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Presence of bacteria in failed anterior cruciate ligament reconstructions
- George Goodman
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1 DOI /s RESEARCH Presence of bacteria in failed anterior cruciate ligament reconstructions Open Access N Luisa Hiller 1,4*, Aakash Chauhan 2, Michael Palmer 3, Sameer Jain 2, Nicholas G Sotereanos 2, Gregory T Altman 2, Laura Nistico 4, Rachael Kreft 4, J Christopher Post 5,6,7 and Patrick J Demeo 2 Abstract Background: Novel microbial detection technologies have revealed that chronic bacterial biofilms, which are recalcitrant to antibiotic treatment, are common in failed orthopedic procedures. Questions: Are bacteria present on failed anterior cruciate ligament (ACL) reconstructions? Is there a difference in the presence or nature of bacteria in failed ACL reconstructions relative to a control set of healthy ACL s? Methods: We used a case control study design, where we analyzed the bacterial composition of 10 failed ACL reconstructions and compared it to 10 native ACL s harvested during total knee arthroplasty. The IBIS Universal Biosensor was used to determine the nature of bacteria on ACL specimens, and fluorescent in situ hybridization (FISH) was used to visualize bacteria in a subset of cases. Results: Bacteria are present in failed ACL reconstructions. Bacteria are present in ACL s harvested during total knee arthroplasty, but the nature of the species differs significantly between experimental and control sets. Twelve genera were detected in the experimental set (in both allografts and s), and in four samples multiple species were detected. In contrast, the control group was characterized by presence of Propionibacterium acnes. Conclusions: We demonstrate the presence of bacteria on failed ACLs surgeries, and open the door to investigate whether and how bacteria and the associated immune responses could possibly contribute to graft failure. Clinical relevance: If microbial pathogens can be linked to failed grafts, it could provide: (1) markers for early diagnosis of abnormal healing in ACL surgeries, and (2) targets for early treatment to prevent additional reconstruction surgeries. Keywords: Anterior cruciate ligament reconstructions, Bacterial composition, IBIS Universal Biosensor, Broad-range PCR and high performance mass spectrometry Background Revision anterior cruciate ligament (ACL) surgery is a challenging problem for orthopaedic surgeons and their patients as long-term outcomes of revision ACL surgery have been shown to be inferior to primary ACL reconstructions (Wright et al. 2012). Failed primary ACL reconstructions can be attributed to traumatic rupture of the graft, technical error, failure of the graft to incorporate, biologic factors, or a combination of the above *Correspondence: 1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Full list of author information is available at the end of the article (MARS Group et al. 2010). When using standard culture to detect infections, revision ACL surgery secondary to infection is reported in less than 1 % of all primary ACL reconstructions (Barker et al. 2010; Burks et al. 2003; Hettrich et al. 2013; Indelli et al. 2002; Katz et al. 2008; Matava et al. 1998; McAllister et al. 1999; Williams et al. 1997); diabetes is a major risk factor associated with post-operative infection (Brophy et al. 2015). When present, deep infection can hinder the ability of the graft to incorporate into the femoral or tibial tunnels, or weaken the structure of the fibers of the graft. A combination of mechanical and biologic factors can cause tunnel lysis or enlargement which leads to long-term instability of the graft (Wilson et al. 2004). Some studies did not observe 2015 Hiller et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
2 Page 2 of 8 a significant difference in the rate of infection between allografts and s, (Burks et al. 2003; Katz et al. 2008), while a large (>2000) patient cohort noted an association between lower rate of infection and s (Brophy et al. 2015). The rate of infections in orthopedic infections has been severely underestimated since clinical infections are usually detected by microbial cultures (Costerton et al. 2011). Studies on culture-negative revision arthroplasties, osteomyelitis, and bone fractures often reveal bacterial DNA and/or direct visualization of bacteria by microscopy (Costerton et al. 2005; Floyed and Steele 2003; Palmer et al. 2014; Tunney et al. 1998) (Gallo et al. 2011; Stoodley et al. 2008). This discrepancy is a result of the differences in metabolism between non-adherent planktonic bacteria and the slime-enclosed communities (termed biofilms) characteristic of chronic infections, which are resistant to growth on agar medium. Bacteria growing in chronic biofilms often do not invade host tissues or release toxins, and consequently can remain undetected for months or years (Costerton et al. 2007). An infectious etiology behind failed ACL reconstruction that has not been evaluated specifically is the presence of bacteria on reconstructed ACL grafts and their potential long-term effects. In this study we applied the Ibis Universal Biosensor, which integrates polymerase chain reactions (PCR) and mass spectroscopy (MS) to detect and characterize bacteria on failed ACL grafts and compared them to native ACL s harvested during total knee arthroplasty. In a subset of cases the bacteria was visualized by confocal microscopy using fluorescent in situ hybridization (FISH) with probes targeted at species-specific regions of the 16S rrna gene. The goal of this study was to test the hypothesis that bacteria are present on failed ACL grafts, and that the bacterial species differ between failed ACL grafts and native ACL s harvested during total knee arthroplasty. Results Description of subjects and detection technology The experimental group was composed of 4 males and 6 females with a mean age of 30 years old undergoing revision surgery for failed primary ACL reconstruction. The primary grafts were three allografts and seven s. The control group was composed of 6 males and 4 females with a mean age of 68 years old undergoing a primary total knee arthroplasty. The material analyzed consisted of tissue, aspirate and/or synovial fluid obtained intra-operatively. Arthroscopic evaluation during revision ACL reconstruction and direct clinical analysis during the total knee arthroplasties did not reveal signs of clinical infection at the time of surgery (Table 1). Microbial detection was performed on the PCR-MS platform using the BAC detection plate. This analysis does not require prior knowledge of bacterial presence and is independent of traditional culture. The PCR primers are targeted at the universally conserved 16s rrna gene and are optimized to classify the most abundant species within the samples. The BAC detection plate is extremely sensitive to Staphylococci since it contains additional primers targeting at this genus. Furthermore, primers targeted at the 23S rrna gene will capture Candida sp and Saccharomyces sp, which were not detected in this study suggesting the ACLs were not infected with these fungal species. Microbial composition of ACLs The PCR-MS identified bacteria in both the experimental and control samples, but the nature of the bacteria detected differed significantly between the sets (Fig. 1). Eighty percent of ACLs from failed grafts (including both the s and allografts) demonstrated evidence of bacterial DNA: Staphylococci (4/10), Streptococci (3/10), Clostridium (2/10), Propionibacterium acnes (2/10) and Treponema denticola (2/10), as well as single cases of Acinetobacter, Enterococcus faecalis, Escherichia coli, Lactobacillus crispatus, Nocardia asteroides, Pseudomonas mendocina, and Shigella flexneri. In contrast, the control group was characterized by presence of Propionibacterium acnes (8/10). Four experimental patients showed evidence of multiple species: one patient with N. asteroides, P. acne, and