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Product Features:-All cards in the set are in high gloss, including jerseys, autographs and parallels-2 Finite fabrics jersey cards per box on average (1:10)-1 Signature card per box on average-3 Rookies numbered to 275 or less per box on average | tomekkorbak/pile-curse-small | Pile-CC |
Q:
Hadoop Administration
I setup a Hadoop 1.2.x version, Two node Cluster. First Node (NameNode, Jobtracker) and Second Node (Secondary NameNode, Datanode, TaskTracker).
How do I Set the map and reduce slots to 4 and 2 respectively for each node
and How do I verify the same from command line.
A:
To set the slots per tasktracker node, limit the maximum number of tasks a node can run simultaneously.
Add these properties to mapred-site.xml
<property>
<name>mapred.tasktracker.map.tasks.maximum</name>
<value>4</value>
</property>
<property>
<name>mapred.tasktracker.reduce.tasks.maximum</name>
<value>2</value>
</property>
You can check the configurations used by jobtracker using
hadoop jobtracker -dumpConfiguration
| tomekkorbak/pile-curse-small | StackExchange |
This needs to be said. The following is not a Pro-Trump article; it is an article to gain some perspective.
The people losing their minds on social media over Trump as President are some of the most lost/spoiled people on the planet.
And they don’t even know it.
These people have obviously never faced a real problem in their lives, which is why they are so worked up about a non-issue.
Let me be clear… anything that you can address by sitting at home whining on your expensive computer on a social media platform is NOT a REAL problem. You are just playing a team sport like fantasy football.
Access to running water/ food… THOSE are real problems.
Right now, as I write this, there are children in developing countries who just arrived at school after starting a 5-HOUR journey at 3AM.
I am not writing that metaphorically. I personally know people who work with these children. Again… these are CHILDREN who wake up at 3AM, walk 5 hours to school because there isn’t another school closer… and then walk back home again.
Do you have a 10-hour commute?
Do you have to walk those 10 hours through mountains or jungle where there are people who could kill or rape you any day of the week?
There are well over 1 billion people in the world who have REAL problems and could use help. The people losing their minds on social media all have resources/ talent/ and energy to help solve these problems.
But they are not.
Instead they are ranting about how “awful” it is in the US.
WAKE. THE. HELL. UP.
Here’s something that could help you get over Trump’s Presidency… looking around at your house which has running water and your fridge which has food in it.
How about looking at your roof/ the walls of your house that stop the weather from washing your home away when it rains.
You want to see real problems? Look at what just happened in Peru a few weeks ago. Flash floods have literally destroyed some people’s entire lives.
Watch the below video of a woman emerging from a mudslide which dragged her for miles.
THAT is a real problem. Not the fact that your particular candidate didn’t win an election.
People all over the world need help. What are you doing to help them? Anything? | tomekkorbak/pile-curse-small | OpenWebText2 |
Introduction
============
In patients with medial knee osteoarthritis with varus deformity, high tibial osteotomy (HTO) provides excellent results[@b1-ksrr-28-283]--[@b4-ksrr-28-283]. Pre-operative planning for appropriate correction gap and angle has a significant impact on the outcomes of HTO[@b5-ksrr-28-283]--[@b7-ksrr-28-283]. One method used for preoperative planning in HTO requires an intraoperative assessment of the lower limb mechanical axis with a radiopaque rod or an electrocautery cord (cable method). Another method involves an assessment of preoperative full-length weight bearing lower extremity radiographs with picture archiving and communication system (PACS)[@b8-ksrr-28-283] or software, such as PreOPlan (Siemens, Munich, Germany/Synthes, Oberdorf, Switzerland) or mediCAD (Hectec GmbH, Altfraunhofen, Germany)[@b9-ksrr-28-283],[@b10-ksrr-28-283]. The cable method allows for real-time monitoring, but it can lead to unsatisfactory results due to the non-weight bearing status during surgery and increase radiation exposure of the surgeon and patient. In contrast, the Miniaci method using PACS calculates the correction angle and gap in weight bearing status, and thus more accurate values can be obtained with less radiation exposure without the need to detect the hip and ankle joint centers.
We hypothesized that correction of an angular deformity based on the weight bearing line would be more accurate and that the extent of preoperative deformity would affect postoperative correction. The purpose of this study was to compare the accuracy of the cable method and Miniaci method using a PACS in determining the correction angle in open wedge high tibial osteotomy (OWHTO) and to evaluate the correlation between the preoperative severity of varus deformity and outcomes.
Materials and Methods
=====================
1. Study Design and Patient Selection
-------------------------------------
This retrospective study analyzed 47 consecutive patients (52 knees) with varus deformity and medial knee osteoarthritis. From January 2007 to March 2013, patients underwent OWHTO using either the cable method (cable group: 20 patients \[20 knees\]) or the Miniaci method using PACS images (PACS group: 27 patients \[32 knees\]). The male-to-female ratio and mean age of patients were similar between groups ([Table 1](#t1-ksrr-28-283){ref-type="table"}). Patients underwent medial OWHTO with the Aescula plate (B. Braun Korea, Seoul, Korea) in the cable group and the TomoFix plate (Synthes, Solothan, Swiss) in the PACS group. The inclusion criteria were as follows: 1) \<65 years of age, 2) body mass index (BMI) of \<30 kg/m^2^, 3) pain in the medial side only, 4) high level of activities except for running and jumping, 5) varus angle of \<15°, and 6) knee flexion of \>90°. The exclusion criteria were as follows: 1) flexion contracture of \>25°, 2) bicompartmental disease, 3) elderly patients with difficulty in mobility, 4) BMI of \>30 kg/m^2^, and 5) subtotal or total meniscectomy in the compartment near the planned osteotomy site. Patients who underwent a partial meniscectomy were not excluded.
2. Preoperative Planning and Surgical Techniques
------------------------------------------------
In the preoperative planning stage, anteroposterior full-length lower limb weight bearing radiography was performed in both groups with a goal of achieving the target alignment passing through the Fujisawa point[@b11-ksrr-28-283]; that is, realigning the mechanical axis of the limb to be located at the 62.5% point from the medial border along the longest medial-to-lateral width of the tibial plateau. Both groups underwent diagnostic knee arthroscopy prior to osteotomy to verify conditions of the medial, lateral, and patellofemoral articular surfaces, which was followed by debridement.
The superficial medial collateral ligament and pes anserinus were completely separated in both groups. In the cable group, a biplanar osteotomy was performed behind the tibial tuberosity. Then, the posteromedial tibial osteotomy site was opened using a chisel and a bone spreader, and the hip and ankle centers were connected using an electrocautery cord under fluoroscopic guidance. The osteotomy site was spread until the electrocautery cord could be placed at the target point on the medial-to-lateral tibial plateau of the knee joint while applying axial load at the foot sole. Then, the metal plate was fixed ([Fig. 1](#f1-ksrr-28-283){ref-type="fig"})[@b1-ksrr-28-283]. The PACS group underwent anteroposterior full-length lower limb radiography with valgus stress applied to the bilateral knee joints under weight bearing condition in order to correct widening of the lateral joint space induced by ligament laxity. Using the preoperative radiograph, the lower limb weight bearing line was drawn (line 1, S). After calculating the 62.5% point from the medial border along the longest medial-to-lateral width of the tibial plateau, an extension line connecting the hip center and the target point (line 2, S′) was drawn. Then, a line connecting the lateral tibial osteotomy site (D) and the center of the ankle joint (line 3, DS) and a line connecting the osteotomy site and S′ (line 4, DS′) were drawn ([Fig. 2A](#f2-ksrr-28-283){ref-type="fig"}). The angle formed by lines 3 and 4 was determined to be the predicted correction wedge angle (α). A predicted osteotomy line (O) was drawn from the proximal extremity of the fibular head to the predicted medial osteotomy site (approximately 4 cm inferior to the medial border of the tibial plateau), and a predicted opening line (O′) was drawn from Line O at the determined correction angle (wedge angle, α). Using the triangle formed, we measured the predicted correction gap (wedge gap, mm) at the cortical bone of the posteromedial tibia ([Fig. 2B and C](#f2-ksrr-28-283){ref-type="fig"})[@b12-ksrr-28-283]. After performing an osteotomy using the same surgical technique as in the cable group, we spread the osteotomy site matching the predicted wedge angle and gap and fixed the metal plate. Allogeneic bone and autologous bone marrow harvested from the anterior superior iliac spine on the same side were mixed and grafted onto the bone defect. The grafted site was covered with the superficial medial collateral ligament, and the pes anserinus was resutured to the periosteal membrane. An active exercise program for joint rehabilitation began in the second postoperative week. The Aescula plate group (cable group) and TomoFix plate group (PACS group) were allowed to commence body weight bearing in the sixth and second postoperative weeks, respectively.
3. Evaluation and Measurement
-----------------------------
The full-length lower limb radiographs obtained preoperatively and at the sixth postoperative week were used to compare the percentage of crossing point of the weight bearing line on the tibial plateau with respect to the medial border. The acceptable postoperative range was set at 62%±5% (range, 57% to 67%), and percentages lower or higher than this range were defined as overcorrection or undercorrection, respectively[@b13-ksrr-28-283]. Pre- and postoperative mechanical femorotibial angles (mFTA) and posterior tibial slope angles were also compared[@b14-ksrr-28-283]. In the PACS group, the preoperatively predicted and postoperatively measured wedge angles and gaps were compared. The wedge gaps were measured as the distance between line O and line O′ at the medial cortex, and the wedge angles as the angle formed by the two lines.
This study was approved by the Institutional Review Board of our institution (no. 2014-09-037-001).
4. Statistical Analysis
-----------------------
A two-sample *t*-test was used to compare the mean values of age, weight bearing line, and mFTA between groups. Chi-square analysis was used to compare the categorical variables between the two groups. Logistic regression analysis was used to evaluate the relationship between the preoperative severity of varus deformity and the outcome of correction based on the location of the weight bearing line on the tibial plateau. Radiographic measurements were performed by two observers. The correlation coefficient between the two observers was 0.79 when calculated by Spearman's rho test. The mean values of two measurements were used for analysis.
Statistical significance was assumed at p\<0.05. All statistical analyses were performed with SPSS ver. 19.0 (IBM Co., Armonk, NY, USA).
Results
=======
The weight bearing line on the tibial plateau was corrected from a preoperative mean of 11.0%±7.0% to a postoperative mean of 47.2%±7.4% in the cable group and from a mean of 12.7%±4.9% to a mean of 59.5%±5.3% in the PACS group (p=0.018). The ratio of undercorrection, acceptable range, and overcorrection was 8:11:1 in the cable group and 3:23:6 in the PACS group. The Miniaci method using PACS images resulted in a significantly higher incidence of correction in the acceptable range than did the cable method (p=0.021) ([Table 1](#t1-ksrr-28-283){ref-type="table"}).
The mFTA was corrected from a preoperative mean of varus 8.9°±3.7° to a postoperative mean of valgus 0.3°±4.0° in the cable group and from a mean of varus 9.0°±3.3° to a mean of valgus 2.9°±2.6° in the PACS group. The postoperative difference between the two groups was significant (p=0.017). The posterior tibial slope angle was increased from 9.5°±2.3° to 10.4°±2.2° (p\<0.001) in the cable group and from 9.8°±2.5° to 10.5°±2.3° (p\<0.001) in the PACS group. The preoperative and postoperative differences were not significant (p=0.675 and p=0.869, respectively) ([Table 1](#t1-ksrr-28-283){ref-type="table"}).
In the PACS group, 18 cases with undercorrection fell short of the target angle by a mean of varus 1.8°±1.9° while 14 cases with overcorrection exceeded the target angle by a mean of valgus 2.6°±2.0°. The overall differences between the preoperatively planned and postoperatively achieved wedge gaps and angles by absolute values were a mean of 3.6±5.2 mm and 2.1°±1.9°, respectively ([Table 2](#t2-ksrr-28-283){ref-type="table"}). There was no significant correlation between the preoperative severity of varus deformity and postoperative outcomes based on the location of the weight bearing line on the tibial plateau (p=0.477).
Discussion
==========
In this study, correction of angular deformity by the Miniaci method using PACS images was more accurate than correction by the cable method in OWHTO.
For osteotomy aimed at mechanical axis realignment, preoperative correction planning is of pivotal importance for successful surgical outcome[@b15-ksrr-28-283],[@b16-ksrr-28-283]. Many authors have presented various pre-operative planning modalities[@b12-ksrr-28-283],[@b17-ksrr-28-283]--[@b19-ksrr-28-283]. Still, the radiopaque line is frequently employed for intraoperative guidance. This method is likely to lead to an unacceptable range of correction after surgery and increase radiation exposure of both the surgeon and patient due to the increased duration of radiography for identifying the hip and ankle joint centers and for verifying the degree of correction. A navigation-based technique has recently been reported, and its accuracy and superiority have been confirmed in clinical trials[@b20-ksrr-28-283]--[@b23-ksrr-28-283]. However, this method involves the use of navigation devices and is applied intraoperatively under non-weight bearing condition, which can yield deviating correction angles after surgery under weight bearing condition. Apart from this accuracy issue, controversy surrounds the cost-effectiveness of the method, which was kindled by research suggesting it has no clinical advantage over conventional methods[@b24-ksrr-28-283].
Preoperative planning can be implemented based on the anatomic axis[@b1-ksrr-28-283], mechanical axis[@b6-ksrr-28-283],[@b12-ksrr-28-283], or the weight bearing line[@b8-ksrr-28-283],[@b10-ksrr-28-283]. The weight bearing line can reflect instability around the knee joint and deviate from the mechanical axis. As demonstrated by the results of this study, significant differences were observed between the cable-based method involving intraoperative fluoroscopic guidance using a radiopaque line and the PACS image-based preoperative planning. The cable group tended to obtain an unacceptable correction because the mechanical axis was measured in a non-weight bearing supine position, whereas the PACS image-based method used the weight bearing line assessed in upright position as a reference. Differences in the mechanical axis between radiographs obtained in preoperative upright (weight bearing) position and those in supine position have been reported to be ≥3°[@b25-ksrr-28-283], presumably due to differences in the knee joint position or lateral laxity under weight bearing condition. To correct such deviations, a valgus stress can be applied during pre-operative anteroposterior full-length lower limb radiography[@b13-ksrr-28-283]. In this study, the cable group had a high ratio of undercorrection, whereas Lee and Moon[@b26-ksrr-28-283] reported the cable method may result in rather overcorrected alignment because the MCL is measured in a state of relaxation. In the current study, axial pressure was applied to the foot sole for simulating the weight bearing state during operation performed using the cable method. This caused decreased lateral laxity, resulting in undercorrection than pre-operatively planned. However, when weight bearing was applied postoperatively, lateral laxity recurred due to muscular action. Moreover, limb rotation during intraoperative fluoroscopy may have resulted in the variation on postoperative weight bearing radiographs and the wide deviation of the correction angle from the target angle in the cable group.
Only 23 cases (71.8%) in the PACS group had the weight bearing line within the acceptable range because the radiographs were not obtained from the same fixed position and differences in bone quality may have contributed to undercorrection during intraoperative gap spreading. Also, the thickness of the saw blade could have affected postoperative undercorrection.
This study had some limitations. First, the number of cases was small. Second, the study did not investigate clinical outcomes. Therefore, a clinical study should be conducted in a large series. Radiographs were not obtained from the same fixed position, which could have undermined the reliability of radiographic measurements considering this can influence postoperative radiographic values. Finally, different plates were used in the two groups. However, taking consideration into the difference in implant stability, weight bearing was allowed at 6 weeks after surgery in the cable group and at 2 weeks after surgery in the PACS group; therefore, the type of plate might not have much influence on correction loss at the time of the sixth postoperative week radiographic evaluation.
Conclusions
===========
In OWHTO, correction of angular deformity based on the Miniaci method using a PACS was more accurate than correction using the cable method. Also this method can be expected to involve less intraoperative radiation exposure. There was no significant correlation between the preoperative severity of varus deformity and outcomes based on the location of the weight bearing line on the tibial plateau. Therefore, the Miniaci method using PACS can be useful in preoperative planning for OWHTO.
**Conflict of Interest**
No potential conflict of interest relevant to this article was reported.
{#f1-ksrr-28-283}
{#f2-ksrr-28-283}
######
Comparative Results of Cable Method and Miniaci Method Using PACS
Variable Cable method Miniaci method using PACS p-value
------------------------- ---------------- --------------------------- ---------
No. of knees 20 32
Age (yr) 55.2±7.7 55±3.9 0.912
Sex (M:F) 5:15 5:27 0.404
Weight bearing line (%)
Preoperative 11.0±7 12.7±4.9 0.511
Postoperative 47.2±7.4 59.5±5.3 0.018
Weight bearing line
Acceptable 11 23 0.021
Undercorrection 8 3
Overcorrection 1 6
mFTA (°)
Preoperative Varus 8.9±3.7 Varus 9.0±3.3 0.213
Postoperative Valgus 0.3±4.0 Valgus 2.9±2.6 0.017
PTS angle (°)
Preoperative 9.5±2.3 9.8±2.5 0.675
Preoperative 10.4±2.2 10.5±2.3 0.869
Values are presented as mean±standard deviation or number.
PACS: picture archiving and communication system, mFTA: mechanical femorotibial angle, PTS: posterior tibial slope.
######
Difference between Preoperatively Predicted and Postoperatively Measured Wedge Angles and Gaps in the PACS Group
Variable Wedge angle (°) Wedge gap (mm)
---------------------------------------------------------- ----------------- ----------------
Undercorrection (\<62.5%, n=18) Varus 1.8±1.9 Varus 3.0±2.3
Overcorrection (\>62.5%, n=14) Valgus 2.6±2.0 Valgus 4.4±7.5
Total[a)](#tfn5-ksrr-28-283){ref-type="table-fn"} (n=32) 2.1±1.9 3.6±5.2
Values are presented as mean±standard deviation.
PACS: picture archiving and communication system.
The overall differences of absolute values.
| tomekkorbak/pile-curse-small | PubMed Central |
Cellular and wireless communication technologies have seen explosive growth over the past several years. This growth has been fueled by better communications, hardware, larger networks, and more reliable protocols. Wireless service providers are now able to offer their customers an ever-expanding array of features and services, and provide users with unprecedented levels of access to information, resources, and communications. To keep pace with these service enhancements, mobile electronic devices (e.g., cellular phones, tablets, laptops, etc.) have become more powerful and complex than ever. With this increase in processing speed, usage, and performance, there is an increased demand for improving the efficiency and processing speed of the mobile devices, which may be achieved via faster and more sophisticated compilers and compiling frameworks.
Generally, application software written for mobile computing devices is compiled into executable code which is what users refer to as “applications.” Developers typically use a compiler to translate code written in higher level programming languages into executable code suitable for target mobile computing devices. Application software may be distributed as pure executable code, in which case the compiling step is typically performed by the developer on what is referred to as a “static compiler.” Application software may also be written so portions of the code are compiled at runtime using software modules within the mobile computing device referred to as a “runtime compiler.” Runtime compilers (e.g., dynamic compilers or Just in Time compilers) translate segments of code into executable native code (e.g., machine code) at the time the code is executed. | tomekkorbak/pile-curse-small | USPTO Backgrounds |
1. Introduction
===============
The amino acid glutamine (GLN) is involved in many processes that are vital to cell function \[[@B1-nutrients-06-04520]\]. The molecular mechanisms of GLN's actions are not entirely clear, but they undoubtedly involve changes in gene and protein expression, protein activity, and intracellular metabolite concentrations \[[@B1-nutrients-06-04520],[@B2-nutrients-06-04520]\]. Enteral and parenteral administration of GLN offers nutritional benefits for critically ill patients and is recommended for use in this population \[[@B3-nutrients-06-04520],[@B4-nutrients-06-04520],[@B5-nutrients-06-04520]\]. Additionally, oral GLN has been used by healthy individuals, particularly athletes, to maintain immune function \[[@B6-nutrients-06-04520],[@B7-nutrients-06-04520],[@B8-nutrients-06-04520]\].
Although oral GLN supplementation offers potential benefits, its low solubility and stability in aqueous solutions limits its blood availability \[[@B9-nutrients-06-04520]\]. Furthermore, approximately 50% of orally administered GLN is extracted by the splanchnic bed in healthy humans \[[@B10-nutrients-06-04520]\]. However, this limitation can be overcome with the use of synthetic, stable, highly soluble glutamine dipeptide (GDP), a dipeptide composed of alanine (ALA) and GLN \[[@B11-nutrients-06-04520],[@B12-nutrients-06-04520]\].
However, the performance of oral GDP in terms of blood concentration compared to oral GLN is controversial. Few studies have investigated the pharmacokinetic responses to oral doses of GDP \[[@B13-nutrients-06-04520],[@B14-nutrients-06-04520],[@B15-nutrients-06-04520]\].
A proper evaluation of the efficacy of GDP compared to GLN requires controlled conditions \[[@B16-nutrients-06-04520]\]. Fasted rats submitted to insulin-induced hypoglycemia (IIH) represent a suitable experimental model to evaluate changes in blood levels of amino acids \[[@B17-nutrients-06-04520],[@B18-nutrients-06-04520]\]. Therefore, we compared the effects of oral GDP, GLN plus ALA, GLN, or ALA on the blood availability of amino acids in hypoglycemic rats.
2. Experimental Section
=======================
2.1. Chemicals
--------------
Insulin detemir (Levemir^®^) was obtained from Novo Nordisk (São Paulo, SP, Brazil). GDP was purchased from Ajinomoto (Ajinomoto North America, NC, USA). GLN and ALA were obtained from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). All other reagents were of the highest purity obtainable.
2.2. Animals
------------
A total of 214 male Wistar rats, weighing approximately 250 g, were housed under a controlled 12 h light/dark cycle and a temperature of 23°C ± 2°C. All rats were fasted for 15 h before initiating the experiments. The experimental protocol was approved by the Ethics Committee of State University of Maringá, Brazil and was in accordance with international law on the protection and use of animals.
2.3. Experimental Protocol
--------------------------
We produced IIH in the rats by the intraperitoneal (ip) injection of insulin detemir (1 U/kg). We chose the dose of insulin on the basis of a previous study \[[@B18-nutrients-06-04520]\]. The insulin was not diluted, and it was injected with an infusion pump. Next, the rats received an oral administration (gavage) of GDP, ALA + GLN, GLN, or ALA immediately after ip insulin injection. An additional control group which received ip saline + oral vehicle was included. The control group furnishes the values before the administration of insulin and the test substances.
All rats were anesthetized with an ip injection of sodium thiopental (45 mg/kg). We collected blood from the vena cava to measure levels of blood glucose \[[@B19-nutrients-06-04520]\] and amino acids \[[@B20-nutrients-06-04520]\].
2.4. Measurement of Blood Glucose and Amino Acid Levels after Oral Administration of Low-Dose GDP (100 mg/kg), GLN (61.9 mg/kg) + ALA (38.1 mg/kg), GLN (61.9 mg/kg), or ALA (38.1 mg/kg)
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
An oral vehicle (IIH + oral vehicle), GDP (IIH + oral GDP), GLN + ALA (IIH + oral GLN + oral ALA), GLN (IIH + oral GLN) or ALA (IIH + oral ALA) was administered to the rats by gavage immediately after ip injection of insulin. Blood was collected 15 min ([Table 1](#nutrients-06-04520-t001){ref-type="table"}), 30 min ([Table 2](#nutrients-06-04520-t002){ref-type="table"}), and 120 min ([Table 3](#nutrients-06-04520-t003){ref-type="table"}) after oral administration to measure glucose and amino acid levels. The dose of GDP was based on a previous study \[[@B18-nutrients-06-04520]\], and the doses of ALA and GLN reflected their proportions in the GDP molecule. An additional control group received ip saline + oral vehicle.
nutrients-06-04520-t001_Table 1
######
Blood glucose (mg/dL) and amino acid (nmol/mL) levels 15 min after intraperitoneal insulin injection (IIH group). The IIH group was divided into 5 subgroups: IIH + oral vehicle (VHC), IIH + oral glutamine dipeptide (GDP), IIH + oral glutamine (GLN) + oral alanine (ALA), IIH + oral GLN or IIH + oral ALA. GDP (100 mg/kg**)**, GLN (61.9 mg/kg) + ALA (38.1 mg/kg), GLN (61.9 mg/kg) or ALA (38.1 mg/kg) or VHC was administered immediately after insulin injection. The Control group received intraperitoneal saline + oral VHC. Data expressed as means ± standard error were analyzed by ANOVA (Newman-Keuls *post hoc* test). **^a^** *p*\< 0.05 *vs.* Control and **^b^** *p*\< 0.05 *vs.* IIH + oral GDP.
Control (*n* = 4) IIH + VHC (*n* = 5) IIH + GDP (*n* = 5) IIH+ GLN + ALA (*n* = 4) IIH + GLN (*n* = 5) IIH + ALA (*n* = 4)
--------------- ------------------- --------------------- --------------------- -------------------------- --------------------- ---------------------
Glucose 108.9 ± 9.0 86.2 ± 8.2 93 ± 3.0 94.2 ± 8.5 89.3 ± 3.2 90.2 ± 3.4
Alanine 704.3 ± 45.0 545.3 ± 66.7 781.1 ± 140.7 551 ± 81.2 556.6 ± 19.7 572.9 ± 89.3
Arginine 68.2 ± 19.0 26.5 ± 0.4 **^ab^** 79.5 ± 5.7 51.8 ± 14.5 57 ± 14.2 32.5 ± 2.1 **^b^**
Glutamine 844.4 ± 104.0 694.9 ± 101.2 660.8 ± 134.6 575.3 ± 121.6 775.3 ± 69.3 565.1 ± 58.2
Histidine 91 ± 1.3 90.9 ± 4.3 91.9 ± 4.0 90.3 ± 6.0 90.5 ± 6.6 82.3 ± 5.4
Isoleucine 114.5 ± 10.2 81.3 ± 10.8 114.5 ± 10.2 76.3 ± 11.0 108.6 ± 6.1 80 ± 5.9
Leucine 222 ± 16.3 165.4 ± 21.2 210.7 ± 26.6 172 ± 30.2 193.9 ± 12.2 164.1 ± 13.9
Metionine 62.6 ± 10.8 53.4 ± 6.6 63.8 ± 5.3 49.2 ± 11.2 53.7 ± 9.2 46.7 ± 3.2
Phenylalanine 94 ± 9.4 80.5 ± 9.2 91.6 ± 12.5 73 ± 11.7 87 ± 5.2 70.6 ± 3.2
Tryptophan 107.7 ± 6.8 117.9 ± 9.1 123.5 ± 15.1 94.9 ± 17.8 111.9 ± 12.9 89.1 ± 6.9
Tyrosine 111.7 ± 22.0 88.2 ± 5.6 95.9 ± 9.6 91 ± 6.8 97.6 ± 7.7 86.4 ± 10.8
Valine 255.6 ± 41.0 207.5 ± 29.7 270.5 ± 27.5 203.4 ± 31.8 263.7 ± 17.7 186.1 ± 15.7
nutrients-06-04520-t002_Table 2
######
Blood glucose (mg/dL) and amino acid (nmol/mL) levels 30 min after intraperitoneal insulin injection (IIH group). The IIH group was divided into 5 subgroups: IIH + oral vehicle (VHC), IIH + oral glutamine dipeptide (GDP), IIH + oral glutamine (GLN) + oral alanine (ALA), IIH + oral GLN or IIH + oral ALA. GDP (100 mg/kg), GLN (61.9 mg/kg) + ALA (38.1 mg/kg), GLN (61.9 mg/kg) or ALA (38.1 mg/kg) or VHC. The Control group received intraperitoneal saline + oral VHC. Data expressed as means ± standard error were analyzed by ANOVA (Newman-Keuls *post hoc* test).
Control (*n* = 4) IIH + VHC (*n* = 5) IIH + GDP (*n* = 5) IIH+ GLN + ALA (*n* = 4) IIH + GLN (*n* = 5) IIH + ALA (*n* = 4)
--------------- ------------------- --------------------- --------------------- -------------------------- --------------------- ---------------------
Glucose 105.9 ± 15.1 71.6 ± 21.3 82.5 ± 21.5 74.0 ± 14.9 68.6 ± 14.9 80.8 ± 10.6
Alanine 272.5 ± 94.4 243.9 ± 55.1 260.8 ± 60.7 304.9 ± 77.9 250.7 ± 31.0 287.5± 86.0
Arginine 66.8 ± 22.6 51.3 ± 19.6 49.2 ± 19.5 54.9 ± 15.1 46.6 ± 12.5 46.7 ± 12.4
Glutamine 367.9 ± 77.3 305.8 ± 57.5 282.3 ± 57.1 339.0 ± 68.8 327.7 ± 76.0 325.2 ± 85.0
Histidine 85.3 ± 7.9 90.2 ± 7.8 88.9 ± 9.5 88.3 ± 5.5 90.6 ± 8.2 91.4 ± 7.2
Isoleucine 83.7 ± 16.5 59.9 ± 17.0 54.6 ± 13.5 52.2 ± 14.2 48.3 ± 13.7 51.0 ± 9.0
Leucine 157.8 ± 33.0 115.5 ± 37.2 103.7 ± 19.9 104.7 ± 22.6 95.1 ± 19.9 101.0 ± 18.5
Metionine 37.3 ± 12.9 35.1 ± 3.4 33.9 ± 5.5 36.8 ± 6.5 37.0 ± 4.2 35.9 ± 11.9
Phenylalanine 69.2 ± 15.2 60.0 ± 12.8 54.3 ± 7.1 59.7 ± 11.0 56.1 ± 8.4 55.1 ± 11.4
Tryptophan 60.3 ± 19.9 76.2 ± 12.3 58.9 ± 11.2 60.9 ± 12.0 74.7 ± 13.0 60.8 ± 19.7
Tyrosine 77.8 ± 13.3 73.3 ± 16.7 74.5 ± 18.6 75.5 ± 18.7 73.0 ± 22.5 83.0 ± 16.5
Valine 180.8 ± 32.8 145.9 ± 34.7 134.7 ± 24.9 140.7 ± 23.5 127.4 ± 19.2 131.6 ± 15.7
nutrients-06-04520-t003_Table 3
######
Blood glucose (mg/dL) and amino acid (nmol/mL) levels 120 min after intraperitoneal insulin injection (IIH group). The IIH group was divided into 5 subgroups: IIH + oral vehicle (VHC), IIH + oral glutamine dipeptide (GDP), IIH + oral glutamine (GLN) + oral alanine (ALA), IIH + oral GLN or IIH + oral ALA. GDP (100 mg/kg), GLN (61.9 mg/kg) + ALA (38.1 mg/kg), GLN (61.9 mg/kg), or ALA (38.1 mg/kg) or VHC was orally administered. The Control group received intraperitoneal saline + oral VHC. Data expressed as means ± standard error were analyzed by ANOVA (Newman-Keuls *post hoc* test). **^a^***p*\< 0.05 *vs.* Control and **^b^** *p*\< 0.05 *vs*. IIH + oral GDP.
Control (*n* = 4) IIH + VHC (*n* = 5) IIH +GDP (*n* = 5) IIH+ GLN + ALA (*n* = 4) IIH + GLN (*n* = 5) IIH + ALA (*n* = 4)
--------------- ------------------- --------------------- ---------------------- -------------------------- --------------------- ---------------------
Glucose 87.9 ± 4.3 18.0 ± 1.7 **^a^** 20.8 ± 7.4 **^a^** 33.3 ± 17.0 **^a^** 23.2 ± 5.9 **^a^** 15.0 ± 1.4 **^a^**
Alanine 607.8 ± 39.8 457.6± 31.3 353.0 ± 33.3 470.4 ± 97.6 464.8 ± 36.7 506.9± 45.5
Arginine 111.8 ± 26.4 103.1± 12.2 101.7 ± 1.9 97.9 ± 18.2 102.9 ± 17.0 94.4 ± 16.5
Glutamine 871.7 ± 93.2 613.3± 38.0 463.7± 68.1 **^a^** 647.5 ±127.9 516.0 ±87.2 **^a^** 459.2 ±65.8 **^a^**
Histidine 88.6 ± 3.3 93.5 ± 4.9 85.3 ± 6.6 85.2 ± 3.7 88.8 ± 2.5 93.5 ± 1.6
Isoleucine 109.1 ± 6.3 58.2 ± 5.6 **^a^** 59.5 ± 6.1 **^a^** 73.7 ± 19.4 **^a^** 53.34 ± 4.4 **^a^** 62.5 ± 2.3 **^a^**
Leucine 196.3 ± 10.9 124.8± 9.6 **^a^** 118.5± 10.3 **^a^** 130.4 ±27.8 **^a^** 109.5 ± 8.9 **^a^** 136.4± 12.5 **^a^**
Metionine 54.1 ± 6.0 38.4 ± 4.0 23.2 ± 3.9 **^a^** 43.9 ± 9.4 **^b^** 45.1 ± 3.7 **^c^** 33.0 ± 3.6
Phenylalanine 85.0 ± 3.4 82.9 ± 4.4 69.8 ± 5.5 80.9 ± 5.6 75.8 ± 3.7 83.7 ± 5.7
Tryptophan 99.0 ± 6.4 175.1±20.5 **^a^** 121.5 ± 11.4 139.2 ± 19.4 156.2 ± 14.0 145.2 ± 11.6
Tyrosine 110.2 ± 10.7 99.7 ± 8.7 99.9 ± 2.4 96.6 ± 15.0 95.7 ± 12.4 109.0 ± 4.2
Valine 295.7 ± 18.9 164.5±10.1 **^a^** 152.4 ± 16.4 **^a^** 196.9 ±34.2 **^a^** 160.3 ±13.4 **^a^** 185.4 ± 6.3 **^a^**
2.5. Measurement of Blood Glucose and Amino Acid Levels after Oral Administration of High-Dose GDP (1000 mg/kg), GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg), or ALA (381 mg/kg)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
An oral vehicle (IIH + oral vehicle), GDP (IIH + oral GDP), GLN + ALA (IIH + oral GLN + oral ALA), GLN (IIH + oral GLN) or ALA (IIH + oral ALA) was administered to the rats by gavage immediately after ip insulin injection. Blood was collected 15 min ([Table 4](#nutrients-06-04520-t004){ref-type="table"}) and 30 min ([Table 5](#nutrients-06-04520-t005){ref-type="table"}) after oral administration to measure glucose and amino acid levels. An additional control group received ip saline + oral vehicle. The doses of ALA and GLN reflected their proportions in the GDP molecule.
2.6. Liver Perfusion Experiments
--------------------------------
The hepatic catabolism of GDP, GLN, and ALA, inferred from liver glucose and urea formation were evaluated. Liver perfusion experiments were conducted in an open system without recirculation of perfusate, as previously described \[[@B17-nutrients-06-04520]\]. The rats were anesthetized with an ip injection of sodium thiopental (45 mg/g). The abdomen was opened by midline incision and a cannula was inserted into the portal vein.
The perfusion fluid, Krebs-Henseleit buffer (KHB; pH 7.4), which was saturated with an oxygen/carbon dioxide (95%/5%) mixture, was pumped (4 ml/g·min of liver weight) through a temperature-regulated (37 °C) membrane oxygenator prior to entering the liver via the cannula inserted in the portal vein.
nutrients-06-04520-t004_Table 4
######
Blood glucose (mg/dL) and amino acid (nmol/mL) levels 15 min after intraperitoneal insulin injection (IIH group). The IIH group was divided into 5 subgroups: IIH + oral vehicle (VHC), IIH + oral glutamine dipeptide (GDP), IIH + oral glutamine (GLN) + oral alanine (ALA), IIH + oral GLN or IIH + oral ALA. GDP (1000 mg/kg), GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg), ALA (381 mg/kg), or VHC was administered. The Control group received intraperitoneal saline + oral VHC. Data expressed as means ± standard error were analyzed by ANOVA (Newman-Keuls *post hoc* test).
Control (*n* = 5) IIH + VHC (*n* = 5) IIH + GDP (*n* = 5) IIH+ GLN + ALA (*n* = 3) IIH + GLN (*n* = 5) IIH + ALA (*n* = 5)
--------------- ------------------- --------------------- --------------------- -------------------------- --------------------- ---------------------
Glucose 134.2 ± 9.2 128.1 ± 6.2 145.8 ± 11.7 126.7 ± 9.9 126.8 ± 5.3 118.1 ± 6.4
Alanine 532.1 ± 23.8 606 ± 104.6 1477 ± 601.2 792.3 ± 150 688.9 ± 65.3 1001.6 ± 172
Arginine 104.2 ± 13.1 146.9 ± 17.6 157.1 ± 32.5 94.3 ± 8.5 120.9 ± 24.2 118.4 ± 12.7
Glutamine 622 ± 33.3 608.2 ± 40.7 1052.7 ± 334 514.2 ± 101 853.7 ± 173 650 ± 14.0
Histidine 91.3 ± 4.5 94.5 ± 10.1 108.4 ± 17.6 87 ± 3.4 91.1 ± 8.0 85.1 ± 2.2
Isoleucine 78.8 ± 4.9 74 ± 5.9 69 ± 8.3 50.1 ± 0.9 65.6 ± 7.2 78.4 ± 3.3
Leucine 149.5 ± 13.2 138 ± 17.9 138 ± 20.6 102 ± 7.8 128.2 ± 14.8 141.8 ± 12.3
Metionine 51.2 ± 4.6 59 ± 7.1 49.2 ± 4.1 38.4 ± 6.5 47.2 ± 5.0 57.3 ± 6.7
Phenylalanine 61.3 ± 3.8 63.7 ± 8.6 67.8 ± 9.0 46.4 ± 3.8 58.7 ± 6.1 58.6 ± 3.5
Tryptophan 73.7 ± 6.0 100 ± 5.9 90.7 ± 3.8 68.9 ± 11.1 88.8 ± 12.1 85.3 ± 5.5
Tyrosine 135.3 ± 14.0 153.4 ± 17.2 210.1 ± 49.3 140.1 ± 28.8 143.8 ± 18.6 177.9 ± 27.5
Valine 193.9 ± 3.9 185.6 ± 8.5 172.2 ± 17.9 136.5 ± 6.3 171.9 ± 22.6 197.5 ± 10.3
nutrients-06-04520-t005_Table 5
######
Blood glucose (mg/dL) and amino acid (nmol/mL) levels 30 min after intraperitoneal insulin injection (IIH group). The IIH group was divided into 5 subgroups: IIH + oral vehicle (VHC), IIH + oral glutamine dipeptide (GDP), IIH + oral glutamine (GLN) + oral alanine (ALA), IIH + oral GLN or IIH + oral ALA. GDP (1000 mg/kg**)**, GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg) or ALA (381 mg/kg) or VHC was administered immediately after insulin injection. The Control group received intraperitoneal saline + oral VHC. Data expressed as means ± standard error were analyzed by ANOVA (Newman-Keuls *post hoc* test). **^a^** *p* \< 0.05 *vs.* Control, **^b^** *p* \< 0.05 *vs.* IIH + VHC and **^c^** *p* \< 0.05 *vs.* IIH + oral GDP.
Control (*n* = 5) IIH + VHC (*n* = 5) IIH + GDP (*n* = 5) IIH+ GLN + ALA (*n* = 4) IIH +GLN (*n* = 5) IIH + ALA (*n* = 5)
--------------- ------------------- ---------------------- --------------------- -------------------------- --------------------- ----------------------
Glucose 102.4 ± 6.3 43.1 ± 6.8 **^a^** 80.1 ± 3.7 **^ab^** 67.4 ± 6.7 **^ab^** 69.2 ±4.2 **^ab^** 55.0 ± 6.8 **^a^**
Alanine 373.3 ± 19.4 358.3 ± 16.7 1318±73.3 **^ab^** 926.3± 90.5 **^ab^** 486.1 ± 0.8 959 ± 116 **^ab^**
Arginine 84.1 ± 14.0 67.1 ± 10.61 99.4 ± 19.1 76.9 ± 415.2 118.5 ± 9.2 76.0 ± 6.5
Glutamine 686.7 ± 10.0 589 ± 31.8 1225±63.1 **^ab^** 842.7± 87.8**^c^** 1006±87.2 **^abc^** 666.8 ± 48.7 **^c^**
Histidine 86.1 ± 5.8 99 ± 3.3 101.6 ± 8.6 92.4 ± 5.8 91.3 ± 5.3 93.4 ± 2.1
Isoleucine 107.1± 17.8 45.9 ± 5.9 **^a^** 55.4 ± 2.5 **^a^** 61.4 ± 6.7 **^a^** 58.4 ± 7.2 **^a^** 44.9 ± 2.4 **^a^**
Leucine 134 ± 5.4 83 ± 8.5 **^a^** 89.9 ± 6.4 **^a^** 97.8 ± 10.7 **^a^** 89.4 ± 10.7 **^a^** 75.6 ± 3.4 **^a^**
Metionine 40.2 ± 4.6 41.1 ± 4.2 36.1 ± 2.2 45.1 ± 3.3 37.4 ± 4.0 38.9 ± 2.9
Phenylalanine 64.5 ± 2.0 55.4 ± 3.1 51.3 ± 2.7 **^a^** 59.6 ± 2.8 51.6 ± 5.1 **^a^** 49.6 ± 0.5 **^a^**
Tryptophan 69.4 ± 1.8 81.6 ± 5.2 68.7 ± 7.0 82.4 ± 5.0 68.2 ± 7.3 80.1 ± 2.3
Tyrosine 97.7 ± 6.5 85.4 ± 5.1 129.9 ± 16.3 115.1 ± 7.1 104.8 ± 21.8 92.6 ± 6.7
Valine 162.6 ± 7.1 113.2 ± 10.2 **^a^** 121 ± 7.1 **^a^** 137.8 ± 11.9 116.2± 12.1 **^a^** 114 ± 5.0 **^a^**
The concentrations of ALA (5 mM), GLN (5 mM), or GDP (5 mM) to be dissolved in the perfusion fluid were chosen on the basis of a previous study \[[@B18-nutrients-06-04520]\]. As shown in [Figure 1](#nutrients-06-04520-f001){ref-type="fig"} and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}, after a pre-infusion period (10 min), ALA, GLN, or GDP was dissolved in KHB and infused over 60 min. This was followed by a post-infusion period (10 min) to allow the return to basal values of the pre infusion period. Samples of the effluent perfusion fluid were obtained at 5 min intervals and the glucose \[[@B19-nutrients-06-04520]\] and urea \[[@B21-nutrients-06-04520]\] concentrations were measured. Glucose and urea formation indicated liver catabolism of ALA, GLN, and GDP. The areas under the curves (AUC) of the infusion periods are expressed as μmol/g.
{#nutrients-06-04520-f001}
{#nutrients-06-04520-f002}
2.7. Statistical Procedures
---------------------------
Data were analyzed by ANOVA (Newman-Keuls *post hoc* test) using Graph-Pad Prism Version 5.0 software (GraphPad Software, Inc., La Jolla, USA). Results are reported as means ± standard error of the means. *p*-values \< 0.05 indicated statistical significance.
3. Results
==========
Oral administration of low-dose GDP (100 mg/kg), GLN (61.9 mg/kg) + ALA (38.1 mg/kg), GLN (61.9 mg/kg), or ALA (38.1 mg/kg) did not change blood concentrations of GLN, ALA, histidine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and tyrosine at 15 min ([Table 1](#nutrients-06-04520-t001){ref-type="table"}), 30 min ([Table 2](#nutrients-06-04520-t002){ref-type="table"}), or 120 min ([Table 3](#nutrients-06-04520-t003){ref-type="table"}) after insulin injection.
In contrast with the low-dose GDP, blood concentrations of GLN tended to be higher 15 min after administration of high-dose GDP (1000 mg/kg) compared to GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg) or ALA (381 mg/kg) ([Table 4](#nutrients-06-04520-t004){ref-type="table"}). However, we observed substantial variability in GLN blood levels, so we repeated the experiments to measure blood amino acid levels 30 min after oral administration of high-dose GDP, GLN + ALA, GLN, or ALA.
After 30 min, IIH rats that received oral GDP (1000 mg mg/kg) showed higher (*p* \< 0.05) blood levels of GLN compared to GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg) or ALA (381 mg/kg) ([Table 5](#nutrients-06-04520-t005){ref-type="table"}).
On the other hand, the blood levels of arginine, histidine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and tyrosine remained unchanged after oral administration of low-dose ([Table 4](#nutrients-06-04520-t004){ref-type="table"}) or high-dose ([Table 5](#nutrients-06-04520-t005){ref-type="table"}) of GDP, GLN + ALA, GLN, or ALA.
Since increased levels of GLN occurred 30 min after the administration of high-dose GDP, GLN + ALA, GLN or ALA, this time period was chosen to evaluate how much the liver catabolism of GDP, GLN or ALA, which were inferred from liver glucose and urea formation, contributed to blood levels of GLN or ALA. From these experiments, we observed more intense liver catabolism during the infusion of ALA ([Figure 1](#nutrients-06-04520-f001){ref-type="fig"}A and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}A for glucose and urea, respectively) and GLN ([Figure 1](#nutrients-06-04520-f001){ref-type="fig"}B and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}B for glucose and urea, respectively) compared to the infusion of GDP ([Figure 1](#nutrients-06-04520-f001){ref-type="fig"}C and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}C for glucose and urea, respectively).
4. Discussion
=============
Hypoaminoacidemia is a common feature of IIH \[[@B22-nutrients-06-04520],[@B23-nutrients-06-04520],[@B24-nutrients-06-04520],[@B25-nutrients-06-04520]\] and involves the inhibition of proteolysis and the stimulation of amino acid uptake in liver and muscle \[[@B26-nutrients-06-04520]\]. We observed decreased (*p* \< 0.05) blood concentrations of branched chain amino acids (BCAA), such as valine, leucine and isoleucine, 30 min ([Table 5](#nutrients-06-04520-t005){ref-type="table"}) after insulin injection, which agrees with results of previous study \[[@B17-nutrients-06-04520]\]. In addition, high-dose oral GDP (1000 mg/kg), GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg), and ALA (381 mg/ kg) did not overcome the reduced blood levels of BCAA produced by insulin injection ([Table 5](#nutrients-06-04520-t005){ref-type="table"}).
Oral GDP (1000 mg/kg) performed better in terms of blood GLN availability than oral GLN (619 mg/kg) ([Table 5](#nutrients-06-04520-t005){ref-type="table"}). This could be because the enterocytes utilize GLN at high rates \[[@B1-nutrients-06-04520],[@B2-nutrients-06-04520]\], which reduces its availability for intestinal absorption. This is reinforced by the fact that parenteral administration of GLN, which overcomes the influence of intestinal metabolism, showed better glycemia recovery than oral GLN in IIH rats \[[@B17-nutrients-06-04520]\].
It should be emphasized that the integrity of the GDP molecule is needed to obtain this effect. Blood GLN availability was lower (*p* \< 0.05) with combined oral ALA (381 mg/kg) + GLN (619 mg/kg) than with oral GDP (1000 mg/kg) ([Table 5](#nutrients-06-04520-t005){ref-type="table"}).
The primary intestinal mechanism for GDP assimilation is its absorption as an intact dipeptide rather than by hydrolysis \[[@B15-nutrients-06-04520]\]. Additionally, it is difficult to estimate exactly how much oral GDP, GLN, or ALA enters the liver. Therefore, we compared the hepatic catabolism of these substances. Since GLN and ALA showed higher blood levels 30 min ([Table 5](#nutrients-06-04520-t005){ref-type="table"}) after the administration of GDP (1000 mg/kg), GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg), or ALA (381 mg/kg), we chose this time period to evaluate how much the liver catabolism of GDP, GLN, or ALA, which we inferred from liver glucose and urea production, contributed to blood levels of GLN and ALA.
It is noteworthy that the increase in liver catabolism was smaller with GDP ([Figure 1](#nutrients-06-04520-f001){ref-type="fig"}C and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}C) than with ALA ([Figure 1](#nutrients-06-04520-f001){ref-type="fig"}A and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}A) or GLN ([Figure 2](#nutrients-06-04520-f002){ref-type="fig"}B and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}B) in rats that received the vehicle (control group) and in rats that received insulin (IIH group). The reason for this difference is the fact that the hepatocytes do not have a transport system for dipeptides; hepatocytes assimilate dipeptides by extracellular hydrolysis from enzymes located on the plasma membranes following its release into the cytosol as ALA and GLN \[[@B27-nutrients-06-04520]\]. Therefore, like enterocytes, hepatocytes showed less intense catabolism with GDP than with GLN.
Less intense catabolism of GDP compared to ALA and GLN occurs not only in the enterocytes \[[@B28-nutrients-06-04520]\] but also in the liver ([Figure 1](#nutrients-06-04520-f001){ref-type="fig"} and [Figure 2](#nutrients-06-04520-f002){ref-type="fig"}). Additionally, blood GDP is rapidly hydrolyzed \[[@B29-nutrients-06-04520]\] and liberated GLN is used for energy production \[[@B30-nutrients-06-04520]\]. These facts help explain why high-dose oral GDP yields superior blood availability of amino acids than oral GLN + ALA, GLN, or ALA.
Taken together, these results have great clinical interest because they help one to understand the performance of oral GDP and oral GLN in terms of blood concentration of GLN.
5. Conclusions
==============
In conclusion, our results indicate that the oral administration of high-dose GDP (1000 mg/kg) displayed better performance than oral GLN (619 mg/kg) + ALA (381 mg/kg), GLN (619 mg/kg), or ALA (381 mg/kg) in terms of blood availability of GLN. No differences were observed in blood availability of ALA or GLN after the administration of low-dose GDP (100 mg/kg).
This research was supported by the Brazilian government (National Council for Scientific and Technological Development (CNPq)), Program of Support for Excellence Centers (PRONEX)/ Araucaria Foundation, and Research Program for the Unified Health System (PPSUS). We also thank the BioMed Proofreading Company for English revision.
Roberto Barbosa Bazotte designed the study and wrote the paper; Vânia C. Minguetti-Câmara, Any de C. R. Marques, Fabiana P. M. Schiavon, and Vanessa R. Vilela conducted the experiments; Marcos L. Brusch revised the manuscript.
The authors declare that they have no conflicts of interest.
| tomekkorbak/pile-curse-small | PubMed Central |
Manager Sir Alex Ferguson explained the decision to leave Rooney on the bench as a tactical one, and the move certainly looked like paying off as United led 1-0 on the night before Nani's red card allowed Real to overturn their hosts and progress into the quarter-finals.
Stoke City striker Owen, who has played alongside Rooney for United and England, believes that only a handful of clubs in Europe would be able to take on one of Europe's highest-paid players and satisfy his ambitions should United decide to sell him.
"Sometimes when you're at the level he is at you don't have many options," Owen told talkSPORT. "If you're a mediocre player you have 20 teams in England and aboard that you can move to. | tomekkorbak/pile-curse-small | Pile-CC |
Posted
by
Soulskill
on Sunday March 11, 2012 @11:53PM
from the rest-in-peace dept.
Dr Herbert West writes:
"According to io9, 'Today is an incredibly sad day for fans of comic books, concept art, and downright anything science fiction. Artist Jean 'Moebius' Giraud, who provided some of the most stunning scifi and fantasy art ever to grace a page, has succumbed to illness at the age of 73.' It's pretty hard to overstate the impact he had on film, comic books, and illustration in general. You can name most any fantasy or science fiction related piece of culture from the last 30 or 40 years, and chances are he provided concept art for it or was involved in some way. Alien, Dune, Heavy Metal, Tron (original AND the new one), The Abyss, Masters of the Universe, The Fifth Element, Willow... the list goes on. With the recent passing of Ralph McQuarrie, it's been a tough week for scifi and fantasy artists."
Regardless, it is pretty clear that many culture and social icons are entering their later years. There isnt two weeks that go by that someone of some significance has passed in the last half year I think.
"Sur l'etoile" was a sci-fi comic book he wrote for Citroen in 1983, but more than just a branding operation and a little gift meant for Citroen employees, it was a very beautiful and poetic piece of work.
Wasn't there also supposed to be a Dune movie with his participation and Jodorowsky's?
He was also famous for Lieutenant Blueberry, his western series he signed under his real name, Jean Giraud.
Really, you think if something is (barely) more than 24 hours old it's not worth posting? If slashdot hadn't posted this article i never even would have heard of the guy, so it definitely counts as news for me.
Moebius was also very much responsible for the look and atmosphere of the original ALIEN film. Of course, everybody remembers H.R. Geiger's contribution of the Alien and it's discovery. But the whole human world was Moebius and Ron Cobb. Cobb is a genius in his own right, but it's clear how much O'Bannon plied him with work Giraud had done for the pre-production of the geat, unmade DUNE.
I think that's supposed to be the Lady Jessica in the sketch of "Feyd from Dune." Feyd was the Sting character; Moebius drew a leggy Veronica Lake-ish blond gal seated on a throne, holding a huge sword. Don't recall Feyd wearing anything gossamer in the film.
Ever since the late 70's when I first saw a copy of "Metal Hurlant" SciFi comic book art has been one of my passions and the name "Moebious" was one of the first I learnt and whose fabulously expressive work I obsessed over. The incredible number of artists he inspired across the world is amazing, from comic/manga artists like Shir Masamune to film makers such as Ridley Scott to authors such as William Gibson. Some years ago I looked across my book cases, upper shelves crammed with works of modern science fiction spanning various genres, lower shelves stacked with western and asian comic art books, and even a couple of shelves of DVDs and I realised a great many of these works are either directly, or with only one degree of separation, influenced by Moebious's work.
yeh been there done that a couple of years ago (though my preferred liquid painkiller is a good rum or a premium Irish or Bourbon ) . Fortunately this was "only just" a broken tooth that needed extraction.
and damn slashdot for not allowing you to edit your own posts (though I understand the reason)
His "Airtight Garage" was the basis for the fantastic architecture of the San Francisco Sony Metreon's original game arcade. (Unfortunately, after years of deterioration and changes in ownership, the Metreon has been torn out and replaced with a Target store.)
His "Airtight Garage" was the basis for the fantastic architecture of the San Francisco Sony Metreon's original game arcade. (Unfortunately, after years of deterioration and changes in ownership, the Metreon has been torn out and replaced with a Target store.)
Jean Giraud was a major reason I stayed with science fiction through my teen years, instead of sacrificing it to peer pressure. The french scifi artists (Moebius, Forrest, Druillet et al) raised so many more posibilities - an alternative view of science fiction. It kept me going when all I was finding was mundane empowerment fare (let's face it - Heinlein was recreating Lot's lot at the time with Lazarus Long).Then the elder brother of a mate showed me this magazine called 'Heavy Metal'... Oh dear... Through Arzarch and The Incal and the Gardens of Aedena, falling off a horse with Lieutenant Blueberry and of course travelling on a train with Jerry Cornelius, he has delighted me with his simplified style. I applaud him and express gratitude at his effort! Merci beaucoup monsieur Gir. I expect this to be a Bakelite trick.
Who's Forrest? Do you mean Jean-Claude Forest? Now that's a hidden treasure. I always had the impression he was completely forgotton apart from Barbarella - which again was known only because of the movie.
I wonder what a child psychologist would have to say about that:) Possibly that your parents should pay more attention to what books they leave lying about. The graphics were by Gillon, which could explain the darkish atmosphere of the book. Masterful artist though. He also draws on a very large format. I've seen originals of his and an original page is about 1m high.
Forest is(was) usually more frivolous in style. Liked to put in a lot of conversation too. Wordy son of a bitch:) Most of it is only availab
I've always thought French science fiction had a peculiar visual aesthetic, but I'm not an expert on it. I mean, I may be basing that idea mainly on The Fifth Element and Another World [wikipedia.org], but... nice to see I'm not the only person to have thought so.
I wouldn't call Moebius representative of french SF comics but maybe it's easier to see the common elements from a distance. There could be a strong sense of visual esthetic in french comics. Or spanish. Italian. Belgian.
To me Moebius feels rather unique. I'm no fan of mysticism but I appreciate Moebius's spirituality, in an ambiguous way:It feels good, and that is valuable, even though I don't take the philosophy behind it seriously from an intellectual point of view.
Moebius started with Major Fatal(Le Garage Hermetique). It was an experiment in freewheeling on intuition.There's no coherent story or style, just playing around and making things that look like a story. Sometimes funny, sometimes just beautiful.http://5.asset [asset.soup.io]
Here's a 1-hour documentary on Vimeo; I've watched most of about half of it so far - even multi-tasking while doing so, it's pretty interesting. So many remembered images from my collection of Heavy Metal magazines! | tomekkorbak/pile-curse-small | Pile-CC |
Al-Qaeda Linked Passenger Attempts to Blow Up Detroit-bound Plane
A passenger, reportedly linked to terrorist organization al-Qaeda, set off an explosive device aboard Northwest Airlines Flight 253 from Amsterdam to Detroit, injuring himself and two other passengers, law enforcement officials said Friday.
The passenger, identified as a Nigerian citizen who tried to blow up the plane but the explosive device failed, told investigators he had acquired the explosive in Yemen, along with instructions as to when it should be used. The Nigerian citizen also claimed, according to ABC, to be an al-Qaeda member. Authorities however, say the credibility of the suspect’s statements are being questioned.
Delta spokeswoman (Northwest is a wholly owned subsidiary of Delta) Susan Chana Elliott said the incident occurred during the plane’s descent in Detroit. The “passenger caused a disturbance” by attempting to ignite what was initially reported to be firecrackers. The man was “subdued immediately,” Elliott said.
President Obama, who is vacationing with his family in Hawaii, was notified about the incident and held a conference call with his national security team.
White House spokesman Bill Burton said the President is monitoring the situation and receiving regular updates. According to Burton, the President has instructed his aides “that all appropriate measures be taken to increase security for air travel”.
“Upon approach to Detroit, a passenger caused a disturbance onboard Northwest Airlines Flight 253. The passenger was subdued immediately and the crew requested that law enforcement meet the flight upon arrival. The flight, operated by Northwest using an Airbus 330-300 aircraft with 278 passengers onboard, landed safely. The passenger was taken into custody and questioned by law enforcement authorities. Delta is cooperating fully with authorities and additional questions should be directed to law enforcement officials who are leading the investigation.” | tomekkorbak/pile-curse-small | Pile-CC |
VoIP Jobs in South Yorkshire
The table below looks at the statistics for VoIP skills in IT jobs advertised for the South Yorkshire region. Included is a guide to the average salaries offered in IT jobs that have cited VoIP over the 3 months to 31 July 2015 with a comparison to the same period in the previous 2 years.
Permanent IT job ads with a match in theCommunications & Networking category
156
152
132
As % of all permanent IT jobs located in South Yorkshire
17.59%
16.29%
18.01%
Number of salaries quoted
136
131
115
Average salary
£40,000
£42,500
£37,500
Average salary % change year-on-year
-5.88%
+13.33%
90% offered a salary of more than
£26,500
£25,000
£22,500
10% offered a salary of more than
£73,000
£49,000
£63,600
UK excluding London average salary
£41,000
£40,000
£40,000
% change year-on-year
+2.50%
-
VoIPJobs Demand Trend in South Yorkshire
This chart provides a 3-month moving total of permanent IT jobs citing VoIP across the South Yorkshire region as a proportion of the total demand within the Communications & Networking category.
VoIPSalary Trend in South Yorkshire
This chart provides the 3-month moving average for salaries quoted in permanent IT jobs citing VoIP across the South Yorkshire region.
VoIPSalary Histogram in South Yorkshire
This chart provides a salary histogram for IT jobs citing VoIP across the South Yorkshire region over the 3 months to 31 July 2015.
VoIPJob Locations in South Yorkshire
The table below looks at the demand and provides a guide to the average salaries quoted in IT jobs citing VoIP within the South Yorkshire region over the 3 months to 31 July 2015. The 'Rank Change' column provides an indication of the change in demand within each location based on the same 3 month period last year.
Location(Use links below to see detailedstatistics and historical trends)
VoIPTop 30 Related IT Skills in South Yorkshire
For the 6 months to 31 July 2015, IT jobs across the South Yorkshire region citing VoIP also mentioned the following IT skills in order of popularity. The figures indicate the number of jobs and their proportion against the total number of IT job ads sampled that cited VoIP.
VoIPTop Related IT Skills in South Yorkshire by Category
For the 6 months to 31 July 2015, IT jobs across the South Yorkshire region citing VoIP also mentioned the following IT skills grouped by category. The figures indicate the number of jobs and their proportion against the total number of IT job ads sampled that cited VoIP. Up to 20 skills are shown per category. | tomekkorbak/pile-curse-small | Pile-CC |
Catalytic converters like it hot
Vienna University of Technology has successfully clarified what it is the required operating temperatures of catalytic converters in cars depend on.
Catalytic converters work poorly if they have not yet warmed up. Tiny metal particles in a catalytic converter require a minimum temperature to function efficiently. At the Vienna University of Technology, thanks to a new measuring method, it has now become possible to examine many different types of these particles at the same time. Reliable information regarding what it is exactly that the efficiency of catalytic converters depends on has thus been obtained for the first time.
Low ignition temperature desired"A large part of the pollutant emissions from an engine are generated immediately after start-up, while the catalytic converter is still cold", explains Prof. Günther Rupprechter from the Institute of Materials Chemistry at Vienna University of Technology. "Only when a certain temperature is exceeded does what is known as catalytic ignition occur and the catalytic converter functions with high efficiency." Complex and expensive catalytic converter heating systems have already been developed in order to reach this critical temperature as quickly as possible. It would, however, save both money and energy to build a catalytic converter that already functions well at the lowest possible temperatures.
Straight or slanting? It all depends on the angleThe critical temperature that the catalytic converter must reach depends on the material used: the precious metals platinum and palladium are used particularly often in catalytic converters. However, the crystallographic orientation of the surfaces of the tiny metal granules also plays an important role. Crystals can be cut in different highly-specific directions – this process is familiar from cut precious stones. Even the surfaces of naturally grown crystals are formed in differing directions and the orientation of these surfaces determines the chemical behaviour of the crystals. "It is apparent that surfaces with differing crystallographic directions require different temperatures for catalytic ignition", explains Prof. Yuri Suchorski, who works with Prof. Rupprechter.
Many measurements in one experimentPreviously, it had hardly been possible to investigate this effect in detail: a catalytic converter is made up of countless tiny granules. "Until now, it has only been possible to measure the superimposed activity of all these differently-oriented granules", says Rupprechter. However, Rupprechter and his team have now been able to use a photoemission electron microscope based on Einstein's famous "photo effect" to analyse the ignition temperatures of the individual metal granules whilst the reaction is occurring. For this, a film was used, on which many tiny crystals – with diameters of only around 100 micrometres – were arranged closely together. The directions of the crystals were randomly distributed, thereby allowing different crystal variants to be investigated in a single experiment.
Under the microscope, the temperature of the film was slowly increased, actually demonstrating that the catalytic ignition took place at different temperatures depending on the direction of orientation. "It is important to us to be able to investigate different crystal grains in close proximity and under identical conditions during a single experiment," explain the researchers. "If you carry out several experiments one after another, it is never possible to perfectly reproduce the external conditions so that the individual measurements are directly comparable."
With the new findings, it is now possible to perform targeted searches for manufacturing processes for catalytic converters with lower ignition temperatures. "We now know that palladium works better than platinum, and we know which crystallographic direction promises the lowest ignition temperature", says Günther Rupprechter. Now it should also be possible to implement these findings in technology, in order to build catalytic converters which take effect in cars as soon as possible after start-up. | tomekkorbak/pile-curse-small | Pile-CC |
Image caption Mr Sarwar (left) complained about comments said to have been made by Labour councillor Davie McLachlan
A Labour Party investigation has found "no case to answer" after a councillor was accused of using racist language about Anas Sarwar.
Mr Sarwar complained about comments said to have been made by Labour councillor Davie McLachlan.
He was alleged to have told Mr Sarwar he could not support him as Scottish Labour leader because "Scotland wouldn't vote for a brown Muslim Paki".
Mr McLachlan said he was delighted the "false allegations" were dismissed.
Mr Sarwar, a Labour MSP, said he was disappointed by the outcome of the investigation.
He had reported that the remarks were made during the contest to succeed Kezia Dugdale as Scottish Labour leader in 2017.
'Difficult process'
Mr McLachlan, who categorically denied the accusation, was suspended by the party.
He had been leader of the Labour group on South Lanarkshire Council.
But Labour's National Constitutional Committee (NCC), meeting in Glasgow, has dismissed the case against Mr McLachlan.
Reacting to the NCC's decision, Mr McLachlan said in a statement: "This has been a tremendously difficult time for my family and me and I'm delighted the NCC hearing panel has come to this conclusion.
"However, it has been a long and difficult process and I don't think my family and I will ever get over the stress this has brought to all of us.
"My reputation and character have been badly maligned by the false accusations that were made against me but there is some consolation for me in the fact that there are many, many people who know for sure that I never have, and never would, harbour racist views."
He added: "I have been a Labour Party member for 35 years and a councillor for 24 years and I now look forward to representing my constituents again as their Labour councillor."
'Wider culture'
But Mr Sarwar said he would wait to make a "considered" response to the outcome.
"I have consistently said that this isn't about one individual," he said.
"This is about challenging a wider culture and we have made great strides in the campaign against Islamophobia.
"Just this week we had Scotland's political parties adopt a working definition of Islamophobia."
Mr Sarwar said he would give the matter more thought and added: "I'm disappointed with the process and outcome and will have more to say about it but I want to give a considered response rather than one made in haste today."
A spokesman for Scottish Labour leader Richard Leonard said: "Richard has said for some time now that he has concerns about the disciplinary process, how properly resourced it is, and whether it delivers fairness to both sides."
A Labour Party spokesman said: "The Labour Party takes all complaints extremely seriously, which are fully investigated in line with our rules and procedures and any appropriate disciplinary action is taken." | tomekkorbak/pile-curse-small | OpenWebText2 |
ValerySquirting free chat
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ValerySquirting statement:
it is so hot that they caress me and kiss my whole body, it warms me when they kiss me in the neck and breasts, i love that they caress my vagina, they give me oral sex and they penetrate me, i love the anal, i like to feel the cock at the bottom of | tomekkorbak/pile-curse-small | Pile-CC |
New York City Says It May Sue Verizon For Failed FiOS Promises New York City officials says they may sue Verizon for failing to live up to the FiOS deployment obligations embedded in the telco's 2008 franchise agreement with the city. A report by the city released back in June found that Verizon has fallen well short of the company's promise to bring FiOS to 100% of the city by 2014.
Of course as we warned back in 2008 , the contract had ample loopholes allowing Verizon to tap dance around (or buy their way through) these obligations. Fast forward seven years and New York City officials are annoyed, and have been looking for ways to pressure Verizon. That has included actually stopping and taking a closer look at Verizon's contracts with the city. But the city also now tells the New York Times they may also consider suing Verizon over its FiOS deployment failures: quote: “We want them to make it available to everyone in every ZIP code and on every block so that everyone can get online, to do research, to do their homework,” said Maya Wiley, the chief lawyer for Mayor de Blasio. “We need our residents to get online.” Ms. Wiley said that her staff was negotiating with Verizon and that the city would prefer not to sue the company for failing to fulfill its obligation. But, she added, “if that’s what we have to do, then that’s what we’ll do." For their part, Verizon continues to insist they did nothing wrong, arguing that by "100%" of the city they meant 100% of homes "passed" with fiber (fiber running somewhere relatively close outside), not necessarily "served." Verizon has also repeatedly tried to blame landlords for restricting access to necessary building internals. For their part, Verizon continues to insist they did nothing wrong, arguing that by "100%" of the city they meant 100% of homes "passed" with fiber (fiber running somewhere relatively close outside), not necessarily "served." Verizon has also repeatedly tried to blame landlords for restricting access to necessary building internals. Even if some of those excuses are true, as we've seen in New Jersey and Pennsylvania, promising to deploy uniform FiOS in exchange for tax benefits and subsidies -- and then failing to deliver -- is longstanding habit for Verizon. Still, New York City's problem is that the 2008 closed-door agreement between then Mayor Mike Bloomberg and Verizon contained all manner of loophole language that made Verizon's bait and switch practices perfectly legal. As such, it should be curious to see if this is empty bravado on the part of the city, or if they're really willing to fire up the lawyers to fight for more even FiOS deployment. Still, New York City's problem is that the 2008 closed-door agreement between then Mayor Mike Bloomberg and Verizon contained all manner of loophole language that made Verizon's bait and switch practices perfectly legal. As such, it should be curious to see if this is empty bravado on the part of the city, or if they're really willing to fire up the lawyers to fight for more even FiOS deployment.
News Jump Comcast Shuts Off Internet for Subs Who Were Sold Service Illegally; AT&T, Verizon Team To Stop T-Mobile 5G; + more news California Defends Its Net Neutrality Law; AT&T's Traffic Up 20% Despite Data Traffic Actually Being Down; + more news Are The Comcast-Charter X1 Talks Dead In The Water?; AT&T May Offer Phone Plans With Ads For Discounts; + more news Europe's Top Court: Net Neutrality Rules Bar Zero Rating; ViacomCBS To Rebrand CBS All Access As Paramount+; + more news Verizon To Buy Reseller TracFone For $7B; 5G Not The Competitive Threat To Cable Many Thought It Would Be; + more news MS.Wants Records From AT&T On $300M Project; Google Fiber Outages In Austin, Houston, Other Texan Cities; + more news States With The Biggest Decreases In Speed; AT&T Hopes You'll Forget Its Fight Against Accurate Maps; + more news AT&T's CEO Has A Familiar $olution To US Broadband Woes; EarthLink Files Suit Against Charter; + more news 5G Doesn't Live Up To Hype, AT&T's 5G Slower Than Its 4G; Cord-Cutting Now In 37% of Broadband Households; + more news FCC Cited False Broadband Data Despite Warnings; ZTE, Huawei Replacement Cost Is $1.87B, But Only $1B Allocated; + more ---------------------- this week last week most discussed
Most recommended from 51 comments
ITALIAN926
join:2003-08-16 8 recommendations ITALIAN926 Member Wrong terminology "Passed" doesnt mean jack-shit for the customer that wants to order. " "served" means they have ordered service. The proper term we are looking for, and Verizon ignores, is that all addresses be QUALIFIED to order service.
cchhat01
Dr. Zoidberg
join:2001-05-01
Elmhurst, NY 5 recommendations cchhat01 Member can we get ex-mayor to testify? why can't the city sue and get bloomberg to testify about what his intention was when the arrangement was made? | tomekkorbak/pile-curse-small | OpenWebText2 |
The EU’s plan to expand its External Investment Plan to finance development projects is not backed up by evidence, writes María José Romero and Isabelle Brachet.
María José Romero is Policy and Advocacy Manager at the European Network on Debt and Development (Eroded). Isabelle Brachet is ActionAid International EU Advocacy Advisor.
In recent years, the EU has come to regard investments by the private sector as the silver bullet when it comes to financing development projects. The latest indication of this is the European Commission’s plan to expand the €4 billion European Fund for Sustainable Development (EFSD) as it prepares the proposal for the next EU budget.
The EFSD is a central pillar of the External Investment Plan (EIP), and it aims to support private investments in Africa and countries around the EU border. But so far there is not enough evidence to suggest that relying on private investments is the best way of reducing poverty or inequality. And there are concerns that it may, in fact, benefit private investors far more than the world’s poorest. Why, then, is the EC considering the expansion of the EFSD?
Civil society organisations across Europe agree that the private sector has an important role to play in delivering the Sustainable Development Goals (SDGs). But governments should put in place the right framework of legislation, regulation and incentives, to make sure commercial considerations are not to the detriment of sustainability and human rights, and that private investors proactively contribute to poverty alleviation and broader development goals such as gender equality.
We do not believe that the EU should divert increasing amounts of scarce Official Development Assistance (ODA) away from vital services that benefit poor communities without proof that using it to subsidise private investments is actually having a positive impact on people living in poverty. What the EU and its member states should do instead, is to ensure their development interventions do not further exacerbate inequalities. They should also change European policies that hamper development. This would include, for example: tackling tax avoidance by European companies that deprive developing countries of resources they need; capping the scandalous fees charged by the money-transfer industry on migrants’ remittances; and stopping the promotion of massive exports of our agricultural products to the detriment of local food systems and food supply chains in Europe and in the Global South.
We already know about many problems that are caused by the use of ODA to encourage private investment. Firstly, when aid is used to subsidise the European private sector, there is a risk that companies in Europe – rather than people living in poverty – will be the biggest beneficiaries. Rigorous safeguards need to be in place to prevent this. Data from the Organisation for Economic Cooperation and Development’s Development Assistance Committee (OECD DAC) shows that a large share of aid already flows back to companies based in donor countries. In the case where ODA is used to ‘leverage’ private finance i.e. subsidise private investments – the risk of aid being used to offer hidden support to donor country companies is even greater, due to the complexity of the transactions involved. As yet, the OECD DAC has not agreed on safeguards to stop this from happening.
Secondly, an evaluation of European Union blending projects (the trend of using ODA to subsidise loans from public finance institutions or commercial lenders) between 2007 and 2014 found that the design and implementation of these generally did not have strong pro-poor dimensions and “gender was rarely targeted”. What’s more, the same evaluation found that in almost half of the cases examined, there was no clear reason for using blending rather than traditional development cooperation. Moreover, encouraging private companies to invest in public services like health and education – which the EC has not ruled out – may further exacerbate inequality, with the imposition of fees eroding universal access, for example.
Thirdly, if aid has to be used to support private companies rather than the non-profit and public sectors, the focus should be on inclusive business models structured to serve local workers and entrepreneurs and the wider communities in developing countries, such as social enterprises and cooperatives. Domestic micro, small and medium-sized enterprises (MSMEs) could also be supported, in line with national development and industrialisation strategies rather than backing multinationals and global value chains. This guarantees that the long-term benefits of investment will reach the women, men and youth of countries in the Global South, instead of corporates in the North.
Finally, a poorly regulated financial sector has been a major contributor to widening economic, social and political inequality and wealth concentration. It is therefore paradoxical to count on the same actors to improve the livelihoods of people living in poverty without appropriate regulation in place. Increasing inequalities results in stark disparities in access to health, education, and housing and hampers people’s equal access to justice and to political participation. Inequality is also an obstacle to global stability.
Instead of expanding the EFSD to cover more regions and involve larger amounts, our governments and MEPs should ensure that ODA is used for what it is intended. Its effectiveness should be measured by its contribution to improving people’s lives in developing countries and those very people should be in the driving seat to define their needs and aspirations. | tomekkorbak/pile-curse-small | OpenWebText2 |
SURPRISE ANAL SEX - girl did not expect that she would be fucked anal | tomekkorbak/pile-curse-small | OpenWebText2 |
Wednesday, May 11, 2005
A Look at My Bear Butt
I'm sick at home today. Some kind of stomach bug. You shall be spared further details.
My bones ache so much that I cramped up when I tried knitting to pass the time. So I have to content myself with photographing what I'm working on.
(That's how bored I am. You know I'm not much of one for photographs of works in progress.)
This is 75% of the body of a teddy bear, which I'm making as a present for C because he asked for it specifically (the only circumstance under which I will knit anything for anybody).
It's the pattern for "Jack" from Sandra Polley's The Knitted Teddy Bear. Making it has been a watershed for me, and I'm not even close to finished.
It's my first piece with yarn lighter than worsted weight.
It's my first piece that involves a lot of three-dimensional shaping on the needles. (The hats I've made have all been of the perfectly circular, pull-on variety.)
It's the first piece I've ever started (meaning, actually cast on) during my commute, and that's where most of the the first half was created.
You knit the body halves starting with the ass end (see illustration) and finish with the shoulder shaping.
I got to the shoulder shaping just before my train pulled into the station near my office. When we arrived, I got off the train but sat on a bench on the platform to finish casting off.
Suddenly, what had been a wonky piece of flat knitting popped into a three-dimensional shape I could recognize: a sloping shoulder, wide hips, a pudgy tummy*, even a humped back (Polley is gratifyingly attentive to the shaping details of old teddy bears).
People who don't knit probably would snort at the application of the adjective "thrilling" to the process.
They will never understand.
Socks? Socks!
I confess to needing another project to balance the endless charcoal stockinette of the bear.
I originally pulled the teddy patterns book off the shelf thinking it might be nice to knit something tawny and light after four projects in black, navy, navy, and gray. When C said he'd like a bear, I was delighted and told him to pick out any one he liked.
He picked the black one.
So, I need something with BRIGHT COLOR in it. And I am dying to try sock knitting.
I know there are sock people who read this blog. Help me out here.
Do you have a favorite basic pattern for a first pair of socks?
How about yarn choice? Brands? Colorways?
These socks are most likely going to be hidden inside boots most of the time (just about the only thing I wear on my feet), so I'm not concerned about sticking to "masculine" shades.
I thought I'd ask here, first, since the service I get in the yarn shops in Chicago often leaves much to be desired, whereas y'all are a veritable font of friendliness.
(Even when some of you are suggesting I made you look like a syphillis advertisement in your new banner, when what I was really trying to do was highlight your captivating good looks. Ahem.)
By the way, if you are one of the seven knitting people in the continental United States who didn't go to Maryland Sheep and Wool, Tricky has done his usual splendid job of writing it up (with copious illustrations).
And and usual, I find myself wanting many of the things (and a few of the people) featured in the photographs.
On that note, back to the couch with me. I have a lighthearted filmfest waiting, consisting of the first two episodes of Fanny and Alexander (the television version) and Akira Kurosawa's Ran.
29 comments:
Sister Sue
said...
I would really, really, really like a scarf (and matching hat?). I know that's boring, but should you decide to oblige, you could pick a happy, bright color (or colors?). I would trust you in color and yarn choice completely (you would never make anything horrid like the furry yellow thing I got for Christmas last year. Remember?) I would wear it to school (and even IN school on cold days--and we have a lot of cold days). I would tell everyone my brother made it for me. My students would love you more than they already do.Maybe for Christmas...? I leave it to you to decide, but now you can't say I never asked. Hope you're feeling better soon (which is probably where this note should have begun. Oh dear. Sorry. And in such a public forum. Alas!)
What you should do is get a copy of Socks Soar on Circular Needles. I've done two pairs on dpn and one on circular, and it goes much, much faster. There's a basic pattern there. The rest of the patterns are all for girls, but once you get the hang of it it's easy to adapt other patterns.
If you like I can mail you a copy of the pattern for the first set of socks I knit.
Actually, there's also a good Patons book of socks. I think it's called Pull up your socks!
Socks are indeed a joy. There is a really good pattern in an old copy of Interweave Knits called Priscilla's Dream Socks, and they are wonderful because the heel is formed by using short rows and you never have to pick up stitches to make the standard squarish looking heel you see on so many sock patterns. That being said, I am currently spending a lot of time with the Nancy Bush book called Knitting on the Road. I think there are a lot of "women's" socks in there, but there are also a lot of socks all inspired by various areas of the world and full of interesting information.
Many people rave and rave and rave about Lorna's Laces and Koigu as being the best of the best of sock yarns. I have had the privilege of touching these yarns, and I can definitely see why they feel this way. And as far as beautiful colorways go, I don't think you can beat Lorna's Laces. Now if only one of the ridiculously stupid yarn shops in my area would carry any decent yarn, I'd be set. I also saw some Cherry Tree Hill once and it seemed rather yummy too. I'm fond of the brightly colored variegated yarns for socks. I feel socks should have character. Oh, and if you are going to use DPNs, be sure to get yourself a pair that is a little shorter. I've been working on 7" DPNs for my current pair of socks and they are too long. I went out and bought some shorter ones for the next pair, as it seems that I am lately obsessed with socks and that doesn't appear to be changing anytime soon. Let me know if you'd like to "look" at that Priscilla's Dream Socks pattern -- I am sure any pattern could be adapted to incorporate that heel.
Don't listen to any of these crazies. Socks should only be done on DPNs. I love Wendy's Toe Up Sock Pattern (www.wendyknits.net) cause it's easy and goes quickly (short rows for the heel and the toe). Besides being an easy pattern, there's a whole tutorial on doing the short rows on Knitty. Though it takes some finagling of numbers (which I could help with if you want), I really like using Cherry Tree Hill Merino Mini for socks. It's a DK weight superwash, the colors are great, and it knits up quick.
DPNs are certainly the way. Don't know how anyone could knit socks without DPNs. And for God's sake buy superwash. (Socks must be able to go in the normal laundry or they're just not worth making - you know how socks are with their wandering ways.)
I have a custom sock worksheet I'd be happy to email you if you want it. You make a gauge swatch, measure your foot, plug in the numbers and it gives you a pattern for a custom-fitted sock!
i'm a sock virgin and just got a pattern from a friend of mine who swears by it. i would go w/ tricky's advice. if anyone knows socks it's gotta be him. shortrows sounds like a good idea, too. i'm not nutty about that boxy square heel and shortrows are nice and round and easy. i prefer circ to dpn's but it seems like dpn's are MADE for socks, so i'd go there first. sorry to hear you're not feeling well. get well soon!
I second the DPN recommendation (in case that wasn't clear in my first post) but like I said, shorter DPNs between 4-6 inches makes for better sock knitting. I have a friend who has 4 inch DPNs and uses them for socks and mittens exclusively. One day I borrowed them to cast on for a pair of mittens, and it made such an amazing difference in how fast I could go.
DPNs? Yuck! I've used both and after I learned to do it on circulars I never went back. This is of course, a matter of great controversy (say that word with a British accent to yourself) and you will find wars, I say, wars(!) about this issue :)
Do go out and by Bordi's book, but start with KnitWit's blog, a wonderful site on socks, http://www.az.com/~andrade/knit/socks.html
I began knitting socks the "usual" way, top-down, finishing with the toe which you have to cleverly pull together without making it too small and lumpy.
I recently started a toe-up sock and I much prefer it. It fits much better and the toe-box is smooth and seam-free. Don't let the provisional cast-on scare you, it's really just simply a crochet chain that you knit into on each side and then rip out, leaving you to knit up on one side and down on the other. It's so easy once you get the hang of it.
So, if I were learning all over again:1. start with a toe-up pattern, maybe from Bordi's book (which is for one sock on two circ. needles)2. consult KnitWit's blog3. move on to doing your next pair as two socks on two circ. needles at the same time, look here! http://www.socknitters.com/2circs/, it's an online class. You could alter it for a toe-up version.
oooh, one more thing! look here for the toe-up, two-circular method: pattern
and, I got this for x-mas this year: sock wizard software and I love it! I can make any pattern I want in any gauge I get with any yarn I have on hand, no more depending on my LYS to figure stuff out for me. I'm not affiliated at all, I just love this software (I have the sweater wizard too).
Sir Edwin, did you mean Knit Wit's blog? Or the site whose URL you provided, which looks like it has more knitting instructions...
Also, Franklin, if memory serves, didn't you once date a guy whose last name was Andrade, or is that my imagination? Is that his website people keep linking you to? Wouldn't that be ironic? Or irritating, or depressing? Or something?
I'm on the third sock of my first pair (basic top down)... no I don't have three feet - it's just that by the time I finished the second sock the first look like crap compared to the second, so I decided to do the first over again. I'm using Lornas Laces on US1 dp (wood). I have rather large hands and find the 7" to be perfect - anything smaller and I think I'd be jabbing myself in the palm all the time.
Inbetween the 2nd and 3rd sock I did start a pair on US3 (like logs after the 1s) using Cascade Fixation. They went much faster and the stretch in there kind of hides those slight imperfections.
I've found that getting thru the first 2" or so on dps if awkward, but once past that it starts to roll along.
I've watched Sir Edwin (aka Loopy) make lots of socks--two at a time on two circular needles--and I can't begin to imagine how much harder it would be to make them with straight needles.
In fact I can't understand why anyone would want to.
I asked Sir Edwin why anyone would want to, and she said it's supposed to be more authentic or traditional or something. Is there a better reason?
Two at a time, folks. C'mon, you can't beat that. Then you'll never have the problem that Diane describes above, or the problem Dr. Faustus had in 2002, when he broke up with a guy for whom he had knitted one sock.
DPN's. Once you master them, sockknitting goes real fast. Plus, if you knit in public, people will think you're wrestling with a porcupine!
http://groups.yahoo.com/group/FirstTimeSocknittersKAL/
Knitty also has a good sock tutorial, and you make a baby sock. The one I use over at my KAL-- you knit a sock on size 6 or 8 dpns, with worsted weight. It's great, because you actually end up with something you can wear.
I have the Knitted Teddy Bear book. The teddies look precious, I just find the patterns to be written in such a way, I have a difficult time translating it to needles. Someday, I'll get it figured out. I was never dyslexic or had learning disabilities until I started to read knitting patterns.
Haven't visited in a bit--loved the duck tale. Socks--I just began to knit socks (after 50+ years of knitting just about everything else including bears). I really like Nancy Bush's patterns (found in her books and in Interweave Knits); they are very well written. I've used DPNs because I had them around, but plan to learn circulars the next time. I'm not interested in the self stipping yarns because I get board doing all stockinette--anything with a challenging stitch pattern to keep me awake until the magical heel turning.
The author of the-panopticon.blogspot.com has written an excellent article. You have made your point and there is not much to argue about. It is like the following universal truth that you can not argue with: Love can hurt. But its always worth the pain. Thanks for the info.
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Super Bowl XLVIII halftime show
The Super Bowl XLVIII halftime show occurred on February 2, 2014 at MetLife Stadium in New Jersey as part of Super Bowl XLVIII and was headlined by American singer Bruno Mars alongside his band The Hooligans with special guests The Red Hot Chili Peppers. The show was produced by Ricky Kirshner and directed by Hamish Hamilton. At the time of airing the halftime show attracted the largest audience in the history of the Super Bowl, attracting 115.3 million viewers. The show was later surpassed by the following year's Super Bowl XLIX halftime show. The performance generated 2.2 million tweets, due to clamoring for tickets to Mars' Moonshine Jungle Tour.
The Super Bowl XLVIII halftime show received two 2014 Primetime Emmy Award nominations for Outstanding Short-Format Live-Action Entertainment Program and Outstanding Lighting Design / Lighting Direction for a Variety Special.
Background
Bruno Mars was confirmed as the lead half time performer for Super Bowl XLVIII in September 2013 during a Fox NFL Sunday broadcast held live in Times Square in New York City. The NFL's entertainment team started working with Mars and his management on the halftime set list around Thanksgiving week and spent the remainder of 2013 mapping out the production and staging, along with the guest list. In January 2014 it was announced that Mars had personally invited Red Hot Chili Peppers to perform as special guests during his performance. On January 24, 2014 during the game’s fourth quarter, Hyundai debuted the spot entitled “Dad’s Sixth Sense”, that featured the 2016 Hyundai Genesis and Mars' single "Count on Me". The song was chosen "for reasons separate and coincidental from Mars’ previously announced Half Time gig".
During rehearsals there were some disagreements between Mars and the NFL, regarding the singer's performance. The latter wanted to show the audience wearing light-up bracelets to which Mars concurred "If you take that camera off me, you're doing yourself a disservice. And what happened? They spent all this money on these things, and it didn't work." Fortunately, Mars and his band rehearsed a lot and it didn't matter if they had "cafeteria lighting" or a budget of $5 million (U.S.), the performance would always be the best they could do. According to the singer, "Every smoke machine and laser light is just a bonus."
Development
The halftime show was produced by Ricky Kirshner and directed by Hamish Hamilton. Mars and his eight-piece band, The Hooligans, were dressed in clothes custom created by Saint Laurent by Hedi Slimane. Prince, Michael Jackson and James Brown used the same style in clothes; Mars worked a retro gold lame jacket paired with a white shirt, black trousers and a matching skinny tie. Mars made history at the Super Bowl XLVIII Halftime Show as the youngest artist ever to serve as the sole NFL Super Bowl Halftime headliner. He opened the show with a drum solo on a kit designed to honor his late mother, Bernadette Hernandez. The artwork was modeled after Mars' arm tattoo dedicated to his mom. During his performance of "Just the Way You Are" he was standing midfield, backed by the biggest firework display in NFL Super Bowl history.
Montreal-based firm PixMob provided audience lighting effects during the show: "video ski hats" with LEDs and an infrared receiver were distributed to attendees of the game. Lighting cues were sent to the hats using infrared signals from around the stadium, transforming the audience into "pixels" of a giant "screen".
Starting with this halftime show, the halftime show logo does not appear in the introduction. As well, the opening was changed to "The National Football League welcomes you to the (sponsor) Super Bowl (Numeral) Halftime Show".
Setlist
"Billionaire" (Intro) (Children's choir)
Drum solo (Bruno Mars)
"Locked Out of Heaven"
"Treasure"
"Runaway Baby"
"Give It Away" (featuring Red Hot Chili Peppers)
"Just the Way You Are" (Tribute to the United States Armed Forces)
Set list adapted from Billboard.
Reception
Critical
Mars' performance received generally positive reviews from critics, while the Red Hot Chili Peppers' appearance received mixed to negative reviews. Mike Bruno of Entertainment Weekly gave a positive review of the halftime performance, complimenting Mars' "impressive" vocal performance and stating that "there's no question the kid is talented". Entertainment Weekly also criticized the Red Hot Chili Peppers' involvement in the performance, stating that there was "nothing wrong" with their performance however that their presence was "random and unnecessary". Matthew Perpetua of Buzzfeed called Mars a "world class entertainer" and stated that Mars' performance was "dazzling" and "pretty awesome". However, Perpetua also stated that the Chili Peppers' presence was "unnecessary" and also noted that the halftime show was not as memorable as the previous year's show, which was performed by Beyoncé Knowles. Lindsey Weber of Vulture stated it was unfair to compare Mars' performance to Knowles' and that the performance was "perfectly Bruno"; however, Weber also said that it was unlikely that people would "still be talking about" the performance the day after. Mikael Wood of Los Angeles Times stated that Mars "delivered" during the performance and showed viewers that he is a "real musician" however felt that the performance did not begin to feel "hopelessly retro" compared to Knowles' performance until the Chili Peppers joined Mars. Wood felt that the Chili Peppers' performance was "stale" and unwanted and stated that Mars did not need any guests to make the performance special.
Chris Chase and Chris Strauss of USA Today gave a mixed review of the show, saying it was "unspectacular" and "instantly forgettable" and felt that Mars' youth and unlengthy repertoire failed to give him the legacy of previous halftime performers. Chase and Strauss stated the performance was "acceptable" however that it would not be remembered.
Red Hot Chili Peppers controversy
Some complaints, including some from fellow musicians, were directed towards Red Hot Chili Peppers bassist Flea and guitarist Josh Klinghoffer who appeared to not have wires connected to their instruments and were accused of faking their performance. A few days following the Super Bowl, Flea responded through the band's website by saying:
Chili Peppers drummer Chad Smith also responded to the controversy through his Twitter page saying: "FYI.... Every band in the last 10 years at the Super Bowl has performed to a previously recorded track. It's the NFL's policy. Period."
Some fans even complained to the U.S. Federal Communications Commission (FCC) about the band's shirtless appearance saying that they were offended and both men and women should wear clothing because children were watching. One person even contacted the FCC complaining sexism and how it was unfair for the Chili Peppers to go topless but Janet Jackson's exposed nipple caused so much backlash.
Ratings
The Super Bowl XLVIII halftime show was once the most watched in the history of the Super Bowl drawing record ratings of 115.3 million viewers, passing the record 114 million who watched Madonna perform two years earlier. This was surpassed by Katy Perry in 2015. However, that number was significantly higher than the 98.88 million viewers reported in the overnight ratings from Nielsen that measure the top 56 markets.
Media
On February 3, 2014 Billboard predicted that retailers had sold Unorthodox Jukebox 40,000 copies in the week ending February 2, representing a 164% gain compared to the previous week, when it sold 15,000, according to Nielsen SoundScan. Despite this effect, the album was already aiming for around 19,000 copies sold in the week ending February 2, thanks in part to Mars' exposure at the 2014 Grammy Awards. Mars was presented the award for best pop vocal album on the Grammys and got additional screen time from his nominations in other categories as record of the year and song of the year. The author of the article added that "the impact of the Super Bowl on Mars' music sales, and on the Billboard charts, will be unique compared to all other halftime headliners in the modern era of Super Bowl halftime entertainment". On the following week it was expect that Unorthodox Jukebox would sell 70,000 to 80,000 copies by the end of the tracking week, coming for the top three on next week's Billboard 200 chart, while his debut album, Doo-Wops & Hooligans, was expected sell around 25,000 copies.
Regarding the tickets sales of his worldwide tour, The Moonshine Jungle Tour, Mars became the fastest entertainer to sell out three concerts at the Blaisdell Arena, in Hawaii. Since the morning of February 3, 2014, the average price for The Moonshine Jungle Tour went up $150 to an average price of $500. Despite existence of some tickets left on the primary market for select shows, many were sold out. Ticketmaster also struggled to keep with demand, having to switch from their interactive seating maps for most events. The price for tickets on the primary market ranges from $49–$100 for most shows. Bigger city shows prices range from $70 to $181. For shows at big venues, the average prices on the secondary market is north of $600 with the cheapest tickets going for $150. At these prices, Mars upcoming tour ranks amongst the most expensive in the country this summer, ahead of One Direction tickets and Jason Aldean tickets, which were two of the highest prices for an American tours during the spring and summer of 2014.
See also
2014 in American music
2014 in American television
List of Super Bowl halftime shows
References
Category:2014 in American music
Category:2014 in American television
Category:2014 in sports in New Jersey
Category:Bruno Mars
Category:Red Hot Chili Peppers
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Category:Television programs directed by Hamish Hamilton (director)
Category:PepsiCo | tomekkorbak/pile-curse-small | Wikipedia (en) |
Do yall think female p0rn stars who already know the reputation of female PICS stars would say otherwise tho? I mean its their bread and butter why would they say something that would hurt their current profession. I do believe that there are some women who are in the sex industry that are truly happy but at the same time I don't think they are happier than ur average woman as a whole. The only 1 up they got on ur average woman is that aside from fetish films they can rest assure that the peen they fugg is gonna be a fatty.
I would be interested in how those numbers compare to the women who arent in p0rn who are also sexually satisfied.
Do yall think female p0rn stars who already know the reputation of female PICS stars would say otherwise tho? I mean its their bread and butter why would they say something that would hurt their current profession. I do believe that there are some women who are in the sex industry that are truly happy but at the same time I don't think they are happier than ur average woman as a whole. The only 1 up they got on ur average woman is that aside from fetish films they can rest assure that the peen they fugg is gonna be a fatty.
I would be interested in how those numbers compare to the women who arent in p0rn who are also sexually satisfied.
They do it all of the time.
Just in the past 3-4 years, (since ive started listening to foxxhole radio/reach around radio; they have PICS stars on all of the time) I've heard interviews of them where they openly talk about the industry. Some go in on aspects that they hate. The stars and relative unknowns.
Howard Stern has PICS stars on his show all of the time. Some talk about their sex abuse. Some talk about daddy issues. Some talk about not liking sex. This could hurt their current profession and add to the belief that sex workers are *&%^$#@Eed up damaged goods, but they speak on it anyways.
I get that what you are getting at. PICS stars doing what it takes (lying on a servery) in the hopes of somehow changing americas view of the industry. But I don't see 177 individual PICS stars, conspiring to answer these kinds of questions in a way to make their profession look good.
1.
What is your sexual orientation?
2.
What was the age of the first time you had sexual intercourse?
3.
Were you a victim of childhood sexual abuse?
4.
How
many different sexual partners have you had in your lifetime? [The PICS
actresses were instructed not to count partners within the industry,
unless it occurred outside of their work.]
5.
How
many different sexual partners have you had during the past 12 months?
[The PICS actresses were instructed not count partners within the
industry, unless it occurred outside of their work.]
6.
Assume
that you are considering a relationship with someone and the topic of
their “sexual history” comes up. What is the ideal number of sexual
partners they should have had?
7.
On a 10-point scale (1 = not concerned at all and 10 = very concerned), how concerned are you about catching an STD?
8.
On a 100-point scale (0 = none and 100 = definite),
if a person had unprotected sex with someone whom they just met, what
would you estimate the probability that they might catch an STD?
9.
On a 10-point scale (1 = not at all and 10 = very much), how much do you enjoy sex?
10.
On a 100-point scale (0 = not likely at all and 100 = definitely), estimate the likelihood that you would use a condom if having heterosexual sex with someone for the first time.
Four data collectors initially recruited participants at the university
using the three criteria; students were given extra credit in a class
for their participation. For those cases that remained unmatched, the
same data collectors were provided access at a regional airport where
they used a screening process to identify individuals who met the
criteria and agreed to participate in the study. There was no incentive
provided to respondents at the airport, although it was believed that
the airport setting would provide an environment with access to a
variety of women who had time available to complete a survey.
So their matched sample pool were mostly college females from an obscure university in Pensylvania, and a few women who answered the survey at an airport. Sorry that doesn't begin to represent all women by any means.
If this study took into account the thousands of women of all ages, statuses, economic backgrounds and such and then compared their findings with 1000 responses from pic actresses, I'd be more impressed.
Four data collectors initially recruited participants at the university
using the three criteria; students were given extra credit in a class
for their participation. For those cases that remained unmatched, the
same data collectors were provided access at a regional airport where
they used a screening process to identify individuals who met the
criteria and agreed to participate in the study. There was no incentive
provided to respondents at the airport, although it was believed that
the airport setting would provide an environment with access to a
variety of women who had time available to complete a survey.
So their matched sample pool were mostly college females from an obscure university in Pensylvania, and a few women who answered the survey at an airport. Sorry that doesn't begin to represent all women by any means.
If this study took into account the thousands of women of all ages, statuses, economic backgrounds and such and then compared their findings with 1000 responses from pic actresses, I'd be more impressed.
I still dont believe their sample was diverse or large enough for it to hold any weight towards my current opinion.
That's cool. But you can toss out pretty much all studies then. But
maybe that's what some in here already do (unless it conveniently
supports whatever issue they are standing on).
I think it's worth repeating that the researchers are not writing these
articles and headlines. Journalist looking for stories are cherry
picking things and posting these (not well written) articles. They are
the ones writing 'happier'.
Seems many reeeeeally want to discredit the surveys for whatever reason.
I don't really care either way. I just don't see why it's such a
stretch for young sexually open people (and i'll pigeonhole PICS stars
into that corner even though there are non-PICS stars who are sexually open) to have better body image, more energy and self esteem
than similarly aged people who may have less time or incentive to focus on their bodies.
You cannot post new topics in this forumYou cannot reply to topics in this forumYou cannot delete your posts in this forumYou cannot edit your posts in this forumYou cannot create polls in this forumYou cannot vote in polls in this forum | tomekkorbak/pile-curse-small | Pile-CC |
Implementing international human rights recommendations to improve obstetric care in Brazil.
In 2011, the UN Convention on the Elimination of All Forms of Discrimination Against Women (CEDAW) issued a groundbreaking decision in the case of Alyne da Silva Pimentel Teixeira versus Brazil involving the maternal death of a young Afro-Brazilian woman. The CEDAW addressed systemic failures in the Brazilian health system that combined to violate Alyne's rights to life, health, and access to maternal health services. Almost 5 years later, after significant back and forth between the concerned parties, a technical follow-up commission was created with the support of the Center for Reproductive Rights, and was welcomed by the government of Brazil. The technical follow-up commission was precedent-setting, seeking to move beyond identifying gaps in "compliance" and concentrate instead on issues that might catalyze re-engagement by national level stakeholders, both governmental and non-governmental, with the aim of advancing CEDAW's recommendations through not only the creation, but also the effective implementation, of policies and programs that promote women's sexual and reproductive rights in practice, including their rights to safe motherhood. Here, the human-rights-based framework of the technical follow-up commission is described, in addition to their findings related to legal and policy frameworks, evidence-based programing, and monitoring and oversight of providers. | tomekkorbak/pile-curse-small | PubMed Abstracts |
”Vår generation kommer att flippa ur” kunde vi läsa idag på Aftonbladet Kultur. Alice Bratthammar och Amanda Ivanović, födda år 1995, gav idag sina ”vittnesmål” om hur det känns att vara ung i Angered år 2013.
Dessa två lärjungar till av Aftonbladet prisade Johannes ”pepparkakskränkt-poet” Anyuru kommer från Angeredsgymnasiet vilket gör texten extra viktig för mig. Angereds socialtjänst har sedan jag kom till Sverige varit mina föräldrar. De har sett till att jag har haft mat i magen, kläder på ryggen och tak över huvudet. Från tre till nitton år gammal, har samma samhällsinstution som många av dessa kravalldeltagare lever på, gett mig möjlighet att vara där jag är idag. Därför blir detta ”jag-bränner-ner-egendom-för-vi-har-ingen-lokal” dokument knappast imponerande.
Jag är inte infödd i den svenska medelklassen, som köper den lögn som försöker spridas via kultursidorna. Ni kan inte plocka sympatipoäng på min okunskap, jag har sett våra förorter, jag har börjat skolan i dem, jag har växt upp i dem och jag hatar dem. Jag kommer ägna hela min politiska kraft till få fler människor att göra samma resa som jag därifrån. Men det dessa kravalligister måste förstå är att denna resa går ej att få snålskjuts på. Du måste ta dig själv i kragen och anstränga dig tio gånger mer än den där innerstadsungen för att ta dig därifrån. Din framtid i förorten kommer knappast bli bättre av att du bränner ner innerstaden.
Aldrig någonsin har Sverige varit rikare än idag, aldrig någonsin har de allra fattigaste haft det bättre i vårt samhälle än idag. Hur väljer dessa ungdomar att utnyttja denna frihet och möjlighet som miljontals människor jobbar ihop dagligen? Jo, de deltar i upplopp, förstör och skrämmer upp omgivningen. Men det värsta är, att de förväntar sig att omvärlden ska tycka synd om dem.
För det första, ni är inte unika, tro inte att de ni gör – aldrig har gjorts förr, att vad ni genomlider – har ingen genomlidit förr. Ni är inte unika, tvärtom, ni är väldigt förutsägbara. bara för att du har en jäkla smartphone och kan göra facebookgrupper betyder det inte att ni är någon gyllene generation. Låt mig dra några exempel för att få ner er på jorden:
Berzelii-kravallerna 1951 då ungdomar samlades i Berzelliparken efter att ha varit ute och roat sig. Kravallerna växte gradvis i styrka under ett antal kvällar efter att polisen gripit misstänkta brottslingar den 20 juli. När anhängare till dessa senare samma vecka kom till parken sände polisen en större styrka som röjde upp i området. Ytterligare ett par stökiga nätter, med gradvis fler och fler deltagare, samt en motsvarad upptrappning från polisens sida och medias uppmärksamhet. Kulmen kom den 26 augusti med polis från fem städer inkallade och dessutom militärpolis, som ställdes mot ca 3 000 civila. Då händelserna senare analyserades kom man till slutsatsen att kravallerna orsakats av flera anledningar, bl.a. en rådande polisstrejk med avsevärt minskad bemanning som följd, men också bristen på sysselsättning för sommarlediga ungdomar.
Vita pursvenska ungdomar vet tydligen också hur man kunde ordna till kravaller (för er SD-troll där ute som skyller kravallerna på invandring), och till skillnad från 90-talisternas kravaller (där 90 procent av tiden gick ut på att stå och kolla in sin facebookfeed) krävdes det fucking jävla militärpolis för att få ordning på torpet.
Hötorgskravallerna ägde rum i augusti 1965 på Hötorget i Stockholm. Dagen för kravallerna samlades flera hundra mods på Hötorget för att umgås. De flesta hade tagit tunnelbanan bort från förorten och ungdomsgården till centrum. Polisen uppfattade situationen som störande av allmän ordning och konstaplar skickades ut för att upplösa folksamlingen. Modsen vägrade flytta på sig och omfattande kravaller utbröt. Flera skyltfönster krossades och bränder anlades på gatan.
Hur självupptagen måste man vara för att tro att man är den första generationen som kravlat ut ur förorten för att åka in till stan och ställa upp med djävulskap? Vad sägs om att göra som resten av folk som inte drar benen efter sig, och ta en taxi ut ur förorten till den nya fräsha bostadsrätten i innerstan med en ordentlig examen eller anställningsbevis i handen? Ska det vara så mycket begärt i ett land med gratis undervisning ända upp till doktorstiteln?
Heta linjen-kravallerna. Fredagen den 17 september 1982 samlades 1000-1500 ungdomar i och omkring Rålambshovsparken i Stockholm efter att ha stämt träff på ett femtontal kända Heta Linjen-nummer*. Till parken anlände då ett 50-tal poliser. Det uppstod kravaller med bland annat stenkastning och fyra poliser skadades. Kommissarie Kjell Andersson uttalade i samband med detta att Heta Linjen måste stoppas om flera upplopp uppstod, och Televerket uppgavs redan vara i färd med att stänga ned linjerna. * Heta-linjen var om man ringde ett nummer som ännu var vakant så blev det automatiskt ett gruppsamtal om någon annan ringde det nr:et
Ungdomar 1982 hade nästintill obefintliga möjligheter att kommunicera, de lyckas ”hacka” en statlig myndighet för att kunna gruppkonversera och drar till med en fet fest och kravaller mitt i stan. 90-talister däremot får kommunkationsmedel nertryckta i handen med oändliga metoder och former för interaktion med hela världen, ett oändligt bibliotek av information och kunskap, vad väljer du och göra? Att beskylla samhället för dina tillkortakommanden.
Vad sägs om att surfa in på Khan Academy och lära dig matte? Eller till Codeacademy och lära dig programmera? Du har alla möjligheter i världen att ta dig ut ur utanförskap, och var inte orolig, du är fantastisk, du är en stjärna och du kan själv.
