This hideous looking strange man could easily navigate through the mud and the dark green scales on his body made him more frightening.
As he shouted in anger, the poisonous miasma in the surroundings all began to gather at their location.
The dense miasma concentrated around the group and continued to condense into a dense fog.
Drops of green poisonous water clattered down all of a sudden.
A poisonous rain fell upon this swamp.
At the same time, Chi Xiao took out a small curtain and expanded it at once, covering the whole group.
The poisonous rain fell on the silver curtain and trailed through the edges, immediately emitting poisonous fumes when falling in the swamp.
After a grunt, Zhua Qi immersed one hand into the swamp and soon, streams of odd green light shot into the mud.
One after another, mud dragons jumped out of the swamp. They were as thick as a human waist and were about ten meters tall, trying to bite their group.
Zhua Qi could not only control the miasma in the Dead Swamp, but could also control the mud.
Shi Yan's expression changed slightly, as he didn't expect themselves facing such a tough enemy right at the beginning of their journey. He was immediately vigilant, circulating his Profound Qi and prepared to attack those mud dragons.
Those mud dragons flew out from the swamps swiftly and moved towards the group from all directions. It was quite magnificent.
The dry land on which the group was currently standing on was continuously sinking into the swamp under Zhua Qi's control.
Xia Xin Yan's eyes turned icy as she decided to use her Incarnation Martial Spirit.
"Zhua Qi! Believe me! I haven't come for you!" Chi Xiao yelled anxiously.
Chi Xiao was fully assured that he could beat Zhua Qi, but the consequence to their group would be dreadful He firmly believed that once they entered into battle, only he and Xia Xin Yan could withstand Zhua Qi's attacks.
"Shi Yan, come here. I will protect you." Xia Xin Yan glanced over him and said without any emotion.
Han Feng and Ku Long were surprised, and looked at Xia Xin Yan in confusion.
So were Wu Yun Lian and Zuo Shi. It was unreasonable that Xia Xin Yan would try to protect Shi Yan.
Shi Yan dared not take the risk, so he hopped through the dry lands to Xia Xin Yan's side.
"Yes. We got a treasure map. And the Gate of Heaven turned out to be located in the Yin Valley!" Chi Xiao had to tell him the truth.
Zhua Qi was stunned, and then giggled cunningly.
As he was giggling, the green miasma in the air began to float away from the group.
As the green miasma dispersed, the poisonous rain stopped as well.
The flying mud dragons turned back to mud and fell into the center of the swamp.
Hundreds of green loaches which were rushing up also stopped as they saw the mud dragons disappear. Under Zhua Qi's order, they went back under the mud as well.
Chi Xiao was relieved and stretched his hand up into the air and shrunk the curtain, finally falling into his palm.
The swamp went back to serenity. Even the green bubbles gradually disappeared. The dangerous swamp suddenly became quiet.
As he was speaking, he looked at Xia Xin Yan.
Chi Xiao could compete with Zhua Qi alone, and with Xia Xin Yan at the Sky Realm, Zhua Qi would have been in great trouble.
Zhua Qi looked towards Xia Xin Yan as he noticed Chi Xiao's gaze, and frowned.
He could feel a stream of forceful power subsiding in her body.
Just from a glance, Zhua Qi noticed that this woman was unmanageable, and she was operating some tricks secretly.
Shi Yan stood by Xia Xin Yan with a gloomy face, still gazing at Zhua Qi vigilantly.
Shi Yan responded casually and hopped through the dry lands where Chi Xiao had tested, to a muddy area ahead, and soon he was ashore.
Zhua Qi grunted, moved his muddy body swiftly in the swamp, and also reached the shore. But half of his body was still in the swamp, for he was still a little suspicious of Chi Xiao.
"Shi Yan, how are you now? The blood of the green loach didn't hurt you? Do you need Zhua Qi's help you remove it?" Chi Xiao asked as he suddenly recalled the incident.
His Immortal Martial Spirit had cleaned the poison before he knew it.
Hearing that, Chi Xiao and Zhua Qi looked at him in surprise.
Only these two knew that a Human Realm warrior would suffer fatigue for at least one day after touching the green loach's blood.
Others didn't know about the blood, so they didn't care much and walked ahead.
Soon, Zuo Shi, Han Feng and others also got out of the swamp and landed ashore.
"Chi Xiao, you really came for the Snake?" Zhua Qi raised his head and asked, while half of his body was still in the mud.
"Behave yourself for the rest of our journey. Take the lead if you want to fight the Snake." Chi Xiao nodded.
"Apart from the Snake, you also want to enter the Gate of Heaven." Chi Xiao grunted.
"Okay." Xia Xin Yan thought for a moment and nodded.
"Well then, you are in." Chi Xiao then replied to him.
"Thin, tall, pale-faced, narrow eyes. He seemed to rarely expose himself to the sun, and trained in a gruesome Martial Skill." Zhua Qi thought for a while and depicted.
Zhua Qi finally landed ashore. His legs were covered with green scales. He seemed not used to walking, for he swayed like a duck.
However, they all had witnessed his ability, they didn't dare make fun of his hilarious walking posture.
As the master of this area, wherever he went, the poisonous miasma dissipated quickly.
There were many pieces of dry land ahead for them to advance smoothly.
They found it much easier to walk after Zhua Qi took the lead. They finished the one-month journey in only eight days.
Eight days later, the group finally entered the Yin Field.


Patio real estate in Toronto is in high demand, but the joy of drinking outdoors doesn't need to break the bank.
For those seeking a slice of sunshine AND affordable drink specials, these are the top destinations for cheap drinks on a patio in Toronto.
Go after work for happy hour deals that include wine bottles for half-price, $6 pints, and $5 mixed drinks between 5 pm and 7 pm.
The backyard patio at this dive bar is the perfect place to pregame on the cheap! Pitchers of Sangria (and other mixed drinks) costs just $13.28 while a pitcher of beer pitcher is priced between $12 and $14.
This well-worn Queen West bar boasts a secluded backyard patio that is always open! Go for $4.50 bottles of domestic and pints from $6.
With drink specials on offer every day of the week, the courtyard at this east side bar is the perfect place to enjoy a drink without breaking the bank. Weekdays from 4 pm to 7 pm a pint of beer costs just $5.75. Meanwhile, cocktails go for $6.42 before 6 pm daily.
The house wine is $1 an ounce while at least a half-dozen different cocktails can be had for $9.25 on the patio of this King West eatery. Compared to neighbouring restaurants, this place is a steal.


Accessibility is ascertained as feature able to enhance the rating of a tourist destination, and Pisa in particular is fairly well placed on the road, railway and airway infrastructure in Italy. This is why Pisa is valuable both as to the ways of getting to this Tuscan city and to the way of heading to other destinations in Italy starting from Pisa.
Getting around in Tuscany should be no problem in general and getting from Pisa to Florence in particular seems to be an even easier task, given this connection is carefully focused on by force of tourist choice to tick off these two Tuscan hotspots. Thus, this route can be covered both by land and by air, buses, trains, cars and planes alike being available to tourists and locals' satisfaction.
There are two major airports servicing the tourist platform of Tuscany, one pertaining to Pisa, and the other to Florence. The International Galileo Galilei Airport of Pisa is only one kilometer from the center of the city, whereas the Amerigo Vespucci Airport (also referred to as the Peretola Airport) of Florence is about 4 kilometers from the center of Florence. It should me mentioned the latter is a smaller venue, and it chiefly deals with domestic flights. Trips by plane from Pisa to Florence seem to be the most timesaving manner of covering the distance, also given the airports are fairly well connected to the cities. Thus, one can always get from Pisa proper to the airport by bus, by train or by car (either the personal vehicle or a rented one), go aboard and then land in the Amerigo Vespucci Airport. At its turn, the latter is, again, connected to Florence mainly by road, a bus operator performing regular routes from the airport to the Santa Maria Novella train station which, for that matter, is located in the very center of the city. This is a fairly convenient way for passengers to be shuttled from the airport to Florence, though alternatives refer to traveling by car (rented or otherwise) or by taxi, which usually does not take more than 15 to 20 minutes.
Getting from Pisa to Florence by bus is, again, an efficient manner of covering the distance. The main bus operator providing trips from Pisa to Florence is CPT (Compagnia Pisana Transporti), the main station from which they start off in Pisa being the one located in Piazza Vittorio Emanuele II, close to the Central Station in Pisa. However, one can always choose to head for Florence directly from the International Airport of Pisa which, again, features a fine range of airport transfers solutions. Tickets can be purchased at the Information Desk, and the buses operated by Terravision reach to Florence in a fairly short time, a benefit complemented by the fact that they also stop in the Santa Maria Novella station, in the center of the city. Details on the hours of departure can be found by visiting www.pisa-airport.com.
Traveling from Pisa to Florence by train is, again, an efficient way of getting around in Tuscany. A first option refers to taking the train from the International Airport of Pisa, five trains connecting the two Tuscan cities each day. Further details can be learned at http://www.pisa-airport.com/aeroporto_di_pisa_221.html. Other alternatives refer to taking the train either from the main train station in Pisa, namely, from the Pisa Central Station, or from the smaller San Rossore Station, the distance being coverable in about 90 minutes by the some 40 trains a day, which is not surprise since Pisa and Florence are the main tourist hubs of Tuscany.
Cars (either rented or otherwise) represent a further manner of traveling from Pisa to Florence. In order to do that, tourists should resort to SS1 Aurelia, the main direct road in and out of Pisa, and then enter either the A11 Mare-Florence motorway or the Pisa-Livorno-Florence expressway.