S. epidermitis; a second with Lactobacillus crispatus and T. denticola; a third with Clostridium, Enterococcus faecalis, Escherichia coli, T. denticola and S. epidermitis; and a fourth with Acinetobacter, Clostrium, Propibacteirum acnes, Pseudomonas mendocina, Staphylococcus hominis, and Streptococcus sp (Fig. 1). Eighty percent of the control group demonstrated evidence of bacterial DNA, but in this set the defining species was Propionibacterium acnes (8/10). In one control patient Acinetobacter and Sneathia were also detected. The gene coding for methicillin resistance was encountered in two patients from the revision ACL group. In four experimental samples and one control sample, where sufficient tissue was available, a secondary method of analysis was applied. These samples were stained by FISH and visualized by confocal microscopy (Fig. 2). Probes were selected based on the species detected by the PCR-MS analysis specifically: Staphylococcus sp, Streptococcus sp., Lactobacillus sp and P. acnes. These probes bind 16S rrna, thus the stained bacteria was metabolically active at the time of fixation and washing. We do not demonstrate a causal link between bacterial presence and ACL failure. However, the presence of bacteria in these failed ACL supports further investigations
3 Page 3 of 8 Table 1 Health and demographics of subject set Study group Estimated inter surgery time Age at time of study Sx Gender BMI Race ACL of interest Symptomology Findings Clinical Impression Failed graft type Reason for failure Experimental 8 33 M 28.0 Caucasian Right Swelling Tenderness, swelling, drainage (blood), instability Experimental F 29.1 Caucasian Right Pain, swelling, stiffness, instability Swelling, pain on No infection Autograft Mechanical No infection Hamstring and BTB patellar Experimental 3 30 M 33.1 Caucasian Right Pain, instability Instability No infection Patellar tendon Experimental 6 22 M 27.4 Caucasian Left Pain Swelling No infection Patellar tendon Experimental 8 17 F 20.1 Caucasian Right Pain Decreased ROM, pain No infection Hamstring Trauma Mechanical Experimental F 25.1 Caucasian Right Pain, instability Pain No infection Allograft Mechanical and technical Experimental 7 46 F 31.8 Caucasian Left Pain, Swelling Swelling, decreased ROM, pain on No infection Allograft Trauma Experimental 1 23 M 29.8 Caucasian Left Pain, Swelling Swelling, Pain on Experimental 7 25 F 20.3 Caucasian Right Pain, numbness, instability No infection Hamstring Pain No infection Posterior tibialis allograft Experimental 4 22 F 23.5 Caucasian Right Instability None No infection Patellar tendon Trauma Control NA 72 M 46.2 Caucasian Left Pain Pain on Control NA 72 F 22.1 Caucasian Right Pain Decreased ROM, pain on Control NA 64 M 37.6 Caucasian Left Pain Decreased ROM, pain on Control NA 61 F 30.3 Caucasian Left Pain Decreased ROM, pain on Control NA 66 F 30.4 Caucasian Left Pain Tenderness, decreased ROM Control NA 63 M 32.7 Caucasian Right Pain Decreased ROM, pain on Trauma Trauma Trauma Mechanical
4 Page 4 of 8 Table 1 continued Study group Estimated inter surgery time Age at time of study Sx Gender BMI Race ACL of interest Symptomology Findings Clinical Impression Failed graft type Reason for failure Control NA 74 M 29.4 Caucasian Right Pain Tenderness, decreased ROM, pain with Control NA 75 F 30.9 Caucasian Left Pain Swelling, tenderness, decreased ROM, pain on Control NA 52 M 31.5 Caucasian Left Pain Swelling, tenderness, decreased ROM, pain on Control NA 70 M 38.9 African Am. Left Pain Tenderness, decreased ROM, pain with NA not applicable, ROM range of, BTB bone-patellar tendon-bone.
5 Page 5 of 8 Fig. 1 Bacterial composition of ACL samples. Left side control set of ACLs from knee arthroplasties. Right side experimental set of ACLs from failed grafts including s (green) and allografts (red). X marks gene for methicillin resistance. Fig. 2 Confocal images of ACLs visualized using fluorescent in situ hybridization (FISH) targeted at 16S ribosomal RNA from Staphylococcus sp (a), Streptococcus (b, d), Lactobacillus sp (c), and P. acnes (e) on four experimental samples (one is co-infected with Streptococcus sp. and P. acnes), and P. acnes on a control sample (f). Red corresponds to the bacteria and blue to reflected light from the tissue.
6 Page 6 of 8 to determine whether there is a role for bacteria in tissue degeneration and/or host inflammation. Discussion The overall ACL revision rate varies in the literature, with the most recent longitudinal long-term results from the MOON group reporting an overall 7.7 % revision rate of the ACL reconstructed knee at 6 years follow-up (Hettrich et al. 2013). Management of septic arthritis following ACL reconstruction requires immediate arthroscopic irrigation and debridement. The rate of removal and replantation of the graft varies in the literature, with recent evidence to suggest that immediate irrigation and debridement with retention of the graft may lead to acceptable results at 5 years of follow-up (Windhamre et al. 2014; Burks et al. 2003; Maletis et al. 2013; McAllister et al. 1999; Schulz et al. 2007; Williams et al. 1997). Currently infections are monitored using standard culture techniques, and are reported in less than 1 % of ACL reconstructions (Barker et al. 2010; Burks et al. 2003; Hettrich et al. 2013; Indelli et al. 2002; Katz et al. 2008; Matava et al. 1998; McAllister et al. 1999; Williams et al. 1997). Our PCR-MS analysis detected bacteria in eighty percent of the experimental samples, suggesting that bacterial presence is ACLs is significantly underestimated by current techniques. This is consistent with studies on other types of orthopedic infections where bacteria were present in culture-negative orthopedic infections (Costerton et al. 2011; Jacovides et al. 2012; Palmer et al. 2014; Stoodley et al. 2011a, b; Mariscalco et al. 2014). The most common culture positive pathogens associated with septic arthritis after ACL reconstruction are S. epidermis and S. aureus (Barker et al. 2010; Burks et al. 2003; Hettrich et al. 2013; Indelli et al. 2002; Katz et al. 2008; Matava et al. 1998; McAllister et al. 1999; Williams et al. 1997; Windhamre et al. 2014; Maletis et al. 2013). We detected these species in 3/10 of the experimental set and none of the control set. The bacterial species identified in the experimental sample are common human flora, and have been associated with both commensal and pathogenic states. Multiple genera identified in the experimental set have been identified in other types of orthopedic infections including Staphylococci, Streptococci, and Treponema. These three genera are oral colonizers, thus it is possible that these oral bacteria gain access to the circulation and establish a local infection when they encounter damaged tissue. The route of entry and its clinical significance remain open to debate and should be addressed in future studies. While bacteria were detected in both experimental and control groups, the species composition varied between these groups. Multiple species of known human pathogens were encountered in the failed grafts. In contrast, arthroplasty samples were characterized by the presence of P. acnes a common skin commensal. These results are consistent with the hypothesis that the activation of either bacterial regulatory pathways and/or host inflammatory responses affects the healing of ligaments and bone that are required in the repair of ACL damage. Alternatively, it is possible that these bacteria are present but do not play a role in graft failure. The present of P. acnes in the control samples from knee arthoplasties underscores the importance of characterizing the specific species and suggests that presence of bacteria alone does not implicate ACL damage. In fact, it is possible that under some conditions certain bacterial species could be beneficial by competing away pathogens or modulating inflammation. The correlation between individual species, host response, and damage is an import next