Specific striatal neuronal subtypes are classified as either phasically or tonically active. Of the four major neuronal subtypes of the striatum, the firing patterns of three have been studied in detail. Spiny projection neurons and fast-spiking interneurons fire phasically in response to synaptic input and are silent when input is absent. Cholinergic (ACh) interneurons are tonically active even in the absence of synaptic input. These ACh interneurons were the only known tonically active neurons in the striatum until recently. We have found that the low-threshold spike (LTS) interneurons also belong to this classification of striatal tonically active neurons, presenting a new aspect to the striatal oscillatory network.

Striatal LTS interneurons are GABAergic interneurons that also contain neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and somatostatin. We used transgenic mice expressing green fluorescent protein (GFP) under control of the nNOS or NPY promoters to help identify LTS interneurons. GFP positive neurons were examined using whole-cell and cell-attached recording techniques to investigate firing patterns and possible mechanisms of such activity. GFP positive neurons displayed the morphological properties characteristic of LTS interneurons, and fired strong rebound bursts at the offset of hyperpolarizing current pulses. LTS interneurons were spontaneously active and this activity persisted in the presence of synaptic blockers. When compared with ACh interneurons, LTS interneurons fired at higher rates and with less regularity. Similar to ACh interneurons, LTS interneurons were capable of firing in a variety of firing patterns, including regular single spiking, irregular single spiking, and bursting combined with periods of regular and irregular single spiking. LTS interneurons were observed spontaneously shifting between these firing patterns. Whole-cell voltage clamp recordings revealed a tetrodotoxin-sensitive region of negative slope conductance in the subthreshold range consistent with a persistent sodium current. An absence of a zero current crossing in the I-V curve combined with a linear F-I curve suggest these interneurons possess a Hodgkin's class 1 oscillation mechanism.

Tonically active neurons in the striatum have been shown to become strongly synchronized with the beta oscillation seen in Parkinson's disease. These strongly synchronized neurons may include LTS interneurons. In addition, both ACh and LTS interneurons are capable of endogenous oscillations in the beta frequency range. Our findings place LTS interneurons in the subtype of tonically active neurons in the striatum and in turn change our understanding of how synchronization may occur in the striatum.


LSU Softball defeats Southeastern 9-1 in Hammond

HAMMOND, La. – The No. 11 LSU softball team struck the first blow early, earning a hit in every inning and exploding for six runs in the top of the sixth in a 9-1 win over the Southeastern Louisiana Lady Lions Wednesday evening at North Oak Park in Hammond.

Coming off of a tough weekend, the Tiger (30-12) offense came to life in a big way, pounding out 12 hits over the course of the seven-inning contest, as seven different LSU players earned hits on the night. Five of the team's hits were for extra bases, ripping four doubles with a triple.

"I liked seeing the offense swing the bat, but I really liked our attitude and approach," head coach Beth Torina said. "From the minute we got to the field they were being positive, trying to fire each other up and I think that's something we started on Monday and carried that into today."

Sandra Simmons led the group, setting a new career high in hits going 4-for-5 with two RBIs and two runs scored. She was one of three players with multiple hits as Bailey Landry went 2-for-2 with a double and Constance Quinn going 2-for-3 with two RBIs and a run scored.

Baylee Corbello (6-0) scattered five hits and held the Lady Lions (19-21) to a single run as she struck out eight in the complete-game victory.

"The one thing she has done is continue to work," Torina said. "Bailey has put the time in during practice and has done everything we've asked her to do, so she showed she was prepared for the day and did a good job."

Caitlyn VanHemelrycke (3-4) suffered the loss after allowing two runs on three hits with three walks in 1.2 innings of work. Taylor Bishop allowed six runs, four earned on eight hits with four walks, with Maidison Vige' pitched the final inning and allowed a run on a hit with a walk in relief.

SLU's Katie Lacour and Kinsey Nichols combined for four of the team's hits on the evening.

Simmons wasted little time in opening the game for the Tigers on a positive note, ripping a double off the wall in center to open the contest. She moved to third after a sacrifice bunt, as Bianka Bell grounded out to drive her in putting LSU up 1-0.

The Lady Lions put up a fight quickly as a leadoff triple put an SLU runner in scoring position in the bottom of the first. An infield grounder kept the runner at bay for the first out, as a walk put runners at the corners. An infield pop-up put two outs on the board, but another walk loaded the bases to put the pressure on, but an inside pitch jammed the batter, forcing a popup to Corbello in the circle to end the inning.

A walk to Elyse Thornhill put an LSU runner on to open the second, moving to second after a sacrifice bunt by Amber Serrett. An infield grounder moved Thornhill to third, but Simmons struck with a single to right to put LSU on top 2-0. An infield single by Landry put runners at the corners, ending VanHemelryck's day, as Bishop came on and got a fly out to end the inning.

A pair of strikeouts put two outs on the board quickly for Southeastern in the bottom of the second, but a walk put one on, as a stolen base and bunt single put runners at the corners. However, another strikeout ended the frame for the home side.

A walk to Sahvanna Jaquish put one on for the Tigers in the third, as after an out Quinn doubled to right center, with a grounder moving the two to second and third. Unfortunately, a fly out to right ended the frame.

Consecutive singles for the Lady Lions in the bottom half of the inning ramped up the pressure for LSU, but a strikeout on a failed bunt put an out on the board. Back-to-back fielder's choice grounders forced out lead runners to end the third.

In the bottom of the fourth with one out, a walk put a SLU runner on, as an error allowed her to advance to second. A hit batter put two on, but a popup and a dart throw to third caught a runner trying to steal third to end the inning.

Jaquish singled through the right side to open the top of the fifth, as after a line out, a walk to Quinn put two on. An unassisted grounder advanced the two, and they would stay at second and third as a grounder to second ended the inning.

A walk to lead off the bottom of the fifth allowed an SLU runner to reach, as on the next at-bat a strikeout ensued, but a wild pitch allowed the runner to move all the way to third. An infield grounder nearly held the runner at third, but after the throw was made to first the runner bolted, just sliding in under the tag to score the run to make it 2-1.

After having the lead cut in half, Emily Griggs roped a triple to the gap in right center to open the top of the sixth. She wouldn't stay there long as Simmons registered her fourth hit of the day to put LSU up 3-1. Consecutive walks following loaded the bases, as Kellsi Kloss hit a high looping single to right to bring in another run to make it 4-1.

Sydney Bourg came on to pinch run at first for LSU, and the wheels would be put in motion for everyone quickly as Quinn posted a two-RBI single off the wall in right to make it a 6-1 ballgame. Quinn bolted for second as the throw sailed high, allowing Bourg to score to make it 7-1, as Quin herself scored on a passed ball to make it 8-1 Tigers.

Taylor Lockwood came on to pinch run for LSU and drew a walk to take first, advancing to second on a wild pitch. She would come around to score on a fly out to right, as an errant throw to third that went in to the dugout allowed her to move up two and score to make it 9-1.

LSU will be back in action on Friday afternoon, taking on the Mississippi State Bulldogs at 5:30 p.m. in Starkville.

For all of the latest news and information on Tiger softball, visit www.lsusports.net/softball. Fans can also follow the program on its social media outlets at www.Facebook.com/lsusoftball along with @lsusoftball and @BethTorina on Twitter and @lsusb on Instagram.


Copy number gain of 1q25 predicts poor progression-free survival for pediatric intracranial ependymomas and enables patient risk stratification: a prospective European clinical trial cohort analysis on behalf of the Children's Cancer Leukaemia Group (CCLG), Societe Francaise d'Oncologie Pediatrique (SFOP), and International Society for Pediatric Oncology (SIOP).
The high incidence of recurrence and unpredictable clinical outcome for pediatric ependymoma reflect the imprecision of current therapeutic staging and need for novel risk stratification markers. We therefore evaluated 1q25 gain across three age- and treatment-defined European clinical trial cohorts of pediatric intracranial ependymoma. Frequency of 1q gain was assessed across 48 ependymomas (42 primary, 6 recurrent) using Affymetrix 500K single-nucleotide polymorphism arrays. Gain of 1q25 was then evaluated by interphase FISH across 189 tumors treated on the Children's Cancer Leukaemia Group/International Society for Pediatric Oncology (SIOP) CNS9204 (n = 60) and BBSFOP (n = 65) adjuvant chemotherapy trials, or with primary postoperative radiotherapy (SIOP CNS9904/RT, n = 64). Results were correlated with clinical, histologic, and survival data. Gain of 1q was the most frequent imbalance in primary (7/42, 17%) and recurrent ependymomas (2/6, 33%). Gain of 1q25 was an independent predictor of tumor progression across the pooled trial cohort [HR = 2.55; 95% confidence interval (CI): 1.56-4.16; P = 0.0002] and both CNS9204 (HR = 4.03; 95% CI: 1.88-8.63) and BBSFOP (HR = 3.10; 95% CI: 1.22-7.86) groups. The only clinical variable associated with adverse outcome was incomplete tumor resection. Integrating tumor resectability with 1q25 status enabled stratification of cases into disease progression risk groups for all three trial cohorts. This is the first study to validate a prognostic genomic marker for childhood ependymoma across independent trial groups. 1q25 gain predicts disease progression and can contribute to patient risk stratification. We advocate the prospective evaluation of 1q25 gain as an adverse marker in future international clinical trials.


Bad news, Into The Woods fans: one of the best songs from the entire musical has been scrapped because sex. Here's hoping you weren't excited about Emily Blunt and Chris Pine singing "Any Moment," because it's gone. Also Rapunzel doesn't die.

In the latest issue of The New Yorker, Stephen Sondheim met with a group of drama teachers to talk all things Sondheim and the stage. Instead, he shattered all their hearts with these reveals about the upcoming Disney adaptation of the beloved musical Into The Woods.

"You will find in the movie that Rapunzel does not get killed, and the prince does not sleep with the baker's wife," he said. The teachers gasped, but Sondheim shrugged. "You know, if I were a Disney executive I would probably say the same thing," he said. A teacher asked what would happen to the song "Any Moment" if the baker's wife remained chaste. "Don't say the song is cut." "The song is cut." The teachers cried out in despair. "I'm sorry, I should say, it's probably cut," Sondheim said. "Stick up for that song!" a teacher called out. "I did, I did," Sondheim said. "But Disney said, we don't want Rapunzel to die, so we replotted it. I won't tell you what happens, but we wrote a new song to cover it."

Look, it's his baby, so he can feel free to let Disney mutilate Into The Woods all he wants. That is life.