Siena is yet another province in Tuscany to amass a reasonable amount of tourist objectives, which is way trips from Pisa to Siena are quite common. The distance between Pisa and Siena are coverable by all land means of transportation.
Taking the bus from the International Airport in Pisa or from the Piazza Vittorio Emanuele II in Pisa is the way to start the trip. Except for a stop in Poggibonsi, the buses departing from the airport need no more than two hours to arrive to Piazza Gramsci in Siena, though other two stops in Siena as such are Siena Nord and Siena P. Rosselli. Further information on this route is to be found by visiting www.pisa-airport.com.
Trains need some 100 minutes to get from Pisa to Siena, this connection being serviced by hourly trains meeting both locals and tourists' need of transportation. The train station in Siena is located in Via Carlo Rosselli, some 2 kilometers from Siena's historical center.
It takes about two hours for cars to get from Pisa to Siena, and the best combination of roads seems to be the following: SS1 Aurelia, followed by the A11 Mare-Florence motorway or the Pisa-Livorno-Florence expressway, and then southwards either SS222 Chiantigiana or the SS2 Siena-Florence motorway.
Trips from Pisa to Rome are quite popular both with tourists and locals. This is why the connection to the capital of Italy is made both by air and by land, which is to say that all roads do lead to Rome.
The only airport in the entire Italy exceeding the International Airport of Pisa in the ability to service the tourist platform of the country is the Leonardo da Vinci Airport (otherwise referred to as the Fiumicino Airport), located some 30 kilometers southwestwards from the center of Rome. The connection between the airport and Rome is assured by public means of transportation – 2 trains with different destinations in Rome, departing every 30 minutes from the airport, and buses shuttling their passenger in the very centre of Rome again every 30 minutes. The 265 kilometer distance between Pisa and Rome can be easily covered in no more than 30 minutes, which is extremely timesaving for anyone who thinks of themselves and busy tourists. Visitors are likely to be directed to Terminal A (which deals exclusively with domestic flights) or, at most, to Terminal B (domestic and international flights alike). Another alternative in terms of landing refers to the second airport servicing Rome, namely, the Ciampino Airport (15 kilometers southwards from Rome) which mainly handles charter and continental flights.
Visitors interested in traveling from Pisa to Rome by bus should rest assured: given both Pisa and Rome are major tourist destinations, there is a fine connection between these two cities by means of buses. From Pisa, tourists must depart from Piazza Vittorio Emanuele II where the main station for intercity buses is located, and they are highly likely to descend nearby Stazione Terminale train station in Rome. All in all, most visitors of Italy prefer traveling by train to traveling by bus when it comes to means of transportation on land. Either way, buses remain an alternative.
Trains are said to be the most convenient way of traveling not only between Pisa and Rome in particular, but in the entire Italy all the same, given both the less pricy tickets and the opportunity of sightseeing. The railway connection between Pisa and Rome is made by about 20 trains a day, and it takes trains about 3 to 4 hours to cover the distance. From Pisa, trains leave from the Central Station and they arrive in Rome in Stazione Centrale.
Those who want to explore Italy at their own pace often resort to cars. In view of getting from Pisa to Rome, one should exit the E80 expressway and then resort to the A12 Rome-Civitavecchia highway.
Venice is serviced by two airports, namely, the Marco Polo Airport (12 kilometers from Venice, in Tessera, very close to Mestre) and the Treviso Airport (25 kilometers from Venice). The connection between the airport and the city of Venice is made by bus and by boat, as far as public transport goes, though tourists also have the opportunity of resorting to taxis and cars for rent (though they should carefully ponder on the fact no kind of car is allowed in Venice as such). Thus, a trip by plane from the International Galileo Galilei Airport of Pisa to the Marco Polo Airport of Venice seems to be the most timesaving manner of getting from the tourist jewel of Tuscany to the Floating City of Italy.
The only way to get from Pisa to Venice by bus is to make a combination of routes covered by various regional buses, which, from the first place, defeats the purpose of a trip one should enjoy to the utmost degree. Thus, if starting off in Pisa, one should head either for Florence or Bologna, and then head for the Province of Veneto where the main reliable stops refer to either Padua or Trieste. Verona might also be considered as a stop in Veneto while traveling for Venice.
It seems there is no direct link between Pisa and Venice as far as traveling by train goes. Most trains stop in Florence (Santa Maria Novella station), from where another train must be taken in view of getting to Venice, most of them via Mestre. This travel takes about 3 hours and 40 minutes, though an even longer alternative refers to a stop and to a change in Bologna (more than 4 hours and 40 minutes). The main train station in Venice is the so-called Santa Lucia, which is conveniently located in Venice (more precisely, in the main city) due to its proximity to the Grand Canal and to the old city of Venice. Further details can be learned by visiting www.trenitalia.com.


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Research article | Open | Open Peer Review | Published: 25 March 2016
Synergistic effect of κ-carrageenan on oxazolone-induced inflammation in BALB/c mice
Wei Wu1,
Feng Wang2,
Xin Gao1,
Tingting Niu1,
Xiaojuan Zhu1,
Xiaojun Yan1,3 &
Haimin Chen1,3
Carrageenan is a traditional ingredient that has been widely used in the food industry. In the present study, we propose a hypothesis that carrageenan is a conditional inflammatory agent. When the intestinal tract is in an "unhealthy" state such as that during bacterial infection or acute inflammation, carrageenan can synergistically enhance the inflammatory response.
BALB/C mice received κ-carrageenan via intragastric administration prior to the induction of oxazolone colitis. Weight changes, survival rate, histologic change, secretion of inflammatory cytokines, ratio of regulatory T cells (Tregs) in peripheral blood, and expression of genes and proteins involved in inflammation and cell proliferation in the colonic mucosa were examined.
Intragastric administration of κ-carrageenan to BALB/c mice prior to the induction of oxazolone colitis resulted in an aggravation of body weight loss, a decrease in the survival ratio, aggravation of colonic inflammation, and decrease in the ratio of CD4 + CD25+/CD4+. The secretion of interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) also significantly increased after κ-carrageenan administration. κ-Carrageenan, together with oxazolone, suppressed the expression of forkhead box p3 (FOXp3) and increased the expression of Toll-like receptor 4 (TLR4), Nuclear factor-κB (NF-κB), and proliferating cell nuclear antigen in the colonic mucosa. These results were confirmed by qRT-PCR and western blot analyses at the molecular and protein levels, respectively.
κ-Carrageenan aggravated oxazolone-induced intestinal inflammation in BALB/c mice. This effect is associated with an activation of the TLR4-NF-κB pathway, a decreased ratio of Tregs, and the induction of Th2-dependent immune responses.
Carrageenan, a high-molecular weight saccharide, is used widely as a thickener, stabilizer, emulsifier, and texturizer in a variety of processed foods (including infant formula, whipped cream, cottage cheese, ice cream, and nutritional supplements) and other products (i.e., pharmaceuticals, cosmetics, and toothpaste) [1–3]. Several international agencies have earlier reported that carrageenan is safe for human consumption [4, 5]. However, investigators later demonstrated that carrageenan induces gastrointestinal ulcerations and cancer in animal models [2, 4]. Carrageenan has been widely used in experimental models of inflammation to assess the activity of anti-inflammatory drugs and to study mediators of inflammation [6]. Therefore, the concept of safety of carrageenan has remained highly controversial.
Current research studies on carrageenan have mainly focused on its effects at the organismal and cellular levels. Animal models such as rats [6], guinea pigs [7], pig [8], mouse [9], and rhesus monkeys [10] have demonstrated that carrageenan induces colorectal tumors and ulcerative colitis. However, several international agencies have concluded that carrageenan is harmless [4, 5]. The US Food and Drug Administration (FDA) has declared food-grade carrageenan as generally recognized as safe (GRAS) for use as a food ingredient [11]. Tobacman et al. conducted extensive research studies to evaluate the safety of carrageenan at the cellular level [2, 12–19]. They observed that exposure of human intestinal epithelial cells to food-grade carrageenan triggered a distinct inflammatory response via the activation of Bcl10 in the NF-κB pathway [20], as well as up regulation of interleukin-8 (IL-8) secretion [16, 20], and identified that the Toll-like receptor 4 (TLR4) is the surface membrane receptor for carrageenan in human colonic epithelial cells. Bernard et al. observed that carrageenan induces tumor necrosis factor alpha (TNF-α) production in human monocytes [21]. Although an upregulation of inflammatory cytokines in human cells occurs following food-grade carrageenan exposure, we found that the increase was actually low, only 2–3-fold higher than that observed in the blank control [16, 20]. Is this difference in expression level enough to cause a strong inflammatory reaction in vivo? Although lipopolysaccharides, which are highly proinflammatory molecules, can increase the secretion of IL-8 and TNF-α in macrophages by a dozen- to a hundred-fold [22], the inflammatory mechanism of carrageenan remains elusive.
In our previous studies, we observed that λ-carrageenan enhances the LPS-induced production of IL-8 at the cellular level [23]. Moreover, when differentiated human colonic epithelial cells were co-cultured with macrophage cells, the epithelial monolayers were significantly and easily damaged by carrageenan. Based on these earlier findings, we hereby propose the hypothesis that carrageenan is a conditional inflammatory agent. When the intestinal tract is in an "unhealthy" state such as that during bacterial infection or acute inflammation, carrageenan can synergistically enhance the inflammatory response.
Therefore, in the present study, an oxazolone (OXA)-induced mild intestinal inflammation model was developed to investigate whether κ-carrageenan can enhance the inflammatory responses of the gut with acute inflammation. Furthermore, the mechanism underlying this inflammatory response was also investigated in relation to the TLR4 pathway.