För det andra, tro aldrig, aldrig att du på något sätt är språkrör för antingen människor som bor i utanförskap eller för den delen din generation. Din generation, precis som flesta av dina grannar, precis som alla tidigare generationer är hårt arbetande, hårt studerande, motiverande och fantastiska individer. Det är på deras axlar detta samhälle står på och kommer stå på. Gör aldrig misstaget att föra deras talan om det är dynga du ska sprida.
Känner du dig förbisedd? Känner du att ”omvärlden” inte ser dina problem? Men ursäkta att folk har svårt att ta ert Canada Goose-beklädda smartphonebestyckade gnäll på allvar, de är faktiskt fullt upptagna med att jobba och betala för den skolgång ni väljer att skolka ifrån. | tomekkorbak/pile-curse-small | OpenWebText2 |
Clinical Implications of Revised Pooled Cohort Equations for Estimating Atherosclerotic Cardiovascular Disease Risk.
The 2013 pooled cohort equations (PCEs) are central in prevention guidelines for cardiovascular disease (CVD) but can misestimate CVD risk. To improve the clinical accuracy of CVD risk prediction by revising the 2013 PCEs using newer data and statistical methods. Derivation and validation of risk equations. Population-based. 26 689 adults aged 40 to 79 years without prior CVD from 6 U.S. cohorts. Nonfatal myocardial infarction, death from coronary heart disease, or fatal or nonfatal stroke. The 2013 PCEs overestimated 10-year risk for atherosclerotic CVD by an average of 20% across risk groups. Misestimation of risk was particularly prominent among black adults, of whom 3.9 million (33% of eligible black persons) had extreme risk estimates (<70% or >250% those of white adults with otherwise-identical risk factor values). Updating these equations improved accuracy among all race and sex subgroups. Approximately 11.8 million U.S. adults previously labeled high-risk (10-year risk ≥7.5%) by the 2013 PCEs would be relabeled lower-risk by the updated equations. Updating the 2013 PCEs with data from modern cohorts reduced the number of persons considered to be at high risk. Clinicians and patients should consider the potential benefits and harms of reducing the number of persons recommended aspirin, blood pressure, or statin therapy. Our findings also indicate that risk equations will generally become outdated over time and require routine updating. Revised PCEs can improve the accuracy of CVD risk estimates. National Institutes of Health. | tomekkorbak/pile-curse-small | PubMed Abstracts |
An academic conference held at the University of Cambridge said that pedophilia interest is “natural and normal for males”, and that “at least a sizable minority of normal males would like to have sex with children, and normal males are aroused by children.”
These sentiments were discussed at a conference that took place last year to discuss the classification of sexuality in the Diagnostic and Statistical Manual of Mental Disorders (DSM), the standard international psychiatric manual used by the legal system.
The conference, which was titled “Classifying Sex: Debating DSM-5″, had featured a number of speakers who spoke in favor of sex with children, which, in essence, is supporting pedophilia.
The American Psychiatric Association (APA), which produces it, had been locked in battle over whether hebephilia should be included as a disorder. Hebephilia is the sexual preference for children in early puberty, typically 11 to 14 year old’s.
The proposal was being discussed because children are going through puberty at a younger age and the current definition of pedophilia is attraction to pre-pubescent children.
One of the attendees, and enthusiastic participant, was Tom O’Carroll, a multiple child sex offender and long time campaigner for the legalization of sex with children and former head of the Paedophile Information Exchange. “Wonderful!” he wrote on his blog afterwards. “It was a rare few days when I could feel relatively popular!”
The Telegraph reproted:
After a fierce battle in the American Psychiatric Association (APA), which produces it, a proposal to include hebephilia as a disorder in the new edition of the manual has been defeated. The proposal arose because puberty in children has started ever earlier in recent decades and as a result, it was argued, the current definition of paedophilia – pre-pubertal sexual attraction – missed out too many young people.
Ray Blanchard, professor of psychiatry at the University of Toronto, who led the APA’s working group on the subject, said that unless some other way was found of encompassing hebephilia in the new manual, that was “tantamount to stating that the APA’s official position is that the sexual preference for early pubertal children is normal”.
Prof Blanchard was in turn criticized by a speaker at the Cambridge conference, Patrick Singy, of Union College, New York, who said hebephilia would be abused as a diagnosis to detain sex offenders as “mentally ill” under US “sexually violent predator” laws even after they had completed their sentences.
But perhaps the most controversial presentation of all was by Philip Tromovitch, a professor at Doshisha University in Japan, who stated in a presentation on the “prevalence of paedophilia” that the “majority of men are probably paedophiles and hebephiles” and that “paedophilic interest is normal and natural in human males”.
O’Carroll, the former PIE leader, was thrilled, and described on his blog how he joined Prof Tromovitch and a colleague for drinks after the conference. “The conversation flowed most agreeably, along with the drinks and the beautiful River Cam,” he said.
Check the FBI Sex Offender Registry Website to search your state and local area for registered sex offenders.
Photos: Google | tomekkorbak/pile-curse-small | OpenWebText2 |
Sharon Ventura
Sharon Ventura, also known as She-Thing, is a fictional character appearing in American comic books published by Marvel Comics. She has used the pseudonym Ms. Marvel and has served as a member of the Fantastic Four and the female wrestlers known as the Grapplers.
Publication history
Created by Mike Carlin and Ron Wilson, the character first appeared in Thing #27 (September 1985).
Fictional character biography
Sharon Ventura met Thing at the time when he was involved with the Unlimited Class Wrestling Federation (UCWF). Inspired by him, she signed up for the Power Broker's program in order to have her strength augmented and join the UCWF. The Power Broker (actually Curtiss Jackson), employed Dr. Karl Malus to create super-powered wrestlers for his competitions. Sharon was unaware of the criminal activities of Jackson or Malus. While boosting their subjects' strength, the pair addicted the subjects to a drug, ensuring their obedience. It has been implied that she was raped while a prisoner of Malus, which caused her to temporarily develop an intense hatred and distrust of men. Sharon managed to break free before Malus administered the drug. She adopted the costume which UCWF minder Ann Fraley (Auntie Freeze) had arranged for her, taking the name Ms. Marvel. Alongside the Thing, she battled the Grapplers and the UCWF wrestlers. She then battled the She-Hulk.
Later, Sharon was used by the Power Broker to determine if the augmentation could be reversed. Alongside Captain America, she battled the Power Broker.
Sharon later joined the Fantastic Four, and alongside the Fantastic Four she battled Diablo. Shortly after joining the Fantastic Four, Sharon was mutated by cosmic rays and took on strength and appearance similar to that of Ben Grimm, a.k.a. the Thing. Although she never officially retired her Ms. Marvel moniker, she became more popularly known as the She-Thing and it is by this name she is most known to comic book fans. She later first encountered Aron the Rogue Watcher, and battled the She-Hulk. With She-Hulk, She-Thing battled Dragon Man. With the Fantastic Four and Frightful Four, she was captured by Aron. They escaped and defeated their clones. Sharon later battled the Hulk. She-Thing was offered the chance to be human again by Doctor Doom while a then-powerless Ben Grimm chose to use one of Reed Richards' machines to revert to the Thing to save She-Thing. Alongside the Fantastic Four, she battled the Time Variance Authority. She-Thing left the Fantastic Four soon after and began working for Doctor Doom. Sharon claimed to be doing this so that Doom would cure Ben as he had her. She was sent by Doctor Doom to spy on the Fantastic Four, where she first met Alicia Masters. With Mister Fantastic and the Thing, she was captured by Aron, but rescued by the Molecule Man and Doctor Doom. Alongside the Fantastic Four, she battled the Secret Defenders. Alongside the Fantastic Four, Lyja, and the Inhumans, she battled Doctor Doom. When she refused to betray her friends in the Fantastic Four to Doctor Doom, Doom spitefully mutated her into a much more monstrous form. After a bout of insanity, she briefly joined the Frightful Four and battered Sue Storm Richards to within inches of her life.
Years later, She-Thing had a guest appearance in Marvel Knights #4, a spinoff of the main Fantastic Four book. The appearance established that she was indeed alive and well in the Marvel Universe and her attire implied she was still residing with Wingfoot. Her absence was poked fun at, as she stated that she could not believe anybody still had her number.
Sharon Ventura was later seen again in her She-Thing form.
During the 2008 "Secret Invasion" storyline, a Skrull impersonating Sharon's "She-Thing" persona is killed by the Skrull Kill Krew. The real Sharon is recovered alive from a downed Skrull ship after the final battle of the invasion. Sharon attends a support group meeting with the others who were replaced by Skrulls.
Sharon has since been revealed as a prisoner in the Raft, with evidence suggesting that she is part of a plot to destabilize the Fantastic Four.
Following the 2015 "Secret Wars" storyline, Ventura has returned to her original human appearance, and has been seen wrestling in her original outfit.
Powers and abilities
As Ms. Marvel, Sharon had superhuman strength and endurance, thanks to augmentation of her physical attributes by Dr. Karl Malus on behalf of the Power Broker. The mutagenic effect due to exposure to cosmic radiation that turned her into the She-Thing later greatly increased her physical attributes and durability.
Sharon is highly proficient in hand-to-hand combat, and skilled at various martial arts, including tae kwon do and American boxing. She is also an expert stuntwoman, scuba diver, skydiver, motorcyclist, mountain climber, skier, lion tamer, and wrestler. She attended a military academy until she was expelled.
As part of Ben Grimm's Fantastic Four, she demonstrated her intelligence by solving complicated situations with her intuition and cunning.
In other media
Video games
Sharon Ventura's Ms. Marvel appearance appears as an alternate costume for Ms. Marvel (Carol Danvers) in Marvel: Ultimate Alliance.
References
External links
Sharon Ventura at Marvel.com
Category:Characters created by Mike Carlin
Category:Characters created by Ron Wilson
Category:Comics characters introduced in 1985
Category:Female characters in comics
Category:Fictional attempted suicides
Category:Fictional professional wrestlers
Category:Fictional sexual assault victims
Category:Marvel Comics characters with superhuman strength
Category:Marvel Comics female superheroes
Category:Marvel Comics mutates
Category:Marvel Comics superheroes | tomekkorbak/pile-curse-small | Wikipedia (en) |
INTRODUCTION
============
The definition of \'elderly\' may vary according to chronological, biological, psychological, and social aspects, however, most developed countries have accepted the chronological age of 65 years as a definition of \'elderly\' or older person, as defined by the World Health Organization (WHO). Advances in medical science have led to an increase in life expectancy, and, consequently, the proportion of elderly people among the total population. In 2013, approximately 12.2% of the Korean population was aged over 65 years.[@B11] With continued expansion of the aging population, and the development of less invasive and high-quality neuroimaging modalities, more elderly patients are expected to develop intracranial aneurysms.
As supportive intensive critical care and neurointerventional treatment options become more advanced, more aggressive treatment of elderly aneurysm patients is being encouraged.[@B1] Aggressive treatment for many conditions is becoming more common, resulting in more positive outcomes.[@B5][@B16][@B20] The aim of our study was to describe intracranial aneurysm treatments in all elderly patients treated in our institute and to observe their clinical course and immediate prognosis under aggressive therapy.
MATERIALS AND METHODS
=====================
This is a retrospective, cross-sectional study from a single institution. The medical records of all patients treated at our hospital from September 2008 to December 2013 were reviewed for selection of patients aged 65 years or older who underwent microsurgical clipping or endovascular management for treatment of an intracranial aneurysm. To reduce variability, a single vascular neurosurgeon performed all microsurgical clippings and endovascular coilings in these patients. Clinical information obtained included age, sex, Hunt and Hess grade (HHG), aneurysm location, Fisher grade (FG), and treatment (microsurgical clipping, endovascular coiling). To determine which risk factors affected the functional outcome, clinical data including hypertension, diabetes, and previous medication were evaluated as well.
All eligible patients were placed in the microsurgical clipping group or the endovascular coiling group. The HHG were evaluated at admission. Clinical outcome at the time of discharge was assessed for each patient using the Glasgow outcome scale (GOS) score: favorable outcome (good recovery, GOS 5; moderate disability, GOS 4) or poor outcome (severe disability, GOS 2-3; death, GOS 1).
Statistical analysis was performed using commercially available software (SPSS version 18, SPSS Inc., Chicago, IL, United States). Univariate comparison of continuous variables with a normal distribution was performed using two sample *t*-tests. All categorical data were assessed by χ^2^ test or Fisher\'s exact test. Logistic regression was used to test bivariate associations between variables of interest and good versus poor outcome. A *p* value \< 0.05 was considered statistically significant.
RESULTS
=======
Patients and characteristics
----------------------------
A comparison of the clinical characteristics of patients treated by endovascular coiling or microsurgical clipping is shown in [Table 1](#T1){ref-type="table"}. A total of 1010 aneurysms of 857 patients were treated during the study period. A total of 183 (18%) aneurysms were treated in 159 (18.6%) elderly patients, including 134 women (84.3%) and 25 men (15.7%) with a mean age of 71.6 years; 108 (67.9%) patients experienced subarachnoid hemorrhage (SAH) and 51 (32.1%) patients had unruptured intracranial aneurysms (UIA); 101 (63.5%) patients were treated by coiling and 58 (36.5%) patients were treated by microsurgical clipping. Fifty three patients presented with a high-grade SAH (HHG IV or V) and 55 patients presented with a SAH of HHG II or III.
The percentage of unruptured aneurysms treated by endovascular coiling was 76.5%. Age was the only significant difference between these two treatment groups. The average age of patients with aneurysms coiled and clipped was 72.3 (65-93) and 70.2 (65-84) years, respectively (*p* \< 0.01). No significant differences based on gender, risk factors, HHG, and aneurysm size were observed between the coiling group and the clipping group.
Aneurysm location
-----------------
There were 164 (89.6%) anterior circulation aneurysms and 19 (10.4%) posterior circulation aneurysms. Forty eight (26.2%) aneurysms occurred in the posterior communicating artery, 43 (26.2%) in the middle cerebral artery bifurcation, and 36 (19.7%) in the anterior communicating artery. Multiple aneurysms occurred in 16 patients. Ninety three (56.7%) of the anterior circulation aneurysms and 18 (94.7%) of the posterior circulation aneurysms were treated by coiling. Seventy one (43.3%) anterior circulation aneurysms and one (5.3%) posterior circulation aneurysm were treated by microsurgical clipping.
Outcome
-------
Of the 113 individuals with GOS scores of favorable outcome (i.e., independence in activities of daily living), 34 (58.6%) individuals belonged to the clipping group and 79 (78.2%) belonged to the coiling group ([Fig. 1A](#F1){ref-type="fig"}). In the ruptured population, 62 (57.4%) patients achieved a favorable outcome. In the population with unruptured aneurysms, 50 (98%) and one (2%) patients achieved an excellent and good outcome, respectively. In the clipping group, there were 46 patients with SAH. Nine (19.6%) patients had an excellent outcome and 13 (28.3%) patients had a good outcome. Fifteen (32.6%) patients experienced moderate and severe disability. In the coiling group, there were 62 SAH patients. Twenty three (37.1%) patients were discharged in excellent status and 17 (27.4%) patients had a good outcome. Thirteen (21%) patients experienced moderate disability or required hospitalization ([Tables 3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}, [Fig. 1B](#F1){ref-type="fig"}).
Despite aggressive treatment, upon discharge, the overall mortality rate was 11.3% (n = 18). Statistical analysis using the binary outcome scale was performed for comparison of favorable outcome with poor outcome in terms of HHG. HHG was divided into binary variables as either good (HHG I, II, III) or not. Binary logistic regression analysis was performed. Analysis for other variables including age, co-existence of other medical diseases such as hypertension and diabetes mellitus, and medications taken such as antiplatelet or anticoagulants was performed using the same method. As shown in [Table 2](#T2){ref-type="table"}, high grade HHG, advanced age, and IVH were strong predictors of poor outcome. [Table 5](#T5){ref-type="table"} shows the causes of mortality in patients with ruptured aneurysms, with the two most common causes being initial hemorrhage (52.6%; 10 of 19 deaths) and pneumonia (26.3%; five of 19 deaths). Sepsis/multiorgan failure (three deaths), myocardial infarction (one death) were other causes of death for patients who experienced ruptured aneurysms. There was no occurrence of death in the unruptured aneurysmal group.
DISCUSSION
==========
In this study, the primary finding was that patients with high grade HHG, increasing age, and intraventricular hematoma (IVH, FG4) had poor clinical outcomes as compared to patients without these factors, which were as follows. The initial clinical presentation of patients is the highest predictive power of the outcome. In our series, the final mortality rate and percentage of severely disabled patients with high grade HHG (HHG IV, V) was 28.3% and 24.5%, respectively. A favorable outcome was achieved in 31% of treated patients, higher than the result from previously published studies.[@B5][@B7] In patients with a poor clinical grade with acute hydrocephalus and significant IVH (FG4), early placement of an external ventricular drainage (EVD) prior to aneurysm securing intervention improved the level of consciousness and short-term outcome.[@B15][@B17][@B19] This study also demonstrated that placement of an EVD improved the clinical grade and outcome.
The secondary finding is that both microsurgical clipping and coiling yielded excellent results in elderly patients with unruptured aneurysm. The overall morbidity and mortality rates were 2% and 0% in each respective treatment modality, similar to those found in the literature.[@B3][@B8][@B12][@B14] Currently, there are no established guidelines for ideal management of UIAs in elderly patients. In the distribution of aneurysm sizes, larger sized unruptured aneurysms were more common in the older age group and the annual incidence of SAH was noted to be three times higher.[@B2][@B6][@B21] Thus, some researchers have suggested that elderly patients with UIAs should be considered for active surgical intervention to improve their outcomes. Advanced age alone has been reported to increase the risk of complications in elderly patients with unruptured aneurysm undergoing microsurgical clipping or endovascular coiling.[@B4][@B10] In the current study, more patients with UIAs were treated by endovascular coiling (76.5%) and the mean age was significantly older than that of the microsurgical clipping group. To our surprise, favorable results have been obtained from both treatments along with our results. Our study supports the idea that surgical treatment of UIAs in the elderly should be advocated in making decisions on the optimal treatment strategy after careful assessment of each individual\'s circumstances.
In the ruptured aneurysm group, nine (47.4%) patients died from \'other\' causes unrelated to SAH. Among the eight patients who died, five died of pneumonia and three died of sepsis and/or multiorgan failure. According to these results, more than one-third of the patients died within the acute period of the SAH, even if it was not due to the hemorrhage. Clinical care of elderly patients is challenging because of a greater chance of pharmacological complications, cardiopulmonary dysfunction, and so on. Elderly patients who survive SAH often face prolonged management in an intensive care unit (ICU), including treatment for multiple potential neurological and medical complications.[@B13][@B18] Therefore, for proper co-management of these patients, in order to handle multiple issues, it is necessary to employ a more specialized neurointensivist dedicated to the ICU.[@B9]
This study is limited by the fact that it was a retrospective review, with a limited number of patients at a single institution and no strictly defined selection criteria for the treatment procedure along with a lack of long-term follow up results. Lack of standard treatment guidelines and sufficient prognostic domestic data concerning treatment of aneurysms in the elderly make it difficult for clinicians when it comes to deciding on the proper treatment modality in such patients. We believe that our study may provide a valuable guide to help in decision making regarding treatment enrollment of elderly patients who could benefit from treatment, despite the known challenges in the management of this age group.
CONCLUSION
==========
According to our data, elderly patients with high grade HHG and IVH are truly associated with poor outcome, so that the indication for aggressive therapy should be considered carefully. To improve outcomes in elderly SAH patients, co-management by specialized neurointensivists who can handle patients with multiple conditions should be considered. According to our data, all elderly patients with UIAs had an excellent outcome, regardless of the treatment modality. Treatment of unruptured intracranial aneurysms can be considered in an optimistic way.
{#F1}
######
A Comparison of the clinical characteristics of elderly patients with intracranial aneurysm
HHG = Hunt-Hess grade; HTN = Hypertension; DM = Diabetes Mellitus; ACA = anterior cerebral artery; Acom = anterior communicating artery; Ant.Cho = anterior choroidal artery; ICA bif= internal carotid artery bifurcation; MCA bif = middle cerebral artery bifurcation; Pcom = posterior communicating artery; PCA = posterior cerebral artery; PICA = posterior inferior cerebellar artery; SCA = superior cerebellar artery; VA = vertebral artery; yrs = years; CI = confidence interval; NS = not significant
######
Multivariate logistic regression analysis to identify factors predictive of a poor outcome as assessed by a Glasgow outcome scale
^\*^indicates significance (*p* \< 0.05)
GOS = Glasgow outcome Scale, HTN = hypertension, DM = diabetes mellitus
######
Management outcome of aneurysm treated by clipping
HHG = Hunt and Hess grade scale
######
Management outcome of aneurysm treated by coiling
HHG = Hunt and Hess grade scale
######
Cause of mortality
An = aneurysm
| tomekkorbak/pile-curse-small | PubMed Central |
843 F.2d 882
46 Fair Empl.Prac.Cas. 907,46 Empl. Prac. Dec. P 37,910, 46 Ed. Law Rep. 67
Irene C. SPEARS, Plaintiff-Appellee,v.BOARD OF EDUCATION OF PIKE COUNTY, KENTUCKY, et al.,Defendants-Appellants.
No. 87-5418.
United States Court of Appeals,Sixth Circuit.
Argued Feb. 12, 1988.Decided March 8, 1988.
Robert L. Chenoweth, Bryan, Fogle and Chenoweth, Frankfort, Ky., for defendants-appellants.
Jennifer B. Coffman, Brooks, Coffman and Fitzpatrick, Lexington, Ky., Walter A. Kamiat, Bredhoff & Kaiser, Washington, D.C., Jeremiah A. Collins, L. Hope O'Keeffe, for plaintiff-appellee.
Before WELLFORD and NELSON, Circuit Judges, and EDWARDS, Senior Circuit Judge.
PER CURIAM.
1
Plaintiff Irene C. Spears, a school teacher in Pike County, Kentucky, brought an action under Title VII of the Civil Rights Act of 1964, 42 U.S.C. Secs. 2000e, et seq., against the county board of education, five of its members, the superintendent of schools, and the superintendent's predecessor. Mrs. Spears alleged that she had been denied promotion on the basis of her sex. The case was tried before a federal magistrate, pursuant to stipulation. On the basis of both direct and circumstantial evidence, the magistrate found that the defendants had failed seriously to consider Mrs. Spears for appointment to any of a number of administrative positions she had sought; that she was objectively better qualified for these positions than the men who received the appointments; and that Mrs. Spear's sex was the motivating factor in the defendants' failure to promote her.
2
The magistrate's determination that the defendants were guilty of unlawful discrimination was not clearly erroneous, in our view, and we shall therefore affirm the finding of liability. We believe that the magistrate did clearly err in his determination of the point at which the defendants' illegal conduct began, however, and we shall therefore remand the case for a recalculation of damages.
3
* Mrs. Spears began working for the defendant board of education in 1972. During the first 13 years of her employment, she was an elementary school teacher. Her supervisors consistently rated her a superior teacher.
4
Shortly after beginning work for the board, Mrs. Spears undertook to improve her qualifications. She obtained a master's degree in 1974 and a degree as an educational specialist in 1978. In 1979 she attained the highest professional rating a teacher could receive in the Commonwealth of Kentucky, a "Rank I Classification." Effective July 1, 1979, the state certified her as qualified to be an elementary school principal and a supervisor of instruction. That autumn Mrs. Spears sought promotion to an administrative position.
5
Nearly unfettered discretion to determine who would receive such promotions rested with defendant James T. Dotson, the superintendent of schools during most of this period in question. Mrs. Spears testified, and the magistrate found, that she made oral requests for promotion to Mr. Dotson each year beginning in 1979. Mrs. Spears testified on direct examination that she first spoke to Mr. Dotson about a promotion "shortly after" she received her principal's certificate, an event that appears to have occurred in September of 1979. On redirect examination she testified that the conversation took place in the fall of that year. In the spring of 1980 and each year thereafter she completed and turned in a written application form that provided: "Your application will be placed on file for consideration when vacancies occur."
6
Mrs. Spears' attempts to improve her position within the school system did not meet with resounding success. Nine administrative positions became available from 1979 to 1984, and each was filled by a man. Mrs. Spears was not seriously considered for any of these positions.
7
Some of the individuals who received the appointments asked for them, and some did not. The positions were not posted. Mr. Dotson was not concerned about whether any of his appointees held a certificate of qualification for the position to which he was appointed. The magistrate found that Mrs. Spears was better qualified for each position than the individual who received the appointment, and there is no reason to believe that the magistrate was in error on this.
8
Mr. Dotson made statements interpreted by the magistrate as demonstrating a discriminatory frame of mind. The magistrate inferred that Mr. Dotson wanted to appoint a man to the co-principalship of Phelps High School in 1979 because of Dotson's statement that he wanted someone who could instill "the discipline of a football coach...." When Mrs. Spears inquired about the principalship at Lookout Elementary School shortly after that position had been filled, Mr. Dotson told her, she testified, that he "needed a man up there."
9
On June 30, 1984, Mrs. Spears filed charges of discrimination with the United States Equal Employment Opportunity Commission and with the Kentucky Human Rights Commission. Charles Wright replaced Mr. Dotson as superintendent of the school system at about this time. In 1985, on Mr. Wright's recommendation, Mrs. Spears became the principal of the Hellier Elementary School. This was one of the lowest paying administrative positions in the school system. Mrs. Spears filed applications for promotion to better paying administrative positions in both 1985 and 1986. Four administrative positions became available for the 1986-87 school year; each was filled by a man less well qualified than Mrs. Spears. Mrs. Spears testified that she considered three of the positions better than hers at Hellier, and she said she would have accepted promotion to any of the three.
II
10
The magistrate viewed Mr. Dotson's statements on the Phelps and Lookout positions as direct evidence sufficient to support a finding of unlawful discrimination. Applying the method of analysis approved in McDonnell Douglas Corp. v. Green, 411 U.S. 792, 93 S.Ct. 1817, 36 L.Ed.2d 668 (1973), the magistrate also found unlawful discrimination on the basis of circumstantial evidence. Mrs. Spears' oral and written submissions to Mr. Dotson and the board constituted applications for jobs that she was qualified to hold and did not receive. Because the board filled the positions with men, a presumption of discrimination arose. Attempting to rebut the presumption, Mr. Dotson testified that Mrs. Spears was passed over only because of the procedure through which vacancies were filled. He testified that when a vacancy occurred he would talk to individuals in the local community, speak with the principals and other administrators of that school and any feeder schools, confer with members of the school board from the locale in question, consider the residence of the potential administrator, and weigh the qualifications and training of that person.
11
The magistrate found the proffered explanation inadequate. He considered Mr. Dotson's testimony suspect because it was contradicted by the testimony of members of the school board; they stated that Dotson had not consulted with them before making his selections. The magistrate found that Dotson's explanation did not overcome the inference of unlawful discrimination that arose from the superiority of Mrs. Spears' qualifications and the credible testimony of Mr. Dotson's discriminatory statements.
12
Mrs. Spears was held to be entitled both to back pay (from a point beginning two years before the filing of her claims with the EEOC and Kentucky's Human Rights Commission) and to front pay, continuing to such time as she might be promoted to a position comparable to that at the Phelps High School, the highest paying of the nine positions for which Mrs. Spears claimed to have been passed over.
III
13
Under Title VII a plaintiff may show unlawful discrimination through either direct or circumstantial evidence, or both. See Blalock v. Metals Trades, Inc., 775 F.2d 703, 706-07 (6th Cir.1985). If a plaintiff attempts to show unlawful discrimination through circumstantial evidence only, and if the defendant introduces evidence of a legitimate reason for its adverse employment decision, the plaintiff may prevail if the finder of fact is persuaded that the proffered reason is not worthy of belief or that unlawful discrimination was more likely the reason for the decision. Sims v. Cleland, 813 F.2d 790, 792 (6th Cir.1987).
14
The defendants in the case at bar contend that there was clear error in the magistrate's finding on liability. See Rule 52(a), Fed.R.Civ.P., and Anderson v. City of Bessemer, 470 U.S. 564, 105 S.Ct. 1504, 84 L.Ed.2d 518 (1985). Defendants suggest, among other things, that Mrs. Spears did not establish her case because she did not "specifically" apply for each of the positions that was filled. Some of those actually appointed also failed to apply "specifically," however, and Mrs. Spears was no less an applicant than they. Her oral requests to Mr. Dotson and her written application forms leave no room for doubt that the defendants were on notice that Mrs. Spears was an applicant.
15
Defendants also suggest that because Mrs. Spears did not have a high school principal's certification, she and the men who received appointments to such positions were "equally unqualified." Five of the positions for which Mrs. Spears was passed over were in elementary schools for which Mrs. Spears did have certification, however, and the defendants never treated certification as a prerequisite to appointment in any event.
16
We are not persuaded that the magistrate erred in rejecting the defendants' claim that they had legitimate, nondiscriminatory reasons for promoting men in preference to Mrs. Spears. First, the magistrate had direct evidence of discriminatory animus in the form of Mr. Dotson's statements. Second, defendants' proffered reasons for rejecting Mrs. Spears were not persuasive; as the magistrate found, Mrs. Spears was better qualified than the individuals who were appointed. Moreover, Mr. Dotson's testimony as to what he actually did in filling these positions was contradicted by the board members. The magistrate's finding on liability was not clearly erroneous.
IV
17
Defendants also argue that the magistrate used an improper benchmark to determine back pay and front pay. Because Title VII limits the award of back pay to "a date [not] more than two years prior to the filing of a charge ...," 42 U.S.C. Sec. 2000e-5(g), defendants contend that the magistrate could not consider the rate of pay for positions for which Mrs. Spears had been improperly passed over more than two years before the filing of her claim. Thompson v. Sawyer, 678 F.2d 257, 291 (D.C.Cir.1982), points the other way, however, and we have recently recognized that Title VII requires that an injured party be placed as nearly as possible in the situation he would have occupied had the wrong not been committed. Shore v. Federal Express Corp., 777 F.2d 1155, 1158-59 (6th Cir.1985). We believe that the magistrate did select the wrong benchmark for determining back pay and front pay, nonetheless, because he used the rate of pay for a position (the co-principalship at Phelps) for which Mrs. Spears was not improperly passed over.
18
Mrs. Spear's evidence indicated that the Phelps position was filled on July 2, 1979. (The man who received the appointment, a Mr. O'Brien, testified that he was actually appointed in 1978.) It was not until the fall of 1979 that Mrs. Spears first expressed an interest in promotion to an administrative position--and by that time Mr. O'Brien's selection for the co-principalship at Phelps had become a fait accompli. Clearly, in our opinion, another benchmark must be selected.
19
Finally, defendants argue that Mrs. Spears is not entitled to front pay because she has already received a promotion. This argument is unavailing; if
20
"an award of back pay does not fully redress a Title VII plaintiff's injuries, and reinstatement is not possible, an award of front pay is sometimes appropriate in order to effectuate fully the 'make whole' purposes of Title VII.
21
* * *
22
* * *
23
"Awards of front pay should be evaluated under the standards applied to all Title VII relief; whether the award will aid in ending illegal discrimination and rectifying the harm it causes."
24
Shore, 777 F.2d at 1159 (citing Sawyer, 678 F.2d at 259). In this case injunctive relief was not available to make Mrs. Spears whole, and the magistrate had discretion to award front pay until such time as Mrs. Spears might be placed in a position comparable to that which she would have occupied had no unlawful discrimination occurred. Her increase in pay by reason of promotion may be taken into account in mitigation of monetary damages.
25
The judgment rendered by the magistrate is VACATED and the case is REMANDED for recalculation of damages on a basis not inconsistent with this opinion.
| tomekkorbak/pile-curse-small | FreeLaw |
Q:
Curly brackets not getting replaced on xargs command
I've been trying to run this command. However, I'm getting some rather unexpected results.
It seems that it's an obvious overlook on my part, but I can't figure it out.
Here's what I tried:
Given that I have a file.txt on the current directory:
find ./ -name *.txt -print0 | xargs --null -I {} echo {}
This successfully returns:
./file.txt
However, if I modify it to echo to a file, like so:
find ./ -name *.txt -print0 | xargs --null -I {} echo 'some string' > {}.alt
The results are not what I would expect:
-rw-r--r-- 1 User 197121 5 Mar 5 20:33 {}
-rw-r--r-- 1 User 197121 908 Mar 5 04:32 file.txt
The desired output would be to have a file named file.txt.alt with some string as content.
If someone could lend a hand, that would be much appreciated.
A:
The shell sets up the redirection before your pipeline is run. Instead, you could use something like
find ./ -name '*.txt' -exec sh -c 'for f; do echo "some string" > "$f".alt; done' sh {} +
| tomekkorbak/pile-curse-small | StackExchange |
Piraeus Apollo
The Piraeus Apollo is an archaic-style bronze dating from the 6th century BC, possibly from the years 530–520 BC, exhibited now at the Archaeological Museum of Piraeus (Athens).
The Piraeus Apollo is a product of the late archaic period (530–480 BC), and is among the few bronzes from that time period have survived. It is also thought to be a very rare survival of a piece that may have been actually used as the cult image in a Greek temple. In the last few decades of the 6th century, the philosophical mystic currents were to have a considerable influence on late archaic art. The logical move from Ionian natural philosophy to metaphysics was the conscious decision of post-archaic mind. In post-archaic period, the illusive imaginative reality was displaced by harmony and symmetry.
The method of interaction and analogy was perfected by Polykleitos in classical period. He used the principle of continuity and in his famous sculptures each member transmitted to the next a part of his existence; therefore there was an harmonious analogy with the rest of the parts. It seems that his canon (κανών:norm, standard) was a standardization which in his opinion led to the ideal form. The discovery of the perfect mathematical relation was a continuous attempt of the Greek architects.
References
External links
Piraeus Apollo (Sculpture), Tufts University
Further Reading
Dafas, K. A., 2019. Greek Large-Scale Bronze Statuary: The Late Archaic and Classical Periods, Institute of Classical Studies, School of Advanced Study, University of London, Bulletin of the Institute of Classical Studies, Monograph, BICS Supplement 138 (London), pp. 97-116, pls 82-126.
Category:Archaic Greek sculptures
Category:Bronze sculptures in Greece
Category:Sculptures of Apollo
Category:Ancient Greek bronze statues of the classical period
Category:Statues in Greece
Category:Sculptures of men in Greece
Category:Sculptures in Piraeus
Category:Archaeological Museum of Piraeus
Category:Archaeological discoveries in Greece
Category:1959 archaeological discoveries
Category:Nude sculptures | tomekkorbak/pile-curse-small | Wikipedia (en) |
Meet TIN MAN (GC3JAY4), the Geocache of the Week that answers the famous question, ‘What about the heart that you promised Tin Man?’
Thanks to Richard E, Tin Man has found his heart just outside of Albany, NY. This difficulty 1.5, terrain 1.5 traditional geocache has treasure hunters falling in love with the the gentle, tender, and emotional metal man.
Richard explains, “I have been a Wizard of Oz fan since childhood, never missing the yearly showing on TV. The Tin Man is my favorite character.” Richard’s love for the movie and geocaching inspired him to build a life-size Tin Man with “a moving heart that is activated by opening the door on his chest.”
The cache is a big hit among Premium Member geocachers, whether or not they consider themselves a fan of The Wizard of Oz. Nearly 80 Premium Members have logged this cache and awarded it 64 Favorite Points since the cache was published less than six months ago.
With Geocaching.com’s request, Richard agreed to temporarily grant access to basic members for an opportunity to view and find this clever creation.
The cache logs are filled with excitement and gratitude from the community. One geocacher who logged “TIN MAN” writes, “What more can be said – just look at the favorite percentage on this cache – 98% at the time of this log. I’d give it two if I could! Without a doubt the most unique cache I’ve ever encountered! Great workmanship and creativity. Very impressive.”
For more information on Geocaching Premium features, such as Premium Caches or Organize (by Favorite Points), please visit Geocaching.com/Premium.
Continue to explore some of the most engaging geocaches around the globe. Check out all the Geocaches of the Week on the Latitude 47 blog or view the Bookmark List on Geocaching.com.
If you would like to nominate a Geocache of the Week, send an email with your name, comments, the name of the geocache, and the GC code to [email protected]
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| tomekkorbak/pile-curse-small | OpenWebText2 |
517 F.Supp. 597 (1981)
Charles Kenneth FOSTER, Petitioner,
v.
Charles G. STRICKLAND, Jr., et al., Respondents.
No. TCA 81-0847.
United States District Court, N. D. Florida, Tallahassee Division.
July 2, 1981.
*598 Steven L. Seliger, Tallahassee, Fla., Richard H. Burr, III, Southern Prisoners Defense Committee, Nashville, Tenn., for petitioner.
Gregory C. Smith, Asst. Atty. Gen., Tallahassee, Fla., for respondents.
*599 MEMORANDUM OPINION DENYING PETITION FOR WRIT OF HABEAS CORPUS
HIGBY, District Judge.
Charles Kenneth Foster challenges the legitimacy of his convictions for first degree murder and robbery, confinement, and death sentence in his habeas corpus petition. On May 26, 1981, when Foster filed his petition, he was scheduled for electrocution June 3, 1981. I stayed his execution because Foster was entitled to and had not received an evidentiary hearing on at least one of his claims, the competency of his trial counsel. June 18, 1981, I held an evidentiary hearing on the issues raised by the petition. In a pre-hearing memorandum and at the hearing Foster's lawyer, with commendable candor and realism, abandoned three of his issues. The abandoned three are: (1) the challenge to the reasonable doubt jury instruction in the guilt phase; (2) the challenge to jury consideration of the felony-murder aggravating circumstance; and (3) the challenge to the jury venire's composition. The remaining issues fall into two categories, one evidentiary issue and strict legal issues, and will be discussed under those headings. I first summarize the bare facts surrounding Foster's conviction to place the various claims in perspective. I will then address each remaining issue.
SUMMARY
There was little doubt from the beginning that early in the morning of July 15, 1975, Foster killed Julian Lanier. Foster and Lanier met the evening before in Tot's Bar where they got acquainted over a few drinks. At Lanier's suggestion Foster agreed to find some women who would hire out for recreational sex. They traveled in Lanier's camper to the Bay Shore Bar where Foster, with Lanier's financial backing, found two women who agreed to their proposition. The foursome drove to a secluded place to party. There the party ended. Foster beat Lanier bloody and then, after talking to him briefly, slit his throat. While Foster and the women were covering Lanier with leaves and branches, Lanier made sounds of life which inspired Foster to slice his cervical spine.
Foster was arrested, given a first appearance, and appointed counsel on July 15, 1975, the day of the murder (P. Ex. 17). Five days later Foster gave a detailed confession to Bay County Sheriff's Office Investigator Joe Coram. A motion to suppress (P. Ex. 11) that statement, submitted on stipulated facts, was denied (P. Ex. 1-D, p. 458). After Foster's counsel filed a Suggestion of Insanity (P. Ex. 14), the court appointed three psychiatrists to examine Foster. The matter of Foster's competency to stand trial was submitted to the court on the basis of the psychiatrists' report (P. Ex. 18). The court found Foster competent to stand trial on September 25, 1975 (P. Ex. 14).
October 1, 1975, through October 4, 1975, Foster was tried by jury. The jury found him guilty and recommended the death sentence. Judge Spear sentenced Foster to death, and the Florida Supreme Court affirmed the conviction and sentence. Foster v. State, 369 So.2d 928 (Fla.1979). The United States Supreme Court denied Foster's petition for certiorari October 1, 1979. Governor Graham signed Foster's death warrant on May 5, 1981. Foster sought a new trial and sentencing proceeding in Florida courts on May 12, 1981, under Florida Rule of Criminal Procedure 3.850. The court denied his 3.850 motion. The Florida Supreme Court affirmed that denial. Foster v. State, 400 So.2d 1 (1981). Shortly afterwards, on May 29, 1981, I stayed Foster's execution. Foster v. Strickland, Jr., 515 F.Supp. 22.
THE EVIDENTIARY ISSUE
One of Foster's habeas issues requires an evidentiary determination. It is his claim of ineffective assistance of counsel.
Ineffective Representation
Foster alleges six instances of ineffective representation: (1) failure to investigate and raise an insanity defense and to present *600 mitigating psychiatric evidence during the trial's sentencing phase; (2) failure to raise Foster's alleged incompetency at trial; (3) mentioning the parole considerations of a life sentence without requesting an appropriate limiting instruction; (4) failure to request an instruction telling the jury to disregard evidence of Foster's criminal past; (5) failure to object to the questions which elicited the testimony about earlier crimes; (6) failure to challenge the jury venire's composition. This last claim I assume included in Foster's abandonment of his challenge in this proceeding to the jury venire, particularly since no evidence was presented on the subject. The others will be individually addressed.
The United States Constitution's guarantee of due process includes a guarantee of effective assistance of counsel. Powell v. Alabama, 287 U.S. 45, 53 S.Ct. 55, 77 L.Ed. 158 (1932) (recognizing the right in certain capital cases); Brooks v. Texas, 381 F.2d 619, 624 (5th Cir. 1967), (footnote omitted), ("An indigent defendant is entitled to the effective assistance of counsel").
Effective counsel does not mean `errorless counsel, and not counsel judged ineffective by hindsight, but counsel reasonably likely to render and rendering reasonably effective assistance.' MacKenna v. Ellis, 280 F.2d 592, 599 (5th Cir. 1960), modified, 289 F.2d 928 (5th Cir. 1961), cert. denied, 368 U.S. 877, 82 S.Ct. 121, 7 L.Ed.2d 78 (1961). This necessarily `involves an inquiry into the actual performance of counsel in conducting the defense ... based on the totality of the circumstances and the entire record.' United States v. Gray, 565 F.2d 881, 887 (5th Cir.), cert. denied, 435 U.S. 955, 98 S.Ct. 1587, 55 L.Ed.2d 807 (1978).
Beavers v. Balkcom, 636 F.2d 114, 115 (5th Cir. 1981).
The Lawyer's Qualifications
The reasonable likelihood of Foster's lawyer, Virgil Mayo, rendering effective assistance cannot seriously be questioned. In 1975 Virgil Mayo had been a member of The Florida Bar for 24 years. All of that time except for about eight months of 1952 was spent as a practicing lawyer. Mayo had been Public Defender of Florida's Fourteenth Judicial Circuit since 1963, the year the legislature created the position. He had tried hundreds of cases. Although Foster is the only defendant tried for a capital offense Mayo has represented since the current death penalty statute was enacted, he represented several capital defendants charged under Florida's earlier law. His record is impressive. Two clients got jury verdicts of acquittal. One got a binding jury recommendation of mercy. The fourth's trial ended in a mistrial and was later plea-bargained out as a manslaughter case. Mayo's experience included more than twelve cases involving psychiatric defenses. He also served three years on the Panhandle Mental Health Board.
This recital of Mayo's experience describes only cases in which he was lead counsel. His experience is more extensive. As Public Defender he has served as an advisor and overseer in hundreds of cases. In addition Mayo was assisted at Foster's trial by a new assistant public defender, Bill Wager. Wager's qualifications and actions during Foster's trial have not been questioned. And I know from having observed Wager in action many times that he is an intelligent, dedicated, innovative, highly competent criminal defense attorney. These facts show Foster was represented by experienced, competent counsel.
Investigation of Psychiatric Defenses
Foster's allegations of ineffectiveness, however, focus not upon his lawyer's qualifications but his conduct. Mayo, Foster claims, did not render reasonably effective assistance. Eight specific actions are claimed to show ineffectiveness. One of the most major is the claim that Mayo did not adequately investigate and present psychiatric defenses for Foster.
Mayo officially took over Foster's case from an assistant on September 10, 1975 (P. Ex. 1-A), although he was aware the case would be his at least a week earlier. Mayo's investigation was thorough. He reviewed the reports of the three appointed *601 psychiatrists. In addition, he reviewed extensive medical records from the Bay County Memorial Hospital (P. Ex. 7), Florida State Hospital (P. Ex. 8), and the Bay County Guidance Clinic (P. Ex. 9). Mayo held at least ten conferences with Foster. His assistant, Wager, met with Foster five additional times. Mayo talked to Foster's mother. To all this Mayo added a valuable personal resource, his long-time residence in the area, his extensive family ties, and his knowledge of Foster.
"An attorney does not provide effective assistance if he fails to investigate sources of evidence which may be helpful to the defense." Davis v. Alabama, 596 F.2d 1214, 1217 (5th Cir. 1979). Mayo's primary alleged investigative failing was not obtaining the medical records. The facts show he did. Foster also alleges Mayo did not talk to an important lay witness, his mother. Again the facts show he did. Mayo did not talk with Foster's siblings. Much of their testimony at the hearing revealed Foster's unsavory history of self-mutilation, rape, alcohol abuse, drug abuse, a fondness for knife fighting, and beating his family members. Mayo was aware of much of this, especially Foster's reputation with a knife, through his community contacts. Most of this information was also in the medical records Mayo reviewed. In short, the facts revealed at the hearing show Mayo's investigation unearthed almost all the information Foster claims Mayo failed to investigate. Foster has not proven his claims of ineffective investigation. Mayo's investigation of possible psychiatric defenses was more than adequate.
Presentation of Psychiatric Defenses
Competency to Stand Trial
Before trial Mayo questioned Foster's competency to stand trial. Three court-appointed psychiatrists examined Foster. All three found Foster competent to stand trial (P. Ex. 4-A). Two of the doctors had treated Foster before. One, Dr. Sapoznikoff, had treated Foster three times; once in 1972, once in 1973, and once in 1974. He was well informed about Foster's medical history, including his hospitalization in the Bay County Memorial and Florida State Hospitals as well as at the Bay County Guidance Clinic. Mayo allowed the judge to decide Foster's competency upon the basis of those reports. That decision is challenged as ineffective.
Foster argues and his experts testified that Mayo should have asked for a hearing and put the psychiatrists on the stand. Mayo did not ask for a hearing because he felt it would be useless. From his experience he believed that most psychiatrists, including these three all of whom he was familiar with, would not change their opinion once it was down in writing. My 15 years of trial experience cause me to agree with Mayo's evaluation of the situation. Over the years I examined a large number of psychiatrists. Once in a while a psychiatrist might vacillate from a written opinion, but I have never seen one retract an opinion. Mayo's submission of the competency issue was a realistic, effective decision.
During the trial Foster took the stand. While he began by recounting a version of the murder blaming the women, he ended confessing:
I believe that she is the one that killed the man because fuck it, I reckon I'll just cop out. I have done it, killed him deader than hell. I ain't going to set up here, I am under oath and I ain't going to tell no fucking lies. I will ask the Court to excuse my language. I am the one that done it. They didn't have a damn thing to do with it. It was premeditated and I intended to kill him. I would have killed him if he hadn't had no money and I know I never told you about it, but I killed him.
(P. Ex. 1-D, pp. 510, 511). Immediately after Foster's dramatic confession Mayo moved for a continuance and for additional psychiatric examinations (P. Ex. 1-D, p. 512). The motion was denied (P. Ex. 1-D, p. 516).
During the trial Foster was taking a mild sedative, Valium. His dosage was ten milligrams three times a day. He took the first dose noon the first day of trial and as *602 prescribed afterwards. Mayo was not aware of this at trial, but Foster informed him of it through a letter to Wager shortly after the trial.
Foster argues Mayo ineffectively raised the competency to stand trial issue when Foster confessed. His focus is on Mayo's failure to notice during trial or raise post-trial Foster's use of Valium. One alleged fact Foster relies on I find did not exist. Foster claims he slept through much of the trial. I find he did not, although he may have laid his head on the table momentarily once or twice.
Perhaps Mayo should have filed a motion for new trial alleging incompetence at trial and informing the court of Foster's Valium use. It would, however, have been a motion destined for denial. Foster had been treated with and seriously abusing much stronger drugs. In fact 30 milligrams a day was the smallest Valium dosage prescribed for Foster in his years of medical treatment. That dosage would have had no effect on Foster, a chronically nervous person, except to relax him a bit. The fact that he had been without Valium at least a month before trial is insignificant. Since Valium is a long-lasting, cumulative drug, his abstinence actually lessened the Valium's effect compared to what it would have been if he had been taking it for some time before the trial. I find Mayo effectively questioned Foster's competency at trial.
The Insanity Defense
Foster claims Mayo's failure to present an insanity defense was ineffective. The decision to not present the insanity defense was Foster's, not Mayo's. In Mayo's opinion the best defense strategies available were seeking a conviction of the lesser included offense, second degree murder, or pleading not guilty by reason of insanity. He explained his strategies and reasoning to Foster. He particularly favored seeking a second degree conviction. His theory was sound. Second degree murder included
The unlawful killing of a human being, when perpetrated by any act imminently dangerous to another and evincing a depraved mind regardless of human life, although without any premeditated design to effect the death of any particular individual ....
§ 782.04(2), Fla.Stat. (1975). The maximum punishment was 30 years' imprisonment. § 775.082(3)(b), Fla.Stat. (1975). Mayo believed that with the psychiatric evidence available, some rather bizarre behavior by Foster before and during the murder, and the circumstances (especially the fact that the victim died while drinking and using a prostitute) he could convince the jury to return a second degree verdict.
Foster rejected the defense. He wanted to "walk or burn." Despite his many arrests Foster had never been convicted, and he was convinced he could talk his way out of jail. It will be my word against the womens' was his reasoning. Mayo pointed out the fallacy of Foster's plan. Two eyewitnesses, he said, would be hard to counter, and Foster's confession would be devastating despite Foster's explanation that the women had just convinced him he killed Lanier. Mayo tried many times to change Foster's mind. But Foster was adamant "Walk or Burn."
Foster rejected an insanity defense for the same reasons. As with the second degree murder strategy, Mayo explained the defense and its consequences. He accurately informed Foster that a not guilty by reason of insanity verdict might lead to some time in a mental hospital. A person acquitted for cause of insanity could be treated as insane and incarcerated "[i]f the discharge or going at large of such ... person shall be considered by the court manifestly dangerous to the peace and safety of the people ...." Fla.R.Crim.P. 3.460 (1975). Given Foster's long history of violence and the brutality of the murder it was highly likely that if acquitted by reason of insanity, he would be confined for a good while in a mental hospital. This possibility of confinement caused Foster to reject an insanity defense.
"Informed evaluation of potential defenses to criminal charges and meaningful discussion with one's client of the realities of *603 his case are cornerstones of effective assistance of counsel." Gaines v. Hopper, 575 F.2d 1147, 1149-1150 (5th Cir. 1978). Informed evaluation of potential defenses and a meaningful discussion of the case's realities are what Mayo gave Foster. Foster insisted on not following Mayo's advice. After properly advising Foster and trying to convince him to follow the advice, Mayo had no choice but to honor Foster's wishes and forego the insanity defense and forego seeking a second degree conviction.
Psychiatric Evidence in the Sentencing Phase
Foster's final major challenge to Mayo's effectiveness questions Mayo's performance in the trial's sentencing phase. Florida's capital punishment statute creates a two-step proceeding. In step one, the guilt phase, the jury determines a defendant's guilt or innocence of the capital felony. If the defendant is convicted of a capital felony, the judge conducts a separate sentencing proceeding, the sentencing phase to determine whether death should be imposed. § 921.141(1), Fla.Stat. (1975). The proceeding is held before a jury which renders an advisory sentence. Eight aggravating and seven mitigating circumstances must be considered. §§ 921.141(2), (5), (6), Fla.Stat. (1975). The judge then considers the jury recommendation, weighs the aggravating and mitigating circumstances, and imposes sentence. § 921.141(3), Fla. Stat. (1975). "In each case in which the court imposes the death sentence, the determination of the court shall be supported by specific written findings of fact based upon the circumstances in subsections (6) [sic] [aggravating circumstances] and (7) [sic] [mitigating circumstances] and upon the records of the trial and the sentencing proceedings." § 921.141(3), Fla.Stat. (1975). The conviction and death sentence are automatically reviewed by the Florida Supreme Court. § 921.141(4), Fla.Stat. (1975).
Upon an appeal from the judgment by a defendant who has been sentenced to death the appellate court shall review the evidence to determine if the interests of justice require a new trial, whether the insufficiency of the evidence is a ground of appeal or not.
Fla.R.App.P. 6.16(b) (1975). See, Boyd and Logue, Developments in the Application of Florida's Capital Felony Sentencing Law, 34 U.Miami L.Rev. 441 (1980), for a thorough review of Florida's death sentence statute.
Foster thinks Mayo was ineffective in the sentencing phase because he did not adequately present two mitigating circumstances: (1) "The capital felony was committed while the defendant was under the influence of extreme mental or emotional disturbance", section 921.141(6)(b), Florida Statutes (1975), and (2) "The capacity of the defendant to appreciate the criminality of his conduct or to conform his conduct to the requirements of law were substantially impaired", section 921.141(6)(f), Florida Statutes (1975).
Mayo presented evidence relevant to these factors. He specifically focused upon only one factor, extreme mental or emotional disturbance. He felt the psychiatric mitigating circumstances would be difficult for an unsophisticated Bay County jury to understand and likely to be ignored as technicalities by a jury outraged by the murder's brutality. Consequently he focused upon seeking mercy. To that end he put Foster's ex-wife on the stand. He felt she would evoke the jury's sympathy while presenting evidence from which he could also argue the psychiatric mitigating factors. She testified about Foster's self-mutilation and suicidal urges and the apparent lack of reason for his actions. Her testimony, albeit on cross-examination, was moving.
He is a very sick person and I have begged for help for him and so has his mother and we could not get any help from nobody. They would rather put him in the electric chair and kill him than send him to an institution where he can get help. He has been to the mental health unit on a whole lot of occasions and they let him out and they give him pills to calm his nerves and the Judge won't do anything. He promised that he would. I have talked to Dr. Mason and *604 he promised he would and I talked to Dr. Cluxton and he promised he would. And Everybody at the Bay County Sheriff's Department knows me and any time I have ever had any occasion to talk to any of them, asking them to do something for him and nobody will. And they still won't because it's much easier to just go ahead and electrocute him and get it over with so they don't have to worry about somebody whose mind is bad. If he had T.B. or anything they would put him in an institution and they would help him where he couldn't hurt anybody but just because his mind is bad people don't understand that. They think it's all right just to go ahead and get him out of society. But why didn't they get him out of society before a crime like this had to be committed.
(P. Ex. 1-E, pp. 625-626).
Mayo also put Dr. Mason, one of the examining psychiatrists, on the stand. Through Mason the jury learned Foster had received psychiatric treatment four times since 1968, that he had been diagnosed paranoid, and was suicidally depressed. Records of a 1970 proceeding to determine Foster's competency were also introduced.
From the evidence introduced Mayo argued for a life recommendation. His argument actually began during voir dire where he asked several jurors whether they would consider mental or emotional disturbance as a mitigating factor in sentencing (P. Ex. 1-B, pp. 131, 133-135, 149). One prospective juror spontaneously mentioned mental disorders during voir dire (P. Ex. 1-B, p. 175). This indicates all the jurors, not just the ones questioned, realized a mental or emotional disturbance was important in sentencing. In any event the jury which unanimously recommended Foster's death included five jurors Mayo had questioned on the subject (P. Ex. 1-B, pp. 133-135; P. Ex. 1-E, pp. 653-654). During voir dire Mayo also emphasized mercy to the jury (P. Ex. 1-B, pp. 130, 132-136, 149-151, 163, 174, 182, 186, 195). The jury was instructed upon all mitigating circumstances (P. Ex. 1-E, pp. 648-649).
Building upon voir dire, the evidence, and the instructions Mayo argued for mercy emphasizing the mental or emotional disturbance mitigating factor as a reason to recommend life (P. Ex. 1-E, pp. 636-639). His use of Foster's ex-wife's testimony was particularly persuasive. He argued:
You know, I would like to almost join that girl that was on that stand in crying out; in crying out as much money as we spend. And it's a shame that I live in a society that thousands and thousands and thousands and literally millions of dollars that are spent that we don't that we don't know much about the mind. You know the reasons that officials haven't done anything I guess, we are treating minds about like the Indians treated boils with some wet moss. We are not even to the stage of treating the mind to where we can lance a boil, much less have penicillin. We know less about the mind than anything else. Yet in our ignorant society the State askes [sic] you to kill a person.
That little girl cried out. She cried out if you are sick with tuberculosis, if you are sick with tuberculosis we treat it and we do. In my generation we have learned how to treat this. And I don't believe that forever we will continue to treat the mind by putting wet moss on it. I think that we are on the threshold of treating the human mind. And I think the reason for twenty-five years of holding a person in this thing with somebody that was compassionate and somebody says well during this period of time, you know, laws could be amended but if we are going to keep them there and I am asking for the life. I am asking for each of you to recommend to this Court that this life be saved because mitigating and extenuating circumstances exist.
(P. Ex. 1-E, pp. 638-639).
A lawyer's effectiveness is evaluated "on the totality of the circumstances and the entire record." United States v. Gray, 565 F.2d 881 (5th Cir. 1978), cert. denied, 435 U.S. 955, 98 S.Ct. 1587, 55 L.Ed.2d 807 (1978). In Foster's case the totality of the *605 circumstances included a new statute. Florida's death penalty law was enacted in 1972. Ch. 72-724, § 9, Laws of Fla. When Mayo tried the case there were only ten Florida Supreme Court opinions interpreting the death penalty law. Alvord v. State, 322 So.2d 533 (Fla.1975); Swan v. State, 322 So.2d 485 (Fla.1975); Slater v. State, 316 So.2d 539 (Fla.1975); Proffitt v. State, 315 So.2d 461 (Fla.1975); Sawyer v. State, 313 So.2d 680 (Fla.1975); Gardner v. State, 313 So.2d 675 (Fla.1975); Spinkellink v. State, 313 So.2d 666 (Fla.1975); Taylor v. State, 294 So.2d 648 (Fla.1974); Lee v. State, 294 So.2d 305 (Fla.1974); State v. Dixon, 283 So.2d 1 (Fla.1973). None of them interpreted the mitigating circumstances. One thing was clear. Jury recommendations of life were very important. See, Swan v. State, 322 So.2d 485 (Fla.1975); Taylor v. State, 294 So.2d 648 (Fla.1974). Supreme court review was in light of the facts, and the jury found the facts. Alvord v. State, 322 So.2d 533 (Fla.1975).
Important evidentiary considerations were also involved. If Mayo introduced Foster's medical history and testimony of his relatives as Foster says he should have, a great deal of highly prejudicial evidence would have come along. The jury would have learned that Foster once took his mother's car from her at knifepoint; that he raped two women, one of whom was his sister-in-law; that he had been arrested a number of times for violent crimes; and that he once told his mother he was going to kill his little brother and cook him for supper.
In light of the law, or lack of it, and the evidence available, Mayo's decision to try talking the jury into a life sentence through use of emotional testimony, psychiatric testimony, and a good old-fashioned appeal to mercy was reasonable. After considering Mayo's handling of the sentencing phase, I cannot conclude it was ineffective. Another lawyer may have done it differently. Another lawyer may have done it better. Another lawyer may have done it worse. But Mayo was not ineffective.
Parole Considerations
From voir dire through closing argument Mayo emphasized to the jury that a life recommendation would mean at least 25 years' imprisonment without parole for Foster. This should not have been done, Foster claims, without a limiting instruction telling the jury that 25 years was the minimum time he would serve. Mentioning parole without a limiting instruction, according to Foster, invited the jury to recommend death in order to prevent him mistakenly being paroled later. However, "[a]dmittedly, Petitioner's [Foster's] former counsel [Mayo] may well have brought the issue of parole before the jury in order to impress upon the jury the severity of a life sentence in Florida ...." Petitioner's Prehearing Memorandum of Law, Doc. No. 18, p. 18, Foster v. Strickland, TCA 81-0847 (U.S.D. Ct.N.D.Fla. June 12, 1981). More plainly put, Mayo's reference to 25 years without parole would discourage the jury from recommending death to avoid early parole. The strategy was well chosen. It may have been better implemented with the addition of a limiting instruction. But it is effective counsel, not perfect counsel Foster is entitled to, and the strategy, as implemented, was effective.
References to Foster's Criminal Record
This issue has two parts. One is Mayo's failure to object to cross-examination questions eliciting testimony about Foster cutting one person and knifing his father and then leaving him in a ditch. The second is Mayo's failure to request a limiting instruction telling the jury to disregard references to Foster's criminal record. Under the existing law Mayo would have been justified in thinking the evidence was admissible. The statute provided for consideration of "any matter that the court deems relevant to sentence" and admission of any "evidence which the court deems to have probative value ... regardless of its admissibility under the exclusionary rules of evidence, provided the defendant is accorded a fair opportunity to rebut any hearsay statements." § 921.141(1), Fla.Stat. (1975). Just *606 two weeks before Foster's trial the supreme court interpreted section 921.141(1) broadly.
There should not be a narrow application or interpretation of the rules of evidence in the penalty hearing, whether in regard to relevance or to any other matter except illegally seized evidence.
Alvord v. State, 322 So.2d 533, 539 (Fla. 1975).
Again, ideally Mayo should have objected to the questions and asked for a limiting instruction. But his failure to do so does not amount to ineffectiveness. This claim highlights the "Catch 22" nature of the ineffectiveness claims. The evidence Foster says Mayo should have objected to is included in the medical records and family member testimony Foster says Mayo should have introduced.
THE NON-EVIDENTIARY ISSUES
The remaining issues raised in Foster's petition may be disposed of as a matter of law. Each will be addressed.
Incompetency at Trial
Foster's position on his incompetency at trial was summarized in the evaluation of his ineffective assistance of counsel claim. Review of the state trial court's determination that Foster was competent to stand trial is foreclosed by Wainwright v. Sykes, 433 U.S. 72, 97 S.Ct. 2497, 53 L.Ed.2d 594 (1977). That decision forbids federal review of a state court decision based upon independent state grounds. Here the independent state ground foreclosing review is abandonment on appeal. Mayo raised and preserved the issue of Foster's competency at trial. The issue was not raised in his appeal to the Florida Supreme Court and was therefore abandoned, as it should have been. Appellant's Initial Brief, Foster v. State, Fla.S.Ct. Case No. 40,380 (filed May 7, 1976) (hereinafter cited App.Int. B.).
Admissibility of Foster's Confession
Foster claims his confession was obtained through violating his right to counsel and should not have been admitted. The issue was raised at trial on stipulated facts and abandoned, as it should have been, on appeal. App.Int.B. Wainwright v. Sykes, supra, forecloses review. In addition the factual findings implicit in the trial judge's ruling, which under Title 28, United States Code, Section 2254(d), are binding on me, support denying the motion because Foster initiated the statement. Edwards v. Arizona, ___ U.S. ___, 101 S.Ct. 1880, 68 L.Ed.2d 378 (1981).
Evidence Constitutionally Insufficient to Prove Robbery
The record supports the state court's factual determination that Foster committed robbery. See, Foster v. State, 369 So.2d 928 (Fla.1979).
Impartial Jury Denied by Exclusion of Juror Smith
The trial court removed Joe Smith from the jury panel because of his objections to the death penalty. Smith said he would never vote for a death sentence or for guilt if the conviction could result in a death sentence (P. Ex. 1-B, pp. 23-25). His statements amounted to "compelling prejudice against the death penalty" or near "resolve to vote against it blindly and in all circumstances" which constitutionally justify a court excluding a juror. Burns v. Estelle, 592 F.2d 1297 (5th Cir. 1979).
Trial Court Mishandling Evidence in Penalty Phase
This argument challenges the admission during the sentencing phase of graphic pictures of the deceased and the court not instructing the jury, on its own motion, to disregard references to Foster's criminal record. The contention is meritless.
"Heinous, Atrocious, and Cruel" Jury Instruction
One aggravating circumstance set forth in Florida's death penalty law and used to justify Foster's death sentence is "[t]he capital felony was especially heinous, atrocious, or cruel." § 921.141(5)(h), Fla.Stat. (1975). This provision was part of Florida's statute *607 when the United States Supreme Court considered and approved it in Proffitt v. Florida, 428 U.S. 242, 96 S.Ct. 2960, 49 L.Ed.2d 913 (1976). Foster claims it is unconstitutionally vague. Proffitt mandates rejection of this claim.
Mitigating Circumstances Jury Charge
Foster's jury was told:
Should you find one or more of these aggravating circumstances to exist it will then be your duty to determine whether or not sufficient mitigating circumstances exist to outweigh the aggravating circumstances found to exist.
(P. Ex. 1-E, p. 648). The statutory mitigating factors were listed next. Then the judge instructed:
If one or more aggravating circumstances are established you should consider all the evidence tending to establish one or more mitigating circumstance and give that evidence such weight as you feel it should receive in reaching your conclusions as to the sentence which should be imposed.
(P. Ex. 1-E, p. 650).
Foster argues these instructions are impermissible under the holding in Lockett v. Ohio, 438 U.S. 586, 604, 98 S.Ct. 2954, 2964, 57 L.Ed.2d 973, 990 (1978) (footnotes omitted), that "the sentencer, in all but the rarest kind of capital case, [must] not be precluded from considering as a mitigating factor, any aspect of a defendant's character or record and any of the circumstances of the offense that the defendant proffers as a basis for a sentence less than death." This issue was not raised at trial, and is, therefore, barred by Wainwright v. Sykes, supra. Also, Florida's law is not like Ohio's, which required a death sentence unless one of three circumstances were proven. Florida's law allows the sentencer to consider and weigh mitigating circumstances as it sees fit.
Findings of Fact by Jury and Judge Justifying Death Sentence
Foster argues that the Constitution requires written findings from judge and jury specifying the mitigating and aggravating circumstances found and considered. Foster was sentenced to death in a procedure approved by the Supreme Court. Proffitt v. Florida, supra. Also, Wainwright v. Sykes, supra, bars this claim since it was not raised at trial.
Florida Supreme Court's Review of Post-Sentencing Psychiatric and Investigative Reports
Foster has alleged the Florida Supreme Court secretly obtained and reviewed psychiatric and/or investigative reports concerning him during its consideration of his appeal. He was one of several prisoners who petitioned the Florida Supreme Court for relief on that ground. The court held a claim for relief had not been stated because its function as a reviewer rather than sentencer made its consideration of such documents irrelevant. Brown v. Wainwright, 392 So.2d 1327 (Fla.1981). A petition for certiorari is pending before the United States Supreme Court. Brown v. Wainwright, cert. pending, U.S.S.Ct. No. 80,6434 (April 3, 1981). I adopt the reasoning of the Florida Supreme Court and hold Foster's allegations do not state a claim for relief.
Florida's Death Penalty is Applied in an Arbitrary and Unconstitutional Manner
This contention merely argues that all the other alleged errors make Foster's death sentence irrational and arbitrary. It is meritless.
SOME FINAL MATTERS
Foster's Counsel
At some point Foster may decide to challenge the effectiveness of his counsel in this proceeding, Richard H. Burr, III. In anticipation of that possibility, I record my observations on Burr's effectiveness. Burr has done a good job. His pleadings have been well written and thorough. The memo he filed was well researched, well written, and quite helpful. Burr clearly took pains to present the law accurately and forthrightly abandoned claims he found unsupportable. *608 In court he was an effective examiner and a gentleman. This decision simply applies the law to the facts. It is no reflection upon his abilities.
Delay
In argument on the Motion for Stay the State complained vociferously of Foster's alleged delay in bringing this petition. Rule 9(a) of the Rules Governing Section 2254 Cases in the United States District Court provides for dismissal of delayed petitions. The State never moved for dismissal under this rule. If its complaints were well founded, a motion should have been made. If the complaints were not well founded, they should not have been made. The comments made at the hearing insinuated Burr or Foster deliberately waited as long as possible to file the petition in order to delay Foster's execution. Such charges about Burr are insults to a capable lawyer, obviously working with a handful of other lawyers to represent the hundreds of people sentenced to death, probably at great personal sacrifice. Such charges about Foster are ludicrous. To imagine a semi-literate indigent plotting his federal litigation strategy is hard. Blame for the delay in this case is more fairly placed upon the Florida Supreme Court which took two years and five months to decide Foster's appeal and the State of Florida which does not provide indigents sentenced to death lawyers for their collateral litigation.
JUDGMENT
Foster's Petition for Habeas Corpus is denied. The stay in this case is vacated.
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The image above shows five “fake” helicopters inserted into a photo using Photoshop CS4. Immediately you can not see the deception but with ELA (Error Level Analysis), you can examine the photo in depth and reveal the scam.
Image Analysis with ELA (Error Level Analysis)
Analysts at all levels can use ELA for image analysis. The program is extensively used in UFO circles to detect fake UFO photos. Also, online media who spread fake news and frequently modifies images can ELA prove to be useful against. ELA is specifically designed to provide quick results and uses algorithms that measure changes in the compression of an image (Error Level Analysis). If there are visible differences in the image after an ELA image analysis, it has probably been digitally modified.
On the ELA website fotoforensics.com you can upload an image or refer to an image URL and put ELA on the error-level analysis immediately. The result will appear a few seconds later on the site with the original image at the top and the results image at the bottom. The resulting image reveals possible differences in the JPEG compression. With JPEG images, the entire image must be about the same level. If a section of the image is at a significantly different error level, it indicates possible digital modification.
Identical edges must have similar brightness in ELA results.
All edges with high contrast should resemble each other with the same intensity. And all edges with low contrast should resemble each other with the same brightness. With an original image, low contrast edges should be almost as bright as edges with high contrast. As for brightness and contrast, the image should be almost the same.
An unedited *.jpg picture (above) downloaded directly from the camera will not give any special visible impact. It will typically be a very dark picture with weak indications of the details in the image. There will be no big differences in brightness and contrast.
Editing in Photoshop reveals digital modification
However, if you load the same *.jpg picture into Adobe Photoshop and start editing it, put a clip of a helicopter from another image taken with another camera into the unedited image, you begin to seriously create visible tracks.
The helicopter in the picture above comes from a picture I took with my Canon EOS 60D. It is inserted into the photo with the Sun Halo and the cigar-shaped UFO taken with a Sony A-100 camera. As expected, the helicopter separates clearly from the rest of the image in ELA results below. The edges of the helicopter have a high contrast and brightness in relation to the surrounding image. Here ELA’s photo analysis revealed digital image manipulation.
ELA’s photo analysis has thus discovered differences in the compression of the image. ELA can see that the inserted helicopter has a slightly different profile regarding compression. It clarifies it in the image with the slightly higher contrast and brightness (error level) around the helicopter.
What ELA photo analysis does not detect
There are also things that ELA photo analysis does not detect. If we take the original photo with the helicopter and load it into Photoshop and make a copy of the helicopter and put it in the same image it comes from, ELA will not see that helicopter No. 2 is false. It is because the compression of the original photo is exactly the same as the clip by helicopter no. 2. The clip comes from the same photo, so ELA detects no difference here, no digital image modification.
In the picture above, the fake helicopter is already inserted in the picture to the left. The one to the right is genuine. And the ELA image analysis below does not provide any useful results. I would be able to insert an entire armada of helicopters without ELA would discover the digital image modification. My fraud would go under the radar here.
However, common sense would probably reveal the deception since it would seem suspicious if a whole fleet of helicopters had the exact same angle in the photo. But what if I take a copy of the real helicopter and rotate it 90 degrees and insert it again?
Again, ELA is not able to see the fraud. Despite the clip with the helicopter is turned 90 degrees, it still reveals no differences in the compression. We need to have a different approach if the Error Level Analysis should be able to detect the fraud.
Digital image modification gives conflicting results
Further digital modification in Photoshop with the fake helicopter enlarged and now twice as large as the real heli, unfortunately, gives a contradictory result, see below. The real helicopter to the right in the picture now stands out more than the fake helicopter to the left in the picture. You will mistakenly believe that the false heli clip is genuine and the real is false…
I actually expected the compression in the enlarged helicopter clip to give clear results, but in this case, the result was the opposite than expected. Only when I reduce the size of the false helicopter in the image, ELA reacts as expected, see below.
The clip with the diminished helicopter to the left in the picture above has a higher contrast and brightness (error level). It obviously does not fit into the rest of the image. We now know the smaller heli has another origin than the original photo…
The picture above (same as the article’s intro image at the top) showing a whole fleet of fake helicopters, visualizes the algorithm behind the error level analysis. Each time the cut of a helicopter decreases in size, the contrast, and brightness increase.
Interim conclusion
ELA image analysis cannot reveal all facets of digital image modification. A JPEG image can easily be modified in Photoshop to an extent without the photo analysis of ELA detecting it. However, we can conclude with the last two images above, that because there are differences in contrast and brightness, the photo must be digitally modified and possibly falsified.
If you feel you can not trust the Error Level Analysis (ELA), you can take a look at the photo’s metadata. It contains information that changes every time a photo is edited in and saved by an image editor like Adobe Photoshop.
The image´s digital fingerprints with EXIF metadata
If you load an unedited photo into Adobe Photoshop and just save it under a new file name without editing it, a subsequent image analysis with ELA will not reveal anything beyond the usual. The image will resemble itself as before it was saved by Photoshop – dark and no big differences in brightness and contrast.
However, a picture will lose some of its quality when it is saved again in an image editor and the compression of the image will, therefore, be slightly changed. This can be seen by the whole picture being slightly brighter in an Error Level Analysis. But the image metadata better known as EXIF data will be able to tell about a photo’s origin. It accompanies the new version of the image (as long as it is not compressed too hard) and is also read by the ELA photo analysis.
EXIF is an abbreviation for Exchangeable Image File Format. These are technical metadata (information) about an image, such as the camera model used, resolution, width, and height of the image, how you have set your optics (fx focal length) and more. And is an image editing program such as Adobe Photoshop used, it will appear from the image’s EXIF data.
Today we have another image data standard called XMP (Extensible Metadata Platform). It is a recently adopted format created by Adobe in 2012 and will also be displayed in the ELA image analysis. It actually shows the same as EXIF data does.
Images on the Internet are compressed
Most images on the internet today are compressed. The reason is obvious. The less a picture fills in the amount of data, the faster it is loaded onto a website. And since smartphones have a fairly low capacity in terms of bandwidth, compression of photos, video, and audio becomes a must to cover the needs of mobile users for fast information on the go.
Nobody wants to sit and wait for minutes before a website fully loads. Loading a website should go quickly, and any webmaster who is ahead on the beat would compress the images before they are posted on a website. The pictures are typically compressed to the utmost, without losing quality. But with optimal compression, the additional EXIF metadata will be lost.
All of my photos on this website are compressed for fast loading. Therefore, EXIF metadata can not be displayed with ELA’s analysis tool or any other EXIF viewer
Basic image metadata
The example from one of my UFO articles shows the analysis of a compressed image. Here, there are no EXIF data, just some basic information. You can view this data by clicking “Metadata” in the box at the top left of the ELA analysis results page
The basic metadata for the example above does not even reveal a date of creation. The line with CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 82 refers to the component of the underlying PHP code on my WordPress website that has helped to render the image into the browser.
CREATOR: gd-jpeg v1.0 is not a program but a library or protocol if you like, see GD Graphics Library. And IJG JPEG v62 refers to the type of compression, see IJG
CNN image reveals digital image modification
Since September 11, 2001, I’ve been aware of CNN modifying both image and video before it reaches the public. For a news media like CNN, it’s important to look good at the public. Therefore, CNN’s images and videos will always review image processing.
The article here shows a picture of Boeing’s new A380 aircraft type with a beautiful blue sky as a background. Right-click on the image and choose to Copy the URL and ask ELA for an image URL analysis. The result of the analysis will reveal obvious digital image modification…
The result image shows a background that is almost black all over. The plane is “sticking out” in a graphic sense. You can simply see the A380 is a separate clip and the background is a separate clip. Here you have obviously wanted a different background.
Although the image has been subjected to compression and has no EXIF data, it obviously reveals digital image modification. So in some cases, an image analysis with ELA can quickly and easily reveal manual modifications.
Final conclusion
ELA’s photo analysis cannot reveal all facets of digital image modification. If a photo has been edited and modified with components from itself, without clips from other photos taken with other cameras, ELA may have difficulty detecting possible digital image modification. And if an image has been subjected to hard compression, the included EXIF/XMP data will not be visible or reveal anything.
So you risk being left with nothing. And if you can not perform a proper image analysis, you can not conclude anything. You should always have at least a copy of the original photo and not a compressed image from the internet. If an original photo cannot be obtained, I can not in my optics perform a proper photo analysis.
What ELA’s image analysis nor will see, is, if you hang an upturned plate resembling a flying saucer in a fishing line, take a picture of it and claim you have a photo of a UFO. ELA is only effective against digital image modification, not real things that are physically present in a photo.
If an image after analysis on the ELA website shows particular differences in brightness and contrast, the picture has undoubtedly been digitally modified.
However, with ELA, you can always stick to one rule of thumb! If an image after analysis on the ELA website shows particular differences in brightness and contrast (as with the image from CNN), the picture has undoubtedly been digitally modified.
Use the free image analysis tool wisely
After each analysis, ELA offers a direct link to the result of the analysis so you do not need to use the free analysis tool again for the exact same image the following day. A direct link to the ELA result can be found at the bottom left of the ELA results page. Remember to use this link in the future. If you abuse the tool, you risk becoming blacklisted.
The direct link in this case indicates the result of an image analysis of one of my UFO photos mentioned in the article Cigar-shaped UFO and airplane in Sun halo above my hometown Odense (Denmark) back in 2009.
ELA results images on this page
Result 1 LINK
Result 2 LINK
Result 3 LINK
Result 4 LINK
Result 5 LINK
Result 6 LINK
Result 7 LINK
ELA guides (tutorials)
Have you already been to the photoforensics.com website where the ELA analysis tool is available, you have probably discovered how much information there is. Most people would fall backwards and abandon the business to understand all aspects of photo analysis. But somewhere you have to start and you can use the tutorials page as well as take up the challenge and solve some tasks or read their FAQs
I use ELA’s image analysis tool sometimes when some users on Twitter try to fool me with their homemade UFO images. Often it is sufficient simply to enlarge the images and reveal their home-forgery. In most cases, the search for digital image modification with error level analysis has been unnecessary. But it is very good to have if you need to investigate for digital image modification.
Dr. Neal Krawetz critical inputs
I was lucky to meet Dr. Neal Krawetz in a Twitter discussion about a Mars image shared by Twitter user @AntonioParis
Dr. Neal Krawetz and @hackerfactor maintain the foto forensics service after Pete Ringwood, which retired his site in 2012. Dr. Neal Krawetz and @hackerfactor continue this free service, which he should be thanked for.
Unfortunately, he was not satisfied with my article here.
Since I am not really a super expert in this field, but Dr. Neal Krawetz is, it´s easier for me to just show you his critical inputs here. You can then draw your own conclusion based on Dr. Neal Krawetz inputs here.
You quoted "Identical edges must have similar brightness in ELA results." Where did that quote come from? It's not in any of my tutorials. Did you read the tutorial on ELA — especially the section titled "Caveats"? — Dr. Neal Krawetz (@hackerfactor) August 16, 2018
Actually, it´s my own quotes…
"image metadata better known as EXIF data will be able to tell about an photo’s origin"
EXIF is one type of metadata. Not all metadata is EXIF, and metadata does not always tell you about a photo's origin. — Dr. Neal Krawetz (@hackerfactor) August 16, 2018
Thanks for that correction!
"EXIF metadata can not be displayed with ELA’s analysis tool or any other EXIF viewer"
ELA evaluate's compression, not metadata. So: Yes, ELA does not display metadata. (Besides learning what the tool does, you might want to use a spell checker.) — Dr. Neal Krawetz (@hackerfactor) August 16, 2018
Yes, I am still learning…
In your section titled "Basic image metadata": See my tutorial on "Common Mistakes" item #3. Your metadata was stripped. It was last-saved at 82% (via JPEG %; supported by metadata but metadata doesn't have to match). ELA shows attribution added = at least 3 resaves. — Dr. Neal Krawetz (@hackerfactor) August 16, 2018
Thanks for that correction!
Allow me to restate your conclusion:
1. You can't detect what isn't there.
2. These tools evaluate structure, but not the content.
3. You don't understand the impact of a resave.
4. You don't understand how to use metadata. — Dr. Neal Krawetz (@hackerfactor) August 16, 2018
Thanks for that punch!
You wrote "If you abuse the tool, you risk becoming blacklisted." True. On the public site, we ban porn, nudity, and sexually explicit content. We also ban commercial use on the public site, anonymous uploads, bots, resource stressers, and attackers. (See the FAQ.) — Dr. Neal Krawetz (@hackerfactor) August 16, 2018
So now everything should be in its right place…
Related reading
Or go back to category UFO research or homepage Real UFO pictures | tomekkorbak/pile-curse-small | OpenWebText2 |
Article excerpt
INTRODUCTION
Laurie Anne Freeman, a world-renowned expert on information technology and Japanese politics and a professor in the Political Science Department at the University of California, Santa Barbara, received extremely positive reviews from her department until she had two daughters and took leaves to be with them. (1) The reviews she received after returning from her leaves were increasingly critical of her research and productivity. (2) Despite family-friendly university policies, including rules that prohibited consideration of leave time when evaluating productivity, the department repeatedly evaluated her earlier than scheduled and compared her unfavorably with professors who had not taken leaves. (3) When Freeman came up for tenure, she had an impressive list of accomplishments including two prestigious fellowships, one book published and one under contract, and invitations to present her work at leading institutions including Harvard and Stanford. (4) Overwhelmingly negative assessments from her department, however, culminated in a unanimous recommendation to deny tenure. (5) But that was not the end of the road for Freeman. (6)
The Chancellor sent Freeman's case back to the Political Science Department for a new tenure review. (7) Again, the department at tacked her scholarly work. (8) This time, however, the Chancellor could not overlook the overwhelmingly positive assessment of experts in her field and her outstanding resume. (9) The Chancellor granted her tenure. (10) Freeman was not satisfied; she filed a charge of sex discrimination with the Equal Employment Opportunity Commission (the "EEOC"), alleging that her decisions to have children and to use the university's family-friendly policy were the real reasons for her tenure denial. (11) On September 6, 2005, Freeman was granted a rare EEOC cause determination. (12) Charlotte Fishman, Freeman's lawyer, said that she thought the cause determination was important because it drew attention to the sex-plus discrimination that women face in academia. (13) Sex-plus discrimination, however, is not limited to academia. Freeman's story highlights the discrimination that women face in the workplace, even at so-called family-friendly institutions.
Despite legislation designed to promote equality for women and mothers in the workplace, including Title VII of the Civil Rights Act of 1964 ("Title VII"), the Pregnancy Discrimination Act (the "PDA"), and the Family and Medical Leave Act (the "FMLA"), discrimination persists. Role-reinforcing stereotypes (14) and the male-centric job model (15) continue to constrain women. The existing statutes are in large part narrowly applied by the courts and, as written, are insufficient to curtail the problem. The passage of the PDA acknowledged that pregnancy discrimination is a problem and began to roll back the paternalistic treatment of pregnant women, (16) but the PDA has not significantly alleviated the problem of pregnancy discrimination. It has been construed narrowly so that in many jurisdictions it covers only discrimination arising from pregnancy itself, as distinct from its side effects. (17) And the PDA does not grapple with many fundamental issues necessary to secure equality for women in the workplace and at home, such as how to structure the provision of childcare and breast-feeding. Women can attempt to pursue these claims as sex-plus claims under Title VII, but that route has proven to be generally unsuccessful. (18) Thus, many women are left unprotected from discrimination in the workplace based on their status as mothers, childcare providers, and producers of breast milk.
According to one possible indicator, the number of charges filed with the EEOC, pregnancy discrimination is on the rise. (19) With more than sixty-eight million women in the workforce, including 72.9 percent of women with children under age eighteen, (20) in recent years the EEOC has seen a thirty-five percent increase in the number of pregnancy discrimination charges filed when compared with the number of charges filed in 1992, (21) even though the United States has seen a nine percent reduction in its birth rate. …
Related books and articles
Books
Academic journals
Magazines
Newspapers
Encyclopedia
Bringing International Human Rights Law Home: Judicial Colloquium on the Domestic Application of the Convention on the Elimination of All Forms of Discrimination against Women and the Convention on the Rights of the ChildDepartment of Economic and Social Affairs.
United Nations, 2000
Playing by Different Rules: If the Supreme Court Upholds Vouchers, Taxpayers May Have to Fund Sectarian Schools That Practice Religious Discrimination, Teach Controversial Material and Shun AccountabilityBenen, Steve.
Church & State, Vol. 54, No. 10, November 2001
Clinched a Deal Playing Golf? That May Be Sexist; European Rules on 'Indirect Discrimination' Could Outlaw Age-Old Ways of NetworkingPatel, Pravina.
The Mail on Sunday (London, England), October 14, 2001 | tomekkorbak/pile-curse-small | Pile-CC |
It’s midnight in Libya, and the math major from UCLA is standing on an overturned pickup truck screaming, “Libya is great!” He has just survived an amateur “drifting” accident – the pickup he was in tipped over on its side, skidding across Benghazi’s Keish Square at 40 miles an hour – and he is jubilant. With his carefully tousled hair and goofy T-shirt (featuring a cartoon bomb that’s crying while it explodes), he looks like a stoner undergrad on spring break, which, remarkably, he is.
“This is wild,” he says.
There are a thousand or so Libyans standing in the overheated square, watching a 21-year-old Korean-American kid from Orange County pledge his allegiance to their country. Not all of them are amused.
A year before, Chris Jeon knew next to nothing about Libya. In the spring of 2011, as Libyans were rallying in Benghazi, igniting a revolution against Muammar Qaddafi, Jeon was a business-minded junior, angling for a high-paying summer internship at BlackRock, the world’s largest asset-management firm. The pay was good, and the internship was a stepping-stone to a career path he’d spent his life gunning for, but it disappointed Jeon almost instantly. Each monotonous day in his cubicle at BlackRock’s San Francisco headquarters showed him how boring his life could be.
So last August, with the rebels advancing on Qaddafi, Chris Jeon flew to Cairo, hitchhiked across the Libyan border, and joined a rebel battalion. From the outside, it was an inexplicable departure: One week he was a finance trainee in a slick San Francisco office tower; the next he was in the stifling desert, dodging mortar fire and going by the name Ahmed Mugrabi Saidi Barga. To Jeon, however, it made perfect sense. Now, five months after the end of the war, Jeon is back in Libya for spring break. He’s abandoned the idea of a career in banking and says he wants to return to Libya to help his friends rebuild their country. But as he stands on the overturned truck, he seems a little dazed. His eyes are wide with adrenaline. He starts chanting in rudimentary Arabic, trying to lead the crowd in a call and response. They’re not going for it.
“I think we should get out of here,” I say, but he ignores me. Somehow the formation of an angry mob doesn’t seem to bother him. Our translator, who’d been watching the rally from the far end of the square, pushes through the crowd to tell us that we need to leave immediately. Jeon doesn’t want to go – he’s taking pictures now – but the translator is insistent. People are demanding to know who we are. We head to the translator’s car and get in. The crowd follows us. Someone shouts that we’re with the CIA. Dozens of men circle the vehicle. Fists start banging on the roof. “Ameriki go home,” someone screams. Jeon just waves.
“They’re so passionate,” he says. “It’s wonderful.”
The translator gets out to reason with the crowd, and someone puts a gun to his head, forcing him back into the car. A large man with a wide, flattened nose climbs into the passenger seat.
“What’s happening?” I yell, in a panic.
“You’re being kidnapped,” the translator says. I look over at Jeon. He’s laughing.
“You gotta love Libya, right?” he says.
Ladera Ranch bills itself as “one of Orange County’s premier master-planned communities.” The development sprouted out of the brush-covered hills of Southern California in 1999 and offered a slice of sunny perfection to anyone who could afford it. The newly paved streets are lined with saplings, American flags hang from porches, and buyers can choose from a handful of elegant home models. The community of approximately 8,000 households has its own schools, freeways, and shopping centers. Soon after Chris Jeon’s family moved there, the local branch of the American Society of Civil Engineers voted it Project of the Year.
The Jeons came to the U.S. from Korea in the 1980s. Chris’ father, Peter, studied at UCLA, racking up an impressive list of degrees: a bachelor’s, a master’s in mechanical engineering, and a medical degree in dental science. His wife, Jane, became a pharmacist, while Dr. Jeon ran his own orthodontics practice in Orange County. Chris was their eldest, a first-generation American, and his parents had high hopes for him.
He didn’t disappoint: National Honor Society, a 4.3 GPA, vice president of the Future Business Leaders of America. When other kids were shotgunning beers in the parking lot behind the cineplex, Jeon was home practicing piano and guitar. “My parents sacrificed a lot so their kids could have opportunities,” Jeon says. He had no intention of wasting them. “I wanted to be the perfect kid for him,” he says, referring to his father.
He wasn’t exactly perfect. In junior high, he assembled a large-enough collection of death metal – Slayer, In Flames, Cryptopsy – to alarm his Catholic parents, who threw it all out one day when he was at school. “I’ve never been so angry,” Jeon remembers. In one of his first acts of rebellion, he slept in his car in the family driveway for a week.
When Jeon arrived at UCLA in 2008, he had the r é sum é; of a classic overachiever. He joined a frat, drinking his first beer in April of his freshman year. He got properly hammered a few times after that but never lost focus on academics or his dream of eventually landing on Wall Street. “I wanted money, status, stock options, all of it,” he says. “To me, that was the pinnacle of achievement.”
But by junior year, he started to feel anxious, stifled – and not just by academics. At the fraternity, there was talk of going to Canc ún for spring break. It would be awesome: They’d chase girls and get wasted just as they had the year before.
“Why don’t we do something different?” Jeon suggested.
Only one of his brothers took him seriously – Ross McCray. McCray had also grown up in Ladera Ranch, in a house with the same floor plan as Jeon’s. They were both math majors whose fathers were doctors. Somehow, they hadn’t met until UCLA.
Together they decided to fly to Seattle for a week and survive on only a dollar. It was something that kids like them would never consider doing. “We were very, very on-track students,” McCray says. “This was like a release valve.”
McCray dubbed it the “one-dollar trip,” and within days, they were in downtown Seattle with nothing more than a single dollar, their driver’s licenses, and a book containing 400 investment-banking interview questions. (Interviews were looming, and they needed to brush up.) They hadn’t counted on it being 40 degrees and were soon freezing and hungry. They started to beg.
For the next week, they slept in parking garages and homeless shelters, panhandling for food and struggling to stay warm. For Jeon, it was a revelation. “Between Ladera Ranch and UCLA, I’d always lived in a bubble,” he says. “It wasn’t the real world.”
A week later, they started interviewing for summer internships with investment banks. It was a hard transition. One interviewer asked Jeon to estimate the number of golf balls that would fit inside a 747. “Who the fuck cares?” Jeon thought. But his family had worked hard to get him to this point. He ran some numbers in his head and said that roughly 15.7 million golf balls would fit in a 747, assuming you didn’t fill the fuel tanks. A few days later, he was offered a job at BlackRock, one of the world’s largest asset management firms.
That summer, Jeon sat in a cubicle for 12 to 18 hours a day in BlackRock’s San Francisco headquarters. He spent weeks preparing a report on the micro- and macroeconomic potential of the medical-insurance industry and researched the balance sheets of Brazilian mining companies. Jeon’s mind wandered. He’d Google things like “most interesting places in the world” and “unexplored frontiers.” He was ready to start living differently.
He went skydiving, but it wasn’t thrills he was looking for. One weekend, he did a dollar trip to Las Vegas, passing himself off as a bellhop at the MGM Grand even though he wasn’t dressed as one. He simply took bags out of open trunks and led guests on meandering, confused journeys through the massive hotel. Somehow he managed to make $20 in tips. He played craps with the money and drank complimentary cocktails on the pit floor. He returned to BlackRock on Monday, bleary-eyed and unshowered, wearing the same suit he’d left in on Friday. His supervisor warned him that his behavior was unprofessional. He didn’t care.
“Each day at BlackRock felt the same,” he says. “But every day on a one-dollar trip lasted so long. There was so much more substance – the emotions were so intense because I was living on nothing. In terms of experience, I felt like I was getting so much more bang for the buck.”
Interns were expected to keep up on how world affairs might influence oil prices and stock indexes – and Jeon became captivated by the scrappy rebels fighting in Libya. At the time, they were advancing on Tripoli with a ragtag army outfitted with Cold War-era rifles and pickup trucks jury-rigged with antiaircraft guns. It’s like a one-dollar war, he thought.
In early August, Jeon went out for sushi with two other interns, Astrid Fernandes and Letian Zhang. He told them that he was thinking of going to Libya to join the rebellion. It was a chance to see something historic before school started, and he wanted to feel what it was like to have different kinds of problems. “No more PowerPoints or crazy-ass spreadsheets,” he said. Libya seemed like the obvious next step in his journey.
“Are you fucking crazy?” Zhang asked.
Zhang took out a pen and started drawing on the place mat. He’d studied math at Stanford and was heading into a statistics-focused sociology Ph.D. program at Harvard. He made a probability map for Jeon. There was a 25 percent chance Jeon would get shot before making it to the front lines. If he did make it, there was another 25 percent chance he’d be killed in the cross fire, since he didn’t speak any Arabic and had no idea what he was doing. He gave his friend a 50 percent chance of dying.
Jeon felt like he was dying already.
A week later, he and Astrid went to a liquor store, where he bought his first pack of cigarettes. He figured the rebels were heavy smokers, and so he wanted to practice. His friend watched him strike a match, take a drag, and break into a fit of coughing.
Chris Jeon landed in Cairo on August 23, 2011. School didn’t start again for another month, and he had told his parents he was going sightseeing in Egypt. He brought one pair of jeans, three shirts, a leather jacket, a pair of Converse, and two condoms. He hopped a bus in Cairo and headed for Saloum on the Egyptian-Libyan border.
The rebels guarding the border were playing FIFA soccer on a PlayStation when he arrived. Jeon waved at them. They glanced at his passport and went back to their video game. “OK, cool,” Jeon said, and simply walked into Libya.
It looked like the moon: empty, burnt-brown desert stretching for mile after mile. The front lines were 500 miles to the west. Jeon didn’t speak Arabic and hadn’t done much research on the region, but he’d read the Wikipedia page on Libya and watched a bunch of YouTube videos documenting the war. He particularly liked one that showed a group of rebels chanting in unison after a victory – he’d never felt that fired up about anything. The momentum had shifted, and Qaddafi’s grip on the country was weakening. With the support of NATO airpower, the rebels were now attacking Tripoli. Qaddafi was in hiding but issued a statement saying the government was ready to “turn Libya into a volcano of lava and fire under the feet of the invaders and their treacherous agents.” Jeon wanted to find the fighting before it was all over.
At the border, Jeon caught a taxi for the rebel capital of Benghazi, where he planned to hitch a ride to the front. But the taxi was stopped at a checkpoint about 10 miles outside the city. Three rebels peered in and motioned the foreigner out of the car. They asked who he was, and Jeon struggled to explain that he was a UCLA student looking for the front lines. One of the rebels asked in broken English if he was a North Korean spy sent by Qaddafi. The taxi took off, stranding him there. The rebels grew impatient: Who was he here to see? Could anybody in Libya vouch for him?
While at BlackRock, Jeon had emailed the only two people in Benghazi who had posted on CouchSurfing.org, a website that helps travelers find free places to stay. One guy had responded that Jeon should call him when he got to Benghazi. Jeon dug out his number and gave it to the rebels. It was the middle of the night, but someone picked up. Jeon could hear yelling on the other end. He figured that this was as far as his Libyan adventure was going to go. The rebel hung up.
“OK, your friend coming,” the rebel said.
A half hour later, a sleek BMW 7 Series sedan pulled up at the checkpoint, blasting Justin Bieber on the radio. A guy was sitting in another’s lap in the passenger seat, even though there was no one in the back. The passenger door flew open, and Ayman Amzain, Jeon’s couch-surfing contact, bounded out. He had long hair and no front teeth.
“Kreeez!” he said in a high-pitched voice and planted kisses on Jeon’s cheeks. He stepped back and took a good look at Jeon.
“I thought you’d be blond,” he said pouting. “And maybe taller.”
Amzain was a 31-year-old medical student who lived with his parents and dreamed of moving to San Francisco. The appearance of a Californian in Libya was probably as close as he’d get to his dream, and he was thrilled that Jeon had come. In fact, he didn’t want him to leave. He tried convincing his new friend to forget going to the war. But after four days of hookah bars and music videos, Jeon grew restless.
“I was living in a cloud of hairspray and Justin Bieber music,” he says. “It was like the revolution wasn’t even happening.”
Amzain reluctantly arranged for a friend to drive Jeon toward the fighting. He wrote a letter in Arabic and told Jeon to show it to anyone who asked questions. The letter read, “Hello. My name is Chris. I am from the United States. Please help me to go to the front lines. Thank you, and thanks to God.”
Amzain kissed Jeon on both cheeks and told him to come back soon.
In early September, Jeon was dropped at the gates of an oil refinery. He could tell that the front lines were close. Pickup trucks mounted with rocket launchers streamed out of the complex, heading west. Tripoli had fallen, but Qaddafi was still at large and unbowed. On the radio that day, he vowed to fight a “long, drawn-out war.”
Qaddafi’s loyalists had concentrated their firepower in the central coastal region, near his hometown of Sirte. For the revolution to succeed, Qaddafi had to be killed or arrested, and many believed that he was hiding in Sirte, the city the rebels were now pushing toward.
As they rolled out of the refinery, each truck blasted a different song: Tupac bled into high-pitched Arabic music followed by the Scorpions. The men onboard wore green camo with red-checkered kaffiyehs over their faces. One of the trucks stopped, and a young rebel stuck his head out of the window.
“Jackie Chan!” he shouted at Jeon and swung the back door open.
“Holy shit, this is really happening,” Jeon thought as he squeezed in among the men, the RPGs, and the AK-47s. Nobody asked who he was or why he was there. They just handed him a grenade, some earplugs, and a cigarette. He was glad he had practiced smoking.
Twenty minutes later, they stopped on the side of the road, where a clump of pickups stood in the open desert. Suddenly, a shell landed nearby and sent a huge plume of dirt into the air. The rebels in Jeon’s car leaped out of the cab, returning fire with the .50-caliber machine gun mounted on the bed of their truck. Others fired cannons and launched rockets. There was no coordination. Everybody just let loose with every weapon they had, aiming in the general direction of the incoming fire. This was way crazier than anything he’d seen on YouTube.
Shells rained down around them. The rebels panicked, scrambling to get into their vehicles. As they sped away, one of the rebels put his hand on Jeon’s chest and felt his heart thudding heavily. Everybody glanced at Jeon and laughed. He looked terrified.
“Where you stay?” the rebel asked.
“Nowhere,” Jeon replied.
“No problem,” he said. “You stay with us tonight.”
Back at the oil refinery, the rebels had commandeered a newly built two-story town house. There were a half-dozen men sleeping in each room. Two members of the battalion had been killed by a rocket that day, so some floor space had opened up. In the living room, Jeon sat on the ground and introduced himself, but the rebels didn’t like his name. Chris was too short and didn’t sound Libyan.
“We give you new name,” announced Mohammed, an 18-year-old from Benghazi.
Shouting broke out as the rebels debated Jeon’s new name. Finally, Commander Absalam, the group leader, held up his hand and pointed at Jeon.
“Chris no more,” Absalam said solemnly. “Now you are Ahmed Mugrabi Saidi Barga.”
The rebels cheered. The name was a mash-up of all the tribal names represented in the room. They would call him Ahmed.
“Ahmed, you smoke hashish?” Jeon’s second day with his battalion was spent brazenly driving into small desert towns in an attempt to flush out pockets of Qaddafi loyalists by drawing enemy fire. No shots had been fired, but the stress was evident as the rebels passed around the dirty plastic soda bottle they used as a water pipe. When it came to Jeon, he hesitated. He had never done any drugs. It was then that he decided to institute what he called a “yeah, man” policy.
“Yeah, man,” he said, taking a hit. “I smoke hash.”
The next night, the battalion drove to a darkened four-story mansion on the sea. Commander Absalam blasted off the mansion’s door handle, and they walked in. The place had been hastily abandoned. Absalam explained that the only people who lived in houses this nice were people who cooperated with the regime. Therefore, everything in the house was now rebel property.
Clothes still hung in the closets, and there was food in the refrigerator. The rebels grabbed the food and rifled through drawers. Jeon saw a toothbrush lying in the master bath and pocketed it. He hadn’t brushed in days, and he could hear his orthodontist father’s voice nagging in his head.
Hanging on the wall of the living room was a shiny new flatscreen TV. Mansur, a rebel in his late twenties, handed Jeon a hammer and an AK-47. Jeon was confused.
“That’s a good TV,” he said. “Why not take it?”
“Qaddafi people, they hurt my father. They hurt my mother,” Mansur said. “This is Qaddafi people. Fuck this house.”
Jeon felt the weight of the gun in his hands and looked around the room. The leather couches were big and new. Chandeliers hung from the ceiling. It reminded him of all the McMansions back in Ladera Ranch.
He took a hammer to the TV, and then aimed the gun. He pulled the trigger, watching as the chandeliers shattered and crashed to the ground.
The next day, Mansur gave him a Russian-made shotgun. He was no longer just an observer. He was becoming part of the katiba, the Libyan word for brigade. He still didn’t speak much Arabic, but that didn’t seem to matter. There was a cheap Casio keyboard in the town house and when they weren’t on patrol, Jeon taught a skinny 17-year-old named Akram how to play Beethoven. In exchange, Akram showed him how to assemble and break down an AK-47. After two days, the Casio was covered in gun grease, but Akram could play “F ür Elise” and Jeon could field-strip the gun in less than 90 seconds.
Akram spoke English and wanted to know everything about Jeon’s life back in California. Jeon showed him photos of Ladera Ranch on his cellphone. Akram couldn’t believe how beautiful it was and wondered why Jeon would ever want to leave such a place. Akram explained that Libya under Qaddafi was hell. A few months earlier, his cousin had spoken out against the dictator and was executed.
“I am fighting for my cousin, for my family, for my country,” Akram said. “I have no fear because death would be better than living the old way.”
Few of the rebels seemed to care why Jeon was fighting, only that he was willing. On Jeon’s fourth day with the brigade, Commander Absalam told him that the mission they were going on was too dangerous. “You’re not ready to be a martyr,” he told him and dropped Jeon at a rebel staging area in the desert.
Two American reporters, Bradley Hope and Kristen Chick, had just arrived. “We were way, way out there,” Hope says. “And then we saw this college kid with a shotgun and a Lakers jersey. It was mind-boggling.”
Jeon explained that he was on summer vacation and “thought it would be cool to join the rebels.” He added that his parents didn’t know he was in Libya and pleaded with the reporters not to mention him, but both wrote articles about the encounter (“At first glance, Mr. Jeon looked like someone who took a wrong turn on their way to the beach or the Santa Monica Pier,” wrote Hope in a Dubai paper, The National). Jeon’s parents learned he was in Libya when people sent them articles online. They frantically started emailing and phoning news organizations in the region.
“Nobody wants their child to be in a war,” Dr. Jeon said. “Plus, school was starting soon.”
An Al Jazeera news crew spotted Jeon at the refinery where he was staying with his katiba. A network correspondent took out her satellite phone and handed it to Jeon. Within a minute, his parents were on the line.
“You have to come home, Chris,” his dad shouted. “You don’t know what you’re doing.”
“I know exactly what I’m doing,” Jeon snapped.
His mother got on the phone. She was crying. She sounded terrible. She begged him to come home. Jeon told her he’d think about it. He hung up and rejoined his brigade.
A couple of days later, the katiba drove into the desert and fired cannons at loyalist positions. Jeon helped load the ammunition. “My lips were cracked and bleeding, I hadn’t brushed my teeth in days, and my face was peeling, but it didn’t matter,” Jeon says. “I was totally happy – happier than I’d ever been.”
He was standing beside a truck, watching his friends fire the cannons, when he heard the whine of an incoming shell. Everybody dove for cover, and the ground shook. Sand rained down, and somebody screamed. When he finally stood, he saw a mangled, charred body lying near the blast. It was Akram.
He stared at the body. Just a few nights before, Akram had played “F ür Elise,” jumping up and down when he did it without a mistake. He was only 17. “Those fuckers,” Jeon kept saying. Somebody told him to get in a truck, and they retreated.
That afternoon, Commander Absalam sent Jeon with five others to relieve a checkpoint in the desert. Nobody talked on the drive out. When they arrived, they chain-smoked and sat wordlessly under a tarp. It was 110 degrees. Jeon saw three trucks appear out of the heat shimmering off the road in the west. They were moving fast, inbound from Qaddafi-held territory. The rebels around him picked up their weapons, cigarettes dangling from their mouths.
As the trucks approached, Jeon saw someone lean out of one of the windows with a gun. It seemed surreal, like a mirage in the desert heat. The rebels around him started yelling, and he heard bullets whiz past. They were under attack.
“Motherfucker,” Jeon hissed, grabbing an AK out of the bed of a truck. He knew how to assemble and disassemble the gun but had yet to fire it in battle. Now he could see the faces of the loyalist forces as they drove off-road, circling the rebels and strafing the checkpoint. He flipped the safety off.
A bullet pierced the leg of a man next to him. The screaming was buried underneath the report of automatic weapons. Jeon was breathing fast. He popped up from behind his vehicle, took aim, and fired at one of the circling trucks. The gun jerked wildly, and he ran out of bullets. He loaded another clip. This time, when he squeezed the trigger, he saw the passenger’s head snap back – blood splattered the inside of the car.