step. Failed ACL reconstructions can be attributed to many causes and our study shows that bacteria are commonly present within reconstructed ACL grafts irrespective of the type of graft used. A chronic, indolent bacterial infection could contribute to a significant weakening of the graft and create a chronic inflammatory environment, which could cause further compromise to the grafts integrity. This hypothesis will require a future study to analyze the cause and effect of chronic bacterial infections on the structural integrity of the ACL graft over time. Our belief is that bacterial presence contributes to failure of ACL reconstructions, which are likely the result of many contributing factors (e.g. mechanic and biologic). If subclinical chronic bacterial infections play a role in ACL graft failure, it will open the door for the development of diagnostics and treatments, targeted at the bacteria or host inflammatory response, to prevent ACL graft failure in the future. Methods Study design and sample preparation Subjects for this study were recruited at the Department of Orthopaedic Surgery at Allegheny General Hospital, and approved by the Institutional Review Board (approval number FWA ). A total of twenty ACLs were collected from 20 patients. The experimental group consisted of ten ACL s from ten patients undergoing revision surgery for failed primary ACL reconstructions. The control group consisted of ten native ACL s from ten patients undergoing total knee arthroplasty without any prior surgery. The average time to revision surgery was 7 years. Specimens were collected under sterile conditions in the operating room, placed immediately into a RNA
7 Page 7 of 8 stabilization agent (RNAlater, Qiagen, Germantown, MD) and stored at 80 C for evaluation with the IBIS T5000 Universal Biosensor System and/or FISH analysis. DNA extraction and IBIS universal biosensor bacteria, antibiotic resistance, and Candida (BAC) detection assay for microbial identification For DNA extraction the ACL was placed into a sterile microcentrifuge tube containing ATL Lysis buffer (Qiagen, Germantown, MD, cat# 19076) and proteinase K (Qiagen, cat# 19131). Samples were incubated at 56 C until lysis. 100 μl of a mixture containing 50 μl each of 0.1 and 0.7 mm Zirconia beads (Biospec cat# z, zx respectively) were added to the samples which were then homogenized for 10 min at 25 Hz using a Qiagen Tissuelyser. Nucleic acid from the lysed sample was then extracted using the Qiagen DNeasy Tissue kit (Qiagen cat# 69506). For microbial detection, 10 μl of each sample was loaded per well onto the BAC detection PCR plate (Abbott Molecular, cat# PN 05N13-01). The BAC detection plate is a 96 well plate which contains 16 primers that survey all bacterial organisms by using the omnipresent loci (e.g. 16S rrna gene sequence), while some are targeted to specific pathogens of interest (e.g. the Staphylococcus-specific tufb gene). The plate also includes primers for the detection of Candida species and some antibiotic resistance markers (e.g. meca, vana, vanb, and KPC). An internal calibrant of synthetic nucleic acid template is also included in each assay, controlling for false negatives (e.g. from PCR inhibitors) and enabling a semiquantitative analysis of the amount of template DNA present. PCR amplification was carried out and the products were desalted in a 96-well plate format and sequentially electrosprayed into a mass spectrometer. The spectral signals were processed to determine the identities of the pathogens and a semi-quantitative determination of their relative concentrations on the ACLs (Ecker et al. 2008). Fluorescent in situ hybridization (FISH) Aliquots of the ACL samples were fixed with fresh 4 % paraformaldehyde and incubate for 2 4 h at 4 C. After the incubation the specimen was spun down and the supernatant removed, this process was repeated twice with Hank s Salt Saline Solution (HBSS). Next, the samples were resuspended in 50 % Ethanol-PBS solution and stored at 20 C for evaluation with FISH. FISH was performed as described by Nistico et al. (Nistico et al. 2014), using species-specific and genus-specific fluorescent 16 s rrna probes. The bacteria targeted and probe sequences selected were: (1) Streptococcus GTG ATG CAA GTG CAC CTT (Kempf et al. 2000); (2) Staphylococcus sp TCC TCC ATA TCT CTG CGC (Trebesius et al. 2000); and (3) Lactobacillus sp CCATTGTGGAAGATTC- CCT (Quevedo et al. 2011). Samples were observed with Confocal Scanning Laser Microscopy (CSLM) imaging using a Leica DM RXE microscope attached to a TCS SP2 AOBS confocal system (Leica Microsystems, Exton, PA) using a 63X (NA1.2) water immersion lens. Authors contributions Conceived and designed the experiments: PJD; performed the experiments: RK, LN, NGS, GTA; analyzed the data: NLH, PJD, JCP, AC, MP; contributed materials/analysis tools: GTA, NGS, SJ, MP; wrote the paper: NLH, PJD, JCP, RK, LN, AC, MP. All authors read and approved the final manuscript. Author details 1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA. 2 Department of Orthopaedic Surgery, Allegheny General Hospital, Pittsburgh, PA, USA. 3 Surgical Operations Squadron, 88th Medical Group, Wright-Patterson Air Force Base, Dayton, OH, USA. 4 Center for Excellence in Biofilm Research, Allegheny Singer Institute, Allegheny General Hospital, Pittsburgh, PA, USA. 5 Department of Surgery, Allegheny General Hospital, Pittsburgh, PA, USA. 6 Drexel University College of Medicine, Philadelphia, PA, USA. 7 Temple School of Medicine, Philadelphia, PA, USA. Acknowledgements The authors would like to thank Angela Finamore for coordinating this study, Tracy Spirk for her work with DNA extractions, Barbara Byers and Kaitlyn DeMeo for their work with confocal imaging, and Leon Hak-Jin Kim for his work on data analysis and presentation. We are also grateful to the generous financial support from the Allegheny Health Network. Compliance with ethical guidelines Competing interests NLH, LN, and RK receive financial support from Allegheny Health Network. NLH is funded by the NIH Grants R00DC No conflicts of interest to disclose. Ethical review committee statement IRB approval letter is attached. Patient samples were collected at the Department of Orthopaedic Surgery, Allegheny General Hospital, Pittsburgh, PA, and all subjects gave their consent to participate in this study. Samples were analyzed at the Center for Excellence in Biofilm Research, Allegheny Singer Institute, Allegheny General Hospital, Pittsburgh, PA. Received: 26 May 2015 Accepted: 3 August 2015 References Barker JU, Drakos MC, Maak TG, Warren RF, Williams RJ, Allen AA (2010) Effect of graft selection on the incidence of postoperative infection in anterior cruciate ligament reconstruction. 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This is the test for the Project for a New American Constitution's "How A Bill Becomes A Law" Series, Lesson #3: Technique.
LESSON 3 TECHNIQUEEdit
This is a timed test, you will be given 60 minutes to complete all of the questions listed below.
Complete all the questions to the best of your abilities, if you are not sure of the answer, skip the question and return to it if you have time, guessing is satanic and for every question you guess on, points will be taken off the total score bringing you that much closer to hell.
Scroll down when you are ready to take the test.
SECTION 1-ESSAY QUESTIONSEdit
Spelling and punctuation mistakes will not be held against the test-taker. Limit your essays to no less than 6,000 words, and write in the style of The Bible, noting where your verses begin and end.
QUESTION 1Edit
Describe what areas of American life could use some reform. Explain why it is important for people to participate in politics and how they can influence those areas needing reform.
QUESTION 2Edit
Describe why only certain people may participate in certain aspects of American political life.
QUESTION 3Edit
Describe how the following people participate in modern American political life:


text:
The Flight of the Mango Flowers: A Memoir of Our Way Out of the Cold War. A Testimony of Pedro Panes and the Early Cuban Exodus. (e-book)
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text:
Size Change
One of the most common reasons for revision surgery is size change. This is a medically unnecessary procedure and should only be performed with sufficient good reason. A size change revision is also totally avoidable. If a patient understands at the time of the first operation that implant size should be based upon both measurements of their own tissue and published data about the ideal size for any given measurement, then it follows that each breast has one "correct" size. Dr. Teitelbaum is a renowned expert for his ability to determine the "correct" size for you.
Sometimes, patients gain or lose weight and therefore need to change their implants. Breastfeeding can also lead to a change in the breasts and the size of an implant that will best fit.
In general, more patients actually want to be revised smaller rather than larger. This finding dispels the myth that patients always wish they had chosen a larger implant size. This desire to reduce the implant size could be because the patient wants a more natural-looking breast, may face discomfort during sports or fitting into fashionable clothes, or even simply wants accommodate her evolving personal relationships and lifestyle. Whatever the reasoning behind the revision, Dr. Teitelbaum will provide you with an exceptional surgery.
Before and After Size Change Photos
Large Images
Capsular Contracture (Right), Implant removed (Left)
This patient had multiple attacks of capsular contracture. The right implant became infected and was removed. Scar tissue was removed from both breasts, and new, larger implants were placed.
Capsular Contracture, Drooping, Wants Larger Implants
The scar tissue made her implants high, hard, and round. Scar tissue was removed and Strattice used to reduce chances of contracture. Larger implants were placed. Her tissue is droopy, but she did not want a lift.
Inferior, Lateral Malposition, Wants Larger
Both implants are too small and narrow, and the left is very low and outside. Larger implants placed in the proper space gave closed the gap between her breasts and made it much more attractive.
Lateral Malposition, Wants Smaller
Her implants have fallen down and out, leaving her upper inner breasts empty, her cleavage wide, and causing her breasts to rub against her arms. Even with smaller implants, by closing the outer pocket she achieves more upper fullness.
Capsular Contracture, Wants Larger
Scar tissue around her breast implants hard, round, and unnatural looking. She wanted more cleavage, soft breasts, and prettier breasts. Removing the scar tissue and placing larger implants improved her cleavage and her shape.
Symmastia, Inferior Malposition (L), Wants Smaller
This patient did not know exactly was wrong. She just knew that they were too big and mishapen. The right was far inward, and the left was inward and low. The pockets were equalized and implants were placed which fit her body.
Rippling, Wants Larger
Her implants were too small and too wide apart. Her breast skin was not filled. A slightly larger ipmlant and removal of scar tissue gave her better cleavage, shape, and treated the rippling.
Wants Larger, Rippling
She is round and far apart, so it looks like a contracture. But it is actually a high profile saline implant (see rippling on side view.) Larger silicone ipmlatns were placed which improved her cleavage, shape, and rippling.
Superior Malposition (Left), Deflation Saline (L), Desires Larger
Her left breast was always too high and recently started leaking and getting smaller. She also wanted to be larger, so larger silicone implants were placed.
Lateral Malposition, Wants Bigger
The left implant was positioned too low and to the side. Moving that inwards would increase upper fill. But she also wanted the right to look more round but not too fake, so slightly larger implants were used.
Inferior Malposition, Wants Smaller
Her implants were positioned to down and in. They were also too big and fake for her vey lean body. The implants were better centered under her nipples and smaller, less highly projecting implants were placed.
Lateral Malposition (Left), Wants Smaller
The left implant was placed too low and to the side. She also just wanted her breasts to be less obvious. So the pocket was corrected and smaller ipmlants placed.
Wants Smaller
Her surgeon talked her into going larger than she wanted, because he told he that everyone wishes they were larger (that is not true.) Clothes were too tight on her bust, so she wanted to go smaller, yet still have full and atractive breasts.
Medial/Inferior Malposition - Symmastia
Concave breast bone and large implants caused symmastia. Strattice was used to reduce and strengthen new pockets, and smaller implants were placed. The left implant was also too low.
Severe Rippling
This patient had such large implants they thinned her tissue. And her muscle was damaged, so she lost coverage over the implants. Note the implant fold at 7 O'Clock on the right breast. Strattice was used to thicken the tissue and pull the muscle down.
Symmastia, Wants Smaller
It is obvious that her implants were simply too wide for her chest. They could not go anywhere but touch in the center. The central pocket was closed, reinforced with Strattice, and smaller implants were placed.
Wants Smaller
These very large implants were placed through the nipple and were too large and uncomfortable for her. Note the distracting scar around her areola. While some Americans use this incision for Asian patients, it is rarely used in Asia.
Symmastia, Wants Smaller
This patient developed symmastia immediately after surgery. Her implant pockets were actdually connected. It is clear that her implants were wider than her chest. She was corrected with smaller implants and a neosubpectoral pocket.
Superior Malposition, Wants Larger
It is hard to know from a picture that this is not capsular contracture. But she was soft; the implants were just too high, and that is why she had the upper bulge. The lower pockets were adjusted and larger implants given as she wished.
Wants Smaller
This very athletic woman liked her implants, but they were just a bit big for her given how thin and active she is. Though they do not appear to be a lot smaller in the photograph, they really changed the way she felt about her breasts.
Wants Larger
She wanted more cleavage, but she didn't want her breast to look round. Her round implants were removed and were placed with larger teardrop shaped impalnts. Note the increase in her cleavage and the overall less round appearance. Subtle, but real.
Drooping (Bottoming Out), Wants Smaller
This patient has had large implants for years, and already had a lift once. But the heavy implants stretched her again. Now she wants a longer lasting result, so the lift was redone and smaller implants placed.
Wants Smaller
Very common story of a woman whose boyfriend paid for and talked her into enormous implants. She developed neck and back pain, and finally wanted implants that were comfortable. Note the loose skin along the inner breast; is from the big implants.
Drooping, Wants Smaller Implants
She "emptied out" after babies and had very large ipmlants placed, which caused her to droop and were always too large. Smaller implants were placed and a lift was done. At 6 months the scars are still very visible, but faded over time.
Wants Larger
If you look carefully, the upper poles of both breasts are a bit concave, and after surgery they are full. She didn't want a big change, only enough to be full. She also wanted her breasts evened out.
Medial/Inferior Malposition - Symmastia and Wants Smaller
Her implants are wide, but they were also placed too close together. Note how that tips her nipples outwards. Strattice was used to reinforce the tissues adn keep the implants more outwards, and smaller implants were placed.
Wants Larger
Her wish was not more size per se, but to be more full on the top and to be closer together in the center. Obviously, that meant having larger implants, though the look itself just looks prettier more than it looks larger..
Wants Smaller, Capsular Contracture, Drooping, Removal
Her breasts always felt too high and disproportionate. She had several failed attempts at treating capsular contracture. She just wanted to be a perky A or B cup, so the implants were removed and she had a lift. Scars are still red here at 3 months.
Inferior Malposition, Larger implants
Whether due to surgeon error or weak tissues, her implants slid downwards. She also wanted to be larger. The lower pocket was closed off and new implants placed. Note the improved nipple orientation just from the implants being in the right place.
Inferior Malposition, Wants Larger
Her saline implants were low, which left her with very little fill of the upper breast. she also wanted them to be a bit fuller and larger. She wanted the smooth upper pole of the 410 breast implant.
Wants Larger, Drooping
She had high profile implants, which are very round, too narrow for her, and have an abrupt step-off on the top. Larger moderate profile implants fixed those problems. She is droppy but does not want a lift.
Drooping, Wants Smaller Implants
This older but athletic woman wanted smaller and perkier implants. After replacing with smaller implants and doing a lift, she looks much better three months after surgery. Scars will fade and shape will improve with time.
Medial/Inferior Malposition - Symmastia and Wants Smaller
Her implants are too wide for her chest, and so they met in the center. The central pockets were closed off and smaller implants were placed.
Wants Larger, Rippling
She had high profile silicone implants in front of the muscle which were too small for her. Going behind the muscle with a larger anatomic implant gave her a much more attractive shape and eliminated her rippling.
Drooping, Wants Smaller, Removal
This woman gradually gained weight, mostly in her chest. Her breast were too large and pendulous. She wanted them smaller and perkier. After the weight gain, she had adequate tissue of her own, so implants were removed and she had a lift.
Drooping, Lateral Malposition, Wants Larger
Her implants were too narrow, the left was too far to the side, she was droopy, and she wanted more cleavage. Larger implants were moved towards the center and a donut lift was performed.
Capsular Contract
Her breasts were very round, hard, far apart, and painful. The scar tissue was removed. Though she looks smaller, the new implants are actually bigger. Note the improvement of cleavage and overall balance of the breasts.