But personally, I'm distraught. Not because this is in abomination, but because I really wanted to see Chris Pine sing "Any Moment." He has a great sort of dickish charm about him that really works for Cinderella's Prince. And, most importantly, this song is just the best song, the best song. THE BEST SONG.

Who hasn't sung, "thisisridiculouswhatamidoinghereiminthewrongstoooooooory" to themselves? It's a classic. This is mostly thanks to the phenomenal Joanna Gleason (who won a Tony for her performance in the classic musical). And it sets up "Moments in the Woods," which is just another fantastic song. What's going to happen to the "Was that me? Was that him? Did a Prince really kiss me?" setup? This is a huge character moment for the Baker's Wife. HUGE. ARGH, I'm so mad about this.

The whole charm of this musical is the sexual crossovers, bad deeds and devious dealings. These characters aren't perfect people, and they didn't get to their happily ever after without a little magic, seduction or flat-out lying. Basically, they make bad decisions. So why would they stop after the book closed? It's an exciting tale that humanizes fantasy creatures in the best way possible, by watching them deal with the consequences of the wishes they make. But now it seems like Disney is taking all the decision-making and repercussions out of the show.

Yes, I'm glad Johnny Depp (the Big Bad Wolf) isn't going to be harassing the very young Lila Crawford (Red Riding Hood), because the casting decisions would have made that AWFUL. But again, the casting decision forever changed the whole dynamic of that dangerous fairy tale world that was setup ever after.

And now Rapunzel isn't going to die. This also cuts all the fire and sadness out of the song "Witch's Lament," which twists into a dastardly sad version of "Children Will Listen" because her child is dead! It also undercuts "Last Midnight." But don't worry, one of the songs Sondheim penned to replace the many cut songs is called "Rainbows." RAINBOWS.

The more I hear about the Into the Woods movie, the more bummed out I get. At this point I have to wonder if any character will die at all. I swear, if the narrator survives, I will rip put all of my hair.

EDIT UPDATE: Thanks to you guys pointing this out I now have a NEW fear. There may not even be a narrator! Simon Russell Beale has been cast as "the Baker's Father" but it says nothing of narrator. So great.


Background
==========

External duodenal fistula is a complication of gastric and kidney surgeries, pancreatitis, and inflammatory bowel disease (IBD). The reported overall morbidity and mortality related to duodenal fistula ranged from 38% to 75% and 7% to 40%, respectively \[[@b1-medscimonit-24-9317],[@b2-medscimonit-24-9317]\].

A few studies have focused on the intra-abdominal bleeding associated with external duodenal fistula, which found that intra-abdominal bleeding accounted for about 10% of complications \[[@b3-medscimonit-24-9317],[@b4-medscimonit-24-9317]\]. The mortality from intra-abdominal bleeding with abdominal infection was approximately 50%, which led to hemorrhagic shock, rectal perforation, and renal injury \[[@b5-medscimonit-24-9317],[@b6-medscimonit-24-9317]\]. Duodenal juice is a mixture of gastric and pancreatic juice and bile, and is more corrosive than juices in the small intestine and colon. Furthermore, the duodenum is surrounded by numerous vessels, that, in theory, gives this site the highest risk for bleeding, with enterocutaneous fistula having the worst prognosis \[[@b7-medscimonit-24-9317]--[@b9-medscimonit-24-9317]\]. The purpose of the present study was to investigate the prevalence of and risk factors for abdominal bleeding in patients with enterocutaneous duodenal fistula.

Material and Methods
====================

Study sesign
------------

This was a retrospective study performed at the Intestinal Fistula Treatment Center, Department of General Surgery, Jinling Hospital, Nanjing, China. From January 2014 to December 2016, patient medical records with external duodenal fistula were reviewed and screened out. All patients were transferred in from other hospitals to treat their duodenal fistula.

Patients in our center with external duodenal fistula were included in the study. The exclusion criteria of the patients were: (1) age \<17 years old, (2) patients with iatrogenic abdominal bleeding, and (3) patients without complete clinical data ([Figure 1](#f1-medscimonit-24-9317){ref-type="fig"}). The prevalence of external duodenal fistula was investigated. According to whether the bleeding occurred after admission, patients were divided into a bleeding group and a non-bleeding group. The risk factors for bleeding were evaluated. The research project protocol was approved by the Ethics Committee of the institution, where the work was performed. All patients provided an informed consent form prior to being included in the study.

Therapeutic principles
----------------------

After admission, the treatment procedure followed the SOWATS treatment guideline proposed by Visschers RG, et al. \[[@b10-medscimonit-24-9317]\]. The SOWATS treatment guidelines include: Sepsis control (the method for sepsis control was puncture and drainage or laparotomy); Optimization of the nutritional state, including the total parenteral nutrition during the transitional period and enteral nutrition (EN) used during the stabilization period; Wound care; Anatomy; Timing of deterministic surgery (3--6 months after the hospital admission); and Surgical strategy.

The treatment was divided into 2 stages. In the first stage, examinations were completed as soon as possible after admission. Intraperitoneal or retroperitoneal infection was diagnosed after CT scans, and puncture and drainage or laparotomies were planned to treat the infections. Enteral nutrition was provided through a nasojejunal tube. The fistula locations were investigated using radiography of the gastrointestinal tract. Surgery was performed during the second stage.

Definitions
-----------

The diagnosis of abdominal bleeding was not difficult. Abdominal bleeding caused by duodenal fistula was signaled by a series of clinical manifestations, including massive bleeding from an abdominal drain, tachycardia, and hemodynamic instability. Organ failure was defined as a score of \>2 using the Marshall scoring system \[[@b11-medscimonit-24-9317]\]. According to this scoring system, acute hepatic failure (AHF) was defined as having a total bilirubin \>60 μmol/L \[[@b11-medscimonit-24-9317]\]. Respiratory failure was defined as having a PaO2/FiO2 \<200mmHg \[[@b11-medscimonit-24-9317]\]. Acute kidney failure (AKF) was defined as having a creatinine \>200 μmol/L \[[@b11-medscimonit-24-9317]\] (or if the patient had continuous renal replacement therapy (CRRT), on admission). According to the location of the effusion, exudation, and necrosis, using CT, retroperitoneal or intraperitoneal infection was diagnosed.

Data collection
---------------

Data on demographic characteristics (including age, sex, and body mass index \[BMI\]), imaging examinations, and etiology were collected on admission. The laboratory examination results and the acute physiology and long-term health evaluation (APACHE) II score were recorded within the first 24 h of admission. Respiratory function, kidney function, and hepatic function were evaluated according to the oxygen index, creatinine levels, and total bilirubin levels, respectively.

Statistical analysis
--------------------

All statistical analyses were performed using the Statistical Package for Social Science (SPSS) version 20.0 for Windows (IBM Analytics, Armonk, NY). A *t* test and a Mann-Whitney U test were used to compare continuous variables. Fisher's exact test was used to compare categorical variables. To identify the potential risk factors for bleeding, all variables with a value P\<0.05 in the univariate analyses were enrolled into a multivariate logistic regression analysis. A P value of \<0.05 was regarded as statistical significance.

Results
=======

Population and prevalence of abdominal bleeding
-----------------------------------------------

A total of 107 patients with external duodenal fistula were treated at our center. Of the 107 patients, abdominal bleeding occurred in 2 patients due to punctures of the viscera or blood vessels, 1 patient did not have complete clinical data, and 7 patients were younger than 17 years of age.

A total of 97 patients were ultimately included in this study ([Figure 1](#f1-medscimonit-24-9317){ref-type="fig"}). There were 34 males (35.05%) and 63 females (64.95%). There were 31 patients in the bleeding group and 66 patients in the non-bleeding group. The prevalence of abdominal bleeding overall was 31.9% (95%CI: 22.5--41.4%). The prevalence of abdominal bleeding in patients with intraperitoneal fistula (stump or bulb fistula, n=40) was 25.0% (95%CI: 11.0--39.0%). The prevalence of abdominal bleeding in patients with extraperitoneal fistula (descending portion or horizontal part of the fistula, n=57) was 36.8% (95%CI: 23.9--49.8%) and there was no significant difference in prevalence of abdominal bleeding between the 2 types (intraperitoneal fistula and extraperitoneal fistula; p=0.218)

Assessment of organ function
----------------------------

A total of 18 cases were diagnosed as AKF, 13 in the bleeding group (2 cases with CRRT on admission and 11 cases with creatinine \>200 μmol/L) and 5 in the non-bleeding group (all the 5 with creatinine \>200 μmol/L; 41.94% *vs.* 7.58%, p\<0.001). Once admitted, 15 cases had AHF: 9 in the bleeding group and 6 in the non-bleeding group (29.03% *vs.* 9.84%, p=0.029). No obvious difference in the incidence of respiratory failure were found between the 2 groups (16.13% (n=5) *vs.* 10.61% (n=7), p=0.441).

Risk factors for abdominal bleeding in patients with duodenal fistula
---------------------------------------------------------------------

The characteristics of patients (general health condition and assessment of organ damage in patients at the time of admission) and the fistulas (locations and the etiologies of duodenal fistula) between the 2 groups are shown in [Tables 1](#t1-medscimonit-24-9317){ref-type="table"} and [2](#t2-medscimonit-24-9317){ref-type="table"}. The results of routine blood and biochemical tests and inflammatory index examinations are shown in [Table 3](#t3-medscimonit-24-9317){ref-type="table"}. Characteristics of the 97 were significantly different patients between the bleeding group and the non-bleeding group in 8 aspects: body temperature of patients on admission, APACHE II score, retroperitoneal infection incidence, C reactive protein (CRP), platelets (PT), albumin (ALB), and incidence of AHF and AKF. Multi-logistic regression analysis was used to identify the risk factors for intra-abdominal bleeding shown in [Table 4](#t4-medscimonit-24-9317){ref-type="table"}. We found that that AKF (OR: 8.462, 95% CI: 1.921--37.28, P=0.005) and retroperitoneal infection (OR: 5.373, 95% CI: 1.504--19.197, P=0.010) were the independent risk factors for abdominal bleeding.

Definitive surgeries and prognoses
----------------------------------

A total of 14 patients died during the treatments, and all died before definitive surgeries were performed. The mortality rate of the bleeding group was 38.71% (n=12), which was higher than that in the non-bleeding group (3.03%, n=2, p\<0.001).

Two patients in the bleeding group and 21 patients in the non-bleeding group achieved spontaneous closure of the duodenal fistula (p=0.005). The surgical procedures for the duodenal fistula included the primary repair of duodenal fistula for most locations expect for the bulb fistula, for which a Billroth II gastrectomy was performed. The length of hospital stay (LOS) of survivors in the bleeding group was 110 (72--176) days, which was longer than it in the non-bleeding group (85 (62--114) days, p=0.045).

