RT-PCR
Total RNA was isolated from an individual whole cornea or separated corneal epithelium/stroma for analysis (as indicated below) using RNA-Stat 60 (Tel-Test), according to the manufacturer's recommendations, and quantitated by spectrophotometric determination (260 nm). One microgram of total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase. The 20-μl reaction mixture contained 200 U of Moloney murine leukemia virus-reverse transcriptase, 10 U of RNasin, 500 ng of oligo(dT) primers, 10 mM dNTPs, 100 mM DTT, and Moloney murine leukemia virus reaction buffer (Invitrogen). Next, cDNA was amplified using SYBR Green Master Mix (Bio-Rad), as suggested by the manufacturer. Briefly, the 20-μl reaction system contained 10 μl of SYBR Green PCR Master Mix, 0.5 μM primers, 2 μl of cDNA (diluted 1/10), and diethyl pyrocarbonate water. All primers for the PCR, except for mBD3 and mBD4, were designed using PrimerQuest (Integrated DNA Technologies) and reported previously (11). Primers for mBD3 and mBD4 were purchased from SABiosciences. Quantitative real-time RT-PCR reactions were performed using the MyiQ single color real-time RT-PCR detection system (Bio-Rad). Optimal conditions for PCR amplification of cDNA were established using routine methods (22, 23). mRNA transcription levels were normalized to the β-actin gene and/or calculated as the n-fold difference of transcription in siRNA for mBD2, mBD3, or both defensins compared with scrambled control treated mice. Data are shown as means ± SEM for relative mRNA levels and means ± SD for n-fold difference, respectively, and represent two individual experiments each with five mice per group per time.
Immunofluorescent staining
Normal uninfected and infected eyes were enucleated (n
n = 5/group/time) at 1 and 5 days p.i. Corneas were homogenized in 0.5 ml of PBS with 0.1% Tween 20. All samples were centrifuged at 13,000 rpm for 5 min and an aliquot of each supernatant was assayed in duplicate for MIP-2, IL-1β, and TNF-α protein per the manufacturer's instruction. The reported sensitivity of these assays is <1.5 pg/ml for MIP-2, <3.0 pg/ml for IL-1β, and <5.1 pg/ml for TNF-α.
Bacterial plate counts
Corneas from siRNA mBD2, mBD3, or both defensins, and scrambled control-treated BALB/c mice were collected (n = 5/group/time) at 1 and 5 days p.i. and the numbers of viable bacteria were quantitated. Individual corneas were homogenized in sterile water containing 0.85% (w/v) NaCl containing 0.25% BSA. Serial 10-fold dilutions of the samples were plated on Pseudomonas isolation agar (Difco Laboratories) in triplicate and plates were incubated overnight at 37°C. Results are reported as number of 105 CFU per cornea ± SEM.
Myeloperoxidase (MPO) assay
A MPO assay was used to quantitate PMN number in the cornea from siRNA mBD2, mBD3, or both defensins, and scrambled control-treated BALB/c mice. Infected corneas (n = 5/group/time) were excised at 1 and 5 days p.i. and homogenized in 1.0 ml of 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich). Samples were freeze-thawed four times and centrifuged at 13,000 rpm for 10 min, and 0.1 ml of the supernatant was added to 2.9 ml of 50 mM phosphate buffer containing o-dianisidine dihydrochloride (16.7 mg/100 ml; Sigma-Aldrich) and hydrogen peroxide (0.0005%). The change in absorbance at 460 nm was monitored for 5 min at 30-s intervals, and the results were expressed as units of MPO per cornea. One unit of MPO activity is equivalent to 2 × 105 PMN (24).
Statistical analysis
The difference in clinical score between two groups at each time point was tested by the Mann-Whitney U test. An unpaired, two-tailed Student's t test was used to determine the significance of viable bacterial counts, MPO, real-time RT-PCR, and protein assays. Data were considered significant at p < 0.05.