Seven-week-old male and female BALB/c mice with weights ranging from 20-25 g were purchased (SCXK 2012–0001, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), housed in the animal center of Ningbo University Medicine College (Ningbo, China), and used in the present study. Five same-sex mice were housed in a cage and maintained under a 12-h light/12-h dark cycle (08:00 AM lights on) with food and water provided ad libitum. Housing and experimental environments were temperature- and humidity-controlled (21 ± 2 °C and approximately 60 %, respectively). All mice were allowed to habituate to the housing environment for 3 days prior to gavage administration. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Ethical Committee of Animal Use and Protection of Ningbo University Health Science Center.
Animal experimental procedure
Eighty mice were divided into eight groups and placed in clear plastic cages with a mesh top under specific pathogen-free conditions, with each experimental group consisting of four males and four females. The experimental groups were designed as follows: the blank group only received a saline solution; the OXA group was treated with 150 μL of 1 % OXA; the carrageenan groups were treated with different concentrations of carrageenan, namely, the low-dose group (1.7 mg/kg, LOW), medium-dose group (8.3 mg/kg, MED), and high-dose group (41.7 mg/kg, HIG); carrageenan + OXA groups received low, medial, and high doses of carrageenan following by the administration of OXA and marked as "LOW + OXA," "MED + OXA," and "HIG+ OXA."
κ-Carrageenan (Sigma Chemical Co., St Louis, MO, USA) was dissolved in saline with vigorous stirring at 37 °C and administered at three different doses, 1.7 mg/kg, 8.3 mg/kg, and 41.7 mg/kg by gavage administration. These doses were decided according to the data of preliminary experiments based on related study [18]. To assess the synergistic effect of carrageenan on OXA-induced inflammation, different doses of carrageenan were gavage administered for two weeks prior to OXA administration.
OXA-induced intestinal inflammation was established as previously described by Heller et al., with minor modifications [24]. To presensitize mice, a 2 × 2 cm area of the abdominal skin was shaved, and 200 μL of OXA [a 3 % (w/v) solution in 100 % ethanol, Sigma-Aldrich, St. Louis, MO] was smeared 5 days before OXA was administered. Five days after presensitization (day 0), the mice were rechallenged intrarectally with 100 μL of 1 % OXA in 50 % ethanol or only 50 % ethanol (vehicle, Blank group) under general anesthesia with isoflurane (Baxter, Deerfield, IL, USA). Intrarectal injection was administered with a polyurethane umbilical catheter. To ensure distribution of the OXA throughout the entire colon, the mice were held head down in a vertical position for 60 s after injection.
The mice were then monitored for symptoms of diarrhea, loss of body weight, and death the next day (day 1). The mice underwent exsanguination by cardiac puncture and the collected blood was used to determine the CD4+CD25+ Treg ratio and cytokine levels by flow cytometry. The colon was also isolated; one portion was used in macroscopic evaluation, whereas the rest was fixed in paraformaldehyde (Merck, Darmstadt, Germany) or glutaraldehyde solution for immunohistochemical and scanning electron microscopy analysis.
Macroscopic evaluation
Macroscopic evaluation of colonic tissues used the following criteria: 0, normal; 1, erythema only; 2, erythema, slight edema, and small erosions; 3, two or more bleeding ulcers and/or inflammation and/or moderate adhesions; and 4, severe ulceration and/or stenosis with dilations and/or severe adhesions.
Histological analysis
The colonic tissues were dissected and washed with Hank's balanced salt solution (containing 10 μg/mL of gentamicin, 100 U/mL of penicillin, and 100 μg/mL of streptomycin). The tissues were then fixed in 10 % natural buffered formalin, embedded in paraffin, cut into tissue sections (5-μm-thick), and stained with hematoxylin and eosin (H&E).
Histological examination was evaluated as previously described [25]. The degree of histologic damage and inflammation was graded in a blinded fashion by expert histologists. The following manifestations were included in the evaluation: the amount of inflammation (0, none; 1, mild; 2, moderate; 3, severe; and 4, accumulation of inflammatory cells in the gut lumen), distribution of lesions (0, none; 1, focal; 2, multifocal; 3, nearly diffuse; and 4, diffuse), depth of inflammation and layers involved (0, none; 1, mucosa only; 2, mucosa and submucosa; 3, limited transmural involvement; and 4, transmural), and nature of mucosal changes (0, none; 1, minimal degeneration; 2, more degeneration; and 3, more necrosis). The overall histologic score was the sum of the four manifestations (maximum score: 15).
Intestinal mucosal morphology
The processing of intestinal segments for scanning electron microscopy was conducted as described elsewhere [26]. Briefly, one segment of the tissue sample was fixed with 2.5 % glutaraldehyde. The segments were washed twice in phosphate buffer (pH = 7.2) and then post-fixed in osmium tetroxide (1 % in phosphate buffer, pH = 7.2, 2 h). After a series of dehydration steps in alcohol from 30 % to 100 %, the segments were critical point-dried (CPD030; BAL-TEC; Balzers; Liechtenstein), mounted on Al stubs, sputter-coated with gold by using a high-resolution fine coater (SCD005; BAL-TEC; Balzers; Liechtenstein), and then examined under a Jeol JSM-6301 scanning microscope (JEOL Ltd., Musashino, Tokyo, Japan). Morphological evaluation of the intestinal mucosal and microvilli was then performed. Scanning electron microscopic images (×200 magnification) were analyzed to measure the histological changes on the epithelial cell surface.
Determination of CD4 + CD25 + Treg ratio by flow cytometry
A total of 2.5 μL of anti-mouse CD3-eFluor450, 0.625 μL of anti-mouse CD4-PE, 0.625 μL of anti-mouse CD25-APC (all from eBioscience, California, USA) were added to 100 μL of peripheral blood. Following incubation at room temperature in the dark for 15–30 min, 1 mL of erythrocyte lysis solution was added to the samples and incubated for 15–20 min. The cells were washed with 2 mL phosphate-buffered saline (PBS) and resuspended in 500 μL PBS. The cells were then analyzed using a Beckman Gallios flow cytometer (Beckman Coulter, Inc., California, USA).
Immunohistochemical analyses
For immunocytochemical analysis, the mouse colonic tissues were fixed with 4 % paraformaldehyde in PBS, embedded in paraffin, and cut into tissue sections (5-μm-thick). The tissues were blocked with 1 % bovine serum albumin (BSA, Nacalai tesque) for 60 min at room temperature. The primary antibodies used in the present study included anti-TLR4 antibody (1:200, ab22048, Abcam, Cambridge, UK), anti-NF-κB antibody (1:200, 8242, CST, Danvers, MA, USA), anti-FOXp3 antibody (1:200, ab54501, Abcam, Cambridge, UK), and anti-PCNA antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies included goat anti-rabbit IgG (PV-9001, ZSGB-BIO, Peking, China) and rabbit anti-mouse IgG (PV-9005, ZSGB-BIO, Peking, China). Negative controls substituted with non-immunized mouse IgG at the same concentrations as that for each primary antibody were employed. Images were captured using a Leitz Dialux 22 Microscope (Leica Microsystems, Wetzlar, Germany) equipped with a QIcam Fast 1394 camera (QImaging, Surrey, BC, Canada). The images were analyzed using the software, Image Pro Plus 6.0 (Media Cybernetics UK, Marlow, UK). The levels of expression of TLR4, NF-κB, FOXp3, and PCNA were determined by measuring the positively labeled areas in relation to the total area.
Analysis of cytokine levels
Serum samples were collected after centrifugation of blood and stored at −80 °C. The levels of proinflammatory cytokines, including interleukin IL-6, tumor necrosis factor (TNF), Th1 (IL-2, interferon IFN-γ), Th2 (IL-4, IL-10), and Th17 (IL-17) cytokines in each serum sample were determined by using the BD Cytometric Bead Array mouse Th1/Th2/Th17 7 cytokine kit (BD BioSciences, NJ, USA) according to the manufacturer's instructions. Samples and standards were analyzed on a FACS Calibur flow cytometer (BD BioSciences, NJ, USA). The concentrations were assessed by using FCAP Array software.
RNA extraction and real-time quantitative reverse transcription-PCR (RT-qPCR) analysis
Total RNA was isolated from scraped colonic mucosa of experimental mice using the TaKaRa RNAiso Plus Reagent (TaKaRa, Dalian, China) following the manufacturer's instructions and then treated with RNase-free DNase I. Then, 2 μg of total RNA was re-transcribed into cDNA using a total reaction volume of 40 μL following standard M-MLV reverse transcriptase protocols (TaKaRa, Dalian, China). The primers of mouse target genes, including TLR4, NF-κB, FOXp3, and PCNA, as well as reference genes β-actin, GAPDH, and 18S-rRNA are listed in Table 1. The corresponding PCR products were sequenced by an ABI 3730 automated sequencer (Invitrogen, Carlsbad, CA, USA). To assess PCR efficiency, 10-fold serial dilutions of TLR4, NF-κB, FOXp3, PCNA, β-actin, Gapdh, and 18S-rRNA plasmid cDNA were used to generate a standard curve for each assay plate. The PCR reaction system included 1 μL of cDNA, 0.4 μM of forward and reverse primers, 10 μL of SYBR Premic Ex Taq II, 7.4 μL of dH2O, as recommended by the manufacturer of SYBR-Green I (TaKaRa, Dalian, China). The cycling conditions were 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 15 s, which were conducted on a Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany). By using a standard curve, PCR efficiency was calculated (Table 1). After the amplification, melting curves were obtained by slow heating from 60 °C to 95 °C at increments of 0.5 °C/s and continuous fluorescence collection, which confirmed that only our specific product peaks were detected. RT-qPCR analysis of the samples was conducted as earlier described. Relative gene expression was analyzed by using the comparative cycle (Ct) value, which was compared using the relative quantification method. The mRNA expression of TLR4, NF-κB, FOXp3, PCNA, GAPDH, and 18S-rRNA were normalized against the expression of β-actin.