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High waisted, back zippered dresses and tops have raglan sleeves; short sleeves A or long sleeves B have elastic in lower casings. D has flared sleeves. A, B has neck binding and bow. C, D has neckbands, and tucked, button trimmed upper fronts.
Links to reviews/blog posts Edit
Sources/Vendors Edit
Gallery Edit
[insert your photos of this pattern made up]
Wishlist Edit
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


text:
- Internships /
- Finance /
- Covance - Student Temp - Global Rewards -Part Time
Similar Internships
FinanceArmstrong World Industries
financeCNO Financial Group, Inc.
Market Research & Analysis InternBurton Hill Capital
Finance RepresentativeReliance Capital Limited
FINANCE OR ACCOUNTING INTERNLundbeck Canada Inc.
West Coast Business Capital InternshipWest Coast Business Capital
FinanceArmstrong World Industries
financeThe Blackstone Group L.P
Covance - Student Temp - Global Rewards -Part TimeBookmark This
3301 Kinsman Blvd.
Madison, WI
Posted: March 02 2016
Always be on the alert for potentially fraudulent job postings online - never send money to a potential employer.
DescriptionPART-TIME OPPORTUNITY FOR STUDENTS - HUMAN RESOURCES, ACCOUNTING, AND FINANCE
To see full job description and to apply online: http://careers.covance.com/job-postings/57419BR/student-temp-global-rewards or go to careers.covance.com and search 57419.
We are currently hiring a student temp for our Global Rewards team in Madison, WI.
The incumbent will work 15 hours per week throughout the entire year. This position requires the ability to work independently with various departments and vendors.
This is considered a part-time temporary position (not an internship).
Covance is one of the world's largest and most comprehensive drug development services companies, with annual revenues greater than $2 billion, employees in more than 60 countries, and more than 12,000 employees worldwide.
ResponsibilitiesDuties for this position include:
•Assists the Corporate US Benefits Department with day to day tasks in regards to the 401k plan and health benefits.
•Audits/monitors eligibility, elections, payroll data, and employee status on a per pay period and monthly basis.
•Reviews and reconciles premium payment reports and assists with SOX auditing as well as external compliance audits.
RequirementsIdeally we are looking for a first or second year college student
interested in a multiple year temp position in Human Resources, Accounting, or Finance. Candidate should be available to work 3 days per week including Thursday and Friday.
► 15 hours per week throughout the entire year
► Requires the ability to work independently with various departments and vendors
► Strong written and verbal communication skills with high proficiency in Microsoft Office (Word, Excel, Powerpoint)
► Attention to detail
► The ability to keep information confidential