Expression of mBD3 and mBD4 in BALB/c vs B6 mice
The distribution of mBD3 and mBD4 in corneal epithelium and stroma of BALB/c and B6 mice before and after infection with P. aeruginosa was tested in BALB/c and B6 mice at 1 and 5 days p.i. using immunofluorescent staining (Figs. 1⇓ and 2⇓). No staining for mBD3 or mBD4 was detected in normal, uninfected BALB/c and B6 corneas (data not shown). At 1 day p.i., mBD3 staining was similar in infected B6 and BALB/c corneas (Fig. 1⇓, A and B), while qualitatively more mBD4-positive cells were detectable in both corneal epithelium and stroma in B6 vs BALB/c mice (Fig. 1⇓, C and D). At 5 days p.i., mBD3 staining of increased intensity was detected in BALB/c vs B6 corneal epithelium, but no difference was seen in stromal staining between the two groups (Fig. 2⇓, A and B). In contrast, qualitatively more mBD4-positive cells were detectable in both epithelium and stroma in B6 vs BALB/c infected corneas (Fig. 2⇓, C and D). Controls (primary Ab replaced with donkey IgG) were similar to SYTOX Green Nuclear staining (Figs. 1⇓, E and F, and 2⇓, E and F). Additionally, mRNA transcription levels of mBD3 and mBD4 in separated epithelium and stroma of normal uninfected and infected BALB/c and B6 corneas were tested at 5 days p.i. using real-time RT-PCR (data not shown) and confirmed the 5 days p.i. immunostaining data.
Silencing mBD3 and mBD4
Because the distribution patterns (mRNA and immunostaining) suggested that mBD3 and mBD4 were differentially expressed in infected BALB/c and B6 corneal epithelium and stroma, the next series of in vivo studies were designed to determine their effects in host defense against corneal infection. First, BALB/c mice were subconjunctivally injected and topically treated with scrambled control or mBD3- or mBD4-specific siRNA to determine whether knock down of either defensin would impair host defense. The results indicated that by 5 days p.i, the cornea of most mBD3 siRNA-treated mice displayed an enhanced level of disease (grade of 3/4), whereas almost all scrambled control-treated corneas showed less opacity/disease (grade of 1/2). Clinical score data (Fig. 3⇓A) showed that mBD3 siRNA-treated mice exhibited increased disease at 3 and 5 days p.i. (both p < 0.001). Representative photographs taken with a slit lamp of control, scrambled (Fig. 3⇓B) vs mBD3 (Fig. 3⇓C) siRNA-treated mice at 5 days p.i. are provided. Treatment with mBD3 siRNA resulted in either perforation (grade of 4, data not shown) or dense opacity covering the entire anterior segment (grade of 3, Fig. 3⇓C) and overall more inflammation than scrambled, control treatment (Fig. 3⇓B) at 5 days p.i.
On the other hand, no significant difference was shown between mBD4 siRNA and scrambled control-treated mice at 1, 3, and 5 days p.i. By 5 days p.i., clinical score data showed that mBD4 siRNA treatment did not significantly change the host response in BALB/c mice after P. aeruginosa infection (Fig. 3⇑D). Representative photographs taken with a slit lamp at 5 days p.i. showed similar corneal opacity/disease in scrambled control (Fig. 3⇑E) vs mBD4 siRNA-treated (Fig. 3⇑F) mice. RT-PCR data confirmed that silencing was significant and specific for mBD3 and mBD4 (Fig. 3⇑, G and I), and that mRNA levels for mBD4 expression in mBD3 siRNA-treated mice as well as mBD3 expression in mBD4 siRNA-treated mice were not changed significantly (Fig. 3⇑, H and J).