Table 1 Oligonucleotide primers used in this work
Preparation of nuclear extracts
To prepare a whole lysate solution, the collected tissues were washed twice with ice-cold PBS and lysed in a lysis buffer (Beyotime, Shanghai, China) that contained 1 mM of phenylmethylsulfonyl fluoride (PMSF) and a protein phosphatase inhibitor cocktail (Roche, Basel, Switzerland) for 10 min on ice. The homogenates were centrifuged at 12,000 rpm at 4 °C for 10 min. Total protein was extracted from the scraped colonic mucosa of the experimental mice using a NE-PER nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA), following the manufacturer's recommendations. Protein concentration was determined using a Bio-Rad DC Protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), following the manufacturer's instructions.
Proteins (30 μg) in the nuclear extracts or in the whole lysate were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes, and probed with antibodies against TLR4, NF-κB, and p65 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by appropriate horseradish peroxidase (HRP)-linked secondary antibodies (CST). The immunoreactive proteins were detected by using WesternBright ECL (Advansta Inc., Menlo Park, CA, USA). The results were quantified by measuring the band intensity relative to that of β-actin using the AlphaView™ Software (Alpha Innotech, San Leandro, CA, USA), and expressing as the relative intensities.
Statistical analyses were performed using the SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA). The results were expressed as means ± SEM. P < 0.05 was considered to indicate statistically significant differences. Macroscopic and histological score analyses were performed by using the student's t-test. Differences in weight loss, cytokine levels, ratio of Treg (CD4+CD25+CD127dim), mRNA expression, western blot data, and average integral optical density of expression of various inflammatory proteins in mouse colonic mucosa among different groups were analyzed by using one-way ANOVA after post hoc testing.
κ-Carrageenan aggravates weight loss and decrease in the survival ratio of mice with OXA-induced inflammation
To determine whether pretreatment with κ-carrageenan affected the damage induced by OXA, weight loss and survival rates of the mouse models were examined. Figure 1a reveals that the percent of weight loss on day 1 was significantly different [F (7, 55) = 30.098, P < 0.01]. No significant differences in weight loss between the Blank group and the carrageenan alone-treated groups on day 1 were detected. The rate of weight loss in mice belonging to the OXA-treated group was significantly lower than that observed in Blank group (P < 0.01), which decreased by about 12 %. Interestingly, pretreatment with κ-carrageenan aggravated the weight loss. The body weights of mice from the LOW + OXA, MED + OXA, and HIG + OXA groups were markedly lower than that of the OXA-treated group on day 1 (P = 0.048, P = 0.001, and P = 0.047), which decreased by about 16.9 %, 21.3 %, and 16.5 % respectively.
The effect of κ-carrageenan on body weight and survival ratio of mice with oxazolone-induced colitis. a Weight changes. b Survival ratio. Mice were treated with different doses of carrageenan, and then intrarectally challenged with 100 μL of 1 % oxazolone. One day after treatment, the decrease in body weight and the mortality rate of the animals were recorded. Pairwise comparisons between groups at various time points were conducted by using two-way ANOVA and the LSD post hoc test. * P < 0.05, ** P < 0.01, compared with that of the Blank group. # P < 0.05, ## P < 0.01, compared to that of the oxazolone-treated group
Consistent with the observed difference in weight change, the Blank and carrageenan-treated groups showed similar survival rates, whereas the OXA-treated mice showed an 87.5 % survival ratio. The survival ratio of the carrageenan + OXA group was lower than that of the OXA-treated groups. Among these, the group that received a medial-dose (8.3 mg/kg) κ-carrageenan for 14 days showed a 37.5 % mortality rate, which was the highest (Fig. 1b), suggesting that medial-dose (8.3 mg/kg) κ-carrageenan significantly aggravated weight loss and decreased the survival ratio due to OXA exposure.
Effects of κ-carrageenan on the severity of colonic damage
Colonic damage scores were significantly higher in OXA-treated mice compared to those of the Blank group mice (P < 0.01). Macroscopic analysis indicated that in the OXA-treated group, inflammation was mainly localized in the distal part of the colon, which shortened and thickened, as well as presented signs of colonic edema and hemorrhagic changes (Fig. 2e).
Effect of κ-carrageenan on colonic damage following oxazolone administration. A representative photograph of the colon encompassing the cecum to the rectum. a Blank group; b LOW group; c MED group; d HIG group; e OXA group; f LOW + OXA group; g MED + OXA group; h HIG + OXA group; i Colonic damage scores 1 day after oxazolone administration. The damage scores were evaluated as described in the "Methods" section. Data are expressed as a column chart. * P < 0.05, ** P < 0.01, compared with that of the Blank group. # P < 0.05, ## P < 0.01, compared to that of the oxazolone-treated group
Carrageenan alone showed no impact on the colons of animals, wherein the colonic mucosa was apparently normal, and symptoms of congestion, edema, and erosion were not detected (Fig. 2b–d). No significant differences in colonic macroscopic scores of mice from the Blank group mice and those of the LOW, MED, and HIG treatment groups were observed. Figure 2f–h reveals that OXA-induced colonic damage was markedly augmented in mice pretreated with different concentration of carrageenan. The colons were more reddish due to the production of a bloody stool compared to those of the OXA-treated mice, suggesting that a more severe intestinal inflammation had occurred in the LOW + OXA, MED + OXA, and HIG-OXA-treated mice (Fig. 2f–g). The colonic damage scores of the LOW + OXA, MED + OXA, and HIG + OXA groups were higher than that of the OXA-induced group (P < 0.05, Fig. 2i). Among these, the MED + OXA group showed the highest score of 3.75. A 1.55-fold higher colonic damage score was observed in the MED + OXA group compared to the OXA-treated group (P < 0.01). These results indicate that carrageenan can synergistically enhance OXA-induced inflammation.
Effects of κ-carrageenan on the histological changes of OXA-induced colonic damage
No histological differences in the effect of κ-carrageenan on OXA-induced colonic damage between the carrageenan-treated groups and the Blank group were observed. The colonic tissue of the Blank group showed normal features such as an adequate number of goblet cells and intact crypts (Fig. 3a). Figure 3e shows the colonic sections of OXA-exposed mice with a lower number of goblet cells and epithelial cells, and an increase in lymphocyte infiltration, accompanied by a mixed inflammatory infiltrate of granulocytes, relative to that observed in mice of the Blank group.
Effect of κ-carrageenan on colonic histological changes and colonic histological scores of oxazolone-treated mice. Representative images of the histological features of the colonic tissue of blank (a Blank group; b LOW group; c MED group; d HIG group; e OXA group; f LOW + OXA group; g MED + OXA group; h HIG + OXA group. HE: ×200, Higher magnifications of the pictures: ×600). i Histological grading of colitis in oxazolone-induced mice. Histological grading was determined as described in the "Methods" section. Data are expressed as a histogram plot. ** P < 0.01, compared to that of the Blank group; # P < 0.05, ## P < 0.01, compared to that of the oxazolone-treated group
Histological analysis showed that carrageenan was effective in aggravating OXA-induced colitis (Fig. 3f–h). The colonic sections from the LOW + OXA, MED + OXA, and HIG + OXA-treated mice revealed mucosal inflammation, which was characterized by lymphocyte cell infiltration, as well as a significant reduction in the number of goblet cells. A 1.22-fold augmentation in histologic score was obtained in the LOW + OXA group (P < 0.05), a 1.32-fold increase in the MED + OXA group (P < 0.01), and a 1.25-fold increase in the HIG + OXA group relative to the OXA-treated group (P < 0.05) (Fig. 3i).
Analysis of histological changes of the colon by using scanning electron microscopy
Intact microvilli, normal epithelial cell surface, and crypts were observed by scanning electron microscopy in the Blank group (Fig. 4a). No significant differences in the epithelial mucosa of the colon between the LOW, MED, and HIG groups and the Blank group were observed, with all groups showing normal features (Fig. 4b–d). However, at higher magnification, damage to the epithelial mucosa surface and disintegration of the microvilli were observed in the colon of the OXA-treated group (Fig. 4e). Figure 4 shows that carrageenan was effective in aggravating OXA-induced colonic damage. The histological changes of the colon were markedly exacerbated in the LOW + OXA, MED + OXA, and HIG + OXA groups (Fig. 4f–h). Scanning electron microscopy showed more pronounced damage to the epithelial cell surface, disintegration of the microvilli, excessive mucus secretion at crypt openings, extensive distribution of ulcers on the colonic surface were observed in the tissues of mice belonging to the MED + OXA group (Fig. 4e).
Scanning electron microscopy of histological changes. a Blank group; b LOW group; c MED group; d HIG group; e OXA group; f LOW + OXA group; g MED + OXA group; h HIG + OXA group. ×200 magnification
κ-Carrageenan administration promoted the OXA-induced decrease in the ratio of CD4+CD25+ regulatory T cells (Tregs)
Treg cells regulate responses of the immune system. Figure 5 reveals that the ratio of CD4+CD25+/CD4+ in the peripheral blood of OXA-treated group was significantly lower than that observed in the Blank group (P < 0.01). No apparent effects of different concentrations of κ-carrageenan alone on the proportion of Treg cells were observed. On the other hand, κ-carrageenan aggravated the decrease in CD4+CD25+/CD4+ cells in the peripheral blood of OXA-treated mice. A 1.206-fold reduction in CD4+CD25+/CD4+ was observed in the LOW + OXA group (P < 0.05) and 1.22-fold decrease in the HIG + OXA group compared to that of the OXA-treated group (P < 0.05). The CD4+CD25+/CD4+ ratio of the MED + OXA group showed 1.49-fold decrease compared to that of the oxazolone group (P < 0.01). These findings demonstrate that OXA causes an immune imbalance in mice, whereas carrageenan augments this imbalance.