After another volley, the attackers sped back to the west, and it was quiet again, except for the growls of the wounded. One of the rebels walked up to Jeon and slapped him on the back.
“You are Libyan now,” the man said.
At dusk, he rolled into the refinery with a contingent from the checkpoint. As the daylight faded and the adrenaline drained out of him, he wondered what he’d just done. “I didn’t feel any remorse,” he said. “But I worried about what my parents would think if they found out.” He remembered his mom crying on the phone and got choked up.
“Problem, Ahmed?” asked Abdul Karim, a rebel friend.
“Mia, mia,” Jeon replied in Libyan slang. It meant he was 100 out of 100, feeling fine.
By morning, he’d decided to go home. Chris Jeon was scheduled for a full load of classes the fall semester of his senior year – linear algebra, differential equations, game theory – but by November, he’d not managed to make it to many. He’d lost interest in math and almost everything else he used to care about. He sat in his off-campus apartment and smoked with the shades drawn. An AK-47 bullet dangled from his neck on a leather necklace – a gift from his brigade. He slept during the day and stayed up late so he could talk to his Libyan friends on Skype.
“I gave him shit because he stopped hitting me up and going out,” said McCray, Jeon’s friend from the one-dollar trip to Seattle. “He just disappeared.”
Jeon’s parents were worried too. Peter Jeon, who’d never done anything more adventurous than golf, for fear of hurting his arm and impacting his orthodontics practice, couldn’t understand why their son had sought out a war zone. “All my friends asked me where Chris had come from,” Dr. Jeon says. “I told them I don’t know.”
In the U.S., many had reacted with horror to his story. Wired.com charged him with “turning someone else’s struggle for freedom into your barstool anecdote” and the L.A. Weekly labeled his trip a “mere summer-vacation thrill.” After all, if it was combat experience he was after, he could have walked into a local recruitment office. But Jeon insists that he was simply looking for a deeper, more direct understanding of the world, from homelessness to war. Besides, he’d forged a deep bond with his rebel friends. “These people treated me like I was part of their family,” he said. “They did so much for me, I have to give back.” In January, he decided to go back to Libya during his spring break. I decided to go with him.
The sun is setting in Benghazi when we touch down in April. I’m already having doubts about the journey. Just before getting on the plane, Jeon asked to put a bottle of vodka in my bag. He said there wasn’t room in his.
“Really?” I asked.
“It’s spring break,” he said. “It’ll be fine.”
The first thing we encounter is a stern-looking customs official sitting beneath a sign that says alcohol is strictly prohibited in Libya. Every bag is X-rayed. Soon, he locates the bottle and is raging mad. He pours the bottle down a drain in front of us.
That night, we walk to Freedom Square, where the revolution started. The buildings around the square are lined with oversize photos of rebels who died in the war, which ended just five months ago. A group of men approach and ask where we are from. When Jeon introduces himself, they throw their arms up, shout, and embrace him. They had heard the stories of the Asian kid from L.A. who had fought on their behalf.
“He is famous here,” Mohammed Al Zawwam tells me, explaining that rebel fighters had spread Jeon’s story. Al Zawwam is a 28-year-old youth organizer and gets choked up the more he talks. “I don’t have words to describe how I feel about what he did. He was fighting very bravely for us. He is amazing.”
The next night, Jeon reunites with five of his rebel friends at a second-floor hookah cafe on the outskirts of Benghazi. It’s a fluorescent-lit, smoke-filled room, and soon after we sit down, two guys near the entrance suddenly attack each other. One of the guys lands a series of rapid jabs to the face before he’s pulled off by the staff. Things calm down, and Jeon’s rebel friends tell him he looks fat. Everybody laughs, and the conversation resumes.
Ebrahem Benamer, a 23-year-old guy with a soul patch, tells me that he initially thought Jeon was a Special Forces commando sent by the U.S. to hunt Al Qaeda. Then he saw that Jeon didn’t know where the safety was on a gun, and he concluded that he was just a guy who wanted to help the revolution.
“We thought American people didn’t care about Libya,” Benamer says. “But after we met Ahmed, we realized we were wrong.”
When the hookahs are smoked through, Jeon gets antsy. He’s heard there’s an informal “drifting” competition in the square, and he wants to check it out. When we arrive, four cars whip past us, skidding sideways. There are no barriers between the 1,000-odd spectators and the cars, which skid perilously close to the crowd. Within an hour, we see two sets of cars crash into each other. A spectator tells me that a few months ago, a car smashed into a pack of people, killing three.
Benamer wants to give it a try in his pickup truck, which is still emblazoned with the spray-painted logo of his brigade. He honks his way through the crowd, stomps on the accelerator, and starts fishtailing across the square. When he comes to a stop near us, Jeon yanks open the back door and leaps in. I follow him.
Benamer peels away before I even shut the door. As I struggle to close it, I notice his AK-47 rattling on the floor. It looks like it’s loaded. “Libya drift 2012!” Jeon shouts beside me. He’s filming himself with a pocket camera. Benamer spins the wheel, forcing the car into long, semicontrolled skids, and then accelerates, gunning for a dramatic slide. We spin sideways at about 40 mph, heeling up on two wheels before the truck crashes on its side. Ten minutes after we crawl from the wreckage, we are taken hostage.
My hands are shaking; my breathing is shallow. The car is surrounded by men with machine guns. They are accusing us of being with the CIA, but my translator is sure they just want to rob us.
“These are very bad people,” he says. “They are going to take us into the desert, and we will not come back.”
I can see some crumbled buildings 50 yards away. Maybe we can make a run for it. I look over at Jeon and see him smiling. “Dude, are you scared?” he asks, laughing. “You look scared.”
He seems to be enjoying himself. I want to shake him and tell him to snap out of it. The only thing he’s worried about is that they might take his camera with the car-crash video on it. “Dude,” he says, “I shoved the memory card up my butt so they won’t take it. I don’t want to lose that video of us crashing.”
Another truck of armed militiamen pulls up, and the new arrivals begin arguing with our captors. My translator explains that this is a different militia. We are driven to a militia compound, where the argument continues for hours. Finally, near dawn, we are released with no explanation.
When we get back to our hotel, Jeon is ecstatic. We all are. I am still worried that a militia might track us down at the hotel, but I also feel the rush of being free. It’s six in the morning, and I’m not tired at all. In fact, I feel enormously alive.
“You see what I’m saying about Libya?” Jeon asks me. “It’s amazing.”
He says he craves the instability. “It’s the total opposite of what I was doing before,” he says. It forces him to take nothing for granted, to live in the moment. I can see the logic, but I still want to get the hell out of here. I book a ticket to Istanbul departing the next night.
Later that day, a youth group in Benghazi stages a protest march in the center of town. They’re demanding that the transitional government explain where all the oil revenue is going. Jeon says he wants to attend, to show his support for the new Libya. When I walk over, I see him standing in the middle of a throng holding an Arabic sign over his head. They’re in the street, and cars honk their way through the protesters. I ask Jeon what his sign says.
“Don’t know,” he says, explaining that he can’t read the Arabic. “I just grabbed one.”
He leaves the protest with the sign, still proudly holding it over his head as we walk down the street. Now that we’re away from the relative safety of the march, I tell him to roll it up. “You have no idea how people will react to whatever’s on it,” I say, stopping on the sidewalk.
“It’s all about risk and reward,” he says.
“Exactly,” I almost shout. People are walking past us, looking suspicious. “The risks outweigh the rewards.”
“But you don’t even know what the reward could be,” he says. “Something cool could happen because I’m holding this. That outweighs the risk.”
I hurry back to the hotel and pack my bag. As I’m checking out, I see Jeon in the lobby. He’s heard about some fishermen nearby who throw explosives into the water and then scoop up the fish that float to the surface.
“I’m going to give it a try,” he says, brightly. “I just have to find someone who will sell me some dynamite.”
For access to exclusive gear videos, celebrity interviews, and more, subscribe on YouTube! | tomekkorbak/pile-curse-small | OpenWebText2 |
this would be a registration nightmare. If it was on white the cloud thing and the text would have to be knocked out of two layers. That's thin ass type. If the paper was yellow the halftones of the cloud would have to line up perfect with the green split. That orange would be on top no matter waht. Three colours. total pain in the ass... | tomekkorbak/pile-curse-small | Pile-CC |
How Nextdoor reduced racist posts - pavel_lishin
http://fusion.net/story/340171/how-nextdoor-reduced-racial-profiling/
======
pilom
Finally! This was one of the reasons I stopped actively using Nextdoor. My
neighborhood in Denver is composed of a large group of elderly white people
(who all seem to use Nextdoor somehow) and then a diverse group of people who
have moved into this lower income neighborhood as the old people have passed
away. The amount of profiling was painful. We had a black mentally handicapped
man get picked up by the police because he asked a neighbor to borrow a ladder
and she posted to nextdoor that she thought her house was getting cased to be
robbed (and the police and Nextdoor have a partnership in our area). Hopefully
this helps some.
~~~
smsm42
> black mentally handicapped man get picked up by the police because he asked
> a neighbor to borrow a ladder
I think there's a problem with the police, not Nextdoor, here. I mean, ok,
somebody asked to check out suspicious person (and police, with their
experience, should know how (non-)reliable such reports usually are) and they
did. But picking the person up - which I understand means detaining the person
- is decision that the police officer responding is making. No reporter can
make such decision. And if this decision leads to innocent man borrowing a
ladder being detained - this is not really reporter's fault, it's the police
being unprofessional.
~~~
dopamean
From my perspective as a black man the problem is someone thinking a black man
is suspicious for no reason other than that he's black.
~~~
rubicon33
You are making an assumption that there was no other reason. OP stated the man
was mentally handicapped. Isn't it possible he was also acting strangely?
~~~
hondo77
A reasonable assumption given that the OP didn't give any other reason. Are
you assuming there was another reason?
~~~
smsm42
We don't know anything about the reasons - we have only third-party
information, which describes that some called the police on a mentally
challenged person who had no ill intent, and that person also was black. Maybe
racism added to the picture - quite possible, maybe not - it's impossible to
tell from just this. It is a fact that there are many cases when black people
are treated with prejudice, it is also a fact that there are many cases where
mentally ill people are treated with prejudice, and it is also a fact that
there are many cases where there are misunderstandings between people not
caused by racism or anything like that. If you're a lone elderly woman and see
somebody going about your property that you don't know and behaving weirdly,
you might feel threatened, whether it's warranted or not. Surely, race _may_
have played a role, but we don't have any real reason to conclude either way
without knowing first-person accounts.
~~~
wcummings
You're trying really hard to justify how this isn't racism. The poster lives
there and knows these people, take it or leave it, your opinion isn't
valuable.
~~~
jasikpark
What he's saying is that we don't know. Innocent until proven guilty, maybe?
~~~
wcummings
This isn't a courtroom, it's OP's anecdote. Stop concern trolling.
------
lagadu
I didn't know what nextdoor was (nor do I live where it exists) but I see what
seems to be a fundamental error:
\- They claim to have reduced "racist posts" by 75%.
\- They say they accomplished this by asking extra questions whenever a racial
descriptor was used.
In other words they simply made it harder to post when using a racial
descriptor than not and claim this is a great reduction in racist posting. As
a software developer I'm well aware of one thing: the more complicated I make
a feature's interface be, the fewer people are going to use it. Without them
having enabled the extra difficulty for all posting regardless of having used
the racial descriptor or not, they're simply looking at people who don't post
more using race not because they're racist but because they're placing an
artificial hurdle associated with that.
Now, if they simply wanted for people not to describe race they should've
simply removed it from the options; they seem to be so proud of effectively
doing that but in a (thinly) veiled way, as I understand it they're saying
that any posts that include race of the individual are racist.
Am I missing something obvious here?
~~~
dfabulich
Nextdoor "suspicious activity" reports were notorious for racist reports that
should never have been made. "Everyone be on the look out! I saw a black man
walking around in our neighborhood, possibly looking for houses to rob."
If the UI helps people realize that a black man walking down the street is not
suspicious activity and should not be reported as such, then it is doing its
job effectively.
~~~
cloudjacker
How exactly does that work, given Google Streetview
~~~
mintplant
I really don't understand what you mean by this.
------
karma_vaccum123
I had to quit Nextdoor. The same small cabal of loudmouths was overwhelming
the forums with strong (yet uninformed) opinions, reflex hatred of anything
signalling change, and a general seething that bordered on xenophobia. I
expect some NIMBYism, but this was outrageously militant hatred of the future
masquerading as quaint pride.
Nextdoor also seems to give people strange priorities for civic engagement.
Winning an argument on Nextdoor doesn't change policy...speaking at
Council/School Board meetings does, or better yet, running in an election.
Lots of people on our local Nextdoor seemed frustrated that the Council were
voting contrary to some conclusion that was reached on the site. They failed
to realize they needed to be making arguments where it mattered - at Council
meetings, not online in some private forum.
~~~
fapjacks
Right, exactly! The one thing Nextdoor taught me is that people are truly just
as awful as you could imagine. I have lived in nice neighborhoods -- as I
imagine a fair amount of HN readers do -- and the blase of outright totally
racist posts just... Well, it was just shocking, and I have to say that I'm
not shocked by very many things. That some people _know_ that they're posting
to their physical _neighbors_ and still saying the things they say... It's
almost like they live on another planet altogether. Now, another very weird
thing is that I've moved into a new neighborhood literally down the street
from my old one, and I was equally shocked to learn that this particular
neighborhood is totally reasonable, and I haven't even seen a single racist
hate-post.
Nextdoor definitely exposed me to the racist, backwards reality of living
among old, wealthy, scared, white people.
~~~
mavhc
Use it to lie to them about when elections are.
The thing that's worse about elections being won by idiots and racists is now
you know that most people around you are idiots, racists, or happy to stand
next to the racist idiots.
~~~
fapjacks
I regret that I can only upvote your comment a single time.
------
TheGRS
Our company recently did a trial of looking for unconscious bias when we are
making decisions, especially in terms of hiring practices. We all took a test
to see if we had any unconscious bias toward gender and then discussed our
results as a group. What really stuck with me is not that we have these sort
of tendencies, but how shocked most people were at their own results. Most men
in the room trended toward a bias toward men and women toward women. That
shouldn't be that surprising really, but many were very disappointed in their
results.
I've been thinking it would be good to do another round of that with the race
test, especially considering how predominately white our workplace is. I don't
believe everyone is actively trying to be racist or sexist (though some
certainly are), but we might not realize that we're doing that on an
unconscious level.
The tests are available here if you'd like to give it a try yourself, doesn't
take very long.
[https://implicit.harvard.edu/implicit/selectatest.html](https://implicit.harvard.edu/implicit/selectatest.html)
~~~
allendoerfer
Inside the German version there is a test for biases against Western or
Eastern Germany. I knew that I had a bias, but I was surprised that I found it
that difficult when moving a bunch of Eastern German cities to one group to
associate negative things to the group with the Western German cities.
The test might not be totally fair, though. Is it actually a bias if it is
based on facts? Soviet architecture, higher unemployment, high rate of
extremism, lower buying power, less people, people moving to the West, the
list goes on and on. I think stereotypes and preconceived notions are bad most
of the time, but I see no reason to get rid of the ones that are actually true
for political correctness.
~~~
Mithaldu
What is the german version of nextdoor? I've never heard of such a thing.
~~~
Jarwain
I think he's referring to the german version of the implicit association test
------
pavlov
This could work on HN too. A simple list of combinations of keywords (such as
"Node.js" and "shit") detected in a comment submission could trigger a
confirmation dialog: "Are you sure this is a meaningful contribution?"
~~~
beat
I'm not sure Node.js is a meaningful contribution, no. :)
~~~
frogpelt
That's the joke.
------
randomanybody
It's interesting how different neighborhoods can be in the same city. In
Seattle, my old neighborhood's board (First Hill) was vastly different than
that of Ballard, to the point where I wasn't sure my friends who lived there
were talking about the same site. The latter sounded like it was filled
entirely with militant, yet simultaneously skittish NIMBYers, while mine was
filled with park get-togethers and lost puppies. And since you could only see
one neighborhood, the other experience was invisible to me.
~~~
techsupporter
I live in the Central District of Seattle and a friend, who made an account
and lets me log in so I can read, lives in Wallingford. The differences are
appalling. The CD has its share of minor crime but reports of suspicious
activity are accompanied by descriptions beyond race or, sometimes, security
camera output. This is mixed in with posts about giveaways, contractor
recommendations, and Metro bus reroutes due to construction.
Meanwhile, my friend's account showed lots of posts like described here. Oh,
and the slagging on the homeless was just vile. I have screenshots of several
examples but I don't want Nextdoor to go all Erica Barnett on me and ban
accounts so I leave them out. But the police department had a lot more
"community forums" on the Wallingford part of Nextdoor than the CD's...
------
hackuser
The Washington Post covering the same story:
[https://www.washingtonpost.com/news/the-
switch/wp/2016/08/26...](https://www.washingtonpost.com/news/the-
switch/wp/2016/08/26/how-this-social-network-for-neighborhoods-is-trying-to-
fix-its-racism-problem/)
\----
And also the WP covering a similar problem in Georgetown (a wealthy district
in Washington):
* The secret surveillance of ‘suspicious’ blacks in one of the nation’s poshest neighborhoods
[https://www.washingtonpost.com/local/social-issues/the-
secre...](https://www.washingtonpost.com/local/social-issues/the-secret-
surveillance-of-suspicious-blacks-in-one-of-the-nations-poshest-
neighborhoods/2015/10/13/2e47236c-6c4d-11e5-b31c-d80d62b53e28_story.html)
* The black man arrested in Georgetown because he looked like a shoplifter
[https://www.washingtonpost.com/local/social-issues/the-
black...](https://www.washingtonpost.com/local/social-issues/the-black-man-
arrested-in-georgetown-because-he-looked-like-a-
shoplifter/2015/10/16/0a93fd00-7409-11e5-9cbb-790369643cf9_story.html)
* Georgetown social network accused of racial profiling is suspended
[https://www.washingtonpost.com/local/georgetown-social-
netwo...](https://www.washingtonpost.com/local/georgetown-social-network-
accused-of-racial-profiling-is-
suspended/2015/10/19/7a8865a2-7674-11e5-b9c1-f03c48c96ac2_story.html)
(Wow. What is the shortest possible Washington Post URL?)
------
daenz
Side note, brilliant marketing. This is such a hot-button topic, that although
I disagree fundamentally with their solution, it will definitely have a lot of
people talking about it.
I wish he had gone into more details about what counts as a "racist post." He
seems to imply that people were just saying "black dude walking around." But
would "black man peering into parked cars" also be flagged? And do those extra
barriers, existing solely because of the word "black", lower the reporting
rate of those kinds of posts?
~~~
clifanatic
Or "man peering into black cars".
------
eknight15
Great to see design changes that have a positive social impact.
I really like Nextdoor, it's one of the few social networks I feel good using.
------
oldmanjay
While I laud the goals, it's sort of disappointing to see such adherence to
the idea that suppressing the expression of racism is going to fix it.
~~~
ap3
A racist crime report will have real world consequences though - it's not just
thoughts in someones head.
It will involve other people and potentially the police.
~~~
beat
Remember, these kinds of racially biased reports can get people _killed_.
That's not insignificant.
~~~
smsm42
Reports don't get people killed. People (usually, police) shooting other
people get people killed. Reporter has no control (unless they specifically
and purposely misrepresent the situation, e.g. falsely reporting somebody
firing a gun when they have none) over police actions. The police should bear
the responsibility for the outcomes, not reporters or online platform or any
other factor that did not actually make a decision to pull the trigger.
~~~
KingMob
Are plane crashes are only caused by gravity, or is that engine that caught
fire somehow related?
Please, don't be pedantic. Police make mistakes, and have their own biases, to
boot. If you call them on some random non-white person in your neighborhood,
and disaster ensues, you're partly culpable.
~~~
smsm42
It's not pedantry. Gravity has no will and no choice, thus blaming gravity is
useless. Police does have will and choice - it's not a law of nature that if
you call the police they'll kill somebody, police is not a gun or any other
mechanism that you just fire. These are people, who have free will, minds and
responsibility for their actions. That's the whole point, only humans can take
blame, and when their choices result in bad results, they _should_ take blame
for their choices, not redirect it to others.
> Police make mistakes, and have their own biases, to boot.
You say it as if it's some kind of excuse. It's funny that for reporter have
biases is a culpable offense, but for the police it comes out as a defense
instead. It doesn't work this way. In fact, random civilians have full right
to be wrong, police are (or supposed to be) trained professionals that are
paid to be better than civilians at this. So for them to behave worse than a
random civilian should be a firing offense, not an excuse.
> If you call them on some random non-white person in your neighborhood, and
> disaster ensues, you're partly culpable.
Nope. The police function is to protect people. If they instead hurt innocent
people, this is their fault, not somebody's that called on their direct and
proper function. Their refusal to perform the function is their fault. Just as
if you walk on the street and get mugged, you are not "partly culpable",
because you could've walked on other street or stayed home - people who mugged
you are fully culpable, because it was their choice to do it.
If you assign blame at the wrong place, they only result would be that people
that need to change won't change, and you get more of the same problem. Which
we are amply witnessing right now.
~~~
KingMob
But the sad reality is whether it's a mistake or prejudice, police wield
deadly force, so calling them always has the possibility of someone dying.
Should they be held to a higher standard? Yes. But many aren't, and until that
happens, thinking otherwise is wishful or naive. If you call the police, and
it spirals out of control, you DO bear some measure of responsibility. Not as
much as the actual police, but it's not zero, either, especially if your call
is racially motivated.
Mugging isn't a counterexample, because the mugger and the muggee have
completely opposite desires in the situation. Nobody calls up a mugger and
invites them to their neighborhood. Whereas, when you call the cops, you're
somewhat in alignment.
------
smsm42
Well, it looks like they define racist post as:
The company decided to define it as descriptions of criminal behavior that
don’t have sufficient description, i.e. “dark-skinned man broke into a car” or
a detailed description of someone, including race, that fails to describe them
doing something sufficiently criminal.
I.e. it is a description that mentions race and lacks either: a) specific
criminal behavior or b) other non-race details.
In this case, no wonder that forcing people to provide these details reduces
number of such posts - it's practically guaranteed by the very definition!
I also wonder about this:
Nextdoor is aware of “two instances of racial profiling that had slipped
through its algorithms in the last few months.”
Nextdoor is a pretty large network. And they have quite a lot of people there,
tens if not hundreds of thousands at least. And only _two_ cases of profiling
in several months, less then one case per month over whole network? It's
either nextdoor users actually are nearly saints in this regard, at least
compared to regular population, or they are humblebragging, or the problem
itself is really minuscule, or they are blind to it.
------
jecjec
Is Nextdoor wise to pursue this end? (pdf)
[http://2kpcwh2r7phz1nq4jj237m22.wpengine.netdna-
cdn.com/wp-c...](http://2kpcwh2r7phz1nq4jj237m22.wpengine.netdna-cdn.com/wp-
content/uploads/2016/03/Color-Of-Crime-2016.pdf)
Synopsis: [http://www.amren.com/the-color-of-crime/](http://www.amren.com/the-
color-of-crime/)
------
randyrand
I understand that it's racial bias to assume that someone is more likely to
commit a crime given the color of their skin, but at the same time given that
thats a reasonable assumption, I also think it's very reasonable to put your
own safety above social progress.
Are my priorities not inline?
------
tptacek
I didn't even know Nextdoor was a thing until I saw this post. Where, once
logged in, would I look to see random people being reported as suspicious by
my neighbors?
~~~
mercutio2
Well, you can't get an account until you validate your postal address, then
you only get access to posts from your neighborhood and adjoining
neighborhoods.
Once you've signed up, if you install the app, you can configure push
notifications about crimes in progress. You can also get emails for urgent
things, if you prefer.
Whether this improves, or diminishes, security and quality of life relative to
reading your local newspaper's coverage, is unclear to me. Even when it's not
subtly racist or ageist profiling, I've never found the alerts to be
actionable.
But the hyperlocal idea is at least an interesting one.
------
jomamaxx
Who decides what 'racism' is?
It's a pretty tricky thing.
Should our corporate overlords be making this decision?
Outside of hate speech, which has some legal parameters, this is pretty tough.
Especially if it's people's personal communications.
Some people think that speaking out against 'Black Lives Matter' is racist,
while others think that 'Black Lives Matter' is in fact racist.
It's a thin line.
------
thescribe
I wonder if they're just scanning for the word "black" or other races too.
Edit:
I answer my own question pretty quickly but it led to a second thought. I'm
curious if the reduction is just people not reporting things because there are
more steps now.
~~~
LeoPanthera
It says right on the article.
"Others actually had their posts scanned for mentions of race (based on a list
of hundreds of terms Nextdoor came up with)"
Edit: You edited your post. Your original comment I was replying to said
something along the lines of "I wonder if they're just scanning for the word
"black" or other races too."
~~~
thescribe
Shoot sorry about that, I hadn't seen your comment. I'll revert my post.
| tomekkorbak/pile-curse-small | HackerNews |
Lewis Hamilton claimed pole position for the Malaysian Grand Prix and in doing so is in the best position to deal a potentially decisive blow to his championship rival Sebastian Vettel, who failed to set a time at Sepang and will start from the back of the grid. Kimi Raikkonen in the sister Ferrari was in second place and the Red Bull of Max Verstappen in third.
Lewis Hamilton joins chorus of support for NFL anthem protests Read more
After going out on the first lap at the last round in Singapore, Vettel suffered a huge setback to his championship hopes. A mechanical problem prevented the German from setting a time in Q1. He trails Hamilton by 28 points and up until the start of qualifying had looked a strong candidate for pole but will now at best be attempting to minimise his losses on Sunday.
Hamilton had struggled with his car all weekend but on the final runs in Q3, put together some exceptional laps. For his first run, a 1min 30.076, he finally had the car hooked up and once again made the most of it. He was unable to improve on the second run but despite an impressive final lap from Raikkonen – the Finn came to within four-hundredths of a second – it was not quite enough.
“Somehow we turned it round, the car felt great, it is a real surprise to be up here,” said Hamilton. “We are going to have a tough battle but I am hoping our car has moved in the right direction for race day.” His ninth pole of the season, takes him to a career total of 70 and is a clear reminder of how strong he has been over the single lap in Sepang. He now has five poles here, equal with Michael Schumacher’s record. The serious work has still to be done however, only once before has he converted pole to a win, in 2014, also his only victory at the race.
Mercedes were expected to be strong in Malaysia, the long straights and sweeping high-speed corners, similar to Silverstone and Monza playing to the strengths of their car and Hamilton, as he had in Britain and Italy, exploited it to maximum effect when it mattered.
It had not come easily, however. The team had brought upgrades to Malaysia but struggled with balance, set-up and a lack of grip in practice. On Friday they were well off the pace, Hamilton was sixth in the second session and the team believed the car had a fundamental issue they needed to solve but he was still over a half a second back in fifth on Saturday morning.
F1 plan to level the financial playing field can’t come soon enough | Giles Richards Read more
Turning it around for qualifying was a mighty effort and once again he proved that he can deliver an inch perfect lap when it matters. He will be taking nothing for granted, however. It was at Malaysia last year that he suffered an engine failure that dealt what was to prove an insurmountable blow to his title hopes. Vettel had an electrical problem in final practice and the team opted to change their engine as a precaution, fitting the new upgraded iteration. They had little time to make the change but had done so in time for the start of the session. On his first run he reported a turbo problem and was forced to return to the pits.
The mechanics struggled to solve the issue but were unable to do so and he could take no further part in the session. He had looked destined for a strong weekend, having been quickest in the second practice session and second on Saturday morning, well clear of Hamilton.
“It’s part of motor racing,” he said. “It’s not ideal, especially when you feel you have got it in the car. It was a bad day but we will make the most of tomorrow.” Having failed to set a time, Vettel will start from the back the grid. Since penalties will make no difference to his starting position, the team are likely to take a raft of new powerunit components, including the turbo, of which he has used his allocated four of this season.
Daniel Ricciardo in the second Red Bull was in fourth, followed by Valtteri Bottas in the Mercedes. The two Force India’s of Esteban Ocon and Sergio Pérez finished in sixth and ninth respectively. McLaren’s Stoffel Vandoorne showed strong form to take seventh, with his team-mate Fernando Alonso in 10th. Renault’s Nico Hülkenberg finished in eighth.
The young French driver, Pierre Gasly, making his Formula One debut having been brought in for this race to replace Daniil Kvyat at Toro Rosso as the team assess his potential for a drive in 2018, proved very impressive. He finished in 15th just two tenths off his team-mate Carlos Sainz, in 14th.
The Williams of Felipe Massa was in 11th, with his team-mate Lance Stroll in 13th and Renault’s Jolyon Palmer in 12th. Romain Grosjean and Kevin Magnussen of Haas were in 16th and 17th respectively, with the two Sauber’s of Pascal Wehrlein and Marcus Ericsson in 18th and 19th. | tomekkorbak/pile-curse-small | OpenWebText2 |
The October 14 suicide by Jamie Hubley, a 15-year-old gay high-school student from Ottawa, has intensified public pressure on schools to protect LGBT students from bullying. In response, the Ontario government will require all state-financed schools to offer extra-curricular clubs to support gay teenagers as part of the province’s new anti-bullying legislation. This includes schools under the jurisdiction of Ontario’s Catholic school boards, which have been reluctant to offer such support programs due to the Roman Catholic Church’s prohibition of homosexuality.
The new directive has been criticized by several faith groups that disagree with state imposition of gay-straight alliances in Ontario schools. In an interview with CBC Radio’s As It Happens, the bombastic, conservative evangelist Charles McVety characterized the initiative as a violation of religious freedom and an affront to the separation of church and state. Despite several prompts from host Helen Mann, he avoided acknowledging that the bullying of gay children was a specific issue, preferring to speak of bullying as a general problem that does not require the creation of such clubs. More significantly, McVety claimed that gay-straight alliances have nothing to do with preventing bullying and are instead a radical and untried social experiment.
McVety is wrong on all counts, but most seriously on the point about church/state separation. Much to my chagrin, Canada does not have a constitutional separation of religion and government, which differentiates it from the United States. However, the concept of political secularism and government neutrality in religious matters is an understood fundamental of Canadian political culture. Nevertheless, the provincial government is not telling the Catholic Church what to do, but is rather instructing the schools for which it is financially responsible and publicly accountable. If there is any abuse of secularism here, it is that the religious schools of one faith group are being funded by the state in the first place.
McVety’s point about the concealed intentions of gay-straight alliances is equally fatuous. Such groups are not created for the promotion of homosexual activity but instead as a safe place for students to go with people whom they can trust. Although young people are bullied for many reasons, children who are gay or perceived to be gay are especially targeted for harassment and intimidation. The result of this bullying is a high rate of depression among the victims, with about a third of gay teens attempting suicide at least once.
It is especially important that gay teens have access to counseling and support at school because it is often the only place where this is possible. While children who are bullied due to their family or religious background can go home to the support of their parents and communities, many gay teens actually fear their parents, especially if they have been raised in a religious household and risk being made homeless. The group environment of gay-straight alliances serves as an ideal means of supporting victims because it reinforces the notion that gay students are not alone and that not every person is out to get them.
Any honest supporter of gay rights is forced to accept that a significant number of Canadians continue to believe that same-sex relationships are not a legitimate or respectable form of love, and that an even greater number oppose the legal recognition of such relationships by the state. Therefore, this remains an issue that we must continue to debate in the public square, but not on the backs of innocent teenagers, who are often unable to deal with their own circumstances, let alone those of a fiery ethical dilemma.
We are quite often reminded that the Christian attitude toward homosexuality is to condemn sexual acts between persons of the same gender while loving the sinners for whom they are. Revolting a piece of casuistry as this is, we ought to hold Christians to their word when they claim to love everyone unconditionally. I would venture to guess that most gay teenagers in middle school and high school are not engaging in homosexual activity, and even if they were, this would not be the reason for which they are picked on.
Gay teens are harassed because they often look and act differently than their peers and have feelings that they do not choose. Many are subjected to violence and intimidation even before they understand what homosexuality means, let alone that they may themselves be gay.
Bullying is a man-made problem with a man-made solution: austere penalties and public embarrassment for the offenders, and uncompromising support for the victims. All that is required is a bit of courage and honesty from school officials and the general population. Choosing to abandon bullied children is not a conservative position, but rather a cowardly and philistine one. Charles McVety can continue to be contemptuous of gay victims of bullying, whose suffering he neither understands nor appreciates, but as long as the public shares responsibility for the upbringing and education of children, we have a duty to ensure that everyone can attend school in a safe and secure environment. | tomekkorbak/pile-curse-small | Pile-CC |
Options for tele-intensive care unit design: centralized versus decentralized and other considerations: it is not just a "another black sedan".
This article seeks assist physicians or administrators considering establishing a Tele-ICU. Owing to an apparent domination of the Tele-ICU field by a single vendor, some may believe that there is only one design option. In fact, there are many alternative design formats that do not require the consumer to possess high-level technical expertise. As when purchasing any major item, if the consumer can formulate basic concepts of design and research the various vendors, then the consumer can develop the Tele-ICU system best for their facility, finances, availability of staff, coverage model, and quality metric goals. | tomekkorbak/pile-curse-small | PubMed Abstracts |
Introduction
============
Biophysical and biochemical investigations of protein structure and function often rely on the availability of a large amount of a protein sample. To meet this demand, proteins are commonly overexpressed by recombinant technology and purified using affinity chromatography techniques.[@b1]--[@b11] During such processes, considerable time and effort is spent to estimate the quantity and purity of the expressed target protein.[@b12] A standard procedure for protein purification involves running PAGE multiple times to measure the expression rate, the concentration of the target protein, and the purities of all the fractions eluted from chromatography to identify the fractions containing the target protein.[@b13] PAGE requires a variety of equipment and involves many time-consuming tasks.[@b14] Herein, we report a complementary method that facilitates expression test and quantification of concentration and purity without running several PAGEs.
The main idea of the new method is to use Photoactive Yellow Protein (PYP), or its truncated version, as a fusion partner with an affinity tag \[[Fig. 1](#fig01){ref-type="fig"}(A)\]. Although making the gene construct of a target protein, one can use PYP with an affinity tag as a tagging partner. PYP is a small (∼14 kDa) and highly soluble protein manifesting in *Halorhodospira halophila* and carries out the first step of phototaxis signal transduction via changing its structure upon absorbing blue light.[@b15]--[@b30] Because PYP appears in only one species, there is no need to make PYP-deficient cells, and thus PYP is appropriate for global tagging. PYP has p-coumaric acid as its chromophore. In the absence of chromophore, the apo PYP has no absorption in the visible region, but the holo PYP bound with the chromophore strongly absorbs in the visible region, exhibiting intense yellow color. This property makes PYP a useful Off/On system[@b31]--[@b33] that we named PYP turn Off/On Label (POOL). During the protein expression, chromophore-deficient apo-PYP is co-expressed with the target protein. The appearance of yellow color after adding a precursor of chromophore to the co-expressed PYP can be used to check the expression of the target protein via visual inspection within a few seconds. In addition, the extinction coefficient of PYP is high and well established (45,500 *M*^−1^ cm^−1^ at 446 nm for the wild type and 53,800 *M*^−1^ cm^−1^ at 460 nm for the E46Q mutant),[@b34] and thus it is possible to detect the exact amount of target protein by measuring the UV--VIS spectrum. Moreover, POOL is capable of estimating the purity of the target protein based on the ratio of absorbance at 280 nm and 460 nm (see below).
{#fig01}
Results
=======
Instant colorimetric inspection of protein expression level and amount of target protein during purification
------------------------------------------------------------------------------------------------------------
The usefulness of POOL was tested for a number of model target proteins. E46Q mutant PYP was used instead of the wild type because the higher extinction coefficient of E46Q[@b35] provides slightly higher sensitivity than the wild type. The first model protein was Small Ubiquitin like Modifier protein (SUMO) that has 98 amino acids (10,780 Da).[@b36],[@b37] A precursor of chromophore, anhydride p-coumaric acid,[@b31] was added to the SUMO-POOL-expressed cell lysate to check the appearance of yellow color. As shown in the [Figure 2](#fig02){ref-type="fig"}(A), prior to the addition of the precursor of chromophore, the cell lysate (1 mL of cultured medium) had no visible color. Upon the addition of the precursor of chromophore, the sample immediately exhibited a yellowish color. One can even approximate the amount of the target protein by comparing the color with those of standard solutions containing PYP at various known concentrations. We could clearly see that the concentration of the target protein is between 10 and 60 μ*M* as shown in [Figure 2](#fig02){ref-type="fig"}(C). To enhance the reliability of the practice, as a control, we added the precursor of chromophore to the *E. coli* lysate in which only the enterokinase[@b38],[@b39] was expressed. The fraction exhibited no visible color, even after the addition of the precursor of chromophore.
{#fig02}
This advantage of POOL is even more pronounced in the protein purification steps. Each fraction from various protein separation procedures, such as affinity chromatography or ion-exchange chromatography, can be directly examined by POOL. [Figure 3](#fig03){ref-type="fig"} shows several eluted fractions after the ion exchange chromatography and a comparison between the results of PAGE and POOL. The presence of strong yellow color in the eluted fractions indicates the presence of the target protein (SUMO in this case), which is double confirmed by PAGE gels and UV--Visible spectra. The fraction without yellow color does not have the target protein and the fraction with yellow color has high concentration of the target protein. The UV--Visible spectra show consistent results; the fraction without yellow color has no peak near 460 nm and the fraction with yellow color has a dominant peak near 460 nm. Therefore one can select the fractions containing the target protein on the basis of the presence of yellow color, and thus can save time because the collection of the fractions can be immediately stopped as soon as the fraction lacking yellow color starts to be eluted.
{#fig03}
Rapid determination of the target protein concentration with a UV--Visible spectrometer
---------------------------------------------------------------------------------------
More accurate and sensitive quantification of the target protein concentration can be achieved by measuring the UV--Visible spectrum instead of the visual colorimetric detection. After the addition of the precursor of chromophore, the lysate containing a POOL fusion protein showed a distinguishable peak near the 460 nm region of the UV--Visible spectrum. The concentration can be easily calculated by using the following simple relation based on Bear-Lambert law,
where *A*~460~ and *B*~460~ are the absorbances at 460 nm after and before chromophore addition and the path length of the cell is assumed to be 1 cm. *B*~460~ is taken into account because *E. coli* extract solution sometimes have absorption at 460 nm. In the SUMO-POOL case, the absorbances at 460 nm of the solution diluted by a factor of 60 were 0 and 0.022, respectively, before and after chromophore addition. Thus, according to Eq. ([1](#m1){ref-type="disp-formula"}), the concentration is 0.0245 m*M,* which is consistent with the result obtained from the colorimetric method. To provide quantitative evidence for the accuracy of the POOL-assisted quantification, we measured the protein concentration by using a commercially available tool, the BCA kit (Pierce, Thermo Scientific, Rockford, IL) and compared the results with those of POOL. For this, we purified SUMO-PYP without chromophore and prepared solutions of various concentrations. Their concentrations were measured by both the BCA Kit and the POOL method. As can be seen in [Figure 2](#fig02){ref-type="fig"}(D), the resulting concentrations show satisfactory correlation with the slope of 0.99. We note that the time consumed for the measurements were ∼1 h and a few minutes for the BCA Kit and POOL, respectively. The concentration determined by POOL shows ∼4% deviation compared with that by the BCA Kit.
Rapid determination of the target protein purity during protein purification
----------------------------------------------------------------------------
After purification by chromatography, the purity, as well as the concentration of the target protein, can be easily estimated by using a UV--Visible spectrometer or a microplate absorbance reader using the following equation,
*A*~460~ is the absorbance at 460 nm, *A*~280~ is the absorbance at 280 nm, *Y* is the molecular weight of PYP or it mutant, and MW is the molecular weight of the target protein. Here, the rather crude approximation that *A*~280~ roughly corresponds to the concentration (mg/mL) of all proteins in the solution is used.[@b40] If one selects several fractions from chromatography where the yellow color begins and ends and measures the absorbance at 280 and 460 nm to identify their corresponding purities, it becomes very easy to determine the fractions containing the target protein with enough concentration and purity. Thus, running PAGE for every single eluted fraction to identify the fractions containing the target protein becomes unnecessary. If a microplate absorbance reader capable of simultaneously measuring the UV--Visible spectra of several wells is used, the entire process would take only a few minutes.
Application of POOL for high-throughput protein expression screening
--------------------------------------------------------------------
We applied POOL to high-throughput expression screening to obtain optimal conditions for protein expression for human calmodulin, mercury binding peptide (MerP),[@b41] and horse myoglobin (Mb). We used a microplate absorbance reader and measured the absorbance at 460 nm to determine the degree of target protein expression and calculated the concentration ([Fig. 4](#fig04){ref-type="fig"}); the testing yielded the results consistent with those obtained by the colorimetric method. [Figure 4](#fig04){ref-type="fig"} shows that the addition of a chromophore precursor induces an immediate color change for the target protein. Therefore, it is possible to obtain an approximate measure of quantity of the expressed protein through the instant colorimetric method and thus to determine the optimal expression condition for the target protein. Under our testing conditions, we can see that the human calmodulin expression reaches its maximum at 18°C with the IPTG concentration of 0.2 m*M* using pQE80L vector. Furthermore, mercury binding peptides are well expressed under all conditions and the horse myoglobin shows a similar trend to that of human calmodulin. These tests prove that the simplistic nature of the POOL method allows convenient high-throughput protein expression screening to obtain optimal conditions for soluble protein expression.
{#fig04}
Application of POOL to eukaryote cells
--------------------------------------
POOL is also applicable to target proteins expressed in eukaryote cells. We checked the expression of the membrane protein CD40 in the mammalian 293 cell line and the truncated TLR3 (toll-like receptor 3 without transmembrane domain) in the insect Sf9 cell line[@b42],[@b43] (Supporting Information [Fig. S4](#SD1){ref-type="supplementary-material"}). In both cases, we could easily perform an expression test simply by adding the chromophore precursor.
Making mini-PYP for POOL
------------------------
Although the full-length PYP is highly soluble and does not interfere with the expression of other proteins, a tag of a smaller size is preferred because it is generally expected to interfere less with the expression of the target protein.[@b44] Through recombinant-biotechnology approaches, we have identified the most basic segment of PYP capable of binding to deprotonated p-coumaric acid and thus being used as a POOL tag. We tested various N-terminal and C-terminal deletion mutants of PYP fused with SUMO ([Fig. 5](#fig05){ref-type="fig"}). First, we produced eight types of N-terminal deletion mutants \[deletion of residues 1--5 (N5), 1--10 (N10), 1--18 (N18), 1--27 (N27), 1--31 (N31), 1--40 (N40), 1--42 (N42), and 1--46 (N46)\] and three types of C-terminal deletion mutants \[deletion of residues 73--125 (C53), 107--125 (C18), and 117--125 (C8)\]. Of all the deletion mutants, three mutants, N15, N10 and N18, show consistent color change, whereas the rest initially show yellow color but gradually lose their color within several minutes. Actually, all the tested mutants can be used to check expressions as the color persisted for at least more than half a minute. We also designed truncated PYP by removing both the N-terminal and C-terminal residues (deletion of residues 1--27 and 107--125 (N27C18)).[@b44] This mini-PYP that has the 28--106 residues of the original PYP can be also used as a POOL tag. However, to obtain an accurate value of concentration and purity, at least the N18 mutant should be used.
{#fig05}
Discussion
==========
The genetically engineered POOL enables us to instantly quantify the concentration and purity of a target protein, thereby aiding the protein expression and purification. This confirms that, with POOL, one can check the amount of protein expression by simply observing the color change, and thus significantly reduces the time required for checking the amount of protein expression. For example, on average the usage of PAGE for checking protein expression requires several hours, whereas the POOL approach requires only a few seconds. UV--Visible absorbance measurement allows determination of the concentration and purity of the target protein within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show an excellent correlation with those by the BCA Kit, which usually takes ∼1 h \[[Fig. 2](#fig02){ref-type="fig"}(D)\]. POOL also allows instant approximation of the amount of protein in the fractions after chromatography by simply comparing colors and easy selection of the fractions containing the highly pure target protein. POOL can be used in eukaryotic cells as well as E. coli. For example, even if the membrane protein CD40 has a low expression rate, we could detect color change in the cell lysate upon addition of the chromophore precursor.
The instant turn/on property of POOL makes it an excellent method for high-throughput expression screening. Conventional methods for high-throughput sample detection[@b45]--[@b49] and quantification such as affinity columns, PAGEs, and immune-detection of dot blots via usage of antibodies[@b50] demand significant time and effort or lack accuracy. Compared to those methods, POOL affords easy fast and accurate high-throughput screening. Using POOL together with a microplate absorbance reader can also yield accurate measurements since a spectrometer possesses high sensitivity. The high sensitivity of POOL eliminates the need for the step of concentration via capture, such as affinity column chromatography. POOL may be comparable to the high-throughput protein expression screening[@b51],[@b52] using GFP as a fluorescence tag. In addition, the turn-on property and the smaller size (see below) of POOL may provide additional advantages.
When compared with the other protein tag techniques, POOL has the following advantages. First, POOL is very soluble, has a small size of about half the size of GFP (26.9 kDa), a well-known tag,[@b53]--[@b55] and does not disturb the expression and solubility of a target protein. Furthermore, even smaller versions of PYP can be employed by utilizing the minimal segment of PYP. For example, POOL sometimes appears to assist the expression rate and solubility (Supporting Information [Fig. S2](#SD1){ref-type="supplementary-material"}) of a target protein. This characteristic of POOL may be associated with high expression rate and solubility of recombinant PYP in the E. coli cells. For example, a recombinant PYP construct can produce about 50 mg of soluble protein per liter of *E. coli* culture.[@b19],[@b56] Second, if we have to tag other proteins that have inherent visible color, such as GFP, LOV2, BLUF, and cytochrome c, it is difficult to distinguish whether the color is due to contamination, or due to other proteins or pigments expressed in the cell, or by the target protein itself. However, using the turn/on property of POOL upon addition of the chromophore, one can precisely distinguish whether the color change is due to the fused tag protein or some other factors. Nevertheless, POOL has the following limitations. First, if the function of the target protein is greatly interfered by external amino acids, POOL, which acts as external amino acids, can cause problems. This problem is also commonly observed in other protein tags. Second, as in the case of other protein tags, we have to use a protease to cleave the target protein from the POOL product. However, this could be overcome by using an autoprocessing enzyme tag.[@b57] Using the POOL approach requires construction of the initial target-POOL fusion protein and cleavage step afterwards. However in the case where a fusion tag such as MBP,[@b58]--[@b61] and TRX[@b62],[@b63] has to be used for the purification purpose, incorporating POOL does not impose any extra effort. In addition, the vectors designed for incorporating any new target protein gene with POOL are already constructed and available (see Materials and Methods). In conclusion we expect that POOL can be a useful method for many applications such as protein expression test, purity check, purification and high throughput protein expression screening.
Materials and Methods
=====================
Cloning for application of POOL
-------------------------------
The gene of a target protein was cloned to a recombinant vector along with the PYP gene in either the N-terminal or C-terminal side. The gene cloning was performed in a manner shown in Supporting Information [Figure S1](#SD1){ref-type="supplementary-material"}. We used the pQE or pET vector systems, but any vector can be used as the base. The PYP gene was inserted into the base vector without the stop codon. In addition to this, a restriction enzyme site, which is used for subcloning both the protease recognition site and the target protein simultaneously, is inserted into either the left or right side of the PYP gene depending on the relative position of the target protein gene with respect to the PYP gene. Any typical protease recognition site can be introduced. We used a protease having a tag (in our case, the his tag) identical to the tag of PYP so that both the protease and the cut PYP can be removed by a single affinity chromatography. We tested the usefulness of POOL by inserting calmodulin, SUMO, horse myoglobin, and mercury binding peptide (MerP) into the pQE80 and pQE30 vectors. All the cloning procedures were conducted using the EZ-cloning kit (enzynomics™) according to the EZ-cloning kit manual. We have made two vectors optimized for cloning (pQE80L + His tag + POOL + protease recognition site + cloning site and pET15b + cloning site + protease recognition site + POOL + His tag) (Supporting Information [Fig. S1](#SD1){ref-type="supplementary-material"}), which can be provided upon request. The details of the cloning experiments performed in this study are described in Supporting Information.
High-throughput protein expression screening
--------------------------------------------
High-throughput protein expression test was conducted by growing small scale cell cultures on two 24-well plates. The expression condition of a target protein was tested by using various combinations of IPTG concentration (1 and 0.2 m*M*), temperature (37 and 18°C) and vector (pQE80 and pQE30). After sonication of cultured cells, the cell lysate was centrifuged (HANIL Science Industrial Co. Ltd, Combi-514R) and the supernatant was transferred to a new 24-well plate. The absorbances at 280 and 460 nm of the supernatant were measured using a microplate absorbance reader (Bio-RAD Laboratories, INC. xMark™). Then the precursor of chromophore was added to the supernatant for inspecting the color change. The concentration of the expressed target protein at each well can be easily estimated by comparing its yellow color with those of the standard reference solutions of known concentrations. For more quantitative measurement of concentration and purity, the absorbance at 460 nm has to be measured again using the microplate absorbance reader.
Mini-photoactive yellow protein expression and quantification
-------------------------------------------------------------
We checked the solubility and the expression of the SUMO-mini-POOL. All the constructs were transformed into the *E. coli* BL21 (DE3) cell line and transferred to a 24-well plate. The subsequent procedures are identical to those described for the preceding section (high throughput protein expression screening).
The authors thank Jie-Oh Lee, Geun-Young Cho and Hee-Sung Park for their helpful discussions.
GFP
: green fluorescence protein
Mb
: myoglobin
MerP
: mercury binding peptide
PAGE
: Poly Acrylamide Gel Electrophoresis
POOL
: PYP turn Off/On Label
PYP
: Photoactive Yellow Protein
SUMO
: Small Ubiquitin like Modifier protein
TLR3
: toll-like receptor 3
Supplementary material
======================
Additional Supporting Information may be found in the online version of this article.
[^1]: Grant sponsor: Research Center Program (CA1201) of IBS (Institute for Basic Science) in Korea.
| tomekkorbak/pile-curse-small | PubMed Central |
cnxps.cmd.push(function () { cnxps({ playerId: '36af7c51-0caf-4741-9824-2c941fc6c17b' }).render('4c4d856e0e6f4e3d808bbc1715e132f6'); });
Turkish president Recep Tayyip Erdogan set off controversy Monday when he said that men and women are not equal, adding that a woman's job is to be a mother."Our religion [Islam] has defined a position for women [in society]: motherhood," Turkey's Hurriyet Daily News quoted Erdogan as saying to an audience of women, including his own daughter, at the Women and Justice Summit hosted by the Women and Democracy Association (KADEM) in Istanbul."You cannot explain this to feminists because they don’t accept the concept of motherhood,” he said.Using another example of mothers, Erdogan went on to say that "[Women's] characters, habits and physiques are different... You cannot place a mother breastfeeding her baby on an equal footing with men," Russia's RTE news reported Erdogan as saying."You cannot bring women and men into equal positions; that is against nature because their nature is different.”Erdogan called instead for "equivalency" between the genders, though emphasized the need for full equality regardless of gender before the law."What is particularly essential is women's equality before justice."This is not the first time Erdogan has voiced his controversial views about women. In the past, he has said that women should have at least three children. He has opposed the use of caesarian sections and abortion. | tomekkorbak/pile-curse-small | OpenWebText2 |
Am Freitag 26 Februar 2010 18:34:35 schrieb Florian Duret:
> It seems that both your suggestions have worked ! Thank you very much.
> But I still can't figure out what went wrong.
Did you change anything besides removing the "do" and "return ()" from the
then-branch and insert the "do" in the else-branch?
If not, you've been bitten by a somewhat less than obvious aspect of layout
- although it's pretty clear with an explanation.
A new layout-block isn't opened by a larger indentation, but by the
keywords do, let, of and where.
So, when you write
... = do
xxx
if blah
then do
foo
bar
else
baz
zap
the "do" after the then opens a new layout-block inside the big do-block,
since you didn't insert an explicit brace. The indentation level thereof
is determined by the 'f' of "foo", bar is indented to the same level as
foo, so it's a new expression in that same block.
The else is indented less than the foo, so that ends the inner layout-block
and we return to the layout-block of the big do-block. The indentation
level thereof is determined by the first 'x', and the "if" is indented to
the same level.
The else, the baz and the zap are all indented further than the if, so they
all belong to the if-expression (as intended).
But since there's no "do" after the "else", all that is on one logical
line, it's parsed as
... = do
xxx
if blah then do { foo; bar } else baz zap
Not what was intended, but it parses just fine.
Now you didn't have
baz
zap
but
baz <- bong
zap
and since we're still having only one logical line for the if-expression,
the parser sees that as
... = do
xxx
if blah then do { foo; bar } else baz <- bong zap
But the syntax is
pattern <- expression
so the parser tries to parse
if blah then do { foo; bar } else baz
as a pattern. But it doesn't conform to the pattern productions, hence the
parse error.
>> My initial goal was to keep the minimum inside the if ... then ... else
> statement. Basically, if the list is empty, then stop. If not, then
> assign the argument to sacFile1, and go on with the rest.
It would be cleanest to have
realWork :: FileName -> IO ()
realWork file = do
sacFile1 <- openBinaryFile file ReadMode
...
main :: IO ()
main = do
argList <- getArgs
case argList of
[] -> putStrLn "No filename ..."
(f:_) -> realWork f
or
if null argList
then putStrLn "..."
else realWork (head argList)
, je pense.
>> Here is what it looks like now:
> module Main () where
>> import System.IO
> import System.Environment(getArgs)
> import Data.Char(intToDigit)
>> import SAC_Type
> import SAC_IO
>> main :: IO()
> main = do
> -- On commence par ouvrir le fichier SAC en mode binaire
> argsList <- getArgs
> if (null argsList)
> then
> putStrLn $ "No filename given to the program.\n $
> ProgramName file.sac"
> else do
> sacFile1 <- openBinaryFile (head argsList) ReadMode
>> position <- hTell sacFile1
> putStrLn $ "Position 1: " ++ [intToDigit( fromInteger
> (position) )]
>> hSeek sacFile1 AbsoluteSeek 440
> position2 <- hTell sacFile1
> putStrLn $ "Position 2: " ++ [intToDigit( fromInteger
> (position2) )]
I don't think that's what you really want:
Prelude Data.Char> intToDigit 440
*** Exception: Char.intToDigit: not a digit 440
perhaps you wanted
putStrLn $ "Position 2: " ++ show position2
?
>> -- A la fin, il faut evidemment le fermer
> hClose sacFile1
>>> Thank you, Danke, 谢谢, merci, etc... | tomekkorbak/pile-curse-small | Pile-CC |
Honestly? I could give two flying shits less, what you scumbag, pedophile loving, pew polishing, demonic shit stains on the underwear of humanity of the Unholy Roman Catholic Cult say, or whether YOU like what I am doing here or not. And I am DAMN SURE YOU are not going to like what I am going to say in this posting, but again, I could give two flying shits less if YOU do or not.
Plain and simple, rabid dogs deserve more mercy than any of you shit stain scumbags of this cult of pedophiles. A rabid dog does NOT know what it is doing. But all your disgusting, demonic, scumbag Pedophile Pimps do. All of you do. Each and every one of you low-life, scumbag pedophiles? Each and every one of your Pedophile Pimps? Each and every one of you retarded, brain-dead, brain-washed, pieces of shit pew polishers who defend them?
ARE ALL DESERVING OF DEATH, AND A DEATH THAT WILL INCLUDE YOU SCUMBAGS BEING TORTURED WITH YOUR OWN TOOLS OF YOUR INQUISITIONS, BEFORE WE CUT YOUR FUCKING HEADS OFF, PUT THEM ON PIKES IN FRONT OF YOUR VATICAN AND YOUR CHURCHES WITH THE WARNING THIS IS WHAT WILL HAPPEN TO ANY OF YOU SCUM WHO MESS WITH OUR CHILDREN.
I agree with your Saint Peter Damian when he said the following all the way back in 1049:
“Saint” Peter Damian
“Listen, you do-nothing superiors of clerics and priests. Listen, and even though you feel sure of yourselves, tremble at the thought that you are partners in the guilt of others; those, I mean, who wink at the sins of their subjects that need correction and who by ill-considered silence allow them license to sin. Listen, I say, and be shrewd enough to understand that all of you alike are deserving of death, that is, not only those who do such things, but also they who approve those who practice them.”
Letter 31, the Book of Gomorrah [Liber Gomorrhianus], containing the most extensive treatment and condemnation by any Church Father of clerical pederasty
YOU ALL CALL YOURSELVES THE ONE TRUE CHURCH OF JESUS, THE ONE HE OFFICIALLY STARTED. YOU CALL YOURSELVES THE HOLY ROMAN CATHOLIC CHURCH AND THEN WITH ALL THAT YOU HAVE DONE TO CHILDREN AND TEENS? RAPING THEM? BRUTALIZING THEM? BEATING THEM? ENSLAVING THEM? USING THEM AS MEDICAL EXPERIMENTS INCLUDING FORCED STERILIZATIONS AND ABORTIONS? AND EVEN MURDERING THEM? THEN YOU GOT THE UNMITIGATED BALLS TO STATE TO ALL OF US HOW YOU ARE THE PROTECTORS AND DEFENDERS OF CHILDREN BECAUSE OF YOUR STANCE AGAINST ABORTION AND CONTRACEPTIVES?
WHO THE HELL DO YOU THINK YOU ARE TRYING TO FOOL OR CON?
THEN? YOU FUCKING ATTACK US? YOU INSULT US? YOU DENIGRATE US? YOU CALL US THE LIARS, THE GOLD DIGGERS OUT LOOKING FOR A PAYDAY?
OR BECAUSE WE SPEAK OUT AGAINST THE CRIMES…NOT THE SINS….COMMITTED AGAINST US, HUNDREDS AND HUNDREDS OF THOUSANDS OF US? HELL MILLIONS OF US ALL THROUGH THE HISTORY OF YOUR DAMNABLE CULT? YOU DARE CALL US ANTI-CATHOLIC BIGOTS AND HATERS OUT TO DESTROY YOUR CHURCH?
Bill “Pig Face” Donohue, degenerate leader of the Catholic League
OR SHIT STAINS LIKE BILL PIG FACE DONOHUE OF THE CATHOLIC LEAGUE SAYS BECAUSE WE DID NOT PUNCH OUR RAPISTS IN THE FACE, THAT MEANS WE WANTED IT, WE ENJOYED IT AND WE ARE HOMOSEXUALS BECAUSE OF IT?
OR THAT WE SEDUCED OUR RAPISTS?
OR ALL THE OTHER EVIL, DISGUSTING, SHITTY THINGS YOU SAY AGAINST US?
HOW FUCKING DARE YOU!!! HOW FUCKING DARE YOU ATTACK US AND DEFEND YOUR CRIMINALS? HOW FUCKING DARE YOU DO THIS TO US? WHO SUFFERED INCREDIBLE HORRORS, PAIN AND SUFFERING BECAUSE YOUR SHIT STAIN PRIESTS, BROTHERS, NUNS AND SISTERS FUCKED US, RAPED US, TORTURED US, BEAT US, BRUTALIZED US AND MURDERED US!!!!
WHO THE FUCK DO YOU THINK YOU ARE? WHY I KNOW WHO THE FUCK YOU ARE.
YOU ARE SCUM, YOU ARE FUCKING DEMONS IN HUMAN FLESH, YOU ARE THE LOWEST FORM OF HUMAN LIFE ON THE EARTH, YOU DESERVE NO RIGHTS, YOU DESERVE NO MERCY, YOU DESERVE NO COMPASSION, YOU DESERVE NOTHING BUT FUCKING DEATH!!
PLAIN AND SIMPLE, YOU GODDAMN PEDOPHILE PIMPS, YOU POPE FRANCIS, YOU POPE EMERITUS BENEDICT XVI, YOU PEDOPHILE CARDINALS, BISHOPS AND ARCHBISHOPS, YOU PEDOPHILE PRIESTS, BROTHERS, NUNS AND SISTERS AND ALL OF YOU SCUMBAG PEW POLISHERS WHO STAND UP AND DEFEND THEM AND ATTACK US? ARE FUCKING DESERVING OF DEATH, JUST LIKE YOUR SAINT PETER DAMIAN PROCLAIMED IN 1049.
SO FUCK YOU ALL.
OH AND I TRULY WISH YOU ASSHOLE, SCUMBAG, PEDOPHILE LOVING AND DEFENDING SHITSTAIN ROMAN CATHOLIC PEW POLISHERS WHO SPEW YOUR SHIT TO ME, WHO THREATEN ME WITH DEATH? WHO SAY I SHOULD BE RAPED? WHO SAY I AND MY SONS SHOULD BE PUT TO DEATH FOR SPEAKING OUT?
WELL I FUCKING WISH, I REALLY WISH, WITH ALL OF MY HEART AND SOUL, WITH ALL OF MY BEING, YOU WOULD HAVE THE BALLS TO SAY YOUR SHIT TO MY FACE, IN PERSON, BECAUSE FUCKING TRUST ME IF YOU DID?
YOU WOULD FIND OUT IF YOUR GOD AND JESUS ARE REAL, CAUSE I WOULD HAVE NO FUCKING PROBLEM….IN BEATING YOU TO DEATH WITH MY FISTS AND FEET RIGHT ON THE SPOT THE MOMENT YOU OPEN YOUR PEDOPHILE LOVING OUTHOUSE PIEHOLES TO ME.
From the Link: https://www.washingtonpost.com/opinions/defiance-and-obstruction-on-child-sex-abuse/2016/04/19/22efc3de-0351-11e6-9d36-33d198ea26c5_story.html
IN THREE years at the helm of the Catholic Church, Pope Francis has been a source of inspiration for millions of faithful around the world. In one critical respect, however, he has fallen short of his own promise: to come fully to terms with decades of child sex abuse by clergymen and the institutional cover granted to them by bishops and cardinals.
Francis has pledged “the zealous vigilance of the Church to protect children and the promise of accountability for all.” Yet there has been scant accountability, particularly for bishops. Too often, the church’s stance has been defiance and obstruction.
In his trip to the United States in the fall, Francis told victims that “words cannot fully express my sorrow for the abuse you suffered.” Yet his initiative to establish a Vatican tribunal to judge bishops who enabled or ignored pedophile priests has come to naught. Not a single bishop has been called to account by the tribunal, which itself remains more notional than real.
Meanwhile, church officials have fought bills in state legislatures across the United States that would allow thousands of abuse victims to seek justice in court. The legislation would loosen deadlines limiting when survivors can bring lawsuits against abusers or their superiors who turned a blind eye. Many victims, emotionally damaged by the abuse they have suffered, do not speak until years after they were victimized; by then, in many states, it is too late for them to force priests and other abusers to account in court.
Eight states have lifted such deadlines, known as statutes of limitations, for victims who are sexually abused as minors. Seven states have gone further, enacting measures allowing past victims — not just current and future ones — to file lawsuits in a finite period of time, generally a two- or three-year window.
In many more states, however, the bishops and their staffs have successfully killed such bills, arguing that it would be unfair to subject the church to lawsuits in which memories and evidence are degraded by the passage of time. Quietly, they also say the church, which has suffered an estimated $3 billion hit in settlements and other costs related to clergy sex abuse scandals nationwide, can ill afford further financial exposure.
A typical case is Maryland, where bills to extend the statute of limitations until the alleged victim turns 38 have failed even to come to a vote, owing to opposition from House of Delegates Judiciary Committee Chairman Joseph F. Vallario Jr. (D-Prince George’s) and the Catholic Church, among others.
In his trip to the United States, Pope Francis praised bishops for what he called their “generous commitment to bring healing to victims” and he expressed sympathy for “how much the pain of recent years has weighed upon you.” Yet by its actions, the church’s “commitment to bring healing” has seemed far from generous. And it seemed perverse to address the bishops’ “pain” when the real suffering has been borne by children.
Dear Roman Catholic Church,
When YOU assholes call what you all did to us? When YOU call crimes such as child abuse, child rape, child slavery and yes, child murder just sins? YOU lessen the impact of these crimes.
Get it through your sick and twisted disgusting heads right now.
1. Raping children is a CRIME. 2. Covering up the rapes of children is a CRIME. 3. Enslaving children in your Magdalene Laundries is a CRIME. 4. Murdering children and victims, by their suicides, is a CRIME. 5. Gang raping and gang beating children, like you all did in your Industrial Homes, like the one at Artane, is a CRIME. 6. Standing up and defending these kinds of CRIMES makes YOU an accomplice.
Asking for victims of your crimes to forgive you is WRONG!!! We did absolutely nothing wrong, YOU DID. Why should we even accept YOUR FAKE APOLOGIES when YOU STILL blame US and attack US for your crimes against US? Why do YOU feel we should forgive YOU when you continue to do the following against us:
1. DENYING US JUSTICE FOR THE CRIMES YOU COMMITTED AGAINST US. 2. BLAMING US FOR THE CRIMES THAT WERE COMMITTED AGAINST US.3. ATTACKING US AS THE EVIL ONES FOR STANDING UP AND CRYING OUT ABOUT THE CRIMES YOU COMMITTED AGAINST US. 4. DECLARING US THE EVIL ONES FOR SPEAKING OUT AGAINST THE CRIMES COMMTTED AGAINST US BY YOUR PSYCHOTIC PEDOPHILE PIMPS, YOUR PSYCHOTIC PEDOPHILE AND ABUSIVE PRIESTS, YOUR PSYCHOTIC PEDOPHILE AND ABUSIVE NUNS. 5. USING YOUR SCUMBAGS PSYCHOS LIKE BILL DONOHUE OF THE CATHOLIC LEAGUE TO INSULT AND DENIGRATE US.
You all deserve to be arrested for your crimes against us. You all deserve to pay for your crimes against us, including the murder of us. For when ANY victim of your pedohiles have committed suicide, that is murder and YOU should be charged for it.
YOU DO NOT DESERVE TO BE CALLED HOLY MEN OF GOD. YOU DO NOT DESERVE DIGNITY AND RESPECT. YOU DO NOT DESERVE ANYTHING LESS THAN TO BE CALLED FOR WHO AND WHAT YOU ARE….
YOU ARE DISGUSTING CHILD RAPISTS. YOU ARE DISGUSTING PEDOPHILES. YOU ARE DISGUSTING PEDOPHILE PIMPS. YOU ARE DISGUSTING CHILD MURDERERS.
YOU HAVE RAPED HUNDREDS OF THOUSANDS OF CHILDREN. YOU HAVE ENSLAVED THOUSANDS MORE IN YOUR LAUNDRIES. YOU HAVE BEATEN AND TORTURED AND BRUTALIZED THEM. YOU HAVE DESTROYED THEIR LIVES. YOU DO NOT DESERVE PRAISE AND WORSHIP FOR THIS, YOU DESERVE CONDEMNATION AND OSTRASIZATION FOR THIS. YOU DESERVE TO BE ARRESTED AND TRIED AND UPON CONVICTION FOR YOUR CRIMES AGAINST HUMANITY AND THE CHILDREN OF THE WORLD, EXECUTED FOR YOUR CRIMES. NO GREATER CALL FOR A DEATH PENALTY PUNISHMENT THAN THE BRUTAL RAPES, BEATINGS, ENSLAVEMENT AND MURDER OF CHILDREN THAT YOU ALL HAVE DONE AND ARE GUILTY FOR SHOULD BE CALLED FOR. YOU DESERVE TO BE EXECUTED ACTUALLY, IN MY OPINION USING THE VERY TOOLS OF THE INQUISITIONS THAT YOU USED TO EXECUTE OTHERS!!!
The Popes Pear
The Judas Chair
Each and every one of the following named individuals, have overwhelming, convincing and clear evidence against, that they were in fact, Pedophile pimps, in that they moved dangerous pedophile priests, from one parish to another, covering up for the rapes and abuses of children by these disgusting pedophiles and then they raped and abused even more children.
THERE IS OVERWHELMING EVIDENCE AGAINST EACH AND EVERY FOLLOWING NAMED INDIVIDUAL, THEY PLACED THE ROMAN CATHOLIC CHURCH, ITS PEDOPHILE PRIESTS AND NUNS, IT’S PSYCHOTIC, ABUSIVE PRIESTS AND NUNS, BEFORE THE SAFETY AND PROTECTION OF CHILDREN.
There is clean and convincing evidence, against each and every following named individual, that they are part of the larger organization, The Roman Catholic Church and that worldwide, have committed atrocities and crimes against the children of the world and humanity that are overwhelming provable:
About 35,000 children and teenagers who were orphans, petty thieves, truants, unmarried mothers or from dysfunctional families were sent to Ireland’s network of 250 Church-run industrial schools, reformatories, orphanages and hostels from the 1930s up until the early 1990s. For six decades, priests and nuns terrorised boys and girls in the workhouse-style schools with sexual, physical and mental abuse.
This does NOT include the crimes against children and humanity, where ever these Roman Catholic Churches institutions were found, from Belgium, France, Italy, Australia, New South Wales, Germany, United States, Canada, and the world over.
EACH AND EVER ONE OF THE FOLLOWING PEDOPHILE PIMPS SHOULD BE ARRESTED AND TRIED FOR THEIR
The integrity of an institution is built on trust between those who lead and those who follow. Since revelations of Gilbert Gauthe’s crimes rocked Acadiana in autumn 1984, chancery officials have erected a wall of silence, offering scant explanation to the Catholic faithful.
A two month investigation by The Times—based on legal documents and interviews with a broad range of sources, including several with active knowledge of the chancery’s inner workings—reveals a chain of events involving a total of seven priests accused of sexual involvements with youths. In most cases, Bishop Gerard Frey and Msgr. H.A. Larroque knew of the priests’ sexual problems and failed to take strong disciplinary action.
The bishop and vicar general have engaged in a cover-up, however it has been a strange one, wedded to a monarchical concept of power, divorced from democratic principles. In refusing to extend the pastoral hand to victims’ families—and deal candidly with the laity and clergy alike—Frey and Larroque have let insurance lawyers dictate their silence. And in a cruelly ironic twist, blunders by those lawyers have deepened the stain of scandal about the diocese. In real dollar terms, the cover-up has been a disaster.
In human terms, the story of a brutal tragedy is emerging, being told through the contours of legal documents and many interviews. According to a consensus of well-placed sources including case workers in Vermilion Parish, Gilbert Gauthe molested at least 100 boys in his years as a priest. Moreover, in New Iberia during the mid-70s, Gauthe and Father Lane Fontenot had sexual encounters with four boys in a ring of common victims, according to sources in the two civil parishes. A damages suit against those two priests on behalf of one victim was filed under protective seal last fall.
Bishop Gerard Frey
The Times has learned that another priest—a close friend of Fontenot’s since seminary days at Immaculata, and who once held an important diocesan position—was sent to a Northern treatment center last spring. Informed sources say he was removed some time after there were reports of sexual involvements with adolescent boys. Because he is not legally charged with sexual offenses, The Times chose not to divulge his identity. “He rationalized celibacy by saying it meant being single and not having a wife,” says a source who knew him.
Yet another priest has been identified by four well-placed sources as a man who seduced youngsters in a parish near Lafayette. He also once held a diocesan position, and was sent off for treatment out-of-state in 1984. Because he is not legally accused or sought for questioning in known litigation, The Times has withheld his identity. In 1985, however, he returned to a parish in another Louisiana diocese. Reached by telephone at that church, the priest said: “I do not wish to be interviewed.” The bishop of that diocese declined to be interviewed.
Banking on Blind Faith
“They knew something was up with Gauthe,” a well-placed source says, “but they thought it would never come out. They thought they could instill the fear in people [who might sue]—that they were up against the church, that they were doomed. The attitude was, ‘We don’t have to say anything.’ They feel they’re impervious because of the people’s faith.”
Diocesan counsel Bob Wright, citing pending civil litigation, refused The Times’ interview requests with the bishop and vicar general. So far, the bulk of $5.5 million in damages—compensating 13 children among nine families victimized by Gauthe—has been paid by different combinations of seven insurance firms. Attorneys Raul Bencomo and Paul Hebert negotiated for the plaintiffs. The diocese has paid 15 to 20 percent of the settlements.
A series of delaying tactics by insurance attorneys has eroded {“Eroded” is perhaps not the correct word. It is difficult to read in our copy.} the leverage of Frey and Larroque to restore confidence in their stewardship. Sources say the churchmen long ago wanted to settle the cases and put the scandal behind them yet remain powerless before the defense lawyers, who demanded silence of them.
As this issue went to press, Wright conceded that insurance lawyers and J. Minos Simon, attorney for the Gastal family, whose son was molested by Gauthe in Henry, were “far apart” on a negotiated settlement. Simon told The Times: “I am expecting a trial. I want to try this case.” Jury selection is slated for Feb. 3 in Abbeville. In the event an 11th-hour agreement is reached—as in the last three Hebert-Bencomo suits—Simon’s clients stand to receive substantially more than the $420,000 per victim which has been paid out in past settlements.
Meanwhile, eight suits against Gauthe, the one involving Gauthe and Fontenot, and three accusing Father John Engbers of pedophilia remain in litigation brought by various lawyers. The defense has not accepted liability in those cases and the prescription issue—whether the suits were filed within a year of when the injuries were sustained—is also unresolved.
The Failure of Forgiveness
Nothing in Gerard Frey’s background prepared him for this. Scion of a prosperous New Orleans family, he was bishop of Savannah, Ga., before replacing the aging Bishop Maurice Schexnayder in 1973. By all accounts a shy man, Frey’s philosophy was to delegate power across mid- and lower-echelons of the diocese and otherwise govern quietly. In the spirit of Vatican II, he encouraged greater involvement among lay people in parish councils and diocesan programs, particularly religious education.
Through the late 1970s, Frey relied on then-Msgr. Jude Speyrer, chancellor, and Alex Larroque as vicar general to handle daily affairs and advise him. When Speyrer became bishop of Lake Charles in 1980, Larroque assumed an even greater position of importance. Sources say that a change came over Bishop Frey in the early 1980s. His shyness gave way to an aloof detachment. He now reportedly spends long stretches at his family’s resort camp in Bay St. Louis, Miss. Larroque manages the diocese’s daily affairs and functions as the bishop’s alter-ego.
“Frey hates confrontations,” another source explains. “I’m convinced the bishop knew all along about the pedophilia, but I don’t think he knew any other way to handle it. In the past, they called in people [when a priest molested a youth], provided counseling, made a settlement with the family, swore them to secrecy, and moved the priest. I think the bishop did a lot of things that were wrong by moving people repeatedly. He has to believe a priest is being straight with him when he says he’ll behave. You don’t have enough priests to go around. He can’t automatically say, ‘I’m sending you off for treatment.'”
This ingrained philosophy of forgiveness—once a priest, always a priest—seems incapable of dealing with addictive pedophiles, who often cannot admit that seducing youngsters is wrong. Moreover, Frey and Larroque’s handling of Gauthe, Fontenot and Engbers runs counter to Article 25 of the Louisiana Criminal Code, which defines accessories after the fact as “any person who, after the commission of a felony, shall harbor, conceal, or aid the offender, knowing or having any reasonable ground to believe that he has committed the felony, and with the intent that he may avoid or escape from arrest, trial, convictions or punishment.”
According to Oliver Houck, law professor at Tulane University and a former federal prosecutor, the statute “does not require that the offender be arrested or convicted. What [a supervisory figure] knew and when he knew it is a question of criminal negligence; what he should have known is the realm of civil negligence.”
Origins of the Cover-Up
How much did diocesan officials know and how long did they cover-up the actions of the accused priests? In civil depositions, Frey says that he learned in 1974 that Gauthe admitted to “impure touches” with a boy in Broussard the year before; Gauthe promised him it would not happen again. In 1976 Larroque ordered Gauthe to see a psychiatrist after he kissed two Abbeville boys in a camper. There was no meaningful follow-up by the chancery, nor was Gauthe reported to law-enforcement officials. And now new information draws sharper focus on that period, when Gauthe and Fontenot were in New Iberia.
Ronald Lane Fontenot, born in Eunice in 1946, was ordained in Lafayette in December 1975. His first assistant pastorship was St. Peter the Apostle in Gueydan. The pastor there was John Engbers. The Times knows of no alleged molestations by Engbers in Gueydan, however sources say Fontenot tried to seduce a young man after hearing his confession there. Three years is the average stay for an assistant pastor. The sources do not know if Fontenot was reported to the chancery while in Gueydan. But after only six months, in June 1976, he was transferred to Our Lady of Perpetual Help in New Iberia where Gilbert Gauthe had been seducing altar boys and other kids on camping trips.
In deposition Msgr. Richard Mouton of Abbeville says he was told that Fr. Joseph Bourque, the New Iberia pastor, wrote to the chancery complaining about Gauthe. Asked about the letter, Father Bourque told The Times: “Any statement I would have to make would come through the chancery office or the [diocesan] attorney.”
But according to a New Iberia family, they reported both priests for molesting altar boys. “It wasn’t but about a month or so later that [Bourque] transferred Father Gauthe and that other [one], Father Lane Fontenot, out of our church.”
Gauthe went to St. Mary Magdalen Parish in Abbeville, but continued relationships with youngsters in New Iberia. As for Fontenot, he lasted only nine months in New Iberia, moving to Our Lady of Mercy in Opelousas in March 1977. There, says a priest who learned of it years later, “he molested many kids.” Why was Fontenot moved out of New Iberia so quickly if Bourque did not request it? Only the bishop can move a priest. In any event, Lane Fontenot climbed the diocesan ladder. By 1982, as Priest in Charge of Spiritual Development for the diocese, he was giving charismatic sermons to Catholic youth rallies.
A disturbing symmetry links Gauthe and Fontenot in another way. In 1975, on recommendation of then-Msgr. Speyrer, Frey named Gauthe diocesan Boy Scout chaplain—while he was in New Iberia, and after Frey knew Gauthe had molested a boy in Broussard. Even if he believed Gauthe was controlling his urges, why put a man like that in a position involving Boy Scouts? If, as the parent claims, Fontenot left New Iberia because of sexual misconduct, why would a man like that be allowed to work so closely with youngsters in Lafayette? Even the philosophy of forgiveness would seemingly be tempered by prudence in personnel assignments.
Fontenot, who lived at Our Lady of Fatima, was hustled out of Lafayette in autumn 1983, six months after Gauthe, when a family accused him of molesting their son. As reported last June, a settlement was reportedly paid to the family. Fontenot followed Gauthe to the Church-run House of Affirmation in a Boston suburb. In 1984, still another priest, Fr. Robert Limoges, left for an undisclosed treatment center after families in Eunice and Lafayette complained to their respective district attorneys.
Trouble with the Lawyers
It is doubtful whether these events would have become publicly known had the bishop in the wake of Gauthe’s June 30, 1983, suspension gone immediately to Henry, told parishioners what Gauthe had done, hired a psychiatrist and offered therapy to families with youngsters molested by Gauthe. “We never planned on suing,” the parent of one of the initial families says. “We just wanted help for our children, and we wanted Church officials to come meet with us and tell everybody what was what. To this day they haven’t done it. That’s what hurts so much.”
Instead, lawyers were called in to represent the Church insurors. The classic defense in high-dollar damages litigation is to delay, negotiate, and keep everything under wraps. This approach melded with the chancery’s strategy of silence on sex abuse, however it is unclear whether the lawyers learned how much Frey and Larroque knew about other priests. Four New Orleans lawyers formed the defense team: Thomas Rayer, counsel for the Archdiocese, Robert Leake, Charles Schmidt, and Gordon Johnson, representing insurors. They have repeatedly declined The Times’ interview requests.
After closed-door negotiations with Bencomo over the first nine Gauthe victims the defense in June 1984 agreed to $4.2 million settlements. That, however, was done without ever taking depositions of Dr. Edward Shwery, the psychologist who wrote the document analyzing the sexual abuse, or Dr. Kenneth Bouillion, the therapist treating the boys.
“I think there’s a feeling that they gave the cart away before the horse,” Wright told The Times recently.
After J. Minos Simon took over the Gastals’ suit, the defense faced a more radical adversary, yet the legal scrimmaging was marked by procedural fumbles opening a swath for Simon’s steamroller. In his discovery phase—the period of pre-trial questioning by lawyers of prospective witnesses to gain information for later use in court—Simon demanded files on 27 priests regarding their sexual conduct. The defense lawyers, instead of mounting an aggressive counter-attack to protect the reputations of other priests, failed to seal the document and continued playing for time. The list was entered as a court document, available to reporters. No Church representative took to the airwaves challenging Simon’s bold charges. Nor did the insurance lawyers appear to take the allegations about other priests very seriously. Leake called it “a fishing expedition.”
At a January hearing in Abbeville to argue the matter nobody for the defense showed up—at the very least, an act of faulty representation for both Church and insurance clients. They missed a hearing on another matter called at their request some time later, and on Oct. 11, 1985, when the Gastal boy’s therapist, Dr. Lyle LeCorgne, accompanied Simon to a deposition scheduled in Wright’s office, once again the defense did not show up. Whether arrogance or ineptitude explains such behavior, the net result worsened prospects for a negotiated settlement in the Gastal suit. And to an attorney like Simon, it had the effect of waving a red flag in front of a charging bull.
As Simon’s aggressive discovery probe began to outline the dimensions of the pedophilia problem in the diocese last year, Frey and Larroque had another force to contend with in attorney Tony Fontana, whose clients, the Butaud sisters, were clamoring to have Father John Engbers removed from Leroy. They alleged that Engbers had molested them when they were children in the mid-1950s. Like defense lawyers, Frey and Larroque stalled—but in a fashion bordering on criminal negligence: for eight months they let an accused child molester remain at the parish in Leroy without telling parishioners or law enforcement authorities.
In June, Judge Bradford Ware ruled Simon had discovery rights to files on pedophile priests. This prompted defense attorney Leake’s verbal stipulation to liability, theoretically halting Simon’s discovery onslaught. By then damage to the chancery’s image had been immense. More sources were leaking information, this time to The Times. Of the priests named in this article, only Fontenot appears on Simon’s list.
Still the defense delayed. Wright, a veteran plaintiffs’ attorney, had long advocated a liability stipulation while Leake resisted. Ironically, Wright became the point man for reporters in Lafayette. When it finally came, the written stipulation dissatisfied Simon—and Hebert and Bencomo on their cases. Simon attacked again, this time after a young man came to his office with allegations against Fr. Lloyd Hebert of Opelousas. The man wanted Fr. Hebert removed. Simon issued a subpoena to depose Hebert, and when that happened Wright stipulated to liability in open court at the end of June, ending Simon’s discovery once and for all, saving the priest from being deposed. Hebert left Opelousas to live with relatives.
At the end of July, Father John Engbers, the seventh priest in this diocese accused of sexual misconduct, fled to Holland.
By mid-autumn, Gauthe had gone to prison but the scandal which began with revelations of his misconduct had become a seemingly bottomless pit. The insurors were paying large sums to Hebert-Bencomo clients in negotiated settlements, but with the Gastals, defense lawyers took the more aggressive step of deposing the parents and child to decide how much their suffering was worth. At the heart of Simon’s case is the issue of consortium—how Gauthe’s sexual invasions altered the family’s life: the boy, his relationship to the parents, the family’s bond to the church.
Fontana on the Offensive
Tony Fontana has six Gauthe suits for which the insurors have not stipulated liability; his discovery powers remain open. With the Engbers suits, he faces a steep incline on the time prescription issue. A devout Catholic, he says: “I feel betrayed by Frey and Larroque. I’m not sure if I want stipulation, because then I have to prove liability—how much Frey and Larroque knew. The damages in these cases are going to come from outrage—not that Gauthe molested kids—but that Frey knew about Gauthe in 1975.” Fontana recently filed a motion to unseal two names of his Gauthe victims, clearing the way for depositions of Frey, Larroque and others.
When Fontana filed interrogatories on the Engbers case earlier this month, he effectively began where Simon left off. Hearings on this matter and the controversial question of prescription—whether, many years after Engbers’ alleged crimes, the Butaud sisters have litigable claims—are scheduled for Feb. 10 in Lafayette. Earlier this month Fontana filed another suit on behalf of two Lake Charles sisters, Brenda Andrepont Gossett and Judy Andrepont Tish, now adults, who claim Engbers molested them when they were children in the early 1950s. A third suit on behalf of an unnamed minor child has also been filed against Engbers and the diocese.
Where Will It End?
Beneath the legalities and mountains of money, dozens of youngsters have been molested and many families bitterly hurt by the chancery’s long policy of silence. Seven priests—Gauthe, Fontenot, Limoges, Engbers, Hebert, the priest in treatment and the priest now in another diocese—have left Lafayette diocesan parishes. The sexual map of their movements includes Broussard, New Iberia, Abbeville, Henry, Esther, Lake Charles, Louisa, Opelousas, Lafayette, Eunice, Gueydan, Sunset, and Leroy. Of them, only Gauthe is known to have been suspended and even he has not been formally defrocked.
What explains the silence by the bishop and vicar general? Why have they resisted candid dialogue with the laity and as pastors begun the healing process?
In one sense, the churchmen acted as their tradition suggested. The Catholic Church is governed by a monarchical sensibility. Appointed by the pope, each bishop is effectively regent of his diocese. Lay people cannot impeach a bishop or elect a new one. The Apostolic Delegate—the Vatican’s ambassador in Washington—acts for the pope in naming new bishops, who retire when they wish or by age 75. Frey is 71.
The trappings of this monarchical structure have for generations been a source of pride to Catholics, who traditionally cherish the milieu of churches, the splendor of choral masses at Advent and at Easter. The bishops in their robes of watered silk, like priests in bright vestments of the seasons, embody sacred links to a spiritual lineage spanning 20 centuries.
Bishop Frey and Msgr. Larroque relied on a royalist defense by ___ring themselves from the truth. But the cover-up was doomed to shatter on a collision course with democracy, the court system and a free press. The issue around which all others pivot is the rights of children. And in this respect, the Church betrayed her own historic commitment to the sanctity of families. Frey and Larroque never realized that. Instead they held fast to a notion that “the Church” must be saved from scandal. But the Church is not marble and mortar, it is a community of faithful people bonded by an ethos of human dignity. In their myopia the two leaders became truly tragic figures, shifting the focus to themselves, and when others who knew too much felt outrage mount, the wall of silence began to crack. Even now, looking through the jagged holes, a terrible question remains: why did it happen here? | tomekkorbak/pile-curse-small | Pile-CC |
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abstract: 'The perturbation theory around the soliton fields of the sin-Gordon model is developed in the coset space. It is shown by explicit calculations that all corrections to the topological soliton contribution are canceled exactly.'
author:
- 'J.Manjavidze$^{a)}$, A.Sissakian$^{b)}$'
title: Quantization of Solitons in Coset Space
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¶ ł § øØ
0.8cm
PACS numbers: 02.30.Cj, 03.65.Db, 02.40.Vh, 31.15.Kb
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The problem of quantization of the extended objects was formulated mainly in the middle of 70-th, see the review paper $^1$ and references cited therein. One starts from the classical Lagrange equation: =0, ł[0.1]{}where, for simplicity, $u(x,t)$ is the real scalar field $^{2}$. If this equation has nontrivial solution $u_c(x,t)$ then the problem of its quantization will arise. One of the first attempts to construct the perturbation theory was based on the WKB expansion in vicinity of $u_c$ $^{3}$.
The Born-Oppenheimer method was adopted also $^{4,5}$. First of all, to construct the quantum mechanics the structure of Hilbert space $\cH$ is postulated. So, it is assumed that the Fock column consist from the vacuum state $|0>$ and from the multiple meson states $|p_1,p_2, ..., p_n>$, $n\geq1$. The ordinary perturbation theory operates just with this ‘meson’ sector only. The $anzats$ $|P_1,
P_2,...,P_l>$ $^5$ for the $l$-soliton state, $l\geq 1$, is introduce also.
It is postulated that the quantum excitations in the soliton sector are described by the excitation of the ‘meson’ field $^4$. Therefore, to construct the perturbation theory, there should also be the mixed states: |P\_1,...,P\_l;p\_1,...,p\_n>, l1, n1, ł[0.2]{}but, at the same time, 0, l1, n+n’0, ł[0.3]{}i.e. it is assumed that the solitons are the absolutely stable field configurations $^1$.
Present paper in definite sense completes the offered in $^{4,5}$ picture. The (1+1)-dimensional exactly integrable sin-Gordon model will be considered to illustrate our result. We will investigate the multiple production of ‘mesons’ by ‘soliton’ and the truth of () will be shown at the end of explicit calculations. In other words, it will be shown that the offered in $^{4,5}$ postulate concerning orthogonality of the ‘meson’ $\cH_m$ and ‘soliton’ $\cH_s$ Hilbert spaces can be proved. We will see that this conclusion follows from exactness of the semiclassical approximation for sin-Gordon model.
It should be noted that the exactness of semiclassical approximation in topological soliton sector of the sin-Gordon model is not ‘beyond the realm’ $^6$. It is well known also that the integrable Coulomb problem is exactly semiclassical. The same we have for the quantum rigid rotator $^{7}$, which is the isomorphic to Poshle-Teller model. The general discussion of the exactness of semiclassical approximation from a geometrical point of view was given in $^{8}$.
It will be crucial for us in many respects to follow the WKB ideology. So, we will consider the ‘meson’ production amplitudes a\_[nm]{}(p,q)=<p\_1,...,p\_n|q\_1,...,q\_m>\_[s]{}, n,m=1,2,... ł[0.5]{}The index $s$ means that the calculations are performed in the ‘soliton’ sector and $p_i$ and $q_i$ are the ‘meson’ momenta. By definition, p\_i\^2=q\_i\^2=m\^2 ł[0.5a]{}since the quantum uncertainty principle leads to the impossibility off mass-shell observation of the field $^{9}$. The ordinary reduction formalism will be used to calculate $a_{nm}$. This means that we will construct the $phenomenological$ $S$-matrix of the ‘meson’ interaction through the ‘soliton’ fields, i.e. we will start from the assumption that the states () exist, and it will be shown at the end of calculations that such $S$-matrix is trivial: a\_[nm]{}(p,q)0, n+m>0. ł[0.6]{}
Offered in the paper formalism allows to prove (). For this purpose we will build the perturbation theory expansion over $1/g$, where $g$ is the interaction constant $^{10}$. This perturbation theory is dual to the theory described in $^{1}$, over $g$, i.e. one can not decompose the definite order over $g$ contribution in terms of the $1/g$ expansion, and vice versa. So, only the summary results of both expansion may be compared.
Following to WKB ideology, to find the corrections to the semiclassical approximation in the vicinity of the extremum $u_c(x,t)$, one should find the solution of the equation for the Green function: $$(\pa^2 + v''(u_c))G(x,t;x',t')=\d(x-x')\d(t-t'),$$ where $v''(u)$ is the second derivative of the potential function $v(u)$. This Green function describes propagation of a particle in the time dependent inhomogeneous and anisotropic external field $u_c(x,t)$. Generally, this problem has not a closed solution. So, for instance, the attempt to solve the problem using the momentum decomposition $^{11}$ leads to the hardly handling double-parametric perturbation theory. To avoid this problem we will build new perturbation theory over $1/g$.
Imagining particles coordinates as the elements of the Lee group, the classical particles motion may be described mapping the trajectory on group manifold. Roughly speaking, this means that the group combination law creates the particles classical trajectory $^{12}$.
Moreover, this program was realized for description of the particle quantum motion $^{13}$. It was shown for essentially nonlinear Lagrangian $ L=\frac{1}{2}g_{\mu \nu}(x)\dot{x}^{\mu}\dot{x}^{\nu} $ that the semiclassical approximation is exact on the (semi)simple Lee group manifold. But this slender solution of quantum problems is destructed in presence of the interaction potential $v(x)=O(x^n)$, $n>2$, since last one breaks the isotropy and homogeneity of the Lee group manifolds $^{10}$. The developed perturbation theory will describe the quantum perturbations breaking isotropy and homogeneity of the group manifold.
Developed formalism contains the following steps $^{10,14}$. (i) We will introduce the manifold $W_G$ of trajectories $u_c$, solving the eq.(). The manifold $W_G$ will be labeled by the local coordinates $(\x,\eta)$, i.e. we will consider $u_c=u_c(x;\x,\eta)$ since $u_c$ should belong to $W_G$ $completely$. (ii) The numbers $(\x,\eta)$ are interpreted as the generalized coordinates of the ‘particle’. Then $u_c(x;\x,\eta)$ will define the external potential for it. The quantum motion of the ‘particle’ may be described noting that $W_G$ is the homogeneous and isotropic manifold, since this case is rather quantum mechanical problem in the ‘flat’ space.
It was shown in $^{14}$ that the WKB model $^3$, where the field excitations in vicinity of $u_c$ are decomposed over the ‘meson’ states, and our model quantum mechanics of the ‘particle’ in the external potential defined by $u_c$, are isomorphic. In other words, we know that the quantum trajectory of the ‘particle’ covers the phase space $(\x,\eta)\in W_G$ densely. But it should be noted also that described in $^3$ model presents the expansion over the interaction constant $g$ and our perturbation theory describes expansion over the $(1/g)$.
In the classical limit (labeled by the index ‘$0$’) the motion of our ‘particle’ must be free $^{14}$, i.e. its velocity should be a constant, \_0=const, \_0=0. ł[0.7]{}This may be achieved expressing the set $\{\eta\}$ through the set of generators of the subgroup broken by $u_c$ $^{15}$. It is evident, such choice of the ‘particles’ coordinate gives the same effect as in the above discussed transformation to the homogeneous and isotropic (semi)simple Lee group manifold $^{10}$, see also $^{16}$. Moreover, we will see that even in the case of nontrivial potential function, one can get to the free ‘particles’ motion, rescaling the quantum sources $^{10,14}$.
Thus, the necessary invariant subspace $W_G$ would be chosen equal to the coset space $G/G_c$: W\_G=G/G\_c, ł[0.7a]{}where $G$ is the symmetry group and $G_c\subset G$ is the classical solutions $u_c$ symmetry group. The problem of quantization of the coset space have a reach history, see e.g. $^{17}$. Described in $^{10,14}$ formalism presents one of possible realization of the coset spaces quantization scheme.
The last one means that we will realize the transformation generated by the classical trajectory $^{14}$: u\_c: (u,p)(x,t)(,)(t) ł[0.8]{}Such construction of perturbation theory in the $W_G$ space require the additional effort noting that the dimension of the original phase space $(u,p)\in T^*V$ is infinite. Therefore, () assumes the infinite reduction since the dimension of coset space $W_G$ is finite $^{18}$. The crucial for us reduction scheme was formulated in $^{14}$.
In other words, quantizing the sin-Gordon soliton fields, the space coordinate would be an irrelevant variable. This is the well known fact, e.g. $^3$, and it leads to the Lorentz non-covariant perturbation theory. It is the consequence of absolute stability of the solitary waves profile, i.e. of conservation of the topological charge. The necessary information concerning this question will be given in Sec.3.
Having the complete theory, one can analyze the perturbations. The crucial point of the new perturbation theory is the statement $^{10}$ that the quantum corrections are accumulated strictly on the boundaries $\pa W_G$ (bifurcation manifolds $^{19,20,15}$) of the $W_G$ space. Therefore, if u\_cW\_G=, ł[0.10]{}then the problem is exactly semiclassical. On other hand, () means conservation of the topological charge: $\pa u_c$ is the flow induced by the quantum perturbations in $W_G$ and if () is not satisfied, then a flow into the forbidden domain with other topological charge, separated by the bifurcation boundary, should exist. So, () is the topological charge conservation law.
Notice, the solution () leads to () since particles production is the pure quantum effect. This will be shown in Sec.4.
The paper is organized as follows. In Sec.2 we will (i) formulate the necessary for us boundary conditions to derive the LSZ reduction formulae, (ii) find the explicit expression for $a_{nm}$, (iii) formulate the mapping into the coset space $W_G$. In Sec.3 we will (i) consider the sin-Gordon model, (ii) discuss necessary in the coset space boundary condition, (iii) remind the structure of the new perturbation theory $^{14}$, (iv) describe ‘meson’ multiple production to show ().
0.5cm [**II. Density matrix on the Dirac measure**]{} 0.3cm
Main point of this section is the attempt to generalize the ordinary for field theory boundary condition: $$u(x\in\s_\infty)=0,$$ where $\s_\infty$ is the remote hypersurface. This boundary condition is used to remove the surface term, and it is necessary to formulate the reduction formalism. We would like introduce the new boundary condition to have a possibility to include the non-vanishing on $\s_\infty$ field configurations and, at the same time, throw off the surface term.
The $(n+m)$-point Green functions $G_{nm}$ are introduced through the generating functional $Z_{j}$ $^{21}$: G\_[nm]{}(x,y)=(-i)\^[n+m]{}\_[k=1]{}\^[n]{}(x\_k)\_[k=1]{}\^[m]{} (y\_k)Z\_j, ł[2.1]{} where $\hat{j}(x)={\d}/{\d j(x)}$ and the generating functional Z\_j=Du e\^[iS\_j(u)]{}. ł[2.3]{}The action S\_j (u)=S(u)-V(u)+dx dt j(x,t)u(x,t), ł[2.4]{} where S(u)=dxdt((u)\^2-m\^2u\^2), m\^20, ł[2.4’]{}is the free part and $V(u)$ describes the interactions. At the end of calculations one should put $j=0$.
To provide convergence, the integral () will be defined on the Mills complex time contour $C_+$ $^{22}$. For example, C\_: tt+i, +0, -t+ł[2.5]{}and after all calculations, one should return the time contour on the real axis putting $\ve=0$.
In a ‘meson’ sector the integration in () is performed over all field configurations with standard vacuum boundary condition: d\^2x \_(u\^u)= \_[\_]{}d\_u\^u=0. ł[2.6]{}It follows from this conditions that u(x\_)=0, pa\_u(x\_)=0. ł[2.6a]{}It excludes a contribution from the surface term since assumes that field disappeared on the remote hypersurface $\s_\infty$. Considering the ‘soliton’ sector this boundary condition require the modification since there is in the $(x-t)$ space such direction along which the soliton field does not disappeared. The integral () would have a formal meaning till this boundary condition will not be specified.
Let us introduce now the field $\vp$ through the equation: - =j(x,t). ł[2.7]{}It is assumed that we can formulate such boundary condition that the surface term may be neglected calculating the variational derivative in (). Then we perform the ordinary shift $u\to u+\vp$ in integral (\[2.3\]). Considering $\vp$ as the probe field created by the source: (x)=d\^2x’ G\_0(x-x’)j(x’), (\^2 +m\^2)G\_0 (x-x’)=(x-x’), ł[2.8]{}the connected Green function $G^{c}_{nm}$ only will be interesting for us: G\_[nm]{}\^[c]{}(x,y)=(-i)\^[n+m]{}\_[k=1]{}\^[n]{}(x\_k) \_[k=1]{}\^[m]{}(y\_k)Z(), ł[2.9]{}where Z()=Du e\^[iS(u)-iV(u+)]{} ł[2.10]{}is the new generating functional.
To calculate the nontrivial elements of $S$-matrix we must put the external particles on the mass shell. Formally this procedure means amputation of the external legs of $G^{c}_{nm}$ and further multiplication on the free particles wave functions. In result the amplitude of $n$- into $m$-particles transition $a_{nm}$ in the momentum representation has the form: a\_[nm]{}(q,p)=(-i)\^[n+m]{}\_[k=1]{}\^[n]{}(q\_k) \_[k=1]{}\^[m]{}\^\* (p\_k) Z(). ł[2.11]{}Here the particles creation operator: \^\*(q)=d\^2xe\^[iqx]{}(x), (x)=. ł[2.12]{}was introduced. The eq.() is the ordinary LSZ reduction formulae. But one should remember that the boundary condition () should be generalized to have a permission for inclusion of the soliton contributions calculating $Z(\vp)$.
Describing particles multiple production it is enough to consider the generating functional: $$\R(\a,z)=
\exp\{-\int
d\O_1(p)\le(\h{\vp}^*_+(p)\h{\vp}_-(p)e^{i\a_+p}z_+(p)\ri.+$$+.\^\*\_-(p)\_+(p)e\^[i\_-p]{}z\_-(p))} Z(\_+)Z\^\*(\_-), ł[2.13]{}where $$d\O_n(p)=\prod_{k=1}^n\f{d^1p_k}{(2\pi)2\e(p_k)}=
\prod_{k=1}^nd\O_1(p_k), ~\e(p)=(p^2+m^2)^{1/2}.$$ Let us calculate $$\le.
\int \f{d^2\a_+}{(2\pi)^2}e^{-iP\a_+} \f{d^2\a_-}{(2\pi)^2}e^{-iP\a_-}
\prod_{k=1}^n \f{\d}{\d z_+(p_k)}\prod_{k=1}^m \f{\d}{\d z_-(q_k)}
\R(\a,z)\ri|_{z_+=z_-=0}$$ Inserting here the definition (), one can find that this expression gives: $$\d(P-\sum^n_{k=1}p_k)\d(P-\sum^m_{k=1}q_k)|a_{nm}(p,q)|^2,$$ where the $\d$- functions are the result of integration over $\a_\pm$. So, the factors $e^{i\a_\pm p}$ in () permit to introduce the energy-momentum shell and the $\d$-functions defines the restriction on the shell. The both restrictions $$P=\sum^m_{k=1}q_k,~P=\sum^n_{k=1}p_k$$ are compatible since the amplitude $a_{nm}$ is translationally invariant. The integration over $P$ gives energy-momentum conservation law.
Notice now that $\R(\a,z)$ is defined through the generating functional $$\R_0(\vp)=Z(\vp_+)Z^* (-\vp_-)=$$=Du\_+Du\_- e\^[iS\_+(u\_+)-iS\_-(u\_-)]{} e\^[-iV\_+(u\_++\_+)+iV\_-(u\_–\_-)]{}. ł[2.13a]{}Then, we can consider the ‘closed-path’ boundary condition: \_[\_]{}d\_u\_+\^u\_+= \_[\_]{}d\_u\_-\^u\_-, ł[2.13b]{}instead of (,). The natural solution of this boundary condition is: u\_+(x\_)=u\_-(x\_)=u(x\_). ł[2.13c]{}It provides cancelation of the surface term on the remote hypersurface $\s_\infty$ independently on the ‘value’ of the field $u(x\in\s_\infty)$.
Considering the system with the large number of particles, we can simplify calculations choosing the CM frame $P=(P_0 =E,\vec
0)$. It is useful also $^{23}$ to rotate the contours of integration over $$\a_{0,k}:~\a_{0,k}=-i\b_k, \Im\b_k =0, k=1,2.$$ Then $\R(\b,z)$ have a meaning of the density matrix, where $\b$ would have, in the some definite case $^{24}$, meaning of the inverse temperature and $z$ is the activity $^{25}$.
It was shown in $^{14}$ that the unitarity condition unambiguously determines contributions in the path integrals for $\R$. Exist the statement:
[*S1. The density matrix $\R(\a,z)$ has following representation*]{}: (,z)= e\^[-i(je)]{}DM(u)e\^[iS\_O(u)-iU(u,e)]{}e\^[N(,z;u)]{}(u)e\^[N(,z;u)]{}. ł[2.14]{}It should be underlined that this representation is strict and is valid for arbitrary Lagrange theory of arbitrary dimensions. The derivation of () is given in Appendix A.
Expansion over the operator: (je)=\_[C\_+]{} dx dt \_[C\_+]{} dx dt (x,t)(x,t) ł[2.15]{}generates the perturbation series. We will assume that this series exist (at least in Borel sense). The variational derivatives in () are defined as follows: $$\f{\d \phi(x,t\in C_i)}{\d \phi(x',t'\in C_j)}=\d_{ij}\d
(x-x') \d (t-t'),~~~ i,j=+,-$$ where $C_i$ is the Mills time contour. The auxiliary variables $(j,e)$ must be taken equal to zero at the very end of calculations.
The functionals $U(u,e)$ and $S_O(u)$ are defined by the equalities: S\_O(u)=(S(u+e)-S(u-e)) +2\_[C\_+]{}dx dte(x,t)(\^2+m\^2)u(x,t), U(u,e)= V(u+e)-V(u-e)-2\_[C\_+]{} dx dte(x,t)v’(u), ł[2.16]{}where $S(u)$ is the free part of the Lagrangian and $V(u)$ describes interactions. The phase $S_O(u)$ is not equal to zero if $u$ have the nontrivial topological charge $^{14}$. We will discusses carefully this question later.
The measure $DM(u,p)$ has the form: DM(u)=\_[x,t]{} du(x,t) (+j(x,t)). ł[2.17]{}The functional $\d$-function in the measure means that the necessary and sufficient set of contributions in the integral over $u(x,t)$ is defined by the classical equation: -=j(x,t), ł[2.17a]{} ‘disturbed’ by the quantum source $j(x,t)$
For further calculation another representation will be useful. If we insert into the integral () $$1=\int\prod_{x,t}dp(x,t)\d(p(x,t)-\dot{u}(x,t))$$ then the measure $DM$ takes the form: $$DM(u,p)=\prod_{x,t} du(x,t)dp(x,t)\t$$((x,t)-) ((x,t)+) ł[2.17b]{}with the total Hamiltonian H\_j(u,p)=dx { p\^2 +(u)\^2 + v(u)-ju }. ł[2.18]{}Last one includes the energy $ju$ of quantum fluctuations. The measure () describes motion in the symplectic space $(u,p)\in V$. But it should be underlined that used expansion is not the Lagrange transformation. So, generally, it is quite possible, considering $x$ as the index of space sell, that not all of $p(x,t)$ are the independent variables. For this reason the measure () has mostly a Lagrange meaning.