In vivo studies of silencing mBD2, mBD3, or both defensins
Our previous studies demonstrated that mBD2 promotes resistance against P. aeruginosa-induced corneal infection. Therefore, we next used siRNA treatment to determine whether mBD2 and mBD3 function together in ocular immune defense. Results indicated that after infection, the cornea of mice treated with siRNA for both defensins displayed an enhanced level of disease when compared with scrambled control or either mBD2 or mBD3 siRNA-treated mice. Clinical scores (Fig. 4⇓A) indicated statistically significant differences at 1, 3, and 5 days p.i. (all p < 0.001) after silencing both defensins. For either mBD2 or mBD3 siRNA treatment, clinical score differences were shown at 3 and 5 days p.i., but not at 1 day p.i., when compared with scrambled controls. Representative photographs taken with a slit lamp of infected eyes at 5 days p.i. are provided for mice treated with scrambled control (Fig. 4⇓B, grade of 1/2), siRNA for mBD2 (Fig. 4⇓C, grade of 3), mBD3 (Fig. 4⇓D, grade of 3), or both defensins (Fig. 4⇓E, grade of 4). Meanwhile, bacterial plate counts (Fig. 4⇓F) and MPO activity (Fig. 4⇓G) were used to detect viable bacteria and PMN infiltration in the infected cornea of mice treated with siRNA for mBD2, mBD3, or both defensins vs scrambled controls at 1 and 5 days p.i. When compared with controls, silencing both defensins led to enhanced bacterial counts and PMN recruitment at 1 and 5 days p.i. (bacteria: p < 0.01, p < 0.001; PMN: p < 0.001, p < 0.001); silencing mBD2 elevated both at 5 days p.i. only (both p < 0.001); silencing mBD3 increased bacterial counts at 5 days p.i. (p < 0.01) and PMN infiltration at 1 and 5 days p.i. (p = 0.02, p < 0.001). Furthermore, when comparing knockdown of both defensins with either mBD2 or mBD3 alone, bacterial counts were elevated at 5 days p.i. (p = 0.02, p < 0.01) and at 1 and 5 days p.i. (all p < 0.001), PMN were increased. RT-PCR data also demonstrated that siRNA treatment for mBD2, mBD3, or both defensins was significant and specific at 5 days p.i. (Fig. 5⇓).
Effects of silencing on proinflammatory cytokines and TLR signaling
Since silencing both mBD2 and mBD3 elevated bacterial load and PMN infiltration in BALB/c corneas earlier after P. aeruginosa infection than did silencing either defensin alone, the next series of studies were initiated to investigate whether the two defensins acted together to regulate host pathogen immune mechanisms. mRNA transcription levels of proinflammatory cytokines and TLR signaling molecules were evaluated at 5 days p.i. by real-time RT-PCR (Table I⇓). At 5 days p.i., the mRNA transcription levels of IFN-γ, MIP-2, TNF-α, IL-1β, TLR4, TLR2, MyD88, and NF-κB were significantly up-regulated by siRNA treatment for mBD2, mBD3, or both defensins, while iNOS levels were significantly up-regulated after silencing mBD2 alone, but down-regulated after silencing mBD3 or both defensins, when compared with controls. No changes in TLR5 and TLR9 transcription were shown between silenced and control groups. Moreover, mRNA transcription levels of selected cytokines/molecules at 5 days p.i. were calculated as the n-fold differences of transcription in mice treated with siRNA for mBD2, mBD3, or both defensins vs scrambled control. The results indicated that silencing both defensins significantly elevated the relative fold increase of IFN-γ, TLR2, MyD88, and NF-κB transcription, when compared with either mBD2 or mBD3 siRNA treatment (data not shown). Additionally, to determine whether mBD2 and mBD3 function together to modulate inflammatory process, protein expression levels of MIP-2 (Fig. 6⇓A), TNF-α (Fig. 6⇓B), and IL-1β (Fig. 6⇓C), key regulators and mediators of inflammatory responses, such as PMN activation, proliferation and chemotaxis, were examined by ELISA at 1 and 5 days p.i. Results suggested that protein levels of these cytokines were significantly up-regulated at 5 days p.i. in siRNA mBD2, mBD3, and both defensins vs scrambled control-treated mice (MIP-2: p = 0.01, p = 0.03, p < 0.001; TNF-α: p = 0.03, p = 0.03, p < 0.01; IL-1β: p < 0.001, p < 0.001, p < 0.001, respectively), as well as when comparing knockdown of both defensins with either mBD2 or mBD3 silencing (MIP-2: p = 0.04, p = 0.03; TNF-α: p = 0.03, p = 0.02; IL-1β: p = 0.02, p < 0.01, respectively).