Ratio of CD4 + CD25+/CD4+ regulatory T cells (Tregs) in the peripheral blood of mice. ** P < 0.01, compared to that of the Blank group. # P < 0.05, compared to that of the oxazolone-treated group
κ-Carrageenan promotes cytokine production in OXA-induced mice colonic inflammation
The levels of seven cytokines in the serum were determined by flow cytometry by using the Mouse Inflammation Cytometric Bead Array (CBA) assay. Of these cytokines, TNF-α and IL-6 as classified as pro-inflammatory cytokines; IFN-γ and IL-2 are Th1 cytokines; IL-4, IL-10 are Th2 cytokines; and IL-17A is a Th17 cytokine.
Interestingly, administration of carrageenan alone resulted in the upregulation of the seven cytokines, except for IL-17A, but no statistical difference (P > 0.05, Table 2). However, OXA treatment induced a significant elevation in the secretion of TNF, IL-6, and IL-10 (P < 0.01). Mice pretreated with medium-dose (8.3 mg/kg) of carrageenan showed a significant increase in the OXA-induced production of IL-10, IL-6, and TNF (P < 0.01). Of the seven cytokines, IL-6 showed the highest increase (3.82-fold) compared to that of the OXA group (Table 2).
Table 2 The levels of the inflammatory cytokines IL-10, IL-17A, TNF, IFN-γ, IL-6, IL-4, and IL-2 in mouse serum (pg/ml, mean ± SEM)
The expression levels of TLR4, NF-κB, PCNA, and FOXp3 mRNA in the colonic mucosa of experimental mice
No differences in the levels of the four proteins were observed between Blank group and κ-carrageenan alone-treatment group. However, OXA exposure and induction of inflammation was associated with a significant increase in the level of mRNA expression of TLR4, NF-κB, and PCNA in mouse colonic tissues. Carrageenan pretreatment resulted in a significant increase in the level of mRNA expression of TLR4, NF-κB, and PCNA compared to that of the OXA control group. However, a decrease in the level of FOXp3 mRNA expression was observed after OXA treatment (P < 0.01), and pretreatment with different doses of κ-carrageenan with OXA resulted in a significantly synergistic decreased in the FOXp3 mRNA expression compared to that of the OXA group (Fig. 6; P < 0.05).
The impact of κ-carrageenan on the expression of inflammatory response-related proteins in the colonic mucosa of experimental mice. 200× magnification
Synergistic effects of κ-carrageenan on OXA-induced protein expression in colonic mucosa of experimental mice
The impact of carrageenan on the expression of TLR4, NF-κB, and PCNA-protein levels in the colonic mucosa of experimental mice was immunohistochemically analyzed. Table 2 shows that κ-carrageenan alone did not apparently affect the expression of TLR4, NF-κB, and PCNA in colonic epithelial cells. The expression levels of TLR4, NF-κB, and PCNA in the colonic epithelium increased after OXA injection (P < 0.01). Mice pretreated with different doses of carrageenan showed a significant increase in the OXA-induced expression of TLR4, NF-κB, and PCNA (P < 0.05). The most significant increase in the expression levels of these proteins was observed in the MED + OXA group. A 1.51-fold increase in TLR4 expression in colonic mucosa was detected in the MED + OXA group (P < 0.01), whereas a 1.14-fold upregulation in NF-κB expression in colonic mucosa was observed in the MED + OXA group (P < 0.01) and a 1.79-fold increase in PCNA expression in the MED + OXA group compared to that of the OXA-treated group was detected (P < 0.01) (Fig. 7, Table 3).
Effect of κ-carrageenan on the level of mRNA expression of TLR4, NF-κB, FOXp3, and PCNA. Real-time quantitative RT-PCR was used to measure the level of mRNA expression of TLR4, NF-κB, FOXp3, and PCNA. * P < 0.05, ** P < 0.01 relative to that of the Blank group. # P < 0.05, ## P < 0.01 compared to that of the OXA-treated group
Table 3 Impact of carrageenan on the expression of various proteins in mouse colonic mucosa (Au, mean ± SEM)
Table 3 shows that the expression levels of Foxp3 expression in the colonic epithelium decreased after OXA injection (P < 0.01), whereas carrageenan treatment significantly decreased the level of OXA-induced expression of FOXp3 (Fig. 6; Table 3; P < 0.05).
κ-Carrageenan enhances oxazolone-induced activation of TLR4 and NF-κB in experimental mice
NF-κB is one of the most important transcription factors that regulate the expression of numerous pro-inflammatory cytokines. We examined whether κ-CGN enhances oxazolone-induced NF-κB activation. Figure 8 shows that treatment with κ-carrageenan alone resulted in a slight increase in oxazolone-induced TLR4 and nuclear levels of the p65 subunit of NF-κB. However, oxazolone significantly increased TLR4 and NF-κB levels (P < 0.01). In addition, pretreatment with different doses of κ-carrageenan markedly promoted the expression of TLR4 and NF-κB in oxazolone-stimulated mice. A 1.78-fold increase in TLR4 expression in colonic tissues was detected in the MED + OXA group (P < 0.01) and a 1.57-fold upregulation in NF-κB expression in the MED + OXA group compared to that of the OXA-treated group (P < 0.01) (Fig. 8, Table 3).
κ-Carrageenan enhanced oxazolone-induced activation of NF-κB. The protein levels of TLR4 and NF-κB p65 were analyzed by western blotting. The data are presented as the density ratio vs. β-actin. **P < 0.01, compared to that of the Blank group. # P < 0.05, ## P < 0.01, compared to that of the oxazolone-treated group
Although the FDA and JECFA consider carrageenan as a safe saccharide [1, 5, 11], studies have shown that it induces ulcerations in animal models, suggests that it poses a carcinogenic risk to humans [1–3, 27]. This finding has sparked controversy and intense debate, and the mechanism underlying this pathological condition has remained uncertain. The present study presents a possible mechanism of carrageenan that involves an inflammatory response.
BALB/c is a mouse strain that is susceptible to OXA-induced inflammation [24, 28]. In this research, we used OXA to establish an acute gut inflammation BALB/c mouse model, which resembles ulcerative colitis in pathology and the immune response inof human. It has been reported that the initial toxic effects of OXA leads to a flooding of the lamina propria and inducing an increase in the secretion of Th2 cytokines IL-4 and IL-5, which in turn leads to further inflammation. [24, 29]. Moreover, rectal instillation of 150 μL of 1 % OXA is enough to lead to an accelerated inflammation lasting for 1–2 days by a Th2 response in the distal half of the mouse colon of the mice, thereby resulting in either rapid recovery or death [24]. Therefore, one day after injection of OXA was decided as the experimental time to detect the aggravating effect of carrageenan on the inflammatory response.
The average daily intake of carrageenan in the infant formula is 0.3 g/L [2], amounting to 240 mg/5.8 kg/day (≈41.7 mg/kg/day). In the typical Western diet, the daily intake of carrageenan is considerably more than 500 mg/60 kg/day (≈8.3 mg/kg/day) [30]. Bhattacharyya et al. reported that a dose of 50 μg/30 g/day (≈1.7 mg/kg/day) carrageenan induces colonic inflammation in IL-10-deficient mice [18]. Therefore, in the present study, we used these concentrations to investigate the effect of carrageenan on OXA-induced inflammation.
Here, we first discovered that κ-carrageenan aggravates OXA-induced inflammation in BALB/c mice by significantly increasing weight loss, lethality rate, and degree of colonic injury. Of particular interest is our finding that mice pretreated with a medial-dose (8.3 mg/kg) of κ-carrageenan for 14 days resulted in a 37.5 % of mortality rate, whereas that of OXA-treated control mice was 12.5 % (Fig. 2). Histological and microscopic evaluation also confirmed that intragastric administration of κ-carrageenan aggravated OXA-induced inflammation in BABL/c mice. Of the three doses of κ-carrageenan used in the present study, the medium dose showed highest score histological scores. The survival ratio of the OXA + MED group was also the lowest. It is possible that the high concentration of carrageenan induces its formation into a gel in the gut of mice, which in turn reduces its contact without other gut content. In addition, Weiner et al. also observed that intragastric administration of a high dose of food-grade carrageen an just directly affected theresulted in the production of soft stools or development of diarrhea, which are common effects of non-digestible high-molecular weight compounds [31]. Therefore, the aggravated effects of carrageenan do not appear to respond to a traditional dose–response curve.
OXA-induced inflammation is often employed in research investigations on the contribution of the Th2-dependent immune response to intestinal inflammation [24]. The present study showed that OXA-induced inflammation is accompanied by the release of various inflammatory cytokines, not only Th2 type cytokines IL-10, but also pro-inflammatory cytokines, TNF-α and IL-6 [24, 28]. Previous reports have revealed that OXA-induced inflammation is characterized by an upregulation of IL-4 [29]; however, in the present study, although an increased level of IL-4 expression was observed in the OXA group, this was not statistically significant. No increase in the levels of Th1 cytokines IFN-γ, IL-2, and IL-17 was observed with OXA and κ-carrageenan treatment. The synergistic effect of κ-carrageenan on OXA-induced changes is also reflected in the secretion levels of inflammatory cytokines. The secretion of pro-inflammatory cytokines TNF-α and IL-6 in OXA-treated mice were all significantly augmented by κ-carrageenan pretreatment. These findings suggest that carrageenan aggravated the OXA-induced inflammation of intestinal tissues.