Discussion
==========

Duodenal fistula is a complication of gastric resections or surgeries of the biliary tract, duodenum, pancreas, and kidney \[[@b4-medscimonit-24-9317]\], which account for 3--14% of all enterocutaneous fistulas \[[@b1-medscimonit-24-9317]\]. Little research has focused on the intra-abdominal bleeding in duodenal fistula cases. However, intra-abdominal bleeding in patients with enterocutaneous fistula and acute pancreatitis was a fatal complication, with mortality rates as high as 46% and 50%, respectively \[[@b5-medscimonit-24-9317],[@b9-medscimonit-24-9317]\]. Duodenal juice is more corrosive than fluid in the small intestinal and colon, and most of the fistulas had an output of over 1000 ml/day. Additionally, the duodenum has more vessels compared with the small intestine and colon. The incidence of bleeding seemed to be higher in duodenal fistulas than in small intestinal and colonic fistulas \[[@b6-medscimonit-24-9317]--[@b8-medscimonit-24-9317],[@b12-medscimonit-24-9317]\], and the prognosis was poorer. As a result, the bleeding caused by duodenal fistulas should be taken seriously. Therefore, our present research is of great significance.

In the present study, 97 patients with external duodenal fistula were included, and the prevalence of bleeding was 31.9%, which was much higher than those published in other articles. In addition, there was no significant difference in prevalence of abdominal bleeding between the intraperitoneal fistulas and extraperitoneal fistulas; (25% *vs.* 36.8%; p=0.218). However, in the present study, the incidence of retroperitoneal infection in the bleeding group was higher than that in the non-bleeding group (61.29% *vs.* 16.67%; p\<0.001). The location of the fistula might not have contributed to the abdominal bleeding and retroperitoneal infection, and more attention should be paid to retroperitoneal infection. The relationship between retroperitoneal infection and the abdominal bleeding prevalence could be the subject of future study.

The AKF and the retroperitoneal infections were the 2 risk factors for the intra-abdominal bleeding in duodenal fistulas. AKF was a common complication of sepsis \[[@b13-medscimonit-24-9317]\]. In the treatment of AKF caused by sepsis, the timing of CRRT was not clear \[[@b14-medscimonit-24-9317],[@b15-medscimonit-24-9317]\], and little research has concentrated on the correlation between creatinine and coagulation function. More studies about hyper-creatinine and bleeding are needed, and arbitrary conclusions should not be made. However, the controversy over the increased risk for bleeding in CRRT and the recent studies focusing on the relationship between renal failure and platelet function would make sense.

Hemodialysis can partially decrease creatinine level and improve acid-base balance, which is indispensable for patients with AKF. However, the heparin used in CRRT could reduce coagulation function, and although citrate can reduce bleeding incidences better than heparin, serum calcium was decreased even further, making bleeding risks still higher in patients needing CRRT compared with patients that do not require CRRT. In addition, some studies on platelet function even suggested that platelet dysfunction, the multifactorial defect of interaction between vessel walls and circulating cells in kidney disease, was able to increase the risk of bleeding \[[@b16-medscimonit-24-9317],[@b17-medscimonit-24-9317]\], while the detailed pathophysiological process is complex and it is not completely understood \[[@b18-medscimonit-24-9317]\]. Moreover, hemodialysis can even contribute to bleeding by the continuously activating platelets, which led to a reduction in platelet numbers and cytokine activation \[[@b18-medscimonit-24-9317]--[@b21-medscimonit-24-9317]\]. In summary, patients with AKF have a higher risk of bleeding.

Most of the duodenum lies in the retroperitoneum. As a result, the retroperitoneal infections are likely a manifestation of poor infection control, meaning that a large amount of duodenal fluid cannot drain adequately. Since many retroperitoneal vessels surround the duodenum, duodenal fluid that does not drain properly can harm and destroy the vascular tissues. This retention of fluid is responsible for the formation of pseudoaneurysms, vascular and intestinal erosions, and consequent hemorrhages, and may be similar to the abdominal bleeding seen in patients with necrotizing pancreatitis \[[@b22-medscimonit-24-9317]\]. Therefore, there is a higher risk of bleeding in patients with retroperitoneal infection.

The present study has certain limitations. First, as the study was retrospective, selection bias exists. Second, hepatic failure could have contributed to the coagulation dysfunction and related bleeding. The criteria for AHF was defined as patients with a score of \>2 using the Marshall scoring system, or in other words, as patients with a total bilirubin \>60 μm/L. However, in patients with duodenal fistula, intestinal fluid loss and intestinal function damage due to poor control of infections has made support of successful enteral nutrition (EN) implementation a difficult task \[[@b23-medscimonit-24-9317]\]. Lack of EN for extended time periods and the use of somatostatin lead to hyperbilirubinemia \[[@b24-medscimonit-24-9317],[@b25-medscimonit-24-9317]\], which can be relieved after EN support \[[@b24-medscimonit-24-9317],[@b25-medscimonit-24-9317]\]. In the present study, there were only 12 patients with EN support at the time of admission. The hyperbilirubinemia probably was related to failed EN support and use of somatostatin. Under these circumstances there may be some deviations in the diagnosis of liver failure according to the Marshall score (cutoff point=60 μm/L). Duodenal fistula combined with bleeding was relatively rare and to the best of our knowledge this study is the first to investigate the risk factors for bleeding in this population. We found that AKF and retroperitoneal infections are predictors of intra-abdominal bleeding.

Conclusions
===========

In the present study, the prevalence of abdominal bleeding in patients with external duodenal fistula was 31.9%. AKF and retroperitoneal infection were revealed to be 2 risk factors for intra-abdominal bleeding in cases of duodenal fistula.

**Source of support:** Departmental sources

**Conflict of interest**

None.

![Flow of patient selection.](medscimonit-24-9317-g001){#f1-medscimonit-24-9317}

######

Characteristics of the patients between the 2 groups.

  Variable                                                                               Bleeding group (n=31)   Nonbleeding group (n=66)   p
  -------------------------------------------------------------------------------------- ----------------------- -------------------------- -------------
  Age, yeas, mean ±SD                                                                    49.63±16.86             45.52±15.59                0.245
  Male, n (%)                                                                            13 (41.96%)             21 (25.76%)                0.108
  BMI, kg/m^2^, mean ±SD                                                                 22.41± 4.60             22.17± 3.79                0.785
  Pulse rate, median (IQR)                                                               95 (82--110)            88 (81--110)               0.226
  Temperature, median (IQR)                                                              37.3 (36.6--38.0)       36.7 (36.5--37.1)          **0.004**
  Enteral nutrition support[\#](#tfn1-medscimonit-24-9317){ref-type="table-fn"}, n (%)   3 (9.68)                9 (13.64)                  0.581
  Previous bleeding, n (%)                                                               4 (12.90)               5 (8.20)                   0.399
  Time interval from fistula onset to admission, days, median (IQR)                      30 (10--53)             26 (16--44)                0.904
  Time interval from admission to bleeding, days, median (IQR)                           5 (3--10)               N/A                        N/A
  APACHE II score                                                                        13.29±4.22              8.98±4.16                  **\<0.001**
  Retroperitoneal infection, n (%)                                                       19 (61.29)              11 (16.67)                 **\<0.001**
  Intraperitoneal infection, n (%)                                                       28 (90.32)              62 (93.94)                 0.521
  Co-morbidities, n (%)
   Hypertensio                                                                           5 (16.13)               7 (10.61)                  0.441
   Diabetes mellitus                                                                     3 (9.68)                10 (15.15)                 0.541
   Chronic nephritis                                                                     1 (3.23)                2 (3.03)                   0.959
   Virus hepatitis                                                                       2 (6.45)                3 (4.55)                   0.692
  AHF, n (%)                                                                             9 (29.03)               6 (9.84)                   0.029
  AKF, n (%)                                                                             13 (41.94)              5 (7.58)                   **\<0.001**
  Respiratory failure, n (%)                                                             5 (16.13)               7 (10.61)                  0.441

Intestinal nutrient solution was fed through the nasointestinal tube and the speed \>20 ml/hour.

AHF -- acute hepatic failure; AKF -- acute kidney failure; APACHE II -- acute physiology and chronic health evaluation II; BMI -- body mass index.

######

Characteristics of the fistulas between the 2 groups.

  Variable                                  Bleeding group (n=31)   Nonbleeding group (n=66)   p
  ----------------------------------------- ----------------------- -------------------------- -------
  Anatomy of fistula, n (%)
   Stump                                    8 (25.81)               20 (30.30)                 0.649
   Bulb                                     2 (6.45)                10 (15.15)                 0.225
   Descending portion                       20 (64.52)              34 (51.52)                 0.229
   Horizontal part of duodenum              1 (3.23)                2 (3.03)                   1.000
  Etiology, n (%)
   Primary repair                           9 (29.03)               24 (36.36)                 0.477
    Perforated ulcer                        2 (6.46)                4 (6.06)                   1.000
    Trauma                                  7 (22.58)               20 (30.30)                 0.429
   Billroth II gastrectomy                  8 (25.81)               20 (30.30)                 0.649
    Perforated ulcer                        1 (3.23)                2 (3.30)                   1.000
    Trauma                                  1 (3.23)                3 (4.55)                   0.761
    Gastric cancer                          6 (19.35)               15 (22.73)                 0.707
   Excision of duodenum tumor               1 (3.23)                4 (6.06)                   0.556
   Biliary tract surgery                    4 (12.90)               3 (4.55)                   0.205
   Acute necrotic pancreatitis              5 (16.13)               9 (13.64)                  0.745
   Kidney surgery                           4 (12.90)               6 (9.09)                   0.565
  Combined with colonic fistula, n (%)      11 (35.48)              17 (25.76)                 0.303
  Combined with intestinal fistula, n (%)   3 (9.68)                8 (12.12)                  0.723

######

Results of laboratory examination.