Cell sources of mBD2 and mBD3 in stroma
To identify the sources of defensins 2 and 3 in the infected corneal stroma of BALB/c mice, dual label immunostaining was used. mBD2 staining was detected in fibroblasts (Fig. 7⇓A), Mφ (Fig. 7⇓C), and PMN (Fig. 7⇓E), while mBD3-specific staining was detected only in PMN (Fig. 7⇓G) but not in the other cell types (data not shown). Controls (Fig. 7⇓, B, D, F, and H), in which the primary Abs were replaced with the same host IgG, were similar to SYTOX Green Nuclear staining (not shown).
In humans, the most important defensins are hBD2 and hBD3. They are mainly expressed in a variety of epithelial cells and play an important role in both innate and adaptive immunity (3, 12, 13, 14, 15, 25). However, little is known regarding the functions of their murine homologs (8, 9), mBD3 and mBD4, especially in the ocular immune defense system.
In this regard, our studies revealed the distribution patterns of these defensins in normal uninfected and infected corneas. Immunostaining and RT-PCR data (not shown) provided evidence that both mBD3 and mBD4 were inducibly expressed and disparately regulated in the corneal epithelium and stroma of BALB/c vs B6 mice after infection, suggesting a potential role for the two defensins in the development of the resistant vs susceptible phenotype. In vivo knockdown studies demonstrated that mBD3, rather than mBD4, promoted host resistance against P. aeruginosa-induced corneal infection. Data to support this tenet included confirming the specificity and selectivity of each knockdown by RT-PCR, as well as photographs taken with a slit lamp and clinical scores that visually showed little difference between mBD4 siRNA vs scrambled control-treated corneas. All of these suggested that mBD4, similar to mBD1 (11), is not required for ocular immune defense against P. aeruginosa. These data were accompanied by similar mRNA distribution patterns (data not shown) of selected proinflammatory cytokines and TLR signaling molecules in infected corneas of mBD4 silenced vs control-treated mice. In contrast, at 5 days p.i., mBD3 siRNA-treated BALB/c mice displayed increased corneal opacity and exacerbated ocular disease, increased bacterial plate counts and PMN recruitment, as well as elevated mRNA levels of selected proinflammatory cytokines and TLR signaling molecules, when compared with controls. Taken together, these results provided direct evidence that mBD3 promoted resistance to P. aeruginosa keratitis by modulating bacterial load, PMN infiltration, inflammation, and TLR activation.
Nonetheless, in vitro studies have reported that defensins may act synergistically in immune defense (5, 16). As a matter of fact, previous studies have revealed that mBD2 is required in ocular defense against P. aeruginosa-induced corneal infection (11). Thus, we tested the interaction of the two defensins by silencing both. This led to a significantly increased disease and enhanced bacterial counts earlier (1 day p.i.) than when silencing either defensin alone, indicating their antimicrobial synergy. Additionally, compared with knockdown of either mBD2 or mBD3 alone, silencing both defensins elevated mRNA levels of TLR2, MyD88, NF-κB, and IFN-γ (calculated as n-fold difference, data not shown), as well as protein expression of MIP-2, IL-1β, and TNF-α at 5 days p.i. Regarding these molecules, IFN-γ is a hallmark regulatory cytokine in inflammation and Th1 responses, which can also induce PMN survival due to its antiapoptotic effect (26, 27, 28). TLRs are critical in innate immune response, and their activation can induce the production of inflammatory cytokines, including MIP-2, TNF-α, and IL-1β, which can modulate PMN activation, proliferation, and chemotaxis (29, 30, 31, 32, 33). Thus, it is logical to predict that mBD2 and mBD3 function together to modulate ocular inflammatory responses through IFN-γ and TLR signaling pathways and their interactions (34).