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Print advertisment created by JWT, United Arab Emirates for Virgin, within the category: Retail Services.
Give your eyes a rest.
Audio Books available at Virgin Megastore.
Executive Creative Director: Chafic Haddad
Senior Art Director: S M Ziyad
Art Director: Tarek Samaan
Illustrator: Milind Aglave
Senior Copywriter: Zahir Mirza
Highest Rated
hey wait a minute, ive seen the same idea somewhere.
Ivan, wasnt there an ad a while back that had big eyeballs?
I know it was an ad for eye drops and it had big eye balls going arround annoying people and it said something like, "irritating eyes"
Now i remember, who else remembers that?
enter your keywords: eyeballs - and you will find a huge number of eyeball-ads
I did and not a single eyeball ad came up.
You have to have eyeballs to steal from David Droga.
Cannes 2000.
why u guys put so much efforts executing such bad idea?
use that time to think better ideas instead..!!
Un poco literal ¿no?
Muy buena de gráfica de todos modos.
Vision Express are using big eyeballs at the moment:
bunch of idiots that said its done, none of the examples have the same concept, they might use the eyeballs, but all three of them have very different concepts.
Popular Reads
Clio Sports
"Say Less, Do More": Words of Inspiration From the 2017 Clio Sports AwardsRead More
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How DDB's Ari Weiss And Keith Reinhard Are Embracing Ad Legend Bill Bernbach's "Rebel Spirit"Read More
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Ward+Robes Designer Gowns Offer Individuality for Hospitalized TeensRead More
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It all started innocently enough. Last day of work before a two-week vacation. I use Rdio all the time for music while programming. Embarrassingly so in fact.
So in preparation for my trip, I was trying to download some of the talks from DjangoCon Europe for my flight for offline enjoyment. Using one of these pieces of software I noticed that it was able to download the current song I was listening to on Rdio.
So 3 flights later and 4 days without reliable internet later. I managed to reproduce the fluke that I randomly ran into.
Here is how I did it.
Poking around.
Once I saw that software I was using to offline videos was downloading Rdio songs. I popped over into the inspector and took a look at the network tab and saw that they are sending a file called full-192.mp3 which mean full song at a bitrate of 192K.
Hmm. Okay.
Rolling up my Sleeves
So I decided to crack out some tools. First I needed to get a better understanding of the communication between Rdio's servers and my machine.
Enter mitmproxy, which is a man-in-the-middle proxy for HTTP. This gave me a bit more of an understanding of the traffic Rdio is sending around and eventually allowed me to script a way to download full mp3 versions of any song on their service.
On to the details.
As you can see in the image above; We were also able to capture the GET request being sent for the full-192.mp3 which returns the entire song being requested. That's all good.
What's awesome is the response below.
Yep, the entire content of the full-192.mp3!!
Once you have reached this point a little (under 10 lines) script is all that is needed to download the full mp3 to your computer.
Hopefully, the Rdio Team gets a handle on this soon.
Edit: The same bug occurs in their "native" application as well on OSX.
Edit 2: My intention here is not to be malicious. It is to point out this bug and see that it gets resolved and hopefully, they will get it fixed soon. I love Rdio as a service.