  Variable                             Bleeding group (n=31)    Nonbleeding group (n=66)   p
  ------------------------------------ ------------------------ -------------------------- ---------
  WBC, (10^9^ cells/L), median (IQR)   10.80 (7.20--12.80)      9.20 (5.90--12.70)         0.477
  CRP, mg/L, median (IQR)              128.10 (43.50--170.00)   31.50 (7.98--87.63)        \<0.001
  PLT, (10^9^ cells/L), mean ±SD       200.23±68.05             237.12±97.87               0.061
  PT, s, mean ±SD                      13.93±1.40               13.23±1.23                 0.015
  APTT, s, mean ±SD                    35.10±6.95               33.87±8.03                 0.47
  ALB, g/L, mean ±SD                   30.41±5.16               33.19±6.18                 0.032
  ALT, U/L, median (IQR)               27.00 (21.00--50.00)     31.00 (22.00--55.00)       0.787
  ALP, U/L, median (IQR)               94.00 (74.00--155.00)    115.60 (84.00--163.50)     0.181
  AST, U/L, median (IQR)               30.00 (20.00--50.00)     30.00 (20.00--50.00)       0.798
  TB, μmol/L, mean ±SD                 56.86±65.34              25.57±28.59                0.015
  CR, μmol/L, mean ±SD                 131.69± 95.79            58.56±24.54                \<0.001

ALB -- albumin; ALT -- alanine aminotransferase; ALP -- alkaline phosphatase; APTT -- activated partial thromboplastin time; AST -- aspertate aminotransferase; CR -- creatinine; CRP -- C-reactive protein; PLT -- bloodplatelet; PT -- prothrombin time; TB -- total bilirubin; WBC -- white blood cell.

######

Logistic regression of risk factors for intra-abdominal bleeding.

  Variable                    OR      CI              p
  --------------------------- ------- --------------- -------
  Temperature                 1.220   0.487--3.056    0.671
  APECHE II score             1.021   0.863--1.208    0.806
  Retroperitoneal infection   5.373   1.504--19.197   0.010
  CRP                         1.007   0.996--1.017    0.159
  PT                          1.154   0.665--2.040    0.622
  ALB                         0.946   0.828--1.0582   0.421
  AKI                         8.462   1.921--37.28    0.005
  AHI                         2.172   0.464--10.171   0.325

AKI -- acute kidney injury; AHI -- acute hepatic injury; ALB -- albumin; APECHE II -- acute physiology and chronic health evaluation; CRP -- C-reactive protein; PT -- prothrombin time.

[^1]: Study Design

[^2]: Data Collection

[^3]: Statistical Analysis

[^4]: Data Interpretation

[^5]: Manuscript Preparation

[^6]: Literature Search

[^7]: Funds Collection

[^8]: Zheng Yao and Zheng Ge are the first authors


Q:

Why does this prime test work?

I have found this Python function for testing whether or not a number is prime; however, I cannot figure out how the algorithm works.
def isprime(n):
   """Returns True if n is prime"""
   if n == 2: return True
   if n == 3: return True
   if n % 2 == 0: return False
   if n % 3 == 0: return False

   i = 5
   w = 2
   while i * i <= n:
      if n % i == 0:
         return False

      i += w
      w = 6 - w

   return True

A:

Let's start with the first four lines of the function's code:
def isprime(n):
    if n == 2: return True
    if n == 3: return True
    if n % 2 == 0: return False
    if n % 3 == 0: return False

The function tests to see if n is equal to 2 or 3 first. Since they are both prime numbers, the function will return True if n is equal to either.
Next, the function tests to see if n is divisible by 2 or 3 and returning False if either is true. This eliminates an extremely large amount of cases because half of all numbers above two are not primes - they are divisible by 2. The same reason applies to testing for divisibility by 3 - it also eliminates a large number of cases.
The trickier part of the function is in the next few lines:
i = 5
w = 2
while i * i <= n:
    if n % i == 0:
        return False

    i += w
    w = 6 - w

return True

First, i (or index) is set to 5. 2 and 3 have already been tested, and 4 was tested with n % 2. So, it makes sense to start at 5.
Next, w is set to 2. w seems to be an "incrementer". By now, the function has tested for all even numbers (n % 2), so it would be faster to increment by 2.
The function enters a while loop with the condition i * i <= n. This test is used because every composite number has a proper factor less than or equal to its square root. It wouldn't make sense to test numbers after the square root because it would be redundant.
In the while loop, if n is divisible by i, then it is not prime and the function returns False. If it is not, i is incremented by the "incrementer" w, which, again, is faster.
Perhaps the trickiest part of the function lies in the second-to-last line: w = 6 - w. This causes the "incrementer" w to toggle between the values 2 and 4 with each pass through loop. In cases where w is 4, we are bypassing a number divisible by 3. This is faster than remaining at 2 because the function already tested for divisibility by both 2 and 3.
Finally, the function returns True. If the function hasn't detected any cases where n is divisible by something, then it must be a prime number.

A:

Except 2 and 3 all prime number can represent using (6*n)+1 or (6*n)-1, where n is 0 to infinite.
This program is working according to this idea.
Using this lines check number can divisible by 2 or 3
 if n % 2 == 0: return False
 if n % 3 == 0: return False

Then we need to check number can divisible by other prime numbers grater than 3.
i = 5
w = 2
while i * i <= n:
   if n % i == 0:
       return False

   i += w
   w = 6 - w

The next prime number is 5. Therefore initial value of i set as 5.
To get all number in set (6*n)+1 or (6*n)-1 alternatively change the value of w (2,4).
And this snippet used to check up to square root of  the number.
 while i * i <= n:

This code is not efficient because some non prime numbers in set (6*n)+1 or (6*n)-1.


Q:

Error when downloading file from amazon s3

I am trying to download files from amazon s3. I am using .NET sdk for that. I have written down follwong code:
GetObjectRequest request = new GetObjectRequest()
    .WithKey("abc/xyz.jpg")
    .WithBucketName(appConfig);

using (GetObjectResponse response = s3Client.GetObject(request))
{
    int numBytesToRead = (int)response.ContentLength;
    if (numBytesToRead > 0)
    {
        int numBytesRead = 0;
        byte[] buffer = new byte32768;
        using (FileStream fstream = new FileStream(@"C:\abc\xyz.jpg", FileMode.Create))
        {
            using (Stream responseStream = response.ResponseStream)
            {
                do
                {
                    numBytesRead = responseStream.Read(buffer, 0, buffer.Length);
                    fstream.Write(buffer, 0, numBytesRead);
                }
                while (numBytesRead > 0);
            }
        }
    }
}

With this code I am able to download file. But now I want to download multiple files. For that first I am listing down all the files which are available on s3 and then store it in arraylist and after that call my download function for each file.
After running list file method when I call download function after using (GetObjectResponse response = s3Client.GetObject(request)) I get exception.
{"Access Denied"}
Stack trace:
at Amazon.S3.AmazonS3Client.processRequestError(String actionName, HttpWebRequest request, WebException we, HttpWebResponse errorResponse, String requestAddr, WebHeaderCollection& respHdrs, Type t, Exception& cause)
at Amazon.S3.AmazonS3Client.handleHttpWebErrorResponse(S3Request userRequest, WebException we, HttpWebRequest request, HttpWebResponse httpResponse, Exception& cause, HttpStatusCode& statusCode)
at Amazon.S3.AmazonS3Client.getResponseCallback[T](IAsyncResult result)
at Amazon.S3.AmazonS3Client.endOperation[T](IAsyncResult result)
at Amazon.S3.AmazonS3Client.EndGetObject(IAsyncResult asyncResult)
at Amazon.S3.AmazonS3Client.GetObject(GetObjectRequest request)
at ScreenshotsUploader.ScreenshotsUploader.downloadFile(AmazonS3 s3Client, NameValueCollection appConfig) in c:\Users\xyz\Documents\Visual Studio 2012\Projects\ScreenshotsUploader\ScreenshotsUploader\ScreenshotsUploader.cs:line 220
at ScreenshotsUploader.ScreenshotsUploader.GetServiceOutput() in c:\Users\xyz\Documents\Visual Studio 2012\Projects\ScreenshotsUploader\ScreenshotsUploader\ScreenshotsUploader.cs:line 55

Status code:
System.Net.HttpStatusCode.Forbidden
What do I need to do to fix this?
I have also tried different approach to download file and got the same error:
Second approach:
string fileToDownload = @"xyzjpg";
string saveToo = @"C:\xyz.jpg";
TransferUtilityDownloadRequest request = new TransferUtilityDownloadRequest().WithBucketName(appConfig).WithKey(fileToDownload).WithFilePath(saveToo);
TransferUtility tu = new TransferUtility(s3Client);
tu.Download(request);

Thanks in advance.

A:

public Stream DownloadS3Object(string awsBucketName, string keyName)
{

    using (var client = Amazon.AWSClientFactory.CreateAmazonS3Client())
    {
        Stream imageStream = new MemoryStream();
        GetObjectRequest request = new GetObjectRequest { BucketName = awsBucketName, Key = keyName };
        using (GetObjectResponse response = client.GetObject(request))
        {
            response.ResponseStream.CopyTo(imageStream);
        }
        imageStream.Position = 0;
        // Clean up temporary file.
        // System.IO.File.Delete(dest);
        return imageStream;
    }
}

pass the value in function get stream path save it in folder with use of below.
SaveStreamToFile(foldername + "/", MainStreamAwsPath);

and than you can apply simple c# code to download that folder.


If you think your student loans are bad, know that it could be far, far worse. Chinese state media reported this week that some online lenders require borrowers to send a nude selfie as collateral. If the They then threaten to post the photo online and send it to their family members if payment isn’t made on time.

The practice largely targets female students who need money to pay for college, and in order to get a higher loan than they otherwise would—reportedly two to five times higher than average—these students must pose naked while holding their IDs and then provide information about their family members. This allows the loan sharks to threaten the release of the photos, and with interest rates that reach as high as 30 percent, those threats can lead borrowers to amass further debt.

In one instance, reported by Computerworld, a student who took out a naked loan worth 500 yuan (the equivalent of $76), struggled to make payments and, having to pull out further loans, saw her debt balloon to 55,000 yuan ($8,352).

The shady scheme appears to have been going on for some time on a lending platform called Jiedaibao, which allows people to make private arrangements to lend or borrow money. And while the company initially told state media that it could not control what lenders asked for as collateral, the recent media exposure may lead to a change in Jiedaibao’s policy. When the Guardian reached out to Jiedaibao on Wednesday, it said they would work with police to investigate the practice the so-called “naked loans.”

Still, that may not be enough to put an end to shady loan schemes, as state-funded student loans are hard to come by, leaving millions of people to find a way to pay for school.