CD4+CD25+ regulatory T cells (Tregs) can regulate and suppress the immune response, thus; therefore, these play an important role in the maintenance of peripheral immune tolerance. The decrease in the number of Tregs often results in a development of various kinds of autoimmune disease such as inflammatory bowel disease (IBD) [32]. The present study showed that the number of Tregs in the peripheral blood of OXA-treated group was lower than that of the Blank group, which further decreased after κ-carrageenan treatment, demonstrating that OXA can cause an imbalance in the immune system of mice, and carrageenan augments this imbalance.
FOXp3 is a transcription factor that plays a major role in the development and maintenance of the immunosuppressive function of Tregs [33]. Galitovskiy et al. observed that the expression of FOXp3 is decreased in the inflamed areas of ulcerative colitis compared to that of the normal colon [34]. This finding is consistentin agreement with the observations of the present study, wherein the intragastric administration of κ-carrageenan significantly decreased the OXA-induced mRNA expression of FOXp3 in the colonic mucosa, indicating that the suppressor activity of FOXp3 might have been abrogated in vivo or was insufficient to counterbalance the acute mucosal inflammation in OXA-treated mice.
Mice pretreated with different doses of carrageenan showed a significant increase in the levels of OXA-induced expression of NF-κB and TLR4. Some studies have demonstrated that carrageenan upregulated interleukin-8 (IL-8) secretion through TLR4, and activated NF-κB in human colonic epithelial cells [16, 20]. Benard et al. also observed that κ-carrageenan induced TNF production via NF-κB activation [21]. Recently, we have beenare exploring the mechanism of underlying the synergistic effect of κ-carrageenan on lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) secretion in HT-29 cells. We found that κ-carrageenan pretreatment increased LPS-stimulated IL-8 secretion. The TLR4-NF-κB pathway was activated, as evidenced by the enhancement of Bcl10 (B-cell CLL/lymphoma 10) expression, IκBα phosphorylation, and nuclear NF-κB localization (Wu, wuweixiehou@163.com). These findings demonstrate that intragastric administration with κ-carrageenan significantly activated intestinal inflammation by upregulating TLR4 and NF-κB in colonic mucosa.
In conclusion, intrarectal injection of OXA in mice causes colonic damage and inflammation. Treatment of mice with κ-carrageenan significantly aggravated OXA-induced inflammation in BALB/c mice. The results of the present study suggest that the synergistic effect of κ-carrageenan might be linked to the induction of an imbalance in the expression of intestinal inflammatory cytokines, which then magnified the existing intestinal inflammation. Therefore, under the intestinal "unhealthy" state, carrageenans may serve as potential inflammatory agents that impart certain harmful effects.
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This work was supported by grants from the NSFC project (81370532), the outstanding (postgraduate) dissertation growth foundation of Ningbo University (py2014002), the Ocean Public Welfare Scientific Research Project, State Oceanic Administration of China (201505033), the K.C. Wong Magna Fund in Ningbo University, and the 151 Talents Project.
Key Laboratory of Applied Marine Biotechnology of Zhejiang Province, Ningbo University, Post Box 71, Ningbo, Zhejiang, 315211, China
, Xin Gao
, Tingting Niu
, Xiaojuan Zhu
, Xiaojun Yan
& Haimin Chen
Department of Clinical Laboratory, Lihuili hospital of Ningbo Medical Center, Ningbo, Zhejiang, 315041, China
Feng Wang
Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo, Zhejiang, 315211, China
Xiaojun Yan
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Correspondence to Xiaojun Yan or Haimin Chen.
WW, FW, and XG performed the experiments; TN and XZ analyzed the data; HC conceived and designed the research; WW and HC drafted the manuscript; and HC and XY edited and revised the manuscript. All authors read and approved the final manuscript.
κ-carrageenan
Oxazolone
Intestinal inflammation

redpajama_set_name: RedPajamaCommonCrawl


Acme PD-108TA2F DP LGA3647 socket P server, 1U rackmount, Black Color.
4 NVMe PCIe ports on motherboard.
Dual 10Gigabit Ethernet Intel c622, RJ45 ports.
VGA port- On-board graphics, AST2500.
remote management -Integrated IPMI 2.0 V. KVM AST2500 dedicated LAN.
8 of 2.5" HDD hot-swap bays w/ backplane( 6 SAS/SATA + 2 SAS/SATA/NVMe option add NVMe cables).


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Fiber forms a geopolitical triangle
Air raids on Chinese-made fiber-optic networks in Iraq reveal and disrupt a marriage of high-speed communications and military strategy, while harkening back to cold war notions of preemptive strikes and deterrence of nuclear war.
BY STEPHEN N. BROWN
Last February, British and American warplanes bombed Iraqi radar and missile sites because they employed fiber-optic systems that vastly improved the defenders' speed of target acquisition and accuracy of counterfire against the planes. President Bush said the situation was "troubling" and "we're concerned about Chinese presence in Iraq," implying that China built the fiber networks, a charge quickly denied by the People's Republic of China. Bush did not say he was surprised. China's People's Liberation Army may be doing for Iraq what the PLA did for itself over the past 10 years-piecing together a nationwide encrypted and integrated communications network that, among other things, commands and controls high-performance computers that lock onto targets and direct counterfire at them. The military term for such a system is C4I.
The PLA's development of its own C4I was discussed by the U.S. Defense Threat Reduction Agency (DTRA) in one of its memos that reached Congress in the summer of 1998. The memo was one of many public evidentiary documents used by some members of Congress to build a case that the Clinton Administration had a reckless policy regarding American exports to China, a policy giving the PLA all it needed to build a C4I. According to the DTRA, "China...has obtained production equipment to manufacture fiber-optic cable and associated technologies...without U.S. licenses....Since 1991, China [h]as sought...a C4I network...essential in warfare to link targeting data with strike assets. The ingredients for this system include high-capacity fiber optics, switching systems, satellite communications systems, and systems integration...[T]his development raises the question...[Is] the ultimate return for U.S. national security...worth this policy approach toward China[?]"
Apparently, Iraq needs a C4I to protect nuclear weapons development: German intelligence has concluded that Iraq's military will have nuclear weapons and delivery vehicles capable of reaching Europe by 2005, according to press reports. Europe is not the likely target of an Iraqi nuclear attack, and the likely target won't wait for an attack, so Iraq needs a C4I to blunt preemptive strikes against its nuclear weapons. But without a C4I, Iraq's nuclear program is vulnerable and without fiber there can be no C4I. The logic of the American-British air raid is abundantly clear, but why would China exacerbate the situation?
One line of speculation goes back to 1999, when China was ready to buy from Israel a "command center in the sky," modeled after the U.S. Air Force's huge and specially equipped C5As that directed air and land battles in the Gulf War. Israel yielded to pressure from the United States and reneged; therefore, Israel's good will was no longer a factor in China's strategic planning. That may explain China's presence in Iraq and other reports suggesting that China is aiding Iran's efforts to build nuclear weapons. Another reason for China's involvement may be its desire to enhance its own power generally. The DTRA memo said China's C4I "will allow for power projection throughout the region [east Asia]"- military-speak for "provide the means to impose one's will or to influence to a great degree."
China's indirect aid of nuclear weapons development in Iraq and Iran, as well as its continuing development of its own nuclear weapons, repeats the historical pattern in which a secondary power tries to nullify an adversary's advantage through "sufficiency." The sufficiency doctrine posits that small forces are just as effective as large ones, and sufficiency is sometimes thought of as an alternative to an arms race.
For example, a nation armed with 100 intercontinental ballistic missiles is just as influential as one armed with 3,000 ICBMs, if the 100 ICBMs cannot be destroyed in preemptive attacks or if all their warheads cannot be destroyed before they reach the targets. Assuming both parties are rational, neither side attacks the other because no one is safe from nuclear retaliation. This nuclear deterrence was practiced in the 1950s by the United States and Soviet Union, but this kind of situation is unstable when allies and other third parties are considered.
Sufficiency invites the smaller force to believe it can paralyze the larger one with the threat of large casualties, then as a reward pick off third parties. This scenario was considered over 30 years ago in the book On Thermonuclear War, authored by Herman Kahn, then of the Hudson Institute, an American think tank that studied nuclear war scenarios. According to Kahn, strategic planners thought the threat of two million American casualties in the United States itself would be enough to dissuade it from defending its NATO allies against a Soviet attack; the theory was never tested, perhaps because the implementing party believed it too risky.
"Sufficiency" is an old doctrine with various permutations going back to the early 1900s when Germany built a navy large enough to challenge the British navy but not big enough to beat it in World War I. "Sufficiency" has never achieved its intended result and has never been shown to have stable or predictable results.
The sufficiency doctrine's tactical advantage-that few missiles and warheads are needed to offset an adversary's advantage-is also its weakest tenet: A "sufficient" force is small enough to invite the development of an antiballistic missile system, just like the one the United States is testing and China vehemently opposes.
In early 1999, when the U.S. Senate approved funds for the development of a national missile-defense system, a spokesman for China's foreign ministry said, "This is counter to the trend of the times of peace and development and contrary to relevant nuclear arms control and disarmament treaties."