The measure () contains following information $^{10,14}$:
[**a.**]{} [*Only the $strict$ solutions of equations -=0, +=0 ł[2.19]{}at $j=0$ should be taken into account*]{}. This ‘rigidness’ means absence in the formalism of the pseudo-solution (similar to multi-instanton, or multi-kink) contributions;
[**b.**]{} [*$\R(\a,z)$ is described by the $sum$ of all solutions of eq.()*]{}, independently from theirs ‘nearness’ in the functional space;
[**c.**]{} [*The field disturbed by $j(x)$ belongs to the same manifold (topology class) as the classical field defined by ()*]{} $^{10}$.
[**d.**]{} [*The consequence of properties [**b.**]{} and [**c.**]{} is the selection rule: quantum dynamics is realized in the coset space of highest dimension*]{} $^{10}$. This, excluding from consideration the pure ‘meson’ sector.
The particles density N(,z;u)=N\_+(\_+,z\_+;u)+N\_-(\_-,z\_-;u), ł[2.20]{}where N\_(\_,z\_;u)=dØ\_1(q)e\^[i\_q]{}z\_(q)|(q;u)|\^2, ł[2.21]{}
The ‘vertex’ $\Ga(q;u)$ is the function of the external particles momentum $q$ and is the linear functional of $u(x)$: (q;u)=-dx e\^[iqx]{} = dx e\^[iqx]{}(\^2 +m\^2)u(x) , q\^2=m\^2, ł[2.22]{}for the mass $m$ field. This parameter presents the momentum distribution of the interacting field $u(x)$ on the remote hypersurface $\s_\infty$ $if$ $u(x)$ is the regular function. Notice, the operator cancels the mass-shell states of $u(x)$.
Generally $\Ga(q;u)$ is connected directly with $external$ particles properties and sensitive to the symmetry of the interacting fields system $^{26}$.
The construction () means, because of the operator $(\pa^2
+m^2)$ and remembering that the external states should be mass-shell by definition $^{9}$, the solution $\R(\a,z)=0$ is actually possible for particular topology (compactness and analytic properties) of $quantum$ field $u(x)$. So, $\Ga(q;u)$ carry following remarkable properties: (i) it directly defines the observables, (ii) is defined by the topology of $u(x)$. Notice that the space-time topology of $u(x,t)$ becomes important calculating integral () by parts. This procedure is available if $u(x,t)$ is the regular function. But the $quantum$ fields are always singular. Therefore, the solution $\Ga(q;u)=0$ is valid iff the semiclassical approximation is exact, i.e. the particle production is the pure quantum effect. Just this situation is realized in the soliton sector of sin-Gordon model.
Let $G$ be the symmetry of the problem and let $G_c$ be the symmetry of the solution $u_c$. Then
[*S2. The measure () admits the transformation: u\_c: (u,p)(,)W=G/G\_c. ł[2.23]{}and transformed measure has the form: DM(u,p)= \_[x,t*C*]{}d(t) d(t) (-) (+), ł[2.24]{}where $h_j (\x,\eta)=H_j(u_c,p_c)$ is the transformed Hamiltonian. h\_j(,;t) =h() -dx j(x,t) u\_c(x;,) ł[2.25]{}and $u_c(x;\x ,\eta )$ is the soliton solution parametrized by $(\x,\eta )$.*]{}
The proof of eq.() is the same as for the Coulomb problem considered in $^{14}$. But the case of the $(1+1)$-dimensional model needs the additional explanations. First of all, one must introduce the functional (u,p)=\_t d\^N (t) d\^N (t) \_[x,t]{}(u(x,t)-u\_c(x;,)) (p(x,t)-p\_c(x;,)), ł[2.26]{}The equalities u(x,t)=u\_c(x;,), p(x,t)=p\_c(x;,) ł[2.27a]{}assume that for given $u(x,t)$ and $p(x,t)$ one can hide the $t$ dependence into the $N$ functions $\x=\x(t)$ and $\eta=\eta(t)$. It is assumed that this procedure can be done for arbitrary $x$. In other respects functions $u(x,t)$ and $p(x,t)$, and therefore, $u_c(x;\x,\eta)$ and $p(x;\x,\eta)$, are arbitrary.
For more confidence, one may divide the space onto the $N$ cells and to each $(u,p)_x$ we may adjust $(\x,\eta)_x$. Quiet possible that $(\x,\eta)$ are $x$ independent. In this degenerate case $\D\sim
(\d(0))^k$, where $k\leq N$ is the degree of the degeneracy. We will omit the index $x$ considering $(\x,\eta)_x$ as the vector of the necessary dimension.
If $(\x,\eta)$ are the solutions of (), then (u,p)=\_t d’(t) d’(t) (u\_c\^’+u\_c\^’)(p\_c\^’+p\_c\^’)= \_c(,)0, ł[2.27]{}where, for instance, $u_c^X=\pa u_c(x;\x,\eta)/\pa X$, $X=\x,\eta$. Notice importance of last condition. If it fulfilled then one may insert into (), with measure (), 1= ł[2.28]{}and integrate over $u(x,t)$ and $p(x,t)$. Notice that the possible infinite factor $(\d(0))^k$ would be canceled in the ratio ().
The Jacobian of transformation
$$J=\int \f{DuDp}{\D_c(\x,\eta)}\prod_{x,t}
\d \le(\dot{u}-\frac{\d H_j (u,p)}{\d p}\ri)
\d \le(\dot{p}+\frac{\d H_j (u,p)}{\d u}\ri)$$(u(x,t)-u\_c(x;,))(p(x,t)-p\_c(x;,)), ł[2.28a]{}is proportional to functional $\d$-functions again. To have the transformation, we should use the last two $\d$-functions. Notice, if the first two $\d$-functions are used to calculate $J$, then last two $\d$-functions realize the constraints. In result, J=\_[x,t]{} (\_c-) (\_c+) ł[2.29]{}It should be underlined that $u_c$ and $p_c$ are $arbitrary$ functions of $\x$ and $\eta$, i.e. on this stage we make the transformation of arbitrary functions $u(x,t)$ and $p(x,t)$ on the new arbitrary functions $u_c(x;\x,\eta)$ and $p_c(x;\x,\eta)$, where, generally speaking, $\x=\x(x,t)$ and $\eta=\eta(x,t)$. Then $\D_c$ is the corresponding determinant.
The expression () can be rewritten identically to the form: $$J= \f{1}{\D_c(\x,\eta)}
\int\prod_{x,t} d\x'(t) d\eta'(t)\t$$$$\t
\d\le(\x'-\le(\dot{\x}-\frac{\d h_j(\x,\eta;t)}{\d\eta}\ri)\ri)
\d\le(\eta'-\le(\dot{\eta}+\frac{\d h_j(\x,\eta;t)}{\d\x}\ri)\ri)\t$$$$\t\d\le(u_c^\x\x'+u_c^\eta\eta'+
\{u_c,h_j\}-\f{\d H_j}{\d p_c(x,t)}\ri)\t$$(p\_c\^’+p\_c\^’- {p\_c,h\_j}+), ł[2.30]{}where $\{,\}$ is the Poisson bracket.
Let us assume now that the auxiliary function $h_j(\x,\eta;t)$ is chosen so that the equalities {u\_c,h\_j}=, {p\_c,h\_j}=-. ł[2.31]{}are satisfied identically. Then, taking into account the condition (), one can find: J=(-) (+). ł[2.32]{}This ends the transformation. Notice that the determinant $\D_c$ was canceled identically.
The transformation specify by the equations () the function $h_j$. It assumes that one can find such functions $u_c=u_c(x;\x,\eta)$ and $p_c=p_c(x;\x,\eta)$, with property (), that () has unique solution $h_j(\x,\eta;t)$.
Let us convert the problem assuming that just $h_j$ is known. It is natural to assume that h\_j(,;t)=H\_j(u\_c,p\_c), ł[2.33]{}then $u_c$ and $p_c$ are defined by the equations () and = =-. ł[2.34]{}It is not hard to see that () together with () are equivalent to incident equations (). This is seen from the following chain of equalities: $$\dot{u}_c(x;\x\eta)=u_c^\x\dot{\x}+u_c^\eta\dot{\eta}=$$$$=
u_c^\x\frac{\pa h_j(\x,\eta;t)}{\pa\eta}-
u_c^\eta\frac{\pa h_j(\x,\eta;t)}{\pa\x}=\{u_c,h_j\}=
\f{\d H_j}{\d p_c(x,t)}$$ and the same we have for $p_c$. Therefore $(u_c,p_c)$ is the classical phase space flow and the space $W_G$, labelled by $(\x,\eta)$, is the coset space $G/G_c$.
In result, new measure takes the form (), i.e. $\x$ and $\eta$ should obey the equations (): = ø() -dx j(x,t) , =dx j(x,t) , ł[2.35]{}where $\o(\eta)\equiv{\pa h(\eta)}/{\pa \eta}$. Hence the source of quantum perturbations are proportional to the time-local tangent vectors $$\int dx \pa u_N(x;\x,\eta)/\pa \eta, ~
\int dx \pa u_N (x;\x,\eta)/\pa\x$$ to the soliton configurations. It suggests the idea $^{14}$ to split the ‘Lagrange’ sources: $$j(x,t) \rar (j_{\x}, j_{\eta})(t).$$
The mechanism of splitting was described in $^{10}$. Resulting operator ${\cal O}(u_c)$, defined in (), has the same structure. But new perturbations generating operator (e\_,e\_;j\_,j\_) =Re\_[C\_+]{} dt {\_ (t)\_(t)+ \_(t)\_(t)}. ł[2.36]{}The measure takes the form: DM(,)=\_[t]{}d(t) d(t) (- ø() - j\_(t)) ( - j\_(t)) ł[2.37]{}The effective potential $U=U(u_c;e_c)$ with e\_c(x,t)=e\_(t) - e\_(t) . ł[2.38]{}
Notice that the space degree of freedom is disappeared from our consideration.
0.5cm [**III. Multiple production in sin-Gordon model**]{} 0.3cm
We would consider the theory with Lagrangian L=(\_u)\^2 + \[(u)-1\] . ł[3.1]{}It is well known that this field model possess the soliton excitations in the (1+1) dimension.
Formally nothing prevents to linearize partly our problem considering the Lagrangian $$L=\f{1}{2}[(\pa_{\mu}u)^2 - \a m^2 u^2] +
\f{m^2}{\la^2}[\cos(\la u)-1 + \a\f{\la^2}{2} u^2]\equiv$$S(u)-v(u) ł[3.2]{}The last term $v(u)=O(u^4)$ describes interactions. Corresponding vertex function is (q;u)=dx dt e\^[iqx]{} (\^2 +m\^2)u(x,t), q\^2 =m\^2. ł[3.3]{}It should be noted here that chosen in () division onto the ‘free’ and ‘interaction’ parts did not affects the equation of motion, see (), and effective potential, see (), i.e. in this sense $\a$ may be chosen arbitrary. But $\a$ will arise in the definition of the mass: one should change $m^2\to\a m^2$ in (). This means that our $S$-matrix approach requires additional, external, normalization condition for the mass shell. We will choose $\a=1$ assuming that $m$ is the measured mass of the ‘meson’.
We assume that $u(x,t)$ belongs to Schwarz space: u(x,t)|\_[|x| =]{}=0 (mod ). ł[3.4]{}This means that $u(x,t)$ tends to zero $(mod \frac{2\pi}{\la})$ at $|x|\rar \infty$ faster then any power of $1/|x|$.
The $\nu$-soliton classical Hamiltonian $h_\nu$ is the sum: h\_()=dr (r) +\^\_[i=1]{}h(\_i), ł[3.5]{}where $\sigma (r)$ is the continuous spectrum and $h(\eta)$ is the soliton energy. Notice absence of the energy of soliton interactions.
The $\nu$-soliton solution $u_\nu$ depends on the $2\nu$ parameters. Half of them $\nu$ can be considered as the position of solitons and other $\nu$ as the solitons momentum. Generally, at $|t|\to\infty$ the $u_\nu$ solution decomposed on the single solitons $u_s$ and on the double soliton bound states $u_b$: u\_(x,t)=\^[n\_1]{}\_[j=1]{}u\_[s,j]{}(x,t)+\^[n\_2]{}\_[k=1]{}u\_[b,k]{}(x,t)+ O(e\^[-|t|]{}) ł[3.5a]{}For this reason the one soliton $u_s$ and two-soliton bound state $u_b$ would be the main elements of our formalism. Its $(\x,\eta)$ parametrizations, i.e. the solution of eq.(), has the form $^{27}$: u\_s(x;,)=-{(mx-)}, = ł[3.6]{} and u\_b(x;,)= -{ }. ł[3.7]{}
The $(\x,\eta)$ parametrization of solitons individual energies $h(\eta)$ takes the form: $$h_s(\eta)=\f{m}{\b}\cosh \b\eta,~~~
h_b(\eta)=\f{2m}{\b}\cosh \f{\b\eta_1}{2}\sin\f{\b\eta_2}{2}\geq 0.$$ The bound-state energy $h_b$ depends on $\eta_2$ amd $\eta_1$. First one defines inner motion of two bounded solitons and second one the bound states center of mass motion. Correspondingly we will call this parameters as the internal and external ones. Note that the inner motion is periodic, see ().
Following to the definition of the Dirac measure one should sum over all solutions of the Lagrange equation, see the property [**b.**]{} in Sec.2. As follows from the equality: $$\sum_{\{u_c\}}=\int_{W_G} d\x_0 d\eta_0 \s(u;\x_0,\eta_0)$$ we should define the density $\s(u;\x_0,\eta_0)$ of states in the element of the coset space $W_G$. The Faddeev-Popov $ansatz$ is used for this purpose $^4$.
In our approach, performing the transformation into the coset space $W_G$, we define the density $\s(u;\x_0,\eta_0)$. Indeed, using the definition: $$\int Dx\prod_t\d(\dot{x})=\int dx(0)=\int dx_0$$ the functional integrals with measure () are reduced to the ordinary ones over the initial data $(\x,\eta)_{0}$.
But it is important here to trace on the following question. One can note that, at first glance, integration over $(\x,\eta)_{0}$ may only give $\R\sim V_{0}^1$, where $V_{0}$ is the zero modes volume, i.e. is a volume of the $W_G$ space. On other hand, as follows from definition of $\R\sim |a_{nm}|^2$, one may expect that $\R\sim
V_{0}^2$. This discrepancy should have an explanation.
Remembering definition of $\R$ as the squire of amplitudes, we should defined the contributions on the whole time contour $C=C_++C_-$, see (), to take into account the input condition that the trajectories $u_+(t\in C_+)$ and $u_-(t\in C_-)$ are absolutely independent in the frame of the ‘closed-path’ boundary condition (): u\_c(x,tC\_+)=u\_c(x,tC\_-), ł[3.8]{}where $\pa C_\pm$ is the boundary of $C_\pm$. Other directions to the $\s_|infty$ are not important here.
Then, if we introduce $(\x,\eta)(t\in C_\pm)|_0\equiv
(\x_0,\eta_0)_\pm$, one should have in mind that, generally speaking, $(\x_0,\eta_0)_+\neq (\x_0,\eta_0)_-$ and the integration over them should be performed independently. This may explain above discrepancy and one should have $\R\sim V_{0}^2$.
It is not hard to see that for our topological solitons the condition () leads to the equalities: (\_0,\_0)\_+=(\_0,\_0)\_-=(\_0,\_0). ł[3.9]{}To see this it is enough to inserting (), or (), into () and take into account that at $t\in \pa C_\pm$ the estimation () is right.
Solution () means that, for arbitrary functional $F(\x,\eta)$, \_[tC\_++C\_-]{}dd()()F(,)= d\_[0+]{}d\_[0+]{}d\_0d\_0 F(\_0,\_0). ł[3.9a]{}Therefore, $\R\sim V_0^2$. We will put out the integrals over inessential variables $\x_{0+}$ and $\eta_{0+}$.
It should be underlined that () is the consequence of the conservation of the topological charge: the solitons by this reason are the stable formation and, therefore, to satisfy the closed path boundary condition, one should have ().
Performing the shifts: \_i (t) \_i (t) + dt’ g(t-t’) j\_[,i]{}(t’) \_i (t) +’\_i (t),\
\_i (t) \_i (t) + dt’ g(t-t’) j\_[,i]{}(t’) \_i (t) +’\_i (t), we can get the Green function $g(t-t')$ into the operator exponent: (ej) =dtdt’(t-t’){’(t’)\_(t)+ ’(t’)\_(t)}. ł[3.10]{}since the Green function $g(t-t')$ of the transformed theory is the step function $^{10}$: g(t-t’)=(t-t’) ł[3.11]{}Such Green function allows to shift $C_{\pm}$ on the real-time axis. This, noting (), excludes doubling of the degrees of freedom.
Notice the Lorentz noncovariantness of our perturbation theory with Green function ().
The measure takes the form: D\^M(,)=\^\_[i=1]{}\_[t]{}d\_i(t) d\_i(t) (\_i- ø(+’))(\_i). ł[3.12]{}The interactions are described by U(u\_;e\_c)=-dx dt u\_ (e\_c -e\_c) ł[3.14]{}with u\_=u\_(x;+’,+’) and $e_c$ was defined in ().
The equations: \_i=ø(\_i+’\_i) are trivially integrable. In quantum case $\eta'_i \neq 0$ this equation describes motion in the nonhomogeneous and anisotropic manifold. So, the expansion over $(\hat{\x'},~\hat{e}_{\x},~\hat{\eta}',
~\hat{e}_{\eta})$ generates the local in time fluctuations of $W_G$ manifold. The weight of this fluctuations is defined by $U(u_\nu;e_c)$.
Expansion of $\exp\{\K(je)\}$ gives the ‘strong coupling’ perturbation series. The analyses shows that $^{14}$
[*S3.Action of the integro- differential operator $\h{\cal O}$ leads to following representation*]{}: (,z)=\_[W\_G]{}{ d(o)R\^(,z)+ d(0)R\^(,z)}. ł[3.13]{}This means that the contributions into $\R$ are accumulated strictly on the boundary ‘bifurcation manifold’ $\pa W_G$. The prove of this important result was given in $^{10,14}$ and we will use it without comments.
We would divide calculations on two parts. First of all, we would consider the semiclassical approximation and then we will show that this approximation is exact.
Performing the last integration we find: (,z)=\^\_[i=1]{} {d\_0 d\_0}\_i e\^[-i ]{}e\^[iS\_O(u\_)]{}e\^[-iU(u\_;e\_c)]{} e\^[N(,z;u\_)]{} ł[5.1]{}where u\_=u\_(\_0 +’,\_0 + ø(t) +’). ł[5.2]{}and ø(t)=dt’(t-t’)ø(\_0 +’)(t’) ł[5.3]{}
In the semiclassical approximation $\x'=\eta'=0$ we have: u\_=u\_(x;\_0 ,\_0 + ø(\_0)t). ł[5.7]{}Notice that the surface term dx\^\_(e\^[iqx]{}u\_)=0. ł[5.8]{}Then d\^2x e\^[iqx]{}(\^2 +m\^2)u\_(x,t) =-(q\^2-m\^2)d\^2x e\^[iqx]{}u\_(x,t) =0 ł[5.9]{}since $q^2$ belongs to the mass shell by definition. The condition () is satisfied for all $q_{\mu}\neq0$ since $u_\nu$ belong to the Schwarz space. Therefore, in the semiclassical approximation $R^c(\a,z)$ is the trivial function of $z$: $\pa R^c(\a,z)/\pa z=0$.
Expending the operator exponent in (\[5.1\]), we find that action of the operators $\hat{\x}'$, $\hat{\eta}'$ create the terms \~d\^2x e\^[iqx]{} (t-t’) (\^2 +m\^2)u\_(x,t) 0. ł[5.12]{}So, generally $R(\a,z)$ is the nontrivial function of $z$.
Now we will show that the semiclassical approximation is exact in the soliton sector of the sin-Gordon model. The structure of the perturbation theory is readily seen in the ‘normal- product’ form: R(,z)=\_\^[N]{}\_[i=1]{} {d\_0 d\_0}\_i\
:e\^[-iU(u\_;/2i)]{}e\^[iS\_O(u\_)]{}e\^[N(,z;u\_)]{}:, ł[6.1]{}where =\_- \_=Ø\_[X]{} ł[6.2]{}and \_[X]{}=dt’ (t-t’)(t’) ł[6.3]{}with the $2N$-dimensional vector $X=(\x ,\eta)$. In eq.() $\O$ is the ordinary symplectic matrix.
The colons in () mean that the operator $\hat{j}$ should stay to the left of all functions. The structure () shows that each order over $\hat{j}_{X_i}$ is proportional at least to the first order derivative of $u_\nu$ over conjugate to $X_i$ variable.
The expansion of () over $\hat{j}_{X}$ can be written using in the form: (,z)=\_\^\_[i=1]{} {d\_0 d\_0}\_i {\^[2]{}\_[i=1]{}P\_[X\_i]{}(u\_)}, ł[6.4]{}where $P_{X_i}(u_\nu)$ is the infinite sum of the ‘time-ordered’ polynomial over $u_\nu$ and its derivatives $^{14}$. The explicit form of $P_{X_i}(u_\nu)$ is unimportant, it is enough to know, see (), that P\_[X\_i]{}(u\_)\~Ø\_[ij]{} . ł[6.5]{}
Therefore, R(,z)=0 ł[6.6]{}since (i) each term in () is the total derivative, (ii) we have () and (iii) $u_\nu$ belongs to Schwarz space.
0.5cm [**IV. Conclusion**]{} 0.3cm
We would like to conclude this paper noting the role of the coset space $G/G_c$ topology. It was shown that if
\(a) $W_G=G/G_c\neq\emptyset$,
\(b) $W_G=T^*V$ is the simplectic manifold,
\(c) $\pa u_c$ is the flow,
\(d) $\pa u_c\bigcap\pa W_G=\emptyset,$\
then the semiclassical approximation is exact.
For this reason, being absolutely stable, ‘topological solitons’ are unable to describe the multiple production processes. This property of the exactly integrable models was formulated also as the absence of stochastization in the integrable systems $^{28}$. The $O(4)\t
O(2)$-invariant solution of $O(4,2)$-invariant theories $^{29}$ is noticeably more interesting from this point of view $^{30}$.
We acknowledged to members of the seminar ‘Symmetries and integrable systems’ of the N.N.Bogolyubov Laboratory of theoretical physics (JINR) for important discussions. We would like to thank V.G.Kadyshevski for fruitful interest to described technique and underling idea. One of us (JM) was partly granted by Georgian Acad. of Sciences.
Appendix. Derivation of eq.([29]{})
===================================
The generating functional () can be written in the form: (,z)=e\^[-(,z;)]{}\_0(), ł[a1]{}where the particles number operator (,z;)=(\_+,z\_+;)+\^\*(\_-,z\_-;), ł[a2]{}and (\_+,z\_+;\_+)=dØ\_1(q)\_+\^\*(q)\_-(q) e\^[-\_+(q)]{}z\_+(q) ł[a3]{}is the produced particles number operator.
The functional $\R_0$ was introduced in (): $$\R_0(\vp)=Z(\vp_+)Z^* (-\vp_-)=$$=Du\_+Du\_- e\^[iS\_+(u\_+)-iS\_-(u\_-)]{} e\^[-iV\_+(u\_++\_+)+iV\_-(u\_–\_-)]{}. ł[a4]{}So, the integration over $u_+$ and $u_-$ is not performed independently: one should take into account the boundary condition (). We can perform in this integrals the linear transformation: u\_(x)=u(x)(x). ł[a5]{}Then the boundary condition () leads to equality: (x\_)=0, ł[a5a]{}leaving $u(x\in\s_\infty)$ arbitrary. Last one means that the integration over this ‘turning-point’ field $u(x\in\s_\infty)$ should be performed, see Sec.3.
Let us extract in the exponents () the linear over $(\phi
+\vp)$ term: $$V_+(u+(\p+\vp)) - V_-(u- (\p+\vp))=$$+U(u,+) +2\_[C\_+]{} dx ((x) +(x))v’(u), ł[a6]{}and S\_+(u+ ) -S\_-(u- )=S\_O (u) -2i\_[C\_+]{} dx(x) (\_\^2 +m\^2 )u(x). ł[a7]{}where $$2\Re\int_{C_+}=\int_{C_+} + \int_{C_-}.$$ Notice that the generally speaking, $S_O(u)\neq 0$ if the topology of field $u(x)$ is nontrivial, see Sec.3.
The expansion over $(\p+\vp)$ can be written in the form: e\^[-iU(u,+)]{}=e\^[Re\_[C\_+]{}dx (x) (x)]{} e\^[i2\_[C\_+]{}dx dt j(x)((x)+(x))]{}e\^[-iU(u,’)]{}, ł[a8]{}where $\h{j}(x),~\h{\vp'}(x)$ are the variational derivatives. The auxiliary variables $(j,\vp')$ must be taken equal to zero at the very end of calculations.
In result, &\_0()=e\^[\_[C\_+]{} dx (x) (x)]{}Du e\^[is\_0(u)]{}e\^[-iU(u,)]{} e\^[i2\_[C\_+]{}dx (j(x)-v’(u)) (x)]{}\
&\_[x]{}(\_\^2 u + m\^2 u +v’(u) -j), ł[a9]{}where the functional $\d$-function was defined by the equality: \_[x]{}(\_\^2 u + m\^2 u +v’(u) -j) =D’e\^[-2i\_[C\_+]{}dx (\_\^2 u + m\^2 u +v’(u) - j)(x)]{}, ł[a10]{}where the prime means that $D'\p$ does not includes the integration over $\p(x\in\s_\infty)$. This condition is not seen in the functional $\d$-function because of the definition: $$\int \prod_x du(x)\d(\pa_\mu u(x))=\int du(x_\mu\in\s_\infty).$$
The eq.() can be rewritten in the equivalent form: \_0()=e\^[-i(j,)]{}DM(u)e\^[is\_0 (u) -iU(u,)]{} e\^[i2Re\_[C\_+]{}dx (x)(\_\^2 + m\^2) u(x)]{} ł[a11]{}because of the $\d$-functional measure: DM(u)=\_[x]{} du(x) (\_\^2 u + m\^2 u +v’(u) -j), ł[a12]{}with (j)=\_[C\_+]{} dx(x)(x). ł[a13]{}Notice at the end that the contour $C_+$ in () can not be shifted on the real time axis since the Green function of the equation $$\pa_{\mu}^2 u + m^2 u +v'(u)=j$$ is singular on the light cone.
The action of operator ${\bf N}(\b,z;\h\p)$ maps the interacting fields system on the physical states. Last ones are ‘marked’ by $z_\pm$ and $\b_\pm$. The operator exponent is the linear functional over $\p$ and this allows easily find ().
[99]{}
$^{a)}$Inst. of Physics, Tbilisi, Georgia $\&$ Lab. of Nuclear Problems, JINR, Dubna, Ru 141980, Russia. E-mail: [email protected]\
$^{b)}$JINR, Dubna, Ru 141980, Russia. E-mail: [email protected]\
$^1$R.Jackiw, Rev. Mod. Phys., [**49**]{}, 681 (1977)\
$^2$The developed below formalism is not manifestly Lorentz covariant. By this reason we decouple the space component $x$ and time $t$. But, if the expression contains the $x$ dependence only, then in this expression $x$ is the 2-dimensional vector.\
$^34$L.Faddeev and V.Korepin, Phys. Rep., [**42C**]{}, 3 (1978)\
$^4$J.Goldstone and R.Jackiw, Phys.Rev., [**D11**]{}, 1486 (1975)\
$^5$R.Jackiw, Phys. Rep., [**22**]{}, 273 (1976)\
$^6$It was noted in the papers of R.Dashen, B.Hasslacher and A.Neveu, Phys. Rev., [**D10**]{}, 4114 (1974), [*ibid.*]{}, 4130 (1974) that the semiclassical bound states mass spectrum of sin-Gordon model coincide with strict one, defined using the isomorphysm among the Thirring fermion fields model and soliton sector of the sin-Gordon model, S.Coleman, Phys. Rev. [**D11**]{} 2088 (1975). This result may be explained as the consequence of stabilizing the quantum perturbations effect of the infinite number of hidden conservation laws in involution presence in the sin-Gordon model, J.L.Gervais and A.Neveu, Phys. Rep., [**23**]{} 237 (1976). One may note also that the factorization of soliton $S$-matrix, see e.g. R.Rajaraman, [*Solitons and Instantons*]{} (North-Holland Publ. Comp., Amsterdam, 1982), and absence of multiple production, A.B.Zamolodchikov and A.B.Zamolodchikov, Phys. Lett., [**72B**]{} 503 (1978), is peculiar for the exactly integrable models. This seems natural since if the soliton may produce the meson, then the soliton $S$-matrix can not be factorizable, as well as the mesons may produce the solitons.\
$^7$I.H.Duru, Phys.Rev., [**D30**]{}, 143 (1984)\
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$^{10}$J.Manjavidze and A.Sissakian, hep-th/0003039, Theor. Math. Phys., [**123**]{}, 776 (2000)\
$^{11}$The eikonal approximation for the path integrals was considered in: S.P.Kuleshov, V.A.Matveev, A.N.Sissakian, M.A.Smondirev, A.N.Tavkhelidze, El. Part. At. Nucl., [**5**]{} 3 (1974)\
$^{12}$E.Cartan, Math. Ann., [**102**]{}, 698 (1930); E.Cartan and E.Schouten, Proc. Kon. Ned. Akad. Wetensch. [**29**]{}, 933 (1926); R.Finkelstein, Rev. Math. Phys., [**1**]{}, 440 (1960); N.H.Ibragimov, [*Groups of Transformations in Mathematical Physics*]{},(Moskow, Nauka, 1983)\
$^{13}$J.S.Dowker, Ann.Phys.(NY), [**62**]{}, 361 (1971)\
$^{14}$J.Manjavidze, Journ. Math. Phys., [**July**]{} (2000)\
$^{15}$V.I.Arnold, [*Mathematical Methods of Classical Mechanics*]{}, (Springer Verlag, New York, 1978)\
$^{16}$J.P.Garrahan and M.Kruczenski, Journ. Math. Phys., [**40**]{}, 6178 (1999)\
$^{17}$G.W.Mackey, [*Induced Representation of Groups and Quantum Mechanics*]{}, (Benjamin, New York, 1969); one can consider also N.Woodhouse, [*Geometric quantization*]{}, (OUP, Oxford, 1980); N.P.Landsman and N.Linden, Nucl. Phys., [**B365**]{}, 121 (1991); N.P.Landsman, Rev. Math. Phys., [**2**]{}, 45, 73 (1991); C.J.Isham, [*Relativity, Groups and Topology II*]{}, eds. B.S.De Witt and R.Stora (North-Holland, Amsterdam, 1984)\
$^{18}$There are the remarkable attempts to construct a geometrical approach to quantum mechanics, e.g. T.W.Kibble, Comm. Math. Phys., [**65**]{}, 189 (1979), J.Anandan, Found. Phys., [**21**]{}, 1265 (1991), and field theory, e.g. N.P.Konopleva and V.N.Popov, [*Gauge fields*]{} (Harward Acad. Publ., Chur-London-New York, 1981). Note that our interpretation is dealing with the classical phase-space flows. By this reason, in contrast with above mentioned approaches, the finite dimensional manifolds only, as in classical mechanics, would arise.\
$^{19}$S.Smale, Inv. Math., [**11:1**]{} 45 (1970)\
$^{20}$R.Abraham and J.E.Marsden, [*Foundations of Mechanics*]{} (Benjamin/ Cummings Publ. Comp., Reading, Mass., 1978)\
$^{21}$A.N.Vasil’ev, [*Functional Methods of Quantum Field Theory and Statistical Physics*]{}, (Leningrad Univ. Press, Leningrad, 1976)\
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$^{24}$J.Manjavidze and A.Sissakian, hep-ph/0003039, to be published in El. Part. [&]{} At. Nucl. (2000)\
$^{25}$J.Manjavidze and A.Sissakian, Phys. Rep., to be published\
$^{26}$Following toy analogy with ferromagnetic may be useful. So, the external magnetic field $\cal{H}$$\sim \bar{\mu}$, where the average spin $\bar{\mu}$ is the magnetics order parameter, and the phase transition means that $\bar{\mu}\neq 0$. Offered $\Ga
(q,u)$ has the same meaning as $\cal{H}$.\
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$^{30}$J.Manjavidze and A.Sissakian, hep-th/9811160
| tomekkorbak/pile-curse-small | ArXiv |
Identification of candidate transcriptional modulators involved in successful regeneration after nerve injury.
Successful regeneration of injured neurons requires a complex molecular response that involves the expression, modification and transport of a large number of proteins. The identity of neuronal proteins responsible for the initiation of regenerative neurite outgrowth is largely unknown. Dorsal root ganglion (DRG) neurons display robust and successful regeneration following lesion of their peripheral neurite, whereas outgrowth of central neurites is weak and does not lead to functional recovery. We have utilized this differential response to gain insight in the early transcriptional events associated with successful regeneration. Surprisingly, our study shows that peripheral and central nerve crushes elicit very distinct transcriptional activation, revealing a large set of novel genes that are differentially regulated within the first 24 h after the lesion. Here we show that Ankrd1, a gene known to act as a transcriptional modulator, is involved in neurite outgrowth of a DRG neuron-derived cell line as well as in cultured adult DRG neurons. This gene, and others identified in this study, may be part of the transcriptional regulatory module that orchestrates the onset of successful regeneration. | tomekkorbak/pile-curse-small | PubMed Abstracts |
Introduction {#Sec1}
============
Colorectal cancer (CRC) is a serious healthcare problem in the world, accounting for \~10% of all cancer cases and deaths^[@CR1]^. It was estimated that more than 1.8 million new cases and 881,000 deaths from CRC occurred in 2018 globally, with a higher incidence rate in Europe^[@CR1]^. The 5-year relative survival rate ranges from higher than 90% in CRC patients with early disease to about 10% in patients with advanced disease^[@CR2]^. Radiotherapy is the cornerstone for the treatment of CRC, along with surgery and chemotherapy^[@CR3]^. However, the existence and development of radioresistance is a great obstacle in the treatment of CRC^[@CR4],[@CR5]^. Over the past decades, accumulating non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been found to be key regulators or potential biomarkers in tumor initiation, progression, and radioresistance of CRC^[@CR5]--[@CR7]^.
LncRNAs longer than 200 nucleotides (nt) in length and miRNAs with the size of about 21 nt are two major families of transcripts that lack protein-coding potential^[@CR8]^. LncRNAs and miRNAs have been extensively studied for their regulatory roles in multiple cancer-related biological processes such as proliferation, apoptosis, and autophagy^[@CR9]--[@CR11]^. Moreover, some evidences disclose that lncRNAs can function as competing endogenous RNAs (ceRNAs) of miRNAs, resulting in the reduction of miRNA levels and increase of miRNA target levels^[@CR12],[@CR13]^.
Homeobox transcript antisense intergenic RNA (HOTAIR), a well-studied lncRNA, has been widely reported as an oncogenic molecule in various cancers such as breast cancer, renal cancer, and nasopharyngeal cancer^[@CR14],[@CR15]^. Previous studies showed that the depletion of HOTAIR could potentiate the radiosensitivity of some cancer cells such as breast cancer cells^[@CR16]^ and cervical cancer cells^[@CR17]^. Moreover, Yang et al.^[@CR18]^ disclosed that HOTAIR knockdown suppressed cell proliferation, migration, and invasion, but promoted cell apoptosis and potentiated cell radiosensitivity in CRC. In this text, the roles and molecular mechanisms of HOTAIR in CRC tumorigenesis and radioresistance were further investigated.
Our present study demonstrated that HOTAIR knockdown reduced cell viability, promoted cell apoptosis, and inhibited cell autophagy by upregulating microRNA-93 (miR-93) and downregulating autophagy-related 12 (ATG12) in CRC. Moreover, HOTAIR loss enhanced radiosensitivity through regulating miR-93/ATG12 axis in CRC cells and CRC xenograft tumor models.
Materials and methods {#Sec2}
=====================
Clinical samples and cell culture {#Sec3}
---------------------------------
Seventy-one patients diagnosed with CRC were recruited from the Affiliated Tumor Hospital of Zhengzhou University between 2012 and 2017. CRC tissues and adjacent normal tissues were collected from these patients through surgery. Partial tissue samples were snap frozen in liquid nitrogen and then stored at −80 °C till RNA extraction. Some specimens were fixed with formalin and embedded with paraffin for immunohistochemistry (IHC) and in situ hybridization (ISH) analysis. Blood samples were collected from 12 patients before or after radiotherapy. Then, plasma was isolated from blood through 10 min of centrifugation at 3000 r.p.m. Our study got approval of the Committees for the Ethical Review of Research at the Affiliated Tumor Hospital of Zhengzhou University and written informed consents from all patients.
Human normal colon epithelial cell line (FHC) and CRC cell lines (HT29, SW20, HCT116, and SW480) were purchased from American Type Culture Collection (Manassas, VA, USA). FHC cells were cultured in Dulbecco's modified Eagle's medium/F12 Medium (Thermo Scientific, Rockford, IL, USA) supplemented with 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/ml cholera toxin (Sigma-Aldrich), 0.005 mg/ml insulin (Sigma-Aldrich), 0.005 mg/ml transferrin (Sigma-Aldrich), 100 ng/ml hydrocortisone (Sigma-Aldrich), 20 ng/mL human recombinant epidermal growth factor (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Thermo Scientific). HT29 and HCT116 cells were cultured in McCoy's 5 A (Modified) Medium (Thermo Scientific) supplemented with 10% FBS (Thermo Scientific). SW620 and SW480 cells were grown in Leibovitz's L-15 Medium (Thermo Scientific) containing 10% FBS (Thermo Scientific). FHC, HT29, and HCT116 were maintained in a humidified incubator containing 95% air and 5% CO~2~ at 37 °C. SW620 and SW480 cells were maintained in an incubator containing 100% air at 37 °C.
Reagents and cell transfection {#Sec4}
------------------------------
Small interfering RNAs (siRNAs) specifically targeting HOTAIR (siHOTAIR\#1, siHOTAIR\#2, and siHOTAIR\#3) and a scramble control (scrambled), siRNA specific for ATG12 (siATG12) and its negative control (siNC), were purchased from GenePharma Co., Ltd (Shanghai, China). MiR-93 mimic (miR-93) and its negative control miR-NC and miR-93 inhibitor (anti-miR-93) and its negative control anti-miR-NC were ordered from Thermo Scientific, lnc. HOTAIR overexpression plasmid was constructed by Sangon Biotech Co., Ltd (Shanghai, China). All these oligonucleotides or plasmids were transfected into CRC cells using Lipofectamine 3000 reagent (Thermo Scientific), referring to the instructions of the manufacturer.
RNA extraction and real-time quantitative PCR {#Sec5}
---------------------------------------------
Total RNAs were extracted from tissues and cells using mirVana miRNA isolation kit (Thermo Scientific) according to the instruction manual. Then, TaqMan miRNA assay (Thermo Scientific) was used to measure miR-93 expression level following the manufacturer's instructions. U6 small nuclear RNA functioned as the internal control to normalize miR-93 expression. For the measurement of ATG12 mRNA level and HOTAIR level, M-MLV Reverse Transcriptase (Thermo Scientific) was used to conduct the reverse-transcription experiments and SYBR™ Green Master Mix (Thermo Scientific) was employed to perform the quantitative PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*) served as the housekeeping gene to normalize the expression of ATG12 mRNA and HOTAIR. The quantitative primer sequences were listed as follows: 5′-ATTGCTGCTGGAGGGGAAGG-3′ (forward) and 5′-GGTTCGTGTTCGCTCTACTGC-3′ (reverse) for ATG12, 5′-CAGTGGGGAACTCTGACTCG-3′ (forward) and 5′-GTGCCTGGTGCTCTCTTACC-3′ (reverse) for HOTAIR, and 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAGAC-3′ (reverse) for GAPDH.
Western blot assay {#Sec6}
------------------
Cells were lysed using ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Then, cells were centrifuged for 15 min at 4 °C at the speed of 12,000 r.p.m. and cell supernatants containing proteins were collected. Next, protein concentration was determined through Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 30 μg of protein samples in each lane were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After the blockage of nonspecific sites using 5% nonfat milk, the membranes were incubated overnight at 4 °C with primary antibodies against ATG12 (Abcam, Cambridge, UK), LC3B (Abcam), Bcl-2 (Abcam), Bax (Abcam), cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Abcam). Next, the membranes were probed for 1 h at room temperature with secondary antibody labeled with horseradish peroxidase (HRP) (Abcam). Finally, protein signals were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories) and quantified using Quantity One software Version 4.1.1 (Bio-Rad Laboratories).
Luciferase reporter assay {#Sec7}
-------------------------
Partial sequences of HOTAIR and ATG12 3′-untranslated region (3′-UTR) containing predicted miR-93 target sites were subcloned into psiCHECK-2 vector by Hanbio Biotechnology Co., Ltd (Shanghai, China), to produce HOTAIR-Wt reporter and ATG12-Wt reporter, respectively. Also, HOTAIR-Mut and ATG12-Mut reporters containing mutant miR-93 binging sites were constructed by Hanbio Biotechnology Co., Ltd. Then, cells were co-transfected with corresponding reporter vector and miR-93 mimic or miR-NC. Forty-eight hours later, cells were collected and lysed for luciferase reporter assay using a dual luciferase reporter assay system (Promega, Madison, WI, USA).
Clonogenic survival assay {#Sec8}
-------------------------
Transfected CRC cells were plated into six-well plates in triplicate and incubated for 24 h at 37 °C. After mock irradiated (0 Gy) or irradiated with different doses (2, 4, 6, 8 Gy) of ionizing radiation (IR), cells were maintained for about 2 weeks at 37 °C, to allow for colony formation. Next, colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet solution (Sigma-Aldrich) for 15 min. Finally, the number of positive colonies containing more than 50 cells was counted.
Cell viability analysis {#Sec9}
-----------------------
Cell viability was analyzed using a Cell Counting Kit-8 (CCK-8) assay kit (Dojindo Molecular Technologies, Rockville, MD, USA) following the protocols of the manufacturer. Briefly, transfected cells were seeded into 96-well plates in triplicate and incubated for 24 h prior to the treatment with sham or 4 Gy of IR. At 24 h after irradiation, 10 μl of CCK-8 solution was added into each well and incubated for 3 h. Finally, cell absorbance was determined at 450 nm.
Cell apoptosis detection {#Sec10}
------------------------
Cell apoptotic rate was determined using an eBioscience™ Annexin V-FITC Apoptosis Detection Kit (Thermo Scientific) referring to the instruction of the manufacturer. Briefly, transfected cells were plated into six-well plates in triplicate and incubated for 24 h prior to the treatment with sham or 4 Gy of IR. At 24 h post irradiation, cells were collected and then stained using Annexin V-FITC and propidium iodide solution. Finally, cell apoptotic rates were measured using a flow cytometry (FACScan; BD Biosciences, San Jose, CA, USA).
IHC assay {#Sec11}
---------
Protein expression patterns of ATG12, LC3 II, and cleaved caspase 3 in tissues were assessed by IHC assay. Briefly, tissue sections were sequentially treated following the procedures of deparaffinage, rehydration, antigen retrieval, endogenous peroxidase blockage, and nonspecific signal blocking. Then, tissue slides were probed overnight at 4 °C with primary antibodies against ATG12, LC3 II, and cleaved caspase 3, and incubated for 1 h at room temperature with HRP-labeled secondary antibody. Next, these sections were visualized using 3,3'-diaminobezidin (DAB) substrate, counterstained with hematoxylin, and imaged. ATG12 expression level was assessed by the scores of positively labeled cells containing brown particles, which was recorded as: 0 (positive rate \< 25%), 1 (positive rate: 25--50%), 2 (positive rate: 51--75%), and 3 (positive rate \> 75%).
ISH assay {#Sec12}
---------
Expression and localization patterns of HOTAIR in tissues were measured through ISH assay kit (FOCOFISH, Guangzhou, China) and custom-made probe following the protocols of the manufacturer. Briefly, after routine ISH treatment, tissue sections were incubated with HRP-conjugated labeling molecule, visualized using diaminobenzidine tetrahydrochloride (DAB) substrate, counterstained with hematoxylin staining solution, and imaged.
In vivo radiation sensitivity experiments {#Sec13}
-----------------------------------------
Animal experiments were conducted with the approval of Institutional Committee for Animal Research of the Affiliated Tumor Hospital of Zhengzhou University and performed following the guide for the care and use of laboratory animals recommended by National Institutes of Health. Lentiviruses containing HOTAIR knockdown fragment (shHOTAIR) and its negative control were customized from Hanbio Biotechnology Co., Ltd. CRC xenograft tumor models were established by subcutaneous injection of 5 × 10^6^ SW480 cells infected with control lentiviruses or shHOTAIR lentiviruses into the flanks of male athymic nude mice (6 weeks old, Laboratory Animal Center of Henan, Zhengzhou, China) with 8 mice in each group. Ten days later, mice were exposed to 10 Gy of IR once. Tumor volume was monitored using a caliper at day 7, 10, 13, 16, 19, 22, 25, and 28 after injection, and calculated using the formula of (length × width^2^)/2. Tumors were resected and weighed at day 28 after injection. HOTAIR and miR-93 levels in CRC xenograft tumors were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. Protein expression patterns of ATG12, LC3 II, and cleaved caspase 3 were estimated by IHC assay.
Statistical analysis {#Sec14}
--------------------
All in vitro experiments were repeated more than three times and animal experiments were conducted once with eight mice in each group. Data were presented as mean ± SD. Difference analysis was conducted using one-way analysis of variance (for multiple data) or Student's *t*-test (for two group data) with *P* \< 0.05 as statistically significant. Survival analysis was conducted using Kaplan--Meier method and log-rank test.
Results {#Sec15}
=======
HOTAIR was highly expressed in CRC tissues and cells, and HOTAIR expression was markedly upregulated in plasma samples from CRC patients after radiotherapy and CRC cells after IR treatment {#Sec16}
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
First, RT-qPCR assay and ISH analysis disclosed that HOTAIR was highly expressed in CRC tissues (*n* = 71) compared with adjacent normal tissues (*n* = 71) (Fig. [1a, b](#Fig1){ref-type="fig"}). ISH analysis also showed that HOTAIR was mainly distributed in the cytoplasm (Fig. [1b](#Fig1){ref-type="fig"}). Moreover, HOTAIR expression was remarkably increased in the majority (57/71) of CRC tissues than that in corresponding normal tissues (Fig. [1c](#Fig1){ref-type="fig"}). CRC clinical samples were divided into HOTAIR high-expression group (*n* = 36) and HOTAIR low-expression group (*n* = 35) with the median value of HOTAIR in CRC tissues as the cutoff point. Subsequent Kaplan--Meier survival analysis disclosed that CRC patients with high HOTAIR expression had a poor prognosis (Fig. [1d](#Fig1){ref-type="fig"}). Also, a notable increase of HOTAIR level was observed in CRC cells (HT29, SW620, HCT116, and SW480) relative to FHC cells (Fig. [1e](#Fig1){ref-type="fig"}). Next, the link of HOTAIR expression and radioresistance was further examined in CRC. As presented in Fig. [1f](#Fig1){ref-type="fig"}, HOTAIR level was markedly increased in most (11/12) of the plasma samples from CRC patients after radiotherapy, compared with those from corresponding patients before radiotherapy. Moreover, IR treatment at the dose of 2--8 Gy induced a dose-dependent elevation of HOTAIR level in SW480 and HCT116 cells (Fig. [1g, h](#Fig1){ref-type="fig"}).Fig. 1HOTAIR was highly expressed in CRC tissues and cells, and HOTAIR expression was markedly upregulated after IR treatment in CRC.**a**, **c** HOTAIR level was determined by RT-qPCR assay in 71 pairs of CRC tissues and corresponding normal tissues. **b** Representative images of ISH analysis for HOTAIR in CRC tissues and adjacent normal tissues. **d** Kaplan--Meier survival analysis of 71 CRC patients according to the difference of HOTAIR expression. **e** HOTAIR expression was measured by RT-qPCR assay in FHC, HT29, SW620, HCT116, and SW480 cells. **f** HOTAIR level was measured by RT-qPCR assay in plasma samples of 12 CRC patients before and after radiotherapy. **g**, **h** HOTAIR level was detected in SW480 and HCT116 cells treated with different doses (0, 2, 4, 6, or 8 Gy) of IR. \**P* \< 0.05.
HOTAIR knockdown potentiated the radiosensitivity of CRC cells {#Sec17}
--------------------------------------------------------------
To further investigate the function of HOTAIR, three siRNAs targeting HOTAIR (siHOTAIR\#1, siHOTAIR\#2, or siHOTAIR\#3) and a scramble control siRNA (scrambled) were synthesized. Following transfection efficiency analyses showed that the transfection of siHOTAIR\#1, siHOTAIR\#2, or siHOTAIR\#3 could trigger the notable reduction of HOTAIR level in SW480 and HCT116 cells compared with that in scrambled group (Fig. [2a, b](#Fig2){ref-type="fig"}). Among these siRNAs, siHOTAIR\#3 (siHOTAIR) was used for the following loss-of-function experiments due to its better knockdown efficiency. Subsequent radiation clonogenic survival assay showed that IR treatment (2--8 Gy) induced a dose-dependent reduction of cell survival fractions in SW480 and HCT116 cells transfected with siHOTAIR or control siRNA (Fig. [2c, d](#Fig2){ref-type="fig"}). HOTAIR knockdown led to the remarkable reduction of survival fractions of SW480 and HCT116 cells when cells were exposed to the same dose of IR (Fig. [2c, d](#Fig2){ref-type="fig"}). Moreover, HOTAIR depletion led to the reduction of cell viability (Fig. [2e, f](#Fig2){ref-type="fig"}), LC3 II/LC3 I ration (Fig. [2i](#Fig2){ref-type="fig"}) and Bcl-2 (anti-apoptosis protein) protein level (Fig. [2i](#Fig2){ref-type="fig"}), and increase of cell apoptotic rate (Fig. [2g, h](#Fig2){ref-type="fig"}) and protein levels of p62 (Fig. [2i](#Fig2){ref-type="fig"}), cleaved caspase 3 (Fig. [2i](#Fig2){ref-type="fig"}), and Bax (pro-apoptosis protein) (Fig. [2i](#Fig2){ref-type="fig"}) in SW480 and HCT116 cells, suggesting that HOTAIR knockdown impaired cell viability, induced cell apoptosis, and inhibited cell autophagy in CRC. Moreover, our data revealed that IR exposure weakened cell viability (Fig. [2e, f](#Fig2){ref-type="fig"}) and induced cell apoptosis (Fig. [2g, h](#Fig2){ref-type="fig"}) and autophagy (Fig. [2i](#Fig2){ref-type="fig"}) in SW480 and HCT116 cells transfected with siHOTAIR or control siRNA. In addition, HOTAIR knockdown triggered the reduction of cell survival fractions and cell viability, and the increase of cell apoptotic rate in IR (4 Gy)-treated SW480 and HCT116 cells, suggesting that HOTAIR loss enhanced radiosensitivity of CRC cells (Fig. [2c--i](#Fig2){ref-type="fig"}). Furthermore, HOTAIR depletion weakened IR-induced autophagy in SW480 and HCT116 cells (Fig. [2i](#Fig2){ref-type="fig"}). In addition, we demonstrated that LC3 II/LC3 I ratio was increased at first and then gradually reduced during 48 h after IR treatment with the highest level at 12 h after IR stimulation in SW480 cells transfected with siHOTAIR or scrambled siRNA (Supplementary Fig. [1](#MOESM1){ref-type="media"}). Protein level of cleaved caspase 3 was remarkably increased at 24 h after IR exposure in SW480 cells transfected with siHOTAIR or scrambled siRNA (Supplementary Fig. [1](#MOESM1){ref-type="media"}). These data suggested that mild autophagy could protect cell from detrimental conditions to a certain extent, whereas serious autophagy could induce cell apoptosis. In a word, these data suggested that HOTAIR knockdown might potentiate radiosensitivity of CRC cells by inhibiting autophagy.Fig. 2HOTAIR knockdown potentiated radiosensitivity of CRC cells.**a**, **b** sw480 and HCT116 cells were transfected with siHOTAIR\#1, siHOTAIR\#2, siHOTAIR\#3, or a scramble control siRNA (scrambled). Forty-eight hours later, HOTAIR level was measured by RT-qPCR assay. **c**, **d** SW480 and HCT116 cells were transfected with siHOTAIR or its scramble control. At 24 h after transfection, cells were exposed to 0, 2, 4, 6, or 8 Gy of IR and then maintained for about 2 weeks. Next, the number of positive colonies were measured using a microscope and the survival fractions were calculated. **e**--**i** SW480 and HCT116 cells were transfected with siHOTAIR or its scramble control for 24 h prior to the treatment with or without sham or 4 Gy of IR. **e**, **f** After 24 h of irradiation, cell viability was measured by CCK-8 assay. **g**, **h** Cell apoptotic rate was estimated using a flow cytometry at 24 h post irradiation. **i** Protein levels of LC3 I, LC3 II, p62, cleaved caspase 3, Bax, and Bcl-2 were determined at 24 h after irradiation. \**P* \< 0.05.
HOTAIR overexpression weakened the radiosensitivity of CRC cells by downregulating miR-93 {#Sec18}
-----------------------------------------------------------------------------------------
Next, bioinformatics analysis by StarBase online database showed that there existed some complementary sites between HOTAIR and "seed sequence" of miR-93 (Fig. [3a](#Fig3){ref-type="fig"}). To further validate this prediction, HOTAIR-Wt reporter containing putative miR-93-binding sites and HOTAIR-Mut reporter containing mutant miR-93-binding sites were constructed and then transfected into SW480 and HCT116 cells together with miR-NC or miR-93 mimic. Transfection efficiency analysis showed that the miR-93 level was markedly elevated in SW480 and HCT116 cells following the transfection of miR-93 mimic compared with miR-NC-transfected cells (Fig. [3b](#Fig3){ref-type="fig"}). Subsequent luciferase reporter assay revealed that miR-93 overexpression markedly reduced the luciferase activity of HOTAIR-Wt reporter in SW480 and HCT116 cells, but had no much influence on luciferase activity of HOTAIR-Mut reporter (Fig. [3c, d](#Fig3){ref-type="fig"}), suggesting that HOTAIR could bind with miR-93 through predicted binding sites. Next, RT-qPCR assay unveiled that HOTAIR knockdown induced the notable increase of miR-93 expression, whereas HOTAIR overexpression led to the remarkable downregulation of miR-93 level in SW480 and HCT116 cells (Fig. [3e, f](#Fig3){ref-type="fig"}). Functional analyses presented that miR-93 overexpression weakened cell viability (Fig. [3i, j](#Fig3){ref-type="fig"}), promoted cell apoptosis (Fig. [3k, l](#Fig3){ref-type="fig"}), and attenuated cell autophagic activity (Fig. [3m](#Fig3){ref-type="fig"}) in SW480 and HCT116 cells without IR treatment. Moreover, enforced expression of miR-93 resulted in the reduction of cell survival fractions and cell viability, and the increase of cell apoptosis in IR-treated SW480 and HCT116 cells, suggesting that miR-93 overexpression improved the radiosensitivity of CRC cells (Fig. [3g--m](#Fig3){ref-type="fig"}). In addition, our data disclosed that HOTAIR upregulation inhibited the increase of radiosensitivity caused by miR-93 in SW480 and HCT116 cells (Fig. [3g-m](#Fig3){ref-type="fig"}). Furthermore, miR-93 overexpression inhibited IR-induced autophagy in SW480 and HCT116 cells, whereas this effect of miR-93 was restored by upregulated HOTAIR (Fig. [3m](#Fig3){ref-type="fig"}). That was to say, HOTAIR overexpression weakened the radiosensitivity of CRC cells by downregulating miR-93 and relieving miR-93-mediated autophagy inhibition.Fig. 3HOTAIR overexpression weakened the radiosensitivity of CRC cells by downregulating miR-93.**a** Predicted complementary sites between HOTAIR and miR-93, and mutant sites in HOTAIR-Mut reporter. **b** SW480 and HCT116 cells were transfected with miR-NC or miR-93 mimic. Forty-eight hours later, miR-93 level was measured by RT-qPCR assay. **c**, **d** SW480 and HCT116 cells were co-transfected with miR-NC or miR-93 mimic and HOTAIR-Wt or HOTAIR-Mut reporter, followed by the detection of luciferase activities at 48 h after transfection. **e**, **f** SW480 and HCT116 cells were transfected with siHOTAIR or its scrambled control, HOTAIR overexpression plasmid or its empty vector. Then, miR-93 level was measured by RT-qPCR assay at 48 h post transfection. **g**, **h** SW480 and HCT116 cells were transfected with miR-NC, miR-93 mimic, miR-93 + Vector, or miR-93 + HOTAIR for 24 h prior to the irradiation of various doses (0, 2, 4, 6, or 8 Gy) of IR. Then, cell survival fractions were measured by radiation clonogenic survival assay at 2 weeks after irradiation. **i**--**m** SW480 and HCT116 cells were transfected with miR-NC, miR-93 mimic, miR-93 + Vector, or miR-93 + HOTAIR for 24 h and then treated with or without 4 Gy of IR for 24 h, followed by the measurement of cell viability, cell apoptotic rate, protein levels of LC3 I, LC3 II, p62, cleaved caspase 3, Bax, and Bcl-2. \**P* \< 0.05.
HOTAIR positively regulated ATG12 expression through miR-93 in CRC cells {#Sec19}
------------------------------------------------------------------------
Next, [microRNA.org](http://microRNA.org) online prediction website was used to identify possible targets of miR-93. Among candidate targets, ATG12 was chosen considering its crucial roles in autophagy and radioresistance (Fig. [4a](#Fig4){ref-type="fig"}). To further demonstrate this prediction, ATG12-Wt reporter bearing miR-93-targeting sites and ATG12-Mut reporter containing mutant miR-93-binding sites were generated. Subsequent luciferase reporter assay showed that miR-93 overexpression mediated the notable downregulation of luciferase activity of ATG12-Wt reporter in SW480 and HCT116 cells, whereas the mutation of miR-93-binding sites in the 3′-UTR region of ATG12 reconstituted luciferase activity (Fig. [3b, c](#Fig3){ref-type="fig"}), verifying that miR-93 could bind with ATG12 3′-UTR by putative binding sites. Moreover, ATG12 mRNA and protein expression was strikingly reduced in SW480 and HCT116 cells following miR-93 overexpression (Fig. [4d--f](#Fig4){ref-type="fig"}). Conversely, miR-93 loss triggered the notable elevation of ATG12 expression at mRNA and protein levels in SW480 and HCT116 cells (Fig. [4d--f](#Fig4){ref-type="fig"}). In addition, ATG12 mRNA and protein expression was significantly reduced in HOTAIR-depleted SW480 and HCT116 cells, but was strikingly increased in HOTAIR-overexpressed cells (Fig. [4g--i](#Fig4){ref-type="fig"}). These data suggested that HOTAIR could act as a ceRNA of miR-93 to sequester miR-93 from ATG12, resulting in the reduction of miR-93 level and increase of ATG12 level in CRC cells.Fig. 4HOTAIR functioned as a ceRNA of miR-93 to regulate ATG12 expression.**a** MiR-93 target sites in ATG12 3′-UTR and mutant sites in ATG12-Mut reporter. **b**, **c** Luciferase activities were measured at 48 h post transfection in SW480 and HCT116 cells co-transfected with ATG12-Wt or ATG12-Mut reporter and miR-93 mimic or miR-NC. **d**--**f** SW480 and HCT116 cells were transfected with miR-NC, miR-93 mimic, anti-miR-NC, or anti-miR-93. Forty-eight hours later, ATG12 mRNA (**d**) and protein (**e**, **f**) levels were measured by RT-qPCR assay and western blot assay, respectively. **g**--**i** SW480 and HCT116 cells were transfected with siHOTAIR or its scramble control, HOTAIR overexpression plasmid or its empty vector. Forty-eight hours later, ATG12 mRNA (**d**) and protein (**e**) levels were measured by RT-qPCR assay and western blot assay, respectively. \**P* \< 0.05.
ATG12 protein expression was markedly upregulated and associated with miR-93 and HOTAIR expression in CRC tissues {#Sec20}
-----------------------------------------------------------------------------------------------------------------
Next, IHC analyses showed that ATG12 protein level was remarkably increased in CRC tissues compared with adjacent normal tissues (Fig. [5a](#Fig5){ref-type="fig"}). Kaplan--Meier survival analysis manifested that CRC patients with high ATG12 expression had a short survival (Fig. [5b](#Fig5){ref-type="fig"}). Moreover, ATG12 protein expression was negatively associated with miR-93 expression and positively associated with HOTAIR level in CRC tissues (Fig. [5c, d](#Fig5){ref-type="fig"}).Fig. 5ATG12 protein expression was markedly upregulated and associated with miR-93 and HOTAIR expression in CRC tissues.**a** ATG12 protein expression pattern in CRC tissues and adjacent normal tissues were detected by IHC assay. **b** Kaplan--Meier survival analysis of CRC patients according to the difference of ATG12 protein scores. **c**, **d** MiR-93 or HOTAIR expression pattern in CRC tissues according to the difference of ATG12 protein scores. \**P* \< 0.05.
ATG12 knockdown improved the radiosensitivity of CRC cells {#Sec21}
----------------------------------------------------------
To further examine the function of ATG12 in CRC, siATG12 and its negative control siNC were synthesized. Following western blot assay demonstrated that the transfection of siATG12 triggered conspicuous reduction of ATG12 protein levels in SW480 and HCT116 cells (Fig. [6a, b](#Fig6){ref-type="fig"}), meaning that siATG12 could be used for the subsequent loss-of-function experiments. Radiation clonogenic survival assay indicated that ATG12 knockdown led to the notable reduction of cell survival fractions in SW480 and HCT116 cells treated with IR (Fig. [6c, d](#Fig6){ref-type="fig"}). Consistently, cell viability was reduced in IR-treated SW480 and HCT116 cells following the depletion of ATG12 (Fig. [6e, f](#Fig6){ref-type="fig"}). Moreover, ATG12 knockdown enhanced IR-induced apoptosis in SW480 and HCT116 cells (Fig. [6g--i](#Fig6){ref-type="fig"}). That was to say, ATG12 loss enhanced the radiosensitivity of CRC cells. Furthermore, ATG12 knockdown alleviated the aggressive phenotypes of CRC cells, as evidenced by the reduction of cell viability and increase of apoptotic rate and cleaved caspase 3 protein level in ATG12-depleted SW480 and HCT116 cells compared with siNC-transfected cells (Fig. [6e--i](#Fig6){ref-type="fig"}). Moreover, LC3 II/LC3 I ratio was reduced and p62 protein level was increased in SW480 and HCT116 cells following the knockdown of ATG12, indicating that ATG12 deficiency inhibited autophagy in CRC cells (Fig. [6i](#Fig6){ref-type="fig"}). Furthermore, ATG12 knockdown suppressed the elevation of cell autophagic activity induced by IR in SW480 and HCT116 cells (Fig. [6i](#Fig6){ref-type="fig"}). Taken together, these outcomes showed that ATG12 knockdown potentiated the radiosensitivity of CRC cells by inhibiting autophagy.Fig. 6ATG12 knockdown potentiated the radiosensitivity of CRC cells by inhibiting autophagy.**a**, **b** Transfection efficiency analysis of siATG12 in SW480 and HCT116 cells through western blot assay at 48 h after transfection. **c**--**h** The effects of ATG12 knockdown on cell radiosensitivity, viability, and apoptosis were measured by radiation clonogenic survival assay (**c**, **d**), CCK-8 assay (**e**, **f**), and flow cytometry analysis (**g**, **h**). **i** The effects of ATG12 silencing alone or in combination with IR stimulation on protein expression of LC3 I, LC3 II, p62, cleaved caspase 3, Bax, and Bcl-2 were measured by western blot assay. \**P* \< 0.05.
HOTAIR knockdown enhanced radiosensitivity of CRC xenograft tumors {#Sec22}
------------------------------------------------------------------
Next, we further demonstrated that CRC xenograft tumors with HOTAIR knockdown and IR treatment grew more slowly than IR-treated control tumors (Fig. [7a, b](#Fig7){ref-type="fig"}). Also, RT-qPCR assay demonstrated that the HOTAIR level was noticeably reduced and miR-93 level was obviously increased in IR-treated CRC xenograft tumors following the introduction of shHOTAIR lentiviruses (Fig. [7c](#Fig7){ref-type="fig"}). Moreover, IHC analyses showed that ATG12 and LC3 II protein expression was markedly reduced and cleaved caspase 3 protein level was strikingly increased in CRC xenograft tumors with HOTAIR knockdown and IR treatment than that in IR-treated control tumors (Fig. [7d](#Fig7){ref-type="fig"}). Based on these data, we might draw the conclusion that HOTAIR knockdown enhanced radiosensitivity by regulating miR-93/ATG12-mediated autophagy in CRC xenograft tumors.Fig. 7HOTAIR knockdown enhanced radiosensitivity of CRC xenograft tumors.**a**--**d** CRC xenograft tumor models were established by subcutaneous injection of SW480 cells infected with control lentiviruses or shHOTAIR lentiviruses into the flanks of mice. Ten days later, mice were irradiated with 10 Gy of IR once. **a** Tumor volume was monitored using a caliper at day 7, 10, 13, 16, 19, 22, 25, and 28 after injection. **b** Tumor weight was measured at day 28 after injection. **c** HOTAIR and miR-93 levels in CRC xenograft tumors were determined by RT-qPCR assay. **d** Protein expression patterns of ATG12, LC3 II, and cleaved caspase 3 were evaluated by IHC assay. \**P* \< 0.05.
Discussion {#Sec23}
==========
CRC is a huge threat for human health and life with the third morbidity and the second mortality in all cancers worldwide^[@CR1]^. Radioresistance is a major reason for the failure of radiotherapy in cancers. An in-depth understanding on radioresistance-related molecular mechanisms contributes to the improvement of radiotherapy outcomes.
In this text, we demonstrated that HOTAIR was highly expressed in CRC tissues and cells, which was in line with previous studies^[@CR18],[@CR19]^. Also, consistent with prior reports^[@CR19],[@CR20]^, our data disclosed that CRC patients with high HOTAIR expression had an unfavorable prognosis. In this text, we further demonstrated that HOTAIR expression was remarkably increased in plasma samples from CRC patients after radiotherapy and IR-stimulated CRC cells. Functional analysis revealed that HOTAIR knockdown resulted in the downregulation of cell viability and increase of cell apoptosis in CRC cells. Similar with our data, prior studies demonstrated that HOTAIR loss weakened the proliferative, migratory, and invasive capacities of CRC cells and induced CRC cell apoptosis^[@CR18],[@CR21],[@CR22]^.
HOTAIR-mediated autophagy has been found to be an essential event in the development and progression of some cancers such as breast cancer^[@CR23]^ and hepatocellular cancer^[@CR24]^. Moreover, HOTAIR could affect chemoresistance by regulating autophagy in endometrial cancer^[@CR25]^ and non-small cell lung cancer^[@CR26]^. Hence, we further investigated whether HOTAIR could control radioresistance by regulating autophagy in CRC. Our present study demonstrated that HOTAIR depletion suppressed autophagy and weakened IR-induced autophagy in CRC cells. Moreover, HOTAIR depletion potentiated radiosensitivity of CRC cells in vitro, which was in accordance with a previous study^[@CR18]^. In summary, these data disclosed that HOTAIR loss improved radiosensitivity of CRC cells by inhibiting autophagy. Similarly, a recent study pointed out that HOTAIR knockdown enhanced radiosensitivity by inhibiting autophagy in pancreatic cancer cells^[@CR27]^. Also, we further demonstrated that HOTAIR loss enhanced IR-mediated anti-tumor effects in CRC xenograft tumors.
MiR-93, a member of miR-106b \~25 cluster^[@CR28]^, has been found to be closely linked with tumor initiation, progression, and radioresistance in some malignancies^[@CR29]--[@CR31]^. Also, previous studies showed that miR-93 could function as a negative regulator of cell autophagy^[@CR32]^. Moreover, bioinformatics analysis by StarBase online website showed that there were several complementary sites between HOTAIR and miR-93. In addition, [microRNA.org](http://microRNA.org) online prediction analysis suggested that ATG12 was a potential target of miR-93. ATG12, a core component of autophagy-related complexes, has also been reported to be implicated in tumor initiation, progression, and radioresistance in some cancers such as cervical cancer^[@CR33]^ and bladder cancer^[@CR34]^. Moreover, miR-93 expression was markedly reduced in CRC tissues and cells, and was negatively associated with HOTAIR expression in CRC tissues (data not presented). In addition, miR-93 expression was notably downregulated in plasma samples of CRC patients after radiotherapy and CRC cells following IR treatment (data not presented).
Bao et al.^[@CR35]^ demonstrated that HOTAIR loss-induced autophagy inhibition led to the reduction of cell viability and increase of cell apoptotic rate by regulating miR-454--3p/ATG12 or miR-454-3p/STAT3 axis in chondrosarcoma. Considering the interrelationships of HOTAIR, miR-93, and ATG12, we further investigated whether HOTAIR could exert its functions by regulating miR-93 and its downstream target ATG12 in CRC.
In this text, we demonstrated that HOTAIR inhibited miR-93 expression by direct interaction and ATG12 was a direct target of miR-93. Moreover, HOTAIR facilitated ATG12 expression in CRC cells, suggesting that HOTAIR functioned as a molecular sponge of miR-93 to induce ATG12 expression in CRC.
Functional analyses presented that miR-93 overexpression weakened cell viability, promoted cell apoptosis, and inhibited cell autophagy in CRC. Similarly, Tang et al.^[@CR36]^ demonstrated that ectopic expression of miR-93 reduced proliferative, migratory, and invasive capacities of CRC cells and hampered CRC xenograft growth by targeting smad7. However, Yang et al.^[@CR37]^ demonstrated that miR-93 overexpression curbed CRC cell proliferation and migration in vitro as well as impeded CRC xenograft growth in vivo, but had no much impact on CRC cell invasion and apoptosis. In addition, a recent report showed that miR-93 overexpression weakened CA3-AS1-mediated anti-proliferation, anti-invasion, and pro-apoptosis effects by inhibiting downstream target phosphatase and tensin homolog expression in CRC cells^[@CR38]^.
Our present study further revealed that miR-93 overexpression potentiated radiosensitivity of CRC cells, as evidenced by the reduction of cell survival fraction and cell viability, and increase of cell apoptotic rate in IR-exposed cells following the upregulation of miR-93. Moreover, ectopic expression of miR-93 inhibited the increase of cell autophagic activity induced by IR in CRC cells. In addition, restoration experiments showed that HOTAIR overexpression abrogated the effects of miR-93 on cell survival, viability, apoptosis, and autophagy in IR-treated CRC cells, suggesting that HOTAIR overexpression weakened radiosensitivity of CRC cells by downregulating miR-93 and relieving miR-93-mediated autophagy inhibition.
Moreover, our study demonstrated that ATG12 protein expression was markedly upregulated in CRC tissues. Moreover, correlation analyses disclosed that ATG12 protein expression was negatively associated with miR-93 expression, but positively associated with HOTAIR expression in CRC tissues. In addition, ATG12 knockdown triggered the reduction of cell viability and cell autophagic activity, and increase of cell apoptotic rate in CRC. Moreover, ATG12 deficiency enhanced cell radiosensitivity and weakened IR-induced autophagy in CRC cells. In line with our results, a recent study showed that IR-induced autophagy inhibited cell apoptosis and reduced cell radiosensitivity in CRC, and miR-214 improved the radiosensitivity of CRC cells through inhibiting ATG12-mediated autophagy in vitro and in vivo^[@CR39]^.
In addition, in vivo experiments demonstrated that HOTAIR knockdown potentiated IR-mediated anti-tumor effects by increasing miR-93 and cleaved caspase 3 expression, and reducing ATG12 and LC3 II expression in CRC xenograft tumors.
Taken together, our data demonstrated that HOTAIR depletion suppressed CRC tumorigenesis in vitro through regulating miR-93/ATG12-mediated autophagy. Moreover, HOTAIR knockdown potentiated the radiosensitivity of CRC cells in vitro and in vivo through upregulating miR-93 and downregulating ATG12-mediated autophagy. Our study provides a deep insight into the roles and molecular basis of HOTAIR in regulating CRC tumorigenesis and radioresistance, and suggests the potential values of HOTAIR in the prognosis, treatment, and radioresistance prevention of CRC. However, our conclusion needs to be further validated by inhibitors or activators of autophagy in vitro and in vivo.
Supplementary information
=========================
{#Sec24}
Supplementary Figure 1 Supplementary Figure legend
Edited by B. Zhivotovsky
**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
These authors contributed equally: Yingqiang Liu, Xijuan Chen
Supplementary information
=========================
**Supplementary Information** accompanies this paper at (10.1038/s41419-020-2268-8).
This work was supported by the Subsidized Project of Henan Medical Science and Technology Research Program (Grant Number 201304060).
The authors declare that they have no conflict of interest.
| tomekkorbak/pile-curse-small | PubMed Central |
The fires expanded by more than 25% and continued to spread as White House announces federal aid
This article is more than 2 years old
This article is more than 2 years old
A seventh person died in a northern California wildfire on Saturday, as a couple of other fast-growing wildfires in the state expanded by more than 25% overnight and continued to spread.
The White House announced on Sunday morning that Donald Trump declared that a major disaster exists in California, a move that makes it easier for residents, businesses, cities and counties to gain access to federal money, food aid and other programs.
How bad are California's extreme fires? A visual guide Read more
“Assistance can include grants for temporary housing and home repairs, low-cost loans to cover uninsured property loses and other programs to help individuals and business owners recover from the effects of the disaster,” the White House said.
More residents were ordered to evacuate their homes this weekend as weary firefighters endured high temperatures and gusting winds.
The Carr fire, about 162 miles (261 km) north of Sacramento, claimed the life of a power company lineman Saturday, according to a CBS affiliate, citing a spokesman for the PG&E Corporation. Neither a spokesman with the electric company nor fire officials were immediately available for comment.
Facebook Twitter Pinterest Firefighters in Redding, in Northern California, attempt to cut open a safe at the request of a resident whose home was destroyed. Photograph: Tracy Barbutes/ZUMA Wire/REX/Shutterstock
The fire, one of the most destructive in California history, had already killed six people, including a great-grandmother and two children, and a firefighter and bulldozer operator.
Two other blazes collectively called the Mendocino Complex burned in Mendocino, Lake and Colusa counties, about 90 miles north of San Francisco. The River and Ranch fires had grown to cover a total of 229,000 acres (92,600 hectares) by late Saturday, and were considered 32% contained.
This year, California wildfires have burned more land earlier in the “fire season” than usual, said Ken Pimlott, Cal Fire director, during a news conference on Saturday.
“Fire season is really just beginning. What seems like we should be in the peak of fire season, historically, is really now the kind of conditions we’re seeing really at the beginning,” said Pimlott.
California governor Jerry Brown, who visited some of the burned areas on Saturday, said: “This is part of a trend, the new normal, that we’ve got to deal with.“
Through last week, California fires had torched about 290,000 acres (117,300 hectares), more than double the five-year average over that same period, according to Cal Fire.
The Mendocino Complex fires cover more than two-thirds the size of sprawling Los Angeles. They have forced the evacuation of more than 20,000 residents and destroyed more than 100 structures.
More evacuations were ordered on Saturday afternoon, but no estimate of people involved was released.
Firefighters had managed to contain 41% of the Carr blaze by Saturday evening, and authorities were allowing some evacuees to return.
Both areas remained under a “red flag warning” issued by the National Weather Service for strong winds, low relative humidity and temperatures topping 90F (32C), all conditions that can drive the growth of wildfires.
So far this year, US fires have burned 5m acres (2m hectares), much more than the 10-year average, according to the National Interagency Fire Center.
Three men were given citations on Saturday for refusing to leave an area where firefighters wanted a tanker plane to make an air drop, causing the plane to be diverted, according to a post on the Lake County Sheriff Office’s Facebook page. The men were watering their marijuana plants, police said. | tomekkorbak/pile-curse-small | OpenWebText2 |
Q:
What directory should I use for the user apache?
I have installed apache from the source with:prefix=/usr sysconfdir=/etc/httpd/ localstatedir=/var.
useradd -g apache -d /usr/apache -s /sbin/nologin apache
A:
If you wonder about the home directory, it should not matter that much, because it will probably not change the behavior of apache at all.
On Debian, the default homedir for the user running apache is /var/www (which is the default DocumentRoot).
Also, if you're on a Debian-based distrib, you should prefer using adduser instead of useradd which is quite low level.
| tomekkorbak/pile-curse-small | StackExchange |
"My brother actually saw Monday was the last day to register, so he dragged me down from my studies, and here I am now, ready to vote for the first time," Lopez said. | tomekkorbak/pile-curse-small | OpenWebText2 |
Q:
How do you create dynamic axis labels using ylab() in ggplot2?
I am making a series of plots in ggplot2, one per column of data, and I'd like to be able to code it so that the Y-axis label changes dynamically to the name of the plotted variable (and some other constant info in the label).
my data from dput():
df <- structure(list(day = structure(c(1L, 1L, 1L, 1L, 1L, 1L, 1L,
1L, 1L, 2L, 2L, 2L, 2L, 2L, 2L, 2L, 2L, 2L, 3L, 3L, 3L, 3L, 3L,
3L, 3L, 3L, 3L), .Label = c("5-Aug", "10-Aug", "17-Aug"), class = "factor"),
station = c(1L, 2L, 3L, 1L, 2L, 3L, 1L, 2L, 3L, 1L, 2L, 3L,
1L, 2L, 3L, 1L, 2L, 3L, 1L, 2L, 3L, 1L, 2L, 3L, 1L, 2L, 3L
), Bug = structure(c(1L, 1L, 1L, 2L, 2L, 2L, 3L, 3L, 3L,
1L, 1L, 1L, 2L, 2L, 2L, 3L, 3L, 3L, 1L, 1L, 1L, 2L, 2L, 2L,
3L, 3L, 3L), .Label = c("Copepod", "Copepod nauplii", "Harpactioid copepods"
), class = "factor"), Mean = c(382.4048896, 1017.171389,
1519.141298, 233.3753491, 167.1086422, 530.7227507, 20.55943305,
482.9992303, 10.49548997, 698.0193699, 5398.533995, 2458.635058,
36.90431458, 124.4956045, 20.8459728, 8.414735929, 4014.649407,
7.279486288, 346.1246378, 5722.82018, 6253.357325, 427.6856315,
1768.984975, 1486.635443, 4.825526305, 46.50462845, 1.07692853
), StErr = c(83.18963396, 100.9187504, 73.45417607, 88.08491141,
44.57580452, 44.03459391, 4.663586058, 112.4238952, 2.551982788,
284.3933876, 3417.741042, 689.5558519, 9.798626545, 49.16103906,
4.268815029, 3.76465482, 1803.977156, 1.328408258, 53.67873047,
1796.827256, 732.573507, 86.56594682, 198.7842421, 229.0132055,
1.129940095, 16.48065742, 0.283417726)), .Names = c("day",
"station", "Bug", "Mean", "StErr"), row.names = c(NA, -27L), class = "data.frame")
I am using the following code to automatically create and save a new, dynamically named .pdf:
library(ggplot2)
df$day<-factor(df$day, levels = c("5-Aug","10-Aug","17-Aug")) #corrects day order
allplots <- ggplot(data=df, aes(x=station, y=Mean)) +
geom_errorbar(aes(ymin=(Mean-StErr), ymax=(Mean+StErr)), colour="black", width=0.1)+
geom_point(size=2) +
xlab(NULL) +
ylab(expression(paste('Copepods,'~'#/m'^3))) +
theme_bw() +
theme(
panel.grid.major = element_blank(),
panel.grid.minor = element_blank()
) +
scale_x_continuous(expand=c(.3,0), breaks=c(1:3), labels=c("In", "FL", "Off")) +
annotation_logticks(sides = "l") +
scale_y_log10() +
theme(axis.text.x=element_text(size=12)) +
theme(axis.text.y=element_text(size=12)) +
facet_grid(Bug ~ day)
plotfun <- function(x,y) {
a <- ggplot(data=x, aes(x=station, y=Mean)) +
geom_errorbar(aes(ymin=(Mean-StErr), ymax=(Mean+StErr)), colour="black", width=0.1)+
geom_point(size=2) +
xlab(NULL) +
ylab(expression(paste('Copepods,'~'#/m'^3))) + #I'd like Copepods to change with variable that is being plotted
theme_bw() +
theme(
panel.grid.major = element_blank(),
panel.grid.minor = element_blank()
) +
scale_x_continuous(expand=c(.3,0), breaks=c(1:3), labels=c("In", "FL", "Off")) +
annotation_logticks(sides = "l") +
scale_y_log10() +
theme(axis.text.x=element_text(size=12)) +
theme(axis.text.y=element_text(size=12)) +
facet_grid(.~day)
pdf(file=paste(y,'pdf',sep="."))
print(a)
dev.off()
}
mapply(plotfun, split(df, df$Bug), levels(df$Bug))
I tried using paste(levels(df$Bug)) within the ylab() but couldn't get it to work. I'm not sure what else to try, and was hoping someone would be able to suggest something I can look into. I would like each Y-axis to read "VariableName, per m^3" with the VariableName taken from the levels of Bug (i.e. Copepod, Copepod nauplii, and Harpacticoid copepods). Does anyone know how to do this? This seems really cool and useful if I can get it to work! Thanks for any suggestions.
A:
I think I can help with the labeling of the ylab of the plots and that seems to be the core of your question. This produces three plots but I encountered various ggplot errors which ( don't understand at all well) and this required that I pull out a bunch of code relating to themes and scales . I later put it back in with a different ordering which seemed to make the ggplot machinery happy.
plotfun <- function(x,y) {
a <- ggplot(data=x, aes(x=station, y=Mean)) +
geom_errorbar(aes(ymin=(Mean-StErr), ymax=(Mean+StErr)),
colour="black", width=0.1)+
geom_point(size=2) +
xlab(NULL) +
ylab( bquote(.(y)~"#"/m^3) )+
scale_y_log10()+
theme_bw()
pdf(file=paste(y,'pdf',sep="."))
print(a)
dev.off()
}
mapply(plotfun, x=split(df, df$Bug), y=levels(df$Bug))
By moving items around (in a manner driven by the error messages I successively got) and adding one +sign I was able to get this version to run:
plotfun <- function(x,y) {
a <- ggplot(data=x, aes(x=station, y=Mean)) +
geom_errorbar(aes(ymin=(Mean-StErr), ymax=(Mean+StErr)), colour="black", width=0.1)+
geom_point(size=2) +
xlab(NULL) +
ylab(bquote(.(y)*","*~"#"/m^3) )+
scale_y_log10() + annotation_logticks(sides = "l") +
theme_bw() +
theme( panel.grid.major = element_blank(),
panel.grid.minor = element_blank() ) + facet_grid(.~day)
pdf(file=paste(y,'pdf',sep="."))
print(a)
dev.off()
}
mapply(plotfun, x=split(df, df$Bug), y=levels(df$Bug))
I'll just post one page:
Also fixed the errors I was geting with the allplots version:
allplots <- ggplot(data=df, aes(x=station, y=Mean)) +
geom_errorbar(aes(ymin=(Mean-StErr), ymax=(Mean+StErr)), colour="black", width=0.1)+
geom_point(size=2) +
facet_grid(Bug ~ day) +
xlab(NULL) +
ylab(expression(Copepods *","* ~'#'/m^3)) +scale_x_continuous(expand=c(.3,0), breaks=c(1:3), labels=c("In", "FL", "Off")) +annotation_logticks(sides = "l") + scale_y_log10() +
theme_bw() +
theme( panel.grid.major = element_blank(),
panel.grid.minor = element_blank() ) +
theme(axis.text.x=element_text(size=12)) +
theme(axis.text.y=element_text(size=12))
print(allplots)
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Learning From the CEOs
Sticking up for one another has worked great for them. How about the rest of us try it too?
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as we began putting this issue together, our interns took a poll among themselves: What’s the difference between a pension and a 401(k)? Upshot: Not one among nearly a dozen twentysomethings really knew the answer. We (both early 40s) were mystified. Granted, no one under 30 thinks about retirement—but how could they not grasp a concept so dominant in the headlines? So key to their ability to survive and thrive in their golden years? Confronted with this generational divide, we consulted a translator: Nicole McClelland, our 28-year-old copy editor. Did she know what these terms meant before we started working on the issue? Nicole put it succinctly. “I guess I would have thought about it like this: A pension is what my grandpa has. A 401(k) is what my dad has. And Social Security—I don’t know, I might get $40 a month from that someday?”
And there, in a nutshell, are two truths commingled with two lies. True, many workers of Nicole’s grandfather’s generation retired with a corporate pension—a company-run “defined benefit” plan that pays a set amount per month based on a worker’s salary and years on the job. And true, those plans have now largely been replaced with the “choice” of 401(k)s, which, we have been told ad nauseam, are just like a pension, only better, since any idiot can make money in the stock market. That last bit was lie number one. Lie number two: that those of us under 50 will never see a dime of our Social Security benefits because the baby boomers will bankrupt the program. Bush & Co. even tried to convince us to turn a big chunk of it over to Wall Street.
We’re lucky that particular bit of ideological overreach went nowhere. But the lessons go deeper than a caveat about worshipping the Dow. For decades now, a driving principle of corporate America has been that the cure for any problem is to pay workers less, make them do more, and get rid of benefits. Product losing traction in the marketplace? Wring concessions out of your union! Supply chain inefficient? Jettison health care! No promise, no social bond was strong enough to withstand the pressure to increase shareholder returns. (And many of those shareholders, ironically, were the rest of us—hoping our 401(k) managers could squeeze out an extra percentage point or two, even as they destroyed the underpinnings of our economy. Talk about cutting off your nose to spite your face.)
We all bought it, for nearly three decades. Somehow we managed to convince ourselves, across generations and income levels, that bad things only happened to other people—or if they did happen to us, it was our own fault. “Personal responsibility” was another way of saying “sorry about your trouble, but shit happens.”
No more. The calamity we now face is egalitarian: Even if you still have a job, your home value has tanked and you’re scared to open your 401(k) statements—or, if you’re our interns’ age, you’re neck deep in student debt and your employment prospects stink. Only one group seems immune: the ceos lining up for bailouts and handing themselves bonuses for a job well done.
And really, should we have expected any different? Never mind all the yammering about competition, merit, may the best man win: The reason top execs have done so well is that they looked out for one another. Consider the aptly named Top Hat pensions David Cay Johnston examines in “Rip-Off Artists“—separately (and very well) funded retirement plans for the guys in the corner offices. How did they manage to create these gilded benefits? By sitting on each other’s boards, scratching each other’s backs, and feeding the world a line about how the “best and brightest” may leave us in the lurch if another $50 million isn’t forthcoming.
The lesson: Solidarity actually works. And it can work, right now, to combat the crisis those financial whizzes created. Back in the ’90s, our generational cohort bought the privatizers’ spin that we were more likely to see a ufo than to collect Social Security. Now we know that fdr‘s legacy has held up better than anything corporate America has come up with. Strengthening and expanding it, as James K. Galbraith points out in “Security Blanket“, could actually serve as a powerful stimulus—and it could mean that today’s twentysomethings won’t depend on the cruel hand of chance for their retirement.
It’s been a long time since angry crowds attacked the wealthy on streets. Those were ugly times, and they feel distant now. We don’t want to go back there. But if corporate America continues to ignore the reality its awful decisions have created, if we have to hear one more time about million-dollar bonuses at a time when tent cities are springing up all over, then things could get really ugly. (aig, finally hip to the zeitgeist, is already warning employees to “avoid wearing any aig apparel” in public.). Here’s hoping that instead, we use our enormous collective strength—as the labor movement did in its heyday, as America almost did after 9/11—to fashion a new set of commitments. And live up to them.
FACT:
Mother Jones was founded as a nonprofit in 1976 because we knew corporations and the wealthy wouldn't fund the type of hard-hitting journalism we set out to do.
Today, reader support makes up about two-thirds of our budget, allows us to dig deep on stories that matter, and lets us keep our reporting free for everyone. If you value what you get from Mother Jones, please join us with a tax-deductible donation today so we can keep on doing the type of journalism 2019 demands. | tomekkorbak/pile-curse-small | Pile-CC |
Det jobbes det ekstra uten betaling hver dag
Måtte stenge E6 da tiltalte og retten var tilbake på ulykkesstedet
Det vil være en meget ulykkelig løsning
– I Trondheim virket det som om streiken til å begynne med kom litt som julekvelden på kjerringa
- Det var brannfarlig, og hjulet kunne ha løsnet
DumDum-Aslak sparket til mot kulturministeren: – Riv deg løs fra det klamme favntaket til Tom Cruise
Nå er det klart hva som skal fylle lokalene etter Obs Bygg på Lade
Bekymret for at noen får for mye gjeld - vil stramme inn på utlån
Saken oppdateres.
Kommersiell reklame på Facebook øker. Stadig flere annonsører sørger for å få plassert reklamen sin i Facebook-brukernes personlige nyhetsstrøm, i tillegg til annonsene som finnes i spalten til høyre på det sosiale mediet.
- Vi mener Facebook skal omfattes av samme regelverk som gjelder for reklame formidlet direkte per e-post og tekstmeldinger. Det er ikke lovlig med mindre mottakeren har samtykket i det, sier Petter Ravne Bugten, fagdirektør for IKT hos Forbrukerombudet.
Det norske Forbrukerombudet har alliert seg med de andre ombudene i Norden, og har nå sendt brev til EU-kommisjonen med spørsmål om den europeiske spam-lovgivningen.
- Vi ønsker å få klargjort rekkevidden av direktivet, og om nyhetsstrømmen på Facebook er en direkte markedsføringskanal på linje med e-post og tekstmeldinger, og om det dermed må kreves samtykke fra mottakeren for hver enkelt reklame som postes, sier Ravne Bugten.
Møte med Facebook
Han viser også til markedsføringslovens regler som gir forbrukeren rett til å reservere seg mot telefonsalg, direkte reklame og uadressert reklame.
- Hensynet bak reglene er at forbrukeren skal kunne styre hvilken reklame han mottar. Vi mener at det samme hensynet må gjøres gjeldende på Facebook. Denne kanalen har klare likheter med e-post ettersom forbrukerne kan nås direkte, sier Ravne Bugten.
Tidligere i november hadde Forbrukerombudet møte med Facebook om saken.
Ny annonseform
Bakgrunnen er den økende mengden reklame, og måten annonsene fremstår både på pc-skjerm og smarttelefon i nyhetsstrømfeltet til facebookerne. Facebooks såkalte sponsede hendelser framstår nå ofte som store reklameplakater for bedrifter, produkter og tjenester innimellom dine venners private kommentarer – ikke bare som mindre logoer med kort tekst til. Særlig på smarttelefonene er disse Facebook-annonsene godt synlige da du må bla forbi flere annonser før du kommer ned til venne-meldingene.
Har du trykket på tommelen opp-symbolet for en bedrift eller produkt, for eksempel Coop, er du blitt «venn» med dagligvarekjeden. Du kan dermed forvente Coop-annonser i nyhetsstrømmen din. Den siste tiden har også venner av venner av Coop fått slike annonser. Dette kaller Facebook for «sponsored stories». Coop har over 90000 «venner» og sprer ikke reklamen sin bare til disse, men også vennene til de 90000.
Få protester til Facebook
Frida Löwengren, talsperson for Facebook Norden, sier at Facebook begynte med sponsede hendelser-annonseringen i nyhetsstrømmen rett over nyttår. Den andre typen «promoted post» der Facebook-brukere får anbefalt et produkt av en «venn» av produktet, har de nylig startet med.
- «Promoted post» er et nytt annonseformat vi nå tester. Bedriftens reklame kan også vises for brukere som ikke er fans av bedriften. Disse annonsene kan ses både på pc-skjermen og mobiltelefonen, og de ser ut som statusoppdateringer men er merket som sponsede hendelser. Facebook tester kontinuerlig nye formater og funksjoner på plattformen, og samler inn in feedback fra brukerne for å finne ut hvordan vi kan forbedre deres opplevelse.
Ifølge Löwengren har ikke Facebook registrert særlig mange reaksjoner mot denne typen annonsering.
- Det som gjør Facebook sosialt, er å se hva venner gjør og liker. Men brukeropplevelsen er viktig for oss, og det er derfor vi tester hva som fungerer. Brukeren skal ikke forstyrres for mye av annonsene, og vi har derfor lagt inn noen begrensninger for hvor mange annonser som postes. Får vi negative reaksjoner, utvikler vi noe annet, sier Löwengren og viser til funksjonen der du kan klikke ned reklamen. Du kan også «unlike» et produkt og firma i lista over det du tidligere har trykket på at du liker. Det er imidlertid ikke mulig å reservere seg mot all kommersiell annonsering på det populære sosiale nettverket.
«Vi følger loven»
- Forbrukerombudet i Norge ønsker samme regler for Facebook som for e-post og sms, at det ikke kan postes reklame i brukerens nyhetsstrøm uten samtykke.
- Facebook følger lover og regler i de landene vi opererer, og fører en kontinuerlig dialog med ulike parter for å sikre at det gjøres, sier Löwengren. | tomekkorbak/pile-curse-small | OpenWebText2 |
Decolorization of molasses and a dye by a newly isolated strain of the fungus Geotrichum candidum Dec 1.
A fungus, Geotrichum candidum Dec 1, newly isolated as a dye-decolorizing microorganism, was used to decolorize molasses and an anthraquinone dye in shaken flasks. A degree of decolorization of molasses of 87% was achieved after 12 days of cultivation, and the maximum rate of decolorization of the dye in the culture broth was obtained in 7 days. The apparent activity of peroxidase in the molasses, which is responsible for dye decolorization, was significantly lower than that of purified peroxidase, due to the inhibition by molasses, but the inhibition was reduced after the fungus was fully grown. As two ultrafiltered fractions of molasses were similarly decolorized by Dec 1, Dec 1 apparently degraded colored substances of a wide range of molecular weights. When Dec 1 was cultivated in a medium in which sucrose in the molasses was hydrolyzed with invertase, the degree of decolorization of molasses, and rate of decolorization of the dye were similar to these obtained above. | tomekkorbak/pile-curse-small | PubMed Abstracts |
Secondary Menu
Blue Light Blocking Glasses From Pill Bottles
So recently I read an article about how wearing orange glasses at night can help you sleep better at night. I wanted to test this for myself, and being the cheap person that I am, I made my own and it took me about an hour and a half. So yeah, that's what this instructable is.
Step 1: What It's Used For/How It Works
The basis of the science is that when you're exposed to sky blue light, it prevents the production of melatonin, which then interrupts your body's sleep cycle. Constant exposure to sky blue light at night-time, most commonly the LED backlight on screens and monitors, tells your body that it is still daytime. One way to reduce blue light at night is to wear orange glasses at night (hmmm I wonder how I would be able to make a pair).
Step 2: Tools and Materials
For this project, you will need:
Pair of Sunglasses (or regular glasses)
Orange Pill Bottles
Big Book
Gloves
Heat Gun
Scissors
Parchment Paper
Sharpie
Tape
Regular Paper
X-Acto Knife (optional)
Hand Saw (optional)
Step 3: What Color of Pill Bottles to Use (Optional)
If you happen to have pill bottles in more than 1 shade of orange, there is a way to test which will work best (if you only have access to 1 color, then move on to the next step).
If you have normal vision and your monitor can display RGB, then the 2 spectra above will have a noticeable difference. The upper one has a normal color range, but the lower one has the color cyan removed. To test which color of pill bottle will work best, look through each pill bottle (so that your vision appears orange) at the 2 spectra. Choose the color of pill bottle where the spectra look the most alike (when looking through said pill bottle).
Step 4: Pop Lenses Out
For this first step, hold the frame in place and push the lens forward, gradually increasing pressure along and around the frame until it comes out. Do this for both of the lenses.
Step 5: Cut Open the Pill Bottles
Use your handsaw to remove the bottom of the bottle by sawing it off. Then, cut through the middle "hot dog" style to cut it in half vertically (only on one side). You can use scissors instead of the hand saw, you just have to cut it open vertically first, and then cut off the bottom.
Step 6: Flatten the Plastic
Flattening the plastic is much easier with two people, one to hold the plastic and one to operate the heat gun.
Before you turn on the heat gun, fold a piece of parchment paper in half and place it anywhere in the book. Line up the crease of the parchment paper with the crease of the book, and leave the book open.
Turn on the heat gun on low, and get someone else to hold the plastic (or the other way around). Make sure to wear gloves. The person holding the heat gun must slowly move it in a circular motion so the heat is distributed throughout the plastic. The person holding the plastic must slowly bend open the plastic as it gets warmer, and once you get it flat enough, place it in the book. How flat you want it depends on how curved the original lenses were. Once it is laying flat inside the book, close the book and stand on it (optional). After a minute or two, you can open the book and take the plastic out.
Step 7: Cut the Plastic Lenses Out
After you've flattened the plastic, trace the original lens on a piece of paper and cut it out. Make sure you press the paper against the lens to include the lens' curve in the tracing. Then, cut out the shape from the flattened plastic using the paper as a template. Remember, it is better to start out cutting it bigger than you need it to be, and the carefully trimming it down to size. As you trim it down to size, compare it to the original lens and check to see if it fits in the glasses frame yet.
Step 8: Pop New Plastic Lenses In
Once it is the correct size, push it in the same way you took it out in the beginning.
Step 9: Enjoy!
Now all you have to do is put these on at night and enjoy sleeping peacefully. If you liked this then vote for it in the contests!
I understand what you're saying, and there are spots on my glasses that are difficult to see through (double vision, slight blurriness, etc.). These spots make the glasses imperfect, but the majority of the plastic is clear and easy to see through. They would only really be "distorted" if you heated the plastic all the way to its melting point.
i'm going to try it with my walgreen's bottles. they're TRIANGULARorange [much nicer than those traditional brownish cylinders]. i believe that they won't need much or any flattening, as the 3 sides are almost flat already.:^D
This is a great instructable! I am definitely going to give this a try, just for the fun of it. I actually have a pair of the "Yellow Driving Glasses" that help with road glare at night. I absolutely love them, but not their price tag (they were about $30 Canadian + 12% tax). But I find they absolutely do work for the night vision/road glare problem.
I have always had huge insomnia issues my whole life, so I wonder if these might help me in the evenings pre-bedtime? I usually shut down any devices at least an hour prior to lights out, and just read a book. Sometimes that works, but many times it doesn't, so here's hoping!
May take me a while, but I'll be sure to let you know how they turn out! And if they work!!
For many years I learn bike drivers uses Yellow lenses todrive at night, not only help with the windshield factor but also help to seebetter at night, is interesting to learn it help with the blue light too.
Oh, I didn't include a solution because when I was making the instructable, I hadn't had enough time to actually test them. I've had some time to test them, so I'll share. I usually put on the glasses around 9 pm until I go to sleep (around 10:30). I fall asleep much quicker since I started wearing these. I don't think it takes days or weeks to start working, just when you start wearing them (I might be wrong about that part). I would recommend trying them. | tomekkorbak/pile-curse-small | Pile-CC |
Srinagar: Jammu and Kashmir Chief Minister Mehbooba Mufti addressing the crowd at Bakshi stadium during the celebration of 70th Independence day in Srinagar on Monday. PTI Photo Srinagar: Jammu and Kashmir Chief Minister Mehbooba Mufti addressing the crowd at Bakshi stadium during the celebration of 70th Independence day in Srinagar on Monday. PTI Photo
Describing the killing of Hizul Mujahideen militant Burhan Wani as one of the thousands of encounters having taken place in the Valley during the last 25 years, the Jammu and Kashmir Chief Minister Mehbooba Mufti on Monday said those instigating young children to indulge in violence were not Kashmiris, but these selfish and vested interests were making them act as their shields during attacks on police stations and security forces’ camps.
“These selfish people with vested interests made these small children lead attacks on police stations and security forces camps virtually making them act as their shield during stone pelting,” she said, addressing a gathering during the inauguration of the first phase of sewerage treatment plant at Bhagwati Nagar here. Over 60 children have been killed and thousands of others, including police and security forces personnel injured.
She also appreciated the police and security forces for exercising restraint in dealing with the situation. “A large number of the injured include police and security force personnel, which is indicative of the extent of restraint observed by them,” she added. “But when you storm into a camp or snatch guns from cops then at one point of time, a police or CRPF personnel feels that he might get killed,” she said, pointing that “at some places, there had been attempts even to torch camps. “This should not have happened,” she added.
READ: PM Narendra Modi expressed ‘deep pain’ over Kashmir unrest: Here’s how politicians reacted
Mehbooba Mufti’s statement assumes significance as it has come a day after Union Finance Minister Arun Jaitley had described stone-pelters in the Valley as a “new form of attack by Pakistan on India’s unity and integrity”. He had ruled out any compromise with separatists and those indulging in violence.
WATCH VIDEO: PM Modi On Kashmir Violence: Analysing His Remarks
Some people talk of 2010, but then people had got angry three civilians were apprehended at one place and killed in a fake encounter, she said, adding there were also allegations at that time about rape and subsequent murder of two girls at Shopian and also instances of children walking on the road getting killed after getting hit by teargas shells. However, nothing of this sort had happened today as neither any innocent civilian was killed nor any excesses took place against any person, she added.
“Three militants were killed during an encounter in an area and no one was at fault in it,” she said, adding that “this has been going on for the last 25 years”. “Thousands of encounters have taken place in which thousands of security forces personnel, police men, militants and even innocents have been killed. But so much of unrest has never been witnessed earlier,” she added.
There were already preparations by vested interests who were looking for an opportunity to push Jammu and Kashmir once again into turmoil as they had realised that the present PDP-BJP government, if it becomes stable, would re-start implementing the agenda of former Prime Minister Atal Behari Vajpayee and former Chief Mufti Sayeed for peace, progress and development from the point it was abandoned in 2004-05, she added. To fuel unrest, they first tried to rake up Handwara and then setting up of one or the other colony in the Valley, but failed to get any response, she added.
“However, they got an excuse into the killing of three militants (including Burhan Wani) during the encounter, she pointed out. Asking opponents of her government to protest if they wanted to do so, she regretted that they, however, have been taking along with them young children to snatch gun from cops inside the police posts and throw petrol bombs, she added.
This has resulted in death of over 60 young children and injuries to thousands of others who, however, had to be at home with their parents. “By taking them along for attacking a police station under curfew restrictions, these vested interests are making these children act as their shield,” she said, recalling that at one point of time her party had fought against security forces, police and even task force using local people as human shield during searches to flush out militants.
“The very same young people whom we had convinced that security forces, police and the task force will not do any harm to them if they did not commit any wrong, these selfish and vested interests by inciting them attack police stations and security forces camps were making them shields,” Mehbooba Mufti said. They are only a handful of people as most of the Kashmirs are peace-loving people who want development. They are wise enough to understand that the freedom they have is not available in countries who claim themselves to be independent, she added.
Invoking Vajpayee, she appealed the Central government and also the Congress leaders in Delhi to work for steering people of Kashmir out of present situation. Ninty five per cent of the Kashmiris are not with the 5 per cent people indulging in violence and it is not proper to punish all of them. They all are our own people, she added.
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© IE Online Media Services Pvt Ltd | tomekkorbak/pile-curse-small | OpenWebText2 |
Role of conserved amino acid residues in the complementarity determining regions on hapten-antibody interaction of anti-(4-hydroxy-3-nitrophenyl) acetyl antibodies.
Monoclonal antibodies (mAbs) specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) were prepared at various times after immunization and the amino acid sequences of VH and V lambda 1 in these mAbs were deduced from cDNA nucleotide sequences. Replacements due to somatic mutation were not found in day 7 mAbs but were found in those of days 14, 84 and 294. The affinity of day 7 mAbs to NP-glycine(NP-Gly) was in the order of 10(4) M-1 and it increased about 8000-fold with time after immunization. The extrinsic circular dichroism (CD) spectrum of the NP-epsilon-aminocaproic acid (NP-Cap)/Ab complex was unique for each mAb, although the spectra were grouped into two types, which tended to shift from one type to another with time, suggesting a variation in the micro-environments around NP-Cap in the combining sites. All these data indicate that the structure of the combining site was altered by somatic mutation; however, the fine-specificity measured by cross-reactivity with hapten analogues did not change significantly with time. We examined the amino acid residues in CDRs responsible for recognition of NP-haptens by comparing the amino acid sequences of anti-NP mAbs. Analyses revealed the presence of several conserved amino acid residues in CDRs of VH and V lambda 1, such as Tyr-32H, and Tyr-60H, in addition to a core segment involving Arg-50H.(ABSTRACT TRUNCATED AT 250 WORDS) | tomekkorbak/pile-curse-small | PubMed Abstracts |
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The world is starting, just barely, to speak up for the drunk girl at the party. | tomekkorbak/pile-curse-small | Pile-CC |
Monday, November 21, 2011
2 more weeks!!