Larval membrane antigens protect Hereford cattle against infestation with Boophilus microplus.
Hereford cattle (Bos taurus) were immunized with antigens solubilized with Triton X-100 from larval membranes of the cattle tick (Boophilus microplus). Based on tick egg production compared to control cattle, vaccinated cattle were protected (78%) against challenge with 2 x 20,000 tick larvae. The soluble Triton X-100 extract of tick larval membranes was further purified by immunoaffinity chromatography, using immunoglobulin ligands (IgG1 and IgG2) from three immune steers, previously vaccinated with membrane antigens from the midgut of partly engorged adult female ticks. Cattle vaccinated with these purified antigens were protected in two separate experiments (80 and 89% respectively), against challenge with 2 x 20,000 larval ticks compared to control cattle. Whole larval membranes used as vaccines in cattle reduced the amount of eggs produced from ticks by 47% compared to control cattle, but this difference was not significant.


1. Introduction
===============

Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV). The disease is characterized by severe diarrhea, vomiting, dehydration, and death, and has a mortality rate of up to 90% ([@bib20], [@bib24]). Since PED was first reported in Belgium and the UK in 1978, the disease has frequently broken out in many swine-raising countries and has led to severe economic losses in Asia, notably in China, Japan and Korea ([@bib5], [@bib23], [@bib16], [@bib10]).

PEDV is an enveloped, positive-sense, single-stranded RNA virus, which belongs to the genus *Coronavirus*, in the family Coronaviridae, order Nidovirales. The genome of PEDV is approximately 30 kb in length. Subgenomic mRNAs of PEDV are transcribed from its genome RNA, and then are translated to produce viral proteins such as spike protein (S, 180--220 kDa), membrane protein (M, 27--32 kDa) and nucleocapsid protein (N, 55--58 kDa) ([@bib9], [@bib12], [@bib1]). The S protein, a glycoprotein peplomer on the viral surface, plays an important role in induction of neutralizing antibodies, specific receptor binding and cell membrane fusion ([@bib8], [@bib17], [@bib18]). These properties make the S protein a suitable candidate for genetic engineering of vaccines and diagnostic reagents.

Previously, we used antisera of PEDV to test the antigenicity of four overlapping fragments spanning the major immunodominant region (residues 1--789) of S protein, and identified one antigenic determinant (S1D: residues 636--789). The anti-S1D antibodies showed neutralizing activity for PEDV-infected Vero cells ([@bib21]). In order to provide some basis for the development of vaccines and diagnostic reagents, it is necessary to map the antigenic epitopes of the S1D region and accurately determine important residues in these epitopes. In this study, one micropanned epitope region of S1D (MER: residues 696--779) was identified by fd-tet phage display using six monoclonal antibodies (McAbs). Further analysis of the MER was carried out by ELISA, Western blotting and Pepscan using the six McAbs. Two novel B cell epitopes, SS2 (residues 748--755) and SS6 (residues 764--771), were identified in the S protein of PEDV.

2. Materials and methods
========================

2.1. Viruses, plasmid, bacterial strains and expression vector
--------------------------------------------------------------

PEDV strain CV777 was kindly provided by Pensaert MB. Plasmid DNA (pMD18-TS1) containing the S gene (1--2367 bp) of PEDV strain CV777 had been constructed previously ([@bib22]). A gene-targeted fd-tet phage display library (S1-phage library) containing DNA fragments ranging from approximately 50 to 400 bp of the gene encoding the S1 region of PEDV S protein was established by our laboratory, and the neutralizing epitope region S1D located in S1 region of PEDV S protein ([@bib21]). *Escherichia coli* strains MC1061 and K91BluKan, and the phage display vector fUSE 1, were generous gifts from Prof. George P. Smith, University of Missouri, Columbia, USA.

2.2. Development of monoclonal antibodies (McAbs)
-------------------------------------------------

The PEDV particles were purified by sucrose density gradient centrifugation. Female 6-week-old BALB/c mice were immunized with 50 μg of purified PEDV emulsified in complete Freund\'s adjuvant (Sigma, St. Louis, MO, USA). At 3-week intervals two boosters of 50 μg of purified PEDV emulsified in incomplete Freund\'s adjuvant (Sigma, St. Louis, MO, USA) were administered, and mice were sacrificed 3 days after the last booster inoculation. Spleen cells from immunized mice were fused with SP2/0 myeloma cells using 50% (v/v) of PEG1450 (Sigma, St. Louis, MO, USA), and the fused cells were cultured in DMEM supplemented with 20% FCS, HAT medium. Positive hybridoma clones were selected by indirect ELISA using purified S1D protein with a glutathione *S*-transferase (GST) tag as coating antigen. The subtype of McAbs secreted by the final hybridoma clones was identified using the SBA Clonotyping™ System/HRP kit (SouthernBiotech, Birmingham, AL, USA). Ascites fluid was produced in primed BALB/c mice with incomplete Freund\'s adjuvant. Reactivity of the McAbs with the native S protein was tested by ELISA and Western blotting. Neutralizing activity of the McAbs was tested according to the methods described by [@bib13].

2.3. Micropanning of the S1-phage library with McAbs
----------------------------------------------------

Ascites fluid containing six strains of McAbs, named 2C4, 3G3, 5F8, 3G5, 6E6 and 3C3, was diluted to 1:200 in 0.1 M NaHCO~3~ (pH 9.6) and 100 μl of the dilution was added to each well of the ELISA plate (Nunc, Roskilde, Denmark). The ELISA plate was incubated at 4 °C for 12 h. The wells were then blocked with 5% skimmed milk at 37 °C for 1 h. After washing three times with TBST (50 mM Tris·Cl pH 7.5, 150 mM NaCl and 0.5% Tween 20), 100 μl S1-phages (10^6^  TUs) were added to each well and then incubated at 37 °C for 1 h. After washing 10 times with TBST, 20 μl elution buffer (0.2 M glycine--HCl, pH 2.2) was added to the wells and incubated at room temperature for 10 min. Twenty microliters of elution buffer was added to blank wells to serve as a mock eluate. Eluted products were mixed with 4 μl neutralizing solution (1 M Tris·Cl pH 9.1); 2 μl of the mixture was used for TUs titration. Remnant elutes were used to infect 20 ml of starved K91BluKan cells, and then binding-phages in culture supernatant were purified by PEG8000/NaCl (2.8%/0.55 M). The binding-phages were used for micropanning as described above. After the third binding-phages were titrated, 10 random clones of each McAb were isolated. The DNA of infected cells from the above single colonies was sequenced with primer: fd-up 5′-TTTTGGAGATTTTCA ACGTG-3′, which anneals at 1562--1581 bp of the fUSE 1 vector, to identify phage containing the correct open reading frame and the correct orientation of the S1D gene.

2.4. Phage ELISA {#sec1}
----------------

Two phage clones, which displayed 696--770 amino acids and 712--779 amino acids, were designated as phage^696^ and phage^712^, respectively. The overlapping region displayed by phage^696^ and phage^712^ overlaid all other micropanned epitopes. Phage^696^ and phage^712^ were purified by PEG8000/NaCl, and their TUs were titrated for standardization. The ELISA plate wells were coated with 50 μl purified phage^696^ and phage^712^ containing 10^8^  TUs/ml in coating buffer (50 mM Tris·Cl, 150 mM NaCl, pH 9.0) and incubated at 4 °C for 12 h. After washing the plate three times with TBST, 200 μl of blocking solution (5% skimmed milk in TBST) was added to each well and incubated at 37 °C for 1 h, and then 100 μl of McAbs from ascites fluid diluted 1/200 was added and incubated at 37 °C for 1 h. After washing three times with TBST, 100 μl of HRP-conjugated sheep anti-mouse IgG diluted 1/8000 in TBST was added, and then incubated at 37 °C for 1 h. After the addition of 100 μl substrate TMB solution and incubation at room temperature for 10 min, the reaction was stopped by 50 μl of 2 M H~2~SO~4~. The absorbance at 450 nm was measured. Wild phage and S1D recombinant protein were used as negative and positive controls, respectively, and the mixtures of the McAbs were used as their primary antibodies.

2.5. Expression of the micropanned epitope region (MER)
-------------------------------------------------------

Seven overlapping mini-fragments, which covered the micropanned epitope regions (2085--2336 bp), were separately synthesized according to the methods described by ([Table 1](#tbl1){ref-type="table"} ) [@bib14]. These mini-fragments were cloned into pGEX-6p-1 vector (Amersham Pharmacia Biotech, New Jersey, USA), and recombinant plasmids were transformed into *E. coli* BL21. The fusion proteins expressed in *E. coli* BL21 were purified according to the manufacturer\'s protocols (Pierce, Rockford, IL, USA). Using the same procedure, GST protein was also purified for the controlled trial.Table 1Synthesized mini-fragments and deduced amino acid sequencesMini-fragment namesSequences of synthesized mini-fragments (5′ → 3′)Deduced amino acid sequences [a](#tbl1fn1){ref-type="table-fn"}S1D1(+) [gatcc]{.ul}ccatgttctttttcagagcaggctgcatatgttgatgatgatatagtg**taa**g^696^PCSFSEQAAYVDDDIV^711^(−) [g]{.ul}ggtacaagaaaaagtctcgtccgacgtatacaactactactatatcac**att**[cagct]{.ul}

S1D2(+) [gatcc]{.ul}gatgatatagtgggtgttatttctagtttgtctaactccacttttaac**taa**g^708^DDIVGVISSLSNSTFN^723^(−) gctactatatcacccacaataaagatcaaacagattgaggtgaaaattg**att**[cagct]{.ul}

S1D3(+) [gatcc]{.ul}tccacttttaacaatacctgggagttgcctggtttcttctaccattct**taa**g^720^STFNNTRELPGFFYHS^735^(−) gaggtgaaaattgttatggaccctcaacggaccaaagaagatggtaaga**att**[cagct]{.ul}

S1D4(+) [gatcc]{.ul}ttctaccattctaatgatggctccaattgtacagagcctgtgttggtg**taa**g^732^FYHSNDGSNCTEPVLV^747^(−) gaagatggtaagattactaccgaggttaacatgtctcggacacaaccac**att**[cagct]{.ul}

S1D5(+) [gatcc]{.ul}cctgtgttggtgtatagtaacataggtgtctgtaaatctggcagtatt**taa**g^744^PVLVYSNIGVCKSGSI^759^(−) gggacacaaccacatatcattgtatccacagacatttagaccgtcataa**att**[cagct]{.ul}

S1D6(+) [gatcc]{.ul}tctggcagtattggctatgtcccacttcaggatggccaagtcaagatt**taa**g^756^SGSIGYVPLQDGQVKI^771^(−) gagaccgtcataaccgatacagggtgaagtcctaccggttcagttctaa**att**[cagct]{.ul}

S1D7(+) [gatcc]{.ul}cttcaggatggccaagtcaagattgcacccatggttactgggaatatt**taa**g^764^LQDGQVKIAPMVTGNI^779^(−) ggaagtcctaccggttcagttctaacgtgggtaccaatgacccttataa**att**[cagct]{.ul}[^1][^2]

2.6. ELISA of the recombinant proteins {#sec2}
--------------------------------------

The ELISA plate wells were coated with 1 μg/ml purified recombinant protein in 0.1 M carbonate buffer (pH 9.6) at 4 °C for 12 h and blocked with 5% skimmed milk at 37 °C for 1 h. After washing three times with PBST, 100 μl of McAbs from ascites fluid, diluted 1/200, were added to the wells and incubated at 37 °C for 1 h. The plates were washed three times and incubated with 100 μl of HRP-conjugated sheep anti-mouse IgG diluted 1/8000 in TBST at 37 °C for 1 h. Procedures of coloration, termination and absorbance measurement were as for the phage ELISA (Section [2.4](#sec1){ref-type="sec"}).