One requisite for a missile-defense system is fast reaction time after an adversary's missile launch, a situation where fiber, optical computing, and optical switching are sure to be vital. These technologies will grow in importance, especially if a missile-defense system looks like a geodesic network, where command and control is distributed over several interlinked sites rather than a network relying on a single and thus more vulnerable site.
Very-high-speed communications and computing are important tools in geopolitical strategy, and an American national missile defense system may be an adequate counter to "sufficient" force levels of China and North Korea. One response from the "sufficiency party" could be building more missiles and warheads, thus creating an arms race, one more proof of the sufficiency doctrine's flaws and an expensive lesson for parties who would rather devote resources to improving their nation's standard of living.
But another response might be movement to a bargaining table, where any effort worth its salt will scale back the proliferation and development of nuclear weapons and their delivery systems, a better alternative than bombing the fiber out of one another.
Stephen N. Brown writes on public policy in telecommunications. He can be contacted by e-mail at policywork@aol.com or telephone: (615) 399-1239.
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University of KwaZulu-Natal Press Release
Science rebel's career inspired by cinema
Seipati Mokhosi
Issued by: University of KwaZulu-Natal
[KwaZulu-Natal, 30 August 2018]
Seipati Mokhosi.
To commemorate National Science Week and National Women's Month, the College of Agriculture, Engineering and Science is honouring its female scientists through a Wonder Women in Science campaign.
These women are regarded as passionate, pioneering and persistent heroines making waves in the field of science. One of the candidates for this year's campaign is Developmental lecturer at the School of Life Sciences, Seipati Mokhosi.
Mokhosi, who grew up in the small village of Monontsha in the Free State, says she conducted her first experiment at the age of six using her grandfather's cows. "I wanted to know if cows ate anything other than grass, so I rolled an orange towards a calf. I was surprised it ate the whole orange," she says.
Mokhosi is doing research for her doctorate, which explores ways to design magnetic nano-therapeutic delivery vehicles to 'encourage' neuronal cells to effectively take up therapeutics. She chose to enter the field of biochemistry after watching the 1992 film Lorenzo's Oil, starring Nick Nolte and Susan Sarandon.
Building a bioeconomy with sugar cane
New radio telescope to probe dark energy, mysterious radio flashes
This film captured the touching journey of parents caring for their son who developed adrenoleukodystrophy (X-ALD), a neurodegenerative disease. Even though they were not scientists, they courageously used all their resources to find a cure. Their efforts contributed immensely to the world of neuroscience.
"This movie was a defining point in my life. I remain fascinated by diseases and disorders of the brain. All my students know about this film because it made such a powerful imprint on me," Mokhosi says.
One of the biggest challenges she has faced as a female scientist is proving that she is just as good as her male counterparts. Traditionally, science has been a space predominantly occupied by men. "I have found that, as a woman, your occupation is often viewed as being part of the status quo and not based on merit. It is up to me to continue to challenge this antiquated notion and shift the mindset."
Mokhosi credits several women who have shaped her into the person she is. These women include her mother and women from her tribe. She also acknowledges Professor Tebello Nyokong, and looks up to and aspires to be like her PhD supervisor, Professor Mogie Singh.
Her advice to budding female scientists is to always be curious and celebrate their individuality. "Keep asking questions, exploring and working hard. You have something unique to offer, and the world is waiting for your uniqueness," she says.
She believes the world will benefit from having more women in leadership roles. "I feel a woman's world-view is often less egocentric than a man's, and often comes from a gentler, caring perspective."
She feels more can be done to make science education relevant as opposed to something done in a lab. "A lot of what we see, experience and use in our households involves science, so these concepts are often not as abstract as we perhaps think. We need to take this awareness to communities, to enlighten and empower them," she says.
Earlier this year, Mokhosi received a National Research Foundation Thuthuka Grant to support her work. Her immediate plan is to complete her PhD and bring quality teaching and research productivity to UKZN. Long-term, she wants to forge meaningful interdisciplinary collaborations around the country and the world to establish a Neuroscience Research Unit.
* In August, the College of Agriculture, Engineering and Science will present articles on each one of its five Wonder Women in Science.
Visit: wwis.ukzn.ac.za to read all the articles.
Words and photograph: Sashlin Girraj
University of KwaZulu-Natal
Nkosinathi Bhekani Dlamini
DlaminiN14@ukzn.ac.za
Home| Newsroom| Contacts| Profile| Events|	University of KwaZulu-Natal| Mail & Guardian


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'Not the contract that you want to accept' | Striking CNH workers reject 'best and final' proposal
55% of workers voted no on the deal.
Author: Cesar Sanchez
Published: 12:23 AM CST January 8, 2023
BURLINGTON, Iowa — It's been 250 days since Case New Holland Industrial workers went on strike, demanding for an increase in wages, affordable healthcare, and more flexibility in time off.
On Saturday, CNH workers gathered to vote on a proposal that the company says its the "best and final offer."
RELATED: 'Last, best and final offer' given to UAW from CNH Industrial
However, some workers like Dustin Spiker think otherwise.
"If you want to accept the bare minimum, I'd say this contract is probably for you," Spiker said. "If you want something more for yourself and your family, this is probably not the contract that you want to accept."
Spiker says it's been a frustrating experience for both sides and wants everyone to make the best decision for themselves.
"I've been with the company for over 10 years, I kind of thought they would have cared a little bit more about getting me back in there and help me provide for my family," Spiker said. "I can't go back to the job that I want to do because they keep offering it to scabs that are willing to do the job."
CNH worker Windy Link said the deal offered changes, but some policies did not change.
"I wasn't disappointed with some of the changes because I knew they probably wouldn't change because they have been with the company for many years." Link said. "It was great to be with my family in the past months, but I am ready to go back to work."
According to Nick Guernsey, President of UAW Local 807, 55% of workers voted no to the offer. Guernsey said meetings among members will be held ahead to see what the process will look like going forward.
CNH Industrial provided News 8 with the following statement:
"CNH Industrial is disappointed to learn that its recently negotiated and improved Last, Best and Final Offer was not approved in the ratification vote conducted by the Union on Saturday, January 7. This Offer for a new contract, which included wage increases of 25% to 38% over the four years, would have allowed the employees and the Company to move forward at the Company's plants in Burlington, Iowa and Racine, Wisconsin."
"The Company strongly encourages the Union to allow the employees to reconsider their position in another vote so that the employees can return to work. While we await the Union's next step, CNH Industrial remains committed to honoring and meeting the needs and demands of our customers and, therefore, we will continue operations at both our Burlington and Racine sites."
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More From News 8
Senate moves to avert rail strike amid dire warnings
CNH Industrial workers still on strike after almost eight months
'Last, best and final offer' given to UAW from CNH Industrial
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If you are looking for an effective way to cool and circulate a large area or an area where space is limited, Schaefer Ventilation can help. Schaefer Ventilation Equipment manufacture some of the highest quality horticulture/greenhouse/hobby house circulation fans in the world. The patented Plant-Air fans and other models are very quiet, highly efficient, and quite durable! The airflow ranges from gentle to powerful and have variable speed capability which allows you to select a perfect airflow pattern. All greenhouses large or small, whether naturally ventilated, mechanically ventilated, cooled, and or heated, will benefit from horizontal airflow. Plants, flowers, fruits, vegetables, shrubs and trees thrive when provided with horizontal airflow.


Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.
We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.
The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.
MEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.


Having fled persecution in Afghanistan as a trainee human rights lawyer, Aishah Saried turned to English FE so she could one day return to make a difference to others in need of help, writes Billy Camden.
Having a voice in places of war and terror can often lead to persecution — and that is what Aishah Saried found as an outspoken trainee human rights lawyer in her native Afghanistan.
She fled the country in 2011 and found asylum in the UK with her three children, Fatemah, Farzin and Mohammed.
But she said she "suffered from humiliation" when she had to bring an interpreter to school events such as parents' evening because she could not speak English.
Determined to change her life from "happening behind a thick glass screen" and to one day return to her home country as a fully qualified human rights lawyer to fight social injustice, Aishah enrolled on an English for speakers of other languages (Esol) course at Birmingham Adult Education Service (BAES) in 2012.
She progressed onto level one and then level two before excelling in English and maths GCSE, meaning she could go on to study a degree in social work at Wolverhampton University.
Her journey is described as "incredible and remarkable" by BAES GCSE tutor Lucy Ellenor (pictured below).
"When Aishah first started she was nervous and timid with it being a new country, but the person I see today is completely different," said Ms Ellenor.
Aishah's achievements were topped off earlier this year when she won an outstanding learner achievement award at BAES.
Ms Ellenor said: "I am beyond words proud of Aishah and how far she has come, she has done incredibly well. She is the sort of student to really make the most about learning opportunity.
And Aishah cannot thank BAES enough for her progress.
"From that first ESOL course to my recent GCSEs I have been treated with nothing but respect and kindness by the staff at BAES," she said.
Aishah added that when she first arrived in the UK she felt "somehow less of a mother than I had been in Afghanistan".
But her children are now "absolutely thrilled" by the changes they have seen in her since she became a learner.
"We now regularly settle down to our homework together and I love it when they ask for my help," said Aishah.


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Volume 5, Number 23 - 2/1/01
Snowpack 60% of Average
2001 looks to be the second drought year in a row
by Rob Shaul
It's not too early for ranchers and irrigators to start thinking about which fields they should focus irrigation efforts on this summer, says Daryle Bennett of the Natural Resource Conservation Service in Pinedale. The lack of snow could also result in a second year of record forest fires in the West.
As of Tuesday, the basin-wide snowpack in the Upper Green River was just 60% of average, reported Mr. Bennett. This time last year, snowpack was 87% of average.