Although both mBD2 and mBD3 contribute to host resistance against bacterial keratitis, the mechanisms by which they do so appear to differ. First, both have antimicrobial activities; however, mBD2 is salt sensitive and preferentially kills Gram-negative bacteria (7), while mBD3 can kill both Gram-negative and -positive bacteria, with much less salt sensitivity (35). Second, in our study, silencing of mBD3, not mBD2, resulted in earlier PMN infiltration (at 1 day p.i.), compared with controls, indicating that in mice, mBD3 may be more important than mBD2 in regulating PMN recruitment. Third, studies described herein provide evidence that iNOS expression was up-regulated by mBD2 silencing, but down-regulated by silencing mBD3 or both defensins. These differences are puzzling, but they may be explained, in part, in the light of other studies suggesting that LPS and IFN-γ regulate iNOS expression through differential activation of TLR4 and JAK/STAT pathways (29, 36). More specifically, regarding TLR4, both defensins have potential LPS neutralization activity (37), but only mBD2 can activate TLR4 (10, 38). This difference may contribute to the disparate outcome in iNOS transcription levels observed after silencing mBD2 vs mBD3 or both defensins.
Additionally, regarding IFN-γ and iNOS expression, other scenarios require consideration. In this regard, iNOS is expressed maximally after an inflammatory stimulus and induces the production of large micromolar quantities of NO, a free radical gas with both signaling and antimicrobial functions (39, 40). NO is an essential mediator of immune defense against bacterial infection, whereas excessive production of NO often results in tissue destruction and pathology (26, 40). Moreover, it has been reported that the synergistic interaction between IFN-γ and NO levels regulates ocular disease outcome in resistant BALB/c mice (26). Thus, because silencing mBD3 or both defensins down-regulates NO, mBD3 may be more important than mBD2 in this outcome.
Additionally, studies have demonstrated that defensins are produced as a functionally inactive preprodefensin form in nature and must undergo posttranslational modification to form a biologically active mature peptide, which is sequentially secreted onto the surface such as an epithelium or immediate surroundings to execute their function (41). Thus, we hypothesize that mBDs at the ocular epithelial surface might be functionally more important early in infection than those in the stroma. Our studies demonstrated that both mBD2 and mBD3 are mainly expressed in the corneal epithelium, which may contribute to their synergy in bacterial killing early after infection. In the stroma, whereas PMN produce both defensins, Mφ and fibroblasts produce mBD2 but not mBD3, which may contribute to their individual and combined effects later in the disease process.
In summary, our studies provide direct evidence that both mBD3 and mBD4 are inducibly expressed and disparately regulated in infected corneal epithelium and stroma of resistant and susceptible mice; that of the two defensins tested by knockdown experiments, only mBD3 is required for host resistance against bacterial infection; and that mBD2 and mBD3 function together to modulate bacterial load, PMN infiltration, production of proinflammatory cytokines, iNOS, and TLR signaling in the infected cornea. These data suggest that the defensins may provide a novel target for treatment of ocular infections induced by P. aeruginosa.
The authors have no financial conflicts of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This study was supported in part by Grants R01 EY016058, EY02986, and P30 EY04068 from the National Eye Institute of the National Institutes of Health.
↵2 Address correspondence and reprint requests to Dr. Linda D. Hazlett, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201. E-mail address: lhazlett{at}med.wayne.edu
↵3 Abbreviations used in this paper: mBD, murine β-defensin; iNOS, inducible NO synthase; PMN, polymorphonuclear neutrophil; p.i., postinfection; siRNA, small interfering RNA; Mφ, macrophage; MPO, myeloperoxidase.