2.7. Western blotting
---------------------

The purified proteins and native S protein of PEDV were separated by SDS-15% polyacrylamide gel. After electrophoresis the separated proteins were transferred to nitrocellulose membrane (NC). The NC membranes were blocked with 5% skimmed milk in PBS at 37 °C for 1 h, and then incubated with 100 μl of McAbs from ascites fluid diluted 1/200 at 37 °C for 1 h. After washing three times with PBS, the NC membrane was incubated with 100 μl of HRP-conjugated sheep anti-mouse IgG diluted 1/2000 in PBS at 37 °C for 1 h. The NC membrane was washed three times with PBST and then coloration was carried out in 3,3′-diaminobenzidine (DAB) substrate solution.

2.8. Analysis of immunogenicity of the epitopes S1D5 and S1D6
-------------------------------------------------------------

The GST fusion proteins of the epitopes S1D5 and S1D6 were purified according to the manufacturer\'s protocols. Female 8-week-old BALB/c mice were immunized with 50 μg of purified fusion proteins emulsified in complete Freund\'s adjuvant (Sigma, St. Louis, MO, USA). At 3-week intervals two boosters of 50 μg of purified fusion proteins emulsified in incomplete Freund\'s adjuvant (Sigma, St. Louis, MO, USA) were administered. Antiserum of GST protein was also prepared for the controlled trial using the same procedures. Three mice were immunized with each protein. The antibody levels were examined by ELISA using the S1D-GST fusion protein as coating antigen (data not shown). To further verify whether antibodies against the epitopes S1D5 and S1D6 could reacted with native S protein of PEDV, an ELISA was carried out using the purified PEDV as coating antigen. In ELISA, antisera of the epitopes S1D5 and S1D6 were diluted 100 times with PBS for detection, other procedures were as for ELISA of the recombinant proteins (Section [2.6](#sec2){ref-type="sec"}). Neutralizing activity of antibodies against the epitopes S1D5 and S1D6 was tested according to the methods described by [@bib13].

2.9. Pepscan of the epitopes S1D5 and S1D6
------------------------------------------

In order to identify core amino acids on the epitopes S1D5 and S1D6, seven polypeptides were synthesized by CL Bio-Scientific Co., Ltd. (XiAn, China). Location and sequences of the synthetic peptides are shown in [Table 2](#tbl2){ref-type="table"} . Six polypeptides (SS1--SS6) with four overlapping amino acids covered the epitope region of S1D5 and S1D6; the SS7 polypeptide comprised all the peptides (SS1--SS6). The Pepscan procedure was as follows. The ELISA plate wells were coated with the synthetic peptides in 0.1 M carbonate buffer (pH 9.6) at 10 μg/ml at 4 °C for 12 h. Other operations were the same as the phage ELISA (Section [2.4](#sec1){ref-type="sec"}).Table 2Sequence of the synthesized peptides corresponding to the epitopes S1D5 and S1D6NamesAmino acids sequences and location on S protein[a](#tbl2fn1){ref-type="table-fn"}SS1^744^PVLVYSNI^751^SS2^748^YSNIGVCK^755^SS3^752^GVCKSGSI^759^SS4^756^SGSIGYVP^763^SS5^760^GYVPLQDG^767^SS6^764^LQDGQVKI^771^SS7^744^PVLVYSNIGVCKSGSIGYVPLQDGQVKI^771^[^3]

3. Results
==========

3.1. Screening of S1-phage library with McAbs
---------------------------------------------

Six McAbs against the S1D region were prepared by lymphocyte hybridoma technique. All six McAbs could reacted with the native spike protein of PEDV and had no neutralizing activity for PEDV-infected Vero cells (data not shown). The S1-phage library was micropanned using the six McAbs against the S1D region. Sixty phage clones, comprising 10 clones selected randomly from the binding-phages of each McAb, were sequenced by the dideoxy method. The result showed that polypeptides displayed by 55 phage clones (with the exception of five meaningless sequences) were focused on amino acids 696--779 (MER) of the S protein. Further analysis of the MER demonstrated that 30 phage clones displayed amino acids 696--770 (SME I) and 25 phage clones displayed amino acids 712--779 (SME II) ([Fig. 1](#fig1){ref-type="fig"}A). The reactivity of the micropanned phage clones with the McAbs was tested by ELISA. The result indicated that phage^696^ and phage^712^, which displayed SME I and SME II, respectively, were both able to react with the six McAbs ([Fig. 1](#fig1){ref-type="fig"}B and C). These results clearly demonstrated that the crucial epitopes on the S1D region were located within amino acids 696--779 of the PEDV S protein.Fig. 1The result of micropanning of the S1-phage library and the reactivity of phage^696^ and phage^712^ with McAbs. Schematic diagram of the micropanned epitope region (MER) located in the PEDV S protein. (A) Gray histogram represents the whole MER, which includes two overlapping regions, SME I and SME II. (B) The result of the phage^696^ ELISA. (C) The result of the phage^712^ ELISA.

3.2. Mapping of the MER with McAbs by Western blotting and ELISA
----------------------------------------------------------------

To further identify epitopes on MER, seven overlapping mini-fragments ([Table 1](#tbl1){ref-type="table"}) were expressed in *E. coli* BL21 with a GST tag, and then these recombinant proteins were purified ([Fig. 2](#fig2){ref-type="fig"}A). The reactivity of the purified proteins with the six McAbs was detected by ELISA and Western blotting, respectively. Using Western blotting, the S1D5-GST protein showed a strong reaction with McAbs 2C4, 3C3 and 5F8 ([Fig. 2](#fig2){ref-type="fig"}B), and the S1D6-GST protein showed a strong reaction with McAbs 3G3, 6E6 and 3G5 ([Fig. 2](#fig2){ref-type="fig"}C). The results of the ELISA were in accordance with those of Western blotting ([Fig. 2](#fig2){ref-type="fig"}D). These results indicated that S1D5 (^744^PVLVYSNIGVCKSGSI^759^) and S1D6 (^756^SGSIGYVPLQDGQVKI^771^) are two linear epitopes of the PEDV S protein.Fig. 2Purification of the recombinant proteins and detection of their antigenicity for McAbs by Western blotting and ELISA. (A) Electrophoresis pattern of the purified recombinant protein on SDS-15% polyacrylamide gel. Lanes: (1) protein marker; (2--8) purified proteins of S1D1-GST, S1D2-GST, S1D3-GST, S1D4-GST, S1D5-GST, S1D6-GST and S1D7-GST, respectively. (B) Western blotting analysis of the recombinant proteins with McAbs 2C4 (B1), 3C3 (B2) and 5F8 (B3). Lanes: (1--9) S1D1-GST, S1D2-GST, S1D3-GST, S1D4-GST, S1D5-GST, S1D6-GST, S1D7-GST, GST and PEDV, respectively. (C) Western blotting analysis of the recombinant proteins with McAbs 3G3 (C1), 6E6 (C2) and 3G5 (C3). Lanes: (1--9) S1D1-GST, S1D2-GST, S1D3-GST, S1D4-GST, S1D5-GST, S1D6-GST, S1D7-GST, GST and PEDV, respectively. (D) Detection of reactivity of the recombinant proteins with six McAbs by ELISA.

3.3. Immunogenicity of the epitopes S1D5 and S1D6
-------------------------------------------------

To detect immunogenicity of the epitopes S1D5 and S1D6, BALB/c mice were immunized using the GST fusion protein S1D5 and S1D6. After immunization three times, antibodies against the epitopes S1D5 and S1D6 could react with native S protein of PEDV in ELISA ([Fig. 3](#fig3){ref-type="fig"} ). But the antibodies induced by the epitopes S1D5 and S1D6 had no neutralizing activity for PEDV-infected Vero cells (data not shown).Fig. 3Reactivity of antisera against the epitopes S1D5 and S1D6 with PEDV by ELISA.

3.4. Minimization of the epitopes S1D5 and S1D6 with McAbs by Pepscan
---------------------------------------------------------------------

In order to identify core amino acids on the epitopes S1D5 and S1D6, Pepscan of seven synthesized peptides ([Table 2](#tbl2){ref-type="table"}) was carried out with the six McAbs. In ELISA, the SS2 polypeptide was recognized by McAbs 2C4, 5F8 and 3C3; the SS6 polypeptide was recognized by McAbs 6E6, 3G5 and 3G3; and the SS7 polypeptide showed a strong reaction with all the McAbs. These data demonstrate that SS2 (^748^YSNIGVCK^755^) and SS6 (^764^LQDGQVKI^771^) are core amino acids of the epitopes S1D5 and S1D6, respectively ([Fig. 4](#fig4){ref-type="fig"} ).Fig. 4Pepscan of the epitopes S1D5 and S1D6 with McAbs.

4. Discussion
=============

PEDV is an important pathogen causing viral diarrhea in the swine industry ([@bib2], [@bib3]). Although much research has been carried out on the general characteristics of PEDV, few reports have been published on the functions of the PEDV structural proteins, especially S protein. Like those of other coronaviruses, the PEDV S protein plays an important role in inducing neutralizing antibodies ([@bib8], [@bib15]). Therefore, the screening and identification of antigenic epitopes on the PEDV S protein is essential for developing epitope-based vaccines and diagnostic reagents.