We have kind of finished our nursery. We still need a couple of letters on the wall but they just need to be painted. Ignore some of of the boxes and things in the pictures. (And a special thanks to my mom who sewed all of the bedding!!! Love you!)I couldn't help but post this next picture. It was the last one that we got at our last appointment. The top of the picture is kind of blurry because there is a shadow, her ambolical cord and her hand in the way. But I cannot get enough of her cute nose and lips! I cannot wait to be able to hold her in my arms! Only two weeks left to my due date but I am hoping she comes before that!
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About Us
We met at BYU-Idaho where we became best friends and soon realized we were are best selves when we were around each other and couldn't be apart! We got married in the Timpanogas Temple on Nov. 2, 2007. Since then we have been enjoying each other and life! | tomekkorbak/pile-curse-small | Pile-CC |
Tuesday, 9 October 2012
Hi there!
For those of you who weren't able to attend last weeks event at Parramatta Library 'Shades of grey' here are some notes on the talks from our three presenters.
Philip Anderson
First up, we had Philip Anderson from Wyong Library who discussed the implementation and maintenance of the library's Gay, Lesbian, Bi and Transgendered collection, which was launched in 2007 with 200 titles. It has now grown to its maximum agreed size of 1400 items which is between 1% and 2% of the total collection. Philip pointed out that the items in this original collection were actually already part of the broader collection, but were not defined or easily identifiable.
Most libraries will already have a GLBT collection under similar circumstances for example, does your library hold Four Weddings and a Funeral? Or Oranges are not the only fruit? Are they consistently identifiable as belonging to a GLBT collection?
The approach that Philip and Wyong Library have taken is to identify the collection virtually. They have created a subject heading of 'GLBT' which is used on every single item in the collection. This means that the titles are spread throughout the collection and across the Library's multiple branches.
After implementation of this system it was identified that gay books had lower borrowing stats than the lesbian books. After further consideration, Philip considered that perhaps this was because of the rather suggestive covers on some of the gay titles, such as the cover of 'The Secret Tunnel' Philip will be watching these stats closely when the library introduces RFID self checkers later this year which may offer borrowers a little more discretion when checking the titles out. Philip has also considered that a higher level of theft of this collection possibly has the same basis of people being a little uncomfortable taking their titles to the counter.
The library has received few complaints about the collection. The issues that have arisen have often involved the staff and their reactions to the collection, rather than the public. All the complaints that have been received involved the DVD collection.
Philip discussed classification and that R18 DVDs are the highest turnover collection at Wyong. The demand is there, and the library is offering items that can be bought over the counter at any store. There have been incidents where customers have taken M rated DVDs from the GLBT collection home and have made a complaint. The library's strategy for dealing with this is to make it clear that responsibility lies with parents or carers to supervise the loans of children according to their own values and explain borrowers who take issue with R18 DVDs or the GLBT collection that the library is providing services to a range of different people in the community.
Philip stresses that in these situations, statistics are your friend and can be used to show both customers and other staff that there is an identified need for these parts of the collection. It is also important to understand classifications and censorship laws and how they work.
Philip is available to answer questions for anyone interested in finding out more at [email protected]
Katherine Mitchell
Next we had Katherine Mitchell who stepped in for Debbie Best who was unable to attend the night. Katherine is the Information Access Librarian at Parramatta and was actually the first Young Adult Librarian in NSW.
Katherine was discussing the way the library has handled some issues with young people in the library who have been disruptive.
Parramatta is a growth LGA and a bit of a melting pot with many young people from different backgrounds and experiences coming together in the one place. The library was dealing with groups of school kids coming in at all times of the day and being disruptive seemingly for the sake of it. The situation reached boiling point when staff were threatened and intimidated and the library management approached Council about the issue.
They were able to get the Lord Mayor on board, who happened to have a big interest in youth issues. The issue of poor behaviour by some young people was then identified as being CBD wide.
Working with the Council, the library was able to forge a network of stakeholders, including the local schools to focus on the issue. Through these contacts a protocol was able to be initiated.
The library learned about the schools system of giving students pass outs to leave the school grounds on free periods. Through the partnerships with the schools, they were in a position to ask the students for their pass outs and to be in contact with the school to check the schedules of the students that were in the library.
This process empowered the staff in the library who were able to see the students as young kids who were doing the wrong thing and who could be sent back to school, rather than the scary, disruptive customers who were causing so much trouble.
Katherine stressed that this network of support from the community is imperative in overcoming such situations and that having all staff on the same page, with a consistent message will help overcome these situations.
Linda Bathur
Last up we had Linda Bathur from Waverley Library discussing some of her experiences and asking us to think critically about difficult situations in the library.
She raised examples of customers she has come across, such as drug users disrupting others with paranoid outbursts, a man who was so in need of a bath he could clear a room not just when he was there, but for hours after, and a homeless man who was very surprised to be asked to leave when staff found him on a bean bag in the children's library, swigging on a beer. Linda also raised the more socially accepted 'difficult customers' such as the mothers whose children are perfect and couldn't possibly be introducing other younger children to porn on the library's computers, and of course the customers who will not pay their overdue fees because they always return their books on time!
Linda questioned our term of 'difficult customer' and wondered whether we are setting ourselves up for further issues based on assumptions with these customers later on. Would it be more appropriate to take the customer out of the equation and call it a 'difficult situation?'
She asked us to consider whether dealing with these situations should be part of our job, which we all agreed was a yes, as libraries pride themselves on being 'the last bastions of free public space and welcoming to everyone.'
So whose problem are these situations? Management or staff? Linda agrees that in many instances frontline staff will try to defuse a situation but if the customer will not back down will then pass the issue up to management. This is often the best way to deal with the situations, but Linda stressed should not be the only way. She pointed out that often managers are no better equipped than the frontline staff in dealing with the issues. The best outcome would be creating workable solutions for all staff through developing strategies, policies and procedures for staff to reduce problems. This could be in the form of a 'customer service charter' or protocols for dealing with homelessness. It is important through such documents to focus on the behaviour that is an issue rather than particular types of people. Training staff to deal with situations and promoting morale and sense of safety are also important as is stressing that no conversation about dealing with difficulties can occur without also discussing good customer service.
Some of the tips Linda left us with are:
listen and apologise for the customers inconvenience, for example 'I'm sorry you have had that experience/ that you are feeling this way'
be diplomatic- 'getting to the bottom of this is just as important to me as it is to you'
go in to 'computer' mode- don't rise to any bait or get emotional
show empathy
show appreciation for customer feedback- 'thank you for bringing this up with us, we will be following it up at our next staff meeting'
Thanks so much to all of our fantastic speakers. I think we all picked up some valuable skills on the night.
Thanks also to all those in attendance, we hope to see you again at the next event!
Sunday, 7 October 2012
On a recent trip to the US I had the pleasure of meeting
with Cynthia Smith and Alyce Jackson of the Peoria Public
Library to talk about Project Next Generation.
‘The goal of Next Generation is to bridge
the digital divide by making recent technologies accessible to students who
have limited access to computers. At the same time, the program strives to
provide a safe and friendly environment filled with positive role models.’
In Peoria, Project Next Generation has been running at
the Lincoln branch since 2002. The Lincoln Branch of the Peoria Public Library
opened in 1911 with a new addition opened in 2011. This new addition included a
state of the art computer lab where the Next Generation session takes place.
The session participants complete homework and work on
projects using a variety of technology. Past projects include a newsletter and
a science fair. Initially the library approached schools to refer students to
the program but now after a decade the program has such a positive reputation
the program is at capacity. The volunteer mentors come from a variety of fields
and guest speakers also come to talk about different aspects of technology.
This area of Peoria has a particular need because without
this program many of these young people would not otherwise have computer
access for academic or personal pursuits. (note the burned out building just outside of the Library's computer lab) But the program goes beyond serving
just the academic needs by supporting whole person. Mentors regularly go beyond
technology training by offering guidance and perspective to these young people.
While talking, I heard the story about one young participant who
came into the program at the bottom half of his class. He was struggling and was particularly
anxious about public speaking and presenting in front of his peers. After
participating in Next Generation his was at the top of his class and also
presented at the Library’s science fair in front of students and adults. I do
not doubt that these results are seen time and time again in this program.
Nitty gritty-
Target students in years 5-8
Volunteers and librarians plan out the
sessions, goals and support.
Sessions are run by tech savvy volunteers
from the local community (librarians help out when needed)
Weekly sessions take place in 10 week blocks during
the school year and some summer classes occur as wellParents sign a contact to promise students
attend sessions
Group sessions are small (no more than 13 participants per session)
The program is run at no cost to participants
For more information see Project Next
Generation. Thank you to Cynthia and Alyce for taking time out of their
busy schedules to show me around and answer so many questions. I found my visit very inspiring. It is amazing to see how one library can have such a postivei impact in their community.
Tuesday, 2 October 2012
The ALIA Sydney Group Committee has been doing some planning for 2013, and there will be a number of vacancies on the committee, which we'll be looking to fill with enthusiastic library and information professionals who would like to get involved.
We'll be inviting people to put in Expressions of Interest (EOIs) for a variety of roles soon, so keep an eye out for this upcoming announcement. In the meantime, if you're interested in finding out more about what the ALIA Sydney Group does, and how our professional development events run, please come along to our next event, which will be held this Thursday 4th October at Parramatta Library and check it out for yourself!
We'll be discussing some of the more challenging and controversial issues facing libraries, from censorship to dealing with difficult customers, but the committee members will be more than happy to talk about what's involved with being an ALIA Sydney group committee member before and after the event.
If you're interested in organising professional development events for LIS professionals, interested in your own career and professional development and would like the opportunity to network, work alongside and meet lots of other LIS professionals, please consider putting in an EOI- we'd love to hear from you. Details will be released shortly.
If you're thinking about coming along to our next event, I've copied the details below:
___________________________
The ALIA Sydney group is heading to Parramatta!
Come
along and join us for a lively evening of discussion around some of the
more controversial issues that libraries are facing. Details below:
Have
you dealt with some tricky situations in the library where there was no
black and white answer? Has someone challenged the inclusion
of something in the library collection? Have you dealt with an unusual
customer?
If
you're interested in unpacking situations like these, developing
strategies to address them and come up with solutions, then come
along to ALIA Sydney's Shades of Grey workshop!
Public
Library Managers including Linda Bathur from Waverley and Debbie Best
from Parramatta who will be talking about their experiences
handling tricky situations including managing the homeless users of the
library, managing young people in the library and maintaining the public
image of their organisation
Following
presentations from our guests you will participate in a workshop to
discuss the issues and brainstorm some ideas so that
YOU can help inform the decisions that affect your library.
Hi there,
We've all heard the stories of employers doing a search on prospective employees and checking Facebook for respectability in photo galleries and wall posts, but I came across something new in the last recruitment I did.
I received an emailed application that referred me to look up the person's LinkedIn page. This was the first time I'd come across this, even though it makes a lot of sense. However, it got me thinking about recruitment and how the use of LinkedIn fits with Council's often strict recruitment guidelines.
I see how LinkedIn is a great tool to use as a resume and to give insight to your career progression. It doesn't however automatically answer the essential criteria of a job advert, which is the most important part of the application (especially when applying for Council jobs).
It's so important to address the criteria thoroughly with clear examples of how you meet each of them.
Referencing LinkedIn in your answers to the criteria is a great example of how you are social media savvy or as an extra 'read on' section, but don't rely on it as your entire application.
The other danger in relying on referring to LinkedIn is that it does add an extra step to someone viewing your application. It's important to make your application clear and easy to read with subheadings for the criteria, for example, to break up the sections. Always remember that the person reading your application may have read 150 other applications before yours, so make it as easy as possible for them. Sending them off to look at a website is an extra step and should be used to complement your application, not replace it at this stage.
It is a great thing to add to your application however and I think including it is a step in the right direction.
I'm interested to see the future of recruitment with sites such as this. Do you think they will one day replace the traditional cover letter and resume combo?
How do you use LinkedIn or sites like it? | tomekkorbak/pile-curse-small | Pile-CC |
A minor victim in the sexual abuse case is now facing social ostracism from her educational institution.
Budging under the pressure from the other residents of the college hostel where she is staying, the hostel authorities seem to have decided to evict the victim. They have already suspended her from the hostel following informal complaints from the residents.
Incidentally, the city police had arrested two persons on charge of sexually abusing the girl, a Scheduled Caste student, on Monday.
The student secretary of the college hostel said they “were not concerned about the girl and she will not be allowed to enter the hostel again. Any attempt to re-readmit the student will be resisted,” the secretary said.
A meeting of the hostel members, held in the presence of a tutor and hostel warden, had discussed the issue in detail, said the secretary.
She said the continuance of the girl in the hostel would bring ill-repute to all the other students staying there. All the members of the hostel were of the view that she should be expelled from the hostel. No person supported her return to the hostel, she said.
Though the hostel committee has not formally written to the college and hostel authorities to expel the victim, they have informally communicated the message to the authorities concerned. The committee was of the unanimous view that she should not be allowed to stay anymore, she said.
While confirming that the student was suspended from the boarding house, hostel warden said a show-cause notice would be served to her following the developments. The girl was suspended for violating the hostel rules. Her father was apprised of the developments as soon as it came to the notice of the college authorities that the student was missing. The warden had filed a police complaint too, he said.
The warden also said that the decision to suspend her from the hostel was communicated to her father. The student would be given an opportunity to explain her stand before taking a final decision, he said.
At the same time, the teacher in charge of victim’s class felt the student should be given another opportunity to return to life and resume her education.
“She was an attentive student and I don’t have any complaints against her as the class teacher. Instead of declaring her a social outcaste and isolating her, society should take steps for rehabilitating the student,” the teacher said.
“Though some students might be harbouring an ill-feeling against her, I am sure they would change their attitude if I appeal to them. When an opportunity arises, I would certainly speak out for rehabilitating the student,” the teacher said.
Budging under the pressure from the other residents of the college hostel where the 17-year-old victim is staying, the hostel authorities seem to have decided to evict the girl. | tomekkorbak/pile-curse-small | OpenWebText2 |
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From The Tombstone Tourist - Musicians (first edition):At the end of October the band took a rare break from touring and recording, and returned to Macon, Georgia. After stopping by the band’s “Big House” to offer birthday wishes to Berry’s wife, Duane took off on his motorcycle. As Duane traveled west on Hillcrest, a pipe truck driven by Charles Wertz entered the intersection of Hillcrest and Bartlett. To avoid a collision, Duane laid his bike down and skidded 90 feet across the intersection’s center line. Hearing the crash, Wertz walked over to the unconscious rider and turned off the still racing engine of the motorcycle. The impact tore the helmet from his head and caused severe liver, chest, and head injuries. Although revived twice, Howard Duane Allman died three hours later, in what is now Middle Georgia Hospital, one month shy of his twenty-fifth birthday.
The following Monday over 300 people attended services in Macon’s Memorial Chapel. With Duane’s guitar case placed in front of the flower-adorned casket, the band took their places, and Gregg sang out the old blues classic The Sky is Crying. Making it clear that the band’s tribute lay in the music, they continued with Stormy Monday, In Memory of Elizabeth Reed, and Statesboro Blues.
The ABB immediately went back on the road, later adding Chuck Leavell on keyboards. Berry also began pulling out of the grief which enveloped him following Duane’s death. Taking an active role in the recording of the Brothers and Sisters album, Barry was becoming the unofficial leader and visionary of the band. Creatively, the band was at their peak.
On a Saturday afternoon, almost one year later to the day and three blocks from Duane’s accident, Berry failed to make the curve on Napier Avenue near Inverness Street, striking the left side of a city bus. Bleeding and visibly shaken, he refused medical treatment. An hour later friends carried him back to the same hospital. He died moments later of head injuries and internal bleeding.
Due to legal and financial disputes, Duane’s remains were still in cold storage, awaiting final instructions from family members. It wasn’t until after Berry’s death that they were buried side-by-side, with matching tombstones, in the Civil War section of an historic Macon cemetery. With a guitar etched in marble, Duane Allman’s tomb reads:
Duane Allman
Nov. 20, 1946
Oct. 29, 1971
I love being alive and I will be the best man
I possibly can. I will take love wherever
I find it and offer it to everyone who will
take it...seek knowledge from those wiser
and teach those who wish to learn from me.
Buried on the right with a matching bass etched into the stone, Berry Oakley’s monument reads:
These guys were the greatest. Their mom's house was directly behind my grandmothers house. We use to sit outside in the summer and listen to them play. For years it was thought that Duane hit a truck carrying peaches, which the Album "Eat A Peach" came from.
Duane Allman and Berry Oakley
No conspiracies here, but still great musicians that "burned out instead of fading away" - wondering if there were any fans out there besides me. The Allman Brothers site Hittin the Note is good, but full of waaayyy too old groupies that really don't know much except that they were cute and rebelious - these were deep guys with deep wells of talent, anyone agree? I am particularly transfixed with the fact that they both died on motorcycles, within blocks of each other in the same city less than 2 years apart..... live on Sweet Melissa....., walking the paths of Rose Hill, A
Duane Allman and Berry Oakley
FYI: Duane Allman's only child is named Galadrielle from Lord of the Rings. I can listen to Live at Fillmore East for hours andhear things I never heard before. If you listen close enough (and loud enough) you can hear him addressing the audience - my favorite is: "here's an old Elmore James tune we'd like to play, called I Must've Did Somebody Wrong - I wonder who...." this was incidentally around the same time he remarked in a radio interview - "I got rid of my old lady, my kid - no old ladies, no kids - just guitars" (I never said he was a super sweet guy), just a genious, taken too soon, but aren't they all.....
I keep remembering things - the album "Eat a Peach" was named after a quote Duane gave in a radio interview shortly before his death - he was asked how he was helping the revolution he said: "there is no revolution, only evolution, so every time I am in Georgia the first thing I do is eat a peach for peace", Gregg always thought he was talking about the female variety - you know (to quote Ronnie VanZant) "I love those Georgia Peaches......"
Actually I've been there a couple of times. The Big House on Vineville houses 6000 sq ft of photos, posters, film footage, audio tapes, gold records, awards, clothing, and instruments. When I was there road manager Kirk West and his wife were still living there (they have since moved and now the whole house is the museum) and he was nice enough but his wife was a beo-tch.
And god don't get me started on the ABB society members who hang at the grave. Jesus they give you major stink eye as if you're not a member of their secret society when in fact they give unemployeed Deadheads a good name. But I digress.
Yes it is worth the trip down to Macon for TBH, the graves, and the Georgia Music Hall of Fame. The museum is as good if not better than the Marley house in Kingston. Another good side trip is to Athens, GA home of the B52s, REM, and others. Great college town.
Seriously, I understand getting sick about certain factions of the ABB society - they care n.o.t.h.i.ng... about the music, the talent, the tragedy of losing brothers and friends - wanna really get sick - go here:
and if you will click on the pictures, at the bottom are posts and comments from freaks that are infatuated with Gregg and his.. uhhh, to put it nicely (and coming from a family with more than one brother over 50 that truely was a "hippie -stoner- rocker" that saw the Brothers more than once in concert) I can't say anything nice, and to borrow a quote - : " if you don't have anyting nice to say, come sit next to me".... all I can say is eeewwww, Gregg would only laugh and I don't even want to know how amused Duane would be.... just look and you will see what I mean... I cannot say anymore without being unnecessarily mean, which is not the way I was raised - to love real southern rock and roll and to bask in the peace it brings me - ahhh the simplicity of it all
Skydog: by Randy Poe with forward by Billy Gibbons (yes, that Billy Gibbons)
Midnight Riders: The Story of the Allman Brothers Band, By Scott Freeman
The Wanee Festival and Bonnaroo
Neither of the latter are hard to find and you can find tickets at www.hittintheweb.com to the Beacon Theater concerts of the ABB most of the time featuring Gregg, Dickey, Butch and Jai, not to mention Warren Hayes of Gov't Mule, Derek Trucks (the reincarnation of DA, in my opinion, and the nephew of Butch Trucks) and Derek's wife Susan Tedeschi, if you are ever going to spend money on any concert and trust me, you could travel 300 miles, and gas and expenses plus the tickets wouldn't cost you as much as 2 tickets to a modernized version of a Rolling Stones "show" these days - it would be the best money you would spend all year. I know this thread is suppossed to be about dead musicians, but I think it is a tribute to Duane and Berry that there be many a pilgrimage to see what they "hath borne to us": a siliquoy to Southern Rock in it's most pure form...
I know what you are thinking and I understand, I thought the same thing - which is why I checked it out when Dickey was ousted. Yes, Betts was fired, and there is alot to be said about what a butthead Gregg Allman can be - but he does appear with them occasionally and only at the festivals I mentioned and at an Atlanta's Peidmont Park gig that also featured The Dave Matthews Band. I try hard not to repeat things I have not made sure are true, and while I will admit I am wrong when I am, go to www.hittintheweb.com for verification of what I said from Gregg himself. When the band was just starting out and moved from Jacksonville to Macon at the behest of Phil Waldon (also RIP) whose Capricorn Records was headquartered there, they used to play free concerts in 1969 in the park to find an audience. While there may be disagreements, there is also a brotherhood that is sadly tied together by tragedy and death, a suspended animation type of thing - check it out, I think you will agree. I have met Warren Hayes and he is an all around pretty sound fellow and I don't think he would be doing what he is doing if he did not know and understand the situation. Dickey has no hard feelings and is still doing his thing the way he wants to. He is pretty hard headed too, so without Duane around to remind them that their "religion", their music, was about brotherhood and peace (ie: referee) it is only natural (and expected by all that know them) that Gregg and Dickey would eventually part ways.
I wish - next summer I am going to go to Macon, on my summer trip to the south - I wish I lived there - Oklahoma kind of sucks a little, I lived in Mt. Pleasant for a while and now go back for a month every summer. My best friend and I are going to go to Macon just for the heck of it - ever been to Mama Louise's in Macon?
Actually I've been there a couple of times. The Big House on Vineville houses 6000 sq ft of photos, posters, film footage, audio tapes, gold records, awards, clothing, and instruments. When I was there road manager Kirk West and his wife were still living there (they have since moved and now the whole house is the museum) and he was nice enough but his wife was a beo-tch.
And god don't get me started on the ABB society members who hang at the grave. Jesus they give you major stink eye as if you're not a member of their secret society when in fact they give unemployeed Deadheads a good name. But I digress.
Yes it is worth the trip down to Macon for TBH, the graves, and the Georgia Music Hall of Fame. The museum is as good if not better than the Marley house in Kingston. Another good side trip is to Athens, GA home of the B52s, REM, and others. Great college town.
I wish (hence the name uwish yes, I can laugh at myself!!) I had seen all that already, I am not old enough - barely - to have seen all I wish I could have. My older brother has a weak heart and is from the SC area, he talks often of going to the hills of Macon and the Rose Hill area, where he was lucky enough to be raised and just slip away. While I know that sounds sad, death is what this group is about, right? I will be in the area this summer and will take your advice, and go to TBH and Athens.
I wish - next summer I am going to go to Macon, on my summer trip to the south - I wish I lived there - Oklahoma kind of sucks a little, I lived in Mt. Pleasant for a while and now go back for a month every summer. My best friend and I are going to go to Macon just for the heck of it - ever been to Mama Louise's in Macon?
Only about a dozen times!
If you have the money, stay at least one night upstairs in the main house of the 1842 Guest House - it is haunted! It is also right next door to the old ABB house that they first occupied.
And when I mean haunted, I mean your doors open, your faucets turn on by themselves, and your wallet and keys get moved. Scared me at first but I got used to it.
If you have the money, stay at least one night upstairs in the main house of the 1842 Guest House - it is haunted! It is also right next door to the old ABB house that they first occupied.
And when I mean haunted, I mean your doors open, your faucets turn on by themselves, and your wallet and keys get moved. Scared me at first but I got used to it.
Is that the house where Linda and Berry lived above Butch Trucks? Who do you think haunts it? I will definetly check it out, do you know if I can make reservations now, or if it is a drop in and get lucky kind of thing? They are also opening up the Lizzie Borden house as a Bed & Breakfast. You can stay in the room she "gave her father 40 whacks" in for extra $$ - I'm not sure about that one, might check it out though.
Is that the house where Linda and Berry lived above Butch Trucks? Who do you think haunts it? I will definetly check it out, do you know if I can make reservations now, or if it is a drop in and get lucky kind of thing? They are also opening up the Lizzie Borden house as a Bed & Breakfast. You can stay in the room she "gave her father 40 whacks" in for extra $$ - I'm not sure about that one, might check it out though.
This was the first house the whole band rehearsed and lived at.
The 1842 is not a drop in kind of place - it is very high end and you can walk down to Rose Hill Cemetery.
The 1842 is not a drop in kind of place - it is very high end and you can walk down to Rose Hill Cemetery.
I am there! That just sounds awesome - the guys from ABB I have met and talked to in the past, Gregg, Dickey (who, I agree with some people might be the Billy Cudrop character in Almost Famous) and Warren have always said they sense Duane and Berry in all they do and everywhere they go - this is a a very strong sense that Gregg, Dickey, Butch and Jai all say is with them every time they play an original ABB song or venue, and Warren insists he feels every time he copies DA's riffs and Derek just plain says he is passing on something above his own inate talent, because he thinks he can feel Duane passing through him, you should if you ever have the chance, talk to Derek, it is truly indescribable how he feels, perceives and puts forth his talent.
Duane Allman and Berry Oakley
No conspiracies here, but still great musicians that "burned out instead of fading away" - wondering if there were any fans out there besides me. The Allman Brothers site Hittin the Note is good, but full of waaayyy too old groupies that really don't know much except that they were cute and rebelious - these were deep guys with deep wells of talent, anyone agree? I am particularly transfixed with the fact that they both died on motorcycles, within blocks of each other in the same city less than 2 years apart..... live on Sweet Melissa....., walking the paths of Rose Hill,
This is not that far from me (Augusta, GA), and I happen to be going to Macon to our corporate office tomorrow so I think I'll nose around a little bit, maybe see if I can take some pics at the grave.
I had a friend tell me that taking pictures of a grave is morbid, but I snapped like hell when we were at Forest Lawn and Hollywood forever....I'm not sure why but I don't find it morbid or disrespectful....maybe I am just sick, which might be why I'm here??? Anyways, if I do get some shots, will share with anyone who's interested.
This is not that far from me (Augusta, GA), and I happen to be going to Macon to our corporate office tomorrow so I think I'll nose around a little bit, maybe see if I can take some pics at the grave.
I had a friend tell me that taking pictures of a grave is morbid, but I snapped like hell when we were at Forest Lawn and Hollywood forever....I'm not sure why but I don't find it morbid or disrespectful....maybe I am just sick, which might be why I'm here??? Anyways, if I do get some shots, will share with anyone who's interested.
Did you make it to Macon, if so did you get any pics, I would love to see them if you did.
Did you make it to Macon, if so did you get any pics, I would love to see them if you did.
I didn't make it down for that trip, but will be going again pretty soon...if you'll PM and REMIND ME so I don't forget, I'll be glad to let you (or anyone else who wants them--just PM me) know when I have pics!!
It's a cool place they are buried at. Macon sure had some great musical talent spring from there....
Please read Midnight Riders: The Story of The Allman Brothers Band by Scott Freeman, it mentions the island as a favorite getaway of the band and is also excelantly (i know- sp?) researched and quoted. I loved it and have read it more than once.
duane and eric were both somewhat put out by layla and assorted love songs languishing in the charts as they thought they had done real good.
Yeah, I remember it didn't do well in sales, but the song Layla still gets a lot of play on the classic rock stations around here, because it was a great tune. I did my part though, I bought the album and still have it! I even play it sometimes. What a shame Duane's talent ended so soon.
GR6-1890
I may look calm. But in my head, I've killed you three times.
They just reran the Intervention "Chuckie" on A&E the other night. Chuckie is the son of Chuck Negron and Julia Densmore (who were both addicted when he was born and thus, he was born heroin addicted). In between Densmore and Negron, Juila had Berry Oakley Jr. and he is a big participant in the episode. He looks just like his father.
Anyone know anything about Julia Densmore? There's not much out there.
They just reran the Intervention "Chuckie" on A&E the other night. Chuckie is the son of Chuck Negron and Julia Densmore (who were both addicted when he was born and thus, he was born heroin addicted). In between Densmore and Negron, Juila had Berry Oakley Jr. and he is a big participant in the episode. He looks just like his father.
Anyone know anything about Julia Densmore? There's not much out there.
I think it is Berry Oakley III, since BO was Jr. Julia Densmore is the ex-wife of The Doors John Densmore and also, while he was married to Cher, had an affair with Gregg Allman.
Scott: it is time to do a Duane/Berry post on the page. I would be interested to see your spin on the while thing. Also, with your super human like ability to track people down, somebody needs to find Dixie. She would have the most interesting perspective on the last few months of DA's life. You can contact Kirk and Kristen West at The Big House website and Randy Poe is the best. You can also contact him via www.allmanbrothers.com through the forum pages. I have his personal email address, but I am not sure I can give it out to the general public, I will give it to you though, as I am sure Randy would take all the help he can get squashing innuendo and rumors spread by people who "know", because they read the Enquirer. You can contact me if you are ever interested. Thanks.. uwish
She had an affair with G. Allman when he was married to Cher? Wasn't she also married to Chuck Negron at that time? Never heard about that, do you have any links? TIA
You are right, I was wrong, it is Berry Oakley Jr., not the 3rd. As far as the reference to the Gregg Allman/Julia Densmore affair, it is completely circumspect, taken from the book Midnight Riders by Scott Freeman. I have not seen this repeated anywhere else, by Randy Poe or Willie Perkins, which make me wonder. Looking back, I should not have repeated the quote, but it is referred to in evasive measure on page 207 of the above mentioned book. Anyone with a perspective on this, let me know. Thanks, Uwish
You are right, I was wrong, it is Berry Oakley Jr., not the 3rd. As far as the reference to the Gregg Allman/Julia Densmore affair, it is completely circumspect, taken from the book Midnight Riders by Scott Freeman. I have not seen this repeated anywhere else, by Randy Poe or Willie Perkins, which make me wonder. Looking back, I should not have repeated the quote, but it is referred to in evasive measure on page 207 of the above mentioned book. Anyone with a perspective on this, let me know. Thanks, Uwish | tomekkorbak/pile-curse-small | Pile-CC |
South American organised crime gangs are travelling to the UK to burgle homes and are sending the multi-million-pound plunder back to Chile.
In the past two years, 75 Chileans have been arrested for breaking into affluent homes in suburbs across the country and Scotland Yard is actively seeking the UK-based leadership of the gang who is coordinating the criminal enterprise, according to a series of exclusive reports by The Times.
Police have arrested thieves following some 200 South American gang-related burglaries in counties in the commuter belt around London including Hertfordshire, Kent, and Surrey but also as far afield as Wales and in the south-west county of Somerset.
It is believed that a new crime gang is being sent to the UK every two weeks, with police gearing up for another series of raids as the coming winter allows the thieves to target homes under the cover of darkness.
There have been 36 arrests made so far this year, and the gang is believed to be active in other European countries such as Spain.
Inspector Ivan Villanueva, a senior police officer in Santiago, Chile, told The Times: “These are Chilean specialist criminals moving abroad to commit crimes.”
“They have identified that certain societies in Europe are more vulnerable to certain kinds of robbery,” he added.
Police Bust ‘Voodoo’ Migrant Sex Slave Ring Which Forced Women ‘To Eat Raw Chicken Hearts’ https://t.co/Ch6Z5NvqTs — Breitbart London (@BreitbartLondon) February 25, 2018
DNA from one burglar was traced to six other robberies across the country. When caught, the thieves are found without identification but the Chilean authorities have been assisting British police where it is found the suspects have criminal records in their home country.
The gang is also believed to be active in Canada and the United States, with The Times reporting that a corruption scandal where police are being paid to scrub criminal records for visa applications to North America was linked to the burglaries.
It is believed that the Chilean nationals — who, like nationals of several other Latin American countries, can travel as tourists to the UK visa-free for up to three months — meet an “embedded contact” in south London after landing where their identification is taken from them and they are handed a burner phone and a car before being dispatched to break into properties.
The thieves make off with jewellery and designer clothing and bags, with one robbery alone worth £400,000 ($514,000).
Police were able to put together the modus operandi of the gang after licence plate tracking had identified the gangsters’ vehicles returning to south London, from where police believe a “Mr Big” is operating.
Thieves who have been caught appear not to have given up the identity of Mr Big or any of his potential UK contacts.
In one case, four Chileans, Alexis Apablaza, 22, Angelo Bustamente, 34, Javier Kurte, 24, and Gustavo Ahumada-Paredes, 19, were arrested after burgling a home in Hertfordshire and gave no comment to police during their interviews. All four pleaded guilty at the earliest opportunity and were sentenced to two years’ imprisonment.
London Gangs More Organised, Driven by Drug Profits, Expanding Criminal Franchises, Report Finds https://t.co/kVd2yRWsaM — Breitbart London (@BreitbartLondon) June 6, 2018
Detective Inspector Tim Court, who is heading the nationwide investigation, said the crimes represent “really sophisticated targeting”.
“The houses were often big, next to golf courses or parkland… We were seeing the burglars making use of garden furniture and tables or insecure ladders to break in on the first floor. They had worked out that lots of people put their alarms on the ground floor only,” he added.
It would appear that the stolen goods are not making their way onto the UK black market, with DI Court noting: “We put the message out to immigration and border force. The big question was ‘where is this stuff going?’ We intercepted a series of boxes going back to Chile. Hundreds of thousands of pounds’ worth of jewellery.”
Other international criminal enterprises are operating in the UK, with British and Spanish police busting a “voodoo” migrant sex trafficking ring in February where criminal gangs were kidnapping women in Nigeria and threatening to use black magic to control them.
Gangs in Britain are also becoming more sophisticated in their methods to sell drugs, with London dealers developing “county lines” to ship their product out of the city and across the country.
Twitter Follow @friedmanpress Follow Victoria Friedman on | tomekkorbak/pile-curse-small | OpenWebText2 |
News and Announcements for Students
Get Involved with Sexual Assault Prevention and Response
Last month, almost 250 students, faculty members and staff members offered their thoughts through an anonymous survey on what Middlebury’s priorities should be for sexual assault education, prevention and response. We write now to share what we’ve learned, and to invite you to join us in advancing the agenda you have helped to shape.
In addition to receiving solid support for the continued development of a confidential sexual assault advocates/allies program, we received strong feedback that what is needed is more: more opportunities for discussion; more facts about what happens on campus; more information about key definitions; and more opportunities to identify and dismantle aspects of our culture—at Middlebury and beyond—that are hospitable to sexual assault, and instead, foster a culture of respect, care and safety.
Our agenda is ambitious, and we can’t do it alone! We invite all interested community members to join us in our work on the projects below.
Sexual Assault Advocacy Program: Working to recruit, select and develop a training agenda for a small group of students, faculty and staff members to provide confidential support to students. To join, please contact Fritz Parker: [email protected]. | tomekkorbak/pile-curse-small | Pile-CC |
Constructed Stormwater Wetland
Constructed Stormwater Wetland
The Constructed Stormwater Wetland (CSW) treats over 42 acres of stormwater runoff on Villanova University’s main and west campus, and is one of the oldest stormwater control measures within the VCRWS. Supported by the EPA section 319 Nonpoint Source National Monitoring Program, the CSW was installed in 1999 and reconstructed in 2011 as a retrofit for a large detention basin located just south of County Line Road and what is now the Villanova Law School.
Detention basins provide little to no water quality improvement for urban stormwater runoff, and are often unable to slow down the flow of water created by impervious surfaces such as asphalt during large storms. Installing a wetland where detention basins currently exist is a very practical, small-footprint method for creating a more environmentally stable transition from urban runoff to local streams.
The CSW was constructed with a series of sedimentation basins and meandering swales that slow down the flow of water and allow for contaminant removal by the dense wetland vegetation. Over its long tenure next to the Villanova Law School, the CSW has become a home for many native plant and animal species. Its vibrant bioactivity, in combination with attentive real-time flow control techniques, creates a system that is very resilient to the flashy peak flows and sudden changes in water quality produced by urban environments.
Adjustable sluice gate at the CSW, May 2017
Constructed wetlands are different from other engineered systems in that they are alive, and have the ability to adapt to their living conditions. Knowing how urban wetlands change over time is an integral part of enhancing their performance. In order to better understand how to improve the efficiency of constructed stormwater wetlands, VCRWS has equipped the CSW with a wide range of monitoring equipment, including flow meters, depth sensors, and automatic samplers. Adjustable sluice gates have also been installed to help control the flow of water.
Ultimately, the Villanova CSW is a very effective study on how outdated, large-scale stormwater control measures in urban environments can be retrofitted for more environmentally sustainable techniques. Advanced flow attenuation and ecological treatment methods will continue to be explored at the CSW as part of the EPA Section 319 Nonpoint Source National Monitoring Program. | tomekkorbak/pile-curse-small | Pile-CC |
Ads support the website by covering server and domain costs. We're just a group of gamers here, like you, doing what we love to do: playing video games and bringing y'all niche goodness. So, if you like what we do and want to help us out, make an exception by turning off AdBlock for our website. In return, we promise to keep intrusive ads, such as pop-ups, off oprainfall. Thanks, everyone!
By Steve Baltimore / April 27th, 2015
Title Omega Quintet Developer Compile Heart / Idea Factory Publisher Idea Factory International Release Date April 28th, 2015 Genre RPG Platform PlayStation 4 Age Rating Teen Official Website
When Idea Factory International announced they were bringing Omega Quintet to the West, I was pretty excited. Being a fan of their other titles, I was very interested to see what they could do on the new PS4 hardware. Was it a big improvement over their last generation titles or a lackluster current generation outing? Let’s find out!
The story centers around a world being consumed by a phenomenon known as the Blare. The Blare transforms the world’s creatures into monsters and slowly eats away the minds of the people. The only ones that can combat the Blare are the majestic Verse Maidens. These rare girls can destroy the Blare with their magical song and dance and are the only defense left for this world’s survival.
While the story is very cliché at times and a bit slow getting started, I still found it to be pretty interesting. You can find lots of hidden documents lying around the world that go into great detail about the girls and world itself. The girls are your standard anime archetypes. You have your tomboy, tsundere, and, of course, the cute little sister character. While this will likely turn some people off, I thought the girls were interesting enough that I wanted to learn more about them, and it didn’t hurt that Aria was so adorable. However, I was not a fan of the manager, Takt. His personality was pretty abrasive and he came off like an ass most of the time. Though I think, deep down, he really did care about the girls. There are plenty of plot twists and a couple of different endings you can obtain, so I felt the story was well done overall.
While the graphics are not the best thing I’ve seen on the PlayStation 4 hardware, they are not the worst, either. The character models are pretty detailed and look nice, but I was more impressed by the environments themselves. They are huge in size. This is a vast departure from other Compile Heart titles. Not only are they big, but they have a lot detail, as well. The game runs silky smooth with only a few frame rate issues. This only happens when there are a ton of monsters on the screen at one time. Combat animations are all wonderful and have some really great effects when performing some of the special attacks.
Being that this one is all about singing and dancing, you would think the soundtrack would be great, right? You would be correct. This has one of the best soundtracks I have heard in a Compile Heart game thus far. There are several nice vocal tracks you can use in PVS Mode, as well as many other wonderful tracks. I really loved the field music, most of it was somber and fit the mood of this dying world very well. The battle themes are really great as well. You gotta get pumped up to kick some Blare ass, after all. The game features both English and Japanese audio. The Japanese audio is top notch, and the dub is pretty good, as well. The English VA folks did a great job bringing the characters to life, but I think more lines should have been dubbed. While most of the main story events are dubbed, very few of the side events are.
Gameplay flows much like what you’ve seen in many other RPGs. You take on the role of Takt, manager of the Verse Maidens. Your base of operations is in the office. From here, you can take on new missions, make equipment and items, do training and many other actions. It is important to walk around the office in between story missions, as events will pop up as icons on the ground. They will show as little icons of the maidens heads. These events will help you get closer to the girls, allowing you to have bigger bonuses when paired with them in battle.
Making items works a lot like in Mugen Souls. Except, instead of selling different types of drop items, you will have to disassemble them. As you disassemble items, you gain Arcanium. There are seven different types of this you can obtain, each represented by a different color. In addition to Arcanium, you will obtain Energy Points or EP from this, as well. Think of EP as money, basically. Some items and equipment can be bought outright with EP while others you will have to disassemble certain items to obtain.
In addition to making items and equipment, you can make different outfits for the maidens wear. Idols have to look great, am I right? In addition to giving them a different look, some outfits can be upgraded further than others. This means that, by spending some EP, you can not only make the costumes more durable so they don’t break in combat as easily, but you will be able to equip more Amps, as well. Amps are accessories that can be equipped to outfits that will give you various combat bonuses and immunity from certain status aliments. The further you upgrade an outfit, the more Amps you can equip on it, but, be warned, if your clothes get damaged in battle, the effects of your equipped Amps will no longer work.
From the office, you can also access PVS or Promotion Video System. This allows you to put on a custom concert with the gals. I thought this was a lot fun and really easy to use. You can pick their dances, the way the camera moves, and even which idol or idols you want to sing each section of the song. You will start off with one song and unlock more as you progress through the game. You can even use your PS Move to control which way the sparkles flow on stage. While this doesn’t do anything useful for the story, I thought it was a great distraction.
More on page 2
About Steve Baltimore Steve started with oprainfall not long after the campaign moved from the IGN forums to Facebook. Ever since, he has been fighting to give all non-mainstream RPGs a fair voice. As the site admin, he will continue to do this and even show there is value in what some would deem "pure ecchi." He loves niche games and anime more than anything... well, except maybe Neptune.
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105 F.Supp.2d 379 (2000)
UNITED FOOD AND COMMERCIAL WORKERS UNION, et al., Plaintiffs,
v.
FLEMING FOODS EAST, INC., et al., Defendants.
No. Civ.A. 95-2587 JHR.
United States District Court, D. New Jersey.
June 29, 2000.
*380 Mark Belland, Tomar, Simonoff, Adourian, O'Brien, Kaplan, Jacoby & Graziano, P.C., Cherry Hill, NJ, for plaintiffs.
Michael McCaney, Heller, Kapustin, Gershman & Vogel, Blue Bell, PA, Timothy Duggan, Stark & Stark, P.C., Princeton, NJ, for defendants.
FINDINGS OF FACT AND CONCLUSIONS OF LAW
RODRIGUEZ, District Judge.
I. Introduction
Plaintiffs, United Food and Commercial Workers Union and Participating Employers *381 Tri-State Health and Welfare Fund (the "Health & Welfare Fund") and United Food and Commercial Workers and Participating Employers Tri-State Pension Fund (the "Pension Fund") (the Health & Welfare Fund and the Pension Fund hereinafter referred to collectively as the "Plaintiffs" or the "Funds") bring this action against Defendant Fleming Foods East, Inc.[1] and Fleming Companies, Inc. (collectively, "Fleming") under the Employee Retirement Income Security Act of 1974, as amended ("ERISA"), 29 U.S.C. § 1145 and for tortious interference with contract.
A bench trial was held on October 19-21, 1998. Having carefully considered the credibility of the witnesses, all of the evidence and the arguments of counsel for the parties, the Court renders the following Findings of Fact and Conclusions of Law in accordance with Federal Rule of Civil Procedure 52(a).
II. Findings of Fact[2]
A. Background
1. The Funds are jointly administered, multi-employer, employee benefits plans governed under the provisions of ERISA and the Labor Management Relations Act ("LMRA"), 29 U.S.C. § 186 et. seq. The Funds provide a comprehensive scheme of medical related and retirement benefits to eligible employees and their dependents who are employed by employers in the retail food industry. The Funds are financed by way of contributions remitted by companies that are signatory to collective bargaining agreements with various Locals of the United Food and Commercial Workers Union.
2. Fleming is one of the world's largest distributors of wholesale food products much of which is distributed to retail food establishments. The company reported sales in 1995 of over $17 billion and net income of $42 million. Fleming is in the business of supplying grocery and other food related products and support services to supermarkets, including such things as retail accounting services, computer services, inventory management, equipment leasing and electronic services. October 19, 1998 Trial Transcript (hereinafter "Tr. I") at 153.
3. Fleming is constantly attempting to increase its business by locating suitable food establishments for acquisition. A small percentage (approximately 25%) of supermarket retailers are able to obtain complete financing elsewhere, but approximately 75% resort to Fleming, generally for inventory financing. Thus, Fleming is also an incidental lender, where the loan function is incidental to its primary mission. Fleming is a lender of last resort.
4. During October 1990 through July 1991, Fleming negotiated with Mayfair Supermarkets, Inc., t/a Foodtown (hereinafter, "Mayfair") for the purchase of five supermarket locations in New Jersey. On July 18, 1991, Fleming and Mayfair executed an Asset Purchase Agreement for the purchase by Fleming of the five supermarket locations for a total purchase price of $7.9 million. The five stores that were the subject of the Asset Purchase Agreement were known as the "Cherry Hill Store," the "Somerdale Store," the "Moorestown Store," the "Haddon Store," and the "Pennsauken Store." The portion of the purchase price allocated to the Pennsauken store was $1,110,000.00.
5. Fleming identified five operators to run each of the five different locations. Carlo DiMichele ("DiMichele") was identified as the operator for the Pennsauken Store. At the time, DiMichele had forty years' experience in the supermarket industry. Contemporaneous with its purchase of the stores from Mayfair, Fleming assigned its rights under the Asset Purchase Agreement to each of the five operators. *382 The rights to the Pennsauken Store were assigned to DiMichele, Carmella DiMichele and Meels, Inc. ("Meels") through an Assignment of Asset Purchase Agreement dated July 18, 1991.
6. DiMichele was to operate the Pennsauken Store through Meels. Meels was a New Jersey corporation formed by DiMichele in August 1989 for the purpose of purchasing a supermarket in Runnemede, New Jersey, which never materialized. DiMichele was the sole shareholder, officer and director of Meels. Meels was not a subsidiary of Fleming and Fleming never owned any shares of Meels stock. Its corporate records were maintained by DiMichele's attorney, John A. Jones. Meels adopted by-laws and held annual meetings of the Board of Directors and shareholders in 1992 and 1993. No representative of Fleming attended these meetings or has ever served as an officer or director of Meels.
7. Neither DiMichele nor Meels had sufficient assets to consummate the transaction with Mayfair for the Pennsauken Store. Therefore, the parties agreed that Fleming would finance the transaction with Mayfair for Meels through loans totaling $1,200,000.00. Contrary to most lending practices, Fleming rather than DiMichele determined the ultimate amount of the loan. See October 20, 1998 Trial Transcript (hereinafter, "Tr.II") at 16; Tr. I at 101; October 21, 1998 Trial Transcript (hereinafter, "Tr.III") at 18-19 (expert opinion of Richard H. Weidner[3]). Also contrary to standard lending practices, Fleming prepared the underlying financial documents on behalf of Meels. Tr. III at 18-19.
8. As early as April 10, 1991, Fleming received financial information from Mayfair on the operation of the Pennsauken Store, including sales figures, gross profits, equipment inventories and other expenses, as well as copies of the union contracts, employee lists and rates of pay. Tr. I at 57-60, 69; Tr. II at 8, 30. This information, together with DiMichele's financial statement and his "operating knowledge of the business," was used by Fleming to prepare an internal financing package (the "Financing Package") containing various financial documents regarding the funding and anticipated operation of the Pennsauken Store. Tr. I at 100; Pl.Exs. 49-54. These documents included a Pro Forma financial statement and a detailed Marketing Plan.
9. The Marketing Plan was prepared by Fleming representatives and arguably incorporated DiMichele's store policies and procedures and his anticipated costs of operation, including labor expenses. Tr. I at 68-69, 100. Although the Marketing Plan, including the Pennsauken Store's labor budget, was completed in May 1991, the new collective bargaining agreement ("CBA") with the United Food & Commercial Workers Union, Local 1360 ("Local 1360") was not ratified until August 1991, approximately three months later. Tr. I at 70-72.
10. The only access DiMichele had to the labor contracts was through Fleming, which had obtained that information from Mayfair in April 1991. Tr. I at 59-60, 62; DiMichele Dep. at 42-46. Negotiations between Local 1360 and each of the five operators took place in the first half of 1991. Attorney Al Fiergang, who had acted on behalf of Fleming in the past, represented the five operators during the negotiations. Tr. I at 109. Representatives of Local 1360 believed that Fleming was playing an active role in the negotiations and was requesting concessions in order to consummate the purchase of the five supermarket *383 locations from Mayfair. As a result of this belief, representatives of Local 1360 advised Fleming of the successor language in the Mayfair collective bargaining agreement upon learning of the impending sale. At the time of the negotiations, Fleming was aware of the labor costs at the Mayfair locations, including the Pennsauken Store. Fleming would benefit, at least indirectly, from a favorable labor contract because such a contract would help operators succeed more. Tr. I at 99, 106-07.
11. Because Fleming would be looking to DiMichele's net worth as collateral for the loan, DiMichele submitted a one-page personal financial statement. This statement was unaudited and unsworn and reflected a personal net worth of $475,000. Tr. I at 71-73; Pl.Ex. 52. Of this amount, $59,000 was tied-up in his pension plan and was unreachable by a creditor. Tr. I at 20, 74-75, 115-16. Approximately one-third of his personal net worth was in the stock of Johnmar Millville, Inc., for which Fleming did not determine a market value because it was not sold on the open market. Tr. I at 115. Fleming never asked for or reviewed DiMichele's tax returns or audited financial statements. Tr. I at 74, 115. Nor did Fleming conduct an analysis or valuation of the net worth of Meels' fixtures and equipment which would serve as collateral on the loan. Tr. II at 20. Based on the financial information available to Fleming, the personal guarantees of DiMichele were "worthless." Tr. III at 23.
12. Nonetheless, the Financing Package was approved by John Traub, Catherine Gold and Mark Batenic because they thought it was "reasonable" and because the total anticipated transfers to Fleming from the Pennsauken Store would be over $43 million. Tr. I at 62-63, 76-78, 110-11; Pl.Ex. 14 (May 28, 1991 Letter from Mark Batenic to Mr. Mangold recommending loan package and noting that "Carlos has a history of running a good, tight operation and is very comfortable with the labor projection which he has included on the pro forma. The store will be operated as a union store and a favorable contract will be negotiated with the new operators and the union."). Although Fleming maintains a written company policy governing the approval of loans for the acquisition of new stores, none of these three individuals were aware of, or relied upon, that policy. Tr. I at 64-65, 110-13. After their approval, the Financing Package was subsequently sent to corporate headquarters where the loan to Meels in the amount of $1,015,000 was approved. Tr. I at 63-64.
13. No arms-length lender, in any capacity, would have approved this loan in light of "the lack of hard collateral, small profit margin, questionable cash flow, lack of personal assets for the individuals guaranteeing the loan." Tr. III at 19-24. Meels never would have been able to obtain credit in the open market or operate without the support it received from Fleming. Tr. III at 71-72 (opinion of Mark Lavinsky[4]).
14. Prior to entering into agreements with Fleming to purchase the Pennsauken Store, DiMichele reviewed these documents with his accountant and attorney. He also visited the store and discussed the store with a Mayfair representative and representatives of Fleming.
B. The Financial Documents
15. The closing on Fleming's five-store acquisition from Mayfair took place on August 8, 1991. Simultaneously with that closing, Fleming and Meels entered into a Loan Agreement which provided for a principal loan in the amount of $1,015,000.00 for the purchase from Mayfair of inventory and fixtures at the Pennsauken Store. Pl.Ex. 37. Under the terms of the Loan Agreement, the loan was to be repaid over a period of seven years, at an interest rate of prime plus three points. *384 The Loan Agreement also provided for a second loan in the amount of $185,000.00 for accounts payable.
16. Pursuant to the terms of the Loan Agreement, Meels agreed to the following obligations:
(a) to maintain minimum life insurance in the amount of $250,000 on all of its officers, Pl.Ex. 37 ¶ 6.11;
(b) to refrain from purchasing or redeeming shares of stock, paying dividends, or permitting any change in the stock ownership of Meels as long as any obligation of Meels to Fleming existed under any of the loan documents, Pl.Ex. 37 ¶¶ 6.30 and 6.31;
(c) to provide Fleming with annual and interim financial statements and weekly sales reports, Pl.Ex. ¶ 6.1(a)-(c); Tr. I at 39-40;
(d) to notify Fleming of any material litigation against it and the occurrence of any default, Pl.Ex. 37 ¶ 6.1(e);
(e) to allow Fleming to inspect the properties of Meels, including financial and accounting records, upon reasonable notice, Pl.Ex. 37 ¶ 6.3;
(f) to refrain from entering into or conducting any business or operation other than the operation of the Pennsauken Store or to make any investments other than those approved by Fleming, Pl.Ex. 37 ¶ 6.5;
(g) to maintain and provide an inventory at the end of every four-week accounting period, or quarterly, using crews acceptable to Fleming, Pl.Ex. 37 ¶¶ 6.3, 6.7;
(h) to permit Fleming, on an unannounced basis, to count the cash on hand, reconcile the actual cash on hand with that reported by Meels and perform a detailed analysis of the items counted, Pl.Ex. 37 ¶¶ 6.3, 6.7;
(i) to maintain hazard insurance and other insurance coverage designating Fleming as mortgagee under a standard mortgage clause; Pl.Ex. 37 ¶ 6.10;
(j) to refrain from incurring any indebtedness in excess of $15,000 not arising out of the ordinary course of business, Pl.Ex. 37 ¶¶ 6.13-14;
(k) to refrain from entering into any lease requiring payment of rent in excess of $15,000 per year without Fleming's consent, Pl.Ex. 37 ¶ 6.16;
(l) to refrain from amending or modifying its organizational documents, Pl.Ex. 37 ¶¶ 6.28-6.29;
(m) to refrain from changing its accounting methods, Pl.Ex. 37 ¶ 6.19;
(n) to refrain from selling its assets serving as collateral, other than through the sale of inventory in the ordinary course of business, Pl.Ex. 37 ¶ 6.32;
(o) to enter into a supply agreement with Fleming, Pl.Ex. 37 ¶ 6.25;
(p) to maintain a minimum inventory level of $340,000 on a FIFO basis, Pl.Ex. 37 ¶ 6.7;
(q) to maintain a minimum ratio of current assets to liability of 2.16 to 1, Pl.Ex. 37 ¶ 6.20;
(r) to maintain a tangible net worth of $215,000, Pl.Ex. 37 ¶ 6.21;
(s) to maintain a debt to tangible net worth ratio of no more than 6.0 to 1, Pl.Ex. 37 ¶ 6.22;
(t) to maintain minimum working capital of $400,000, Pl.Ex. 37 ¶ 6.23; and
(u) to refrain from compensating employees at a rate higher than prevailing rates in the area, Pl.Ex. 37 ¶ 6.33.
17. Simultaneously with execution of the Loan Agreement, Meels executed a Promissory Note to Fleming as evidence of the $1,015,000.00 loan. Pl.Ex. 2.
18. In accordance with the terms of the Loan Agreement and to ensure that Fleming would realize the annual transfers projected in the Financing Package, Fleming and Meels entered into a supply agreement (the "Supply Agreement"), whereby Meels was required to maintain a "Teamwork Score" of 55%. Pl.Ex. 95. This score was determined by dividing the total purchases of Fleming products by the total retail sales at the Pennsauken Store. Pl. Ex. 95. This had the effect of requiring Meels to purchase more than half of its inventory from Fleming. Tr. I at 76-78, *385 134. Fleming would not have financed Meels' acquisition of the Pennsauken Store without the Supply Agreement. Tr. I at 76-77, 124.
19. Also in accordance with the terms of the Loan Agreement and as security for the loan, Fleming and Meels executed a Security Agreement whereby Fleming retained a security interest in the assets of Meels. Pl.Ex. 35.
20. Meels, DiMichele and Carmella DiMichele entered into a Lease Agreement with Mayfair for lease of the land and building where the Pennsauken Store was located for a term of seven years. The Lease Agreement was guaranteed by Fleming pursuant to a Guaranty of Lease Agreement with Mayfair. Pl.Ex. 41. As consideration for Fleming's guaranty, Meels, DiMichele and Carmella DiMichele entered into a Guaranty and Indemnification Agreement with Fleming whereby Meels agreed to pay Fleming 5% of the rental costs of the subject property and to hold Fleming harmless from any claims brought by Mayfair against Fleming as a result of Fleming's guaranty. Pl.Ex. 42. In effect, Meels forfeited any defenses that it had to the transaction in the event of default.
21. In further accordance with the Loan Agreement, Meels executed a Leasehold Mortgage whereby it granted Fleming a mortgage covering its leasehold interest. Pl.Ex. 34; Tr. I at 82-83. Fleming and Mayfair simultaneously executed an agreement granting Fleming curative rights, effectively allowing Fleming to control the location for the period of the lease if the Pennsauken Store under Meels failed. Pl.Ex. 36; Tr. I at 83. After Meels and DiMichele shut down operations, Fleming brought in another operator to run the Pennsauken Store. Tr. I at 83-84.
22. Fleming, through the foregoing financial agreements, controlled operations at the Pennsauken Store because the different requirements detailing how business should be conducted and "the extent of the reporting" required. See Tr. III at 27. The terms of the Loan Agreement "got into the nitty gritty, the operations of the company. It just went to the whole fabric of the operation." Tr. III at 28. The Supply Agreement created a conflict of interest and took away one of Meels' few controllable costs by requiring that it maintain an ongoing minimum inventory of $350,000, with 55% of that coming from Fleming. Tr. III at 28-29. Together with the lease arrangement, Fleming controlled the property, the inventory, the equipment and essentially "all of the underlying assets and ... [Meels was] the interchangeable part of the transaction." Tr. III at 27-32.
C. Operation of the Pennsauken Store
23. Subsequent to the August 8, 1991 closing, DiMichele operated the Pennsauken Store through Meels under the trade name of "Shop `N Bag." This is a licensed trademark owned by Fleming, who also uses the name "Thriftway" in a similar manner. DiMichele's decision to use the Shop `N Bag trade name made it mandatory for DiMichele to participate in Fleming's directed advertising and pricing for the Shop `N Bag group of stores. Tr. I at 163. The retailers who comprise the Shop `N Bag advertising group meet once a week to select prices for advertised specials from Fleming's suggested retail price zones. Tr. I at 162-63. Each meeting is attended by a Fleming marketing coordinator who provides the retailers with Fleming's core ad and suggested price zones. Tr. I at 163-64. Thus, in any given week, Fleming essentially controlled the price of certain goods.
24. Fleming assigned sales counselors to monitor the Pennsauken Store's operations. The position of sales counselor is a management position and requires individuals with specialized knowledge or retail operations, merchandising, advertising and products. Tr. I at 138 and Pl.Ex. 77. According to the company's job description, the function of a sales counselor is "[t]o obtain the sales goal set for the territory by counseling retail members in store *386 operations and merchandising programs, to insure their growth, profitability and support." A sales counselor visits Fleming supplied stores on a regular basis and serves as a liaison between Fleming and the operator. A sales counselor gives advice and education but has no power or authority to instruct operators as to how to conduct their business. Sales counselors visited the Pennsauken Store sixty-eight times during the approximately thirty-one months it was operated by Meels. Tr. I at 143.
25. The Fleming employees assigned to monitor the Pennsauken Store were not limited to sales counselors. Fleming also sent a diverse array of specialists to monitor the Pennsauken Store, including such things as frozen food specialists, deli specialists, produce specialists, meat specialists, bakery specialists, technology specialists and specialists in retail merchandising. Tr. I at 152-55. These specialists were used when a sales counselor identified a problem with operations at the Pennsauken Store. Tr. I at 142-43. It is unclear how frequently Fleming specialists visited the Pennsauken Store because, unlike sales counselors, they did not keep log books or records showing their visits to stores. Tr. I at 140, 146.
26. Flemings sales counselors and specialists monitored the Pennsauken Store's operations through the quarterly financial statements and weekly sales reports submitted by Meels. These allowed Fleming to monitor Meels' compliance with the Pro Forma and Supply Agreement, and operations in general. Tr. I at 88, 120. Thus, as early as December 1991, Meels was in violation of the Loan and Supply Agreements. Tr. I at 135-36. Similarly, Fleming learned and objected to DiMichele's purchase of a vehicle without Fleming approval. Pl.Ex. 25.
27. As a result of the frequent and regular reporting and on-site monitoring, Fleming was continuously aware of problems at the Pennsauken store and was able to analyze them. For example, in December 1991, Fleming attributed them to problems with salary, advertising, expense controls and shortfall in capital. Pl.Ex. 26; Tr. I at 118, 135-37. As a result, a sales counselor was sent to the Pennsauken Store to "assist" in raising the Teamwork Score up to the score required under the Supply Agreement and to help DiMichele get his wage percentage back in line. Tr. I at 136, 150. Problems at the Pennsauken Store escalated to the point where Fleming prepared a "retail action plan" through its sales counselors and specialists to improve the Pennsauken Store's sales, profits, customer patronage, and store operations. Tr. I at 143-46; Pl.Ex. 106. A retail action plan can be requested by a sales counselor, owner or store manager. Tr. I at 155. At another point in time, a Fleming sales counselor indicated that a "full store reset" had to be done. A reset is ordered by a sales counselor, but requires the agreement of the retailer. Essentially, a reset involves changing the store's configuration and remerchandising its products by replacing discontinued items with new ones. Tr. I at 147, 154-55. This is accomplished by a sales counselor communicating the reset to other vendors in the community, who in turn come in and "reset" their part of the store. Although Fleming can order one, outside vendors cannot. Tr. I at 159.
28. Despite the problems at the Pennsauken Store, the first time Meels wrote checks to Fleming drawn on insufficient funds was in March 1994. Moreover, during the period Meels was in operation, it paid Fleming approximately ten million dollars. Tr. III at 71.
29. Flemings extensive on- and off-site monitoring, together with terms of the financial documents, in particular the Loan and Supply Agreements, allowed Fleming to exert enormous, essentially de facto, control over Meels and its operations at the Pennsauken Store. See Tr. III (testimony of Weidner and Lavinsky). Although technically Meels was a separate entity, these financial arrangements resulted in Meels operating essentially to accomplish *387 the business objectives of Fleming. See Tr. III (testimony of Weidner and Lavinsky).
30. DiMichele was required by Fleming to make an initial equity contribution of $200,000.00. Despite this requirement, DiMichele's equity contribution was $25,000 short. Tr. I at 116. As of February 1992, he had not taken any action to rectify the deficiency. Fleming had knowledge of it and did not take action either. When he finally did rectify it, he did so by borrowing money from his existing corporations to satisfy his obligation with Fleming, which Fleming ultimately learned. Tr. I at 117-18. Thus, out of the $200,000 equity contribution, he borrowed $100,000 from his other corporations. Tr. I at 117; Pl.Ex. 26.
31. Given the nature of the business, even if DiMichele had timely paid his initial contribution of $200,000, there would have been insufficient capital to sustain the operation. Tr. III at 61-65. Meels was grossly undercapitalized from its inception. By March 1992, Meels was already in violation of the current ratio required under the Loan Agreement. Tr. III at 66. It was insolvent in less than a year. Tr. III at 61-71.
32. As of September 1992, the Pennsauken Store was not operating profitably. Tr. I at 122. Nonetheless, Fleming extended an additional $500,000 to DiMichele (through Mella, Inc.) to open another store in Folcroft, Pennsylvania. Tr. I at 108, 123. This additional $500,000 was secured by the same collateral as the Pennsauken loan. Tr. I at 123. At the time of the loan, the Pennsauken Store had already lost significant amounts of money, and the personal guarantees of DiMichele and Carmella DiMichele and Meels were worthless. Tr. III at 25-26. No arms length lender would have approved this loan. Tr. III at 26. Ultimately, Folcroft also failed and Fleming has never been able to collect its debt. Tr. I at 108.
33. After Meels took over operation of the Pennsauken Store, DiMichele suffered from health problems and was frequently absent for an extended period of time. Tr. I at 102. Even before his illness, however, DiMichele's son Cubby primarily managed the operation. Tr. I at 147-48, 172. DiMichele also suffered a loss of capital as a result of litigation of a sexual harassment lawsuit. Tr. I at 102. Although Fleming was aware of these facts, all of which technically constituted defaults under the Loan Agreement, it took no action.
34. In 1994, Fleming and DiMichele discussed restructuring the loan. Ultimately, Fleming decided not to restructure them because "at the time, the stores were so marginal that even if a case broke down or somebody spilled something they would have lost money again. There was just no reason to go any further with this." Tr. I at 121-22. When the decision was made to close the Pennsauken Store, Fleming did not obtain a list of other creditors or discuss payment to other creditors. Tr. I at 124-25. DiMichele voluntarily surrendered any rights to the store to Fleming. Although DiMichele owes Fleming the balance of the loan proceeds from the Pennsauken Store, Fleming has not taken any action to enforce the personal guarantees executed by DiMichele.
35. As a result of its other financial obligations, Meels did not pay contributions to the Funds, resulting in a substantial delinquency and thereby compelling the Funds to commence legal action to collect the delinquent amounts. The Plaintiff Funds obtained a judgment for delinquent contributions in this Court at Civil Action No. 94-1873(SSB) (consolidated with Civil Action No. 94-1966) in the sum of $54,750.33 against Meels. To date, no part of the judgment has been satisfied.
36. Since the closing of the Pennsauken Store `N Bag, Fleming has obtained a new operator, Browns Supermarkets, Inc., which also operates the store as a Shop `N Bag. Despite the fact the new operator obtained outside financing, it has a supply agreement with Fleming similar to that between Meels and Fleming. Tr. I at 131.
*388 III. Conclusions of Law
1. This Court has subject matter jurisdiction and jurisdiction over the parties. In addition, venue in this District is proper.
A. Liability Under ERISA § 515.
2. Plaintiffs bring this suit under the Employee Retirement Income Security Act of 1974 ("ERISA") § 515, 29 U.S.C. § 1145, which requires an "employer" to make contributions to an employee benefit plan pursuant to the terms of an applicable collective bargaining agreement, and further imposes liability on an "employer" for unpaid contributions. An employer is defined as "any person acting directly as an employer, or indirectly in the interest of an employer, in relation to an employee benefit plan; and includes a group or association of employers acting for an employer in such capacity." 29 U.S.C. § 1002(5). Fleming asserts that it cannot be held liable for the delinquent contributions under the CBA because it does not qualify as an employer under this definition. The Funds acknowledge that technically Fleming was not a party to the CBA and that there is no liability under ERISA § 515 unless a person is a party to the collective bargaining agreement. The Plaintiffs contend that Fleming is nonetheless liable for the delinquent contributions because it was the alter ego of Meels. See Board of Trustees, Sheet Metal Workers' Nat'l Pension Fund v. Elite Erectors, Inc., 212 F.3d 1031, 1037-38 (7th Cir.2000) (noting that "[e]fforts to pierce the corporate veil ask a court to hold A vicariously liable for B's debt ... [whereas] a contention that A is B's "alter ego" asserts that A and B are the same entity").
3. The Third Circuit has not expressly set forth the standard for piercing the corporate veil on an alter ego theory in an action to recover delinquent contributions under § 515 of ERISA. However, it is settled that federal law governs liability for breach of a labor contract between a union and employer, including liability based on a theory of corporate veil piercing. American Bell, Inc. v. Federation of Telephone Workers of Pennsylvania, 736 F.2d 879, 886 (3d Cir.1984); see also United States v. Pisani, 646 F.2d 83 (3d Cir. 1981).
4. As a general rule, the corporate entity should be upheld unless specific, unusual circumstances call for an exception. Zubik v. Zubik, 384 F.2d 267, 272 (3d Cir.1967), cert. denied, 390 U.S. 988, 88 S.Ct. 1183, 19 L.Ed.2d 1291 (1968). A court should disregard the corporate existence only when it appears that "a corporation was an artifice and a sham to execute illegitimate purposes and [an] abuse of the corporate fiction and immunity that it carries." Kaplan v. First Options of Chicago, Inc., 19 F.3d 1503, 1520-22 (3d Cir.1994) (quoting Wheeling-Pittsburgh Steel Corp. v. Intersteel, Inc., 758 F.Supp. 1054, 1058 (W.D.Pa.1990)), aff'd in part, 514 U.S. 938, 115 S.Ct. 1920, 131 L.Ed.2d 985 (1995). "The alter ego concept is a `tool of equity'" which should be utilized by the courts only on clear and convincing evidence of "fraud, illegality or injustice, or when recognition of the corporate entity would defeat public policy or shield someone from public liability for a crime." Kaplan, 19 F.3d at 1521, 1522 (quoting Carpenters Health & Welfare Fund of Philadelphia v. Kenneth R. Ambrose, Inc., 727 F.2d 279, 283-84 (3d Cir.1983)).
5. When determining whether the facts of a particular case warrant piercing of the corporate veil based on an alter ego theory, a court should engage in a fact-specific inquiry in light of the following factors:
First is whether the corporation is grossly undercapitalized for its purposes. Other factors are ... failure to observe corporate formalities, non-payment of dividends, the insolvency of the debtor corporation at the time, siphoning funds of the corporation by the dominant officers or directors, absence of corporate records, and the fact that the corporation is merely a facade for the operations of the dominant stockholder or stockholders. *389 Pisani, 646 F.2d at 88 (quoting DeWitt Truck Brokers, Inc. v. W. Ray Flemming Fruit Co., 540 F.2d 681, 686-87 (4th Cir. 1976)). This list is not exhaustive, nor must all of the above factors be present to warrant a finding of alter ego liability. American Bell, Inc., 736 F.2d at 886-87. Moreover, the Third Circuit has recognized that alter ego liability may be premised on some "less precise notion that the [two distinct] corporations simply acted interchangeably and in disregard of their corporate separateness." American Bell, Inc., 736 F.2d at 886-87 (quoting Publicker Indus., Inc. v. Roman Ceramics Corp., 603 F.2d 1065, 1070 (3d Cir.1979)). For example, the Third Circuit has found that alter ego liability may be appropriate where "the controlled corporation acted robot- or puppet-like in mechanical response to the controller's tugs on its strings or pressure on its buttons." Culbreth v. Amosa (Pty) Ltd., 898 F.2d 13, 15 (3d Cir.1990) (considering alter ego liability under Pennsylvania and New Jersey law which require showing of "pervasive domination"). Thus, a court should impose alter ego liability where the facts of a case "present an element of injustice or fundamental unfairness." Pisani, 646 F.2d at 88.
6. Applying these principles and the Pisani factors to the facts of this case, the Court finds that the Plaintiffs have shown through clear and convincing evidence that Fleming was the alter ego of Meels and is liable to the Funds for Meels' delinquent contributions. In so finding, certain arguments raised by Fleming warrant express discussion. Fleming argues, in part, that (a) it cannot be held liable under an alter ego theory of liability because it had no ownership interest in Meels. Thus, traditional tests to determine alter ego status are inappropriate in this case; and (b) even under the Pisani analysis, Plaintiffs adduced insufficient evidence that Fleming functioned as an alter ego of Meels. As support for this argument, Fleming focuses primarily on its lack of a formal ownership in Meels, the fact that no representative of Fleming was ever an officer, director or shareholder of Meels and Meels' adherence to the corporate formalities. Fleming further notes that none of its representatives had the authority to sign checks on the account of Meels, to instruct Meels as to which of its creditors should be paid or given priority, and that its representatives were not present at the business premises on a daily basis.
7. Fleming's arguments are unpersuasive because they ignore the underlying purpose of the alter ego concept and are unsupported by the law, principles of equity and the facts of this case. At the outset, the Court notes that Fleming has not identified any authority that expressly states that alter ego liability may not be imposed on one entity because it lacks a formal ownership interest in the second. Nor has Fleming cited to any relevant precedent which holds that "ownership" is a determinative factor in the alter ego analysis, or states that the absence of an ownership interest requires a stricter standard. It is true that the natures of the alter ego concept and the principle of corporate veil piercing from which it derives are such that the issue of alter ego liability has arisen primarily in cases involving shareholders, parent and subsidiary corporations or successor corporations. At its core, however, the alter ego theory is designed to prevent injustice and fundamental unfairness. Whether it applies turns on the unique facts of a specific case. To make formal ownership a prerequisite to alter ego liability as Fleming suggests would serve to elevate form over substance and runs counter to the fundamental principles underlying such an equity doctrine.
As the Court has already described in its findings of fact, Fleming created a complicated and elaborate financial structure to enable Meels to operate the Pennsauken Store as a separate and independent corporate entity. In reality, however, Meels was little more than a "puppet" that allowed Fleming to aggressively pursue its own goals with virtual immunity. Contrary to its assertions, Fleming effectively *390 controlled the day-to-day operations at the Pennsauken Store. To allow Fleming to circumvent its ERISA obligations because it technically lacked an ownership interest in Meels or because Meels pursued all corporate formalities would be tantamount to condoning Fleming's manipulation of the corporate form to avoid its liabilities and obligations under ERISA. This, the Court will not do. The facts of this case present exactly the sort of injustice, fundamental unfairness and public policy concerns that the alter ego concept was designed to redress. Accordingly, the Court finds that Fleming was the alter ego of Meels and that it is liable to the Funds for Meels' delinquent contributions pursuant to ERISA § 515.
B. Tortious Interference With Contract
1. The Funds further allege that Fleming tortiously interfered with contractual relations in violation of Section 301 of the LMRA, 29 U.S.C. § 185(a). The Third Circuit has held that a claim of tortious interference with a labor contract is one arising under federal common law over which the district court has jurisdiction. Wilkes-Barre Publishing Co. v. Newspaper Guild of Wilkes-Barre, Local 120, 647 F.2d 372, 381 (3d Cir.1981), cert. denied, 454 U.S. 1143, 102 S.Ct. 1003, 71 L.Ed.2d 295 (1982); Young v. West Coast Industrial Relations Ass'n, 763 F.Supp. 64, 76 (D.Del.1991), aff'd, 961 F.2d 1570 (3d Cir. 1992).
2. To support a claim of tortious interference with a contract, a plaintiff must prove: (1) actual interference with a contract; (2) that the interference was inflicted intentionally by a defendant who is not a party to the contract; (3) that the interference was without justification; and (4) that the interference caused damage, 214 Corp. v. Casino Reinvestment Development Authority, 280 N.J.Super. 624, 656 A.2d 70, 72 (1994) (citing Norwood Easthill Associates v. Norwood Easthill Watch, 222 N.J.Super. 378, 536 A.2d 1317 (1988)); Restatement (Second) of Torts § 766. An essential element of a cause of action for tortious interference with a contractual relationship is the requirement that the defendant's interference be malicious, which requires a showing not only that the interference was done "intentionally" but also that it was "without justification or excuse." East Penn Sanitation, Inc. v. Grinnell Haulers, Inc., 294 N.J.Super. 158, 682 A.2d 1207, 1213 (1996) (citing Printing Mart-Morristown v. Sharp Electronics Corp., 116 N.J. 739, 563 A.2d 31 (1989)), cert. denied, 148 N.J. 458, 690 A.2d 606 (N.J.1997).
3. In this case, the Plaintiffs have not shown by a preponderance of the evidence that Fleming "intentionally" and "without justification or excuse" caused a breach of the valid collective bargaining agreement between the Funds and Meels. The Funds argue that Fleming caused them harm and tortiously interfered with the CBA when it "directed" Meels to cease operations.[5] However, they have not produced any probative evidence that would suggest that Fleming "directed" Meels to shut down without justification or excuse. As set forth in the findings of fact, Meels was in default of the loan documents from the time of their execution. Nonetheless, Fleming devoted significant energy to improving operations at the Pennsauken Store. The events of default increased steadily in magnitude, however, to the point where Meels was writing checks drawn on insufficient funds. At that point, Fleming determined that "[t]here was just no reason to go any further...." Findings of Fact ¶ 32. The Plaintiffs have pointed to nothing in the record to suggest that the alleged interference with the CBA was anything more than an unfortunate, but incidental, result of a valid business decision to cease operations and close a business that was no longer viable.
*391 IV. Conclusion
1. The Plaintiff Funds have proven by clear and convincing evidence that Fleming was the alter ego of Meels. Accordingly, Fleming is liable to the Funds for Meels' delinquent contributions under ERISA § 515.
2. The Plaintiff Funds have not shown by a preponderance of the evidence that Fleming tortiously interfered with the valid CBA between the Funds and Meels in violation of Section 301 of the LMRA, 29 U.S.C. § 185(a).
NOTES
[1] Fleming Foods East, Inc. no longer exists. Fleming Companies, Inc. is the successor by merger to the obligations of Fleming Foods East, Inc.
[2] Factual findings that do not contain citations to the record have been stipulated by the parties or are undisputed.
[3] Where cited, the Court adopts as fact the opinion of Plaintiff's banking expert, Richard Weidner. The Court found Mr. Weidner to be a very credible witness who performed an exhaustive review of the record in this case, including the financial documents and depositions. At trial, Mr. Weidner exhibited a thorough knowledge and understanding of these materials and of relevant banking and lending practices. The Court notes that it does not adopt the opinions of Robert J. Bifalco, Defendant's expert witness, as he testified to a review of only limited portions of the Loan Agreement.
[4] Where cited, the Court adopts as fact the expert opinion of Mark Lavinsky. Mr. Lavinsky was a credible witness who exhibited extensive knowledge of and familiarity with the financial documents in this case and with the accounting field in general.
[5] There is also nothing probative in the record to support the argument that Fleming tortiously interfered with the CBA by compelling Meels to pay Fleming before other creditors. Meels' failure to pay the contributions was a symptom of the financial arrangement between Meels and Fleming.
| tomekkorbak/pile-curse-small | FreeLaw |
Ask HN: Apple TV or Mac Mini? - winanga
I'm looking at buying an Apple TV or Mac Mini to use as a home media centre. Which one would you recommend? And have you tried running Linux on either of them?
======
brk
I have several of each running for the same application.
I'd recommend the Mini. It does everything the ATV does, and much more, for
not a lot of extra $$$.
The ATV is a nice little device, but you're fairly limited in what you can
really do with it (no Netflix streaming, for example). One nice this about the
ATV is that you can stream audio TO it from iTunes on another machine, I use
that feature a lot.
~~~
winanga
Thank you @brk
| tomekkorbak/pile-curse-small | HackerNews |
High TSH and low T4 as prognostic markers in older patients.
To examine the association between thyroid profile and morbidity/mortality (MM) in hospitalized older patients. This is a retrospective study of patients over the age of 60 yr admitted to the Dr. Cesar Milstein Hospital between 2009 and 2010 and who had thyroid function tests (TFT). The patients were grouped as per their thyroid tests and their clinical characteristics and MM was associated with their TFT. High MM was defined as mortality, intensive care unit (ICU) requirement or prolonged hospital stay (>18 days, 75th percentile), and mortality assessed during an 18-month follow-up period after their hospital discharge. Out of 2599 older patients admitted to our hospital, 7% had TFT performed for various reasons. The patients who had TFT were mostly women and presented in a more serious clinical condition compared to the rest of the patients. The patients were grouped as per their thyroid values as follows: 61% of them had a non-thyroidal illness, 25% were euthyroid,7% had overt hyperthyroidism, 5% overt hypothyroidism and 1% had subclinical hyper- or hypothyroidism. The hypothyroid patients had a worse clinical outcome compared to the others. Patients with increased MM exhibited higher TSH and lower TT4 (p<0.005). Short-term MM (OR=2.0,95%CI=1.1-3.6, p<0.01) was associated with the decrease of TT4 adjusted by age, sex, T3 and TSH, while for long-term MM the increase in TSH (OR=1.6,95%CI 1.1-2.3, p<0.05) was also significant. Among hospitalized older patients who had TFT tests, low TT4 and high TSH were associated with a worse prognosis. We propose that TFT be used as an additional tool in assessing MM in elderly hospitalized patients. | tomekkorbak/pile-curse-small | PubMed Abstracts |
Pennsylvania Railroad class GG1
The PRR GG1 was a class of electric locomotives built for the Pennsylvania Railroad (PRR), in the northeastern United States. Between 1934 and 1943 General Electric and the PRR's Altoona Works built 139 GG1s.
The GG1 entered service with the PRR in 1935 and later ran on successor railroads Penn Central, Conrail and Amtrak. The last GG1 was retired by New Jersey Transit in 1983. Most have been scrapped, but sixteen are in museums.
Technical information
Body and mechanical
The GG1 was long and weighed . The frame of the locomotive was in two halves joined with a ball and socket joint, allowing the locomotive to negotiate sharper curves. The body rested on the frame and was clad in welded steel plates. The control cabs were near the center of the locomotive on each side of the main oil-cooled transformer and oil-fired train-heating boiler. This arrangement, first used on the PRR's Modified P5 class, provided for greater crew safety in a collision and provided for bi-directional operation of the locomotive. Using Whyte notation for steam locomotives, each frame is a 4-6-0 locomotive, which in the Pennsylvania Railroad classification system is a "G". The GG1 has two such frames back to back, 4-6-0+0-6-4. The related AAR wheel arrangement classification is 2-C+C-2. This means one frame mounted upon a set of two axles unpowered (the "2") and three axles powered (the "C") hinged with the ball and socket to another frame of the same design (the +). The unpowered "2" axles are at either end of the locomotive. The GG1 was equipped with Leslie A200 horn.
Electrical and propulsion
A pantograph on each end of the locomotive body was used to collect the 11,000 V, 25 Hz alternating current (AC) from the overhead lines. In operation the leading pantograph was usually kept lowered and the trailing raised to collect current, since if the rear pantograph failed it would not strike the forward pantograph. A transformer between the two cabs stepped down the 11,000 V to the voltages needed for the traction motors and other equipment. Twelve GEA-627-A1 traction motors (AC commutator motors, not AC induction motors ) drove the GG1's diameter driving wheels on six axles using a quill drive. The power required was such that were used, with two motors driving each axle. The traction motors were six-pole field, 400 volts, 25 Hz rated each at . The motors were frame-mounted using quill drives to the sprung driving wheels providing a flexible suspension system across a relatively long locomotive frame which allowed full wheel weight to rest on the rail for good traction regardless of track condition. A series wound commutator motor's speed is increased by increasing the applied voltage to the motor thus increasing the current through the motor's armature which is necessary for increasing its torque, thus increasing motor speed. The engineer's cab had a 21 position controller for applying voltage to the motors. Four unpowered leading/trailing wheels were mounted on each end of the locomotive.
Steam generation for heating
At the time of the GG1's introduction, railroad passenger cars required steam from the locomotive to operate heating equipment. The GG1 had an oil fired boiler to provide this steam to the train's "steam line."
History
Beginning in the early 1910s, the Pennsylvania received the FF-1 but decided it was too slow for passenger trains and was relegated to heavy freight service. In the mid 1920s, they received the L5 electric which had third rail power supply at the time. When the Pennsylvania built the O1 and the P5, they chose the P5 over the O1 for its ability and power on the rails. After a grade crossing accident with the P5, the cab was moved to the center and was designated P5a. Pennsylvania still searched for the ultimate electric since the P5 did not track well at high speeds and were wondering if the P5a could be improved even further. Soon enough, the Pennsylvania was in luck and found two contacts as early as 1932. The mechanical design of the GG1 was based largely on the New Haven EP3, which had been borrowed earlier from the New Haven Railroad by the PRR to compare it to its current standard electric locomotive, the P5a. In 1933, the PRR decided to replace its P5a locomotives and told General Electric and Westinghouse to design prototype locomotives with the following specifications: a lighter axle load and more power than the P5a, a top speed of at least , a streamlined body design and a single (central) control cab.
Both companies delivered their prototypes to PRR in August 1934. General Electric submitted the GG1 and Westinghouse submitted the R1. The R1 was essentially "little more than an elongated and more powerful version of the P5a" with an AAR wheel arrangement of 2-D-2. Both locomotives were tested for ten weeks in regular service between New York and Philadelphia and on a test track in Claymont, Delaware. Because the R1's rigid wheelbase prevented it from negotiating sharp curves and some railroad switches, PRR chose the GG1 and ordered 57 additional locomotives on November 10, 1934. Of the 57, 14 were to be built by General Electric in Erie and 18 at the Altoona Works. The remaining 20 locomotives were to be assembled in Altoona with electrical components from Westinghouse in East Pittsburgh and chassis from the Baldwin Locomotive Works in Eddystone. An additional 81 locomotives were then built at Altoona between 1937 and 1943.
On January 28, 1935, to mark the completion of the electric line from Washington, D.C. to New York City, PRR ran a special train pulled by PRR 4800 before it opened the line for revenue service on February 10. It made a round trip from D.C. to Philadelphia and, on its return trip, set a speed record by arriving back in D.C. 1 hour and 50 minutes after its departure from Philadelphia.
In 1945, a Pennsylvania GG1 pulled the funeral train of President Franklin D. Roosevelt from Washington, D.C.'s Union Station to New York City's Pennsylvania Station.
In the mid-1950s, with declining demand for passenger train service, GG1s 4801–4857 were re-geared for a maximum speed of and placed in freight service. They initially retained their train heating steam generator, and were recalled to passenger service for holiday season mail trains, and 'Passenger Extras' such as those run for the annual Army–Navy football game in Philadelphia.
Timetable speed limit for the GG1 was 75-80 mph until October 1967 when some were allowed 100 mph for a couple of years; when Metroliner cars were being overhauled in the late 1970s, GG1s were again allowed 100 mph for a short time when pulling Amfleet cars on trains scheduled to run 224.6 miles from New York to Washington in 3 hours, 20–25 minutes.
On June 8, 1968, two Penn Central GG1s pulled Robert F. Kennedy's funeral train.
Shell design
The first designer for the GG1 project was industrial designer Donald Roscoe Dohner, who produced initial scale styling models, although the completed prototype looked somewhat different. At some point, PRR hired famed industrial designer Raymond Loewy to "enhance the GG1's aesthetics."
Although it was thought until 2009 that Loewy was solely responsible for the GG1's styling, Dohner is now understood to have contributed as well. (Dohner's GG1 designs influenced the modified P5as, which debuted before the GG1 — not, as was thought, the other way around.) Loewy did claim that he recommended the use of a smooth, welded body instead of riveted one used in the prototype. Loewy also added five gold pinstripes and a Brunswick green paint scheme.
In 1952, the paint scheme was changed to Tuscan red; three years later, the pinstripes were simplified to a single stripe and large red keystones were added.
Incidents
On January 15, 1953, train 173, the overnight Federal from Boston, was approaching Washington behind GG1 4876. The train passed a signal north of Union Station between , and the engineer decreased the throttle and started applying the brakes. When the engineer realized that the train was not slowing down, and applying the emergency brake had no effect, he sounded the engine's horn. A signalman, hearing the horn and noting the speed of the 4876, phoned ahead to the station master's office. 4876 negotiated several switches without derailing, at speeds well over the safe speed limits and entered the station at around . The train demolished the bumping post, continued through the station master's office and into the concourse where it fell through the floor into the station's basement. Thanks to the evacuation of the concourse, there were no fatalities in the station, or aboard the train. A temporary floor was erected over the engine, and the hole it created, for the inauguration of President Dwight D. Eisenhower. 4876 was eventually dismantled, removed from the basement and reassembled in Altoona. It survives in the B&O Railroad Museum in Baltimore.
The accident was determined to have been caused by a closed "angle cock", a valve on the front and rear of all locomotives and rail cars used in the train's airbrake system, on the rear of the third car in the train. The handle of the angle cock had been improperly placed and had contacted the bottom of the car. Once it was closed the air brake pipe on all the cars behind the closed valve remained at full pressure, keeping the brakes released on those cars while the brakes on the locomotive and first three cars were applied in emergency. This was the only major wreck involving a GG1 in the entirety of its 59-year career.
The only major electro-mechanical breakdown of the GG1 was caused by a blizzard which swept across the northeastern United States in February 1958. The storm put nearly half of the GG1s out of commission. Exceptionally fine snow, caused by the extreme low temperatures, was able to pass through the traction motors' air filters and into the electrical components. When the snow melted it short circuited the components. On about 40 units, the air intakes were moved to a position under the pantographs.
Disposition
In 1968 the PRR with its 119 surviving GG1s merged with the New York Central Railroad to form Penn Central. After its creation in 1971, Amtrak purchased 30 for $50,000 each, and leased 21, of which 11 were for use on New York and Long Branch commuter trains. Amtrak renumbered the purchased GG1s #900–929 as a solid block, later renumbered with a prefix "4". The leased units with conflicting numbers were then renumbered 4930–4939, except 4935 which kept its old PRR/PC number.
Amtrak attempted to replace the GG1s in 1975 with the General Electric E60, but they were not a success: a derailment during testing had to be investigated (the E60 used the same trucks as the P30CH diesel then in service with Amtrak), which delayed acceptance, and the hoped-for service speed was never achieved (timetable limit was 90 mph, then 80, then 90).
It was not until Amtrak imported two lightweight European locomotives – X995, an Rc4a built by ASEA of Sweden and X996, a French design – that a replacement was found. The ASEA design, initially nicknamed the "Swedish swifty", or the "Mighty Mouse"—and later referred to often as the "Swedish Meatball"—was the winning design. Electro-Motive Diesel, then a part of General Motors, was licensed to build it and it became the basis of the AEM-7. With AEM-7s on hand, Amtrak finally replaced its GG1s. GG1 service on Amtrak ended on April 26, 1980.
Penn Central went bankrupt in 1970 and its freight operations were later assumed by government-controlled Conrail, which used 68 GG1s in freight service until the end of electric traction in 1980.
The last GG1s in use were some of the 13 assigned to New Jersey Transit (#4872–4884) for its North Jersey Coast Line between New York and South Amboy (the former New York and Long Branch) that ran until October 29, 1983, thus retiring the locomotive after 49 years of service.
Preservation
Fifteen production locomotives and the prototype were preserved in museums. None are operational, as they had to have their main transformers removed due to the presence of PCB's in the insulating oil.
PRR/PC/CR 4800 — Railroad Museum of Pennsylvania, Strasburg, Pennsylvania (nicknamed "Old Rivets" due to it being the only GG1 to have been built with a riveted body)
PRR/PC/CR 4859 — Transportation Center, Harrisburg, Pennsylvania (designated Pennsylvania state electric locomotive in 1987)
PRR/PC/CR/NJT 4876 — B&O Railroad Museum, Baltimore, Maryland
PRR/PC/CR/NJT 4877 — United Railroad Historical Society of New Jersey, Boonton, New Jersey (nicknamed "Big Red")
PRR/PC/CR/NJT 4879 — United Railroad Historical Society of New Jersey, Boonton, New Jersey
PRR/PC/CR/NJT 4882 — National New York Central Railroad Museum, Elkhart, Indiana (currently painted in Penn Central colors)
PRR/Amtrak 4890 — National Railroad Museum, Green Bay, Wisconsin
PRR 4903/Amtrak (4)906 — Museum of the American Railroad, Frisco, Texas (pulled Robert Kennedy's funeral train with GG1 4901 from New York to Washington on June 8, 1968).
PRR 4909/Amtrak 4932 — Leatherstocking Railway Museum, Cooperstown Junction, New York (purchased by The Henry Ford)
PRR 4913/Amtrak (4)913 — Railroaders Memorial Museum, Altoona, Pennsylvania
PRR 4917/Amtrak 4934 — Leatherstocking Railway Museum, Cooperstown Junction, New York
PRR 4918/Amtrak (4)916 — Museum of Transportation, St. Louis, Missouri (was once the property of the Smithsonian Institution).
PRR 4919/Amtrak (4)917 — Virginia Museum of Transportation, Roanoke, Virginia
PRR 4927/Amtrak 4939 — Illinois Railway Museum, Union, Illinois
PRR 4933/Amtrak (4)926 — Central New York Chapter of the National Railroad Historical Society, Syracuse, New York. It has been cosmetically restored and is on display at the NYS Fairgrounds Historic Train Exhibit.
PRR 4935 / Amtrak 4935 — Railroad Museum of Pennsylvania, Strasburg, Pennsylvania (nicknamed "Blackjack")
In popular culture
During the mid-1930s, the art of streamlining became popular, especially with locomotives, as it conveyed a sense of speed. While other railroads were introducing streamlined trains, like the Union Pacific's M-10000 or the Chicago, Burlington and Quincy Railroad with the Zephyr, the PRR had the GG1. The GG1 has "shown up over the years in more advertisements and movie clips than any other locomotive." It was also featured in art calendars provided by PRR, which were used to "promote its reputation in the public eye." It has appeared in the films Broadway Limited in 1941, The Clock in 1945, Blast of Silence in 1961, the 1962 version of The Manchurian Candidate, and Avalon in 1990 in the PRR paint scheme. Two GG1s appear in the 1973 film The Seven-Ups—a black Penn Central locomotive and a silver, red and blue Amtrak locomotive. A Penn Central GG1 also appears in another 1973 film The Last Detail. PRR GG1 4821 appears briefly in the 1952 film The Greatest Show on Earth, pulling the Ringling Bros. and Barnum & Bailey Circus Train into Philadelphia's Greenwich Yard, with the movie's director Cecil B. DeMille narrating the scene of their arrival. Near the end of the 1951 film Bright Victory GG1 #4849 is shown pulling into the station.
A GG1 and the Congressional were featured on a postage stamp as part of the United States Postal Service's All Aboard! 20th Century American Trains set in 1999.
The PC games Railroad Tycoon II, Railroad Tycoon 3, Sid Meier's Railroads!, Train Fever and Transport Fever allow for players to purchase and operate GG1 locomotive engines on their train routes. The GG-1 is also available with the default Trainz Simulator Games in recent years, and they have been made available as add-ons for Railworks, Train Simulator by Dovetail Games, and Microsoft Train Simulator.
"The Magnificent GG1" video by Mark I documents the locomotive's history.
Model GG-1s have been produced in O, S, HO, and N scales by Rivarossi, Bachmann, Tyco, Lionel, MTH, USA Trains, Kato, and other manufacturers.
References
Sources
Further reading
External links
GG1: An American Classic, 1984 documentary on the last run of the GG1 at YouTube
Category:General Electric locomotives
Category:2-C+C-2 locomotives
Category:11 kV AC locomotives
GG1
Category:North American streamliner trains
Category:Passenger locomotives
Category:Electric locomotives of the United States
Category:Railway locomotives introduced in 1934
Category:Conrail locomotives
Category:Amtrak locomotives
Category:Standard gauge locomotives of the United States
Category:Raymond Loewy | tomekkorbak/pile-curse-small | Wikipedia (en) |
Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins.
Many plant proteins, particularly those found in foods and pollen, are known to act as sensitizing agents in humans upon repeated exposure. Among the cereal flour proteins involved in asthmatic reactions, those members of the alpha-amylase inhibitor family which are glycosylated, polypeptides, BMAI-1, BTAI-CMb*, and WTAI-CM16* are particularly reactive both in vivo and in vitro. We show here that these major glycoprotein allergens carry a single asparagine-linked complex glycan that contains both beta 1-->2 xylose and alpha 1-->3 fucose. Evidence is presented that the xylosyl residue and, to a lesser extent, the fucosyl residue are key IgE-binding epitopes and largely responsible for the allergenicity of these and unrelated proteins from plants and insects. Our results suggest that the involvement of xylose- and fucose-containing complex glycans in allergenic responses may have been underestimated previously; these glycans provide a structural basis to help explain the cross-reactivities often observed between pollen, vegetable food, and insect allergens. | tomekkorbak/pile-curse-small | PubMed Abstracts |
Introductory review
===================
\"On the Origin of Species\", which was published in 1859 authored by Charles Darwin, laid the foundation of theory of evolution. Modern evolutionary biology has been now illustrated through genetic variations among individuals. Today, studies showed that genetic alterations can cause diseases, yet the driver mutations that cause complex diseases such as cancer remain unclear. Disease-driving genomic alterations can be inferred from comprehensive studies of genomic data and gene network analysis.
It has been known that many identified mutation-based drug targets have unwanted side effects of inhibitor treatment that often cause resistance to drugs. Studies of IDP (intrinsically disordered proteins) showed that due to differences in the post-translational circuitry such as the phosphorylation networks, where phosphorylation sites are typically within IDP regions of motifs that are dynamically unconserved during evolution. Genetically altered cells such as cancerous cells often have aggregated mutations in the target kinase. Evolutionary divergence of phosphorylation and functional alterations in protein kinases are likely correlated and evolutionary conserved kinase substrate interactions in phosphoproteins are more likely mutated in cancer. Genomic mutation-induced rewiring of the signalling networks can be prone to complex diseases such as cancer. It is important to identify IDP and evolutionary conserved networks that affect diseases for the identification of underlying disease mechanisms and more effective drug targets.
Equally important are the identification of bidirectional promoters and the regulation of genes associated with mutations or dysregulation in cancer that are enriched with bidirectional promoters. Those genes such BRCA1, BRCA2, BARD1, FANCA, FANCF, FANCB, FANCD2, P53, ERBB2, and CHEK2 work together as a group sharing regulation by bidirectional promoters and were found prevalence in ETS family factors. Identification of biological roles in bidirectional promoters is essential to our understanding of the regulatory mechanisms of bidirectional promoters and how they can regulate cancer genes. While common transcription binding factors were found in these genes, co-expression networks in human cancer can often be used to infer the underlying disease mechanisms from bidirectional promoters in combination with gene expression profile.
Furthermore, advances in high throughput RNA sequencing technologies have generated large volumes of gene expression data from RNA-Seq in addition to the microarray DNA-chip data. Genomic mutations in disease-causing genes or structural variations in chromosomes can disturb signaling pathways that impact the expression of a set of genes performing certain biological functions. Integrating differentially expressed genes and pathways can lead to discovering higher-level disease-associated networks. The Mid-South Bioinformatics Centre (MBC) and Joint Bioinformatics Ph.D. Program of University of Arkansas at Little Rock and University of Arkansas for Medical Sciences (UALR-UAMS) are particularly interested in promoting education and research advancement in comprehensive translational bioinformatics studies. To promote synergistic education and research in translational bioinformatics, the International Society of Intelligent Biological Medicine (ISIBM) provided academic sponsorship to the 2014 International Conference on Bioinformatics and Computational Biology. MBC works with UALR-UAMS in promoting cutting-edge translational bioinformatics research.
The 2014 International Conference on Bioinformatics and Computational Biology received large pool of hundreds of paper submissions. All submitted papers were peer reviewed by the conference program committee members (<http://www.world-academy-of-science.org/worldcomp14/ws/conferences/biocomp14/committee>) and invited external experts. Six papers selected to this special *BMC Bioinformatics*supplement represent current trends in the cutting-edge translational bioinformatics research and were chosen from large submissions by the review committee chaired by ISIBM President Dr. A. Keith Dunker (Founding Director and T. K. Li Professor for Medical Research of Indiana University School of Medicine Centre for Computational Biology and Bioinformatics) based on peer-reviews. ISIBM Vice-President Dr. Hamid R. Arabnia (Editor-in-Chief of Journal of Supercomputing, Professor of Computer Science at University of Georgia), Vice-President Dr. Dong Xu (Editor-in-Chief of International Journal of Functional Informatics and Personalised Medicine, Associate Editor-in-Chief of IEEE/ACM Transactions on Computational Biology and Bioinformatics, James C. Dowell Professor and Chair of Computer Science Department at University of Missouri), Secretary-General Dr. Yunlong Liu (Associate Editor of BMC Genomics, Associate Professor of Molecular and Medical Genetics and Director of Bioinformatics Core at Indiana University School of Medicine, Indiana University Purdue University Indianapolis), Dr. Zhongxue Chen (Associate Editor of BMC Genomics and Director of Study Design and Data Analysis Consulting Centre of Indiana University at Bloomington), Dr. Xiang Qin (Assistant Professor of Molecular and Human Genetics at the Human Genome Sequencing Centre at Baylor College of Medicine), along with Dr. Weida Tong (HHS/FDA/NCTR Director of Bioinformatics and Biostatistics and Professor of Bioinformatics by courtesy at University of Arkansas at Little Rock, and core faculty of joint bioinformatics Ph.D. program), and Dr. Youping Deng (Associate Editor of BMC Research Notes, Associate Professor of Medicine and Director of Cancer Bioinformatics and Biostatistics of Rush University Medical Centre in Chicago) also served on the review committee. External experts were invited to review the submitted papers and the committee finally select these six significant papers \[[@B1]-[@B6]\] for the BMC Bioinformatics supplement based on peer-reviews.
In this *BMC Bioinformatics*supplement, Yang and Elnitski\'s laboratories performed a series of in-depth investigations and identified bidirectional promoters and conservations of this type of promoters utilizing orthologous mapping in human and mouse genomes \[[@B1]\]. They incorporated data generated by cap analysis gene expression (CAGE), and validated most of 5\' end of UCSC Genome Browser annotations that were used in the study. Their studies of coordinated expression of bidirectional gene pairs were significant as the conserved bidirectional promoters in humans have been implicated in complex diseases such as cancer. While mouse has been used as a standard model animal for investigating a variety of diseases in humans, compassion with human genes revealed that bidirectional promoters regulate significant amounts of genes in mammalian genomes, especially regulate disease associated genes. Understanding mechanisms of these promoters based on their functional roles and evolutional patterns can provide valuable resources to further understanding of gene regulation, transcriptional mechanisms and their roles in the disease transformation. In addition, Yang and Elnitski\'s laboratories found that the gene expression mediated through bidirectional promoters can influence many biological processes such as histone modification. Furthermore, correlations between bidirectional promoters and lncRNAs (long non-coding RNAs) identified by Yang are considered as highly significant because such findings can facilitate the identification of functions of lncRNAs in connection with the regulatory roles of bidirectional promoters. This important work offers deeper insights into the regulatory roles of bidirectional promoters in connection with lncRNAs in complex diseases.
Equally impressive is the work of Yang\'s laboratory to systematically investigate TCGA (The Cancer Genome Atlas) real cancer data. The advent of high-throughput next-generation sequencing technologies marked the beginning of a new era for personalized medicine research. The impact of having an individual genome and personalized genomic data in hands generates high demands of developing more powerful computational approaches to handle massive information imbedded in the big data, which will obviously generate profound effect on how data-intense biomedical research shall be conducted toward the improvement of human health and lifesavings. Yang\'s laboratory utilized TCGA RNA-Seq data from more than 500 kidney renal clear cell carcinoma (KIRC) patients to investigate genes and pathways that were significantly altered in the disease \[[@B2]\]. The laboratory identified 186 genes with significant differential expressions between normal and disease samples. They found four-subtypes of the kidney cancer, which were consistent with the results of recent publications. In addition, an intelligent SVM (support vector machine) based supervised classifier was built using the identified differentially expressed genes to predict unknown samples. The intelligent machine can effectively distinguish cancer samples from non-cancer samples with high accuracy. By integrating differentially expressed genes with pathway analysis, the authors revealed several putative pathways disrupted in the disease. Their results not only confirmed a number of previously reported disease pathways in literature, but also identified new roles of pathways in the disease that has not been well studied yet. Furthermore, based on the differentially expressed genes between tumor and normal tissue samples, results from their network analysis suggested that combining differentially expressed genes, pathways and networks can infer the upstream regulators, which can ultimately help identifying disease causal genomic mutations. The research was a part of the plenary invited talk entitled \"Integrative systems biology approaches to identify disrupted pathways in disease development\" (<http://www.world-academy-of-science.org/worldcomp14/ws/keynotes/invited_talk_yang> ). The integrative methods presented from Yang\'s laboratory demonstrated that combining differentially expressed genes, gene networks and biological pathways have provided powerful approaches to further reveal underlying disease mechanisms and effective drug targets.
Effectively combining information from individual studies is critical to assess rare variants in GWAS (genome-wide association study). Chen\'s laboratory and collaborators developed a novel statistical approach using the inverse of the p-value as the shape parameter in the gamma distribution to more effectively combing p-values from individual studies \[[@B3]\]. This approach can adaptively choose the shape parameter of the gamma distribution for each individual study, since the flexibility in choosing the parameters allows effectively combing p-values for either homogenous or heterogeneous individual studies. This is considered as an improved method to assess the genomic variants association with diseases, especially to handle the heterogeneous cases in complex diseases. Chen\'s laboratory and collaborators demonstrated that the performance of their new method outperformed existing approaches when the effects among the studies are more heterogeneous. The newly developed approach has been tested favourably in genome-wide association study.
Intrinsically disordered proteins (IDP) play important roles in many biological processes that include post-translational modifications, entropic chain spring-based restoring forces, flexible linkers, signal transduction, protein aggregation and coupled folding and binding. Dunker\'s laboratory developed new the IDP-Hydropathy scale using the C-H (charge-hydropathy) plot as the classifier based on sets of sequences that fold into 3D structure as compared to collections of sequences that do not fold \[[@B4]\]. The method provided a measurement of how various amino acids contribute to protein folding using the property of hydropathy. For many years, Dunker\'s laboratory has taken a lead in classifying proteins that either fold into 3D structures or do not fold into any structure intrinsically. The paper certainly aided to the advancement of this very important but not widely known field. Dunker\'s laboratory provided a new approach to accurately classify structured and disordered proteins based on hydropathy using the C-H plot. The authors reported 19 different hydropathy scales including Kyte & Doolittle scale. They compared the predicting accuracy of the C-H plot method using different hydropathy scales. They used support vector machine (SVM) to train the classifier that discriminates structured proteins from disordered proteins. The weights produced by the SVM are then used as a new hydropathy scale for the C-H plot. Their new hydropathy scale was used to boost the predictive power of the classifier. They concluded that their IDP-Hydropathy would likely be the best scale to use for any type of algorithm developed to predict protein disorder.