- Received July 6, 2009.
- Accepted October 19, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.


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Glare on unabated illegal arms entry
There was a constant flow of illegal arms into Assam for militant outfits, criminal gangs and poachers, according to police records.
The records reveal that 2,171 cases were registered under the Arms Act in the state since 2014.
According to the records, the total number of cases registered in 2014 under Arms Act was 580, followed by 511 in 2015. In 2016, there were 423 cases and in 2017, 370 cases were recorded under the act. At least 287 cases were registered last year.
Police attributed the declining trend in cases to the successful counter-insurgency operations with most outfits joining the peace parleys. Sources, however, claimed that arms smugglers continue to supply to small-time criminal gangs and poachers in the state.
"Nagaon district has emerged as one of the hot favourites for gunrunners. With a number of criminal gangs active and the presence Kaziranga National Park in the district, smugglers are supplying firearms to them and poachers," a source said.
On Monday, Nagaon police apprehended a person from Batadrava and recovered a 9mm factory-made pistol and five live ammunition from his possession. On Friday, in a joint operation by police and Assam Rifles, Mofidul Islam was arrested from Salakati Pathar under Dhing police station in the district. A 7.65mm pistol, one magazine, four bullets and one county-made grenade were recovered from Islam.
Earlier, Nagaon police, in a series of raids, had arrested six persons with firearms from Kathiatoli. They gang had shot a petrol pump manager, Raju Jain, last month and looted lakhs of money.
Nagaon superintendent of police Shankar Barata Raimedhi said, "Since January this year, we have arrested 36 people and recovered 25 firearms and 75 live ammunition. We will continue our drive against illegal arms."
In Assam, there are some 25,000 licensed arms, out of which there are over 1,200 licensed firearms in the city alone. Most of these belong to industrialists, businessmen, politicians, private security agencies and a few sports personalities. The 9mm and 7.62mm pistols are found to be common.


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Oakland-based Egyptian American artist Sarah Farahat invites women of mixed race to join her for a picnic and conversation in the OMCA gardens. Power can look like many things and sometimes she is assumed or unrecognizable. The artist welcomes self-identified mixed race women to take a moment together to relish the power of in between-ness: of slippage, of fuzzy, frizzy, straight, unruly, freckly, caramel, obsidian, ochre and alabaster. Together we will ask questions, speak story and meet one another in-between Here and There.
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Darrell Keith Jones
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Darrell Keith Jones Jr., 34, Beechy Ridge, Bickmore, entered into rest Friday, January 6, 2012. Born February 20, 1977, in Wichita Kansas. He was the son, of Darrell Keith and step-mother Janey Jones of Bickmore. Mother, Gwin Payne of Wichita Kansas. Keith was also preceded in death by his wife Misty. He was correctional officer for the state of West Virginia. In addition to his parents, Keith is survived by his daughters, Alayna Paige and Kaylee Autumn Jones of Beechy Ridge, Bickmore, brothers, Harry Jones of Fresno, Calif, Danny Downtain and sister, Kimberly Bell both Wichita Kansas. Funeral service will be held 10:00 Tuesday, January 10, 2012, at Bentree Church of God, Bentree, with Minister Mike Holcomb, Minister Mike Coleman and Jonathan Brown officiating. Burial will follow in the Tanner family cemetery, Beechy Ridge. Friends may call 6:00 to 8:00 P.M. Monday, at Wilson-Shamblin-Smith Funeral Home, Clay. In lieu of flowers the family would like donations to be made to the Hubbard Hospice House 1001 Kennawa DR. Charleston, WV 25311 On-line condolences may be sent to www.carlwilsonfuneralhome.com Wilson-Shamblin-Smith Funeral Home is honored to be serving Jones Family.
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