In this study, phage display and Pepscan methods were adapted for mapping epitopes of the neutralizing epitope region (S1D, residues 636--789) of the PEDV S protein. First, one epitope region (MER) was identified on the S1D region of PEDV S protein by phage display. Epitopes could not be determined accurately because the inserts expressed on the surface of the recombinant phages were overlength (approximately 200--300 bp). Second, more accurate identification of the epitopes was performed based on the method described by Hua, and two linear epitopes, S1D5 and S1D6, were determined in the MER ([@bib14]). Antibodies against the epitopes S1D5 and S1D6 could react with native S protein of PEDV in ELISA, but the antibodies induced by the epitopes S1D5 and S1D6 had no neutralizing activity for PEDV-infected Vero cells. The epitopes S1D5 and S1D6 are 16 amino acids in length and have four overlapping amino acids. Generally, linear epitopes are short stretches of the primary structure of proteins and consist of six to nine or more continuous amino acid residues. The Pepscan method was used to identify core amino acids on epitopes S1D5 and S1D6, the result showed that the core amino acids were located at amino acids 748--755 (SS2) and amino acids 764--771 (SS6), respectively. It was found that epitopes SS2 and SS6 were adjacent to epitope S1P3 (residues 697--742) in the primary structure of S protein. The epitope S1P3 was identified previously by phage display using native antisera against PEDV ([@bib22]). This result suggests that epitopes SS2, SS6 and S1P3 represent a considerable immunodominant region of PEDV S protein.

In the case of PEDV, the S protein could not be cleaved into S1 and S2 subunits, due to a lack of proteolytic cleavage sites ([@bib8], [@bib19], [@bib26]). However, the S1 domain (residues 1--789) and the S2 domain (residues 790--1383) have been artificially defined by the conserved nonamer and the GxCx motifs at the position of S protein cleavage sites in coronavirus group II members ([@bib11]). Based on bio-informatics analysis of the S protein structure of other coronaviruses, the S1 domain is thought to form the spherical head of the S protein, and is easily accessible to neutralizing antibodies. The S2 domain forms the stem portion and trans-membrane structure of the S protein, and it seldom induces antibodies because of the inaccessibility of this region in intact virions ([@bib12], [@bib25], [@bib7]). The epitopes SS2 and SS6 are presumed to be located in the spherical head of the S protein of PEDV.

Recently, Chang reported one neutralizing epitope region (residues 499--638) on the S protein of PEDV ([@bib4]). In addition, Cruz recently identified a mocking epitope (^1368^GPRLQPY^1374^) that has antigenic similarities with PEDV neutralizing epitopes ([@bib6]). The epitopes SS2 and SS6 reported here are located in different regions of these epitopes, as identified formerly, and they represent two novel B cell antigenic epitopes of PEDV S protein. The results of this study will provide some basic information for further characterization of the antigenic structure of PEDV S protein, and identified epitopes have a potential use for developing diagnostic reagents and effective vaccines for PEDV.

We thank Prof. George P. Smith for provision of the phage display kit. This work was supported by a grant from the National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute (HVRI), The Chinese Academy of Agricultural Sciences (CAAS).

[^1]: *Note*: (+) sign denotes sense strands and (−) sign denotes anti-sense strands. At 5′ and 3′ terminal of each sense strand there is a sequence of "gatcc" (underlined) and "g" (shading), respectively. And at 3′ and 5′ terminal of anti-sense strand there is a sequence of "g" (shading) and "cagct" (underlined), respectively. When the sense and anti-sense oligo-nucleotides annealed they would form a cohesive BamHI site at 5′ terminus and a cohesive SalI site at 3′ terminus. At the end of mini-fragment encoding gene there is an additional taa sequence to form a stop codon (bold).

[^2]: Location of the polypeptides encoded by these mini-fragments based on the sequence of S protein of PEDV strain CV777 (GenBank accession no. [AF353511](AF353511)).

[^3]: Location of the synthesized peptides based on the sequence of S protein of PEDV strain CV777 (GenBank accession no. [AF353511](AF353511)).


Q:

iframe background image showing fine in Firefox but not in IE

Why IE not showing BG mage like firefox in Iframe?
I do not have access of iframed page.
any CSS or javascript solution

A:

As well as adding the CSS style background-color:transparent; to the iframe document's body element, you will also need to add the allowtransparency attribute to the iframe element in the containing document.
See http://msdn.microsoft.com/en-us/library/ms533072(VS.85).aspx for more information.
If you can't modify the iframe's document then you are out of luck.  Maybe there's another source you can use for the data that provides it in a different format such as XML or JSON?


Does this application include a request for confidentiality for any portion(s) of the data contained in this application pursuant to 47 CFR § 0.459 of the Commission Rules?:

Is the equipment in this application part of a system that operates with, or is marketed with, another device that requires an equipment authorization?

Enter any text that you would like to appear at the bottom of the Grant of Equipment Authorization:

If there is an equipment authorization waiver associated with this application, has the associated waiver been approved and all information uploaded?:

WILLFUL FALSE STATEMENTS MADE ON THIS FORM ARE PUNISHABLE BY FINE AND IMPRISONMENT (U.S. CODE, TITLE 18, SECTION 1001), AND/OR REVOCATION OF ANY STATION LICENSE OR CONSTRUCTION PERMIT (U.S. CODE, TITLE 47, SECTION 312(a)(1)), AND/OR FORFEITURE (U.S. CODE, TITLE 47, SECTION 503).

The applicant must certify that neither the applicant nor any party to the application is subject to a denial of Federal benefits, that include FCC benefits, pursuant to Section 5301 of the Anti-Drug Abuse Act of 1988, 21 U.S.C. § 862 because of a conviction for possession or distribution of a controlled substance. See 47 CFR 1.2002(b) for the definition of a "party" for these purposes.

Applicant/Agent Certification:

I certify that I am authorized to sign this application. All of the statements herein and the exhibits attached hereto, are true and correct to the best of my knowledge and belief. In accepting a Grant of Equipment Authorization as a result of the representations made in this application, the applicant is responsible for (1) labeling the equipment with the exact FCC ID specified in this application, (2) compliance statement labeling pursuant to the applicable rules, and (3) compliance of the equipment with the applicable technical rules. If the applicant is not the actual manufacturer of the equipment, appropriate arrangements have been made with the manufacturer to ensure that production units of this equipment will continue to comply with the FCC's technical requirements.

Authorizing an agent to sign this application, is done solely at the applicant's discretion; however, the applicant remains responsible for all statements in this application.

If an agent has signed this application on behalf of the applicant, a written letter of authorization which includes information to enable the agent to respond to the above section 5301 (Anti-Drug Abuse) Certification statement has been provided by the applicant. It is understood that the letter of authorization must be submitted to the FCC upon request, and that the FCC reserves the right to contact the applicant directly at any time.


# Configure the Google Cloud firewall
resource "google_compute_firewall" "default" {
  name        = "ngx-http-fw-rule"
  network     = "default"
  description = "Allow access to ports 80 and 8080 on all NGINX instances."
  allow {
    protocol = "tcp"
    ports = [
      "80",
      "8080",
    ]
  }
  source_ranges = [
    "0.0.0.0/0",
    "130.211.0.0/22",
    "35.191.0.0/16",
  ]
  target_tags = [
    "nginx-http-fw-rule",
  ]
}

# Create a static IP address in Google cloud
resource "google_compute_address" "default" {
  name = "ngx-network-lb-static-ip"
}

# Create a target pool to balance incoming connections to the load balancer instance group
resource "google_compute_target_pool" "default" {
  name = "ngx-network-lb-backend"
  health_checks = [
    google_compute_http_health_check.default.name,
  ]
  session_affinity = "CLIENT_IP"
}

# Forward incoming connections to port 80 to the Google network balancer
resource "google_compute_forwarding_rule" "port_80" {
  name       = "ngx-network-lb-frontend-80"
  target     = google_compute_target_pool.default.self_link
  ip_address = google_compute_address.default.address
  port_range = "80"
}

# Forward incoming connections to port 8080 to the Google network balancer
resource "google_compute_forwarding_rule" "port_8080" {
  name       = "ngx-network-lb-frontend-8080"
  target     = google_compute_target_pool.default.self_link
  ip_address = google_compute_address.default.address
  port_range = "8080"
}


Q:

"Line of sight" path across navigation mesh

I want to calculate the line of sight in a navigation mesh.
Consider the image below, the yellow line is the result of only A* and the red line is the result of a line of sight" algorithm that uses the yellow line as input. Now the unit can move directly without “zig-zagging”.
What is an algorithm to calculate that "line of sight"?

A:

You are looking for a funnel algorithm.
Here you are a simple one
http://digestingduck.blogspot.com.es/2010/03/simple-stupid-funnel-algorithm.html
Basically, the algorithm identify edges as portals, and build a funnel that is tested against the vertex of the edges to check if they are inside funnel or not.
In step A the funnel is built with the start position and the portal crossed by yellow line.
In step B the next portal is checked, the upper vertex is inside the funnel, so the funnel upper line now pass though it. But the bottom vertex is out of the funnel because red line in under green line, so bottom line will not pass though it, it will continue passing through the bottom vertex of previous portal.
As you can check the funnel will be smaller, and smaller, until step F, where the funnel is not possible to be build, because red line makes a bad  funnel, so the upper vertex is choosed as new start point and a new funnel would be build if the end point is not in that mesh.

Realize that this kind of algorithm lets a simple solution to the model size problem too, because you can consider that the portals are smaller by the 2xradius of your model.

A:

There's a simple technique that can be used with generated paths using this line of sight idea. Basically, you want to walk the path, and at each node, "look back" two the node before the last to see if it's visible. If the node before last is visible, you can remove the last node (since you have line of sight between your current node and the node before last, the last node, being an intermediate node, is not required).

A Gamasutra article has the following pseudo code example:
checkPoint = starting point of path
currentPoint = next point in path
while (currentPoint->next != NULL)
if Walkable(checkPoint, currentPoint->next)
// Make a straight path between those points:
temp = currentPoint
currentPoint = currentPoint->next
delete temp from the path
else
checkPoint = currentPoint
currentPoint = currentPoint->next

This algorithm does as you want, where the Walkable function is essentially a line-of-sight function, but slightly improved to also include situations where the path is visible, but not walkable (i.e. pits, traps, restricted zones).


Within every person lies a great story and behind each designer bag lies a fascinating story. Why is this bag created, how is it created and where does the inspiration come from?

The Le pliage is Longchamp’s secret weapon; it’s one of the few affordable and beautiful designer bags in the world, made from great craftsmanship and durable materials. But I have lost counts; I can’t remember how many versions of the Le Pliage bag exist today, here are a few more featured on our site – the Le Pliage Heritage Tote Bag, Le Pliage de Noel Bag, Le pliage Losange Tote or the Le Pliage Cuir Bag.