Lu and Deng\'s laboratories and collaborators performed differentially expressed gene analysis using microarray and genome-wide expression profile of Type II diabetes (T2D) \[[@B5]\]. Using blood samples from healthy humans, pre-diabetic and diabetes patients, they identified 79 differentially expressed genes with fold change larger than 2. They built a discriminant model using expression levels of 79 differential genes in combination with clinical factors that include age, sex, and race to achieve over 91% accuracy in diagnosing / predicting status of the T2D (normal people, pre-diabetic patients and T2D patients). Their Gene Ontology (GO) analysis revealed a collection of significant GO term associated with the differential genes. Their work can provide a combined molecular and pedigree analytic method that could potentially lead to an effective screening tool for identifying overall health or illness of humans and predicting progression of the disease development. The pairwise analysis presented in the paper is significant and innovative. Combining gene expression, pathway and network analysis can reveal underlying molecular mechanisms for better preventing, diagnosing and treating the disease.
United States Foods and Drug Administration (FDA) has been the leader in drug toxicity studies, Tong\'s laboratory at FDA assessed safety of drugs and the impact on human health from drug toxicity \[[@B6]\]. Medical drugs are not natural products and often cause damages to livers and human health. Unfortunately, toxicity of many drugs has not been fully studied. The investigators from United States Foods and Drug Administration and University of Arkansas at Little Rock fully understand the importance of a comprehensive assessment of drug safety and toxicity. They developed a method to systematically search the literature and gathered information together to present the risk of accurate liver failure, which can be a fatal consequence of certain drugs. This research opens a new opportunity to comprehensively identify potential outcomes of certain drugs using acute liver failure as case studies. Results from the research can be potentially useful in future personalized genomics and individualized healthcare investigations.
Conclusion
==========
Integrating multi-layer genomic data has helped to reveal many underlying molecular mechanisms. In particular, identifying roles of bidirectional promoters with cancer-related genes using different genome-scale data can systematically assess genomic mutations and gene expression that are associated with dysfunctional regulations in cells and/or malignant transformation, while combining gene expression and pathway analysis with gene networks using systems biology approaches can help revealing underlying disease mechanisms and link pathways to disrupted gene networks in disease development. The cutting-edge research presented in this BMC Bioinformatics special supplement represents the current development of computational approaches in different bioinformatics studies. Developing approaches to combine the information from different data helps the advances in translational bioinformatics, which ultimately facilitate revealing the landscape of complex disease mechanisms.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
AKD, YD, HRA, XQ, LW, ZC, ZL, YL, AN, JSL, JYY, WT, and DX organized external peer-reviews, contributed to reviews and participated in the selection of five high-quality research papers for the special BMC Genomics supplement based on peer-reviews. MQY and WY wrote the article which was read and approved by all authors.
Acknowledgements
================
The research at MBC was supported by the National Institutes of Health (NIH) and Arkansas Science and Technology Authority (ASTA). MQY was supported by NIH/NIGMS 5P20GM10342913 and ASTA Award \# 15-B-23. In addition, XQ was supported by NIH/NHGRI 5U54HG003273-11.
Declaration
===========
The funding for publication of the article has come from the MidSouth Bioinformatics Centre, and the Joint Bioinformatics Ph.D. Program of University of Arkansas at Little Rock and University of Arkansas for Medical Sciences with NIH/NIGMS 5P20GM10342913 and ASTA award \# 15-B-23.
This article has been published as part of *BMC Bioinformatics*Volume 15 Supplement 17, 2014: Selected articles from the 2014 International Conference on Bioinformatics and Computational Biology: bioinformatics. The full contents of the supplement are available online at <http://www.biomedcentral.com/bmcbioinformatics/supplements/15/S17>.
| tomekkorbak/pile-curse-small | PubMed Central |
Water insoluble salts such as calcium and magnesium carbonates or silicates or sulfates commonly referred to as limescale, but also barium sulfate, calcium oxalate, calcium phosphate, iron oxide and the like are readily formed in watery solutions when the conditions are right and may each represent particular challenges in relation to their removal.
Limescale or limestone is the hard, off-white, chalky deposit found in kettles, hot-water boilers and the inside of inadequately maintained hot-water central heating systems. It is also often found as a similar deposit on the inner surface of old pipes and other surfaces where “hard water” has evaporated.
These types of limescale differ slightly due to their origins. The type found deposited on the heating elements of water heaters, laundry machines, etc. has a main component of calcium carbonate, precipitated out of the (hot) water. Hard water contains calcium (and often magnesium) bicarbonate and/or similar salts.
Calcium bicarbonate is soluble in water, however at temperatures above 70° C. the soluble bicarbonate is converted to poorly-soluble carbonate, leading to deposits in places where water is heated. Local boiling “hot spots” can also occur when water is heated, resulting in the concentration and deposition of salts from the water. Likewise calcium sulfate is a common component of fouling deposits in industrial heat exchangers, due to its decreased solubility with increasing temperature. Silicate containing laundry and automatic dishwashing products may cause a calcium or magnesium silicate deposit, which is especially difficult to remove (in contrast to calcium carbonate) from glassware.
The type found on air-dried cooking utensils, dripping taps and bathroom tiling consists of calcium carbonate mixed with all the other salts that had been dissolved in the water, prior to evaporation. It can also be found on taps and water reservoirs (such as in the toilet) where hard water has been continually running through and has deposited calcium carbonate.
The presence of limescale presents several problems. Other than being unsightly and harder to clean, limescale can impair the operation of various components or damage them. In kettles, limescale acts as an insulator, impairing heat transfer. Additionally, it can damage the heating element, which overheats due to accruing limescale. Limescale can build up inside tubing, thus reducing water flow and necessitating higher electrical consumption for the circulation pumps, and eventually blocking the tubing. Expresso machine manufacturers recommend to descale the machine (depending on the water hardness) every month or trimester in order to avoid bitter taste development, machine malfunction and slowing down.
Other types of deposits formed by insoluble salts are beerstone and milkstone. Calcium oxalate forms a major component of beerstone, a brownish precipitate that tends to accumulate within vats, barrels and other containers used in the brewing of beer. Beerstone is composed of calcium and magnesium salts and various organic compounds left over from the brewing process; it promotes the growth of unwanted microorganisms that can adversely affect or even ruin the flavor of a batch of beer. Calcium oxalate is also formed during carbonation of raw sugar beet juice before it undergoes crystallization. First, the juice is mixed with hot milk of lime (a suspension of calcium hydroxide in water). This treatment precipitates a number of impurities, including multivalent anions such as sulfate, phosphate, citrate and oxalate, which precipitate as their calcium salts and large organic molecules such as proteins, saponins and pectins, which aggregate in the presence of multivalent cations.
Milkstone is a layer of scale mainly formed by cations like calcium and magnesium originating from both milk and hard water. Besides giving the equipment an unclean appearance, milkstone could harbour and protect micro organisms always present in raw milk and ready to multiply at a high rate. Since milk products are some of the most perishable major foods, cleaning and sanitization in that industry generally require the highest standards. The main part of milk residu is easily removed by rinsing with water. However, the last part comprising the milkstone is often harder to get rid of.
Several methods and products have been developed in order to remove some or all of these different types of deposits by insoluble salts, such as limescale.
Generally, different types of descaling agents are used to remove deposits by insoluble salts. Descaling agents are either acids or complexing agents or both in one (e.g. carboxylic acids). They remove insoluble deposits such as limescale by respectively dissolving the limescale and or complexing its cationic constituents. Acids used as descaling agents can be either mineral acids or organic acids. Below in table 1, the properties of some organic and mineral acids that are used or can be potentially useful for descaling are shown.
TABLE 1Properties of some organic and mineral acids used or potentially useful for descaling.SolubilitySolubilityTrivial namewatercalcium saltphysicalof acid20° C.g/100 mlformSourcingdescalingpKasmellCompatibilitylabeloxalic 14% 0.0007powderPetro1.3/4.3++C, Xnmaleic>40%2.9powderPetro1.9/6.3+−Xi/Xnmalonic>90%No datapowderPetro2.9/5.7+−Xntartaric+−60% 0.04powderFerm 3/4.3++Xifumaric 64%1.4,2%bpowderPetro 3/4.4++Xicitric 60%5%(1.Ca)powderFerm−3.1/4.8/6.4++++Xi0.09%(2-3.Ca)malic>80% 0.8%bpowderPetro+3.4/5.1+−−−Xnformic100%17 liquidPetro++3.8−−−−C, Xnglycolic100% 1.2%powderPetro−−3.9+−−−C, Xnitaconic 9.5%No datapowderFerm3.9/5.1++Xilactic100%7%,3.1%bliquidFerm+3.8++++Xigluconic>50%3,3%bpowderFerm3.9−−Xnsuccinic 7.7% 0.004%bpowderpetro/ferm4.2/5.6++C, Xnglutaric 50%SolublepowderPetro4.3/5.4++XiAcetic100%33.8 liquidpetro/ferm+4.8−−−−C, XnLactideaInsol.aaPowderFerm/++++Xiphosphoric100% 0.03liquidMin++ 2.2/6.8/12.4+−C, Xnsulfamic 29%No datapowderMin++0.1+−+−C, Xn, Nhydrochloric>40%75 liquidMin−9.3 −−C, T, Nsulfuric100%0.3liquidMin−3 +−C, TbLactide is a dimeric ester rather than an acid but readily hydrolyses to lactic acid. Data obtained from presentations by Purac and complemented with various literature data. Properties listed include the solubility in water (pH 7, 20° C.), the solubility of the calcium salt (mono, di, tri-salts,bas % anhydrous at 25° C.), their physical form, descaling effectiveness (Purac data), pKa value(s), smell, overall material compatibility (Purac data) and labeling according to EU legislation.
Table 1 documents among other characteristics the water solubility of di- and tri-salts of polybasic carboxylic acids which tends to be (very) limited as compared to that of monocarboxylic acids, with maleic and glutaric acids as an exception to this apparent rule. No literature data were found regarding the calcium salt of itaconic acid. Whereas the monocalcium salt of citric acid is water soluble (5%), the disalt and trisalts are only sparingly or practically insoluble (0.09 g/l).
The majority of acids commercially used for descaling are mineral acids such as phosphoric, sulfamic, hydrochloric and sulfuric acid (cf table 1). These are however classified as corrosive to the skin and the eyes and as environmentally hazardous or in case of phosphoric acid represent a substantial eutrophication potential. Moreover they tend to be either fuming or cause a pungent smell and their overall material compatibility is limited.
Organic acids have one, two or three carboxyl groups (note the pKa values in table 1) and are usually less aggressive which is why acetic, citric and formic and more recently glycolic and lactic acid found their way to the market.
Organic acids can be sourced from fermentation or from petrochemical synthesis. Citric and lactic acid for example are obtained by fermentation from renewable feedstock (typically molasses). The fact that many of these organic acids suitable for descaling action are renewable is increasingly considered an environmental advantage as illustrated in life cycle analyses. However, some of these organic acids still show disadvantages.
For example, the iron and calcium salts of citric acid are said to be less soluble than those of glycolic acid, so they may precipitate onto the treated surfaces, diminishing cleaning effectiveness of citric acid.
Acetic and formic acid have a pungent smell that is hard to cover with fragrance, which is a serious disadvantage.
Acetic acid, which may be sourced from fermentation or from petrochemical synthesis, is renowned for its corrosivity to copper which leads to the formation of toxic copper acetate (a fungicide) thus rendering acetic and vinegar unsuited for descaling coffee and expresso machines which often have a copper mounting tube for hot water or steam. Acetic acids will thus also be unsuited for all other surfaces comprising cupper.
Furthermore, the descaling activity of many organic acids is quite weak. Many organic acids either show efficiency in fast descaling or in descaling upon prolonged contact, but not both. Moreover a limited number of organic acids is available as a solid. These are huge disadvantages as they put a restriction onto the development of descaling agents that offer an overall better efficiency.
There is a need for a descaling agent which is renewable, and which shows a better efficiency than the existing products.
One specific application of descaling agents is their use in toilet blocks, since toilets often suffer from severe insoluble salt deposits. Traditionally toilet blocks in the past where designed to mask odors and have a slight cleaning effect in the toilet. The two main types manufactured and marketed up until the late 1980's were the so called rim and the in-cistern blocks, applied in the toilet bowl and the water cistern respectively. During the 1990's several new developments have been marketed, with the liquid rim containers coming on the market which has greatly increased the flexibility and number of ingredients available to formulate with, and the solid block formulations have also been expanded with products that have special properties (i.e. lime scale inhibition, bleaching, cleaning efficiency, etc.).
There are different challenges to the formulation and manufacture of toilet and cistern blocks as these are dependent on most of the ingredients being supplied as practically water free chemicals, otherwise they might have a negative influence on the chemical properties of the block as well as the stability and compatibility with other ingredients included. The main manufacturing process for such blocks is by extrusion of a pre-made dry mixture of all the ingredients. A crucial property for the manufacture of solid blocks however has been the extrusion properties of the anionic surfactants and in particular dry LAS (Linear Alkyl Benzene sulfonate). The sodium salt of LAS in dry form is available as a very hygroscopic powder, which means that precaution has to be taken in terms of handling and storage, but it is also this product characteristic that makes it an excellent main ingredient in formulating solid extrude toilet blocks. The hygroscopic nature of LAS ensures that once the final product is exposed to water in the toilet bowl or in the cistern it will create an outer layer or membrane that slows down the overall solubility of the block thereby imparting a controlled release of all the active ingredients in the block (source: Toilet block introductory Leaflet by Unger, 2008). Formulating rim and in-cistern blocks among others implies selecting solubility retarding or “matrix” ingredients with a melting point at or just above the extrusion temperature, which upon cooling will form a homogenous solid block that will gradually and evenly set free its actives over time, typically during several weeks for 50 to several hundreds of flushes, more typically up to 500-800 flushes. Such formulas contain 25-50% LAS (typically 40%), 0-8% fatty alcohol sulfate (mainly C12-14, some C16 in cistern blocks) or 0-5% highly ethoxylated fatty alcohol (e.g. C16-18 with up to 50 mol ethylene oxide), 0-3.5% Coconut monoethanolamide, 1% foam enhancing fatty alcohol ether sulfate, 0.05% paraffin oil, 5-6% fragrance and dyes and sodium sulfate as a filler. Low amounts of acids (e.g. 2% lactic acid or 10-20% citric acid anhydrate) have been incorporated as well as polymers. US2007191245A1 for example describes the use in toilet blocks of polysuccinimide for preventing or dispersing urine scale.
Effervescent toilet descaling tablets form an alternative approach for descaling, targeting fast tablet disintegration (as opposed to toilet blocks) but long contact times (e.g. overnight). They are produced by tabletting and always contain an acid (usually sulfamic or citric acid) for dissolution of the immersed limescale and a carbonate source for the effervescent system (sodium carbonate, bicarbonate, percarbonate, . . . ). Formulating such tablets is all about finding the balance between fast dissolution on one hand and tablet strength and stability on the other. Low moisture content is of paramount importance, especially when the formula contains percarbonate bleach. A typical formula contains 1-2% lauryl sulfoacetate or FAS, 1% FAEO C16-18 8EO, 40-50% citric or sulfamic acid, 20-30% sodium carbonate, some polyethylene glycols, fragrance, dye, and sulfate as a filler. Some formulations additionally contain about 2% percarbonate bleach.
Products dedicated to periodical cleaning and descaling of automatic dishwashing machines usually are based on citric acid and a small amounts of FAEO (e.g. C9-11, 4EO), and additionally may contain some corrosion inhibitor, solvents, PEG, phosphonates, fragrance and dye.
There is a need for a renewable low moisture and stabile descaling agent that can be used in descaling block or tablet formulations and has a better efficiency than the existing descaling agents used in toilet blocks and tabs.
It is an object of the present invention to provide a new descaling agent which is made of renewable material and which shows a better overall efficiency than the descaling agents known from the prior art.
It is also an object of the present invention to find a toilet block comprising a descaling agent, which is made of renewable material, is low moisture and stabile with a better efficiency than the existing descaling agents used in toilet blocks. | tomekkorbak/pile-curse-small | USPTO Backgrounds |
Eating Real Food for a Healthy Lifestyle
Monthly Archives: November 2011
I was feeling a little guilty for revealing in yesterday’s post that I used to think that brussels sprouts were the work of the devil. To atone, I am dedicating this post to the little green fellas (and to Wade Jennings who claims that not even bacon can make brussels sprouts appealing). On their own they are still not my favorite veggie so a little something something is necessary to give them a boost. The special somethings in this case are sautéed mushrooms and shallots and a little bacon. Bacon makes everything better (this is a proven fact) and there are few things that I like better in this world than sautéed mushrooms. How can adding both be anything less than fab? Although I may never enthusiastically embrace brussels sprouts like a long-lost relative in a tear jerking Folgers Coffee commercial, it is good enough to make me want to give them a hearty whack on the back like an old college drinking pal I don’t mind seeing every now and then. Seriously, this is pretty darn good. For those that don’t care for the taste of sprouts, cutting them into smaller pieces lets more of the other flavors permeate and you get less brussel sproutiness. And yes, sproutiness is a word.
Cook bacon to the desired degree of doneness (I like mine crispy). When done, remove from pan and set aside to cool. When cool, break bacon into smaller pieces.
Remove all but 1 Tbsp of the bacon grease from the pan. Add 1 Tbsp of the butter, shallots, mushrooms, and garlic to pan and saute on medium high heat until shallots become translucent and mushrooms start to brown.
Mushrooms & Shallots
Ready for the sprouts!
Add remaining 1 Tbsp of butter and brussels sprouts.
w/ sprouts added
Cook, stirring occasionally, until sprouts are tender (about 8 to 10 minutes). When tender, add bacon, stir to mix, and heat for a few more minutes to spread flavor. Salt and pepper to taste & serve.
I find breakfast to be the hardest meal to eat paleo — or perhaps just the most boring. Somehow I always seem to fall back on eggs and/or bacon. While I love to eat me some bacon, a woman can not live on bacon and eggs alone. Consequently, I have been looking for a good recipe I could use for breakfast that incorporated more veggies and isn’t egg based.
The concept of vegetables for breakfast is relatively new for me. When I was a kid the only vegetables I liked were broccoli, corn, and the occasional iceberg lettuce salad. Beans and carrots were yucky, and brussel sprouts were surely the work of the devil. In hind sight, part of the lack of appeal may have been that the only vegetables that were ever prepared at home were dumped from a can or boiled/steamed to the point of mushiness. My grandma may have been an excellent cook, but veggies were boring afterthoughts.
Not too surprisingly many of my vegetable prejudices carried into adulthood. It’s really only been in the last 5 or 6 years, as I’ve tried to eat healthier, that I’ve begun exploring the wide world of produce. Some things I liked right away. Other tastes took a while to grow on me. Squash has been one of the latter for me. Everyone seemed to like acorn squash— except me. If anyone tries to convince you that spaghetti squash works well in place of spaghetti, they are lying to you.
Then one day, a light shone down from heaven, angels sang, and I discovered the Sweet Dumpling squash. It’s super sweet and creamy flesh made it the perfect “gateway” squash for me. Although I must confess, it’s really not such a good gateway since I still usually tend to grab a Sweet Dumpling rather than other varieties.
Sweet Dumpling Squash
Well, that was a bit of a long segue to get to today’s squash recipe. I found my current inspiration while flipping past the Food Network the other day. Guy Fieri, the spiky haired host of about 27 Food Network shows, was making a stuffed acorn squash that sounded totally yummy — and oddly breakfast appropriate. The squash, cabbage, onions, and bell peppers gave my breakfast a dose of veggies that I had been missing, and the turkey sausage gave it the necessary protein plus some zip and a little traditional breakfast appeal.
My Finished Squash
I substituted Sweet Dumplings for the acorn squash (of course). Your favorite squash would work also, however, if you’ve never tried these little gems, I strongly suggest you give them a go. This dish reheats well in the microwave, so, if you are like me, you could make a batch and enjoy them for several days. While I am making this as a breakfast entrée, you could clearly enjoy it for any other meal.
This dish takes a little bit of pre-planning because you need to make the sausage 8 to 24 hours ahead of time, but it’s worth it. The results were super! I’m not typically a fan of turkey used as anything other than plain old turkey, but this homemade sausage was totally tasty and is something I am going to incorporate in other recipes as well. For the time challenged, you could probably substitute your favorite pre-made paleo-friendly turkey, chicken, or pork sausage with good results.
One additional thought — Although I kept them in the recipe, the toasted squash seeds didn’t really add much for me (and tended to get stuck in my teeth) so I’ll probably omit them in the future unless I’m making them for guests and want them to look fancy-schmancy. They do taste good on their own though, so roast them up and enjoy! If you want a little extra crunch or nuttiness, some toasted pecans might be a good alternate. I plan to try that next time.
Below is my modified version of Guy Fieri’s recipe to make it more paleo friendly and incorporate a few personal changes. You can link to his original recipe above.
Ingredients:
For the turkey sausage:
2 tsp. thyme leaves (dry)
2 tsp. rubbed sage (dry)
2 tsp. fennel seeds (This was my addition. I couldn’t imagine sausage without fennel. It tastes fabulous, but you could omit it if you aren’t a fennel fan.)
2 tsp. sea salt , plus more to taste
1 tsp. freshly cracked black pepper, plus more to taste
1/4 tsp. cayenne pepper
1 lb. lean ground turkey
For the squash and vegetables:
3 squash, cut in half (I used sweet dumpling, but acorn or your favorite squash would work too)
To make the turkey sausage, combine the thyme, sage, fennel, salt, pepper, and cayenne in a medium bowl. Add the turkey and mix well. Cover and refrigerate for 8 to 24 hours.
To prepare the squash , preheat the oven to 375 degrees. Trim the ends off the squash so that it will sit flat. Scrape the seeds and membranes from inside the squash halves (if you go through the end, don’t worry). Set the seeds aside. Place the squash halves cut side up on a baking sheet, drizzle with 2 tbsp. olive oil, and sprinkle with 1 tsp. of the salt and the pepper. Next time I’m also going to grate a little fresh nutmeg on them too.
Separate the seeds from the membranes and rinse well. Dry the seeds with a paper towel. Place the seeds on a separate baking sheet or prepare a separate foil sheet for them to roast on.
Place the squash in the oven. Roast the squash for 30 to 35 minutes, or until golden around the edges and a knife can be inserted easily into the flesh. Remove from the oven but leave the squash on the baking sheet.
Roasted Squash Halves
Place the seeds in the oven. Stir the seeds every 5 minutes and check them for doneness after 10 to 20 minutes (mine were done in 10 minutes); you want them to be crisp and golden brown. Be warned that seeds started popping everywhere in my oven. Never having roasted seeds before, I’m not sure how to combat that problem. Watch for flying seeds when you open the oven. Always remember, safety glasses are your friend! Oven mitts and a large spatula can be used for defense in a pinch. Now where was I? Oh yes — remove the seeds from the oven and sprinkle them with the remaining 1/2 tsp. salt. Turn the oven off, but leave the door closed to keep the heat in — if necessary, we’ll slide the squash back in to re-warm it later.
Heat a large skillet over medium-high heat and add 1 tbsp. coconut oil. When hot, add the turkey, making sure to leave large chunks, about 1 inch across. Let the chunks brown, then turn and cook through, 8 to 10 minutes. Transfer to a plate and keep warm.
Cooked Turkey Sausage
(While everything is cooking in this next step, you may want to slip the pan of squash back into the still warm oven to warm them a smidge before stuffing. It’s not necessary to turn the oven back on if it’s still warm. Careful not to over cook the squash.)
In the same pan, melt the grass-fed butter over medium-high heat. Add the cabbage and cook until it starts to color and wilt, about 4 minutes. Add the peppers and onion and cook for 6 to 8 minutes, until the onions are soft and the peppers are still a little al dente. Add the turkey sausage and the garlic. Cook for 2 to 4 minutes more to blend the flavors. Adjust the seasoning with salt and pepper if necessary.
Sauteed Veggies & Sausage Ready to Fill the Squash
Divide the turkey mixture among the squash halves. Sprinkle with the roasted squash seeds, garnish with a bit of parsley, serve, and enjoy!
I’ve been wanting to try something in my “new” rotisserie, so I thought I’d start with an easy roasted chicken. I recently received this awesome vintage Sunbeam carousel rotisserie on Free-cycle. I love mid-century design in general, but this one looks like something out of Star Wars to boot. It’s like having my own personal kitchen droid! I have been affectionately calling it R2 (yes, I am a geek). Not only is he cute as a button, but R2 makes damn good chicken!
Vintage Sunbeam carousel rotisserie a.k.a R2 my kitchen droid
The drip pan doesn’t detach from this rotisserie and I was afraid it was going to be a pain in the butt to clean, but it wasn’t too bad. The drippings didn’t burn at all. They just poured right out and cleaned up nicely with a sponge. I saved the juices because I’m going to attempt to make Paleo Herb Gravy from the Nom Nom Paleo site using some of the drippings —- plus I paid a small fortune for an organic free-range chicken and I’m not about to waste any part of it! If you don’t have a rotisserie, check to see if your mom or grandma has one sitting around in the basement and give it a whirl. You might even get lucky and find a vintage R2 of your own! If you prefer, you could just roast it in the oven, but that’s not nearly as much fun.
Remove the giblet bag if there is one. If you like giblets, more power to you; enjoy them however you like. I can’t stand organ meat, but I save them to add to my next batch of chicken stock or to cook up for the dog. He thinks they are the bee’s knees.
Rinse chicken and pat dry, inside and out, with a paper towel. Set chicken aside on a plate.
In a small bowl combine the coconut oil and dry herbs. Moosh it together with the back of a spoon (or your fingers) to break apart the coconut oil and fully mix in the herbs. Rub the herb mix all over the chicken. Be sure to rub some under the skin on the breast as well. If you are using a bigger bird, you may need more herbs and oil.
Place the onion and garlic inside of the chicken and place chicken in your rotisserie or roasting pan and adjust roasting time and temp for your bird size and cooking method. R2 cooked my 4.25 pound chicken in about 1 hour. Based on Sunbeam’s cooking chart I set my timer for about 1 hour 10 minutes, but that was just a smidge too long. The chicken was still juicy but starting to dry a touch. One hour would have been perfect.
Overall, the flavor was fabulous and the chicken practically fell off the bones. The skin on the breast was nice and crispy — probably from the herb/oil mix I put under the skin. I definitely rate my first rotisserie chicken as a success! I’m sure this is just the beginning of a long, beautiful relationship between me and R2. I’d rather be starting a long, beautiful relationship with Harrison Ford, but that’s a story for a different blog.
My name is Trish and I’m a paleo lifestyle newbie. That sounds a little like I should be standing up in front of a support group or a 12 step program of some sort. Although, I suppose blogging is a little like creating your own support group —- so welcome aboard! Coffee and kale chips are in the back of the room, feel free to stand up and comment as the spirit moves you.
The goal of my blog is to document my experiences in paleo cooking and share my recipes and results with you. I love to cook and experiment with new recipes. Who knows where I’ll end up from day-to-day. You will see a variety of influences in my cooking. I mostly learned to cook by watching my grandmother. She was your traditional meat and potatoes sort of lady. Her kitchen yielded lots of hearty dishes like beef stew, pork and sauerkraut, stuffed cabbage, and chicken paprikas —- most of which lend themselves very well to paleo, so you will likely see me transform her recipes into total paleo versions. In recent years I’ve also become very interested in Indian and Middle-Eastern cooking. The aroma of the herbs and spice blends in Indian cooking are so complex and sexy I can’t resist! I have been trying to incorporate them into my more traditional cooking as well. Sometimes my fusion experiments go well. Other times… | tomekkorbak/pile-curse-small | Pile-CC |
leadpipe wrote:If Waiters has any chance in developing into the kind of player you find on winning team, well, throw that chance out the window if you bring Shabazz in there to do stupid shit along with him.
And yes, exactly.
The Waiters and Kyrie mixture is volatile enough, Shabazz is fucking napalm to it.
He plays hard, he'll D (he's going to end up only guarding two guards though, which is a problem with Waiters), but he doesn't pass, he isn't efficient and he isn't athletic enough to really be a force.
JJN wrote:2. There is no reason to believe that Shabazz will be anything more than a scorer.
OK. Sign me up. casue I seen he has mad skeelz to do that.
If all you want is a scorer, then you take McLemore. Shorter than Shabazz, but way more efficient and way more athletic. Additionally, he won't be the worst passing player to be taken in the first round since Michael Beasley (who averaged over 1 assist per game his freshman year, something Shabazz did not).
The Waiters and TT rumors are centered around real bona-fide NBA players and if you do trade Waiters for a LA or Love you draft Porter or Oladipo then, not the single most selfish player in college basketball.
There is a reason teams become the Sacramento Kings and teams like the Warriors make their leap with guys like Barnes and Thompson around Curry.
Olapido* & Otto are good. If the Cavaliers could get out from under Waiters and slip into some Pido, that'd be a nice swap, and I think he carries a little more upside than Otto, as much as Doris Burke & I love Otto. Shabazz is poop. McLemore is pretty much Harrison Barnes with a superior school choice. DX has Trey Burke as a Top 5 prospect? WTF? I think Cody Zeller has some untapped potential, especially considering Creen went all 3-jacky Matta/Oden on him (and without an injury excuse). Shabazz really is poop. I'd be surprised that Olynyk is so low if it weren't for the Bad Hair Zag burden he's carrying into the NBA, courtesy of Adam Morrison. But he's a 7-footer with a legit offensive game--how is that a mid 1st prospect? Plumlee (especially) and Dieng are stiffs. The Adams guy from Pitt is an interesting project. Nerlens Noel is, of course, the most irresistible combination of Name + Hair + BLOX that I can remember, so clearly you have to take him if you can.
A poisonous roster can ruin just about anyone in the NBA. Reke has actually gone backwards from his rookie year, badly and that roster/collection of idiots is a big part of it. Similar to what happened last year once Miles and Speights refused to try for Scott anymore and called him or for making them practice too much then the youth on the team rebelled as well, you need a healthy environment to grow a large collection of young players in this league.
e0y2e3 wrote:PS: Shabazz was not a hyper elite scorer this year and hasn't been "elite" since he was shitting on kids younger than him in HS
How do you single him out for that in HS?
I liked what i saw of bazz alot in the all star games, so I have bias. He looked pretty athletic to me. IDK what to think about 1 year in college ball, really. I like his 18 / 5 line alot, he was recognized with some nice post season honors, and all that is said about him is probably true. I just can't help but wonder if that's all he'll ever be.
A 19 year old lying about his age and beating up on 17/18 year olds matters far more than an 18 year old being viewed as having elite upside because he is 18. The age situation with Shabazz only matters to me in that it clouded his ceiling as viewed coming out of college. A lot of his hype was centered around "holy crap this kid is 18 and is killing every other 18 and 19 year old out there". Now he's played against kids his own age and hasn't shown nearly as well.
The age issue doesn't matter hugely to me, but from the perspective of it added a ton to his pre-college hype it needs to be considered.
HoodooMan wrote:Olapido* & Otto are good. If the Cavaliers could get out from under Waiters and slip into some Pido, that'd be a nice swap, and I think he carries a little more upside than Otto, as much as Doris Burke & I love Otto.
Porter has more upside than Oladipo. Otto is 2 years younger and never played AAU ball, which Victor has, meaning he hasn't access to higher level competition and coaching for as long (this is also the argument for the upside of McLemore). Additionally, Victor can't handle the ball. At all. Otto isn't going to be torching fools, but he isn't a turnover waiting to happen on anything other than a straight drive.
HoodooMan wrote:McLemore is pretty much Harrison Barnes with a superior school choice.
McLemore has had basically one year (this year at Kansas) of good coaching outside his AAU play, where they played him at the 4. He is really efficient, much more than Barnes was (10% better true shooting and 0.2 points per play higher), and he can actually translate his crazy athleticism into plays, something Barnes can only do on defense. McLemore has a long way to go, but he is one of the few players with star potential in this draft.
HoodooMan wrote:DX has Trey Burke as a Top 5 prospect? WTF?
Burke's stock had been increasing all year, he has really improved his performance since last season, and then he went crazy in the tourney. This propelled him into the lottery, probably in the 8-13 range. Then Marcus Smart went back to school, pushing all point guards up a slot. Smart could have contended for the no. 1 pock depending on who landed it. Burke isn't an option there, but teams need PGs. He isn't Kyrie Irving, but he could be Ty Lawson or Kemba Walker. I think he will have a solid, if unspectacular career, probably a Mo Williams who doesn't wilt in the moment.
HoodooMan wrote:I'd be surprised that Olynyk is so low if it weren't for the Bad Hair Zag burden he's carrying into the NBA, courtesy of Adam Morrison. But he's a 7-footer with a legit offensive game--how is that a mid 1st prospect?
Not athletic, not a great rebounder, not a great defender. He might be very good offensively, but he will probably get destroyed on the other end.
HoodooMan wrote:Plumlee (especially) and Dieng are stiffs. The Adams guy from Pitt is an interesting project. Nerlens Noel is, of course, the most irresistible combination of Name + Hair + BLOX that I can remember, so clearly you have to take him if you can.
The last two years have gone a long way towards proving my thesis that defense prowess resides in awesome hair. I've been gathering experimental evidence by growing a unibrow and flat top myself, then going to the Y and crushing the shot of anyone who comes near the rim. I'm starting to grow a thick mustache and soul patch a la Artis Gilmore, and have noticed that the more it grows, the more my face smooshes and shifts towards resembling Dikembe Mutombo.
<-- not going to get into a pedo v. Potter upside debate, as tomayto tomahto.
<-- also not going to get into a debate defending McLemore v. Barnes, because "can't beat anyone off the dribble" is can't beat anyone off the dribble. Barnes w/ a good PG was also way more efficient than Barnes w/out a good PG and really, really, really impacted his numbers.
In a dream world the Cavs get #1 and land Nole and then draft one of the huge upside foreign SFs at 19 and they live up to their potential.
I don't see any feasible way these two things happen, but that honestly is the best way to turn the team around.
In the real world you probably are ending up with Otto and then either a high upside Euro to stash at 19 (not a terrible idea since having too many rookies and second year players makes team uncoachable) or a high upside center to D-League.
BTW: I'd really like to see them stash a few foreign guys this year and bring no more than two rookies too camp. If they want to unload those 2nd rounders to move up again I'd be fine w/ that but stashing picks is probably the best course of action. Power in numbers and stuff.
We could always trade up as well, given that we have 2 early 2nd round picks. I kind of hope that we actually keep our second rounders and take some more seasoned role players or a role player and a high upside guy, rather than moving them for more 2nd rounders down the road. Speights, Walton, Livingston, Ellington, Gibson, and Casspi could all be gone. Bringing in some vet FAs and taking someone like Nate Wolters to replace Gibson or Mike Muscala to replace Speights provides you with a kid who has gone all the way through college and been a leader on a team already, despite it being at a lower level. You can also run them through your training camp to introduce them to how you want to play, but let them season in the D league.
I also like Glen Rice out of the D league, I think he could be that JR Smith type of guy that comes and just gets really hot from everywhere. Athletic and a great shooter, and seems to have turned his life around. He also already has a year of pro experience, and has bounced back from a bunch of (self caused) adversity. I would definitely interview the shit out of him before taking him though.
Could do worse with a high 2nd round pick. Seems to be more efficient than Smith. 3rd in WS/48, 4th in Off Rat, 5th in True Shooting and eFG%, T-3rd in PPP. 18/8/2.5 as a starter and 25/9.5/4.2/2/2 in the D league playoffs. That can't be worse than the shit salad we have been throwing out there the last three years.
Oh I didn't mean anything at all about Rice, I meant (seriously) that 2013 being the year JR Smith became a huge asset might be the most unbelievable thing in the history of the NBA. Even more so than TT learning to dunk at age 20.
JJN wrote:Porter has more upside than Oladipo. Otto is 2 years younger and never played AAU ball, which Victor has, meaning he hasn't access to higher level competition and coaching for as long (this is also the argument for the upside of McLemore). Additionally, Victor can't handle the ball. At all. Otto isn't going to be torching fools, but he isn't a turnover waiting to happen on anything other than a straight drive.
I can't really argue this too hard, because I really like them both a lot. Otto's roll changed a lot from his freshman year to this past, but during his first year at Gtown, his BBIQ was Kyrie-level off the charts for a CBBer. Then he comes in this year with a much improved outside shot on top of all that...I really like Otto a lot. But there's an awful lot to like about Olapido*, too, and the primary reason I give him the edge in upside over Otto is athleticism. I don't think either of them are likely to be stars, but I think Olapido* has a little more flash. Eh.
JJN wrote:McLemore has had basically one year (this year at Kansas) of good coaching outside his AAU play, where they played him at the 4. He is really efficient, much more than Barnes was (10% better true shooting and 0.2 points per play higher), and he can actually translate his crazy athleticism into plays, something Barnes can only do on defense. McLemore has a long way to go, but he is one of the few players with star potential in this draft.
It doesn't surprise me that he's statistically ahead of HB, since he didn't have HB's early struggles in college. But I just think they're strikingly similar prospects. Both kind of look like NBA athletes, but it often feels like a tease, because it seems you rarely see it. Nice outside shots (BM's a little > HB's), neither of them very strong off the dribble, both nice defenders, both frustratingly passive on offense. And because it seems the ideal program to enter as That Player is Kansas, since as a 2/3 at KU all you're really being asked to do is 1) play defense, 2) make the right pass, and 3) make open 3s--this system fit feels to me like the greatest difference between the two as prospects. But, then, who can blame HB for not wanting to go to Kansas, when he grew up in fucking Iowa.
JJN wrote:Burke's stock had been increasing all year, he has really improved his performance since last season, and then he went crazy in the tourney. This propelled him into the lottery, probably in the 8-13 range. Then Marcus Smart went back to school, pushing all point guards up a slot. Smart could have contended for the no. 1 pock depending on who landed it. Burke isn't an option there, but teams need PGs. He isn't Kyrie Irving, but he could be Ty Lawson or Kemba Walker. I think he will have a solid, if unspectacular career, probably a Mo Williams who doesn't wilt in the moment.
I get all that about the tourney helping his stock and whatnot, but Burke just screams College PG Star/NBA Nobody to me. I think he was a talented player, but often not a very good PG. Even when he was being shut down by the likes of Pido, he looked for his own shot far more often than he bothered to get his (rather talented) teammates involved--and drive & kick seemed to be his only understanding of how to ever accomplish that (part system, but part--WTF?). And who else is to blame for that offense looking as rudderless & ugly as it often did this past year, despite all that talent?
JJN wrote:The last two years have gone a long way towards proving my thesis that defense prowess resides in awesome hair. I've been gathering experimental evidence by growing a unibrow and flat top myself, then going to the Y and crushing the shot of anyone who comes near the rim. I'm starting to grow a thick mustache and soul patch a la Artis Gilmore, and have noticed that the more it grows, the more my face smooshes and shifts towards resembling Dikembe Mutombo.
e0y2e3 wrote:Oh I didn't mean anything at all about Rice, I meant (seriously) that 2013 being the year JR Smith became a huge asset might be the most unbelievable thing in the history of the NBA. Even more so than TT learning to dunk at age 20.
Gotcha. There is one thing that could make this year even stranger, which is ZBo leading a team to a championship. Griz probably have a good a shot at beating Miami as anyone, especially with a destroyed Chicago showing how much physical play takes Miami out of their game.
HoodooMan wrote:
JJN wrote:Porter has more upside than Oladipo. Otto is 2 years younger and never played AAU ball, which Victor has, meaning he hasn't access to higher level competition and coaching for as long (this is also the argument for the upside of McLemore). Additionally, Victor can't handle the ball. At all. Otto isn't going to be torching fools, but he isn't a turnover waiting to happen on anything other than a straight drive.
I can't really argue this too hard, because I really like them both a lot. Otto's roll changed a lot from his freshman year to this past, but during his first year at Gtown, his BBIQ was Kyrie-level off the charts for a CBBer. Then he comes in this year with a much improved outside shot on top of all that...I really like Otto a lot. But there's an awful lot to like about Olapido*, too, and the primary reason I give him the edge in upside over Otto is athleticism. I don't think either of them are likely to be stars, but I think Olapido* has a little more flash. Eh.
I really like Oladipo. He is a tremendously hard worker and is likely to improve his game, and I would really have no issue with him being the pick. The thing about Oladipo is that he is already physically much closer to his peak than Porter is. Porter's athleticism should benefit from the NBA strength training he will receive. He will never be as athletic as Oladipo, but he should be athletic enough. (I would also note that I am worried that Oladipo's shooting was a fluke, as he took under 2 3pt shots per game, and previously only hit 30% and 20% from deep. Porter shot nearly the same percentage on one and a half more 3's per game this season.)
What worries me about Oladipo is his upside specifically for this team. 6'5 is just really short for a SF, which is where he would need most of his minutes unless we trade Waiters (and this creates a mess of other issues with who will create other than Kyrie). Yes, he is really athletic and long, but he is short for the position. Even though Iggy is listed at 6'6", his in shoes measurement was 6'6.75".
When Hardwood Paroxysm (links below) looked at what combine measurements are most indicative of success in the NBA, they found that height only seemed to be really important at the SF position, and that athleticism at the 3 is a nice add on, but not indicative of success. Athleticism is important for SGs in terms of success though.
Of course this doesn't mean that Oladipo or Porter will or won't be successful just because they measure a certain way. What this does say to me though is that if you have two players who you see as having the same value, you should be pick the taller, more skilled player if you are drafting to fill your SF spot, and take the more athletic player if you are looking to fill the 2-guard. Porter is definitely taller, and I think there is little argument that he is more offensively skilled than Oladipo is.
JJN wrote:What worries me about Oladipo is his upside specifically for this team. 6'5 is just really short for a SF, which is where he would need most of his minutes unless we trade Waiters (and this creates a mess of other issues with who will create other than Kyrie). Yes, he is really athletic and long, but he is short for the position. Even though Iggy is listed at 6'6", his in shoes measurement was 6'6.75".
I agree that Pido is a SG. I'm not sure I agree that Dion Waiters' presence on the roster should impact the Cavaliers' decision, but I assume they do and it will.
Mostly, I just hope it's one of Noel/Pido/Porter, and not more out-of-left-field BS.
I agree that if they see a SG as the best prospect that they should select him, but with the dearth of talent on this team, two players who play the same position seems like a bit of a luxury that we can't afford. If Pido is their guy, I won't be upset that they took him, I just don't think there is a meaningful difference between Porter and Pido in terms of production.
I also doubt that they are going to change Waiters's role for anyone in this draft. They liked him enough to take him over a lot of players who were considered better picks last year, and he rated really highly in college based on the analytics magic. So yes, he might ultimately end up coming off the bench, but I think that they are going to give him quite a bit of run as a starter before deciding to make changes.
One name that I have seen popping up a bit the last few days for our #19 pick has been Sergey Karasev out of Russia. Already a 3 year pro at 19, and has been playing really well against high level competition. Not likely to slow down anyone, and couldn't leap a rock in a single bound, but this kid can shoot (49% on 4.6 threes per game).
Maybe I'm confused here. It sounds to me that you guys think Pido and Porter are the same player or same position. Pido is a 6'5 shooting guard who shot 44% on 3s this year, but 20% and 30% the previous two years. Pido shot 75% at the FT line. Waiters shot 35% on 3s and 81% on FTs during his 2 years at Cuse. Pido certainly improved his game this past year and shot up draft boards. And he is more athletic than Waiters and more of an impact player. But aren't their games similar in style? I mean, if I have Waiters, and I want to upgrade the 2-gd spot, I want him to do something differently than Waiters can... namely shoot. In that regard, isnt McLemore the better upgrade? McLemore shot 42% on 3s (but is considered a better shooter) and 87% on FTs
Porter is 6'8 with a wingspan > 7'0. He shot 42% on 3s and 78% on FTs.
If we think Pido is a SF then this is a no brainer, you draft Porter. If Pido is a 2Gd, you'd have to consider McLemore first. If we're not sure what position Pido is, that concerns me as well.
Perhaps we aren't including McLemore in the conversation because we thing he will be gone. But if its between Porter and Pido, I am all in on Porter.
"The nose of the bulldog has been slanted backwards so that he can breathe without letting go." -- Winston Churchill
This teams needs, regardless of position are aggressive interior defender that can score a little and perimeter defender that can hit his open threes. I don't care if those players at C/PF or SF/SG.
And if Oladipo really has a 6'11 reach that makes things interesting.
And being a first guard off the bench in the NBA is no longer a bad thing, you've had guys like Jason Terry, Jamal Crawford and now JR Smith make very good careers out of it that have also included a lot of notoriety. And I'm not even touching on Manu and Harden being sixth men....
OldDawg wrote:Maybe I'm confused here. It sounds to me that you guys think Pido and Porter are the same player or same position.
We are basically arguing if it Pido can play SF most of the time, and whether or not we should take him over Porter.
e0y2e3 wrote:This teams needs, regardless of position are aggressive interior defender that can score a little and perimeter defender that can hit his open threes. I don't care if those players at C/PF or SF/SG.
I don't disagree.
e0y2e3 wrote:And being a first guard off the bench in the NBA is no longer a bad thing, you've had guys like Jason Terry, Jamal Crawford and now JR Smith make very good careers out of it that have also included a lot of notoriety. And I'm not even touching on Manu and Harden being sixth men....
I don't disagree here either, but most of those teams already had an established starting lineup. Terry didn't start coming off the bench until Kidd was in Dallas, Crawford was a starter nearly every season until he got to Atlanta, and the Thunder and Spurs each had two stars in their starting lineups. I don't really mind the idea of Waiters coming off the bench, but I think we have too many holes in the starting lineup to worry about moving him around yet.
You don't win in the NBA without 7-8 high quality players. Right now unless that player is a PG you still take the best player available the fits your needs, regardless of position.
The Cavs desperately need defense and threes. Like that defense is abysmal and CJ Miles being your best deep threat is a laughable joke.
Having two SGs and still having two major holes on the roster is no worse than having a SF and SG with no back-up SG (especially with how ball dominant Waiters is and the already obvious chemistry issues between him and Kyrie).
e0y2e3 wrote:This teams needs, regardless of position are aggressive interior defender that can score a little and perimeter defender that can hit his open threes. I don't care if those players at C/PF or SF/SG.
e0y2e3 wrote:You don't win in the NBA without 7-8 high quality players. Right now unless that player is a PG you still take the best player available the fits your needs, regardless of position.
The Cavs desperately need defense and threes. Like that defense is abysmal and CJ Miles being your best deep threat is a laughable joke.
Having two SGs and still having two major holes on the roster is no worse than having a SF and SG with no back-up SG (especially with how ball dominant Waiters is and the already obvious chemistry issues between him and Kyrie).
And who is the best player available?Most say Noel and McLemore are the top two. Porter, Pido, Bennett and a couple others seem to share time at the 3rd pick spot. If we are picking, and the guys we are choosing from are basically even, then you go on greatest need.
I don't hear any buzz about McLemore on here. Do we all think Pido is better? Or do we all think McLemore will be gone?
"The nose of the bulldog has been slanted backwards so that he can breathe without letting go." -- Winston Churchill
e0y2e3 wrote:This teams needs, regardless of position are aggressive interior defender that can score a little and perimeter defender that can hit his open threes. I don't care if those players at C/PF or SF/SG.
e0y2e3 wrote:I don't think I've seen a single person, nor do I think McLemore has the upside (on D and 3) as Pido and Otto.
Not sure where you are getting that, but it just isn't true.
I am going by Mocks I've looked at. Most have Noel and McLemore 1-2 in the draft, with a couple even having McLemore #1. I know we don't all agree with mocks, but there is pretty decent consensus on the top two spots.
I don't know if his skill set is what you say fits the Cavs needs, I am just saying that according to most mocks, he's rated ahead of everyone but Noel.
I'll be honest, the only guy of the group I've seen a ton of is Pido, since I watched a bunch of Big 10 games this year.
A quick google sampling of the top 3 picks according to some NBA mocks:
Again, as a D and 3 guy is the key point here. That is what the Cavs desperately need. McLemore is a solid starting 2G prospect that will have too much overlap with Waiters. Taking guys that fill needs while not worrying about SF/SG is the key to all of this. Well that and Pido being long enough to actually be a shut down defender on a team's best scorer, not just a SG.
e0y2e3 wrote:Again, as a D and 3 guy is the key point here. That is what the Cavs desperately need. McLemore is a solid starting 2G prospect that will have too much overlap with Waiters. Taking guys that fill needs while not worrying about SF/SG is the key to all of this. Well that and Pido being long enough to actually be a shut down defender on a team's best scorer, not just a SG.
I don't know as much about some of these prospects as some of you guys do. My impression is that Pido's offensive game has too much overlap with Waiters' game. My impression is that both of their games is more bent towards scoring off the bounce. Pido took less that 2 threes per game. He did shoot 44% on those threes, but as a sophomore he shot 21% on threes. His athleticism lends itself to solid perimeter D, but I worry about him as a SF.
I am only stating these stats because I don't see Pido being a sure-thing upgrade as on offensive three-point shooter. McLemore's 3-pt production is clearly better. However, if you are prioritizing defense over three-point shooting (and I don't disagree), then the discussion changes.
What I really like about Pido is that he really seems to be a competitor. The Cavs need that. When you watch the NBA playoffs, teams that do well, have a couple of tough SOBs that know how to compete. McLemore's performance in the NCAA tourney scares me about his competitiveness. I haven't seen enough of Porter to make an evaluation on that. If we take Pido, I agree with you that we bring Waiters off the bench.
I said earlier, that if we were to take a guard, I was pulling for Smart, as he can play both the PG and SG spots and give us depth there, considering the fragility of Kyrie. But shockingly, he's staying in college.
Personally I would still be surprised if the Cavs take a guard with their 1st pick in the draft.
Last edited by OldDawg on Mon May 13, 2013 9:58 pm, edited 1 time in total.
"The nose of the bulldog has been slanted backwards so that he can breathe without letting go." -- Winston Churchill
Pido is nothing like Waiters who plays more like a combo guard/Wade slasher than anything. Pido scores opportunistically.
Also Waiters is the definition of inefficient chucker and won't be anything as a basketball player until he stops.
And again, you only draft a guard if you think he can guard the teams #1 scorer, be it a SF or SG. They need a lock down defender that can shut down the other teams top wing player, I don't give two shits what position said player plays.
A lot is going to come down to Pido's combine measurements.
Unless you guys aren't paying any sort of attention, BTW, the NBA is trending incredibly small. Everyone but Memphis that is. Even the Spurs run small a good bit now.
If Ds & 3s are really your only priorities, then McLemore is a solid option. Probably better than Porter, I guess. And one's preference between him & Oladipo--again, if your only priorities are Ds & 3s--is likely only decided by which of D or 3 you want a little more. (Hopefully, Mike Brown's assumed preference would win out here.)
Again, the problem with McLemore is he can only ever guard SGs. As I've said before, I don't care which position Pido plays, but I really, really care how long is he is that he can guard the teams best player.
And I don't see elite lock-down defender form McLemore and with how incredibly (like worst ever) bad the Cavs D was last year the D matters more than the 3 right now.
It never occurred to me that Oladipo would/could play SF in the NBA, but if the league's going as small as you say, OK, I guess.
I don't think McLemore has the mentality necessary for elite lockdown defender, but he's athletic/solid enough there, and he has a really pretty shot. So if you tip the scales to 3 over D...
I'd say Oladipo is the best defender of the three by a wider margin than McLemore is the best long range shooter, but again, if those are really your only priorities, these are the two you're looking at, I'd guess. Which would be a shame, I think, because there's so much to love about Otto Porter, too. | tomekkorbak/pile-curse-small | Pile-CC |
Guys... It's not gonna happen, idk why people think that putting a petition on change.org will change things.
Probably true. But anyone can express its wishes about it. No matter what's the result.
In the other hand, not even express it is absolutely wrong. And leaves no chance for others know. Leaves no possibility at all. So that's why I express my opinion about it and thanks to @deutch1976 for this post.
If I am to sign up to something, giving away my details and exposing myself to a potential spam attack or worse, at least I want it to be not for a complete naught. This is an utterly pointless gesture.
__________________In particle accelerators atoms are indeed not only touching each others. But banging together in a massive explosive orgasm.
-- nieldk in a TMO post
If I am to sign up to something, giving away my details and exposing myself to a potential spam attack or worse, at least I want it to be not for a complete naught. This is an utterly pointless gesture.
For such things i have a fake identity ;-)
Its not complete pointless. If there are a lot of signatures - there is a chance, that it will be used by some Manager to show it to his CEO, that here is still a lot interest. | tomekkorbak/pile-curse-small | Pile-CC |
Heller v. New York
Heller v. New York, 413 U.S. 483 (1973), was a United States Supreme Court decision which upheld that states could make laws limiting the distribution of obscene material, provided that these laws were consistent with the Miller test for obscene material established by the Supreme Court in Miller v. California, 413 U.S. 15 (1973). Heller was initially convicted for showing a sexually explicit film in the movie theater which he owned, under New York Penal Law § 235.0 which stated that and individual “is guilty of obscenity when, knowing its content and character, he 1. Promotes, or possesses with intent to promote, any obscene material; or 2. Produces, presents or directs an obscene performance or participates in a portion thereof which is obscene or which contributes to its obscenity."
Heller appealed this ruling to the supreme court, claiming that his first amendment rights had been violated due to the broad nature of New York’s obscenity laws. The defendant also claimed that his 14th amendment rights had been violated due to the fact the film was seized prior to him receiving a hearing of any kind. The Supreme Court ruled in favor of Heller in a 5-4 decision, with the majority decision delivered by Justice Burger. The Court found the procedure by which the film was seized to be constitutional, but ruled in favor of Heller in order to afford New York a chance to bring their obscenity laws in line with the guidelines established by the Supreme Court in Miller v. California. The Dissenting opinions were written by Justice Douglas as well as Justice Brennan, the latter of which was joined by Justice Stewart and Justice Marshall. These dissenting opinions argued that the obscenity laws that Heller was convicted under were themselves unconstitutional and thus the seizure of the film was unconstitutional. This case was one of several cases that the Burger court ruled on concerning obscenity laws in the early 1970s.
Background
Heller was the manager of the New Andy Warhol Garrick movie theater in Greenwich Village, New York city. On July 29, 1969, the Andy Warhol film Blue Movie was displayed, a film which depicts a couple engaged in sexually explicit acts and discussing various subjects ranging from athletes foot the Vietnam War. Three police officers saw part of this screening of the film and based on their observations an assistant district attorney requested that a judge of the New York criminal court see the film. On July 31, 1969 a judge accompanied by a police inspector saw the film and deemed that the film was “obscene as I saw it then under the definition of obscene, that is [in] . . . section 235.00 of the Penal Law.". The judge thus issued John Doe warrants for the arrest of the not only the theater manager, but also the projectionist and ticket tacker. The judge and police inspector both stated that they did not recall seeing any signs that limited admission to film to only adults. The theater manager as well as the ticket taker and projectionists were arrested and the film seized. There was no pretrial claim that the copy of the film seized as evidence was the only copy.
The case came to trial in the New York City Criminal Court on September 16, 1969, before 3 judges. Heller provided several “experts” to testify that the film “had social, literary, and artistic importance” and also testified that in addition to signs restricting the admittance of anyone under 17 the staff had been instructed to not allow in those under 18. Heller argued that the seizure of the film violated his 14th amendment right and that the New York state obscenity laws under which the film was seized violated the first amendment because they were vague and more broad than necessary. Heller also argued that films shown exclusively to consenting adults in private are free speech protected by the constitution.
The defendant was found guilty by the New York City Criminal Court, a decision that was upheld on appeal by both the Supreme Court of the State of New York and the New York Court of Appeals. The Court of appeals cited the Supreme Court’s ruling in Lee Art Theatre v. Virginia its decision, which stated that a judge could not issue a warrant for the seizure of a film based only the word of an officer. The Court of appeals felt that because the judge had in fact gone to see the film himself he had sufficient grounds to constitutionally issue a warrant for seizure of the film even without a formal hearing.
Majority Opinion
The Majority opinion was delivered by Justice Berger and joined by justices White, Powell, Blackmun and Rehnquist. It held that the procedure by which the film was seized and the seizing of the film were constitutional and violated neither the first or fourteenth amendment. Burger held that Heller’s assertion that films show only to consenting adults in private was protected was incorrect as the court ruled Paris Adult Theatre I v. Slaton 413 U.S. 49 (1973). that the privacy granted in the home does not necessarily extend to the theatre and that even consenting adults can be subject to governmental restrictions for the “legitimate state interests at stake in stemming the tide of commercialized obscenity".
Furthermore, the New York Court of Appeals applied correctly the precedent set in Lee Art Theatre v. Virginia, which ruled that as long as the judge’s procedure prior to issuing the warrant was "designed to focus searchingly on the question of obscenity”, the judge did not have to even see the film himself. By doing so the Judge had made every effort to determine the question of obscenity for himself. Also the protection of seizure set forth in Quantity of Books v. Kansas 378 U.S. 205 (1964), only applied to obscene materials being seized for the expressed purpose of being destroyed.
As long as there was another copy of the film was available, the seizure for the purpose of evidence was okay, and if there was no other copy as long as the defendant could be allowed to make another pending a judicial decision it would still be constitutional. Since the record failed to show there were no other copies and there was no pretrial motion to make more copies the State was allowed to seize the film. However, the case was remanded in order to afford the New York Court of Appeals to make changes to the underlying definition of obscenity according to the guidelines laid out by the court in the recently decided Miller v. California.
Dissenting Opinion
There were 2 dissenting opinions in this case. Justice Douglas dissented on the grounds that he would have preferred to outright reverse the decision as he felt as though the underlying obscenity law for which Heller was charged was unconstitutional and violated the first amendment. Justice Douglas referenced his dissenting opinion in United States v. 12 200-ft. Reels of Film, in which he stated that “I know of no constitutional way by which a book, tract, paper, postcard, or film may be made contraband because of its contents”, as well as his dissenting opinion in Miller v. California in which he argued that the court should not try to define obscenity by itself as there are no constitutional guidelines for what should be considered obscene.
Justice Brennan dissented as well, joined by justices Stewart and Marshall, arguing that the obscenity law was too broad to be considered constitutional and thus there could be no grounds to seize the film. Justice Brennan also referenced his dissent in Paris Adult Theatre I v. Slaton in which he stated that the difficulty in precisely defining material as obscene is what makes it impossible to outright ban obscenity.
Subsequent Developments
During the time period between when the court agreed to hear Heller v. New York they ruled on Miller v. California, a ruling which established that states could make obscenity laws, provided they follow the guidelines of the Miller test. The Miller test contained 3 parts and became the standard for determining whether material was obscene. The 3 parts were:
Whether "the average person, applying contemporary community standards", would find that the work, taken as a whole, appeals to the prurient interest,
Whether the work depicts or describes, in a patently offensive way, sexual conduct or excretory functions specifically defined by applicable state law,
Whether the work, taken as a whole, lacks serious literary, artistic, political, or scientific value.
The Court also asked for the New York Court of Appeals to make sure that their definition of obscenity was concurrent with standard set in Miller v. California. The New York Court of Appeals later confirmed in a 5-4 decision that New York Penal Law § 235.0 was constitutional as it was already written, even after considering the new obsceneity definition put forth in Miller v. California.
Heller set the precedent that a judge who had seen a movie considered to be obscene could issue a warrant for its seizure without having to hold a hearing on whether or not the content was truly obscene, as long as there were other copies of the film were available, and in the case that no other copies existed, as long as the defendant was allowed to make copies prior to a formal hearing. This was an expansion on the precedent set in Lee Art Theatre v. Virginia that a judge must determine probable cause of something being obscene, by more explicitly saying that a judge having seen a film himself was sufficient grounds for the judge issuing a warrant to seize a film based on obscenity charges. This precedent was cited in New York v. P. J. Video, Inc., 475 U.S. 868 (1986), another obscenity case in which films were seized on charges of being obscene. In this case the owners of a video store tried to claim that there was a higher standard for determining probable cause in obscenity cases than an investigator simply watching the film. The supreme court ruled against PJ Video, citing there decision in Heller v. New York that an official watching the film themselves was sufficient probable cause for the seizure of a film.
This case is an interesting example of one section of free speech where American courts and European courts agree to an extent, as shown in the landmark case Handyside v United Kingdom (5493/72), where the seizure of materials deemed to be obscene was by the government was found to be permissible by the European Court of Human Rights, much like it was in this case and in New York v. Heller and New York v. P.j. Video. In Handyside v United Kingdom, Handyside had books seized for being obscene, not completely unlike Heller's film which was seized, although some key differences appear in the 2 cases, such as the intended audiences of each. However, the fact remains that in both these cases the government was able to seize material due to it being obscene, while both attempting to prevent, to an extent, the over labeling of materials as obscene.
See also
List of United States Supreme Court cases, volume 413
Miller test
References
External links
Category:1973 in United States case law
Category:United States obscenity case law
Category:United States Supreme Court cases
Category:United States Supreme Court cases of the Burger Court | tomekkorbak/pile-curse-small | Wikipedia (en) |
Q:
How to annotate the difference between blended vowels and non-blended vowels
Similar to this question about consonants, I'm wondering how you annotate with IPA (or any other system if IPA doesn't support it) the difference between blending vowels together (morphing between multiple vowels) and discretely shifting between them.
For example, the word "wow" is really like 3 different vowel sounds from what it seems. So I would maybe do /ʊɛʊ/. But this is blending the sounds together.
A different way to do it to demonstrate non-blending is to use glottal stops, /ʊʔɛʔʊ/, or "ʊ'ɛ'ʊ". But that is essentially putting a pause between the sounds which is the extreme.
In the middle, in between blending and putting pauses between the sounds, is just shifting from one to the next without blending. A way to replicate this is to pronounce each long, and transition to the next quickly, so "ʊʊʊʊʊʊʊɛɛɛɛɛɛɛɛɛʊʊʊʊʊʊʊʊ". If you do this quickly but not so quickly as to morph or blend them together, then it's not a glottal stop and not a blend, so it's a ________ I don't know, a discrete shift.
Wondering how to annotate this in IPA. It seems then there are 3 types:
blending
discretely shifting
glottal stops
Wondering how to write all 3. If there are more ways to do it than these 3 types, that would be good to know too!
A:
There are a couple of ways that the IPA can annotate such differences. The first is a tiebar: u͡a (unicode 0361) or u͜a (unicode 035C). Use of a tiebar is usually taken to indicate "these two vowels are part of a single phoneme" (i.e., they form a diphthong), which technically indicates something about their behavior in the phonological patterns of the language, but usually also means they are pronounced more "blended" (to use your term).
A way to indicate that the vowels are more "discrete" is to explicitly mark them as members of separate syllables, using a period (as in /a.u/, or if the second syllable is prominent, /aˈu/ or /aˌu/).
A third way one might (ab)use the IPA to denote a "blended" vs "discrete" vowel sequence would be to mark one of the vowels as an onglide or offglide, using /j/ (in place of /i/), /w/ (in place of /u/) or /ɰ/ (in place of /ɯ/). Personally I don't think that's a very good approach, for a couple of related reasons:
Technically those symbols represent consonants, not vowels, and so /aj/ ought to be considered a V-C sequence, which makes it no longer just a vowel.
In speech the glide-vowel or vowel-glide transition is often quite "blended" and may be indistinguishable from a diphthong solely on auditory or acoustic grounds. In other words, the question of whether English "pie" is transcribed as /pa͜i/ (two phonemes, CV) or /paj/ (three phonemes, CVC) depends more on larger-scale patterns of how the [aɪ] sound(s) behave in different contexts, rather than on how blended or discrete the sounds are (e.g., if English "pie" follows a phonological rule that is followed by other words that are unambiguously CVC and not by words that are unambiguously CV, you might argue on that basis that /paj/ is correct).
As a final aside: when vowels are adjacent in speech with continuous voicing, they will never be fully "discrete". There will always be a period of transition in the vowel quality, because when the jaw/tongue/lips move to make the second vowel gesture, the size and shape of the air mass in the oral cavity changes too, and that change is never instantaneous.
| tomekkorbak/pile-curse-small | StackExchange |
Abnormal puberty in paediatric Cushing's disease: relationship with adrenal androgen, sex hormone binding globulin and gonadotrophin concentrations.
Paediatric Cushing's disease is frequently associated with abnormal puberty. We addressed the hypothesis that prepubertal patients show excessive virilization and pubertal patients show suppression of LH and FSH secretion. Serum androstenedione (A4), dehydroepiandrosterone sulphate (DHEAS), testosterone (T), and sex hormone binding globulin (SHBG) were determined at diagnosis and converted to standard deviation scores. LH, FSH concentrations were also determined. Severity of CD was assessed from the sleeping midnight cortisol concentration. Puberty was staged and excessive virilization defined as advance in pubic hair stage for breast stage or testicular volume (TV). Twenty-seven CD patients (17 male, 10 female), median age 13.4 years (range 5.9-17.8) were studied. In the CD group as a whole, A4, DHEAS, T standard deviation scores (SDS) values were normal. SHBG SDS values (n = 19) were low (median -1.93, -4.32-0.86) correlating with BMI (r = -0.49). A4, DHEAS, T, SHBG, LH and FSH did not correlate with midnight cortisol, but A4 and T SDS correlated with ACTH at 09.00 h (both r = 0.51). Thirteen patients (11 male, 2 female) had excessive virilization with increased A4 (P = 0.033), DHEAS (P = 0.008), testosterone (P = 0.033) and decreased SHBG (P = 0.004) compared with subjects without excessive virilization. Pubertal boys (TV > or = 4 ml) (n = 7) and girls (breasts > or = stage 2) (n = 8) had low median LH and FSH. Boys had an LH concentration of 1.2 mU/l (0.3-3.5), FSH, 0.9 mU/l (0.2-6.4) and median T SDS, -1.95 (-3.8-4.65), while girls had an LH concentration of 1 mU/l (0.3-7.4). Many patients had abnormal puberty and excessive virilization associated with increased adrenal androgens and decreased SHBG. Pubertal patients had low LH and FSH suggesting impaired pituitary-gonadal axis function. | tomekkorbak/pile-curse-small | PubMed Abstracts |
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ETHERNET PORT CONFIGURATION: 26 Gigabit ports
FLEXIBILITY FROM UPLINK PORTS: 4 x 1G SFP ports (all 4 are shared)
WHISPER-QUIET DESIGN: Reduces fan noise to protect your home office or business environment.100Mbps backward compatiblity on all SFP ports | tomekkorbak/pile-curse-small | OpenWebText2 |
At least 31 killed in India's northeast, Bangladesh by heavy rains
GUWAHATI, India/DHAKA, Bangladesh (Reuters) - At least 17 people have been killed by heavy rains and flooding in the northeastern Indian state of Assam, while at least 14 people have died in Bangladesh, with millions of people also displaced, officials said on Saturday.
Persistent heavy rains this week have caused widespread disruption across South Asia, with at least 68 people killed in Nepal by flash floods and landslides.
Although rainfall had decreased as of Saturday, officials in Assam, a tea-growing and oil-rich state, added water levels across rivers were still overflowing.
"Overflowing rivers are flooding new areas, making things worse," said Keshab Mahanta, Assam's water resource minister. | tomekkorbak/pile-curse-small | Pile-CC |
Dungeons & Dragons will include a recently playtested druid subclass to its upcoming Guildmaster's Guide to Ravnica publication.
Lead rules designer Jeremy Crawford confirmed that an official version of the "Circle of Spores" druid would be available in D&D's new campaign setting book, which will explore one of the worlds of Magic: The Gathering.
The Circle of Spores was liked by enough people that it will appear in "Guildmaster's Guide to Ravnica" this November! #DnD #MagicTheGathering //t.co/HaBIDReQXZ — Jeremy Crawford (@JeremyECrawford) August 16, 2018
The subclass, which focuses on decay and the ability to grow life from death, was first introduced as playtest material back in January of this year. Druids who join the Circle of Spores gain a number of interesting necromantic spells, including animate dead and chill touch. They can also spend their reaction dealing slight bits of poison damage to those around them, which adds a bit of danger to anyone who comes near them.
Another core feature of Circle of Spores druids is the ability to buff their attacks and hit points using their "Symbiotic Entity" ability. A Circle of Spores druid can expend their Wild Shape abilities to infuse themselves with magical spores, giving them temporary hit points, and adding extra poison damage when making melee attacks or using their halo of spores ability.
At higher levels, circle of spores druids can also animate the dead using their fungal infestation, giving them temporary minions to command, and can resist different types of effects and become immune to critical hits as fungal spores gradually takes over their ability.
The Circle of Spores druids is one of our favorite recently playtested subclasses in Dungeons & Dragons and we're happy that it will be included in an official publication soon. Soon, players will be able to become fun guys (and gals) while exploring a fantasy world filled with danger and intrigue.
Guildmaster's Guide to Ravnica will be released on November 20, 2018.
Let us know what you think about the Circle of Spores druid in the comment section or by hitting me up at @CHofferCbus on Twitter! | tomekkorbak/pile-curse-small | OpenWebText2 |
Some Really Cool Food Apps to Download
Some Really Cool Food Apps to Download
From making reservations to getting nutrition advice, there's a food app for every person and diet
August 23rd, 2012by Daryl Nelson of ConsumerAffairs
in News
In the world of eating out, you have your casual diners, your every-now-and-then-food enthusiast, and your hardcore foodie types. But the one thing they all have in common is a way to find and keep up with the eateries that fit their taste and pocketbook.
Way back when - which was really just a few years ago - it was a bit harder to know where the best restaurants and eateries were. A person either had to count on word of mouth to find a good place, see some sort of advertisement or review in the newspaper, or run into that ambitious restaurant owner who talks you into entering his establishment when you walk by.
Today it's a lot easier to find out where the hot spots are through a bevy of new smartphone apps that do everything from allowing you to see restaurant deals in real time, to making reservations and inviting friends to come dine with you, like Foursquare for example.
Foursquare
The company, founded by two buddies and based in New York City, gives the user suggestions on where to eat based on their preferences and past restaurant choices.
For example, if you've been frequenting a lot of Chinese restaurants, and you saved each location in the app, it will tell you about that new Szechuan place that just opened up by your house.
Foursquare will also give you deals and discounts for all the different types of eateries you've been visiting, and it will help you coordinate a restaurant dinner party, or simply let your friends know where you're eating and invite them to join you for a bite.
And of course there's a review component to the app, so anybody can be a food critic with a built-in audience whenever they feel like it.
This past June, Foursquare relaunched the app after the company redesigned some of its features and highlighted its social media usage.Â
Although some of its initial user numbers have slightly decreased since the relaunch, it's still one of the more popular free food apps for Androids, iPhones and iPads.
Around Campus
The Around Campus app has a very specific area of usefulness, as it gathers all of the restaurant and bar deals from college campus planners and relevant Internet sources.
The app is ideal for the college kid with little to no money, or those people who happen to live in or near a college town.
The company says there are over 300 campuses that currently access the service, and the app almost serves as a personal deal expert that does all of the legwork for you by consolidating coupons and cheap places to eat.
The app also allows you to keep abreast of other businesses in the area that may be frequented and helpful to the average college student.
Around Campus is free to access and available on iOS and Androids, and with new restaurants popping up by the second, these types of apps can only help consumers decide what's best out of a sea of choices.
Big eaters
According to research conducted by the National Restaurant Association, the entire restaurant industry will rake in nearly $632 billion before the year is over, which shows people are still eating out in droves, despite the economy, and app makers can barely keep up with the growing consumer interest.
Many of the app makers are competing with each other and trying to separate themselves by creating a unique service niche that has yet to be thought of by other companies.
Thryve
Thryve is one of those apps that provide a niche service. Its creators made the app to serve as a nutritional coach to help you avoid foods that your body has a low tolerance for.
Users keep track of what they're eating and document how each food item made them feel.
Afterwards, the app suggests other foods to both eat and avoid, so if you that particular beverage or vegetable is making your stomach churn, the app will lead you to other options so you can hopefully enjoy your meal instead of feeling discomfort.
Another useful electronic assistannt that focuses on health is Nutritionix. It helps users eat a little better by providing useful menu information for popular eating locations. Although it's really a database and not an app, Nutritionix can still be accessed on your mobile device.
So if you would like to check the nutritional information of a particular restaurant you're going to, the app will individually breakdown each item, just in case you wanted to know what's really in that cheese dish at Red Lobster.
Mobilecious
Mobilecious is pretty cool too. For those who have immersed themselves in the food-truck craze, this nifty little app could turn into your best friend.
Simply use it to find the particular food truck you crave in real time, and you can also check out what's on each truck's menu. The app also lets you know all of the RV's deals and coupons that are being offered.
Moblilecious can be downloaded for free, and is only available for the iPhone and iPad.
If you're one for an upscale dining experience, and live in San Francisco, Los Angeles, or New York, Blackboard Eats may be just right for you.
The app uses staff members from the food magazine Gourmet to make suggestions, and provides deal offers, reviews and helps users find certain types of restaurants or cuisines. The app is free and can be accessed on both the Android and iOS.
All of these apps are great for foodies as well as the person who just loves to restaurant-hop. It doesn't matter if your taste-buds crave a laid back meal in a moderately priced restaurant, or a high-end dish that requires dressing up a little.
Either way, it's certainly easier than driving around aimlessly searching for the perfect place to eat. | tomekkorbak/pile-curse-small | Pile-CC |
Watch also in iWant or TFC.tv
Tens of thousands turned up in the streets of Hong Kong overnight to push for democracy. Ryan Chua reports among those who participated in the protests were Filipinos based in the territory.
ANC Alerts, September 30, 2014 | tomekkorbak/pile-curse-small | OpenWebText2 |
Issues that warrant investigation regarding infidelity
Perhaps you and your spouse got married in a Washington church or courthouse more than 10 or 20 years ago. Then again, maybe you’re among the newlyweds of the state. Either way, it’s not impossible that your relationship has withstood numerous challenges as it is common for most married couples to hit some rough spots in their journeys. However, if your relationship is teetering on the brink of divorce and you suspect infidelity is a causal factor, you’re definitely not alone in your struggle.
Signs often exist that a spouse is being unfaithful. Your marriage is unique, however, so even though you have issues in common with other couples, it doesn’t necessarily mean you have the same problems. If you have evidence that your spouse is cheating on you, it’s always a good idea to learn more about resources that are available in your area to provide support.
Do any of these issues sound familiar?
As mentioned earlier, hitting a rough spot in your relationship isn’t uncommon for married couples. If you have children, parenthood can bring added joy but also added stress to your marriage. However, if you experience one or more of the issues included in the following list, you may want to further investigate the situation:
Does your spouse seem especially secretive? If he or she tries to conceal cellphone conversations or computer use, it may be a sign of trouble.
If you’re used to your spouse arriving home at approximately the same time every day and his or her schedule has suddenly changed, you may want to inquire more about it.
Is money missing from your jointly owned bank account? This is often a sign that a spouse is cheating and using money from an account to fund an affair, such as paying for dates or buying gifts for a lover.
When you ask your spouse about time away, money or other issues, does he or she become defensive or combative? This is often a sign of infidelity.
Has your sex life changed? Whether you and your spouse are less intimate than you used to be or have experienced an increase in your private time together, if you’re struggling in your marriage, these issues could be a sign that your spouse is having an affair.
Does your partner blame you for every problem in your relationship? Spouses who cheat often feel guilty and then project those feelings onto their spouses because they want to blame someone else for their problems.
Marriage isn’t easy. Neither is divorce. The good news is that you don’t have to go through such times alone. There are counselors, ministers and experienced attorneys who can provide guidance and support as needed. You may also have friends or relatives who have gone through similar experiences. Discussing your concerns in a private setting may help you formulate a plan and can also help you learn more about how to protect your and your children’s interests if you decide to sever your marital ties. | tomekkorbak/pile-curse-small | Pile-CC |
This is why the Catholic church upholds this as an ideal. And it does so with great wisdom. But, as Ross concedes, the question is whether this ideal should rest on its own laurels or needs to be elevated by law and doctrine to the highest level of human relationship, and also, in order to achieve this ideal, actively exclude others - both in the religious and the secular sphere?
We know the answer in the religious sphere. The church - even in its current High Ratzinger phase - opts for inclusion over exclusion. It allows the infertile to marry. It does not remove the Sacrament of Matrimony from those who do not produce kids. It even annuls countless marriages, many of which have been consummated, in enormously large numbers. It marries those past child-bearing age. It treasures adopted kids, even though they violate Ross's parent-procreating "microcosm of civilization" ideal. And that's only the Catholic church. The Protestant churches freely allow divorce and contraception - breaking both the monogamy and the procreative elements of Ross's ideal (which is to say all of it). So in the religious sphere, the Church breaks its own ideal with regularity, and the other churches have long since given almost all of it up. And yet the Catholic church still insists that its ideal be enforced as an act of civil exclusion in the secular sphere, even on people who are atheists.
On what conceivable grounds, if you pardon the expression? Look at how diverse current civil marriages are in the US. The range and diversity runs from Amish families with dozens of kids to yuppie bi-coastal childless couples on career paths; there are open marriages and arranged marriages; there is Rick Santorum and Britney Spears - between all of whom the civil law makes no distinction. The experience of gay couples therefore falls easily within the actual living definition of civil marriage as it is today, and as it has been now for decades. To exclude gays and gays alone is therefore not the upholding of an ideal (Britney Spears and Larry King are fine - but a lesbian couple who have lived together for decades are verboten) so much as making a lone exception to inclusion on the grounds of sexual orientation. It is in effect to assert not the ideal of Catholic Matrimony, but the ideal of heterosexual superiority. It creates one class of people, regardless of their actions, and renders them superior to another.
Ross's view is increasingly, therefore, one faction of one religion's specific definition of Matrimony out of countless arrangements that are available for cohabitation in civil society and world history. It's a view freely breached within his own church itself. And it has already been abandoned as a civil matter in some of the most Catholic countries on earth, including Spain and Argentina. And heterosexuals-only marriage is only a microcosm of civilization if you exclude all other relationships from civilization - friendship, citizenship, family in the extended sense, families with adopted, non-biological children, etc. | tomekkorbak/pile-curse-small | OpenWebText2 |
Q:
How would you do "who's that Pokemon?" with AI?
How would you take an image like this
and determine that as "Pikachu"? I'm basically not sure what I should search to learn how to do something like this, or if there's an API or tools of some sort that already does it. I looked at options like https://www.clarifai.com/demo, however they don't have the "models" or "trained classifiers" for something specific to my needs, so I think I'd have to build it on my own, and am not sure where to begin.
Any guidance would be appreciated. A high-level overview would be fine.
A:
Personally, I would advise using machine learning for this project. In high concepts, what you would do is feed a python program thousands of pictures of different pokemon. The program will analyze them and find patterns and store those patterns.
You can then submit other pokemon images and the python script will return the likely-hood of what the pokemon is.
The library that you use is called tensorflow. Google has a great guide for image recognization use with tensorflow. There they explain all the code used to recognize images and all the steps to setup tensorflow. Here is the link.
The good thing with tensorflow is that creating different objects for different pokemon is not hard. All you need to do is create a folder and name it then name of the pokemon. Then just drag in pictures of the pokemon from multiple different angles. You will need LOTS of pictures for this to work and hours of free time. Tensorflow does take time to learn the patterns especially since you will have A LOT of pokemon, you will need a lot of images.
If you want to speed up the process, make sure to have a fast computer!
Have fun with tensorflow! and good luck on your project!
| tomekkorbak/pile-curse-small | StackExchange |
Q:
Trouble doing simple parse in pyparsing
I'm having some basic problem using pyparsing. Below is the test program and the output of the run.
aaron-mac:sql aaron$ more s.py
from pyparsing import *
n = Word(alphanums)
a = Group( n | Group( n + OneOrMore( Suppress(",") + n )))
p = Group( a + Suppress(".") )
print a.parseString("first")
print a.parseString("first,second")
print p.parseString("first.")
print p.parseString("first,second.")
aaron-mac:sql aaron$ python s.py
[['first']]
[['first']]
[[['first']]]
Traceback (most recent call last):
File "s.py", line 15, in <module>
print p.parseString("first,second.")
File "/Library/Python/2.6/site-packages/pyparsing.py", line 1032, in parseString
raise exc
pyparsing.ParseException: Expected "." (at char 5), (line:1, col:6)
aaron-mac:sql aaron$
How do I modify the grammar in the test program to parse a list of comma separated names terminated by a period? I looked in the docs and tried to find a live support list, but decided I was most likely to get a response here.
A:
The '|' operator creates a MatchFirst expression, in which the alternatives are evaluated until there is a first match.
Pyparsing works purely left-to-right, applying parser expressions to the input string as it can. The only lookahead that pyparsing does is whatever you write into the parser.
In this expression:
a = Group( n | Group( n + OneOrMore( Suppress(",") + n )))
Let's say n is just a literal "X". If this parser was given the input string "X", it would obviously match the leading, lone n expression. If given the string "X,X,X", it would still match just the leading n, because that is the first alternative in the parser.
If you turn the expression around to:
a = Group( Group( n + OneOrMore( Suppress(",") + n )) | n)
then to parse "X" it would first try to match the list, which will fail, and then match the lone n. To parse "X,X,X", the first alternative will be the list expression, which will match.
If you want the longest alternative to match, use the '^' operator, which gives an Or expression. Or will evaluate all the given alternatives, and then select the longest match.
a = Group( n ^ Group( n + OneOrMore( Suppress(",") + n )))
You can also simplify this using the pyparsing helper method delimitedList. Parsing lists of the same expression separated by commas is a common case, so rather than see people have to reinvent expr + ZeroOrMore(Suppress(",") + expr) over and over, I added delimitedList as a standard pyparsing helper. delimitedList("X") would match both "X" and "X,X,X".
| tomekkorbak/pile-curse-small | StackExchange |
Ira Trivedi is an author and journalist based in New Delhi. She is the author of Nikhil and Riya and India in Love: Marriage and Sexuality in 21st century India. The opinions expressed in this commentary are hers.
(CNN) On New Years Eve, women were allegedly molested en masse by groups of men on the streets of Bangalore, a South India city famous for its software and tech industries -- India's answer to Silicon Valley.
Claims of this "mass molestation" came almost exactly four years after the tragic gang-rape of a young woman on a moving bus in New Delhi brought the issue of sexual violence and women's safety to the fore in India, and around the world.
Bangalore is one of India's most cosmopolitan cities, known for its diversity, scientific attitude and technical prowess. Young people from across India flock there for jobs.
The tech industry in particular has been known to be woman-friendly, with 30 per cent of its work force made up of women. That this sort of incident allegedly took place in the heart of Bangalore -- where 1500 police men were deployed to control revelers -- is not just a clear failure of governance, but also an indication that patriarchy still triumphs in a country n which complex cultural changes are underway.
This generational clash of tradition versus modernity is perhaps best explained by some of the political responses to this horrific incident. G. Parameshwara, the Home Minister of Karnataka -- the state in which Bangalore is in -- blamed the problem on young people who "try to copy westerners, not only in their mind-set but also in their dressing." The result, he said, was "some disturbance, some girls are harassed, these kinds of things do happen."
Read More | tomekkorbak/pile-curse-small | OpenWebText2 |
All relevant data are within the manuscript and its Supporting Information files.
Introduction {#sec001}
============
Lentil (*Lens culinaris* Medik.) is one of the earliest domesticated crops and is sometimes referred to as one of the ancient grains in modern times \[[@pone.0223419.ref001]\]. This high-value crop is an excellent source of high quality plant protein for human nutrition. Average annual global production of lentil was reported to be 5.07 million metric tonnes from 2010 to 2017. Production is concentrated in Canada, India, Turkey, United States of America and Australia, which contributed to a combined total of more than 75% of the world's lentil production \[[@pone.0223419.ref002]\]. The Australian lentil industry produces an average of 292,000 tonnes annually and production is centred in the Southern temperate cropping zones of South Australia and Victoria \[[@pone.0223419.ref003]\]. Lentil ascochyta blight (AB) is a major threat to lentil production worldwide and is caused by the necrotrophic ascomycete fungal pathogen *Ascochyta lentis*, a member of the *Didymellaceae* \[[@pone.0223419.ref004]\]. *A*. *lentis* produces necrotic lesions on the above ground parts of the plant resulting in stem damage, loss of leaves and pod discoloration \[[@pone.0223419.ref005]\]. In severely infected crops, necrotic lesions on leaves and pods lead to reduced yield and poor seed quality \[[@pone.0223419.ref006]\]. Pathogen populations persist in lentil cropping areas by growth on plant residues and stubble at the start of the next growing season and adaptation and distribution of *A*. *lentis* are facilitated through sexual recombination between compatible mating types and the production of windborne ascospores. Average annual yield and grain quality losses in Australia amount to A\$0.9 M and control costs add a further A\$15.3 M penalty to the Australian lentil industry \[[@pone.0223419.ref007]\].
As is the case for ascochyta blight of other pulse crops, *A*. *lentis* is difficult to control. Management strategies include cultural practices, fungicide treatment of seed and by foliar application \[[@pone.0223419.ref008]\], and use of resistant cultivars \[[@pone.0223419.ref009]\]. Plant breeding for AB resistance has provided some degree of protection \[[@pone.0223419.ref009],[@pone.0223419.ref010]\], but the durability of plant resistance has often been challenged by the ability of pathogen populations to evolve new virulence forms or levels of aggressiveness \[[@pone.0223419.ref011]\]. Adaptation of pathogens at the population level often leads to novel pathotypes that can result in breakdown of resistance genes. Changes in aggressiveness and shifts in cultivar specificity or virulence are a constant challenge for growers and breeders \[[@pone.0223419.ref012]\]. Thus, many studies have aimed to investigate the interaction of *A*. *lentis* with different lentil genotypes \[[@pone.0223419.ref013]--[@pone.0223419.ref016]\]. Studies on the early infection process on detached leaf samples have revealed that resistant lentil genotypes recognised and reacted faster to pathogen infection \[[@pone.0223419.ref013]--[@pone.0223419.ref016]\]. Recognition of the pathogen triggers a cascade of cellular and molecular responses. Defense signalling pathway and key defense response genes have been identified through transcriptome profiling of lentil genotypes with non-allelic AB resistance genes to *A*. *lentis* \[[@pone.0223419.ref017],[@pone.0223419.ref018]\]. Genes associated with pathogen recognition as well as genes that comprise salicylic acid, abscisic acid and jasmonic acid transduction pathways were differentially expressed in a genotype-dependent manner \[[@pone.0223419.ref017]\]. On the pathogen side, *A*. *lentis* isolates exhibited delayed and reduced formation of infection structures in resistant varieties compared to susceptible varieties. Histopathological studies using differential staining and monitoring of the production of reactive oxygen species (ROS) have been widely used for studying plant-pathogen interactions including *A*. *lentis* in lentil \[[@pone.0223419.ref014],[@pone.0223419.ref019]\]. Diaminobenzidine (DAB) staining is effective for detection of host responses where ROS are produced by host cells and Trypan Blue stain enables specific staining of mycelium and other fungal structures on plant surfaces. However, staining techniques are most suited to observation of fungal development on host surfaces that pertain to the early colonization phase of infection. The detection of fungal invasion of epidermal and mesophyll layers that characterize late stages of the infection cycle for fungal pathogens is limited by the inefficient staining of fungal structures inside intact plant tissues.
The use of fluorescent reporter proteins, such as green fluorescent protein (EGFP), is a powerful, non-destructive strategy for the study of fungal pathogens and the interaction with their respective hosts. Fluorescence microscopy, and the more sensitive confocal laser scanning microscopy (CLSM), enable the observation of fluorescent fungal structures, both on the surface and inside infected tissues. Various plant pathogenic fungi have been transformed with constructs for the expression of GFP, including *Leptosphaeria maculans* \[[@pone.0223419.ref020]\] *Sclerotinia sclerotiorum* \[[@pone.0223419.ref021]\], *Parastagonospora nodorum* \[[@pone.0223419.ref022]\] and *Ascochyta rabiei* \[[@pone.0223419.ref023]\]. This approach has allowed all infection stages of early colonization, intracellular growth and sporulation to be observed *in planta* and analysed non-invasively in living cells and tissues. The combination of fluorescent tagging and advanced microscopy offers new possibilities for studying pathogen-host interactions.
Information on the development of transformation methods for members of the *Didymellaceae* family is very limited. Previous studies have reported the transformation of the hygromcyin resistance gene and the *gfp* gene into *A*. *rabiei* using *Agrobacterium tumefaciens*-mediated transformation (ATMT) \[[@pone.0223419.ref023],[@pone.0223419.ref024]\], but none so far has been reported for *A*. *lentis*. Here, we describe the first application of ATMT in *A*. *lentis* and report the use of an EGFP-expressing *A*. *lentis* strain to characterize the interaction of *A*. *lentis* with lentil host genotypes that differ in susceptibility and pathogen responses. Furthermore, this study demonstrates that we can monitor AB disease progression in lentils for the full infection cycle using CLSM.
Materials and methods {#sec002}
=====================
Materials, microbial strains and plasmid vector {#sec003}
-----------------------------------------------
*Ascochyta lentis* Kewell strain (named *Al*Kewell hereafter) \[[@pone.0223419.ref012],[@pone.0223419.ref014]\] was used as a recipient strain for transformation and was grown in half strength Potato Dextrose Agar (½ PDA) (BD Difco, Sparks, MD, USA). Sambasivam et al. (2017) \[[@pone.0223419.ref014]\] described responses of lentil accessions ILL6002 and ILL7537 to *Al*Kewell as moderately resistant and resistant, respectively. In our bioassay conditions, at 14 days after inoculation, *Al*Kewell produces more distinct disease responses in these lentil accessions with 30% leaf area damage and no disease, respectively. Maintenance and spore preparation of *A*. *lentis* isolates were performed as described by Davidson et al. \[[@pone.0223419.ref012]\]. The AGL1 strain of *A*. *tumefaciens* was used for ATMT. The *Escherichia coli* strain One Shot OmniMAX 2^TM^ T1 (OM2) (Invitrogen, Carlsbad, USA) was used for cloning and construction of plasmids. Plasmids were maintained in OM2. All reagents were from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated.
Resistance level of *Ascochyta lentis* Kewell to hygromycin B {#sec004}
-------------------------------------------------------------
The *Al*Kewell strain was grown in ½ PDA plates supplemented with hygromycin B (Invitrogen, Carlsbad, USA) at concentrations: 0, 50, 100, 150 and 200 ng.μL^-1^ and incubated at 25°C in the dark. Hyphal growth was monitored daily until the colony covered two-thirds of the plate. Growth was totally inhibited at 50 ng.μL^-1^ and this concentration was used for selection of putative transformant colonies.
Plant material {#sec005}
--------------
Lentil accessions that differ in the disease response to *Al*Kewell, ILL6002 and ILL7537 were used in this study. Lentil accessions ILL6002 and ILL7537 were susceptible and resistant controls, respectively. Lentil varieties were obtained from Seednet (Horsham, Australia) and from Pulse Breeding Australia (Horsham, Australia). Three lentil seeds were sown 1 cm deep in 10 cm pots filled with soil (UWA mix, Richgro, Jandakot, WA, Australia) (five replicates per host genotype-isolate combination). Plants were grown for two weeks in a growth chamber maintained at 18 ± 2°C with a 12/12-hour photoperiod. LED lights were Lumigrow Lumibar 200W (LumiGrow, Emeryville, CA, USA) and S-tech LED 108D18-12 18W tubes (S-tech, Canning Vale, WA, Australia).
Plasmid construction {#sec006}
--------------------
The binary vectors used for ATMT were constructed using parts of the two plasmids, pEAQ-HT-DEST1 \[[@pone.0223419.ref025]\] and pGpdGFP \[[@pone.0223419.ref020]\]. A 4.8 Kb fragment containing the ColEI, OriV, a kanamycin resistance gene (*nptIII*) and *trfA*, left and right border sequences were PCR-amplified from pEAQ-HT-DEST1 (kindly provided by G. Lomonossoff and F. Sainsbury, John Innes Centre, UK) using the primers JD278 and JD287 ([S1 Table](#pone.0223419.s001){ref-type="supplementary-material"}). The 4 Kb fragment containing the *trp*C promoter, hygromycin resistance gene (*hph*), *gpd*A promoter, *egfp* gene and *trp*C terminator were amplified from pGpdGFP \[[@pone.0223419.ref020]\] (obtained from R. Oliver, Curtin University) using primers JD292 and JD293 ([S1 Table](#pone.0223419.s001){ref-type="supplementary-material"}). The PCR of both fragments was carried out in 20 μL reactions containing 1 μL Phusion U Hot Start DNA polymerase (2 U/μL) (Thermo Fisher Scientific), 4 μL 5x HF buffer and 0.2 μM of each primer. Amplification/cycling conditions were: denaturation for 30 s at 98°C, 35 cycles of 10 s at 98°C, 20 s at 55°C and 3 min at 72°C, final elongation for 10 min at 72°C. The PCR products were digested with the USER^TM^ enzyme mix (NEB, Ipswich, MA, USA) according to manufacturer's instructions to remove deoxyuracil residues. Briefly, PCR products of equal concentration were mixed and 8 μL of the mixture were combined with 1 μL of 10x Cutsmart buffer and 1 U of USER enzyme (NEB, 1000 U/mL). The reaction mixture was incubated for 20 mins at 37°C followed by incubation at 25°C for 20 minutes. The resulting vector, pATMT-GpdGFP ([S1 Fig](#pone.0223419.s003){ref-type="supplementary-material"}), was transformed into competent OM2 *E*. *coli* cells and plated onto LB + kanamycin plates (30 ng. μL ^-1^). Putative transformants were screened using PCR to select for constructs having the correct arrangement of components. Verification of the final construct was determined by Sanger sequencing across the junctions between fragments. The binary vector, pATMT-GpdGFP, was produced in OM2 *E*. *coli* and *A*. *tumefaciens* AGL1 was transformed by electroporation of competent AGL1 cells.
*Agrobacterium tumefaciens*-mediated transformation (ATMT) of *Ascochyta lentis* {#sec007}
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AGL1 harboring pATMT-GpdGFP vector was grown in LB medium containing 50 μg.mL^-1^ rifampicin (Astral Scientific, Gymea, NSW, Australia) and 30 μg.mL^-1^ kanamycin (Astral Scientific, Gymea, NSW, Australia), at 28°C and 250 rpm shaking, until approx. OD~600nm~ 0.5. The culture was washed twice with induction medium (IM: Minimal media salts, 40 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH5.3), 10 mM glucose and 0.5% \[w/v\] glycerol. MM salts contain 2.05 g K~2~HPO~4~, 1.45 g KH~2~PO~4~, 0.15 g NaCl, 0.5 g MgSO~4~·7H~2~O, 0.1 g CaCl~2~·6H~2~O, 0.0025 g FeSO~4~·7H~2~O and 0.5 g (NH~4~)~2~SO~4~ per litre) and resuspended at approx. OD~600nm~ 0.5 in the same medium containing 200 μM acetosyringone \[[@pone.0223419.ref026]\]. Spores of *Al*Kewell from a one week-old ½ PDA plate were washed twice with water, filtered through sterile cotton wool inside a five ml plastic syringe, and resuspended in water to a final concentration of approx. 2 x 10^7^ spores.mL^-1^. Equal volumes of *Al*Kewell spore suspension and the AGL1 transformant carrying pATMT-GpdGFP were co-cultivated on a filter membrane (cellulose nitrate 0.45 μm, Whatman^TM^, Buckinghamshire, UK) on IM plates \[[@pone.0223419.ref026]\] containing 200 μM acetosyringone for two days in the dark at 25°C. Following co-incubation, membranes were transferred to ½ PDA plates containing 50 ng.μL^-1^ hygromycin B, 50 ng.μL^-1^ cefotaxime (Astral Scientific, Gymea, NSW, Australia) and 30 ng.μL^-1^ streptomycin (Astral Scientific, Gymea, NSW, Australia) and incubated at 25°C in the dark until colonies appeared. Colonies that appeared 7--10 days after incubation were transferred to fresh selection plates containing antibiotics. The putative transformants were maintained on ½ PDA plates containing antibiotics. The stability of *A*. *lentis* transformants resistant to hygromycin B was tested by sub-culturing five times on ½ PDA plates without hygromycin B, after which transformants were transferred to PDA plates containing hygromycin B (50 ng.μL^-1^).
Putative *A*. *lentis* transformants were screened for positive EGFP expression by monitoring fluorescence using a Nikon A1+ Confocal cell imaging system (Nikon, Tokyo, Japan) using filters at 488 nm excitation and 525/50 nm emission. EGFP-fluorescent transformants were stored at -80°C as a spore suspension in 15% glycerol until further analysis. All experiments were initiated from stored cultures.
Molecular characterization of *Al*Kewell82-GFP {#sec008}
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From multiple colonies resistant to antibiotics, the stable transformant, *Al*Kewell82-GFP, was grown in yeast extract dextrose liquid media with constant shaking at 180 rpm for 72 hours at 22°C in the dark. Mycelium samples were frozen with liquid nitrogen and ground to a fine powder. Approximately 500 mg of powdered mycelium was used for DNA extraction using a modified cetyltrimethylammonium bromide (CTAB) method \[[@pone.0223419.ref027]\]. Briefly, DNA samples were dissolved in 10 mM Tris-HCl, pH 8 and purified with AMPure XP magnetic beads (Agencourt, Beckman Coulter, Brea, CA, USA) following manufacturer recommendations. DNA concentration was determined using a Qubit® 2.0 fluorometer (Invitrogen, Carlsbad, USA) with the dsDNA Broad range assay kit (Thermo Fisher Scientific) and NanoDrop Spectrophotometer (Thermo Fisher Scientific), and DNA quality was assessed by agarose gel electrophoresis (1%).
To validate and to characterize the integration site of the GFP expression cassette into the genome of *Al*Kewell82-GFP, we carried out Illumina sequencing on total DNA from the *Al*Kewell82-GFP strain. Sequencing was performed by Novogene on an Illumina HiSeq (Illumina, San Diego, CA, USA). Raw 150 bp paired end reads were trimmed with Trimmomatic version 0.38 \[[@pone.0223419.ref028]\] and *de novo* assembled with SPAdes version 3.13.0 \[[@pone.0223419.ref029]\]. Reads were mapped back against the *Al*Kewell82-GFP *de novo* assembly using HISAT2 version 2.1.0 \[[@pone.0223419.ref030]\]. The contig with the GFP cassette insertion site was determined using BLASTn with the *egfp* nucleotide sequence against the *de novo Al*Kewell82-GFP assembly. The insertion site was confirmed by PCR with primer pairs JD425 and JD381 and JD427 and JD321 ([S1 Table](#pone.0223419.s001){ref-type="supplementary-material"}).
Evaluation of growth and sporulation of *Al*Kewell82-GFP {#sec009}
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The stably fluorescing transformant *Al*Kewell82-GFP was chosen for further studies with respect to virulence, growth rate and sporulation. Characterization of the EGFP-expressing *A*. *lentis* transformant was undertaken using spore suspension harvested from a one-week old plate as described above. The spore concentration was determined using a haemocytometer and adjusted to approximately 1 x 10^6^ spores.mL^-1^. A sterile circular (0.5 cm diameter) paper filter disk (Whatman^TM^, Buckinghamshire, UK) was dipped in the spore suspension and placed in the middle of a ½ PDA plate without hygromycin B and allowed to grow in an incubator with 12/12-hour near ultra-violet light/dark cycles at room temperature. Mycelial growth was monitored regularly until the colony covered half of the plate. Measurements were reported as the diameter of the mycelial area. Spores and mycelia from the plate were collected and mounted on a glass slide for microscopic examination to assess EGFP expression *in vitro* using CLSM.
Virulence of *Al*Kewell82-GFP was assessed on the susceptible lentil accession ILL6002 and compared to untransformed *Al*Kewell. Whole plant infection assay was performed as described by Davidson et al. \[[@pone.0223419.ref012]\] with modifications. Briefly, spores were prepared as described above. Two-week old plants were spray-inoculated until run-off, making sure of an even distribution of inoculum by spraying plants all together as one group. Inoculated plants were kept in a chamber with misting to ensure the chamber environment was under high humidity. The whole chamber was misted for five seconds every two hours for nine days. The room temperature was maintained at 18°C with a 12/12-hour photoperiod. Macroscopic lesions appeared 6--8 days post infection (DPI). Disease symptoms were evaluated at 14 DPI and scores were reported as % leaf area damage (LAD). Three independent experiments were carried out for each lentil genotype.
Confocal laser scanning microscopy of infected lentil accessions {#sec010}
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Development of fungal structures and disease progression of *Al*Kewell82-GFP were monitored over a period of 14 days. Samples were collected at 1, 3, 5, 7, 9, and 14 DPI. A minimum of three leaves were collected per lentil genotype for each time point. At early time points up until day 5, there were no observable symptoms on either accession. However, at later time points symptoms were observed in accordance with the natural progression of the disease as recorded in which approximated 30% leaf area damage as evidenced by tissue necrosis at 14 DPI. The whole leaf was mounted on a microscope slide with 20% glycerol and covered with a cover slip. Fluorescence images were acquired using an inverted Nikon A1+ confocal microscope with NIS-Elements confocal software (Nikon Instruments, Tokyo, Japan). Images were collected at digital scan resolution (pixel dwell = with 1024 resolution using a 20x Plan Apo dry objective). Sequential laser scanning was performed using four lasers: 405 nm (450/50 filter), 488 nm (525/50 filter), 561 nm (595/50 filter) and 640 nm (700/75 filter) to view nuclei, GFP, and chloroplast and cell wall autofluorescence. The top and bottom positions of the field of view for each sample were set using the NIS-Elements microscope control software and multiple images were taken in Z-series. Three-dimensional representations and 2D Z-projections were produced using Fiji ImageJ software, version 1.51w \[[@pone.0223419.ref031]\].
Propidium iodide staining {#sec011}
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Leaf tissues from infected lentil accession ILL6002 was stained with propidium iodide (PI) at a concentration of 10 μg.mL^-1^ as described by Jones et al. \[[@pone.0223419.ref032]\]. Observation of PI-stained leaves was carried out using Nikon A1+ confocal microscope with 561 nm excitation and 595/50 nm emission. Images were processed using ImageJ, version 1.51w \[[@pone.0223419.ref031]\].
Histochemical detection of hydrogen peroxide (H~2~O~2~) {#sec012}
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The production of H~2~O~2~ by ILL6002 in response to *A*. *lentis* WT and *Al*Kewell82-GFP was detected using the 3,3-diaminobenzidine (DAB) uptake method as described by Sambasivam et al. \[[@pone.0223419.ref014]\]. Briefly, a minimum of three infected lentil leaf tissue samples were collected at one DPI for each isolate and incubated in DAB solution (1 mg.mL^-1^) for approximately eight hours in the dark at room temperature. Tissues were then cleared by immersion in ethanol:glacial acetic acid solution (1:2 V/V) for at least 24 hours to clear the chlorophyll. Fungal structures were then stained with Trypan Blue (0.05%; Sigma-Aldrich) for 10 mins at room temperature \[[@pone.0223419.ref014]\]. Stained leaf samples were mounted on a microscope slide with 20% glycerol and covered with cover slip. Production of ROS was detected as a reddish-brown coloration while fungal structures appeared blue. Samples were examined with Olympus BX51 microscope (Olympus, Tokyo, Japan) fitted with a digital camera. Images were captured using the Olympus DP controller image capture software.
Results {#sec013}
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Construction of the pATMT-GpdGFP binary vector and transformation of *Ascochyta lentis* {#sec014}
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For the development of a method for genetic transformation of *A*. *lentis* using A*grobacterium tumefaciens*-mediated transformation (ATMT), we constructed a new binary vector, pATMT-GpdGFP, using parts of the two plasmids, pEAQ-HT-DEST1 \[[@pone.0223419.ref025]\] and pGpdGFP \[[@pone.0223419.ref020]\]. The regions essential for replication in *E*. *coli* were amplified from pEAQ-HT-DEST1 which included the ColEI and OriV replication origins, a kanamycin resistance gene (*nptIII*), the plasmid replication initiator protein trfA, as well as the left and right border sequences necessary for *Agrobacterium*-mediated integration into the target fungal genome \[[@pone.0223419.ref025]\]. The transgene components containing the hygromycin resistance gene (*hph*) and e*gfp* gene expression cassettes were amplified from pGpdGFP. Both fragments were amplified using primers containing short overlapping tails that allow for targeted and directional fusion of fragments using USER^TM^ cloning \[[@pone.0223419.ref033]\]. PCR fragments were digested with USER^TM^ enzyme mix to remove deoxyuracil residues incorporated into the primers. Subsequent dissociation of the remnant short single-stranded DNA upstream from the dU site produced single-stranded ends for annealing to adjacent PCR fragments in the final construct. The resulting T-DNA was flanked by left and right borders and contained the hygromycin selectable marker with the hygromycin B phosphotransferase resistance gene (*hph*) driven by the *Aspergillus nidulans* tryptophan C promoter (P*trp*C), and the reporter gene *egfp* under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter (P*gpdA*) ([S1 Fig](#pone.0223419.s003){ref-type="supplementary-material"}) The plasmid sequence of pATMT-GpdGFP can be found in the Supporting Information ([S1](#pone.0223419.s008){ref-type="supplementary-material"} and [S2](#pone.0223419.s009){ref-type="supplementary-material"} Files).