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Sailing with Paradoxe Crois is a great way to see the island from the sea as well as mingle with other vacationers like yourself as this company specializes in large group tours.
Typical excursions include sailing to Petite Terre and Marie Galante. The Petite Terre trip leaves at 8:00 a.m. and returns at 5:30 p.m. and features snacks and drinks on the boat, a walking tour of the island, lunch on the beach, and a rum tasting on the way back. The Marie Galante trip, meanwhile, leaves at 7:30 a.m. and returns at 5:00 p.m. with all of the same activities.
Paradoxe II is an 82 foot catamaran.
Office hours are 8:00 a.m. to noon and 4:00 p.m. to 7:00 p.m. daily.
Paradoxe Croisieres is located near the middle of Saint-Francois, a city on Grande-Terre; it is to the east of Pointe A Pitre.
Location: Saint-Francois, Grande-Terre
Phone: (059) 088-4173
Website: http://www.paradoxe-croisieres.com/
Email: email@example.com
Help us improve! We welcome your corrections and suggestions.


text:
In the moment she felt surprised that he would enter her without protection. She gasped at the unfamiliar warmth of his skin touching her most sensitive of places. He sunk the familiar inch into her quickly and stopped. Her hips tried to rock more of him inside her, but he held her down with both hands to keep her still.
"You want me," he whispered in her ear, sliding his lips down to her neck and biting it before she could reply.
"Please, yes!" she gasped.
"Aren't you afraid I might cum in you without pulling out first?" He seemed amused for a moment beacause she had to take a few deep breaths before replying.
"I-I trust you. This feels so good, I trust you. I'm your woman. Now, please."
"Please what?"
Please what indeed. He felt so big. Too big. Like he'd been hiding half his girth in the condom the whole time, or he'd exchanged his old dick for a newer one a half size bigger. The bastard.
She remembered the first few weeks of their courtship, the romantic dates that had melted into seductions that put her in touch with her submissive side. He'd broken her in each night, just like a new pair of shoes. Broken her stubbornness and stretched her pussy to accommodate his mass. Now when the doors were locked and the windows shut, she was lovingly obedient, attentive, and wet. She'd surprised him one evening by wishing she had a collar to wear around her neck when they were alone. The next day he'd bought one for her and she wore it the same night. That night she had begged him to cut himself loose and stop being so gentle with her tiny pussy.


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Creating fluid zone within Fluent
Hi ,
Does anyone know if we can create a new fluid zone within "Fluent" that covers a part of the fluid zone of the entire geometry? I want to find out the "volume integral" of a custom-field function I have defined. I want calculate the volume integral of the function for only a part of the fluid zone. It would be great if anyone suggest a solution for this problem.
Thanks, Suresh
Re: Creating fluid zone within Fluent
Yes. Use Grid/Separate (cells) to create a new fluid or solid zone. You can use the Adapt functions to precisely mark a specific zone can be separated.
You should ask your questions in the Fluent forum for a better response :)
|All times are GMT -4. The time now is 19:54.


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Beautiful Squeeze Spiral Solar Desk Lamp
Here's one solar lamp that recharges itself using clean energy, solar power that is. It does not come in the shape of a traditional lamp which makes use of a single lamp, but comes in a spiral form that illuminates light along its entire spiraled body.
This spiral lamp called 'Squeeze' is a lamp that comes in a double sided strip of acrylic shaped into a spiral. On the outer side just beneath the translucent hued surface lies a strip of solar panels that capture the energy from the sun, while the inner side comes with the light emitting source, which is an LED stripe running along the spiral.
With one side capturing energy and the other side giving out light, this is definitely a clever way to make use of every available space on the surface of the solar lamp to capture solar energy and for producing light. This spiral shape using an LED strip instead of the usual rounded light bulb breaks the tradition of how lamps look and does give one a feeling of freshness when incorporated into the décor of a room.
A beautiful-looking solar lamp that will fit the style of a modern home or office.
The Squeeze spiral solar desk lamp retails for $990 on Etsy.


text:
Brian Baldinger suspended six months by NFL Network for bounty comments
NFL Network analyst Brian Baldinger has been suspended six months from his role after telling a local radio station that the Eagles should put a bounty on anyone trying to stop Cowboys rookie running back Ezekiel Elliott, according to ProFootballTalk.
Baldinger's suspension is without pay.
"This is the guy that we've got to take out of the game," Baldinger said on Philadelphia's 97.5 The Fanatic. "There's got to be 10 guys that want to hurt him every single play. In fact, we may even put a little bounty on Ezekiel Elliott."
Baldinger, a former NFL offensive lineman, played for the Cowboys from 1982 to 1987 and then the Eagles in 1992 and 1993.
• Just how many teams are actually good this season?
The Eagles limited Elliott to 96 yards, his fewest since Week 2. He has 799 yards and five touchdowns on the season.
The NFL has strengthened its stance against bounties after a scandal involving the New Orleans Saints from 2009–2011.


text:
Hot Springs Village Blog
Danville & East Gate "Free-Exit" Upgrades
HSVPOA to install "free exit" hardware at the Danville and East Gates.
Danville Gate – March 7, 2017
The exit side of the Danville gate will be closed on March 7th for installation of "free exit" hardware. Only emergency vehicles will be allowed to exit from the entrance side of the gate, during this time. In the event of thunderstorms in the area, the alternate closure date will be March 14, 2017. Please plan to use alternate routes.
East Gate – March 8, 2017
HSVPOA IT Department will be installing "free exit" hardware at the exit side of the East gate on March 8th. During this time, exit traffic will be re-directed through one of the entrance lanes. In the event of thunderstorms in the area, the alternate closure date will be March 15, 2017. Please obey all traffic signs and use caution while driving in work zones.
Once the hardware is installed on the Danville and East gates, vehicles will be able to exit these gates without an electronic gate access card. The goal of the "free-exit" upgrade is to improve member and visitor experience. Security at all gates will continue to be monitored with cameras.
Schedules for installation of "free exit" hardware at other HSVPOA gates will be announced.
Events At The Village
Visit The Village
West Gate Visitor Center
121 Cordoba Center
Suite 300


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|Add to
As divided into two prime categories - reclining loveseats and stationary loveseats - The Classy Home features all the most wanted styles and material components of loveseats that you have always dreamed for. Offering that justifiable comfort not just for one person, but for two or more, all loveseats here at The Classy Home serve the top purposes of painless and restful sitting experiences. Due to the wide range of selections available, you will never have to think twice about searching somewhere else as every style, every color, and every personality of loveseats that you need have been readily made available here at The Classy Home for your prime satisfaction!