Now as an extension of the Le Pliage Collection, we present the Longchamp Le Pliage Neo Fantaisie Bags. And just like each Le Pliage Bag, there is a reason for its existence. The bag is not made from thin air, in fact it has been inspired by something.

The Le Pliage Neo Fantaisie Bag is characterized by two motifs representing the banks of the river Seine. La Rive Doite is a bold nail-motif print, demonstrating a look of the Colonnes de Buren, which is a contemporary art on the Place du Palais Royal.

Currently, there are three designs available; in beige with dots, in red and black. The shape is almost exactly like the original Le Pliage Tote bag, but the materials are stronger and more beautiful. Measuring 10’ x 9’ x 6,25’ inches,

The Le Pliage has been taken to the next level. Re-made, re-shaped and entire re-designed with new leathers and colors. The ultimate feminine tote bag with a touch of chic’ness, presenting the Longchamp Le Pliage Heritage Bag Ad Campaign.

Uniquely and practical – you can carry it over your shoulder, on your arm, cross body or in your hand, it’s all possible. The Le Pliage Heritage Collection bears the witness of Longchamp’s tradition and expertise in the field of leather craftsmanship.

Special attention has been invested in the details, like the slightly curved trompe-l’oeil flap and handles, exposing the sophistication and timeless elegance all over the design.

This post is the extension of Alexa Chung’s Ad Campaign featuring the Heritage Bag. It’s never too early to learn more about beautiful handbags. The vivid images are shown clearly, giving you a view of how it’s like to carry such beauty in your daily life.

Unlike the original Le Pliage Bag, the Heritage Bag holds shape. It’s crafted with the idea of creating more space than any woman needs. The signature Longchamp has been embellished on the front and it comes with a shoulder strap.

This is quite literally the most hideous bag I’ve seen in a long time. In fact, with its grotesquely mangled facial features complete with bloodshot eyes, bad teeth and huge bubbling warts, Jeremy Scott’s latest collaboration with Longchamp is just ugly.

Simply known as the Longchamp Le Pliage ‘Madballs’, it’s another collectible Le Pliage tote that fans will snap up in an instant, the roomy and lightweight canvas tote a boon for those who like over-shopping at the airport (I always pack one of these in case it happens to me, which is pretty often), or for the gym and quick weekend getaways. And like every Longchamp and Jeremy Scott collaboration, once they are sold out they’re gone, with something else completely fun and wacky when the new season rolls by.

One of the 2 designs available (the other one is even worse), only 15 pieces of each design will be available in Singapore, and with the launch date slated for mid-August, I’ll be back with another update, along with the price and which of the Longchamp boutiques you should specifically hit for this limited edition collab.

The Le Pliage Heritage line of bags is as illustrious and timeless as ever. It isn’t any wonder that the luxury house Longchamp is also banking on the said collection, because it really does rake in the dough, and why not? The designs, the artisan flair and quality materials used on these pieces surpass expectations and raise the bar in bag architecture.

The Le Pliage Heritage Luxe Shoulder Bag is another addition to this growing family, and it is indeed a stunning piece! A true must-have, this is a bolder, more fearless take on the classic design by the brand. It has a mix of leather and fur on its exterior, which is very elegant to look at. If you’re looking for a bag that’s bound to take you places, then this is it!

It comes with an adjustable shoulder strap, which allows you to let it sit beautifully on your shoulders. Also, for the organized diva, there’s more to take in – one front pocket, two small front flap pockets and one large zipped back pocket can be found inside.


Outfitted with an ultra modern technical setup and skilled team of professionals, we are engaged in providing seamless PICO PLC System. This is available in 120V/240V ac, 24V ac, 12V and 24V dc versions at comprehensive prices.


Praying for our Enemies Podcast

The main thing that I remembered from that prayer was the emotional aspect of it. All of the sudden, I began weeping for the woman as I prayed. I felt so much compassion for her. I felt sad for her, by the fact of what must she feel like on the inside to be so mean, and so rude and hateful, and at such an older age, too. It was sad really. I felt sorry for her. After I prayed, I just let it all go. It was like it was released and flooded out by the compassion.

One afternoon at work, I had a colleague stop by my classroom to visit me for the first time. I would see her at lunch as we ate with each other and other colleagues at times. I thought it was a nice surprise. I didn’t normally have visitors.

However, the reason for her visit was not a social call, it was a mission to collect gossip – and it was gossip on me. She was a forthright Russian woman who came fearlessly down to collect what she could. Bold. She came to confront me about what was being told about me.

She was there to find out if the rumors of me ratting out another colleague to administration for her smoking during her lunch break were true. I was disgusted and pissed-off at her coming to me with this. It was not true at all. I was bothered and disappointed about the whole thing, and I let her know it. I thought I was going to have a nice visitor, and I’m being ridiculed by people for something that never occurred. I expressed my disgust to her while also hoping to find out why I was being targeted, how did they pin me as the “rat-er” in this error of accusation?

I was piecing it all together in my mind as she spoke. I was also thinking of how I saw “the ratted-out” with these other woman with whom I used to have lunch when I was in a different area who I found were all of the sudden treating me poorly when I’d see them in the halls, for no apparent reason. These women were being mean to me with their looks and snippy remarks, and behaviors. I didn’t understand it, until this. I surmised that is why I was being treated rudely by them – these women had chosen to erroneously believe in what I’d done, and they were standing with their friend in solidarity against me. Oh well.

I never told that colleague out-rightly that I was innocent of what I was being accused. Perhaps I should have righted her, but I didn’t. All that I could think of was how ridiculous this whole thing was – it was all so high school. Pun intended. I was surprised that it was all going on.

I would let them keep their beliefs, at least for a while. If the woman wanted to know the truth, she could come to me herself. I let that go.

My colleague left with little more certainty than she had come with. I deliberately left her confused almost making it seem like I had done it because I refused to admit anything either way. I may have done the wrong thing there. Either way, they would likely have thought what they wanted to despite me telling them the truth.

I thought about the situation after she left. I was mad that I was being targeted unfairly, and being marked by their meanness, really. It was stupid, really, too. I don’t like injustices, either.

I then thought, too, about another injustice that was occurring simultaneously, again, toward me, in part. One of the women in the mix who’d been rude to me, was, separately, unfairly making these “rulings” about a going-away party she was hosting for another colleague. She was ostracizing from the party everyone who did not contribute to it financially. I didn’t even see the original email. She dis-invited all who did not contribute. I went to explain the situation to her in person. She ignored me. She was, at one time, so kind and respectful to me. I was figuring it was all a result of this rumor-mill, and was bothered by it.

I thought about it more after the colleague left. I was pissed about it. I did not like the unfair treatment I was getting, nor that colleague coming to my classroom like that bringing all that garbage. I was hurt and bothered. This was right on top of the emails from this rude colleague which compounded my “righteous anger.” I was feeling accursed by the damning.

As I thought about all that had happened, I was reeling. I was then reminded, quickly, of the duty of Christian’s to pray for their enemies. That was the thought that came to my mind. Pray for your enemies. I didn’t resist too much at all because that’s what we’re to do. There’s got to be some logic to it if we’re told to do it, right? So I, began to pray. That is the only reason I did it.

Jesus instructs Christians in Matthew 5:43 – 44: “You have heard that it was said, ‘Love your neighbor and hate your enemy.’ But I tell you: Love your enemies and pray for those who persecute you”.

The main thing that I remembered from that prayer was the emotional aspect of it. I recall that as I was praying, the only woman I prayed for was the one who’d sent me the emails who I felt really hurt by; the whole situation itself other than that seemed to fall by the wayside because it was all so trivial, despite it pissing me off.

All of the sudden, I began weeping for the woman as I prayed. I felt so much compassion for her. I felt sad for her, by the fact of what must she feel like on the inside to be so mean, and so rude and hateful to be that way toward me, and at such an older age, too. It was sad really. I felt sorry for her. After I prayed, I just let it all go. It was like it was released and flooded out by the compassion.

While I prayed, God handled it. I did my part, and actually got peace from it. I got some perspective, too. It was healing, my prayer, and all was well from then.

1 Peter 3:9 in another instruction by Jesus tells us: “Do not repay evil with evil or insult with insult, but with blessing, because to this you were called so that you may inherit a blessing.”

I got a blessing with my prayer for that woman that afternoon. I got compassion for her. I got free of the whole worry and “wrongs that were being done to me.” I was no longer angry or bothered, at all.

I eventually came clean sometime over that year to the woman who thought I ratted her out. I told her I never did it, and she accepted it. I just wanted her to know the truth.

I learned that my obedience to the instruction of praying for my enemies freed me up, and I also trust that it helped that woman, too. That’s how it works.

I believe we are to pray for others because their hearts aren’t right with God, and we can help them by praying that God change them to be more pleasing to Him, too. It’s important to remember too, that, according to Ephesians 6:12, ”We wrestle not against flesh and blood, but against principalities, against powers, against the rulers of the darkness of this world, against spiritual wickedness in high places.”

They clearly need help. That’s why they are acting that way. They are love-less in some area, right? According to scripture, however, it’s a spiritual issue. People who are hurting, hurt people. So, we can pray for them for Jesus to help them – to free them up from their loveless state – whatever it may be. They are not right and need help. We do too. If we take on that bitterness that ensues from wrongs committed because we do not understand, we may be subject to perpetuating and perhaps worsening the situation. We are to pray for our enemies, simply. We are also going to get a blessing by doing it because we become freed in it. At least that is my experience. We are free from our accusation and self-righteous judgement, maybe also resentment and hurt, as well.

I found a blessing in the prayer offering. I received compassion, replacing my indignation, and it freed me. I clearly needed a miracle too. A treasure of obedience is clearly a blessing, always. Thank you, Lord.

Thank you for helping me and this woman. Thank you, also, for letting me get a blessing for the obedience to your word. I got to experience the “why,” at least in part, of why we are to pray for our enemies. I’m grateful for the Holy Spirit for this experience for I believe He brought to my attention the thought to “Pray for my enemies.” I am grateful to Christ for helping me see His instruction in practice and the blessing that flowed from that. The bible says that grace and truth are from Jesus Christ. I believe by reverencing Him by praying the word, as He commanded, that He bestowed his grace upon me that day for, my colleague, in the form of compassion for her and the grace to let go of the hurt I felt from her and for giving me peace.

I am grateful to God for giving us Jesus, my brother, to have Him give us the words of life that lead us to walk in paths of righteousness. Thank you for showing me the blessing of praying for our enemies – a double win – and for always leading me in your ways. Thank you for helping me, and for helping my colleague.