For the method development of ATMT in *A*. *lentis*, we first determined the sensitivity of the fungal isolate to different concentrations of hygromycin B (50, 100, 150 and 200 ng.μL^-1^). Fungal growth was not observed at 50 ng.μL^-1^ hygromycin B and this concentration was therefore used to select putative transformants in subsequent experiments. Transformation was achieved by combining *A*. *tumefaciens* AGL1 carrying pATMT-GpdGFP with *Al*Kewell spores, and co-culturing on nitrocellulose membranes on solid media containing acetosyringone without antibiotic to enable recovery of fungal transformants. The membrane containing the co-cultures was then transferred onto a selection plate containing hygromycin B, cefotaxime and streptomycin. Colonies that appeared 7--10 days after co-incubation were subcultured onto fresh selection plates to ensure that the recovered transformants were not false positives. Colonies resistant to hygromycin B were selected for further analysis, including PCR detection of both the hygromycin B selectable marker and *egfp* genes, and the ability to express EGFP both *in vitro* and *in planta*, tested using fluorescence microscopy. Several single spore isolates showed fluorescence with varying degrees of intensity. However, only stable *Al*Kewell-GFP transformant that maintained a high level of EGFP expression after repetitive subculture on a non-selective medium was chosen for further studies.
The *A*. *lentis* transformant, *Al*Kewell82-GFP was chosen for spectral confocal microscopy and plant-pathogen interaction studies because it showed strong and uniform green fluorescence. Expression of EGFP was clearly evident at the different stages of fungal development, including vegetative hyphae and pycnidiospores, when grown on PDA containing hygromycin B ([S2A Fig](#pone.0223419.s004){ref-type="supplementary-material"}). Moreover, we monitored EGFP expression of *Al*Kewell82-GFP *in planta*, where we observed strong green fluorescence in mycelia at three DPI ([S2B Fig](#pone.0223419.s004){ref-type="supplementary-material"}) and pycnidiospores that formed at 14 DPI on the susceptible lentil accession, ILL6002 ([S2C Fig](#pone.0223419.s004){ref-type="supplementary-material"}).
Molecular analysis of *egfp* gene integration into the fungal genome {#sec015}
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The insertion of T-DNA into the genome in *Al*Kewell82-GFP was verified by Illumina whole genome sequencing. A total of 4.43Gbp of data was *de novo* assembled resulting in a genome assembly of 41.4 Mbp, with an average read coverage of 107x ([S2 Table](#pone.0223419.s002){ref-type="supplementary-material"}). Using BLASTn with the coding sequence for the *egfp* gene against the *de novo Al*Kewell82-GFP assembly identified the contig with the T-DNA insertion, which revealed the integration of not only the T-DNA but also the vector backbone in a single site in the genome. The insertion site was found to be in the middle of a gene that is annotated by InterProScan as a glycosyltransferase. The glycosyltransferase gene in *Al*Kewell contains six exons where the first three exons were annotated as a glycosyltransferase domain and the last three exons encode a domain of unknown function (DUF). In *Al*Kewell82-GFP, the binary plasmid containing the T-DNA interrupted this gene where the third exon (ctg2018) was potentially fused to *trfA* as predicted by Genemark, while the DUF (ctg251) was annotated as a separate gene. The orientation of the binary plasmid insertion is depicted in **[Fig 1A](#pone.0223419.g001){ref-type="fig"}**. From the number of reads mapping to and around the insertion site, we suspect that three copies of the complete plasmid and four full copies of *egfp* and the *gpdA* promoter driving its expression have been integrated into the genome. The average sequencing depth for *A*. *lentis* genomic sequence for *Al*Kewell82-GFP was 107x, and for the vector backbone and for the *egfp* expression cassette sequences, 300x and 400x, respectively ([S3 Fig](#pone.0223419.s005){ref-type="supplementary-material"}). We validated the integration of the T-DNA into the genome of *Al*Kewell82-GFP by amplifying the flanking regions of the *A*. *lentis* genome and a region in the plasmid using PCR (**[Fig 1B](#pone.0223419.g001){ref-type="fig"}**). The 917 bp and 707 bp bands confirmed the integration of the plasmid from the *trfA* gene into the *Al*Kewell82-GFP genome.
{#pone.0223419.g001}
To determine the effect of multiple in-tandem insertion and the disruption of the putative glycosyltransferase gene, *Al*Kewell82-GFP was further characterized to ensure that it did not phenotypically vary from the wild-type, *Al*Kewell. Radial mycelial growth and sporulation of *Al*Kewell82-GFP and *Al*Kewell showed no difference when grown on ½ PDA plates after seven days of incubation (**Figs [2A and 2B](#pone.0223419.g002){ref-type="fig"} and** [S4](#pone.0223419.s006){ref-type="supplementary-material"}). Similarly, the ability of *Al*Kewell82-GFP to cause necrosis and to form pycnidia on ILL6002 14 days post infection was comparable to *Al*Kewell-WT (**Figs [2C and 2D](#pone.0223419.g002){ref-type="fig"} and** [S5](#pone.0223419.s007){ref-type="supplementary-material"}).
{#pone.0223419.g002}
Finally, *Al*Kewell82-GFP virulence on the susceptible ILL6002 and resistant ILL7537 lentil accessions was evaluated. A disease severity of 35% on ILL6002 by *Al*Kewell82-GFP was found to be not significantly different from the untransformed *Al*Kewell at 34%, and both strains were not virulent on ILL7537 (**[Fig 2E](#pone.0223419.g002){ref-type="fig"}**) as evidenced by the absence of any observable disease symptoms.
Fungal development during early stages of host infection {#sec016}
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We monitored the infection cycle of *Al*Kewell82-GFP under our standard seedling infection assay conditions on the susceptible variety ILL6002. We followed fungal development using confocal microscopy from the time of inoculation until 14 DPI. Conidial germination occurred within 24 hours upon making contact with the leaf surface, consistent with previous studies \[[@pone.0223419.ref013]--[@pone.0223419.ref015]\]. Analysis of confocal microscope optical sections showed that spores adhered on the cuticle at the leaf surface and occasionally around the trichomes (**[Fig 3A and 3B](#pone.0223419.g003){ref-type="fig"}**). Germ tubes emerging from the spores were typically unipolar, but two germ tubes originating from occasional spores were also observed. The germ tube was observed to extend to varying lengths before forming an appressorium (**[Fig 3C](#pone.0223419.g003){ref-type="fig"}**). However, not all germ tubes produced an appressorium with some continuing to grow from the apex to form extended hyphae. Within 3--5 days after inoculation, infection hyphae grew long and thin on the host surface and attempts to penetrate the host were observed throughout the early growth stage up to five DPI (**[Fig 3D](#pone.0223419.g003){ref-type="fig"}**) without any macroscopic signs of infection or necrosis.
{#pone.0223419.g003}
Penetration of the host leaf surface was evident as early as 24 hours post inoculation. Penetration structures were identified by the localised swelling of hyphal tips to form appressoria (**[Fig 3C](#pone.0223419.g003){ref-type="fig"}**). However, the most commonly observed mode of entry was penetration of the host surface without the formation of an obvious appressorial structure. Alternative penetration structures adopted the form of a simple unlobed structure that was slightly swollen at the tip. These penetration structures were most often found near the groove or at the junction between epidermal cells (**[Fig 4A](#pone.0223419.g004){ref-type="fig"}**). The response of ILL6002 to *Al*Kewell82-GFP was indicated by the presence of elevated white autofluorescence signals around the appressoria and the hyphal tips of the green fluorescent fungi. White fluorescence results from the co-localization of multiple signals from all detector channels. The fluorescence signal at the site of penetration indicates the release of phenolic compounds from the host due to disruption of the cell protoplasm \[[@pone.0223419.ref015],[@pone.0223419.ref022]\]. We confirmed the localised host response using propidium iodide (PI) that stains dead cells. It was evident from the red fluorescent signal (wavelength range: 595/50 nm), that there are dead plant cells at the site of penetration or possibly damage to the cuticle or epidermal layer that has allowed the propidium iodide stain to penetrate tissues and label cell walls \[[@pone.0223419.ref032]\]. Taken together, fluorescence signals with or without propidium iodide stain, clearly indicate that *A*. *lentis* mycelia have attempted, and in some cases succeeded in penetrating the host tissue (**[Fig 4B](#pone.0223419.g004){ref-type="fig"}**). In addition, formation of reactive oxygen species (ROS), a known host defense response, was detected using DAB stain by light microscopy within 24 hours of infection (**[Fig 4C and 4D](#pone.0223419.g004){ref-type="fig"}**).
{#pone.0223419.g004}
Fungal entry through the stomatal opening was also observed (**[Fig 4E](#pone.0223419.g004){ref-type="fig"}**). The germ tube grew at random without moving directly towards the stomata. Interestingly, Z-projections of confocal optical sections from the leaf surface to beneath the epidermis (approx. 10 μm) revealed that entry through the stomata did not show elevated white autofluorescence around the stomata which suggests that the pathogen was able to enter the host without the plant eliciting/producing phenolic compounds or a cell death response.
Cellular reaction of susceptible and resistant lentil varieties to *Al*Kewell82-GFP {#sec017}
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To investigate the interaction between *Al*Kewell82-GFP and different lentil genotypes, we performed a whole plant infection assay on the susceptible ILL6002 and resistant ILL7537 accessions (**[Fig 5](#pone.0223419.g005){ref-type="fig"})**. We divided the infection cycle into three phases as early, mid and late stages. The early phase consists of the conidial adhesion, germination and entry into the host that occurs from 0--5 DPI. The mid-phase refers to the first sign of the necrotic lesion on the susceptible variety where colonization of the epidermal layer was observed by confocal and light microscopy; from 6--9 DPI. The late stage refers to extensive necrosis and sporulation on the susceptible variety, typically from 10--14 DPI.
{#pone.0223419.g005}
During the early stage of infection, *Al*Kewell82-GFP germinated and formed hyphae on the surface of both ILL6002 and ILL7537. Penetration attempts on both lentil genotypes were evident as elevated white fluorescence signals around the various infection structures. These fluorescence signals were localised within the area of attempted penetration sites, which indicates that these were likely hypersensitive responses by the host plants. Nevertheless, no discernible difference in response to *Al*Kewell82-GFP from either of the lentil genotypes was observed (**[Fig 5](#pone.0223419.g005){ref-type="fig"} early**). Hyphal growth continued on the surface of both lentil genotypes for the next three days and except for the localised HR, host cells remained intact and no visible signs of necrotic lesions were observed for either genotype.
At seven DPI, coincident with the first signs of macroscopic necrotic lesions in ILL6002, hyphal morphology around the lesion changed to a thicker, irregularly septated structure. In addition, increased hyphal growth and mycelial proliferation on the surface as well as colonization of the epidermal cells were observed. It appears that following the collapse of the epidermal layer, the pathogen continued to colonize the mesophyll, as we could clearly discern green fluorescent hyphae colonizing the plant cells below the sunken epidermal layer. On the other hand, long and thin filamentous hyphae were detected on the surface of ILL7537 (**[Fig 5](#pone.0223419.g005){ref-type="fig"} mid**) with no apparent further differentiation in the hyphal structure and no colonization of epidermal or mesophyll tissues. At nine DPI, severe infection was clearly visible in the susceptible, ILL6002 leaves, manifested by the characteristic concentric black spots and sporulation on the leaf surfaces. Confocal images confirmed the extensive hyphal growth accompanied by formation of pycnidia (**[Fig 5](#pone.0223419.g005){ref-type="fig"} late**). At this stage, ILL7537 remained free from necrotic lesions and *Al*Kewell82-GFP was almost completely absent from the surfaces of leaves. The differences in *A*. *lentis* colonization is further illustrated by viewing surface growth of hyphae and the colonization of internal leaf tissues by construction of separate Z-projections at the same leaf location for the two lentil accessions (**[Fig 6A and 6B](#pone.0223419.g006){ref-type="fig"}**). The distinct hyphal morphologies of surface, and epidermal and mesophyll-localised mycelium are clearly shown by selective optical sectioning. In **[Fig 6B](#pone.0223419.g006){ref-type="fig"}. i**, *A*. *lentis* has grown through a stoma on the resistant lentil, ILL7537, and neither a white fluorescence signal nor continuation of hyphal extension beyond the base of the stomatal cavity was observed (**[Fig 6B](#pone.0223419.g006){ref-type="fig"}. ii)**.
{#pone.0223419.g006}
Discussion {#sec018}
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During plant-pathogen interaction, a well-orchestrated set of events occurs between the host and the pathogen that ultimately determines the progression and outcome of plant disease. The primary aim of this study was to characterise the disease progression of *A*. *lentis* during the full infection cycle from spore germination on the plant surface through to full colonization of the mesophyll and the production of asexual conidia, using a fluorescently-labelled *A*. *lentis* strain. In addition, differences in disease progression in resistant and susceptible lentil genotypes were explored. To help visualize and fully investigate the initiation and development of ascochyta disease in lentil, a fluorescent strain of *A*. *lentis* was produced using protocols developed for the genetic transformation of *Aspergillus awamori* \[[@pone.0223419.ref026]\]. This is the first report of the transformation of *A*. *lentis* and the use of an EGFP-expressing *A*. *lentis* strain to investigate the AB infection cycle in lentil. Successive stages of the infection process in whole plants were followed using CLSM up to 14 DPI, the stage at which macroscopic lesions indicate invasion and extensive fungal proliferation within infected lentil tissues. The use of fluorescently-labelled *A*. *lentis* enabled the investigation of the processes that occur inside the infected epidermal and mesophyll tissues that might otherwise be challenging to observe using histochemical dyes such as Trypan blue that stain fungal structures inside plant tissues with less efficiency and resolution than on the plant surface. Differences in the efficacy of the histochemical stain lactophenol cotton blue and the GFP-labelling approach have been demonstrated for *Colletotrichum truncatum* \[[@pone.0223419.ref034]\] where GFP shows higher resolution for labelled fungal cells in infected capsicum specimens than stained infected samples. In addition, CLSM enables imaging of invading mycelium through different optical planes, and image reconstructions across multiple optical sections to be produced. The results presented here illustrate the usefulness of the combination of a fluorescently labelled fungal isolate and CLSM for studies of lentil-*A*. *lentis* interactions.
ATMT has been widely adopted and proven to be an efficient tool for genetic transformation in filamentous fungi \[[@pone.0223419.ref035],[@pone.0223419.ref036]\]. Several studies have reported the use of ATMT to transform fungal pathogens to express reporter proteins such as GFP and DsRed \[[@pone.0223419.ref023],[@pone.0223419.ref037],[@pone.0223419.ref038]\]. For the genetic transformation of *A*. *lentis* in this study, a new binary plasmid, pATMT-GdpGFP, was developed. USER^TM^ cloning technology facilitated the single step fusion of the respective components of the desired construct, that were derived from donor plasmids pEAQ-HT-DEST1 \[[@pone.0223419.ref025]\] and pGpdGFP \[[@pone.0223419.ref020]\]. The resulting plasmid contained the fungal GpdA and TrpC promoters that drive the expression of the *egfp* gene and the selectable hygromycin phosphotransferase gene (*hph*), respectively. The transformant, *Al*Kewell82-GFP, was able to express EGFP both *in vitro* and *in planta* ([S2 Fig](#pone.0223419.s004){ref-type="supplementary-material"}), which confirms that the GpdA promoter from *A*. *nidulans* is recognised by *Al*Kewell. As ATMT is known to insert transgenes randomly \[[@pone.0223419.ref039]\], the insertion site and number of T-DNA copies integrated into the genome of *Al*Kewell82-GFP were assessed by genomic sequencing. Approximately 3.5 tandem repeats of the whole binary plasmid inserted into the *Al*Kewell82-GFP genome, of which four full copies of T-DNA fragment remained intact ([Fig 1](#pone.0223419.g001){ref-type="fig"}). Tandem T-DNA insertion has been reported previously in the screening of *F*. *oxysporum* f. sp. *lycopersici* T-DNA mutants \[[@pone.0223419.ref039]\]. In addition, different patterns of T-DNA integration have been reported such as multiple T-DNA integration, inverted repeats, abortive T-DNA integration and the presence of non-T-DNA or binary vector for other fungal pathogens \[[@pone.0223419.ref020],[@pone.0223419.ref023],[@pone.0223419.ref039]\]. DNA sequencing of *Al*Kewell82-GFP showed a single insertion site within the intron of a gene annotated by InterProScan as a glycosyltransferase. The transformant remained stable after growth on agar plates without selection pressure and exhibited wild type growth characteristics ([Fig 2](#pone.0223419.g002){ref-type="fig"}). More importantly, there was no evidence that T-DNA integration had led to disruption of the normal capacity for the strain to infect lentil and to cause disease. Whole genome sequencing for characterizing transformants and assessing transgene copy number is more informative than the conventional Southern blotting method, with the ability to describe the exact location of insertion and the nature of the DNA inserted into the target organism. It is very likely that other *A*. *lentis* isolates would also be amenable to genetic manipulation using ATMT.
To gain insight into the basis of the full AB infection process, disease progression on susceptible and resistant lentil genotypes was monitored using CLSM, focusing on the development of infection structures, penetration and subsequent colonization of the host (Figs [3](#pone.0223419.g003){ref-type="fig"}--[6](#pone.0223419.g006){ref-type="fig"}). Our research approach using GFP-labelled *A*. *lentis* follows the study of Sambasivam et al. (2017) \[[@pone.0223419.ref014]\] where early infection structures in the first 48 hours after *A*. *lentis* inoculation were characterized on detached lentil leaves. We observed that using the whole-plant infection assay, spore germination and development of infection structures on the leaf surface occurring over the first three days after inoculation exhibited no difference in germination rate between resistant and susceptible lentil genotypes ([Fig 5](#pone.0223419.g005){ref-type="fig"} early). We suspect that the swollen hyphal structures at sites, indicated by associated fluorescence signals from adjacent host cells, are playing a role in penetration. However, these structures could possibly be hyphopodia that are more specialized towards nutrient uptake from the host rather than penetration \[[@pone.0223419.ref022]\]. Differences in plant cuticle properties between host species such as rice and maize, and in this case, lentil may have a bearing on the size and shape of penetration structures of plant pathogenic fungi and that the small hyphal swelling that we observe for *A*. *lentis* may be sufficient to breach the comparatively weak barrier of the lentil plant surface. Occasionally, direct penetration attempts of host cells were noted, similar to the penetration strategies documented for other necrotrophic filamentous fungi, such as *A*. *rabiei* \[[@pone.0223419.ref023]\] and *P*. *nodorum* (formerly named *Stagonospora nodorum*) \[[@pone.0223419.ref022]\]. It seems that the preferred mechanism of host entry is through the junction between the epidermal cells, either through direct germ tube penetration or formation of an appressorium. Stomatal penetration was also observed but there seems to be no preference for stomatal entry. This suggests that at least for this strain of *A*. *lentis*, stomatal invasion is a random process similar to *Zymoseptoria tritici*. \[[@pone.0223419.ref040],[@pone.0223419.ref041]\], and in contrast to the observations reported by Roundhill et al. (1995) \[[@pone.0223419.ref013]\] in the infection of lentil cultivars, Laird and Invincible, where stomatal penetration of *Ascochyta fabae* f. sp. *lentis* (syn: *A*. *lentis*) was not observed.
Recognition by the host was evident during the early stage of infection where attempts by *A*. *lentis* to enter the host cells trigger a hypersensitive response (HR) and the release of reactive oxygen species (ROS). Sambasivam et al. (2017) \[[@pone.0223419.ref014]\] showed that the resistant genotypes elicited an earlier HR response as a defense mechanism against the invading *A*. *lentis* pathotypes using DAB staining. In our results, ROS was evident from brown DAB staining around both *Al*Kewell82-GFP and wild type *Al*Kewell on ILL6002 ([Fig 5C--5D](#pone.0223419.g005){ref-type="fig"}). Host cell death was confirmed in propidium iodide staining of ILL6002 cells attacked by *A*. *lentis*. In addition, the presence of white autofluorescence signals from epidermal cells at the site of invading *A*. *lentis* was a clear indicator of the host response by both the susceptible ILL6002 lentil and the resistant ILL7537 (Figs [3](#pone.0223419.g003){ref-type="fig"}--[4](#pone.0223419.g004){ref-type="fig"}). It can be inferred that the white autofluorescence is a result of the phenolics and phenolic-conjugates released by the host as a defense response. Different phenolic compounds emit light at different wavelengths, and in this case, the white autofluorescence is a co-localization of light from the four detector channels and not from any one signal in particular. In the infection of barley by *Blumeria graminis* f. sp. *hordei* (*Bgh*) the host produces the defensive structures called papillae. These consist of the cell wall polysaccharides arabinoxylan and cellulose deposited at the site of fungal penetration of the epidermal cell wall \[[@pone.0223419.ref042]\]. Phenolic compounds accumulate at the site of penetration and the papilla forms at this site through polysaccharide deposition and cross-linking, and cell-wall strengthening by the incorporation of phenolics into the polysaccharide matrix \[[@pone.0223419.ref042]\]. We speculate that similar processes are occurring during pathogen attack of lentil by *A*. *lentis*. Chowdhury et al. \[[@pone.0223419.ref042]\] report that the level of autofluorescence attributed to phenolics at the site of penetration for *Bgh* in barley was not different for effective papillae that could ultimately prevent infection, and ineffective papillae that were unsuccessful in resisting the pathogen. Our results where penetration-site autofluorescence, which we speculatively ascribe to phenolics, was similar for both susceptible and resistant lentil accessions, were consistent with the findings for the proposed model for pathogen-associated papillae in barley \[[@pone.0223419.ref042]\]. Evidence of papilla formation as an early host defense response to *A*. *lentis* infection was reported by Khorramdelazad et al. \[[@pone.0223419.ref018]\]. In the transcriptome analysis of ILL6002 and ILL7537, the authors reported the upregulation of xyloglucan endotransglucosylase/hydrolase (XTH), and the differential regulation of laccase diphenol oxidase (PPOI) and Exocyst subunit 70A1 (*EXO70A1*), genes involved in cell wall restructuring and papilla formation. These genes were overexpressed in both lentil genotypes which suggests that a structural defense response is part of the host artillery to prevent pathogen spread \[[@pone.0223419.ref018]\]. Papilla formation has been reported to function as a barrier and may act as a first line of defense to the invading pathogen \[[@pone.0223419.ref042]\]. Our results show that changes in autofluorescence signals detected by CLSM at the site of attempts at penetration in resistant lentil and successful penetration in susceptible lentil genotypes, are highly sensitive indicators of plant defense responses towards *A*. *lentis* in histology studies of lentil-pathogen interactions. With minimal manipulation of infected leaf samples, the use of EGFP-labelled *A*. *lentis* and the fluorescence-based CLSM method allows both fungal cells and fluorescent plant responses to be observed concurrently.
During the early stage of infection, both susceptible and resistant lentil varieties remained asymptomatic where leaves appeared healthy and no macroscopic lesions were observed for the first 6 days. A long latent phase is also observed for the hemibiotrophic wheat pathogen *Z*. *tritici* \[[@pone.0223419.ref043]\]. The production of numerous proteases by *Z*. *tritici* during the proposed hemibiotrophic phase indicates that the pathogen may live on plant resources by metabolizing the apoplastic proteins and starch released from chloroplasts. In addition, host-adapted cell wall degrading enzymes are produced during early stages to avoid the basal wheat defense. The long latent phase observed in this study supports the idea that *A*. *lentis* is a hemibiotroph where the pathogen is asymptomatic for a prolonged duration after inoculation and then switches to the necrotrophic phase to colonize the host \[[@pone.0223419.ref013],[@pone.0223419.ref015]\].
Colonization of the mesophyll of the susceptible lentil genotype coincides with the onset of macroscopic lesions, usually 6--8 days after inoculation. Microscopically, the onset of necrotrophic phase is characterized by the collapse of the leaf tissues (Figs [5](#pone.0223419.g005){ref-type="fig"}--[6](#pone.0223419.g006){ref-type="fig"}). At this point, fungal hyphae invade the sub-epidermal layer and the mesophyll cells start to collapse, leading to visible signs of necrosis. Fungal morphology changes to enlarged and thicker structures with irregular septation and increased vacuolation. This is in contrast to colonization by the pathogen on the resistant lentil genotype, where surface fungal hyphae remained thin and did not form enlarged and complex subcellular structures. In addition, there was minimal proliferation on the leaf surface of the resistant lentil accession and further colonization of the mesophyll was not observed.
Our observations indicate that both resistant and susceptible lentil genotypes can recognise *A*. *lentis*, as evidenced by similar autofluorescence signals at penetration sites that are likely associated with phenolic compounds. Other defense responses are upregulated upon recognition of the pathogen by the host and these have been shown to differ in resistant and susceptible lentils. Transcriptome profiling during early stages of infection performed by Khorramdelazad et al. \[[@pone.0223419.ref018]\] on the same lentil genotypes used in this study, ILL6002 and ILL7537, infected with *A*. *lentis* revealed the differential expression of early plant defense response genes such as PR2, PR4 and PR10. Several other protein kinase receptors involved in pathogen recognition and early signalling, such as leucine-rich repeat (LRR) receptor-like kinase and calmodulin domain protein kinase (CDPK) were also differentially regulated. This is in agreement with the transcriptome analysis done by Sari et al. \[[@pone.0223419.ref017]\] on lentil varieties with non-allelic R genes, CDC Robin and 964a-46. In both studies, *A*. *lentis* was able to evade recognition by the susceptible host and continued to colonize the mesophyll. Degradation of the host cells provides nutrients to the invading pathogen that leads to the spreading hyphal growth during the asymptomatic phase followed by the transition to necrotrophy and the subsequent formation of pycnidia. Release of pycnidiospores serves as inoculum that enables infection of neighbouring plants and signals the start of the next infection cycle.
Detached leaf assays are often used to study early *in planta* growth stages of plant pathogenic fungi because they are convenient and often yield uniform results. However, Dadu et al. \[[@pone.0223419.ref016]\] observed a significant difference between detached leaf assays and whole plant inoculations for *A*. *lentis* in lentil. The authors observed that excision of leaves enhanced the susceptibility to *A*. *lentis* regardless of the resistance status of the lentil host genotype. Detached leaf assays for *Arabidopsis thaliana* similarly showed a distinct reaction by the hemibiotroph *Colletotrichum* spp \[[@pone.0223419.ref044]\] that was different to the plant response normally observed for whole plant infections. Excision of leaves may activate hormone defense-related signalling and may trigger leaf senescence \[[@pone.0223419.ref044]\]. Thus, the whole plant infection method reported here replicates the natural state of plant infection and interaction between pathogen and host.
Here we have presented new genetic transformation and microscopy methods that build upon previous work to further our understanding of ascochyta blight disease progression in lentils. The development of a transformation protocol for *A*. *lentis* opens the door to introducing other foreign genes and reporter tags to study molecular mechanisms underlying pathogenesis. This will also enable the study of virulence mechanisms using reverse genetics approaches by means of a targeted gene replacement or gene knock-out strategies to study the functions of particular genes. Furthermore, *Agrobacterium*-mediated transformation is a powerful technique to create a library of strains carrying random T-DNA insertions and mutations. Overall, the use of EGFP-expressing, and potentially other fluorescently labelled *A*. *lentis* strains, together with confocal microscopy will be valuable for studying interaction mechanisms among complex fungal communities in plant host systems. New strategies for observing plant-pathogen interactions will be useful in the search for resistant genotypes in lentil germplasm collections and in future breeding programs for evaluating the effectiveness of *R* gene-mediated resistance. Understanding processes and resistance mechanisms operating in lentil during its interaction with *A*. *lentis* is of utmost importance for the development of effective and durable ascochyta blight-resistant lentil cultivars.
Supporting information {#sec019}
======================
###### List of primers.
(DOCX)
######
Click here for additional data file.
###### Summary of genome assembly statistics for Illumina sequencing of *Al*Kewell82-GFP.
(DOCX)
######
Click here for additional data file.
######
Schematic diagram of the transformation vector (A) pATMT-GpdGFP amplified from pEAQ-HT-DEST1 and pGpdGFP and (B) the T-DNA region. Illustration was prepared using SnapGene Viewer (GSL Biotech; available at snapgene.com).
(EPS)
######
Click here for additional data file.
###### Expression of EGFP in *Al*Kewell82-GFP.
\(A\) *Al*Kewell82-GFP grown on ½ PDA solid media; (B) *Al*Kewell82-GFP mycelia on ILL6002 at three DPI; (C) *Al*Kewell82-GFP pycnidia containing pycnidiospores on ILL6002 at 14 DPI.
(EPS)
######
Click here for additional data file.
###### Integration and coverage of pATMT-GpdGFP in *Al*Kewell82-GFP.
(EPS)
######
Click here for additional data file.
###### Mycelial growth of *Al*Kewell and *Al*Kewell82-GFP on ½ PDA plates.
(EPS)
######
Click here for additional data file.
###### Formation of pycnidia on ILL6002.
(EPS)
######
Click here for additional data file.
###### Sequence of *Al*Kewell82-GFP transgene geneious format.
(GENEIOUS)
######
Click here for additional data file.
###### Sequence of *Al*Kewell82-GFP transgene FASTA format--text file.
(TXT)
######
Click here for additional data file.
This research is a Curtin University and Grains Research and Development Corporation co-investment---Research grant CUR00023. pEAQ-HT-Dest1 was a gift from Prof. George Lomonossoff and Dr. Frank Sainsbury from John Innes Centre, Norwich, UK and pGpdGFP came from Prof. Richard Oliver. We acknowledge the use of microscopy facilities at the Curtin Health Innovation Research Institute (CHIRI) with the assistance of Dr. Connie Jackaman and Dr. Rob Steuart. We also thank Dr. Lars Kamphuis and Prof. Richard Oliver for critical reading of the manuscript and useful suggestions.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
| tomekkorbak/pile-curse-small | PubMed Central |
1. Introduction {#sec1-pathogens-09-00201}
===============
Poultry farming is a profitable agricultural business globally, in Bangladesh; livestock and poultry contribute approximately 1.47% of the total GDP of the country \[[@B1-pathogens-09-00201]\]. In the last decade, tremendous development has been achieved in this sector in Bangladesh \[[@B2-pathogens-09-00201]\]. Poultry products including eggs and meat are excellent sources for humans' daily protein requirement. The advancement of poultry production is being seriously hampered by management factors, such as biosecurity measures and infectious diseases \[[@B3-pathogens-09-00201]\]. Among the infectious diseases, salmonellosis caused by MDR *Salmonella* is a major constrain for poultry industries in Bangladesh \[[@B4-pathogens-09-00201]\] and is a worldwide epidemic causing millions of illness and deaths annually \[[@B5-pathogens-09-00201]\].
The chicken-adapted *Salmonella* serovar Gallinarum biovar *Pullorum* and Gallinarum are responsible for avian salmonellosis, e.g., pullorum disease and fowl typhoid, respectively \[[@B6-pathogens-09-00201]\]. In poultry, Salmonellosis is often associated with high mortality up to 90%, thus causing severe economic losses \[[@B7-pathogens-09-00201]\]. *S. enterica* serovar Typhimurium normally colonizes the cecum and is commonly found asymptomatic in cloaca in older chicks \[[@B7-pathogens-09-00201]\]. Day old chicks infected with *Salmonella* may show intestinal pathology and inflammatory responses \[[@B8-pathogens-09-00201]\]. Although birds have no obvious disease symptoms, *S. enterica* serovar Typhimurium can cause systemic metabolic changes in birds affecting production \[[@B9-pathogens-09-00201]\]. Human salmonellosis outbreaks are mostly associated with the consumption of poultry products contaminated with *S. enterica* serovar Typhimurium and *S. enteritidis* \[[@B10-pathogens-09-00201]\]. Therefore, the occurrence of *Salmonella,* particularly *S. enterica* serovar Typhimurium, in broiler farms is a public health concern as they can transmit to the food chain.
Modern food animal production, specifically broiler chicken production, is directly dependent on the use of antibiotics as growth promoters \[[@B11-pathogens-09-00201]\]. Antibiotics are also used in broiler chicken for treatment purposes. The escalated use of antibiotics causes the development of resistance through selection pressure \[[@B12-pathogens-09-00201]\]. Antimicrobial resistance (AMR) is an increasingly serious public health challenge \[[@B13-pathogens-09-00201]\]. It is speculated that if measures are not taken properly, by 2050 AMR will cause 300 million human deaths, financial losses will be equivalent to 100 trillion USD and an 11% fall in animal production \[[@B14-pathogens-09-00201]\]. By far, most of the affected countries will be the low and middle-income countries (LMICs) in Africa and Asia \[[@B15-pathogens-09-00201]\]. Bangladesh is located in the World Health Organization's Southeast Asia region which is assessed as having a high risk of AMR \[[@B16-pathogens-09-00201]\].
Bacteria can acquire resistance genes through mobile genetic elements such as plasmids, transposons, integron, and insertion sequences (IS) elements \[[@B17-pathogens-09-00201]\]. Due to their mobile nature, these genetic elements can easily spread horizontally across many species of bacterial populations \[[@B18-pathogens-09-00201]\]. Humans can get exposed to antibiotic resistant bacteria through consumption of contaminated meat and eggs, or through direct transmission from colonized animals or manure and litter \[[@B19-pathogens-09-00201]\]. Because of their zoonotic nature, there are possibilities for the transmission of MDR *Salmonella* from poultry to humans throughout the food chain \[[@B20-pathogens-09-00201],[@B21-pathogens-09-00201],[@B22-pathogens-09-00201]\]. In recent years, the development of MDR among foodborne pathogens, such as *Salmonella* spp., have been associated with an increase in human mortality, and longtime hospitalization due to therapy failure \[[@B22-pathogens-09-00201]\].
The isolation of *Salmonella* from poultry was previously reported in Bangladesh, however, none of these studies focused on the molecular detection of resistance genes \[[@B23-pathogens-09-00201],[@B24-pathogens-09-00201],[@B25-pathogens-09-00201],[@B26-pathogens-09-00201]\]. However, resistance genes were detected in *Escherichia coli* isolated from cloacal swabs of boiler chicks in Bangladesh \[[@B27-pathogens-09-00201],[@B28-pathogens-09-00201]\]. Moreover, a study in Bangladesh, detected resistance genes in *E. coli* and *Salmonella* from dairy farms \[[@B29-pathogens-09-00201]\]. Another study detected integrons in *Salmonella* from chicken feces \[[@B30-pathogens-09-00201]\]. Therefore, we investigated the prevalence of resistance genes including integrons responsible for multidrug resistance in *S. enterica* serovar Typhimurium in broiler farms using different sample types (cloacal swabs, litter and feed).
2. Results {#sec2-pathogens-09-00201}
==========
Among the 100 samples analyzed, 35 (35%) were found to be positive for the presence of *Salmonella* as evidenced from the isolation on selective media, followed by Gram staining, biochemical tests, and *invA* gene-targeted PCR ([Figure 1](#pathogens-09-00201-f001){ref-type="fig"}). The prevalence of *Salmonella* was significantly (Chi-square test, *p* = 0.017) higher in cloacal samples (48%, n = 24) compared to all types of samples. The confirmed 35 isolates were inoculated into motility indole urea medium and 30 samples were found to be motile, suggesting that these motile *Salmonella* would be *S. enterica* serovar Typhimurium. Motile and non-motile *Salmonella* were also differentiated by PCR amplification of *fliC* gene specific for motile *S. enterica* serovar Typhimurium. All 30 motile isolates were also found to be positive for the *fliC* gene, confirming them as *S. enterica* serovar Typhimurium. The detection of *S. enterica* serovar Typhimurium was found to be the highest significantly in cloacal sample type (95.8%, n = 23) as compared to litter and feed samples (Chi-square test, *p* = 0.034).
From the antibiogram study, it was found that the isolated *Salmonella* were highly resistant to tetracycline (97.1%) followed by chloramphenicol (94.3%), ampicillin (82.9%), and streptomycin (77.1%), while the highest susceptibility rate was observed against cefixime (100%), followed by ertapenem (94.3%) and ciprofloxacin (85.7%) ([Table 1](#pathogens-09-00201-t001){ref-type="table"}). *Salmonella* from cloaca always showed a peak of resistance against all antibiotics and a statistically significant relationship was found for tetracycline, chloramphenicol and ampicillin. All 35 identified *Salmonella* were MDR with the highest 80% (n = 28) against three antibiotics classes, and only two isolates showed MDR phenotype against five antibiotic classes ([Table 2](#pathogens-09-00201-t002){ref-type="table"}).
It was found that 34 (97.1%) out of 35 isolates were positive for tetracycline resistance gene *tetA*. The prevalence of *floR*, *bla~TEM-1~*, and *aadA1* in *Salmonella* were 94.3%, 82.9%, and 77.1%, respectively, from different sources ([Table 3](#pathogens-09-00201-t003){ref-type="table"}). *Salmonella* isolated from cloacal swabs showed a statistically significant peak prevalence for all the resistance genes similar to their phenotype. Among the 35 isolates, 7 (20%) were found positive for class 1 integron gene *intl1*. Although cloacal isolates showed the highest prevalence of *intl1*, no statistically significant relationship was found when compared with isolates originated from litter and feed. These seven isolates carried multiple resistance genes ([Table 4](#pathogens-09-00201-t004){ref-type="table"}).
3. Discussion {#sec3-pathogens-09-00201}
=============
Avian salmonellosis is a major threat for poultry industries since it is capable of causing heavy economic losses through mortality and reduced production. *Salmonella* are also important foodborne zoonotic pathogens. In this study, among the samples analyzed, 35% (35/100) were found to be positive for the presence of *Salmonella* as confirmed by PCR. Earlier, Al Mamun et al. \[[@B26-pathogens-09-00201]\] reported the prevalence of Salmonellosis in poultry as 23.53%, lower than the present findings, and Mahmud et al. \[[@B23-pathogens-09-00201]\] reported a prevalence of 37.9% in Bangladesh, which is quite similar to the current findings. The prevalence of *Salmonella* was significantly the highest in cloacal swabs (48%). Previously a study in Bangladesh, showed the highest rate of *Salmonella* occurrence in cloacal swabs (32%) among different samples of poultry \[[@B31-pathogens-09-00201]\] and another study found 48% *Salmonella*, similar to our result \[[@B25-pathogens-09-00201]\]. Litter (25.7%) was also found to be contaminated with *Salmonella* in the present study, whereas Islam et al. \[[@B32-pathogens-09-00201]\] reported 66.6% *Salmonella* in litter from broiler farms. *Salmonella* could be either motile or non-motile. The motile *Salmonella* are mostly associated with food products and are the major causes of salmonellosis in humans worldwide \[[@B33-pathogens-09-00201]\]. In the current study, 30 out of 35 *Salmonella* isolates were confirmed as motile *Salmonella*, i.e., *S. enterica* serovar Typhimurium by growing on motility indole urea agar and by *fliC gene* detection through PCR. Barua et al. \[[@B24-pathogens-09-00201]\] reported the presence of motile *Salmonella* in commercial broiler chicken farms as 11%. Another study in Bangladesh revealed 15.91% *S. enterica* serovar Typhimurium among the identified *Salmonella* \[[@B25-pathogens-09-00201]\]. Detection of *S. enterica* serovar Typhimurium in poultry implies the possibility of *S. enterica* serovar Typhimurium transmission via poultry originated food and may lead to foodborne illness in human \[[@B22-pathogens-09-00201]\].
These observed variations in the prevalence of *Salmonella* may be linked with various management factors such as biosecurity, hygiene, and sanitation of the farms. The presence of *Salmonella* spp. in cloacal swabs of healthy broiler chicken provides the evidence of persistent intestinal colonization of *Salmonella* spp. of the individual bird \[[@B34-pathogens-09-00201]\]. Moreover, their presence in cloacal swabs and poultry litter indicates that poultry droppings may act as vehicles for shedding *Salmonella* spp. to other birds \[[@B34-pathogens-09-00201]\]. In addition, poultry litter aeration can be a vital risk factor for spreading pathogens like *Salmonella* and can contaminate farm environments, causing birds to be at risk \[[@B35-pathogens-09-00201]\]. It is interesting to note that in this study, 13.3% of feed supplied to the broiler chicken were also found to be contaminated with *Salmonella* spp. Detection of *Salmonella* in poultry feed in Bangladesh is not uncommon since earlier Malorny et al. \[[@B36-pathogens-09-00201]\]; Islam et al. \[[@B32-pathogens-09-00201]\] and Al Mamun et al. \[[@B26-pathogens-09-00201]\] also reported presence of *Salmonella* in poultry feed. Our findings suggest that contaminated feed could be a potential source of *Salmonella* in poultry.
AMR is an escalating global health problem \[[@B37-pathogens-09-00201]\]. Treatment of infections caused by MDR bacteria are expensive and may be fatal. AMR can affect sustainable development goals (SDGs), especially those targeting hunger, health and economic growth \[[@B38-pathogens-09-00201]\]. MDR *Salmonella* has emerged as a major public health issue worldwide \[[@B39-pathogens-09-00201]\]. One of the classical examples of such public health issue is MDR *S. enterica* serovar Typhimurium phage type DT104, which was found to be resistant to several antibiotics, namely ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline \[[@B40-pathogens-09-00201]\]. The present study revealed that 97.1% to 77.1% of the isolated *Salmonella* showed resistance against commonly used antibiotics, namely tetracycline, ampicillin, streptomycin, and chloramphenicol. An alarming result was that all the tested isolates in the present study were found to be MDR. Development of antibiotic resistance in these bacteria could be a result of several factors including strong selective pressure resulting from the indiscriminate use of antibiotics \[[@B41-pathogens-09-00201]\]. Moreover, unpublished data suggested that, in many cases, these antibiotics are used as a growth promoter and are added to the poultry feed and water, respectively, by feed companies and the farmers. There is evidence of antibiotic residues in commercial feed in Bangladesh, acts as subnormal dose which also accelerates the emergence of antibiotic resistance \[[@B42-pathogens-09-00201]\]. Cloacal isolates always showed a higher resistance against different antibiotics than litter and feed isolates, which indicates that feces may play a vital role in the spread of resistant bacteria within the farm environment, including feed stored in the poultry shed.
The present study reported the detection of *tetA* (97.1%), *floR* (94.3), *bla~TEM-1~* (82.9%) and *aadA1* (77.1%) responsible for specific antibiotic resistance. The presence of several resistance genes in different samples may be due to integron that was detected in this study. Integrons are mobile genetic elements. They play important role in the transfer of clusters of genes including antibiotic resistance. Integrons could also be transferred horizontally among *Salmonella* serotypes \[[@B43-pathogens-09-00201],[@B44-pathogens-09-00201]\]. *Salmonella* carrying class one integron *int1* were found positive for multiple resistance genes. In this study, 20% isolates were found positive for the presence of the *int1* gene, revealing the molecular basis of observed multidrug resistance \[[@B45-pathogens-09-00201]\]. These factors may facilitate the emergence of MDR pathogens. In many cases, resistance patterns are observed against those antimicrobials that are frequently used in veterinary practices \[[@B45-pathogens-09-00201],[@B46-pathogens-09-00201]\].
MDR *Salmonella* are important to the etiology of bacterial foodborne associated deaths, particularly in the LMICs \[[@B47-pathogens-09-00201]\]. The WHO recognized *Salmonella* as one of the pathogens having severe impacts on human health \[[@B48-pathogens-09-00201]\]. Poultry and poultry products have been frequently reported to be involved in the outbreaks of salmonellosis \[[@B49-pathogens-09-00201]\]. Alarmingly, all the *Salmonella* isolated in the present study were found to be MDR including fluroquinolone-resistant *Salmonella*. The detection of MDR *Salmonella* in broiler chicken as evident in this study is of great public health concern. It is possible that these MDR *Salmonella* may transmit into the food chain in broiler meat and lead to serious illness in humans. Detection of mobile genetic elements, such as integron in these isolates make the situation more aggravated. Integrons are directly associated with resistance to antibiotics \[[@B50-pathogens-09-00201]\] and there are possibilities of their transmission to other bacterial species horizontally inside the broiler chicken gut. The present findings highlight the importance of practicing strict biosecurity, hygienic measures, proper litter management and safe storage of feed to reduce the load and spread of MDR *Salmonella* in broiler chicken and to ensure consumers' health and safety. In addition, the implementation of one-health measure in Bangladesh is critical to early monitor and detect AMR in pathogens of zoonotic potential and to reduce the imprudent use of antibiotics in poultry farms.
4. Materials and Methods {#sec4-pathogens-09-00201}
========================
4.1. Ethics Statement {#sec4dot1-pathogens-09-00201}
---------------------
The experimental procedures and protocols used in this study were approved by the Animal Welfare and Experimentation Ethics Committee of Bangladesh Agricultural University (approval number AWEEC/BAU/2018(20)
4.2. Collection of Samples and Isolation of Salmonella {#sec4dot2-pathogens-09-00201}
------------------------------------------------------
A total of 100 samples (50 cloacal swabs, 35 litter samples, 15 feed samples) from five broiler (*Gallus gallus domesticus*) farms located in Mymensingh, Bangladesh were randomly collected in July 2017 and samples were transported immediately to the Department of Microbiology and Hygiene, Bangladesh Agricultural University, while maintaining sterile and cold chain conditions. From each farm, 10 cloacal swabs, 7 litter samples and 3 feed samples were collected. Isolation and identification of *Salmonella* from the collected samples were based on culture on selective media (XLD) (Hi Media, India), Gram staining and biochemical test as was previously described \[[@B29-pathogens-09-00201]\].
4.3. Extraction of Bacterial Genomic DNA {#sec4dot3-pathogens-09-00201}
----------------------------------------
For the PCR, the genomic DNA was extracted using the boiling method \[[@B51-pathogens-09-00201]\]. In brief, initially a pure bacterial colony was mixed with 100 µL of distilled water in an Eppendorf tube followed by boiling for 10 min. After boiling the Eppendorf tube, it was immediately kept on ice to have cold shock followed by 10 min of centrifugation at 10,000 rpm. Finally, the supernatant was collected and used as a DNA template for PCR.
4.4. Molecular Detection of Salmonella {#sec4dot4-pathogens-09-00201}
--------------------------------------
*Salmonella* was detected by PCR targeting the *invA* gene as previously described \[[@B52-pathogens-09-00201]\]. Motile *Salmonella* were detected by PCR targeting the *fliC* gene specific for *S. enterica* serovar Typhimurium \[[@B53-pathogens-09-00201]\]. Primers specific for *invA* and *fliC* genes are listed in [Table 5](#pathogens-09-00201-t005){ref-type="table"}. PCR was done in the final 25 µL containing nucleus free water (5.5 µL), master mixture (Promega) (12.5 µL), forward and reverse primer (1 µL each) and DNA template (5 µL). The PCR thermal profile consisted of 95 °C and 5 min for initial denaturation, followed by 29 cycles of denaturation at 95 °C for one-minute, variable annealing temperature ([Table 5](#pathogens-09-00201-t005){ref-type="table"}) for one-minute, elongation at 72 °C for one- minute and a final extension at 72 °C for 10 min. After completion, the PCR products were analyzed by running in 1.5% agarose gel electrophoresis.
4.5. Antimicrobial Susceptibility Testing {#sec4dot5-pathogens-09-00201}
-----------------------------------------
The antibiogram phenotype were determined by the Kirby-Bauer disk diffusion method \[[@B54-pathogens-09-00201]\] against seven commonly used antibiotics classes, namely penicillin (ampicillin-25 µg), cephalosporin (cefixime-5 µg), amphenicol (chloramphenicol-30 µg), fluoroquinolones (ciprofloxacin-5 µg), carbapenem (ertapenem-10 µg), aminoglycoside (streptomycin-10 µg) and tetracycline (tetracycline-30 µg). The antibiogram test was performed by disk diffusion method on Mueller-Hinton agar (Hi Media, India) plates with a concentration of bacteria equivalent to 0.5 McFarland standard, incubated for 18--24 h aerobically at 37 °C. The results of the antibiogram test were recorded as sensitive, intermediately sensitive, or resistant and the diameters of the zones of inhibition were compared with the diameters of interpretative tables provided by the Clinical and Laboratory Standards Institute (CLSI) \[[@B55-pathogens-09-00201]\]. *Salmonella* isolates that were found to be resistant to multiple antimicrobials (at least 3 classes of antibiotics) were considered as MDR \[[@B56-pathogens-09-00201]\].
4.6. Detection of Antibiotic Resistance and Class 1 Integron Gene {#sec4dot6-pathogens-09-00201}
-----------------------------------------------------------------
All the MDR phenotypes were subjected to PCR for the detection of *aadA1*, *bla~TEM-1~*, *floR,* and *tetA* genes responsible for resistance against streptomycin, beta lactam antibiotic, amphenicol (chloramphenicol), and tetracycline respectively, and class 1 integron gene *intl1* \[[@B57-pathogens-09-00201],[@B58-pathogens-09-00201],[@B59-pathogens-09-00201],[@B60-pathogens-09-00201]\]. The list of primers used to detect these genes are presented in [Table 5](#pathogens-09-00201-t005){ref-type="table"}.
4.7. Statistical Analysis {#sec4dot7-pathogens-09-00201}
-------------------------
All the data was incorporated in Excel sheets (MS-2010) and analyzed by SPSS software (SPSS-24.0). Descriptive analysis was performed to calculate prevalence and the Chi-square test was performed to determine the level of significance. A *p*-value less than 0.05 (*p*-value \< 0.05) was considered as statistically significant.
We are very much grateful to the farmers for giving access to the samples. The authors would also like to thank Department of Microbiology and Hygiene, Faculty of Veterinary Science Bangladesh Agricultural University, Mymensingh for the support during the research.
Conceptualization, M.R., K.N.H.N. and T.R.; methodology, S.B.A., M.M. and M.R.; software, R.A., M.H. and A.S.; validation, M.R., A.S. and M.E.E.Z.; formal analysis, A.S. and T.R.; investigation, S.B.A., M.M., R.A. and M.H., resources, S.B.A., M.M. and M.R.; data curation, R.A., M.H. and A.S.; writing---original draft preparation, S.B.A., M.R., A.S. and K.N.H.N. writing---review and editing, M.R., M.E.E.Z., T.R., A.N. and A.S.; visualization, S.B.A. and A.S. supervision, M.R., K.N.H.N. and T.R.; project administration, M.R.; funding acquisition, M.R.; critical revisions and writing, M.R., M.E.E.Z., A.N., and T.R. All authors have read and agreed to the published version of the manuscript.
This study was supported in part by research fund from the Bangladesh Agricultural University Research System (BAURES) (Grant number: 2018/581/BAU).
The authors declare no conflict of interest.
{#pathogens-09-00201-f001}
pathogens-09-00201-t001_Table 1
######
Antibiotic resistance patterns of *Salmonella* in broiler farm in this study.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------
Sample (n) Antibiotic Resistance Pattern (%)
------------------- ----------------------------------- --------- -------- -------- -------- ------- -------- ------- -------- ------- ------- ------- ----- ----
Cloacal Swab (24) 24 (100) \<0.001 24\ 0.010 23\ 0.011 21\ 0.012 4\ 0.772 2\ 0.615 0\ nc
(100) (95.8) (87.5) (16.7) (8.3) (0)
Litter (9) 9 (100) 8\ 5\ 6\ 1\ 0\ 0\
(88.9) (55.6) (66.7) (11.1) (0) (0)
Feed (2) 1 (50) 1\ 1\ 0\ 0\ 0\ 0\
(50) (50) (0) (0) (0) (0)
Total (35) 34 (97.1) 33\ 29\ 27\ 5\ 2\ 0\
(94.3) (82.9) (77.1) (14.3) (5.7) (0)
-----------------------------------------------------------------------------------------------------------------------------------------------------------------
Note: TE, tetracycline; C, chloramphenicol; AMP, ampicillin; S, streptomycin; CIP, ciprofloxacin; ETP, ertapenem; CFM, cefixime; nc, not computed; n, no. of positive *Salmonella*.
pathogens-09-00201-t002_Table 2
######
Multidrug resistance profile of *Salmonella* used in this study.
--------------------------------------------------------------------------------------------------------------------------------------------------------
Isolates No. of Antibiotic (Class) Multidrug Profile No. of Isolates (%) Prevalence of MDR %
---------------- ------------------------------------------------------- ----------------------------------- --------------------- ---------------------
*Salmonella*\ 1 (1) Any one of the tested antibiotics 0 100
(n = 35)
2 (2) TE-AMP or other combination of two antibiotic classes 0
3 (3) TE-AMP-C 28 (80)
TE-S-C 26 (74.28)
4 (4) C-AMP-S-TE 24 (68.57)
CIP-AMP-TE-S 5 (14.28)
5 (5) C-AMP-S-TE-CIP 1 (2.87)
C-AMP-S-TE-ETP 1 (2.87)
--------------------------------------------------------------------------------------------------------------------------------------------------------
Note: TE, tetracycline; AMP, ampicillin; C, chloramphenicol; S, streptomycin; CIP, ciprofloxacin; ETP, ertapenem; MDR, multidrug resistance.
pathogens-09-00201-t003_Table 3
######
Prevalence of antibiotic resistance genes in *Salmonella* in broiler farm in the present study.
---------------------------------------------------------------------------------------------------------------------------------
Sample (n) Antibiotic Resistance Gene (%)
------------------- -------------------------------- --------- -------- -------- -------- ------- -------- ------- ------ -------
Cloacal Swab (24) 24 (100) \<0.001 24\ 0.010 23\ 0.011 21\ 0.012 6\ 0.517
(100) (95.8) (87.5) (25)
Litter (9) 9 (100) 8\ 5\ 6\ 1\
(88.9) (55.6) (66.7) (11.1)
Feed (2) 1 (50) 1\ 1\ 0\ 0\
(50) (50) (0) (0)
Total (35) 34 (97.1) 33\ 29\ 27\ 7\
(94.3) (82.9) (77.1) (20)
---------------------------------------------------------------------------------------------------------------------------------
Note: TE, tetracycline; AMP, ampicillin; C, chloramphenicol; S, streptomycin; n, no. of positive *Salmonella*.
pathogens-09-00201-t004_Table 4
######
Profiles of class 1 integron gene *intl1* carrying *Salmonella* in the present study.
Isolate number Resistance Genes MDR Profile
---------------- ----------------------------------- ----------------
CS3 *terA, floR, bla~TEM~-~1~, aadA1* C-AMP-S-TE
CS11 *terA, floR, bla~TEM~-~1~, aadA1* C-AMP-S-TE-ETP
CS19 *terA, floR, bla~TEM~-~1~* C-AMP-S-TE
CS22 *terA, floR, bla~TEM~-~1~, aadA1* C-AMP-S-TE
CS41 *terA, floR, bla~TEM~-~1~, aadA1* C-AMP-S-TE-CIP
CS43 *terA, floR, bla~TEM~-~1~, aadA1* C-AMP-S-TE
L8 *terA, floR, aadA1* C-AMP-S-TE
Note: CS, cloacal swab; L, litter, MDR, multidrug resistance; C, chloramphenicol; TE, tetracycline; AMP, ampicillin; S, streptomycin; CIP, ciprofloxacin; ETP, ertapenem.
pathogens-09-00201-t005_Table 5
######
PCR primers with sequence used in this study.
Gene Primer Sequence Amplicon Size (bp) Annealing Temperature Reference
---------------- -------------------------- -------------------- ----------------------- -------------------------------
*invA-F* CGGTGGTTTTAAGCGTACTCT T 796 58 \[[@B52-pathogens-09-00201]\]
*invA-R* CGAATATGCTCCACAAGGTTA
*Flic-C-F* CCCGCTTACAGGTGGACTAC 433 58 \[[@B53-pathogens-09-00201]\]
*Flic-C-R* AGCGGGTTT TCGGTGGTTGT
*aadA1-F* TATCAGAGGTAGTTG GCGTCAT 484 55 \[[@B57-pathogens-09-00201]\]
*aadA1-R* GTTCCATAGCGTTAAGGTTTCATT
*bla~TEM-1~-F* CATTTCCGTGTCGCCCTTAT 793 56 \[[@B58-pathogens-09-00201]\]
*bla~TEM-1~-R* TCCATAGTTGCCTGACTCCC
*floR-F* AACCCGCCCTCTGGATCAAGTCAA 548 62 \[[@B57-pathogens-09-00201]\]
*floR-R* CAAATCACGGGCCACGCTGTATC
*tetA-F* GGTTCACTCGAACGACGTCA 577 57 \[[@B59-pathogens-09-00201]\]
*tetA-R* CTGTCCGACAAGTTGCATGA
*intl1 A-F* GGCATCCAAGCAGCAAGC 2000 55 \[[@B60-pathogens-09-00201]\]
*intl1 A-R:* AAG CAG ACT TGA CCT GAT
| tomekkorbak/pile-curse-small | PubMed Central |
Barrista Mike Breach of Smile To Go Creates Caffeinated Masterpieces
Boutique coffee shops have set themselves apart from larger chains through more personable service and creative drinks, but barrista Mike Breach has taken this up a notch with his latte fine art. An incredible feat of skill and patience that could rival the masterpieces of Leonardo da Vinci and Picasso, Mike Breach demonstrates that detailed portraits can be depicted as easily with milk foam and espresso as it can be with pencils and paintbrushes.
Using a bamboo skewer as a paintbrush, Mike Breach has portrayed rock stars, extraterrestrials, artists and some of the most brilliant minds. He reveals, "I kind of want to be like Willy Wonka with coffee- make it interesting and fun for people; take the pretentiousness out of it, take the seriousness out of it." | tomekkorbak/pile-curse-small | Pile-CC |
Meaglin Surion
Meaglin Surion was General soon to be crowned king of Trevek that was until Trevek was attacked by Herobrine and his demonic minions on that night most of the city was destroyed and Meaglin and his great friend Lord Colm seeked revenge (i will add more when the story develops i want no SPOILERS you will know what happens soon enough).
Meaglin's early infant years are a mystery all there is to tell is that he was given to a middle class Human family on the outskirts of Trevek on a late Autumn morning he was not a full human or full elf it was illegal in Minecraftia for a Human and an elf to have a sexual relationship so they didn't want Meaglin to be away so they gave them to a Human family. no one ever knew him had any idea that he was half elf half human. But his lust for knowledge, his quick reflexes and agility made him a local celebrity.
Meaglin Surion went to a private school where he learnt the rules of magic politics law astrology and mysteries of the over world and the nether world myths and legends of many kinds in Minecraftia.
It was when he was 16 he met his friend Colm Hogg he was near to death from starvation and dehydration Meaglin immidietly helped this dieing stranger therefore starting a great friendship while Colm was 15 at the time he was not well educated and didn't know how to read or write Meaglin taught him how to do both. although Colm had no family he said they all died from famine Colm did not cry or weep at this as if his soal had been killed along with them Meaglin felt sorry for Colm and let him live at home with him he got new clothes for him in the Market district and Meaglin said I always wanted to be a soldier Meaglin said why Colm said because my father was one he was a sergeant but he had died after the tower he was in collapsed there was no investigation as to why it collapsed all the soldiers died but they all had a proper burial which was a good thing but they never found his dads body and it troubled him a lot so Meaglin said so do you want to join i heard they are recruiting, Colm said yes please i would like that and meaglin thought why the hell not so they both recuited in the army. | tomekkorbak/pile-curse-small | Pile-CC |
[Pre-operative, operative and postoperative complications in 2266 cases of transurethral resection of the prostate].
Pre-operative and operative complications in 2266 patients having undergone transurethral resection of prostate (TURP) for the past 20 years at Kitasato University Hospital were analyzed. They consisted of 2008 benign prostatic hyperplasia and 258 prostate cancer patients. Seven hundred and fifty four patients showed some of physical disorders prior to TUR:hypertension in 147 cases, diabetes mallitus in 87, ischemic heart disease in 46, chronic obstructive lung disease in 41 and others. Operative and postoperative complications of TURP were seen in 308 cases (13.6%). Perforation of the prostatic capsule was seen in 100 cases (4.4%) and bladder perforation into intraperitoneal cavity in 6 cases (0.3%). Transurethral fulgulation for postoperative hemorrhage was conducted on 79 cases (3.5%). Hyponatremia lower than 130 mEq/L was noted in 14 cases (0.6%). Severe urinary tract infection leading to bacteremia was observed in 9 cases (0.4%). Postoperative epididymitis was evident in 20 cases (0.8%). There was postoperative urinary incontinence in 19 cases, 3 of which was treated with Teflon-paste injection successfully. One patient had to undergo AMS-800 artificial sphincter implantation. The number of postoperative urethral stricture patients requiring urethral dilatation or internal urethrotomy was 12 (0.5%) and postoperative bladder neck contracture was seen in 20 cases (0.9%). One patient (0.04%) who developed DIC after profuse postoperative hemorrhage died on the 37th postoperative day. The efficiency of TURP depends not so much on the skill of cutting as on the speed and accuracy of orientation and haemostasis. The quick recognition of anatomical landmarks will assure effective and safe resection. | tomekkorbak/pile-curse-small | PubMed Abstracts |
---
abstract: 'GTPase molecules are important regulators in cells that continuously run through an activation/deactivation and membrane-attachment/membrane-detachment cycle. Activated GTPase is able to localize in parts of the membranes and to induce cell polarity. As feedback loops contribute to the GTPase cycle and as the coupling between membrane-bound and cytoplasmic processes introduces different diffusion coefficients a Turing mechanism is a natural candidate for this symmetry breaking. We formulate a mathematical model that couples a reaction–diffusion system in the inner volume to a reaction–diffusion system on the membrane via a flux condition and an attachment/detachment law at the membrane. We present a reduction to a simpler non-local reaction–diffusion model and perform a stability analysis and numerical simulations for this reduction. Our model in principle does support Turing instabilities but only if the lateral diffusion of inactivated GTPase is much faster than the diffusion of activated GTPase.'
author:
- Andreas Rätz
- Matthias Röger
title: Turing instabilities in a mathematical model for signaling networks
---
Introduction {#sec:intro}
============
GTP-binding proteins (GTPases) are crucially involved in many processes in cells such as membrane traffic, cellular transport, signal transduction, or cytoskeleton organization [@TaSM01; @JaHa05]. Common to the diverse families of GTPase is the cycling between an active and an inactive state. Besides the activation-inactivation cycle there is also a spatial cycle: in the cytosol almost all GTPase is inactive whereas the active state is only present at the membrane. Reaction and diffusion processes both in the cytosolic volume and on the membrane surfaces as well as membrane attachment and detachment contribute to the proper function of GTPase molecules. For different GTPase localization into subcellular compartments has been observed and has been recognized as crucial for its function. Cluster formation of activated small GTPase Cdc42 precedes the budding of yeast [@PaBi07], other small GTPase of the Rho-subfamily are known to form micro-domains on continuous membranes [@PfAi04; @SRNR00]. Such a transition from a homogeneous distribution to a polarized state is often key for the formation and maintenance of complex structures. The emergence of localized structures is typically driven by a continuous input of energy [@NiPr77]. Turing [@Turi52; @Murr90] pioneered models for symmetry breaking by diffusion-driven instabilities. These are based on a slowly diffusing self-activator and a highly diffusive antagonist [@KoMe94]. Self-activation is typically present by some kind of feedback. In activator–substrate-depletion type Turing mechanisms the production of the activator induces a decrease of the substrate. Diffusion-driven instabilities typically require large differences in the diffusion coefficients of the activator and its antagonist. In many biological applications this is not realistic and Turing type mechanisms can therefore not explain symmetry breaking events. In our context, however, cytosolic diffusion is typically much faster than lateral diffusion. Coupled systems of 2D and 3D reaction–diffusion processes might therefore be a candidate for a realistic Turing mechanism.
Distinct mathematical models for the GTPase cycle have been proposed and analyzed, with diverse conclusions. A general model for signaling molecules in a cell that cycle between a non-recruiting cytosolic state and a recruiting membrane-bound one has been evaluated in [@AAWW08]. There the emergence of cell polarity has been demonstrated for an intrinsically *stochastic* mechanism for self-activation by positive feedback. A corresponding deterministic model in contrast has been shown not to produce any heterogeneous pattern. However, the deterministic PDE model in [@AAWW08] does not directly treat processes in the cytosol as all variables are membrane bound and all reactions are local. A complex PDE model that accounts for chemical reactions, membrane-cytoplasm exchange and diffusion is given in [@GoPo08]. There a scaling factor accounts for the different volume of a thin 3D membrane layer and the inner volume. Variables representing membrane bound molecules and variables representing cytosolic quantities however both have the same domain of definition. Numerical simulations and a linear stability analysis show that the model allows for a Turing mechanism. The formation of micro-domains in a GTPase cycle model are also studied in [@BDKR07]. Here the equations are formulated on a flat membrane surface. It is shown that no Turing pattern can occur unless an extra flux term is included. This flux term accounts for interactions between GTPase and membrane proteins and represents a phase separation type energy gradient. As an alternative explanation for the emergence of cell polarity in GTPase mediated processes a ‘wave-pinning’ mechanism is proposed in [@MoJE08]. A two component system for the nucleotide cycle is suggested with a Hills type non-linearity that leads to a bistability. Domains are formed by emerging traveling waves that are stopped by a decreased supply of non-activated GTPase.
Our goal is to introduce a model for the GTPase cycle with an improved coupling of processes with different dimensionalities. We investigate whether a Turing type instability – of activator–substrate depletion type – could possibly explain the localization of activated GTPase on the membrane. In Section \[sec:model\] we will first formulate our mathematical model and derive a reduction that only incorporates membrane-bound active and membrane-bound inactive GTPase. We perform a stability analysis and numerical simulations for this reduction. The explicit dimensional coupling in the full model is still reflected by the appearance of a *non-local* term. We show in Section \[sec:turing\] that for this model Turing patterns are possible. Our numerical simulations in Section \[sec:numerics\] confirm this result and shed some light on the kind of patterns that are supported by our model and the influence of changes in different parameters. We develop here a general numerical scheme that can be extended to general membrane geometries and to more involved coupling laws. In Section \[sec:no-turing\] we investigate – even for a more general class of similar models – whether for equal diffusion constants of activator and substrate, *i.e.* activated and non-activated GTPase, Turing pattern are possible. Our results will finally be discussed in Section \[sec:discussion\].
Acknowledgment {#acknowledgment .unnumbered}
==============
We would like to thank Roger Goody and Yaowen Wu from the Max-Planck institute for molecular physiology for introducing us to the biochemistry of signaling networks.
Model Description {#sec:model}
=================
Mechanistic description of the GTPase cycle
-------------------------------------------
Here we briefly review the key steps of the GTPase cycle as indicated in Fig. \[fig:cycle\]. Chemically, the difference between the active and inactive state of the GTPase is that in the active state *guanine-tri-phosphate* (GTP) is bound whereas *guanine-di-phosphate* (GDP) is bound in the inactive state. Only activated GTPase interacts with downstream effectors. Activation of a GTPase is by exchange of GDP by GTP, inactivation by hydrolysis and dephosphorylation of GTP to GDP. Both processes are intrinsically very slow and need the catalyzation by a GEF (*guanine exchange factor*) and GAP protein (*GTPase activating protein*), respectively [@GuAG05; @BoRW07]. Cytosolic GTPase can only be found in complex with a displacement inhibitor (GDI) that prevents the binding of GTPase to the membrane. As the affinity of GTPase towards GDI is much higher when GDP is bound, predominantly the inactive state occurs in the cytosol [@GoRa05]. How GDP-bound GTPase is released from the complex with GDI and how it associates to the membrane is less clear, mediation by a GDI displacement factor (GDF) has been proposed as a possible mechanism [@Pfef03]. For several GTPase positive feedback loops have been identified that support the activation of GTPase. Activated GTP-Rab5 is known to recruit a cytosolic GEF-effector complex (Rabex5 and Rabaptin5) to the membrane and to increase the activity of the GEF [@GrON06]. A similar feedback loop has been found for activated Cdc42 GTPase [@WAWL03]. In the following we formulate a mathematical model that reflects the key features of a GTPase cycle. Our main focus is on the treatment of the dimensional coupling and less on a detailed description of the reaction kinetics.
![\[fig:cycle\] The GTPase reaction cycle: The activation of GDP-bound GTPase is either catalyzed by GEF (lower semi circle) or by an effector–GEF–GTP-GTPase complex (upper semi circle). The inactivation of GTP-bound GTPase is catalyzed by GAP. Further reactions that are depicted are GDP-GDPase–GDI complex formation/dissociation and effector–GEF–GTP-GTPase complex formation/dissociation. See the text for additional information.](./GTPaseCycle5AR-1.pdf){width="95.00000%"}
The mathematical model
----------------------
We restrict ourselves to the investigations of processes in the cytosol and at the outer plasma membrane only. Inner organelles with additional membrane boundaries could be included as well. The cytosolic volume of a cell is represented by a bounded, connected, open domain $B \subset {\mathbb{R}}^3$ and the cell membrane by the boundary of $B$ that we assume to be given by a smooth, closed two-dimensional surface $\Gamma := {\partial}B$ without boundary. In addition we fix a time interval of observation $I:=[0,T] \subset {\mathbb{R}}$. We formulate a system of PDE’s for the following unknowns: $$\begin{aligned}
V &: \overline B \times I \to{\mathbb{R}}&&\text{concentration of cytosolic
GDP-GTPase (in complex with GDI)},\\
v &:\Gamma \times I \to {\mathbb{R}}&&\text{concentration of membrane-bound GDP-GTPase},\\
u &:\Gamma \times I \to {\mathbb{R}}&&\text{concentration of membrane-bound GTP-GTPase},\\
m &:\Gamma \times I \to {\mathbb{R}}&&\text{concentration of membrane-bound,
effector--GEF--GTP-GTPase complex},\\
g&:\Gamma \times I \to {\mathbb{R}}&&\text{concentration of membrane-bound GEF.}\end{aligned}$$ We prescribe initial conditions at time $t=0$, $$\begin{aligned}
&V(\cdot, 0) \,=\, V_0,\quad v(\cdot, 0) \,=\, v_0,\quad
u(\cdot, 0) \,=\, u_0,\quad m(\cdot, 0) \,=\, m_0, \quad
g(\cdot, 0) \,=\, g_0,\\
&V_0\,:\, B\,\to\, {\mathbb{R}},\quad v_0,u_0,m_0,g_0\,:\,\Gamma\,\to\,{\mathbb{R}}.\end{aligned}$$ The coupling condition between cytosolic and membrane processes involves a Neumann boundary condition for $V$ that is specified below. Physical units are given by $$[V] = \frac{\text{mol}}{\text{m}^3}, \quad
[u] = [v] = [m] = [g] = \frac{\text{mol}}{\text{m}^2}.$$ Much more extended sets of variables could be considered here. In particular we do not explicitly take into account the effector and GAP concentrations. Catalyzation of the inactivation process will be described implicitly.
### Reaction Kinetics
We assume simple mass action kinetics or a Michaelis–Menten type law for catalyzed reactions. For the change of concentration of the above variables due to reactions we prescribe the following equations. Our choices here are similar to the more general model in [@GoPo08].
The concentration of membrane-bound GDP-GTPase is decreased by the activation process, which is catalyzed by both GEF and the effector–GEF–GTP-GTPase complex. For the corresponding rates we assume that they are proportional to the GDP-GTPase concentration and the concentrations of the catalysts. Vice versa GDP-GTPase is produced by the inactivation of GTP-GTPase. Since we have not taken the GAP concentration into account, we here assume a Michaelis–Menten law for the kinetics. The change of $v$ due to activation and inactivation we therefore describe by $$\begin{aligned}
\label{eq:v_kin}
[{\partial}_t v]_{\text{reaction}} &= - k_1 v g - k_2 v m + k_3 \frac{u}{u
+ k_4} \quad \text{on} \quad \Gamma
\times I.\end{aligned}$$ For the change in $u$ we have in addition to the processes above the production of $u$ by dissociation of the effector – GEF – GTP-GTPase complex and the loss due to the formation of this complex. We model this by the equation $$\begin{aligned}
\label{eq:u_kin}
[{\partial}_t u]_{\text{reaction}} &= k_1 v g + k_2 v m - k_3 \frac{u}{u + k_4} -
k_5 ug + k_{-5}m
\quad \text{on} \quad \Gamma \times I.\end{aligned}$$ Correspondingly complex formation and dissociation lead to the following laws for the concentration of the complex and GEF (or rather a GEF–effector complex as we do not take explicitly into account the effector). $$\begin{aligned}
\label{eq:m_kin}
[{\partial}_t m]_{\text{reaction}} &= k_5 u g - k_{-5} m \quad
\text{on} \quad \Gamma \times I,\\
\label{eq:g_kin}
[{\partial}_t g]_{\text{reaction}} &= - k_5 u g + k_{-5}m \quad
\text{on} \quad \Gamma \times I.\end{aligned}$$ Units for the reaction rates $k_i$ are given by $$[k_1] = [k_2] = [k_5] = \frac{\text{m}^2}{\text{mol}\cdot\text{s}}; \quad
[k_3] = \frac{\text{mol}}{\text{m}^2\text{s}} ; \quad
[k_4] = \frac{\text{mol}}{\text{m}^2} ; \quad
[k_{-5}] = \frac{1}{\text{s}}.$$
### Simplified Kinetics
For later use in the mathematical analysis we use a quasi–steady state approximation for the complex formation to do a first reduction of our kinetic laws. We assume $$m = \frac{k_5}{k_{-5}}ug \label{eq:m-stst}$$ and use GEF–conservation $$m + g = \text{const} =: \bar{g}_0, \label{eq:gef-cons}$$ where $[\bar{g}_0] = [g] = \frac{\text{mol}}{\text{m}^2}$. If we take initial data $m_0=0$ we have $\bar{g}_0=g_0$. Equations , yield $$\begin{aligned}
m &= \frac{K_5ug_0}{1 + K_5u},\\
g &= g_0\left(1 - \frac{K_5u}{1 + K_5 u}\right)\end{aligned}$$ with $K_5:=\frac{k_5}{k_{-5}}$ and $[K_5] =
\frac{\text{m}^2}{\text{mol}}$. From this, one obtains simplified rate equations for the change due to reactions $$\begin{aligned}
[{\partial}_t v]_{\text{reaction}} &= - k_1 v g_0\left(1 - \frac{K_5u}{1 +
K_5 u}\right) - k_2 v \frac{K_5ug_0}{1 + K_5u} + k_3 \frac{u}{u
+ k_4} \quad \text{on} \quad \Gamma \times I,\\
[{\partial}_t u]_{\text{reaction}} &= k_1 v g_0\left(1 - \frac{K_5u}{1 +
K_5 u}\right) + k_2 v \frac{K_5ug_0}{1 + K_5u} - k_3 \frac{u}{u +
k_4} = - [{\partial}_t v]_{\text{reaction}}
\quad \text{on} \quad \Gamma \times I.\end{aligned}$$
### Diffusion
We describe cytosolic diffusion of the (inactive) GTPase in $B$ by the standard Laplace diffusion operator $\Delta$ and a diffusion constant $D>0$. Lateral diffusion on the membrane is described by the Laplace–Beltrami–operator ${\Delta_{\Gamma}}$ (which is the generalization of the ordinary Laplacian to surfaces [@DiHS10]) and diffusion constants $d_u$ and $d_v$ for the active and inactive membrane-bound GTPase concentrations, respectively. $$\begin{aligned}
\label{eq:V_diff}
[{\partial}_t V]_{\text{diffusion}} &= D \Delta V \quad \text{in} \quad B
\times I,\\
\label{eq:u_diff}
[{\partial}_t u]_{\text{diffusion}} &= d_u {\Delta_{\Gamma}}u \quad \text{on} \quad
\Gamma \times I,\\
\label{eq:v_diff}
[{\partial}_t v]_{\text{diffusion}} &= d_v {\Delta_{\Gamma}}v \quad \text{on} \quad \Gamma
\times I.\end{aligned}$$
### Membrane attachment and detachment
We describe the association and dissociation of inactive GTPase at the membrane by a flux boundary condition for $V$ at $\Gamma$ $$\label{eq:flux1}
-D \nabla V \cdot \nu = q \quad \text{on} \quad \Gamma,$$ where $\nu$ denotes the outer normal to $B$ at $\Gamma$. For the flux $q$ we formulate a constitutive equation: membrane attachment is treated as a reaction between cytosolic GTPase and a free site on the membrane and modeled by a Langmuir rate law [@Kell09]. Detachment is taken proportional to the inactive GTPase concentration, which together gives the equation $$\label{eq:flux2}
q = b_6 \frac{|B|}{|\Gamma|}V (c_{\max} - u - v)_+ - b_{-6}v.$$ Here $c_{\max}$ denotes a saturation value and $b_6, b_{-6}$ are sorption coefficients. By $ (c_{\max} - u - v)_+$ we denote the positive part of $c_{\max} - u - v$ as adsorption stops when the saturation value is reached. $|B|$ and $|\Gamma|$ denote the $3$-dimensional volume of $|B|$ and the $2$-dimensional surface area of $\Gamma$, respectively. In $\frac{|B|}{|\Gamma|}V$ has to be understood as the trace of the cytosolic GDP-GTPase concentration and has units $\frac{\text{mol}}{\text{m}^2}$. The units of the various coefficients are given by $$[D] = [d_u] = [d_v] = \frac{\text{m}^2}{\text{s}}; \quad
[b_6] = \frac{\text{m}^2}{\text{mol} \cdot \text{s}}; \quad
[b_{-6}] = \frac{1}{\text{s}}.$$
### Reaction, Diffusion, and attachment/detachment
Taking reaction and diffusion into account, one obtains the following model, which is the basis of further considerations $$\begin{aligned}
\label{eq:V-dim}
{\partial}_t V &= D \Delta V \quad \text{in} \quad B
\times I,\\
\label{eq:u-dim}
{\partial}_t u &= d_u {\Delta_{\Gamma}}u + k_1 v g_0\left(1 - \frac{K_5u}{1 +
K_5 u}\right) + k_2 v \frac{K_5ug_0}{1 + K_5u} - k_3 \frac{u}{u +
k_4}\quad \text{on} \quad
\Gamma \times I,\\
\label{eq:v-dim}
{\partial}_t v &= d_v {\Delta_{\Gamma}}v - k_1 v g_0\left(1 - \frac{K_5u}{1 +
K_5 u}\right) - k_2 v \frac{K_5ug_0}{1 + K_5u} + k_3 \frac{u}{u
+ k_4} + q\quad \text{on} \quad \Gamma
\times I\end{aligned}$$ with the flux conditions , . The model satisfies conservation of GTPase in the form $$\frac{\rm{d}}{\rm{d} t} \left(\int_B V {\, {\rm{d}} x}+ \int_\Gamma (u + v)
{\, {{\rm{d}}}\sigma(x)}\right) =0,$$ where ${\, {{\rm{d}}}\sigma(x)}$ denotes integration with respect to the surface area measure. This equation confirms that the total number of cytosolic inactive plus membrane-bound active and inactive GTPase is constant over time.
### Non–Dimensionalization
For $x \in \overline B$ and $t \in I$, we introduce non-dimensional coordinates $\xi$ $$\begin{aligned}
\xi := \frac1{R}x, \end{aligned}$$ where $R > 0$ denotes a typical length, e.g. half the diameter of the cell. We represent this length as $R=\sqrt{\gamma}\,\mathbb{I}$ with $\gamma>0$ and $\mathbb{I}=1\text{m}$ denoting the unit length. Furthermore, we use a dimensionless time $$\tau := \frac{d_u}{R ^2} t.$$ This leads to transformed domain $\tilde B := \{\xi \in {\mathbb{R}}^3 : R
\xi \in B \}$, $\tilde \Gamma := {\partial}\tilde B$ and time interval $\tilde
I := [0,\frac{d_u}{R ^2}T]$. Non-dimensional Rab concentrations are defined through $$\tilde V := \frac{R}{c_{\max}} V , \quad
\tilde u := \frac{1}{c_{\max}} u, \quad
\tilde v := \frac{1}{c_{\max}} v.$$ We introduce dimensionless quantities $$\begin{gathered}
a_1 := \frac{\mathbb{I} ^2}{d_u}k_1g_0, \quad
a_2 := \frac{1}{K_5c_{\max}}, \quad
a_3 := \frac{k_2}{k_1}a_1, \quad
a_4 := \frac{\mathbb{I} ^2}{d_u c_{\max}} k_3, \quad
a_5 := \frac{k_4}{c_{\max}}, \\
a_6 := \frac{\mathbb{I}^2 b_6}{d_u} c_{\max}\frac{|B|}{|\Gamma|R},\quad
a_{-6} := \frac{\mathbb{I} ^2 b_{-6}}{d_u},\quad
d := \frac{d_v}{d_u}, \quad
\tilde D := \frac{D}{d_u}.\end{gathered}$$ Note that $\frac{|B|}{|\Gamma|R}$ is scale invariant in the sense that multiplying the system size by a constant factor $\alpha>0$ does not affect this value. In particular all above constants are independent of the system size, which is solely represented by the dimensionless quantity $\gamma$. With these definitions, one easily verifies $$\begin{aligned}
\label{eq:V_tilde}
{\partial}_\tau \tilde V &= \tilde D \Delta_\xi\tilde V \quad \text{in} \quad
\tilde B \times \tilde I,\\
\label{eq:u_tilde}
{\partial}_\tau \tilde u &= {\Delta_{\tilde \Gamma}}\tilde u + \gamma\Big(\left(a_1 + (a_3 -
a_1)\frac{\tilde u}{a_2 + \tilde u} \right)\tilde v - a_4
\frac{\tilde u}{a_5 +\tilde u}\Big)\quad \text{on} \quad
\tilde \Gamma \times \tilde I,\\
\label{eq:v_tilde}
{\partial}_\tau \tilde v &= d {\Delta_{\tilde \Gamma}}\tilde v + \gamma\Big(-\left(a_1 + (a_3 -
a_1)\frac{\tilde u}{a_2 + \tilde u} \right)\tilde v + a_4
\frac{\tilde u}{a_5 +\tilde u} + \tilde q\Big)\quad \text{on} \quad \tilde
\Gamma \times \tilde I\end{aligned}$$ with the flux condition $$-\tilde D \nabla_\xi \tilde V \cdot \tilde \nu = \gamma \tilde q \quad
\text{on} \quad \tilde \Gamma \times \tilde I$$ with $$\label{eq:q_tilde}
\tilde q =
a_6 \tilde V (1 - (\tilde u + \tilde v))_+ - a_{-6} \tilde v.$$
### Reduction
We further reduce the non-dimensional model of the previous section. Our reduction is motivated by the observation that the cytosolic diffusion coefficient is much larger than that of the lateral diffusion on the membrane [@PBLH04]. We thus assume $\tilde V$ to be spatially constant, i.e. $\tilde V =
\tilde V(\tau)$ depends only on time but not on the $\xi$ variable. If we also assume that the initial concentration of cytosolic GTPase is spatially homogeneous the concentration is then for positive times determined by GTPase conservation, $$\label{eq:V_tilde2}
\tilde V(\tau) = \bar{V}_0 - c \int_{\tilde \Gamma} (\tilde u + \tilde v)(\xi,\tau) {\, {{\rm{d}}}\sigma(\xi)}$$ where $c := |\tilde B|^{-1}$ and where $\bar{V}_0$ is given by the initial conditions, $$\begin{gathered}
\bar{V}_0 \,=\, \tilde{V}(0) + c \int_{\tilde \Gamma} (\tilde u + \tilde v)(0,\xi) {\, {{\rm{d}}}\sigma(\xi)}.\end{gathered}$$ In particular $\bar{V}_0=\tilde{V}(0)$ if initially no membrane-bound GTPase was present. In the following, we then consider the system – of reaction diffusion equations on $\tilde \Gamma \times \tilde I$ including the flux and the conservation law .
The fully coupled system converges in the limit $D\to\infty$ to this reduced model. However, no estimates for the difference between solutions to the respective models are at present available. The ratio between cytosolic and lateral membrane diffusion reported in the literature [@PBLH04] is of order $10^2$. Numerical experiments for the full system with diffusion coefficients $d=D=10^3$ showed qualitative agreement with the reduction.
Turing pattern {#sec:turing}
==============
In this section we investigate the stability properties and the possibility of Turing-type pattern formation for the dimensionless reduced model derived above. In the following we drop all tildes and denote the space and time variables by $x$ and $t$ respectively. This yields the system $$\begin{aligned}
\partial_t u \,&=\, \Delta_\Gamma u + \gamma f(u,v), \label{eq:u}\\
\partial_t v \,&=\, d\Delta_\Gamma v + \gamma \left(-f(u,v) + q(u+v,v,V[u+v])\right) \label{eq:v}\end{aligned}$$ where $$\begin{aligned}
f(u,v)\,&=\, \left(a_1 + (a_3 - a_1)\frac{u}{a_2 + u} \right) v -
a_4\frac{u}{a_5 +u},\\
q(u+v,v,V)\,&=\, a_6 V (1 - (u + v))_+ - a_{-6} v,\end{aligned}$$ and where $V[u+v]$ is the *non-local* functional $$\begin{gathered}
V[u+v]\,=\, V_0 - c \int_{\Gamma} (u + v) {\, {{\rm{d}}}\sigma(x)}, \label{eq:V}\end{gathered}$$ with $V_0$ given. For convenience we also define $$\begin{gathered}
g(u,v)\,=\, -f(u,v) + q(u+v,v,V[u+v]).\end{gathered}$$ System , has to be solved on $\Gamma\times I$. Our particular interest is to understand the effect of the non-local term in system , on the stability properties. We can not use a predefined set of ‘realistic’ parameter values: first our model is very general and applies to several specific cases with different set of parameters; second kinetic rates etc. are difficult to obtain experimentally. We therefore rather investigate whether *in principle*, i.e. for *some* parameter values, our model allows for stationary states, whether stationary states of substrate–depletion type exists, and whether Turing type instabilities are possible. It is quite difficult to guess parameters that allow for example for Turing pattern formation. We therefore derive conditions for the parameters that are sufficient to ensure certain behavior, in particular showing that the *Turing space* is not empty. With such a set of parameters identified it is possible to explore the boundaries of the Turing space and then compare whether the parameter ranges are reasonably close to available estimates for ‘realistic’ values.
To start with our stability analysis we first observe that spatially homogeneous solutions of , satisfy the ODE system $$\begin{aligned}
\partial_t u \,&=\, \gamma f(u,v), \label{eq:ode-u}\\
\partial_t v \,&=\, \gamma \left(-f(u,v) + q_0(u+v,v)\right) \label{eq:ode-v}\end{aligned}$$ where $$\begin{gathered}
q_0(u+v,v)\,=\, a_6 \left(V_0 -c|\Gamma|(u+v)\right)(1 - (u + v))_+ - a_{-6}v.\end{gathered}$$ We set $g_0(u,v)\,=\, -f(u,v) + q_0(u+v,v)$. The set of values for $u,v$ described by $$\begin{gathered}
{\mathcal A}\,:=\, \big\{ u,v \,\geq\, 0 \,: \, u+v \leq \min\{1,m\}\big\},\quad m\,:=\,\frac{V_0}{c|\Gamma|}\end{gathered}$$ is an invariant region for , , *i.e.* if the initial data are in this set the solution does not leave it. In fact we observe that at the boundaries of ${\mathcal A}$ we obtain $$\begin{gathered}
f(0,v)\,\geq\, 0,\quad g_0(u,0)\,\geq 0 \quad \text{ for all }
0\,\leq\,u,v\,\leq \min\{1,m\},\\
f(u,v)+g_0(u,v) \,\leq\,0\quad\text{ for all } u,v\geq 0,\,
u+v=\min\{1,m\}. \end{gathered}$$ By these inequalities the conclusion follows.
Under suitable conditions on the data we next show the existence of a stationary spatially homogeneous state $(u_*,v_*)\in{\mathcal A}$ for , . This means that $(u_*,v_*)$ has to satisfy $$\begin{aligned}
0\,&=\, f(u_*,v_*), \label{eq:f=0}\\
0\,&=\, g_0(u_*,v_*). \label{eq:g=0}\end{aligned}$$ The first equation is satisfied if and only if $$\begin{gathered}
v\,= v[u]\,:=\, \frac{a_4 u(a_2+u)}{(a_5+u)(a_1a_2+a_3u)}. \label{eq:def-vu}\end{gathered}$$ We compute $$\begin{gathered}
\label{eq:v-prime}
v'[u]=0 \quad\Leftrightarrow\quad \big(a_1a_2+a_3(a_5-a_2)\big)u^2 + 2a_1a_2a_5u +a_1a_2^2a_5\,=\,0.\end{gathered}$$ If we assume $$\begin{aligned}
a_2\,&> a_5, \label{eq:ass1}\\
2 a_1 a_2 \,&<\, a_3(a_2-a_5) \label{eq:ass2}\end{aligned}$$ we find that $v[\cdot]$ has a unique positive stationary point $u_0$, $$\begin{gathered}
u_0\,=\, \frac{a_1 a_2 a_5}{a_3(a_2-a_5)-a_1a_2} + a_2\sqrt{a_1a_5}\frac{\sqrt{(a_3-a_1)(a_2-a_5)}}{a_3(a_2-a_5)-a_1a_2}.\end{gathered}$$ In particular we have $$\begin{gathered}
v'[u]\,<\, 0\quad\text{ for all }u>u_0. \label{eq:est-v-prime}\end{gathered}$$ By , we estimate $$\begin{aligned}
u_0\,&\leq\, \sqrt{a_1a_5}a_2 \Big( \frac{2\sqrt{a_1a_2}}{a_3(a_2-a_5)} + 2\frac{1}{\sqrt{a_3(a_2-a_5)}}\Big)\label{eq:est-u0}\\
&\,\leq\, 4\frac{\sqrt{a_1a_5}a_2}{\sqrt{a_3(a_2-a_5)}}. \notag\end{aligned}$$ For the maximum value $v_0:=v[u_0]$ we obtain $$\begin{gathered}
v_0\,=\, a_4\frac{a_2+2 u_0}{a_1a_2+a_3(a_5+2u_0)}.\end{gathered}$$ Using and a short calculation yields $$\begin{aligned}
v_0 \,&\leq\, \frac{a_2a_4}{a_1a_2 +a_3a_5}\,\leq\, \frac{a_2a_4}{a_3a_5}, \label{eq:upper-v0}\\
v_0\,&\geq\, \frac{a_4a_5}{a_1a_2+a_3a_5}\,\geq\, \frac{a_4a_5}{a_3a_2}. \label{eq:lower-v0}\end{aligned}$$ If we then choose $$\begin{aligned}
\frac{a_2a_4}{a_3a_5}\,&<\, \frac{1}{4}\min\{m,1\}, \label{eq:ass3} \\
a_1\,&<\, 2^{-8}\frac{a_3^2(a_2-a_5)^2}{a_2^2a_5}(\min\{m,1\})^2 \label{eq:ass4}\end{aligned}$$ we deduce by , that $u_0+v_0<\frac{1}{2}\min\{m,1\}$.
In order to satisfy we need to find $u>0$ with $u+v[u]<\min\{m,1\}$ such that $$\begin{gathered}
0\,=\, a_6 \left(V_0 -c|\Gamma|(u+v[u])\right)(1 - (u + v[u])) - a_{-6}v[u]\,=:\,\Phi(u).\end{gathered}$$ We evaluate $$\begin{gathered}
\Phi(u_0)\,>\, \frac{a_6}{4}V_0 - a_{-6}\frac{a_4a_5}{a_3a_2}\end{gathered}$$ and assuming $$\begin{gathered}
\frac{a_{-6}}{a_{6}}\,\leq\,\frac{V_0}{4}\frac{a_3a_2}{a_4a_5} \label{eq:ass5}\end{gathered}$$ we see that $\Phi(u_0)>0$. On the other hand there exists $u_1>u_0$ such that $u_1+v[u_1]=\min\{m,1\}$ and we observe that $$\begin{gathered}
\Phi(u_1)\,<\, 0.\end{gathered}$$ Since $\Phi$ is continuous we obtain from the intermediate-value Theorem that there exists $u_0<u_*<u_1$ such that $\Phi(u_*)=0$. But this implies that $(u_*,v_*)\in{\mathcal A}$, $v_* = v[u_*]$, is a stationary point of ,. In summary we have proved the following Proposition.
\[prop:stat-sol\] Assume that the conditions $$\begin{aligned}
a_2\,&>\, a_5, \label{cdt:1}\\
4a_2a_4\,&<\, a_3a_5\min\{m,1\}, \label{cdt:2}\\
4a_4a_5a_{-6}\,&<\, V_0 a_2a_3a_6, \label{cdt:3}\\
a_1\,&<\, \min\Big\{ \frac{a_3(a_2-a_5)}{2a_2}\,,\,2^{-8}\frac{a_3^2(a_2-a_5)^2}{a_2^2a_5}(\min\{m,1\})^2 \Big\}\label{cdt:4}\end{aligned}$$ are satisfied. Then there exists a stationary spatially homogeneous solution $(u_*,v_*)\in{\mathcal A}$ of , .
The stationary point $(u_*,v_*)$ is under suitable assumptions on the data linearly stable against spatially homogeneous perturbations. For a brief summary of the classical stability analysis and of the Turing mechanism for two-variable reaction–diffusion systems we refer to Appendix \[app:turing\].
\[prop:stab\] Assume that - hold and that moreover $$\begin{aligned}
2a_4(a_2-a_5)\,&<\, a_3a_5^2, \label{cdt:5}\\
a_{-6}\,&<\, a_6 c|\Gamma||1-m| \label{cdt:6}\end{aligned}$$ are satisfied. Then $(u_*,v_*)$ is a stable stationary point of , . This system is in $(u_*,v_*)$ of activator–substrate-depletion type, where $u$ acts as an activator and $v$ as substrate.
We show that the stability conditions , are satisfied. We first observe that since $a_1<\frac{a_3}{2}$ by $$\begin{aligned}
\partial_v f(u,v)\,&=\, a_1 + (a_3 - a_1)\frac{u}{a_2 + u} \label{eq:pd-f-v}\\
&>\, 0\quad\text{ for all }u>0. \label{eq:del-v-f}\end{aligned}$$ For the function $v[\cdot]$ defined in we have $f(u,v[u])=0$. This yields $$\begin{gathered}
0\,=\,\partial_u f(u,v[u])\,=\, (\partial_u f)(u,v[u]) + (\partial_v f)(u,v[u])v'[u].\end{gathered}$$ Since $u_*>u_0$ we deduce from that $v'[u_*]<0$ and obtain $$\begin{gathered}
\partial_u f(u_*,v_*)\,=\, - \partial_vf(u_*,v_*)v'[u_*]\,>\,0. \label{eq:del-u-f}\end{gathered}$$ Furthermore we have $$\begin{aligned}
\partial_u q_0(u,v)\,&=\, -a_6 c|\Gamma|\big(1+m-2(u+v)\big)\,<\,0,\label{eq:del-u-q0}\\
\partial_v q_0(u,v)\,&=\, -a_6 c|\Gamma|\big(1+m-2(u+v)\big)-a_{-6}\,<\,0. \label{eq:del-v-q0}\end{aligned}$$ By - the stationary point $(u_*,v_*)$ is of activator–substrate-depletion type. To check the criteria for Turing type instabilities we need to estimate combinations of derivatives. We first compute $$\begin{gathered}
\partial_uf \,=\, \frac{a_2(a_3-a_1)}{(a_2+u)^2}v - \frac{a_4a_5}{(a_5+u)^2}.\end{gathered}$$ Evaluating this expression at $(u,v)=(u_*,v_*)$ and using we deduce $$\begin{aligned}
\partial_uf \,&=\, \frac{a_2(a_3-a_1)a_4 u}{(a_2+u)(a_5+u)(a_1a_2+a_3u)}- \frac{a_4a_5}{(a_5+u)^2}\notag\\
&\leq\, \frac{a_2 a_3a_4}{(a_2+u)(a_5+u)a_3}- \frac{a_4a_5}{(a_5+u)^2} \notag\\
&=\, \frac{a_4(a_2 -a_5)u}{(a_2+u)(a_5+u)^2}. \label{eq:pd-f-u}\end{aligned}$$ We thus obtain in $(u,v)=(u_*,v_*)$ that $$\begin{aligned}
\partial_uf + \partial_v g_0 \,\leq\,& \frac{a_4(a_2 -a_5)u}{(a_2+u)(a_5+u)^2} -\Big(a_1 + (a_3 - a_1)\frac{u}{a_2 + u}\Big) \notag\\
&-a_6 c|\Gamma|\big(1+m-2(u+v)\big)-a_{-6}\notag\\
\leq\,& \frac{a_4(a_2 -a_5)u}{(a_2+u)(a_5+u)^2} -a_3\frac{u}{a_2 + u}\,
<\, 0 \label{eq:turing-cdt1}\end{aligned}$$ by . This verifies condition . We next estimate in $(u,v)=(u_*,v_*)$ $$\begin{aligned}
&\partial_uf\partial_vg_0 - \partial_vf\partial_ug_0 \notag\\
=\, &\partial_uf(-\partial_vf + \partial_vq_0) - \partial_vf(-\partial_uf + \partial_uq_0)\notag\\
=\, & \partial_uq_0(\partial_uf-\partial_vf)-a_{-6}\partial_uf\notag\\
\geq\,& -a_6 c|\Gamma|\big(1+m-2(u+v)\big)\Big(\frac{a_4(a_2 -a_5)u}{(a_2+u)(a_5+u)^2} - a_3\frac{u}{a_2 + u}\Big) - a_{-6}\frac{a_4(a_2 -a_5)u}{(a_2+u)(a_5+u)^2}\notag\\
=\,& -\frac{u}{(a_2+u)(a_5+u)^2}\Big(a_6 c|\Gamma|\big(1+m-2(u+v)\big)\big(a_4(a_2 -a_5)-a_3(a_5+u)^2\big)+a_{-6}a_4(a_2 -a_5)\Big) \label{eq:Tu-hilf}\end{aligned}$$ By and since $1+m-2(u+v)\geq |1-m|$ the term in the brackets in the last line is estimated by $$\begin{aligned}
&\,a_6 c|\Gamma|\big(1+m-2(u+v)\big)\big(a_4(a_2 -a_5)-a_3(a_5+u)^2\big)+a_{-6}a_4(a_2 -a_5)\\
\leq\,& -a_6 c|\Gamma||1-m|a_4(a_2 -a_5)+a_{-6}a_4(a_2 -a_5)\,<\, 0,\end{aligned}$$ where the last estimate follows from . Together with the last equation implies $$\begin{gathered}
\partial_uf\partial_vg_0 - \partial_vf\partial_ug_0 \,>\,0,\end{gathered}$$ and therefore holds. Thus the ODE system is linearly stable.
We next evaluate the response of the full reaction–diffusion system to perturbations of the spatially homogeneous solution $(u_*,v_*)$ in direction of arbitrary smooth functions $\varphi,\psi:\Gamma\times(0,T)\to{\mathbb{R}}$. In particular we have to linearize the non-local functional $V=V[u+v]$ that occurs in the source term $q$ in . With this aim we consider a variation $(u_s,v_s)$ of $(u_*,v_*)$ in direction of $(\varphi,\psi)$, $$\begin{gathered}
u_s,v_s \,:\, \Gamma\times (0,T)\,\to\,{\mathbb{R}},\quad s\in (-1,1),\\
u_s\big|_{s=0}\,=\, u_*,\quad v_s\big|_{s=0}\,=\, v_*,\quad \frac{\partial}{\partial s}\Big|_{s=0}u_s\,=\, \varphi, \quad \frac{\partial}{\partial s}\Big|_{s=0}v_s\,=\,\psi.\end{gathered}$$ The corresponding linearization of $V$ is then given by $$\begin{aligned}
\frac{d}{ds}\big|_{s=0} V[u_s+v_s]\,=\, -c\frac{d}{ds}\big|_{s=0} \int_\Gamma (u+v)\,{\, {{\rm{d}}}\sigma(\xi)}\,=\, -c\int_{\Gamma} (\varphi + \psi) {\, {{\rm{d}}}\sigma(\xi)}. \label{eq:lin-V}\end{aligned}$$ For the linearization of , we therefore obtain $$\begin{aligned}
\partial_t \varphi \,&=\, \Delta_\Gamma \varphi + \gamma \partial_u f(u_*,v_*)\varphi + \gamma\partial_vf(u_*,v_*)\psi, \label{eq:u-lin}\\
\partial_t \psi \,&=\, d\Delta_\Gamma \psi + \gamma \big(-\partial_u f(u_*,v_*)\varphi -\partial_v f(u_*,v_*) \psi +\partial_1q(u_*+v_*,v_*,V_*)(\varphi+\psi)\big)+ \label{eq:v-lin}\\
&\qquad \qquad + \gamma\Big(\partial_2q(u_*+v_*,v_*,V_*)\psi- c \partial_3q(u_*+v_*,v_*,V_*)\int_{\Gamma} (\varphi + \psi) {\, {{\rm{d}}}\sigma(\xi)}\Big). \notag\end{aligned}$$ We next decompose the direction of perturbation $(\varphi,\psi)$ in $L^2(\Gamma)$ in a part that is spatially homogeneous and a part that is orthogonal to the constants. Since spatially homogeneous perturbations have already been analyzed in Proposition \[prop:stab\] it suffices to assume that we have $$\begin{gathered}
\int_\Gamma \varphi {\, {{\rm{d}}}\sigma(x)}\,=\, \int_\Gamma \psi{\, {{\rm{d}}}\sigma(x)}\,=\, 0.\end{gathered}$$ Equation shows that for such directions $V$ is unchanged to first order. Therefore, with respect to variations in direction of functions that are orthogonal to the constants, the linearizations of , coincide with that of the system $$\begin{aligned}
\partial_t u \,&=\, \Delta_\Gamma u + \gamma f(u,v), \label{eq:u-het}\\
\partial_t v \,&=\, d\Delta_\Gamma v + \gamma \left(-f(u,v) + q_1(u+v,v)\right) \label{eq:v-het}\end{aligned}$$ in $(u_*,v_*)$, where $$\begin{aligned}
q_1(u+v,v)\,&=\, q(u+v,v,V_*)\,=\, a_6 V_* (1 - (u + v)) - a_{-6} v, \label{eq:def-q1}\end{aligned}$$ $V_*=V[u_*+v_*]$ For convenience we define $$\begin{gathered}
g_1(u,v)\,=\, -f(u,v) + q_1(u+v,v).\end{gathered}$$ Thus we see that the non-local term in the full system , leads to a difference in the linearization with respect to homogeneous or heterogeneous perturbations, that can be understood as a change (from $q_0$ to $q_1$) in the source term. Below we show that a range of parameters exists where $(u_*,v_*)$ is an unstable stationary state. We first need some estimates on $u_*,v_*$ to prepare the proof.
\[lem:estimates\] Assume , , , and $$\begin{gathered}
a_1a_2\,<\, \frac{a_3}{1+a_3^2}. \label{cdt:7}\end{gathered}$$ Then $$\begin{aligned}
v_*\,&<\, \frac{a_4 a_2}{a_3a_5}, \label{eq:upper-v-star}\\
u_*\,&>\, \frac{1}{2}\min\{1,m\}, \label{eq:lower-u-star}\\
v_*\,&>\, \frac{a_4(a_2+1)(a_3^2+1)\min\{1,m\}}{2(a_5+1)(a_3^2+2)a_3}, \label{eq:lower-v-star}\end{aligned}$$ holds.
By and $v_*<v_0$ we deduce . From $q_0(u_*,v_*)=0$ we obtain that $(u_*,v_*)$ is a solution of $$\begin{gathered}
0\,=\, a_6c|\Gamma|\big(m-(u+v)\big)\big(1-(u+v)\big) - a_{-6}v. \label{eq:def-uv}\end{gathered}$$ If we denote by $u[v]\in (0,\min\{1,m\})$ the solution of for given $0<v<\min\{1,m\}$ we observe that $u$ is decreasing in $v$. By , we have $v_*<v_1:=\frac{1}{4}\min\{1,m\}$ and therefore $u_*> u_1$, $u_1:=u[v_1]$. By and we get $$\begin{aligned}
u_1\,&=\, \frac{1}{2}(m+1-2v_1) - \sqrt{\frac{1}{4}(m+1-2v_1)^2 -(m-v_1)(1-v_1)+ \frac{a_{-6}v_1}{a_6c|\Gamma|}}\\
&\geq\, \frac{1}{4}(2\max\{1,m\}+\min\{1,m\}) - \sqrt{\frac{1}{4}(m-1)^2 + \frac{1}{4}|1-m|\min\{1,m\}} \\
&=\, \frac{1}{2}\min\{1,m\},\end{aligned}$$ which proves . Next we obtain from $f(u,v)=0$ for $(u,v)=(u_*,v_*)$ that $$\begin{aligned}
v\,=\, \frac{a_4u(a_2+u)}{(a_5+u)(a_1a_2+a_3u)}\,\geq\, \frac{a_4(a_2+1)u}{(a_5+1)(a_1a_2+a_3u)}\,\geq\, \frac{a_4(a_2+1)(a_3^2+1)u}{(a_5+1)(a_3^2+2)a_3},\end{aligned}$$ where we have used and . By this yields .
\[thm:turing\] Let $(u_*,v_*)$ be the stationary state found in Proposition \[prop:stat-sol\] and let the parameters in , satisfy all the conditions -. If in addition $$\begin{aligned}
a_1\,&<\, \min\Big\{\frac{\min\{1,m\}a_3}{2a_2(a_3^2+1)}\,,\, \frac{\min\{1,m\}^2a_3(a_2-a_5)}{4a_2(a_2+1)^2(a_3^2+1)}\Big\}, \label{cdt:8}\\
d\,&>\, \frac{2\big(a_3(a_3^2+2)+(a_2+1)(a_3^2+1)(a_6V_0 +a_{-6})\big)(a_3^2+2)(a_5+1)^2}{\min\{1,m\}a_3^2a_4(a_2-a_5)(a_3^2+1)},\label{cdt:d1}\\
d\,&>\, \frac{4(a_3^2+2)^2(a_5+1)^2\big(a_3^2a_4(a_2-a_5)\min\{1,m\}+4(a_3^2+2)a_6V_0(a_2+1)(a_5+1)^2\big)}{a_3^3(a_3^2+1)a_4^2(a_2-a_5)^2\min\{1,m\}^2}\label{cdt:d2}\end{aligned}$$ then there exists $\gamma>0$ such that $(u_*,v_*)$ is linearly unstable.