The low level of snowpack this late in the winter means another drought this summer, predicts Mr. Bennett. He says it would have to be an "incredible spring" with lots of snow for the snowpack to get above 80% of average. He does say that a rainy late spring and summer could help.
Last year's drought will compound the problem. The ground is a lot drier this year than it would normally be, says Mr. Bennett, and will soak up a lot of the runoff in the spring, further reducing irrigation and river flow levels.
As dry as it is, it's been worse. Mr. Bennett looked at decades-old records of snowpack levels for this time of year, and found several years that had lower levels, including 1977, 1968, and 1963. More recently, at this time in 1994, the basin-wide snowpack level was just 44% of average. <


Encouragement - PENTECOSTAL CHURCH ROCK OF SANCTUARY INC.
Encourage the underprivileged (Isaiah 1: 17)-Learn to do good. Seek justice. Help the oppressed. Defend the orphan. Fight for the rights of widows.
We the body of Christ should be an encouragement to the underprivileged. We should be doing good to everyone. We should want justice for all. We should help those who are oppressed. Those who deserve fair treatment. We should be defending the orphans. They are innocent, and should be protected. We should fight for the rights of widows. They also are disadvantaged. We should display love for them, as we would love ourselves, and our families.
God encourages us (Isaiah 40: 31)-But those who wait on the LORD will find new strength. They will fly high on wings like eagles. They will run and not grow weary. They will walk and not faint.
God gives us encouragement in that, in Him, we will find new strength. When we feel we cannot go on, the LORD gives us strength to keep going. To press on. To live as His Word commands. We will fly on wings like eagles; meaning that we will soar above trials, tribulations, illness, disadvantages. We will be free from slavery to sin, and high in spirits, on His path. We will run and not grow weary; we will never quit fighting the good fight of faith, we will not give up, because we know that He is our reward. We will walk, and not faint; our faith will remain forever in Him. In his ways, we have peace. In His ways, we have strength. In Him, we have comfort. In Him, we are victorious!!
The Holy Spirit encourages us (Acts 9: 31)-The church then had peace throughout Judea, Galilee, and Samaria, and it grew in strength and numbers. The believers were walking in the fear of the LORD and in the comfort of the Holy Spirit.
When we are in the Holy Spirit, we have encouragement in that we are peaceful in our hearts. We are able to grow in strength and numbers because we are able to support and encourage one another to remain faithful to Him, and to one another. When believers walk in the fear (respect) of the LORD, they find comfort in the Holy Spirit, and need not fear for their lives, nor the lives of their family. They walk in confidence, knowing that they are pleasing the living God. That His wrath and judgement will not fall upon them at any given moment, they are confident that they are saved, and that they are in His favor.
Encourage your neighbor (Romans 15: 2)-We should please others. If we do what helps them, we will build them up in the LORD.
We should encourage our Brothers and Sisters in Christ. We should live in a way that respects them, and pleases them. If we do this, we are not only honoring God, we are honoring ourselves, and we are honoring our Brothers and Sisters. By doing this, we will build them up. We will encourage them to live in the same way. They will be confident in their walk with Christ. This is very pleasing to the LORD.
The Bible encourages us (Romans 15: 4)-Such things were written in the Scriptures long ago to teach us. They give us hope and encouragement as we wait patiently for God's promises.
As we know, the Bible is inspired by the Holy Spirit (God with us). It is written to give us encouragement on our journey in life. It was written to guide us, to teach us the correct way. It is designed to give us hope, and encouragement as we wait patiently for God's promises to be fulfilled. We should be reading it regularly, and growing from it continually, so that we will not grow weary and faint. We should do this daily, so that our faith remains strong.
Our position in Christ encourages us (Philippians 2: 1)-Is there any encouragement from belonging to Christ? Any comfort from his love? Any fellowship together in the Spirit? Are your hearts tender and sympathetic?
The way that we make God truly happy is by agreeing wholeheartedly with each other(the Church), loving one another, and working together with one heart and purpose. This is why our position in Christ encourages us. We know that we belong to Him. We know that we have His love. We know that we are living in unity with one another. We know that our hearts are tender and sympathetic to those who do not know him, or who do not have such a close relationship with Him. So, we love them, pray for them, teach them, and encourage them to do the same. That is our mission in Christ. We are encouraged by our position. We know it is good.
We should encourage each other (1 Thessalonians 4: 18)-So comfort and encourage each other with these words.
We should be encouraging one another with the words written in Scripture, as we know that First, all the Christians who have died will rise from their graves. Then, together with them, we who are still alive and remain on the earth will be caught up in the clouds to meet the LORD in the air and remain with Him forever. So, we should find encouragement in this, knowing that our lives, hard work, and dedication is not in vain. Our reward is that we will reign with the LORD forever! Amen!
Encourage those who are weak and afraid (1 Thessalonians 5: 14)-Brothers and Sisters, we urge you to warn those who are lazy. Encourage those who are timid. Take tender care of those who are weak. Be patient with everyone.
In 1 Thessalonians 5: 12 it reads: Dear Brothers and Sisters, honor those who are your leaders in the Lord's work. They work hard among you and warn you against all that is wrong. Think highly of them, and give them your wholehearted love because of their work. And remember to live peaceably with each other. We see in this verse that we should be appreciating God's instruction through our leaders. They are set in that position to look after you. We should encourage those who are weak and afraid. Warn those who are lazy, and don't want to do their parts as members of the body of Christ. We should remind them that they have a responsibility. We should encourage those who are timid, teach them not to be shy, teach them that they have the Spirit of the LORD, and that they need not fear. They should feel confident that with Christ, they will be able to accomplish any task or mission they are assigned to. We should be helping all believers and new Christians... (baby Christians). We should have patience with all. We should not give up on any of them. Hope with and for them. Pray with and for them.
Encourage elderly people (1 Timothy 5: 1-4)-Never speak harshly to an older man, but appeal to him respectfully as though he were your own father. Talk to the younger men as you would to your own brothers. Treat the older women as you would your mother, and treat the younger women with all purity as your own sisters. The church should care for any widow who has no one else to care for her. But if she has children or grandchildren, their first responsibility is to show godliness at home and repay their parents by taking care of them. This is something that pleases God very much.
We should take care in how we speak to the elders of the Church. The same is true in any setting. We should respect them as if they were our own father. We should treat the younger men as we would our brothers, with love, respect, and humility. We should be doing the same with the women, as if they were our mothers or sisters. This is what pleases God. We should also care for the widows; for they are as vulnerable as a child. (Especially the ones who have no relatives to care for them). When their children and grandchildren are able to care for them, they honor God very much. Their hearts make him proud!! This touches God's heart!
Encourage others not to sin (Hebrews 3: 13)-You must warn each other every day, as long as it is called "today", so that none of you will be deceived by sin and hardened against God.
What this means is that we should never be tired of poking at our Brothers and Sisters in Christ, or believers across the world. This pleases God when we care enough about them to remind them even if they get tired of hearing it from us. We should continue to remind them that they should fear God, and not become comfortable in sin, or disobedience. In verse 12 it reads: Be careful then, Brothers and sisters. Make sure that your own hearts are not evil and unbelieving, turning you away from the living God. We know that those who are deceived by sin, have hearts that become hardened against God like in the story of Moses, and the Pharaoh. We know that king Pharaoh's heart became hardened against God, and this resulted in God's wrath and punishment over Egypt, there was death, plagues, pestilence, and severe punishment as a result. Sin separates man from God. We become God's enemy when we remain in sin. This of course, is not good.
Encourage others to love (Hebrews 10: 24)-Think of ways to encourage one another to outbursts of love and good deeds.
Verse 23 reads: Without wavering, let us hold tightly to the hope we say we have, for God can be trusted to keep His promise. Verse 25 reads: Let us not neglect our meeting together, as some people do, but encourage and warn each other, especially now that the day of His coming back again is drawing near. Here we see that God warns His Church to love and encourage one another with good deeds. We should be holding on tightly to the hope we have, knowing that God will keep His promise to His people. We should continue to meet at Church, waiting for His return. We know that all the prophesies written in Scripture are being fulfilled right before our very eyes, wars, rumors of wars, hunger, pestilence, earthquakes, floods, etc... We need to continue now more than ever to live in the way He instructed us to.
May God bless you this and everyday, that your faith and hope grow ever stronger. May you be encouraged by the Spirit of our LORD behind this study-that you continue to be an inspiration to others around you. God bless you!
Let us pray: "Heavenly Father, we thank you for this day and a new opportunity to love one another as your Word commands, that we be given new strength to fly like an eagle and shed your light on others so that they too may come to know you. Bless our Pastors, Missionaries, Evangelists, and Members across the globe so that they too may continue to have increased faith and strength. That they are a blessing to others, and that your Holy Spirit continue to comfort and guide us all for your glory! Amen!"


I need to find out how to figure the dimensions for a balloon canopy that will cover a space of 10x10. I have the columns and need the formula.
With dimensions I suppose you want to know how long the swags will have to be and how many balloons you need, right? This depend on what type of canopy you have in mind - the main types are full canopy, open canopy and criss-cross canopy.
There is an excellent section about balloon canopies at the BalloonHQ. And here's an article with instructions for a criss-cross canopy. It contains sketches which should help you in figuring out the dimensions for your own design.
You could also buy a balloon canopy with instructions at Ebay. This set comes with 75 high quality link-o-loon balloons, ceiling clips and easy to follow instructions for just under $25.
Hope that helps and we would love to see a photo of your balloon canopy in our new Photo Gallery. Submission is free and easy and you can even win an Amazon voucher.