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Wilson Artisan Wineries Offer Scholarships
Children of Sonoma County Vineyard Workers Scholarship
Application Deadline Extended:
5 p.m. March 1, 2017
Scholarships of $750 to $2,000 will be awarded to children of Sonoma County vineyard workers. Scholarships are open to high school seniors continuing their education and to students who are
already in a college, trade, or technical school.
Wilson Artisan Wineries provides these annual scholarships because Ken and Diane Wilson believe the children of Sonoma County vineyard workers should not be limited by financial hardships in their pursuit of educational opportunities. This year Wilson Artisan Wineries will award more than $20,000 in scholarships to deserving students.
Download Scholarship Application:
English: Children of Vineyard Workers Scholarship - English.pdf
Spanish: Children of Vineyard Workers Scholarship - Spanish.pdf
Completed applications are to be mailed to:
Antoine Favero
1400 Lytton Springs Road,
Healdsburg, CA 95448
For more information contact:
Wilson Family Winery - 707-385-2261


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Listen to free music by Gfmgf
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station.
GFMGF – Закрой рот открой глаза| play
Jun 15, 2010 at 1:23 pm via YouTube | AMAZON | ITUNES
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text:
Adafruit Perma-Proto Half-sized Breadboard PCB - 3 Pack!
Customers have asked us to carry basic perf-board, but they never liked the look of most basic perf: its always crummy quality, with pads that flake off and no labeling. Then they thought about how people actually prototype - usually starting with a solderless breadboard and then transferring the parts to a more permanent PCB. That's when they realized what people would really like is a proto board that makes it easy!
This proto-board is the PCB you always wish you had, but never realized it! Adafruit took the basic layout of a half-sized breadboard and turned that into a beautiful PCB. The top side has a white silkscreen, and the same markings you're familiar with, to make transferring components easy. The bottom has the 5-hole pad design that matches a classic breadboard, with 4 power bus lines on the sides, and no mask so you can easily cut traces when necessary. Adafruit used 1.2mm diameter drill holes so even parts with big leads will fit. All holes are thru-plated for strength - these wont peel off with rework. The finish is a gold plate - you won't get oxidation like with bare copper perf! There are also two mounting holes so you can attach the PCB to your project box. They'll also fit nicely in an Altoids-sized mint tin
These are so handy, they decided to put them in a pack of 3. Once you use a Perma-Proto board, you'll never go back!
Technical Details
- 30 rows of double 5-hole rows
- 4 power rails with positive/negative markings
- 3.2" x 2.0" (81mm x 51mm), 0.063" thick FR4
- 1.2mm / 0.047" drill holes
- 2 x 0.125" or 3.2mm mounting holes 2.9" apart
- Breadboards for Beginners
- Remote Control with the Huzzah + Adafruit.io
- DIY Bluetooth Gamepad
- Pro Trinket as a USB HID Mouse
- Collin's Lab: Breadboards & Perfboards
- Trinket-Powered Conference Room Occupancy Display
- Secret Knock Activated Drawer Lock
- Trinket Powered Analog Meter Clock
- PianoGlove
- Reverse Geocache Box


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Kidz R People Too
Alameda, CA
To view the State's license info—inspections, citations, and complaints—click the License number below. Also see About Childcare & Preschool Reviews.
Parent Reviews
Parents, have you used this organization's services? Sign in to post a review.
I'm recommending Kidz R People Too Family Daycare in the city of Alameda; which is owned and operated by Ms. Elizabeth Rucker. Ms. Rucker is extremely personable and attentive. All of the children she facilitates, love her and her assistants. If you're a family looking for a loving, fun, upbeat, structured daycare this is the perfect place for your child. Ms. Rucker nurtures the children so beautifully while also teaching them independence.
My child has been enrolled in this in-home family daycare from 3 months old and is now 3 years old. His vocabulary is extensive, his personality continues to flourish, he has amazing table etiquette, and is extremely independent. He loves daycare so much!
Jan 2013
Just wanted to recommend the daycare provider that I use part-time for my 16 month old - Kidz R People Too run by Elizabeth Rucker.
It's a small family day care (only about 4 or 5 kids), but is set up and run like a preschool - so, in my opinion, the best of both. My son loves the two caregivers there and they love him. So glad I found them. They are super affordable and usually have at least one or two openings.
Anyway, in case you're looking for Alameda (West End) day care - she doesn't do a ton of advertising, so I wanted to post here. Heather


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Design as Art
Product details
- An illustrated journey into the artistic possibilities of modern design translated by Patrick Creagh and published as part of the 'Penguin on Design' series in Penguin Modern Classics.
- Bruno Munari was among the most inspirational designers of all time, he was described by Picasso as 'the new Leonardo'.
- Munari insisted that design should be beautiful, functional and accessible.
- It is within this book that his ideas are set out about visual, graphic and industrial design looking at how it plays a role in objects we use everyday.
- He turns his illuminating gaze to lamps, road signs, typography, advertising and more.
- This book will enlighten the reader to think; how do we see the world around us?
- Paperback, 224 pages


text:
A cute Inn. Loved all the flowers hanging and in pots outside of the room. The price was very affordable and was super happy with the room. Very clean and you can tell the owners take great pride in there Inn. Would highly recommend if...More
Getting you more information on this room
Getting you more information on this room
"With 101 being right there, I think any room would be fine - there's a hedgerow out front to quell some..."


text:
Your shopping cart is empty
01 Apr 1010 Back to the bad old days?MEMBER 65 writes about his experiences last week:
"What is going on at the ATO?
Australian Business Register closed for maintenance since at least last Thursday. Client requires ABN urgently
Country client rings ATO for balance of account. Told 90 minute delay.
Agent rings 131080 re a SMSF. Told 45 minute delay.
Hope we are not going back to bad old days."


text:
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This section is showing information as of the end the 2016-17 season. To join our email list and get notified when we launch the 2017-18 College Football section (about a week before the season starts), register for a free account.
S Mississippi at TX El Paso : Travel Analysis
Saturday Oct 29, 2011 8:00 pm -
El Paso, TX
Odds: Southern Mississippi by 10, Total Points: 59