We show that the conditions , stated in Appendix \[app:turing\] for the instability of , are satisfied. Let us start with by estimating the different partial derivatives. $$\begin{aligned}
\partial_uf(u_*,v_*)\,&=\, \frac{a_2(a_3-a_1)a_4u(a_5+u)-a_4a_5(a_1a_2+a_3u)(a_2+u)}{(a_2+u)(a_5+u)^2(a_1a_2+a_3u)}\\
&=\, \frac{a_4\Big(a_3(a_2-a_5)u^2-a_1a_2(2a_5u+u^2+a_2a_5)\Big)}{(a_2+u)(a_5+u)^2(a_1a_2+a_3u)}.\end{aligned}$$ We next observe that by , and $$\begin{aligned}
a_1a_2(2a_5u+u^2+a_2a_5)\,&\leq\, a_1a_2(1+a_2)^2\,\leq\, \frac{a_3}{1+a_3^2}(a_2-a_5)u^2,\notag\\
a_1a_2 \,&\leq\, \frac{a_3}{1+a_3^2}u.\label{cdt:7a}\end{aligned}$$ We therefore deduce that $$\begin{aligned}
\partial_uf(u_*,v_*)\,&\geq\, \frac{a_4(a_3-\frac{a_3}{1+a_3^2})(a_2-a_5)u^2}{(a_2+u)(a_5+u)^2(\frac{a_3}{1+a_3^2}+a_3)u}\notag\\
&=\, \frac{a_4a_3^2(a_2-a_5)u}{(a_2+1)(a_5+1)^2(a_3^2+2)}\notag\\
&\geq\, \frac{a_4a_3^2(a_2-a_5)\min\{1,m\}}{2(a_2+1)(a_5+1)^2(a_3^2+2)}.\label{eq:lower-dfu}\end{aligned}$$ Next we estimate $$\begin{aligned}
\partial_v g_1(u_*,v_*) \,&=\, -\Big(a_1 + (a_3 - a_1)\frac{u}{a_2 + u}\Big) -\Big(a_6 \big(V_0-c|\Gamma|(u+v)\big) +a_{-6}\Big)\\
&\geq\, -\frac{a_1a_2+a_3u}{a_2+u} -\big(a_6V_0 +a_{-6}\big)\\
&\geq\, -\frac{a_3(a_3^2+2)}{(a_2+1)(a_3^2+1)}-\big(a_6V_0 +a_{-6}\big),\end{aligned}$$ where we have used . Together with we deduce from that holds.
Next we verify the condition , *i.e.* $$\begin{gathered}
D\,:=\,(d\partial_uf+\partial_vg_1)^2 -4d(\partial_uf\partial_vg_1-\partial_vf\partial_ug_1)\,>\, 0. \end{gathered}$$ We compute $$\begin{gathered}
\partial_uf\partial_vg_1-\partial_vf\partial_ug_1\,=\, \partial_uf\partial_vq_1 -\partial_vf\partial_uq_1\end{gathered}$$ and obtain for the left hand side $$\begin{aligned}
D\,&=\,d^2(\partial_uf)^2 +2d\Big(-\partial_uf(\partial_vf+\partial_vq_1)+2\partial_vf\partial_uq_1\Big) +(\partial_vg_1)^2\\
&\geq\, d\Big[ d(\partial_uf)^2 -2\Big(\partial_uf\partial_vf-2\partial_vf\partial_uq_1\Big)\Big].\end{aligned}$$ Moreover $$\begin{aligned}
\partial_vf\,&=\, \frac{a_1a_2+a_3u}{a_2+u}\,\leq\, \frac{a_3(a_3^2+2)u}{(a_2+u)(a_3^2+1)}\,\leq\, \frac{a_3(a_3^2+2)}{(a_2+1)(a_3^2+1)},\\
-\partial_uq_1\,&=\,a_6V_*\,\leq\, a_6V_0.\end{aligned}$$ This yields $$\begin{aligned}
D\,&\geq\, d(\partial_uf)^2\Big[ d - \frac{2a_3(a_3^2+2)}{(a_2+1)(a_3^2+1)(\partial_uf)} - \frac{4a_3(a_3^2+2)a_6V_0}{(a_2+1)(a_3^2+1)(\partial_uf)^2}\Big].\end{aligned}$$ We then deduce from and . The conclusion now follows from [@Jost07].
The above conditions ensure that perturbations with eigenvectors of the Laplace–Beltrami operator on $\Gamma$ corresponding to eigenvalues in a certain interval are unstable. As this interval scales linearly with $\gamma$ we obtain a range of values for $\gamma$ where a Turing instability exists.
We conclude this section with some comments on the implications of the conditions derived above.
By Theorem \[thm:turing\] parameters satisfying - and - belong to the *Turing space* where diffusive instabilities exist. Some of these conditions can be easily interpreted. The requirement $a_2>a_5$ concerns the Michaelis–Menten constants appearing in the catalyzed reactions: the affinity of GEF towards activated GTPase (forming the GEF–GTP-GTPase–effector complex) has to be higher than the affinity of GAP towards activated GTPase. Several conditions require $a_1$ to be (much) smaller than $a_3$, which means that activation by the GEF–GTP-GTPase–effector complex is stronger than activation by single GEF molecules, which in fact has been reported for example in the case of Rab5 GTPase [@LMRR01]. The conditions , for a Turing instability are most critical, as a substantially larger lateral diffusion coefficient for inactive GTPase compared to the lateral diffusion coefficient for active GTPase is required. We investigate below whether this condition is due to the particular choices of kinetic and sorption laws or rather a general feature of the kind of (reduced) model that we are considering. Finally, the condition on $\gamma$ requires a certain minimal size of the system to allow for a Turing instability.
Stability analysis for equal lateral diffusion {#sec:no-turing}
==============================================
As in most applications no substantial difference in the diffusion coefficients of the GDP-bound and GTP-bound GTPase is present we investigate in this section the possibility of Turing pattern in , for the case $d=1$ of equal lateral diffusion. The non-locality of our model – the remnant of the full 2D-3D coupling in our reduction – changes the classical stability analysis. Therefore, in contrast to the classical case, Turing pattern for $d=1$ might become possible. However, we show here that in our simple model this is not the case.
The set-up in this section is as follows. We assume a system of the general form $$\begin{aligned}
\partial_t u \,&=\, \Delta_\Gamma u + \gamma f(u,v), \label{eq:u-gen}\\
\partial_t v \,&=\, \Delta_\Gamma v + \gamma \left(-f(u,v) + q(u+v,v,V[u+v])\right) \label{eq:v-gen}\end{aligned}$$ where $u,v$ denote GTP-bound and GDP-bound GTPase concentrations, respectively, and where $V[u+v]$ represents the cytosolic (inactive) GTPase concentration that is given by the mass conservation condition . The nonlinear terms $f,q$ account for the activation/deactivation processes and from the attachment/detachment of GTPase at the membrane. For $q$ we assume that $$\begin{gathered}
\partial_1 q\,\leq\, 0,\quad \partial_2q\,\leq\, 0, \quad \partial_3q\,\geq\, 0,\label{cdt:q}\end{gathered}$$ which are natural condition with respect to the interpretation of $q$ as the flux induced by ad- and desorption of GTPase at the membrane. As before, the system , has to be solved on $\Gamma\times I$.
We assume a spatially homogeneous stationary point $(u_*,v_*)$ of the ODE reduction of , , $$\begin{aligned}
\partial_t u \,&=\, \gamma f(u,v), \label{eq:u-gen-ode}\\
\partial_t v \,&=\, \gamma \left(-f(u,v) + q_0(u+v,v)\right) \label{eq:v-gen-ode}\end{aligned}$$ where $$\begin{gathered}
q_0(u+v,v)\,=\, q(u+v,v,V_0(u+v)),\quad V_0(u+v)\,=\,V_0 -c|\Gamma|(u+v).\end{gathered}$$ We set as before $$\begin{gathered}
g(u,v)\,:=\, -f(u,v) + q(u+v,v,V[u+v]),\qquad g_0(u,v)\,=\, -f(u,v) + q_0(u+v,v).\end{gathered}$$ The main result of this section is the following theorem.
\[thm:no-turing\] Assume that $(u_*,v_*)$ is of activator–substrate depletion type, that is $$\begin{aligned}
\partial_uf(u_*,v_*)\,&>0\, & \partial_vf(u_*,v_*)\,&>\, 0, \label{eq:acsub1}\\
\partial_ug_0(u_*,v_*)\,&<0\, & \partial_vg_0(u_*,v_*)\,&>\, 0. \label{eq:acsub2}\end{aligned}$$ Then no Turing type instability of , exists.
The conditions , to ensure that $(u_*,v_*)$ is a stable stationary point of , are $$\begin{aligned}
0\,&>\, \partial_u f +\partial_v g_0\,=\, \partial_u f -\partial_v f + \partial_1 q + \partial_2 q +\partial_3qV_0', \label{eq:Tu-gen-1}\\
0\,&<\, \partial_u f\cdot\partial_vg_0 -\partial_vf\cdot\partial_ug_0\,
=\, \partial_u f(\partial_1 q+\partial_2q+\partial_3qV_0') - \partial_v f (\partial_1 q+\partial_3qV_0'). \label{eq:Tu-gen-2}\end{aligned}$$ This corresponds to the stability of , in $(u_*,v_*)$ with respect to spatially homogeneous perturbations. Therefore, for the instability with respect to general perturbations it is sufficient to consider perturbations in direction of functions perpendicular to the constants. As above we observe that such perturbations leave $V[u,v]$ unchanged. The respective linearization corresponds to that of the system $$\begin{aligned}
\partial_t u \,&=\, \Delta_\Gamma u + \gamma f(u,v), \label{eq:u-gen-het}\\
\partial_t v \,&=\, d\Delta_\Gamma v + \gamma \left(-f(u,v) + q_1(u+v,v)\right) \label{eq:v-gen-het}\end{aligned}$$ in $(u_*,v_*)$, where $$\begin{aligned}
q_1(u+v,v)\,&=\, q(u+v,v,V_*),\quad V_*=V[u_*+v_*]=V_0(u_*,v_*). \label{eq:def-q1-gen}\end{aligned}$$ We set as before $g_1(u,v)\,=\, -f(u,v) + q_1(u+v,v)$. Then the conditions , for the instability of , with respect to spatially heterogeneous perturbations yield $$\begin{aligned}
0\,&<\, \partial_u f+ \partial_v g_1
\,=\, \partial_u f-\partial_vf + \partial_1 q +\partial_2 q, \label{eq:Tu-gen-3}\\
0\,&<\, (\partial_u f +\partial_v g_1)^2 - 4 (\partial_uf\cdot\partial_vg_1-\partial_vf\cdot\partial_ug_1) \notag\\
&=\, (\partial_u f -\partial_v f -\partial_1 q +\partial_2 q)^2 -4\partial_uf \cdot\partial_2 q + 4\partial_1q\cdot\partial_2q.\label{eq:Tu-gen-4}\end{aligned}$$ By our assumptions , we observe that the last condition is automatically satisfied. On the other hand, we obtain from that $$\begin{gathered}
0\,<\, (\partial_u f-\partial_vf)(\partial_1 q+\partial_3qV_0')
+\partial_u f \partial_2q. \label{eq:Tu-gen-2b}\end{gathered}$$ By and we deduce that $\partial_u f-\partial_vf\,=\,-(\partial_1 q +\partial_2 q)\,>\,0$. Using again and using that $V_0'\leq 0$ this yields that the first term on the right-hand side in is nonpositive. But we also find by , that the second term is nonpositive. This is a contradiction. Therefore the system has no Turing-type instabilities.
Numerical Approach {#sec:numerics}
==================
We use a finite element discretization similar to the one described in [@LaVo10]. It is implemented in the adaptive finite element toolbox AMDiS [@VeVo07].
Discretization
--------------
Following the surface finite element method described in [@DzEl07], we choose a triangulated discrete approximation $\Gamma_h$ of the membrane $\Gamma$ and a triangulation $\mathcal
T_h$. We split the time interval $[0,T]$ by discrete time instants $t_0 < t_1 < \dots < t_{M}$, from which one gets the time steps $\Delta t_m := t_{m + 1} - t_m$, $m = 0, 1, \dots, M-1$. Given initial conditions $u(\cdot,0) = u_0$, $v(\cdot,0) = v_0$ with $u_0,v_0 \in
H^1(\Gamma_h)$ and time discrete solutions $u^{(m)}, v^{(m)} \in
H^1(\Gamma_h)$, $m = 1, \dots, M$, we linearize all nonlinear terms $$\begin{aligned}
f(u^{(m+1)}, v^{(m+1)}) \approx f(u^{(m)}, v^{(m)}) + \nabla
f(u^{(m)}, v^{(m)}) \cdot
\begin{pmatrix}
u^{(m+1)} - u^{(m)}\\
v^{(m+1)} - v^{(m)}
\end{pmatrix}\end{aligned}$$ and $$\begin{aligned}
q(u^{(m+1)}, v^{(m+1)}, V^{(m+1)}) \approx q(u^{(m)}, v^{(m)},
V^{(m)}) \\ + \nabla_{(u,v)}
q(u^{(m)}, v^{(m)}, V^{(m)}) \cdot
\begin{pmatrix}
u^{(m+1)} - u^{(m)}\\
v^{(m+1)} - v^{(m)}
\end{pmatrix}.\end{aligned}$$ We introduce test functions $\eta^u, \eta^v \in H^1(\Gamma_h)$ and end up with a weak formulation and semi-implicit time discretization for $u^{(m+1)}, v^{(m+1)} \in H^1(\Gamma_h)$ of , $$\begin{aligned}
\frac1{\Delta t_m}&\int_{\Gamma_h}u^{(m+1)}\eta^u
+ \int_{\Gamma_h}{\langle {\nabla_{\Gamma}}u^{(m+1)},{\nabla_{\Gamma}}\eta^u \rangle_{\Gamma_h}}
- \gamma\int_{\Gamma_h} \nabla f(u^{(m)}, v^{(m)}) \cdot
\begin{pmatrix}
u^{(m+1)}\\
v^{(m+1)}
\end{pmatrix}
\eta^u\\
&= \frac1{\Delta t_m}\int_{\Gamma_h}u^{(m)}\eta^u
+ \int_{\Gamma_h} F_e(u^{(m)}, v^{(m)}) \eta^u
\quad \forall \eta^u \in H^1(\Gamma_h)\\
\frac1{\Delta t_m}&\int_{\Gamma_h}v^{(m+1)}\eta^v
+ d\int_{\Gamma_h}{\langle {\nabla_{\Gamma}}v^{(m+1)},{\nabla_{\Gamma}}\eta^v \rangle_{\Gamma_h}}
+ \gamma\int_{\Gamma_h} \nabla f(u^{(m)}, v^{(m)}) \cdot
\begin{pmatrix}
u^{(m+1)}\\
v^{(m+1)}
\end{pmatrix}
\eta^u\\
&+ \gamma\int_{\Gamma_h} \nabla q(u^{(m)}, v^{(m)}, V^{(m)}) \cdot
\begin{pmatrix}
u^{(m+1)}\\
v^{(m+1)}
\end{pmatrix}
\eta^u\\
&= \frac1{\Delta t_m}\int_{\Gamma_h}v^{(m)}\eta^v
- \int_{\Gamma_h} F_e(u^{(m)}, v^{(m)}) \eta^v
+ \int_{\Gamma_h} Q_e(u^{(m)}, v^{(m)}, V^{(m)}) \eta^v
\quad \forall \eta^v \in H^1(\Gamma_h),\end{aligned}$$ where $$\begin{aligned}
F_e(u^{(m)}, v^{(m)}) &:= \gamma f(u^{(m)}, v^{(m)}) - \gamma \nabla
f(u^{(m)}, v^{(m)}) \cdot
\begin{pmatrix}
u^{(m)}\\
v^{(m)}
\end{pmatrix}\\
Q_e(u^{(m)}, v^{(m)}, V^{(m)}) &:= \gamma q(u^{(m)}, v^{(m)},V^{(m)}) -
\gamma\nabla_{(u,v)} q(u^{(m)}, v^{(m)}, V^{(m)}) \cdot
\begin{pmatrix}
u^{(m)}\\
v^{(m)}
\end{pmatrix}.\end{aligned}$$ Furthermore, the non-local relation for $V^{(m)}$ is treated explicitly by $$V_h^{(m+1)} = V_0 - \frac1{|B_h|}\int_{\Gamma_h}(u^{(m)}+v^{(m)})$$ for given $V_0$ and the inner $B_h$ of $\Gamma_h$. To discretize in space, let $\mathbb V_h$ the finite element space of globally continuous, piecewise linear elements. In addition, with $(\psi_i)_i$ the standard nodal basis of $\mathbb V_h$ and $u_h^{(m+1)},
v_h^{(m+1)} \in \mathbb V_h$ we write $u_h^{(m+1)} = \sum \limits_i
U_i^{(m+1)}\psi_i$ and $v_h^{(m+1)} = \sum \limits_i V_i^{(m+1)}
\psi_i$ with $U_i^{(m+1)}, V_i^{(m+1)} \in {\mathbb{R}}$. Furthermore, we define ${{\boldsymbol U}}^{(m+1)}=(U_i^{(m+1)})_i$ and ${{\boldsymbol V}}^{(m+1)}=(V_i^{(m+1)})_i$. This leads to the linear system of equations $$\begin{gathered}
\begin{pmatrix}
\frac1{{\Delta t_m}}{{\boldsymbol M}} + {{\boldsymbol A}} - {{\boldsymbol F_u^{\text{impl}}}}
& \frac{1}{{\Delta t_m}} {{\boldsymbol M}} - {{\boldsymbol F_v^{\text{impl}}}} \\
{{\boldsymbol F_u^{\text{impl}}}} - {{\boldsymbol Q_u^{\text{impl}}}}
& \frac{1}{{\Delta t_m}} {{\boldsymbol M}}
+ d {{\boldsymbol A}} + {{\boldsymbol F_v^{\text{impl}}}} - {{\boldsymbol Q_v^{\text{impl}}}}
\end{pmatrix}
\begin{pmatrix}
{{\boldsymbol U}}^{(m+1)} \\
{{\boldsymbol V}}^{(m+1)}
\end{pmatrix}\\
=
\begin{pmatrix}
\frac{1}{{\Delta t_m}} {{\boldsymbol M}} {{\boldsymbol U}}^{(m)} + {{\boldsymbol F^{\text{expl}}}} \\
\frac{1}{{\Delta t_m}} {{\boldsymbol M}}{{\boldsymbol V}}^{(m)}
- {{\boldsymbol F^{\text{expl}}}} + {{\boldsymbol Q^{\text{expl}}}}
\end{pmatrix}\end{gathered}$$ with $$\begin{aligned}
{{\boldsymbol M}} &= (M_{ij}) &M_{ij} &= (\psi_i, \psi_j)_{\Gamma_h},\\
{{\boldsymbol A}} &= (A_{ij}) & A_{ij}&= (\nabla \psi_i, \nabla
\psi_j)_{\Gamma_h},\\
{{\boldsymbol A}}^2 &= (A_{ij}^2) & A_{ij}^2 &= (A({\nabla}\phi_h^{(m)})\nabla
\psi_i, \nabla \psi_j)_{\Gamma_h}, \\
{{\boldsymbol F_u^{\text{impl}}}} &= ((F_u^{\text{impl}})_{ij}) & (F_u^{\text{impl}})_{ij} &=
(\partial_uf(u_h^{(m)},v_h^{(m)}) \psi_i, \psi_j)_{\Gamma_h},\\
{{\boldsymbol F_v^{\text{impl}}}} &= ((F_v^{\text{impl}})_{ij}) & (F_v^{\text{impl}})_{ij} &=
(\partial_vf(u_h^{(m)},v_h^{(m)}) \psi_i, \psi_j)_{\Gamma_h},\\
{{\boldsymbol Q_u^{\text{impl}}}} &= ((Q_u^{\text{impl}})_{ij}) & (Q_u^{\text{impl}})_{ij} &=
(\partial_uq(u_h^{(m)},v_h^{(m)},V_h^{(m)}) \psi_i, \psi_j)_{\Gamma_h},\\
{{\boldsymbol Q_v^{\text{impl}}}} &= ((Q_v^{\text{impl}})_{ij}) & (Q_v^{\text{impl}})_{ij} &=
(\partial_vq(u_h^{(m)},v_h^{(m)},V_h^{(m)}) \psi_i, \psi_j)_{\Gamma_h},\\
{{\boldsymbol F^{\text{expl}}}} &= (F^{\text{expl}}_{i}) & F^{\text{expl}}_{i} &=
(F_e(u_h^{(m)},v_h^{(m)}), \psi_i)_{\Gamma_h},\\
{{\boldsymbol Q^{\text{expl}}}} &= (Q^{\text{expl}}_{i}) & Q^{\text{expl}}_{i} &=
(Q_e(u_h^{(m)},v_h^{(m)}), \psi_i)_{\Gamma_h},\end{aligned}$$ where $(\cdot, \cdot)_{\Gamma_h}$ denotes $L^2$-scalar product. The above linear system has to be solved in every time step, which is done by a stabilized bi-conjugate gradient method (BiCGStab).
Numerical Results
-----------------
First, we present numerical results reproducing the results of the stability analysis in Section \[sec:turing\]. To be more precise, we choose a set of parameters fulfilling the conditions of Theorem \[thm:turing\] sufficient for instability, which is the basis of our further numerical investigations: $$\label{eq:parameters}
d=1000;\;a_1 = 0; \; a_2 = 20; \; a_3 = 160; \; a_4 = 1; \; a_5 = 0.5; \; a_6
= 0.1;\; a_{-6} = 1;\; \gamma = 400.$$ Furthermore, we consider the unit-sphere $\Gamma = S^2$ and random initial conditions $u_0, v_0: \Gamma_h \to [0,0.02]$ and $V_0 =
10$. Fig. \[fig:basic\] shows the corresponding discrete solutions $u_h, v_h$ at different times. A stationary pattern with a single spot appears.
![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/u00_0.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/u00_5.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/u05_0.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/u25_0.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/legend_u.jpg "fig:"){width="3.50000%"}\
![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/v00_0.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/v00_5.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/v05_0.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/v25_0.jpg "fig:"){width="14.00000%"} ![\[fig:basic\]*From left to right: the discrete solutions $u_h,v_h$ for $t=t_0=0$, $t = 0.5$, $t = 5$, and $t
= 25$.*](./figures/basic/legend_v.jpg "fig:"){width="3.50000%"}
### Varying Parameters
Based on the choice of parameters we investigate the influence of varying parameters. First, we observe that doubling the parameter $a_2$ leads to spatially homogeneous stationary solutions, whereas halving $a_2$ leads to stationary patterns with two maxima for $u_h$ (see Fig. \[fig:a2\]).
![\[fig:a2\]*From left to right: the discrete solutions $u_h,v_h$ for $a_2=40$ (left) and $a_2=10$ (right) $t = 25$.*](./figures/a240/u25.jpg "fig:"){width="22.00000%"} ![\[fig:a2\]*From left to right: the discrete solutions $u_h,v_h$ for $a_2=40$ (left) and $a_2=10$ (right) $t = 25$.*](./figures/a240/v25.jpg "fig:"){width="22.00000%"} ![\[fig:a2\]*From left to right: the discrete solutions $u_h,v_h$ for $a_2=40$ (left) and $a_2=10$ (right) $t = 25$.*](./figures/a210/u25.jpg "fig:"){width="22.00000%"} ![\[fig:a2\]*From left to right: the discrete solutions $u_h,v_h$ for $a_2=40$ (left) and $a_2=10$ (right) $t = 25$.*](./figures/a210/v25.jpg "fig:"){width="22.00000%"}
Additionally, we observe that halving the parameter $a_3$ leads to spatially homogeneous stationary solutions, whereas doubling $a_3$ leads to stationary patterns with two maxima for $u_h$ (see Fig. \[fig:a3\]).
![\[fig:a3\]*From left to right: the discrete solutions $u_h,v_h$ for $a_3=80$ (left) and $a_3=320$ (right) $t = 25$.*](./figures/a380/u25.jpg "fig:"){width="22.00000%"} ![\[fig:a3\]*From left to right: the discrete solutions $u_h,v_h$ for $a_3=80$ (left) and $a_3=320$ (right) $t = 25$.*](./figures/a380/v25.jpg "fig:"){width="22.00000%"} ![\[fig:a3\]*From left to right: the discrete solutions $u_h,v_h$ for $a_3=80$ (left) and $a_3=320$ (right) $t = 25$.*](./figures/a3320/u25.jpg "fig:"){width="22.00000%"} ![\[fig:a3\]*From left to right: the discrete solutions $u_h,v_h$ for $a_3=80$ (left) and $a_3=320$ (right) $t = 25$.*](./figures/a3320/v25.jpg "fig:"){width="22.00000%"}
Another interesting behavior can be seen by varying the diffusion constant $d$. While the estimates and lead to a condition $d \gtrsim 790$ sufficient for instability, an exact computation yields a maximal diffusion constant $d_c \approx 101$ satisfying equality in one of relations , . This is reproduced in Fig. \[fig:d\] showing stability for $d=100$ and instability for $d=105$.
![\[fig:d\]*From left to right: the discrete solutions $u_h,v_h$ for $d=100$ (left) and $d=105$ (right) $t = 25$.*](./figures/d100/u25.jpg "fig:"){width="22.00000%"} ![\[fig:d\]*From left to right: the discrete solutions $u_h,v_h$ for $d=100$ (left) and $d=105$ (right) $t = 25$.*](./figures/d100/v25.jpg "fig:"){width="22.00000%"} ![\[fig:d\]*From left to right: the discrete solutions $u_h,v_h$ for $d=100$ (left) and $d=105$ (right) $t = 25$.*](./figures/d105/u25.jpg "fig:"){width="22.00000%"} ![\[fig:d\]*From left to right: the discrete solutions $u_h,v_h$ for $d=100$ (left) and $d=105$ (right) $t = 25$.*](./figures/d105/v25.jpg "fig:"){width="22.00000%"}
Comparison with ‘realistic’ parameter ranges
--------------------------------------------
A full set of realistic parameters is not available, but there are several *in vivo* and *in vitro* measurements giving some estimates or average values. For the case of the Cdc42 GTPase cycle in yeast cells we have collected data from [@GoPo08], [@Jilk03], [@KaCh07], and [@GaGJ07] and evaluated our model for the following set of values, $$\begin{aligned}
&k_1 := 1.056831769\cdot 10^{-8} ,\quad k_2 := 0.1056831769\cdot
10^{-5} ,\quad k_3 := 946.2243938\\ & k_4 := 18.92448788 ,\quad k_{5}
:= 0.1056831769\cdot 10^{-2} ,\quad k_{-5} := 0.3 ,\quad b_6 :=
0.3170495307\cdot 10^{-1} \\& b_{-6} := 0.133 ,\quad g_0 :=
37848.97575 ,\quad d_u := 2.5 \cdot 10^{-15} ,\quad c_{max} :=
47311.21969 ,\\ &V_0 := 4.894264108\cdot10^{10}, \end{aligned}$$ where the units are as given in Section \[sec:model\].
We find for these values that a homogeneous stationary state of activator–substrate-depletion type in fact exists. For a Turing-type instability we need a lateral diffusion for activated GTPase of order $10^{-8}\text{m}^2\text{s}^{-1}$, which is unrealistically large. Nevertheless assuming such a value we obtain a condition on the spatial scale: We find that $R$ has to be at least of order $10^{-6}\text{m}$, which is close to the typical diameter of a yeast cell. We therefore see that the critical condition is in fact the large difference in diffusion.
The uncertainty in the parameter values is quite large: there are not enough data for individual proteins available and the parameters chosen above are therefore a composite of available data. Many measurements are taken *in vitro* and are only an estimate for *in vivo* conditions. Comparing different sources and different GTPases one may find variations up to order 10 in the parameters. Within the set of parameter values allowing for Turing instabilities we find values that are within the range of realistic values, except that the ratio $d$ of lateral diffusion values has to be of order at least $10^2$. Such a large difference in free lateral diffusion for active and inactive GTPase seems unrealistic. However, heterogeneities of the cell membrane may lead to differences in the ‘effective’ diffusion speed, see the discussion below.
Discussion {#sec:discussion}
==========
The GTPase cycle presents an example for a coupled system of processes in the inner volume and on the outer membrane. We have proposed a mathematical model in the form of a fully coupled PDE system. A two-variable reduction yields a *non-local* reaction–diffusion system on the membrane. With the interest in finding mechanisms that support the emergence of cell polarity we have investigated pattern forming properties. We have shown that the reduced model in principle supports Turing type diffusive instabilities, but – as for general local RD systems – needs large differences in diffusion constants. In numerical simulations we have confirmed our theoretical findings and have explored the type of pattern produced and the influence of parameter changes. Both the formulation of the model and the numerical schemes are prepared to investigate more complex models and to incorporate additional features.
In similar but different models diffusive instabilities have been shown to exist [@BaJo05], [@GoPo08], or not to exist [@AAWW08], [@BDKR07] (unless a phase separation force is added). Particularly interesting is a comparison with the work of Goryachev and Pokhilko [@GoPo08]. Their model is more detailed in the set of variables they consider and also accounts – at least partially – for the different dimensionalities of the processes: the membrane is thought as a thin compartment with positive volume and the cell as an adjacent bigger compartment. Concentrations on the membrane and in the inner cell are weighed with a factor that accounts for the different sizes of the volumes. A major difference to our work is that the mathematical analysis in [@GoPo08] treats all variables on one common domain of definition (for both cytosolic and membrane-bound quantities). In our approach on the other hand we distinguish explicitly between the cytosolic GTPase variable $V$ defined in $B$ and the membrane variables defined on $\Gamma$, which then makes laws for fluxes from the cytosol to the membrane necessary and meaningful. Nevertheless there are more similarities between these two approaches. Also in [@GoPo08] a non-local reduction to a two-variable system is given. The substrate there is represented by the sum of cytosolic and membrane bound GTPase and inherits a higher diffusion constant than the solely membrane bound active form. In view of pattern forming properties their model then produces Turing instabilities, in more realistic parameter ranges than our model.
One of the main features of our reduced model is that large differences in the lateral diffusion constant for active and inactive GTPase are required to obtain a Turing instability. Mathematical models with a simpler (but more artificial) dimensional coupling on the other hand do support diffusive instabilities. However, in these studies differences in lateral diffusion were put more directly into the model and Turing instabilities then appear as a consequence of this model design. Our analysis demonstrates that it is not clear whether large cytosolic diffusion is sufficient for cell polarization by a Turing mechanism. To clarify this more detailed studies are necessary. The absence of realistic Turing patterns in our model might be a consequence of our reduction to the infinite cytosolic diffusion limit. This leads to constant cytosolic GTPase concentration, whereas the Turing mechanism intimately relies on spatial heterogeneity. We have performed numerical experiments to compare the behavior of the fully coupled system and the reduced model. For large but finite cytosolic diffusion our simulations for the full model agreed qualitatively with corresponding simulations for the reduction. However, it is difficult to numerically explore the Turing space of the fully coupled model and error estimates for differences of solutions to the reduced model and the three-variable system are at present not available.
In our model various properties of the GTPase cycle in living cells have been neglected that in principle may contribute to polarization. In a recent paper by Butler and Goldenfeld [@BuGo11] it was shown that the inclusion of intrinsic noise in reaction-diffusion systems can lead to the formation of Turing-type ‘quasipatterns’ for parameter values substantially different from the Turing space for the corresponding noise-free system. The heterogeneity of the plasma membrane might influence polarization in living cells: Microdomains with different lipid composition are present, and active and inactive forms of GTPase possibly associate with different preferences to distinct microdomains. For the case of Ras GTPase in [@LSSS05] a severe reduction in Ras mobility has been observed upon activation. This effect might introduce a difference in diffusion sufficient for Turing patterns. Finally, other mechanisms different from Turing pattern formation might be responsible for cell polarization. Possible candidates are a wave pinning mechanism [@MoJE08], or an intrinsically stochastic mechanism that relies on a particle based approach (as proposed in [@AAWW08]) instead of a continuous model.
A better understanding of symmetry breaking properties in models where systems of different dimensionalities are coupled remains important. We have proposed a more detailed coupling model that presents a good basis for future extensions. A stability analysis of the fully coupled model and the inclusion of possible additional contributions to cell polarization are interesting tasks for further studies that might lead to a more complete picture of the origin of cell polarization.
Turing instability {#app:turing}
==================
Since in our GTPase cycle model the classical conditions for Turing type pattern have to modified by the non-locality of the source term, we briefly outline the analysis of the classical Turing mechanism. We follow here [@Jost07 Section 5.3]. Let a spatial domain $\Omega\subset {\mathbb{R}^n}$ be given and consider a system of reaction–diffusion equations $$\begin{aligned}
\partial_t u\,&=\, \Delta u + \gamma f(u,v), \label{eq:rd-1}\\
\partial_t v\,&=\, d\Delta v + \gamma g(u,v), \label{eq:rd-2}\end{aligned}$$ where $u=u(x,t)$, $v=v(x,t)$, $d>1$, $\gamma>0$ and where $f:{\mathbb{R}}^2\to{\mathbb{R}}$, $g:{\mathbb{R}}^2\to{\mathbb{R}}$ are given functions. We complement , first by initial conditions $$\begin{gathered}
u(x,0)\,=\,u^0(x),\qquad v(x,0)\,=\, v^0(x),\end{gathered}$$ for given $u^0,v^0:\Omega\,\to\,{\mathbb{R}}$, and second by zero Neumann-boundary data $$\begin{gathered}
\nabla u\cdot\nu_\Omega u\,=\, \nabla v\cdot\nu_\Omega\,=\, 0\qquad\text{ on }\partial\Omega,\end{gathered}$$ where $\nu_\Omega$ denotes the outer normal of $\Omega$. With this boundary conditions the following analysis carries immediately over to the case that the spatial domain is given by a compact closed smooth hypersurface in ${\mathbb{R}^n}$, with the only difference that the Laplace operator has to be replaced by the surface Laplace–Beltrami operator.
The Turing mechanism is described by a stationary point $(u_*,v_*)$ that is spatially homogeneous and linearly stable under spatially homogeneous perturbation, but that is linearly unstable under heterogeneous perturbations. We therefore consider now $(u_*,v_*) \in{\mathbb{R}}^2$ with $$\begin{gathered}
f(u_*,v_*)\,=\,0,\qquad g(u_*,v_*)\,=\, 0.\end{gathered}$$ For spatially homogeneous solutions , reduce to the ODE system $$\begin{aligned}
\partial_t u\,&=\, \gamma f(u,v), \label{eq:rd-ode-1}\\
\partial_t v\,&=\, \gamma g(u,v). \label{eq:rd-ode-2}\end{aligned}$$ The condition of linear stability then reduces to the conditions that the trace of the Jacobian $D(f,g)$ is negative and that the determinant of $D(f,g)$ is positive, *i.e.* $$\begin{aligned}
\partial_uf(u_*,v_*) + \partial_vg(u_*,v_*)\,&<\, 0, \label{eq:rd-Tu1}\\
\partial_uf(u_*,v_*)\partial_vg(u_*,v_*) - \partial_vf(u_*,v_*)\partial_ug(u_*,v_*)\,&>\,0. \label{eq:rd-Tu2}\end{aligned}$$ The linearization of , in $(u_*,v_*)$ for perturbations in direction of arbitrary smooth functions $\varphi,\psi:\Omega\times(0,T)\to{\mathbb{R}}$ is given by the system $$\begin{gathered}
\partial_t\begin{pmatrix} \varphi\\ \psi \end{pmatrix} \,=\, \begin{pmatrix} 1 & 0 \\ 0 &d\end{pmatrix}\begin{pmatrix} \varphi\\ \psi \end{pmatrix} + \gamma \begin{pmatrix} \partial_u f(u_*,v_*) & \partial_v f(u_*,v_*) \\ \partial_u g(u_*,v_*) & \partial_v g(u_*,v_*)\end{pmatrix}\begin{pmatrix} \varphi\\ \psi \end{pmatrix}. \label{eq:rd-lin}\end{gathered}$$ We next take the complete orthonormal basis $(w_j)_{j\in{\mathbb{N}}_0}$ of $L^2(\Gamma)$ given by eigenvectors of the Laplacian with respect to zero Neumann boundary data, $$\begin{aligned}
-\Delta w_j \,&=\, \lambda_j w_j\quad \text{ in }\Omega, \label{eq:eigen}\\
\nabla w_j\cdot \nu_\Omega \,&=\, 0 \quad\text{ on }\partial\Omega, \label{eq:eigen-bdry}\end{aligned}$$ for $j=0,1,2,\ldots$ and where $\lambda_0=0<\lambda_1\leq\lambda_2\leq\ldots$ denote the corresponding eigenvalues. By the orthonormality condition and , we have $$\begin{aligned}
\|w_j\|_{L^2(\Omega)}\,&=\,1\quad &\text{ for all }j\in{\mathbb{N}}_0, \label{eq:app-modes}\\
\int_\Omega w_j w_i \,dx \,&=\, \int_\Omega \nabla w_j\cdot\nabla w_i \,dx \,=\, 0 \quad &\text{ for all }i,j\in{\mathbb{N}}_0, i\neq j. \label{eq:app-onb}\end{aligned}$$ We then decompose $\varphi(\cdot,t),\psi(\cdot,t)$ with respect to this orthonormal basis, $$\begin{aligned}
\varphi(x,t)\,&=\, \alpha_0(t)w_0 + \sum_{j\in{\mathbb{N}}} \beta_j(t)w_j(x),\\
\psi(x,t)\,&=\, \beta_0(t)w_0 + \sum_{j\in{\mathbb{N}}} \beta_j(t)w_j(x).\end{aligned}$$ Inserting this representation in , and taking the $L^2(\Gamma)$ scalar product with $w_i$ by the orthonormality and the equation decomposes into linear systems $$\begin{gathered}
\partial_t\begin{pmatrix} \alpha_i\\ \beta_i \end{pmatrix} \,=\, -\lambda_i \begin{pmatrix} 1 & 0 \\ 0 &d\end{pmatrix} \begin{pmatrix} \alpha_i\\ \beta_i \end{pmatrix} + \gamma \begin{pmatrix} \partial_u f(u_*,v_*) & \partial_v f(u_*,v_*) \\ \partial_u g(u_*,v_*) & \partial_v g(u_*,v_*)\end{pmatrix}\begin{pmatrix} \alpha_i\\ \beta_i \end{pmatrix} \label{eq:app-alpha-beta}\end{gathered}$$ for $i=0,1,\ldots$. The case $i=0$ corresponds to the case of spatially homogeneous perturbations. In order to have an instability of , we therefore need that for an $i\in{\mathbb{N}}$ is unstable. This gives the necessary conditions [@Jost07 Theorem 5.3.1] $$\begin{aligned}
d\partial_uf(u_*,v_*) + \partial_vg(u_*,v_*)\,&>\, 0, \label{eq:rd-Tu3}\\
\big(d\partial_uf(u_*,v_*) + \partial_vg(u_*,v_*)\big)^2 -4d \Big(\partial_uf(u_*,v_*)\partial_vg(u_*,v_*) - \partial_vf(u_*,v_*)\partial_ug(u_*,v_*)\Big)\,&>\,0. \label{eq:rd-Tu4}\end{aligned}$$ In order to have an instability under these conditions it is sufficient that there exists an eigenvalue $\lambda_i$, $i\in{\mathbb{N}}$, such that $$\begin{gathered}
\mu_-\,<\,\lambda_i\,<\, \mu_+\end{gathered}$$ where $\mu=\mu_\pm$ are the roots of the quadratic equation $$\begin{gathered}
d\mu^2 -\gamma \big(d\partial_uf(u_*,v_*) + \partial_vg(u_*,v_*)\big)\mu + \gamma^2 \big(\partial_uf(u_*,v_*)\partial_vg(u_*,v_*) - \partial_vf(u_*,v_*)\partial_ug(u_*,v_*)\big) \,=\, 0.\end{gathered}$$
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<span style="font-variant:small-caps;">Wedlich-Soldner</span>, Roland ; <span style="font-variant:small-caps;">Altschuler</span>, Steve ; <span style="font-variant:small-caps;">Wu</span>, Lani ; <span style="font-variant:small-caps;">Li</span>, Rong: Spontaneous Cell Polarization Through Actomyosin-Based Delivery of the Cdc42 GTPase. *Science* 299 (2003), Nr. 5610, 1231-1235. <http://dx.doi.org/10.1126/science.1080944>. – DOI 10.1126/science.1080944
| tomekkorbak/pile-curse-small | ArXiv |
Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typing Clostridium difficile strains.
Clostridium difficile is a Gram-positive sporulating anaerobic bacillus which causes pseudomembranous colitis. Nosocomial acquisition of this bacteria has proved frequent, and epidemiological markers are needed to recognize and control common-source outbreaks. We therefore compared the results of pulsed-field gel electrophoresis (PFGE) after restriction with SmaI or NruI, random-amplified polymorphic DNA (RAPD) using 3 10-mer oligonucleotides, and ribotyping to differentiate between 30 unrelated strains of C. difficile belonging to 8 serotypes. The strains were separated into 26 different types by PFGE, 25 by RAPD, but into only 18 types by ribotyping. Median percentages of similarity between strains ranged from 27 in the PFGE assay to 90 in the ribotyping assay, but there was good agreement between the 3 methods for the clustering of strains. PFGE was more time-consuming than RAPD but its patterns were easier to analyze. | tomekkorbak/pile-curse-small | PubMed Abstracts |
All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Johne's disease is a chronic disease of cattle, causing major economic losses to the dairy and beef industry. The economic impact of Johne's disease is estimated to reach into the millions of dollars annually. A United States Department of Agriculture (USDA) study showed a loss of approximately \$200 per cow each year with an annual economic loss of between \$200 million to \$250 million dollars to the U.S. dairy industry \[[@pone.0154685.ref001]\]. In addition, Johne's disease can afflict sheep and various ruminant and non-ruminant wildlife species \[[@pone.0154685.ref002], [@pone.0154685.ref003]\], providing additional reservoirs for the disease causing pathogen, *Mycobacterium avium* subsp. *paratuberculosis* (MAP).
An immunologic reagent was developed in the early 1900s for skin testing of cattle as a simple way to determine exposure to MAP. This reagent, referred to as Johnin, consisted of heat-concentrated culture filtrate proteins obtained after long-term MAP culture in defined conditions. Advances to the production process with the addition of a protein precipitation step led to an improved product, termed purified protein derivative or PPD. When PPD was injected into the skin of a cow, a large erythema due to a delayed-type hypersensitivity (DTH) response measured 72 hours later would indicate MAP exposure. Testing methods that involve the use of PPD include skin testing \[[@pone.0154685.ref004], [@pone.0154685.ref005]\] and more recently it has been routinely incorporated as a stimulating antigen in the gamma interferon test for Johne's disease \[[@pone.0154685.ref006]--[@pone.0154685.ref011]\].
The National Veterinary Services Laboratories (NVSL) has been involved in *M*. *bovis* PPD and MAP PPD production since the early 1970's. All production lots must be tested for potency in guinea pigs prior to distribution and use for skin testing cattle. Historical production methods consisted of obtaining floating MAP cultures in Povitsky bottles, which was how the reference field lot 9801 was prepared, but have more recently transitioned to using Erlenmeyer flasks. Distribution of MAP PPD domestically is minor in comparison to *M*. *bovis* PPD and is used in the field on a limited basis. Contributing to the reduced demand for MAP PPD is the requirement for producers to use either serological assays or fecal culture, or a combination of both for determining a herd's eligibility in the USDA Voluntary Johne's Disease Control Program, which is no longer funded. With the emphasis on culture and serology as the approved testing methods, the number of U.S. dairy herds skin tested is projected to decrease. In contrast to lower domestic requests, the NVSL has had increased requests for MAP PPD from India and the United Kingdom. Having a consistent, well-characterized MAP PPD as part of an intradermal skin testing program or as an antigen in the IFN-γ assay is important for more accurate detection of preclinical stages of infection \[[@pone.0154685.ref010], [@pone.0154685.ref012]\], and may increase confidence for use of intradermal skin testing.
Proteomic analysis has been conducted on MAP PPD preparations previously \[[@pone.0154685.ref013], [@pone.0154685.ref014]\]. An analysis of PPD products from various laboratories and facilities revealed that the proteomic composition was highly similar and that variability observed was due to production protocols specifying differing MAP strains as well as the age of the MAP cultures at harvest \[[@pone.0154685.ref014]\]. The identified proteins in each PPD preparation varied greatly which may be related to its complexity, production processes or the MAP isolate used. Since the 1980s, the NVSL has used a single strain of MAP (ATCC 19698) for its PPD production, and that strain has not changed significantly with time due to defined culture limits that are set at five passages. Only six proteins were identified in the USDA NVSL PPD \[[@pone.0154685.ref014]\], but it is hypothesized that MAP PPD contains hundreds of proteins. Another study did not examine PPD produced from the ATCC 19698 strain \[[@pone.0154685.ref013]\], but did identify 156 proteins in the MAP 3+5/C strain.
The objectives of this investigation were to expand on the quantity of proteins identified in the NVSL MAP PPD preparations and determine if removal of the heating step improves the reagent. Furthermore, we examined if recombinant proteins could potentially replace PPD. Importantly, NVSL MAP PPD suspensions that have been used in the field for decades were selected for identification of the specific protein components. In addition, proteins were identified in other MAP PPD preps to determine which proteins were consistently detected by mass spectrometry. Specific proteins identified by mass spectrometry were further evaluated using potency and IFN-γ assays to determine reactivity as possible diagnostic reagents. Information from this study can be used toward improved production methods for a well characterized and consistent PPD as well as reveal specific proteins useful for *in-vitro* testing. Use of an improved reagent could also improve cell mediated immune response tests for earlier detection of MAP infected animals.
Materials and Methods {#sec002}
=====================
Bacteria, PPD Preparation and Source {#sec003}
------------------------------------
MAP strain ATCC 19698 was used in all the MAP PPD production processes. PPD lots produced at the NVSL in 1998 (9801), and ten years later in 2008 (0802, 0803A) were produced by traditional production methods involving autoclaving of production flasks containing floating cultures with subsequent protein precipitation using trichloroacetic acid (TCA) as described previously \[[@pone.0154685.ref015], [@pone.0154685.ref016]\]. Lot 9801 currently serves as a reference lot and has been used in the field for cattle skin testing purposes since its production in 1998. Lot 0902 was produced by an alternative production method involving sterile filtration through a 0.2 μm membrane (Millipore) in place of autoclaving followed by protein precipitation using TCA. Lots 0902A and 0902B, which comprised the larger PPD 0902 bulk preparation, were harvested on different days from the same culture inoculum. In production of large PPD batches (lots), multiple concentrated harvest bulks are commonly combined to reduce the number of potency tests conducted and allow for larger final product release volumes. Due to the alternative production process used for PPD 0902 these two smaller bulks were held as reserve on each harvest date to evaluate possible differences in this revised procedure. Purity testing was conducted in accordance with the Code of Federal Regulations, 9CFR part 113.26.
Protein concentration assay {#sec004}
---------------------------
Protein measurements of the final PPD preparations were conducted utilizing the Pierce microplate BCA protein assay procedure according to the manufacturer's directions (Thermo Scientific), with one modification. The compatibility reagent contained within the assay kit was not used because there were no interfering compounds present within the PPD preparations. Assay plates were read on a VersaMax microplate reader at a wavelength of 562 nm. Protein concentrations were calculated using SoftMax Pro software from a standard curve of bovine serum albumin protein standards.
SDS-PAGE and immunoblot analysis {#sec005}
--------------------------------
Pre-cast 4--12% Novex Bis-Tris gels (Invitrogen, 12 well, 1 mm thickness) or precast 10% Novex Bis-Tris gels (Invitrogen, 12 well, 1 mm thickness) were used for SDS-PAGE separation of PPD lots. MAP PPDs, *M*. *bovis* PPD, and *Mycobacterium avium* subsp *avium* (*M*. *avium*) PPD were standardized to approximately 1.0 mg/ml before loading 15 μg/well onto SDS-PAGE gels. MAP PPD's were prepared by the addition of 175 μl of sample buffer (4x NuPage LDS Sample Buffer, ThermoFisher Scientific) to 325 μl of PPD for each 500 μl of preparation and then heated at 70°C for ten minutes before loading onto gels.
Electrophoresis was conducted in an Invitrogen XCell SureLock Mini-Cell system at a constant current of 125 mA for 35 minutes. Invitrogen SeeBlue Plus2 prestained molecular weight standards served as markers for molecular weight determination. Gels were stained with Invitrogen Novex Colloidal Blue Stain according to the manufacturer's directions.
Pre-cast 4--12% Novex Bis-Tris gels (Invitrogen, 1 well, 1-mm thickness) were used for immunoblot analysis. A single, long well was loaded with 500 μl of reduced sample to provide an even distribution of antigen across the entire gel. Electrophoresis was conducted in an Invitrogen XCell SureLock Mini-Cell system at a constant current of 125 mA for 35 minutes.
Electrophoretic transfer of proteins onto nitrocellulose was performed using the Invitrogen XCell II Blot Module and Invitrogen NuPAGE transfer buffer at 160 mA for 1 h. After transfer, membranes were blocked with Sigma Western Blocker solution at room temperature for 1--2 hours with rocking. Membranes were washed three times with Trizma-Buffered Saline plus 0.5% Tween 20 (TBS-T). Cattle sera were diluted 1:20 in Phosphate-Buffered Saline plus 0.5% Tween 20 (PBS-T) and incubated with the membranes at room temperature on a rocker platform for approximately 60 min. Membranes were washed three times with TBS-T.
Membranes were incubated for approximately 60 minutes at room temperature on a rocker with peroxidase labeled rabbit anti-bovine IgG (Jackson Immunoresearch Affini-Pure) diluted 1:2000 in PBS-T. Membranes were then washed three times with TBS-T. Membranes were developed in Sigma TMB Substrate according to the manufacturer's directions.
Cattle Sera {#sec006}
-----------
Whole blood samples were obtained from cattle housed at the NVSL and also from naturally infected animals on dairy farms located around the US. The dairy farm blood samples were collected by herd veterinarians from traditional dairy cow breeds managed within privately owned herds. Herd owner consent was given for participation in sample collection for later use by the NVSL. Blood was collected via jugular puncture using standard animal welfare handling methods into blood collection bags and later processed for serum. Sera from these animals were used in the annual nationwide NVSL Johne's disease serology proficiency test program, and thus represent a well-characterized and standardized serum set. Naturally infected cattle were confirmed serologically positive on both the Parachek^®^ (ThermoFisher Scientific) and IDEXX MAP ELISA assays ([Table 1](#pone.0154685.t001){ref-type="table"}). Positive cattle were also confirmed positive by fecal culture on Herrold's Egg Yolk with Mycobactin J (HEY-MJ). Negative sera were from negative control source animals within the NVSL and also from MAP negative confirmed production herds participating in the USDA Voluntary Bovine Johne's Disease Control Program. Negative animals were confirmed by bacterial culture and serology using the same diagnostic tests as the positive cattle.
10.1371/journal.pone.0154685.t001
###### Cattle Sera used in this study.
{#pone.0154685.t001g}
Lane in [Fig 2](#pone.0154685.g002){ref-type="fig"} Animal ID Serological Status[^a^](#t001fn001){ref-type="table-fn"} IDEXX ELISA Value Prionics ELISA Value Fecal Culture Status[^b^](#t001fn002){ref-type="table-fn"}
----------------------------------------------------- -------------------- ---------------------------------------------------------- ------------------- ---------------------- ------------------------------------------------------------
1 Lindenhoff 90 (PA) Pos 0.712 0.465 Moderate
2 Meyer 1688 (PA) Pos 1.895 1.34 Moderate
3 Calf1 (IA) Pos 2.233 1.599 Neg
4 \$900 (IA) Pos 2.55 1.073 Moderate
5 1266SS CA (CA) Pos 1.914 0.749 High
6 2075 (PA) Pos 1.938 1.681 High
7 874 (MN) Pos 1.565 0.822 Neg
8 34 (MN) Pos 2.875 3.443 Low
9 Horst 256 (PA) Pos 1.046 0.5125 High
10 834 (IA) Pos 1.049 0.234 Low
11 E22 (IA) Pos 1.92 1.28 Moderate
12 Charity (WI) Pos 2.146 2.975 Low
13 Cow 5 (IA) Pos 1.506 0.979 High
14 Meyer 70 (PA) Pos 2.57 2.982 Low
15 Lindenhoff 90 (PA) Pos 0.712 0.465 Moderate
16 Lizzie (IA) Neg 0.006 0.036 Neg
17 1823 (IA) Neg 0.158 0.089 Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 790 Neg 0.189 N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 2407 Neg 0.260 N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 2222 Pos 2.32 N/A Moderate
N/A[^c^](#t001fn003){ref-type="table-fn"} 8339 Pos 1.93 N/A Low
N/A[^c^](#t001fn003){ref-type="table-fn"} 1211 Neg 0.036 N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 1044 Neg 0.079 N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 4 Neg[^d^](#t001fn004){ref-type="table-fn"} N/A N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 11 Neg[^d^](#t001fn004){ref-type="table-fn"} N/A N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 1081 Neg 0.036 N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 212 Neg 0.067 N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 61 Neg[^d^](#t001fn004){ref-type="table-fn"} N/A N/A Neg
N/A[^c^](#t001fn003){ref-type="table-fn"} 1392 Neg[^d^](#t001fn004){ref-type="table-fn"} N/A N/A Neg
^a^ Serological status determined by IDEXX and Parachek ELISA test results.
^b^ 1--10 CFU/gr feces = low shedder; 11--50 CFU/gr feces = moderate shedder; \>50 CFU/gram of feces = high shedder
^c^ Serum samples used in IFN-γ testing only.
^d^ Based upon previous IFN-γ testing.
Recombinant Protein Production {#sec007}
------------------------------
The cloning, production, and purification of recombinant MAP proteins was performed as previously described \[[@pone.0154685.ref017]\]. Briefly, maltose binding protein (MBP) fusions of MAP predicted coding sequences were constructed in *Escherichia coli* DH5α using the pMAL-c2 vector (New England Biolabs, Beverly, MA). Primers designed from the reading frame of each coding sequence contained an *Xba*I site in the 5' primer and a *Hind*III site in the 3' primer for cloning. Ligation of the DNA fragments was followed by transformation into ClearColi BL21(DE3) (Lucigen) and selection on Luria-Bertani (LB) agar plates containing 100 μg/ml ampicillin. PCR screening of ampicillin resistant colonies was conducted with primers used to amplify the sequence from MAP DNA.
Each MBP fusion protein was overexpressed in *E*. *coli* by induction of 1-L LB broth cultures with 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma Chemical Company, St. Louis, MO) for 2.5 hr with shaking at 37°C. Cells were harvested by centrifugation and resuspended in column buffer for sonication. Affinity chromatography purified protein fractions were eluted and analyzed using a NanoDrop spectrophotometer at 280 nm. The most concentrated fractions were pooled and dialyzed at 4°C against 1.5 L phosphate buffered saline. Purified proteins were finally aliquoted and stored at -20°C for future use.
Mass Spectrometry {#sec008}
-----------------
Proteomics experiments were accomplished using previously published methods \[[@pone.0154685.ref018]\]. Total protein concentrations were determined using the Biorad Protein Assay Kit (BioRad, Hercules CA), along with a known BSA standard. Briefly, 100 μg of each sample was resuspended in 250 μl PBS, 3 μl of 10% sodium deoxycholate acid, and 1 μl of 2% SDS. Samples were heated at 90°C for 20 minutes, then cooled on ice for 5 minutes. Next, 2 μl of 50 mM tris-(2-carboxyethyl) phosphine and 1.5 μl fresh 1M iodoacetimide were added and the samples were incubated 1 hour in the dark at room temperature. An equal volume of methanol was added to the protein sample. Proteins were then digested using 10 μg of trypsin gold (Promega) and incubated overnight at 37°C. Formic acid (10 μl) was added, the sample vortexed, and then centrifuged (14,000 x g for 10 minutes at 4°C) to precipitate detergents. Supernatants were transferred to clean tubes and then dried in a vacuum centrifuge. Peptides were dried in one tube, and held at -80°C until use.
Samples were injected onto nano-LC chromatography using a Proxeon Easy-nLC (ThermoFisher Scientific, West Palm Beach, FL) connected to the mass spectrometer. The chromatography used a trapping column (Proxeon Easy-Column, 2 cm, ID 100 μm, 5um, 120A, C18) and an analytical column (Proxeon Easy-Column, 10 cm, ID 75 μm, 3um, 120A, C18). The gradient using a mobile phase A (95% H2O: 5% acetonitrile and 0.1% formic acid) and mobile phase B (5% H~2~O: 95% acetonitrile and 0.1% formic acid). The gradient was: 0% B for 3 minutes, 0%-8% B from 3--5 minutes, 8--18% B from 5--85 minutes, 18--30% B from 85--100 minutes, 30--90% B from 100--105 minutes, and held at 90% B from 105--120 minutes at continuous flow rate throughout the gradient of 300 nl/min.
The analytical column was connected to a PicoTip Emitter (New Objectives, Woburn, MA; FS360-75-15-N-20) cut to size. The column and Emitter were attached to a LTQ OrbiTrap Velos Pro (ThermoFisher Scientific, West Palm Beach, FL) mass spectrometer using the Proxeon Nanospray Flex Ion Source. The capillary temperature was set at 275°C and spray voltage was 2.8 kV. The mass spectrometer used a data dependent method. In MS mode the instrument was set to scan 300--2000 m/z with a resolution of 30,000 FWHM. A minimal signal of 20,000 could trigger MSMS and 10 consecutive MSMS were possible. The activation type used was HCD. The normalized collision energy was set to 35 and repeat mass exclusion was set to 120 seconds.
Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer version 1.4.0.288 (ThermoFisher Scientific, San Jose, CA, USA; version 1.3.0.339). All MS/MS samples were analyzed using Sequest assuming digestion with trypsin. Sequest was set up to search a combined FASTA database of *Mycobacterium avium* complex (taxon \#120793) and *Mycobacterium tuberculosis* complex (taxon \#77643) databases generated in July of 2013. Sequest was searched with a fragment ion mass tolerance of 0.800 Da and a parent ion tolerance of 10.0 PPM.
Guinea Pig Potency Testing of PPDs {#sec009}
----------------------------------
Conventional guinea pigs weighing between 450 and 600 grams were paired into cages and acclimated for 1 week to the National Centers for Animal Health vivarium housing conditions prior to testing. Guinea pigs were sensitized by injecting a Johne's bacterin preparation (NVSL lot \#0601) consisting of 23 grams, wet weight, of heat-killed MAP cells homogenized in 180 ml of mineral oil.
Prior to inoculation, the Johne's bacterin was further diluted 1:4.55 in mineral oil and held at 45°C on a hotplate. Each guinea pig was inoculated intramuscularly with 0.1 ml of the warmed suspension. Control guinea pigs were inoculated intramuscularly with 0.1 ml of phosphate chloride buffer. The sensitization period after inoculation was 35 days after which PPD lot numbers 9801, 0802, 0803A, 0902A were prepared for animal inoculation. Each preparation was diluted to 10 ug/ml and 2 ug/ml in phosphate chloride buffer (34 mM Na~2~HPO~4~, 86 mM NaCl).
After the sensitization period and prior to inoculation all guinea pigs had both sides shaved. The shaved area was divided into two equal blocks. Each block contained four randomized injection sites resulting in eight total injections. This resulted in replicate injections of each PPD in each guinea pig. Guinea pigs were injected intradermally with a volume of 0.1 ml of the designated MAP PPDs. No adverse affects resulted from the procedures used and animals were monitored daily by the animal care staff.
A total of 12 guinea pigs (6 guinea pigs for each PPD concentration) were used for PPD inoculation. An additional 4 guinea pigs were used as negative non-sensitized controls. Twenty-four hours post-injection, test responses were measured to the closest square millimeter by measuring the greater and lesser dimensions of the erythema.
All animal work was approved by the NVSL Institutional Animal Care and Use Committee under protocol numbers 2202 and 2738.
Guinea Pig Potency Testing of Recombinant Proteins {#sec010}
--------------------------------------------------
Forty-seven MAP proteins identified by mass spectrometry were recombinantly expressed in *E*. *coli* for further evaluation in the guinea pig potency assay. Eighteen guinea pigs, sensitized as described previously, were used in the screening of selected proteins.
After the 35-day sensitization period and prior to inoculation, all 16 guinea pigs had both sides shaved. There was a single row of four injections sites blocked out on each side of each animal. This allowed for the injection of 6 proteins and two control proteins in each animal. Seven pair of guinea pigs each received six proteins (42 proteins total) and the final pair of animals received five proteins. One guinea pig in each pair also received either PPD 9801 or PPD 0902, whereas the second guinea pig received the MBP/LacZ control peptide diluted to 10 μg/ml in phosphate chloride buffer. This configuration enabled each of the 47 recombinant proteins to be injected twice. Guinea pigs were injected intradermally with a 0.1-ml volume of the designated antigen.
Twelve recombinant proteins were evaluated further in a second guinea pig potency test. Thirty guinea pigs were sensitized as previously described and four guinea pigs served as non-sensitized control animals. Recombinant proteins were randomized and assigned to respective guinea pig groupings. PPD 9801 served as a positive control and phosphate chloride buffer served as a negative control. Suspensions were randomized utilizing a Microsoft Excel randomization program. Sensitized guinea pigs were assigned to four groups of five animals per group.
After the sensitization period and prior to inoculation all guinea pigs had both sides shaved as before. Each side contained four randomized injection sites resulting in eight total injections for each animal. Each sensitized animal received replicate inoculations of two recombinant proteins divided between the left and right sides. Each animal also received one inoculation of lot 9801 and one inoculation of phosphate chloride buffer. Paired non-sensitized control animals received one inoculation of designated recombinant proteins as well as control suspensions. Guinea pigs were injected intradermally with a 0.1-ml volume of the designated MAP recombinant protein or control suspension. Guinea pigs were housed with 5 animals per cage with each of the five animals from different treatment groups within a cage. Test responses were again measured to the closest square millimeter 24-h post injection. Recombinant protein measurements were compared against the positive control within each animal.
Gamma-interferon Testing Using Recombinant Proteins {#sec011}
---------------------------------------------------
Evaluation of recombinant MAP proteins was conducted using blood from 6 Johne's positive cattle, 2 *M*. *bovis* sensitized cattle, and 4 non-infected control cattle. Approximately 40--50 ml of blood were collected from each animal into vacutainer blood tubes containing sodium heparin. Blood samples were held at room temperature prior to stimulation.
Between 6--7 hours after collection, blood tubes for each individual animal were mixed well and combined into a sterile 50 ml conical tube. For each animal, 1.5 ml aliquots of whole blood were dispensed into each well of a 24-well tissue culture plate. Blood was cultured either alone as non-stimulated controls or with pokeweed at a concentration of 10 μg/ml to serve as a positive control. Whole blood was also cultured with designated mitogens at 10 μg/ml. Mitogens used for evaluation were as follows: MAP PPD 9801 (PPD-J), *M*. *bovis* PPD (PPD-B), *M*. *avium* PPD (PPD-A), MAP_1997, MAP_4143, MAP_1138c, MAP_3567, MAP_3061c, MAP_2121c, and MAP_3651c. For all mitogens, 100 μl was dispensed into each designated well and each mitogen was cultured in duplicate wells.
Plates were covered and incubated at 37°C for 21--22 hours. Plates were centrifuged at 500 x g for 15 minutes and the plasma from each well was harvested and refrigerated. Plasma samples were tested for IFN-γ production over the next 72 hours.
The IFN-γ ELISA was conducted according to the manufacturer's directions (BOVIGAM^®^, ThermoFisher Scientific). Samples from each well were incubated in duplicate for one hour at room temperature in a 96-well plate that is pre-coated with anti-bovine IFN-γ antibody. Plates were washed six times with wash solution and then incubated with an anti-bovine IFN-γ-horseradish peroxidase conjugate for 30 minutes at room temperature. Plates were again washed six times with wash solution and incubated with peroxidase substrate for 30 minutes at room temperature. Enzyme stopping solution was added to each well and the plates were read at A~450~ and A~650~ nm.
Statistical Analysis {#sec012}
--------------------
The PPD mean skin test responses from the guinea pig potency test were compared by ANOVA. Differences were considered statistically significant at a P value of \< 0.05.
Results for the recombinant protein guinea pig potency testing were evaluated by calculating the treatment group mean erythema value using the measurements for each individual animal comprising the treatment group. Paired comparisons calculating the difference between the response of the treatment to the response of the positive control for each animal were completed. The five injections (same protein) were then averaged and ranked based on the average differences. A positive difference indicates the average response from the treatment is higher than the average response from the positive control. For this difference data a 95% confidence interval was also calculated for each treatment group.
Results {#sec013}
=======
PPD Potency Testing in guinea pigs {#sec014}
----------------------------------
Potency testing of the traditional and alternate PPD production lots resulted in significant differences (*P* \< 0.05) when compared against the reference standard (lot 9801; [Table 2](#pone.0154685.t002){ref-type="table"}). Traditional lot 0803A and alternate lot 0902 resulted in the greatest skin test responses and all responses showed a larger erythema as PPD concentration increased. The average area of the erythema at the injection sites for lot numbers 0803A and 0902 were much greater than the average area for the reference standard (261% and 177%, respectively,). Traditional PPD lot 0802 resulted in a measurement that was 79% of the average area compared to the reference standard. An acceptable potency test result for new production lots must have a response that is a minimum of 75% compared to the reference response. Using these criteria, all three PPD lots demonstrated acceptable potency, although 0802 is marginal. These data suggest that the potency was not adversely affected by removal of the autoclave step.
10.1371/journal.pone.0154685.t002
###### Guinea pig potency skin test responses at 24 hours post-inoculation of MAP PPD.
{#pone.0154685.t002g}
PPD Concentration PPD 9801 PPD 0802 PPD 0803A PPD 0902
------------------- ----------------------------------------------------- --------------- ---------------- ----------------
10 ug/ml 143.22 ± 48.82 93.63 ± 15.59 264.25 ± 39.72 202.07 ± 39.63
2 ug/ml 45.52 ± 32.13[^a^](#t002fn001){ref-type="table-fn"} 53.45 ± 15.49 228.41 ± 41.91 132.50 ± 67.79
^a^ PPD 9801, 2 ug/ml sample data, contained one inoculation of one guinea pig which had no measureable skin reaction. This resulted in an elevated standard deviation value. All guinea pigs were sensitized with MAP Strain 19698 killed cells.
Values represent the average response (mm^2^ ± standard deviation) of six guinea pigs used for evaluating each PPD concentration.
SDS-PAGE analysis of PPD from different production methods {#sec015}
----------------------------------------------------------
Aberrant migration or protein smearing was observed by SDS-PAGE analysis of reference PPD Lot 9801; and this resulted in the lack of any discernable protein bands ([Fig 1](#pone.0154685.g001){ref-type="fig"}). This result is similar to that observed in other laboratories studying *M*. *bovis* and *M*. *tuberculosis* PPDs \[[@pone.0154685.ref019], [@pone.0154685.ref020]\]. Smearing was also observed on lanes containing lots 0801A, 0801B, 0802, and 0803A from the traditional production method ([Fig 1](#pone.0154685.g001){ref-type="fig"}). Similar protein smearing characteristics have also been noted in *M*. *bovis* and *M*. *avium* PPD preparations at the NVSL (unpublished data). The appearance of these "dirty" gel tracks ([Fig 1](#pone.0154685.g001){ref-type="fig"}) suggest the autoclaving step may have contributed to this unusual migration pattern, because PPDs produced by the alternate production method each yielded distinct protein bands ([Fig 1](#pone.0154685.g001){ref-type="fig"}, lanes 6 and 7). Serial dilution of PPD lot 9801 also failed to display distinct protein bands ([Fig 1](#pone.0154685.g001){ref-type="fig"}), suggesting that the smearing was not due to overloading. Protein bands less than 28 kDa were observed in the *M*. *bovis* PPD despite significant smearing, but no protein bands were observed in the *M*. *avium* PPD preparation ([Fig 1](#pone.0154685.g001){ref-type="fig"}). Collectively, these data suggest autoclaving modified this reagent by denaturation and/or hydrolysis.
{#pone.0154685.g001}
Humoral immune response against PPD preparations {#sec016}
------------------------------------------------
The affect of autoclaving on the antigenicity of PPDs was next examined. Immunoblots using cattle sera ([Table 1](#pone.0154685.t001){ref-type="table"}) yielded distinct differences between lot 9801 and lot 0902. Immunoreactive protein bands were present in lot 0902, but absent in the reference lot 9801 ([Fig 2](#pone.0154685.g002){ref-type="fig"}). Serum from cow 34 in lane 8 had the highest antibody titer to MAP ([Table 1](#pone.0154685.t001){ref-type="table"}) and reactivity was predominant throughout regardless of the PPD production method. However, other sera readily detected protein bands in the alternate PPD but not the traditional PPD. At least one protein band was observed in the alternate PPD from 7 of 15 positive cattle sera. Lanes 4, 11, and 12 containing alternate PPDs all appear to react with a protein of similar size ([Fig 2](#pone.0154685.g002){ref-type="fig"}). These results suggest that removing the autoclaving step in the production process may preserve antigenic properties.
{ref-type="table"}.](pone.0154685.g002){#pone.0154685.g002}
Analysis of PPD preparations by Mass Spectrometry {#sec017}
-------------------------------------------------
Mass spectrometry analysis was performed on three PPDs, including the reference (9801) and alternative lots (0902A and 0902B). The total number of proteins identified among all three PPDs was 194, 78% of which were unique to an individual PPD ([Fig 3](#pone.0154685.g003){ref-type="fig"}). All 194 proteins, including peptide matches and molecular weights are listed in [S1 Table](#pone.0154685.s001){ref-type="supplementary-material"}. Ten proteins were detected in all three PPD suspensions ([Fig 4](#pone.0154685.g004){ref-type="fig"}). These 10 proteins may represent the best candidates for development of a consistent recombinant PPD and are listed at the top of [S1 Table](#pone.0154685.s001){ref-type="supplementary-material"}. MAP_3840 had the greatest number of peptide matches among all the PPDs, and was also one of the most common proteins observed among PPDs analyzed in the Wynne et al., 2012 and Santema et al., 2009 studies. Furthermore, it was identified as a cell surface protein using a trypsin shaving technique \[[@pone.0154685.ref021]\]. These results suggest that MAP_3840, which encodes a 70-kDa heat shock protein, is present in high abundance in MAP. Also present among all PPDs were MAP_1588c and MAP_1589c, which are tandemly located on the K-10 genome \[[@pone.0154685.ref022]\] and have been examined as potential diagnostic antigens long ago \[[@pone.0154685.ref023]\]. Furthermore, both have been reported as a stress protein \[[@pone.0154685.ref024]\] and MAP_1589c was also found to be surface located \[[@pone.0154685.ref021]\].
{#pone.0154685.g003}
{ref-type="supplementary-material"}).](pone.0154685.g004){#pone.0154685.g004}
Potency Testing of recombinant proteins in guinea pigs {#sec018}
------------------------------------------------------
Forty-seven of the 194 proteins identified by mass spectrometry were cloned, expressed and purified in *E*. *coli*. Five of these purified proteins are among the 10 PPD proteins identified from all three preparations. All 47 recombinant proteins are highlighted in red in [S1 Table](#pone.0154685.s001){ref-type="supplementary-material"} and were used for potency testing to determine if they substantially contribute to the skin test response observed with PPDs. Seven proteins showed reproducible skin test responses that were stronger than or equivalent to the reference lot 9801 ([Table 3](#pone.0154685.t003){ref-type="table"}, highlighted in bold). The strongest skin test response among all recombinant proteins was elicited by MAP_1997, which is annotated as an acyl-carrier protein ([Table 3](#pone.0154685.t003){ref-type="table"}). This protein has not been previously described as an antigen in the literature, although it was listed among surface exposed proteins in MAP \[[@pone.0154685.ref021]\]. Known antigens that are among these seven proteins include the major membrane protein \[[@pone.0154685.ref025], [@pone.0154685.ref026]\] and the LrpG protein \[[@pone.0154685.ref013], [@pone.0154685.ref027]\]. Seventeen additional proteins produced a measureable skin test response in one of two replicate intradermal inoculations. The MBP/Lac-Z control protein showed no visible reactivity at any inoculation site. Two of the 10 PPD proteins present in all three preparations generated positive skin test responses with MAP_4143 showing the second strongest response.
10.1371/journal.pone.0154685.t003
###### Positive skin test responses in a guinea pig potency test of 24 MAP recombinant proteins from 47 total proteins tested.
{#pone.0154685.t003g}
Protein Description Locus Tag Area Measurement (mm^2^) Standard Deviation[^a^](#t003fn001){ref-type="table-fn"}
-------------------------------------------------------- --------------- -------------------------- ----------------------------------------------------------
**Acyl carrier protein** **MAP_1997** **231.52** **87.70**
**Elongation factor Tu** **MAP_4143** **209.08** **124.13**
**Electron transfer flavoprotein (Beta-subunit) FixA** **MAP_3061c** **192.36** **87.68**
**Putative uncharacterized protein** **MAP_2694** **171.72** **56.83**
**Putative acyl-CoA dehydrogenase** **MAP_3651c** **166.53** **77.51**
**LprG protein** **MAP_1138c** **161.79** **50.09**
**Major membrane protein 1** **MAP_2121c** **128.90** **11.11**
Peroxisomal multifunctional enzyme type 2 MAP_3567 242.51 N/A
Chaperone protein DnaK MAP_3840 221.81 N/A
Putative uncharacterized protein MAP_3692c 148.58 N/A
Lipoprotein LprC MAP_2497c 141.95 N/A
Superoxide dismutase (Fragment) MAP_0187c 131.90 N/A
Fructose-bisphosphate aldolase class-I MAP_4308c 122.33 N/A
LppZ protein MAP_3041 104.28 N/A
DNA-binding protein HU MAP_3024c 68.78 N/A
Peptidyl-prolyl cis-trans isomerase MAP_1693c 48.52 N/A
Fatty acid desaturase MAP_2698c 44.25 N/A
Antigen 85-B MAP_1609c 41.96 N/A
ATP synthase subunit alpha MAP_2453c 41.49 N/A
Serine/threonine protein kinase MAP_0016c 38.83 N/A
Wag31 protein MAP_1889c 36.72 N/A
Putative uncharacterized protein MAP_2020 29.59 N/A
Peroxiredoxin MAP_1589c 26.12 N/A
6-phosphogluconolactonase MAP_1174c 16.32 N/A
MBP/Lac-Z \-\-\-- 0.00 N/A
PPD 9801 \-\-\-- 149.14 10.82
PPD 0902 \-\-\-- 241.84 37.60
^a^ For the proteins which resulted in skin test responses for both inoculation sites the values represent the average response (mm^2^) and the standard deviation from two guinea pigs.
For the 17 proteins, which exhibited only one positive skin test reaction the value represents the response from the single reactive inoculation and the standard deviation is not available (N/A).
Twelve recombinant proteins that showed the largest skin test responses listed in [Table 3](#pone.0154685.t003){ref-type="table"} were evaluated further in a second guinea pig potency test. Even the four best proteins were still not considered significantly different from the reference 9801 PPD based upon results from the ranking by difference calculations ([Table 4](#pone.0154685.t004){ref-type="table"}). Those recombinant proteins were MAP_1997, MAP_4143, MAP_3567, and MAP_1138c. The only recombinant protein that elicited responses greater than the reference PPD was MAP_1997, but this difference was not significant.
10.1371/journal.pone.0154685.t004
###### Guinea pig potency testing of MAP recombinant proteins ranked by subtracted differences.
{#pone.0154685.t004g}
MAP Protein (Locus tag) Rec Protein Mean MAP PPD 9801 Mean Difference[^a^](#t004fn001){ref-type="table-fn"} CI Upper Range[^b^](#t004fn002){ref-type="table-fn"} CI Lower Range[^b^](#t004fn002){ref-type="table-fn"} Guinea Pig ID's
------------------------- ------------------ ------------------- -------------------------------------------------- ------------------------------------------------------ ------------------------------------------------------ -------------------------
MAP_1997 328.93 248.89 80.04 212.00 -51.93 306, 312, 318, 324, 530
MAP_4143 199.68 277.11 -77.43 0.83 -155.69 304, 310, 316, 322, 528
MAP_3567 192.05 272.39 -80.33 88.62 -249.28 305, 311, 317, 323, 529
MAP_1138c 173.70 264.41 -90.72 24.25 -205.68 301, 307, 313, 319, 325
MAP_3061c 106.80 277.11 -170.32 -73.52 -267.12 304, 310, 316, 322, 528
MAP_3651c 72.57 256.51 -183.94 -132.90 -234.99 302, 308, 314, 320, 526
MAP_2121c 74.40 272.39 -197.98 -63.20 -332.77 305, 311, 317, 323, 529
MAP_2497c 50.25 248.89 -198.65 -176.95 -220.34 306, 312, 318, 324, 530
MAP_3692c 34.73 243.82 -209.08 -141.21 -276.95 303, 309, 315, 321, 527
MAP_2694 42.20 256.51 -214.31 -133.99 -294.64 302, 308, 314, 320, 526
MAP_3840 47.95 264.41 -216.46 -92.78 -340.14 301, 307, 313, 319, 325
MAP_0187c 15.31 243.82 -228.51 -184.33 -272.68 303, 309, 315, 321, 527
^a^Differences were calculated by subtracting the MAP protein skin test response from the PPD skin test response for each animal and then averaging the calculated differences for all animals in each treatment group.
^b^Upper and lower range values represent 95% confidence interval (CI) calculations.
Gamma-interferon Testing using MAP recombinant proteins {#sec019}
-------------------------------------------------------
Seven recombinant proteins having the best performance in the guinea pig potency test were further evaluated as mitogens in an IFN-γ stimulation assay. The proteins were evaluated against Johne's positive animals (\#790, \#2407, \#2222, \#8339, \#1211, and \#1044) based upon results of serological and fecal shedding, *M*. *bovis* sensitized animals (\#4 and \#11), and negative control animals (\#1081, \#212, \#61, and \#1392). MAP_3651c and MAP_3567 demonstrated the greatest IFN-γ stimulation within the Johne's positive animals with responses in some animals being greater than the stimulation values from MAP PPD 9801 ([Fig 5](#pone.0154685.g005){ref-type="fig"}). MAP_1997, which resulted in the greatest skin test response in the guinea pig potency test, showed the least IFN-γ stimulation. Each MAP recombinant protein resulted in negative stimulation values against samples from the *M*. *bovis* sensitized animals ([Fig 5](#pone.0154685.g005){ref-type="fig"}). All negative control samples remained negative for IFN-γ stimulation using MAP recombinant proteins or PPD suspensions, indicating good specificity.
{#pone.0154685.g005}
In summary, recombinant proteins can be equivalent or a more potent immunological reagent in skin testing than PPD based on guinea pig potency test results. In addition, these recombinant proteins may serve as mitogens in an IFN-γ assay resulting in cytokine stimulation comparable to or greater than that from MAP PPD.
Discussion {#sec020}
==========
This study had two objectives with the overall goal of analysing a little studied but very important antigen used in Johne's disease diagnosis and research. One objective was to compare the effect of autoclaving on MAP PPD preparations and measure this affect by SDS-PAGE, immunoblot analysis and guinea pig potency testing. The data collectively suggest that removal of the autoclaving step may enhance antigenicity and prevent protein degradation, but does not negatively affect the potency. A second objective was to define the components present in each preparation through a proteomic analysis and determine proteins commonly present in the different production lots. A subset of the identified PPD proteins was then selected for evaluation in a guinea pig potency test to determine skin test responsiveness in sensitized animals. In addition these proteins were further evaluated to determine their use as antigens in IFN-γ testing. These objectives worked toward the long-term goal of obtaining a more consistent and equally potent PPD that may be composed entirely of recombinant proteins. This reagent could then circumvent the problems inherent in obtaining potent PPDs. Finally, a "recombinant PPD" may avoid cross reactivity issues in cattle vaccinated against *M*. *bovis* or MAP.
The use of high pressure heat inherent in the autoclaving process results in lysis and the appearance of cytosolic proteins that may not be present when autoclaving is eliminated. The presence of the 70-kDa heat shock protein (MAP_3840) in both traditional and alternate PPDs argues against this idea since it is considered a cytosolic protein. However, because this protein is present in high abundance in MAP, it appears at some level in all cell fractions including membrane and secreted fractions \[[@pone.0154685.ref021], [@pone.0154685.ref028]\]. Since secreted or surface proteins should be the first antigenic components encountered in an infected animal, this may allow for more specific responses as well as earlier detection. Multiple studies have shown that many strong antigens are also secreted proteins in MAP \[[@pone.0154685.ref029], [@pone.0154685.ref030]\]. The interest in secreted antigens has primarily involved their significance for use in enzyme-linked immunosorbent assays \[[@pone.0154685.ref028], [@pone.0154685.ref031]\]. In addition, the 10 proteins identified by mass spectrometry and common to all three NVSL PPD preparations were also identified as part of the culture filtrate protein composition characterized by Facciuolo et al \[[@pone.0154685.ref028]\]. In total there were 58 proteins in the NVSL PPD preparations identified by mass spectrometry that were in common with the Facciuolo et al. study. Furthermore, of the seven recombinant proteins resulting in the greatest skin erythema in the guinea pig potency test, four of those proteins were also identified in the culture filtrate found by Facciuolo et al. \[[@pone.0154685.ref028]\]. Three of the four proteins, which include MAP_1997, MAP_4143 and MAP_3061c, were also the most reactive in the guinea pig potency test, with MAP_4143 being one of the proteins present in all three PPDs. Therefore, any work toward development of a consistent, recombinant PPD should start with inclusion of MAP_4143 among other proteins.
Potency testing of four MAP PPD lots (9801, 0802, 0803A and 0902) was conducted to ensure that PPDs produced by both methods retained acceptable potency in sensitized guinea pigs. Replacing the autoclaving step with a sterile filtration process would avoid protein degradation, but it was uncertain how this change might affect the potency. Results from potency testing suggest there was no significant effect on potency regardless of whether the culture material was subjected to autoclaving or sterile filtration. This is the first study to examine effect of heating on PPD performance in guinea pig potency testing. However, our results suggest that age of the PPDs might have an effect on potency because the skin test responses where much stronger in the recently made PPDs (0803A and 0902) compared to the reference lot made in 1998. Thus either potency of the new PPDs is significantly better or the age of the reference PPD resulted in decreased potency over time.
Mass spectrometry analysis of PPD preps has revealed several protein identities in these complex protein mixtures. In a separate study, proteomic analysis of *M*. *bovis*, *M*. *avium* subspecies *avium*, and MAP derived PPD suspensions identified 156 proteins among all PPDs \[[@pone.0154685.ref013]\]. Three of these proteins (MAP_1718c, MAP_3515c, and MAP_1138c) were further analyzed in a serum antibody ELISA with MAP1138c (LprG) emerging as the strongest antigen in high fecal shedding cattle. MAP LprG was also previously identified from a MAP gene fusion library screening study and found to be antigenic in bovine paratuberculosis serum samples by Western blot analysis \[[@pone.0154685.ref032]\]. Our study also identified LprG and it was among the strongest stimulators in guinea pigs ([Table 3](#pone.0154685.t003){ref-type="table"}). However, LprG showed no significant reactivity in a lymphocyte proliferative assay \[[@pone.0154685.ref013]\] decreasing its consideration for use in cell-mediated immune response based assays. LprG elicited IFN-γ responses in eight of nine sheep that had been vaccinated with an attenuated MAP strain in the study by Dupont et al. \[[@pone.0154685.ref032]\]. Using LprG as a mitogen in the IFN-γ assay 50% of Johne's positive cows, and all *M*. *bovis* sensitized and MAP negative animals were identified correctly, whereas in the Santema et al. \[[@pone.0154685.ref013]\] study there was no significant difference between positive and negative animals. While results varied for use as an antigen for *in-vitro* IFN-γ stimulation, its ability to elicit immunological responses in both humoral and cell-mediated assays may be indicative that MAP LprG is a highly expressed protein during all stages of Johne's disease and could prove to be a more versatile candidate for diagnostic assay improvements.
A total of 194 proteins were identified among all preparations with 110 of these identified from lot 9801. This greatly adds to a previous study which identified only six proteins in this same NVSL lot 9801 \[[@pone.0154685.ref014]\]. It is interesting to note that reference PPD 9801 had the most proteins identified by LC-MS/MS, yet was migrating as a smear in denaturing protein gels and did not appear antigenic by Western blot analysis or as potent by guinea pig sensitization. This shows that while proteins were modified in the heating process, it did not destroy peptides, which are essential for MS identification. Fewer proteins were identified in the alternate PPD lots, with 67 and 74 proteins being identified in each production lot. Although a number of proteins were identified in two of the three preparations analysed, only ten proteins were detected in all three preparations. Combining the results from the Wynne et al. \[[@pone.0154685.ref014]\] and Santema et al. \[[@pone.0154685.ref013]\] studies shows the number of proteins consistently appearing in PPD preparations among all three studies is only three. They include MAP_4143, MAP_1595, and MAP_3840. The total number of MAP proteins identified at least once in any of the three studies was 214, which suggests a starting point for the complete PPD proteome of MAP. Although some variability may be attributed to the methodology used for protein identification, the small overlap of proteins nonetheless further suggests the inconsistencies that can be encountered in PPD production processes and the need to improve such processes to reduce or eliminate such variability.
MAP recombinant proteins used as antigens in an IFN-γ assay showed five proteins as potential candidate antigens. MAP_3651c and MAP_3567 were the strongest antigens with Johne's positive samples and they correctly identified four of the six animals as positive. MAP_3061c, MAP_1138c, and MAP_4143 were comparable to each other, and also identified three of the same four animals identified by MAP_3651c and MAP_3567. These results were comparable to animals identified as positive using MAP PPD 9801 with differences noted for two animals. Cow \#2407 was identified as positive by MAP PPD 9801, but was negative by all individual proteins. In contrast, cow \#8339 was negative using MAP 9801, but was positive by five of the seven individual proteins. These results were in contrast to the guinea pig potency test results in which MAP_3651c and MAP_3061c did not stimulate skin test responses comparable to MAP PPD 9801. Also in contrast to the strong skin test response in the guinea pig potency test, MAP_1997 was not a strong mitogen in the IFN-γ stimulation assay. These contrasting results between the two tests demonstrate that neither test is a good predictor of success in the other and may be due to the additional immunological responses ongoing at skin test sites that involve more than IFN-γ alone.
There are a number of false positive tuberculin skin test results, particularly in developing countries, due to BCG vaccination and non-tuberculous mycobacteria \[[@pone.0154685.ref033]\]. The issue of false positive skin tests has been a long-term concern due to the conserved antigenic characteristics of mycobacteria. This situation is similar in the veterinary field with caudal fold tests using *M*. *bovis* PPD and skin tests with MAP PPD giving false positive results if animals are exposed to other mycobacteria. By developing a recombinant PPD-type reagent, it may now be feasible to not only avoid false positive reactions but to have a consistent reagent that could be evaluated in the gamma-interferon assay as well.
Conclusions {#sec021}
===========
Our results suggest that autoclaving during PPD preparations, though performed for decades, is an unnecessary step in PPD production. Over 100 proteins have now been identified from NVSL PPDs that have been used in field studies for decades. Results from this study have also identified a number of proteins that are reactive to skin testing in sensitized guinea pigs and as antigens in IFN-γ assays. These proteins may contribute to improvements for a well-defined, consistent PPD for diagnostic purposes that may not cross react with TB skin testing. Further research is needed to confirm the DTH responses of these proteins and examine specificity characteristics to differentiate MAP immunological responses from *M*. *bovis* infected animals.
Supporting Information {#sec022}
======================
###### Proteins identified by mass spectrometry from *Mycobacterium avium* subsp. *paratuberculosis* PPD lot numbers 9801, 0902A, and 0902B.
(DOCX)
######
Click here for additional data file.
The authors thank Janis Hansen and the National Centers for Animal Health Animal Resources Unit for their technical assistance with this study.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: RTC COT TAR JDL JRS JPB. Performed the experiments: RTC JDL RO. Analyzed the data: RTC TAR JDL JRS JPB. Contributed reagents/materials/analysis tools: COT TAR JDL JRS JPB. Wrote the paper: RTC COT JPB.
| tomekkorbak/pile-curse-small | PubMed Central |
1942: Joint Strike
1942: Joint Strike is a video game developed by Backbone Entertainment for the Xbox 360's Xbox Live Arcade and PlayStation 3's PlayStation Network. It was released in 2008. It is the remake of the original 1984 video game 1942.
Despite the name, the game is an amalgamation of various elements of the 19XX series. For example, it includes health meter and bomb system from 1943: The Battle of Midway; charge-fire, land-based battle sections; and rank increases from 1941: Counter Attack, fighter lineup, bomb-based end-level bonus, level rank system from 19XX: The War Against Destiny.
Gameplay
Reception
The game received "average" reviews on both platforms according to the review aggregation website Metacritic. IGN commented on the short length of the game, "spotty" online play and poor value for money, but praised the graphical overhaul. Anthony Gallegos of 1UP.com stated that while the game was short, this added to its 'arcade' appeal. As of year-end 2010, the game has sold over 111,000 units. Sales at year-end 2011 were over 119,000 units.
References
External links
Developer Backbone page
Category:2008 video games
Category:Capcom games
Category:Anti-war video games
Category:Vertically scrolling shooters
Category:World War II video games
Category:Cooperative video games
Category:Video games developed in Canada
Category:Video game remakes
Category:Xbox 360 Live Arcade games
Category:Xbox 360 games
Category:PlayStation 3 games
Category:PlayStation Network games | tomekkorbak/pile-curse-small | Wikipedia (en) |
Having more women calling the shots in Venezuelan politics may be the way to move faster towards a democratic transition. You shouldn’t expect different results if you always do the same thing, so why not have more women at the table when it comes to the big issues for Venezuela?
Regarding this topic, Venezuela is an atypical case in the region:the issues discussed about women in politics in other countries include quota or parity laws, campaign financing, leadership training and equitable media coverage; in Venezuela, they get lost (or lose priority) in the context of the most complex humanitarian crisis the region has experienced. However, the crisis can also be an opportunity to create better conditions for women’s political participation, now and in the future.
When thinking about Venezuelan women in politics, we should all understand that…
We need more women in Venezuelan politics, and a gender perspective in all decisions;
We need to strengthen Venezuela’s legislative framework to facilitate the previous point, and;
We must have women at the table where decisions are made.
Let me go quickly over these points.
We need more women in Venezuelan politics.More numbers. And a gender perspective in policy decisions, especially as we face a democratic transition.We know that women comprise 50% of the population and 50% of the electorate, and if regional patterns are consistent (which they generally are), they should be 50% of the main political parties in the country. That’s50-50-50. However, we have one female leader of a political party and only 19% female representation in the National Assembly.
Granted, Latin America is the second region in the world after the Nordic countries with the highest representation of women in Parliament (with around 30%), but Venezuela is far from that critical mass most countries in the region have achieved.
Additionally, we need to make sure women’s issues are mainstreamed in all decisions, in all discussions. A quick review ofPlan Paísreveals how little attention has been paid to women’s issues (per sector). So we need more women. We need their views.
And we need to strengthen Venezuela’s legislative framework to facilitate female participation.Of course Venezuelans need to work on their patterns of socialization, working against stereotypes, on improving education and teaching equality and human rights as the basis of structural changes, but institutions are key and we can start paving the way for women’s representation learning from other Latin American countries.
We must reinforce Venezuela’s legislative frameworks.We need to learn from other countries, and design a parity law that can increase female representation for public office.
Countries of the region have been trying quotas for years as a temporary affirmative action measure, usually at 30%. The Interamerican Commission of Human Rights’ stance on quotas also supports their implementation, and research shows that quotas work. Moreover, a few selected countries (Bolivia, Costa Rica, Mexico, Nicaragua and, more recently, Chile) have even adopted parity measures as a permanent feature of their legislation, with the specific purpose of ensuring female representation as part of the political candidates’ lists.
Only three countries in the region don’t exercise a quota: Venezuela, Guatemala and Uruguay.
The first quota measure in Venezuela dates back to 1998, specifically Article 144 of the Citizens’ Suffrage and Participation Law, which required political parties to include 30% of women in their lists of candidates.
This 1998 quota Article was declared unconstitutional by the National Electoral Council and repealed by the Supreme Tribunal of Justice in a ruling dated May 15th, 2000, on the basis of a supposed contradiction with the 1999 Constitution that prohibits discrimination based on race, sex, creed or social status.
In 2005, the CNE issued an administrative measure and approved a 50% parity for men and women. The problem is that, since it’s just an administrative measure, its application depends on the CNE’s whims, and it carried very limited effectiveness—the CNE allowed parties to place women in losing districts, or as substitutes in the lists, so their representation was actually very limited.
We must reinforce Venezuela’s legislative frameworks.We need to learn from other countries, and design a parity law that can increase female representation for public office.
This means that we need to ask more from Venezuela’s democratic leaders. We must demand women at the table where decisions are made. It matters in terms of numbers (symbolic representation) and in terms of content (ensuring their perspectives, views, and opinions are also considered);we see media coverage of international missions, we go to meetings and discussions in the main cities where diaspora Venezuelans and political exiles are giving their all to help a transition to democracy. We salute and respect that. However, the ones calling the shots are usually men, and that’s not right; we need more women in decision-making spaces, contributing with their views, improving decisions. Especially in Venezuela’s road to democracy.
So having women at the table goes beyond morality and fairness, it’s beneficial to peace and democracy.In the long run, we improve the quality of decisions, we achieve a stronger democracy and, as Madeleine Albright so eloquently said, women in power “can be counted on to raise issues that others overlook, to support ideas that others oppose, and to seek an end to abuses that others accept.”
So we all win.
* Views are personal. They do not represent those of the Organization of American States. | tomekkorbak/pile-curse-small | Pile-CC |
Q:
jExcel getting cell content missing
I read excel file correctly.For example my cell content is 0,987654321 in excel file.When I read it with jExcel api i reads just few chars of cell.For example 0,987.
here is my read excel code part:
Cell A = sheet.getCell(1, 1);
String stringA = A.getContents().toString();
How can I fix that problem.I want all content of cell.
A:
getContents() is a basic routine to get the cell's content as a string. By casting the cell to the appropriate type (after testing that it's the expected type) you get access to the raw numeric value contained, like this
if (A.getType() == CellType.NUMBER) {
NumberCell nc = (NumberCell) A;
double doubleA = nc.getValue();
// this is a double containing the exact numeric value that was stored
// in the spreadsheet
}
The key message here is: you can access any type of cell by casting to the appropriate subtype of Cell.
All this and a lot more is explained in the jexcelapi tutorial
| tomekkorbak/pile-curse-small | StackExchange |
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Federal Agency for Tourism (Russia)
The Federal Agency for Tourism (Rosturizm) is a federal executive body of the Russian Federation, created by Presidential Decree No. 1453 of November 18, 2004. The Federal Agency for Tourism is responsible for rendering state services, managing state property and performing law-enforcement functions in the field of tourism. It is under the jurisdiction of the Ministry of Culture of the Russian Federation. Ministry of Culture of the Russian Federation manages the activities of the Federal Agency for Tourism. Activities of the Federal Agency for Tourism guided by The constitution of the Russian Federation, Federal laws, Decrees of the President and the Government of the Russian Federation, International treaties, and Normative legal acts of the Ministry of Culture. Federal Agency for Tourism carries out its activities in cooperation with other federal executive bodies, executive authorities of the subjects of the Russian Federation, local self-governing bodies, public associations and other organizations. The Federal Agency for Tourism is a legal entity, has a seal with the image of the State Coat of Arms of the Russian Federation and its name, other seals, stamp, and letterhead, as well as bank and other accounts opened. It get funds for its operation and project implementation from the federal budget. The agencies head office is in Moscow.
History
The Council for Foreign Tourism under the Cabinet of Ministers of the USSR established In April 1991 at the same time the State Committee for Foreign Tourism abolished. The Ministry of Physical Education and Tourism formed in January 1992 it had a separate department for tourism. Its responsibility was to manage and develop the domestic and foreign tourism sector. The Ministry of Culture and Tourism of the Russian Federation created on 27 March 1992. On the basis, the Ministry of Culture of the RSFSR abolished. A Provisional Interdepartmental Commission on Foreign and Domestic Tourism of the Russian Federation formed on 20 April 1992. New state policies were adopted to develop tourism sector such as; Domestic and Foreign tourism integrated into a single framework, a network of small hotels created, etc.
The Committee for Tourism of the Russian Federation (Roskomuturizm) established in September 1992. Its responsibility was to develop a strategy for the development of tourism in Russia, implementation of state policy of tourism, organization, development, and implementation of federal targeted and interstate programs. Training, retraining, and upgrading human resources in the tourism sector, establishing new bodies of tourism management in the territories, regions, autonomous entities and city of Moscow and St. Peters burg was also in its responsibility. The Roskomuturizm transformed into the Russian Federation Committee on Youth, Physical Education, and Tourism on 25 April 1994. The responsibility of the Committee was developing a state strategy, Inter-regional and Inter-sectoral coordination in the field of Physical Education and tourism, preservation/development of tourist centers and administrating national tourism sector. In August 1994, it unified with the Russian Federation Committee on Physical Education and reformed to the State Committee of the Russian Federation for Physical Education and Tourism (CFT of Russia).
June 1999 the State Committee of the Russian Federation for Physical Education and Tourism converted into the Ministry of Physical Education, Sports and Tourism. The tourism department in the new ministry headed by a deputy minister; at that time by Vladimir Igorevich Strzhalkovsky. On 17 May 2000 the Ministry of the Russian Federation for Physical Education, Sports and Tourism converted as the State Committee of the Russian Federation on Physical Education, Sport, and Tourism. At the same time, Ministry of Economic Development and Trade also formed with a Department of Tourism. The State Committee of the Russian Federation for Physical Education, Sports, and Tourism transformed into the State Committee of the Russian Federation for Physical Education and Sports on 29 April 2002. The Federal Agency for Physical Education, Sports, and Tourism (Rossport) established on March 12, 2004, initially it administered by the Ministry of Health and Social Development of the Russian Federation.
On 18 November 2004, the Federal Agency for Physical Education, Sports, and Tourism, divided into two agencies. One was The Federal Agency for Tourism of the Russian Federation; under the direct jurisdiction of the Government of the Russian Federation And the other was Federal Agency for Physical Education and Sports; under the administration of the Ministry of Health and Social Development of the Russian Federation. The Ministry of Sport, Tourism, and Youth Policy established in 2008 and The Federal Agency for Tourism of the Russian Federation with the Federal Agency for Physical Education and Sports of the Russian Federation transferred under its authority. On 21 May 2012, the Ministry of Sport, Tourism and Youth Policy renamed as The Russian Ministry of Sports. On 22 May 2012, the Federal Agency for Tourism of the Russian Federation was transferred, under the jurisdiction of the Ministry of culture.
Functions
Realizing the state strategy of tourism with the priority oriented planning and activities. Formation and maintenance of a single federal register of tour operators. Developing and implementing strategic plans on issues related to tourism industry within the jurisdiction of the Russian Federation. Information support of tourism, including assistance in the creation and functioning of tourist information centers, navigation and orientation systems in the field of tourism. It provides Information to travel agents, tour operators, and tourists about safety threats in a country or place to visit. It promotes the tourism product of the Russian Federation in the domestic and world tourism markets. Manage the procurement of goods, works, and services for the agency or any other agencies related to it as per the demand. It is the Owner of the federal properties in the tourism sector and can practices ownership rights on such properties. Act as the Federal public authority to federal institutions and federal unitary enterprises subordinate to the agency. Conduct economic analysis of the activities of subordinate state unitary enterprises. Auditing financial and economic activities of the organizations subordinate to the Agency. Inaugurate Scientific, technical and innovative programs and projects in the field of tourism. It cooperates with the authorities of foreign states and international organizations. It represents on behalf of the Government of the Russian Federation and the Ministry of Culture of the Russian Federation in international organizations in the tourism field. The agency opens representative offices outside the Russian Federation to promote tourism. Provide related supports, ensures timely and complete consideration of oral and written appeals of citizens, adoption of decisions on them and responding to applicants. Ensure protection of information constituting a state secret. Organizes and provides training for the human resources of the agency and organizations under its authority. Arrange additional professional education of Agency employees. It carries out work on acquisition, storage, accounting and use of archival documents produced in the course of the Agency's activities. The agency organizes congresses, conferences, seminars, exhibitions and other events. Take measures to support small and medium-sized businesses, aiming development, including the implementation of relevant departmental target programs. Register new tour operators in the single federal register of tour operators and issues certificates of registration. Performs functions to manage state property and provide public services in the tourism sector. Every year the Rosturizm attend exhibitions in Madrid, Berlin, London, Paris, etc.to represent the Russian Federation to attract both professional participants of the tourist market and potential tourists.
Rights to exercise power
It has the following rights to exercise power: Request and receive information necessary for making decisions on matters within the competence of the Agency. Give explanations to legal entities and individuals on issues within the competence of the Agency. It has the awarding right of departmental decorations of the Ministry of Culture of the Russian Federation. The agency has the right to have a heraldic sign - emblem, flag, and pennant, established by the Ministry of Culture of the Russian Federation in consultation with the Heraldic Council. It has the right to exercise administrative power in the tourism sector. It has the right to receive federal budget funds. It can appoint scientific and other organizations, scientists and specialists to study issues related to the scope of the Agency's activities. To establish coordinating, advisory and expert bodies (councils, commissions, groups, colleges), including interdepartmental ones. Establish mass media for the publication of official announcements, placement of other materials on the issues within the competence of the Agency.
Structure
The Federal Agency for Tourism headed by a head appointed and dismissed by the Government of the Russian Federation on the proposal of the Minister of Culture of the Russian Federation. The head of the Federal Agency for Tourism is personally responsible for the fulfillment of the powers conferred on the Agency and the implementation of state policy in the established sphere. The head of the Federal Agency for Tourism has deputies. The government of the Russian Federation on the proposal of the Minister of Culture of the Russian Federation appoints and dismisses the deputy heads. From 15 January 2015, the head of the agency is Mr. Oleg Petrovich Safonov. The Government of the Russian Federation determines how many deputy heads will appoint. Currently, it has four deputy heads. They are Konyushkov Alexey Alekseevich, Koeneyev Sergey Evgenievich, Korolov Nikolay Vadimovich and Skoryy Roman Petrovich. Structural subdivision of the agency is the five Directorates. They are Directorate of State Tourist Projects and Safety in Tourism, Legal Directorate, Directorate of International Cooperation, Directorate of Domestic Tourism and State Target Programs, Administrative Directorate. This directorate has many departments.
Responsibility and power of the head and deputy heads
Responsibility and power of the head
Head of the Agency distributes duties between his deputies. Represent the Minister of Culture of the Russian Federation, nationally and internationally. He drafts regulations on the Agency. Propose the maximum number of employees and remuneration fund for Agency's head office. Propose candidacies for the posts of deputy heads of the Agency. Drafting annual plan and forecast indicators of the Agency's activities, preparing reports on their implementation. The right to award departmental decoration of the Ministry of Culture of the Russian Federation to agency employees subordinate organizations, as well as other persons carrying out activities in the sphere of the agency. Approve the structural divisions of the Agency. Appoints and dismisses Agency employees.Take executive decisions related to federal civil service in the Agency. He approves the structure and staffing of the head office of the agency within the limits of the labor remuneration fund and the number of employees. Provide cost estimates for the maintenance of the Agency for the corresponding period of Federal Budget. Submit proposal to the Ministry of Finance on the drafting of the federal budget for the relevant year.
Responsibility of the deputy heads
General responsibility of the Deputy heads is to help the head of the Agency to accomplish agency goals. All of them have different roles assigned by the head of the agency.
See also
Intourist
General References
Regulation on The Federal Agency for Tourism
Detailed information on Federal Agency for Tourism
References
External links
Category:Federal executive bodies of Russia
Category:Government agencies of Russia
Category:Tourism in Russia
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