As a corollary, these genes are not involved in the formation of

As a corollary, these genes are not involved in the formation of isochorismic acid from chorismic acid. In addition, we have shown that trpE2 is involved in the conversion of chorismic acid to isochorismic acid (Table 1). The gene product of trpE2 thus corresponds to ICS and would be equivalent

of PchA in P. aeruginosa (Gaille et al., 2002; Kunzler et al., 2005). In this study, the targeted mutagenesis has elucidated the roles of trpE2, entC and entD genes in the conversion of salicylic acid from chorismic acid. Hence, salicylic acid seems to have only one function, although its involvement in the recognition of iron and its transfer cannot be ruled out completely. However, since we observed the salicylate nonauxotrophy of the mutants, the most viable explanation for this is that the gene products of salicylate biosynthesis interact with other proteins of the mycobactin pathway, making the conversion of salicylate to mycobactin and carboxymycobactin Sorafenib nmr less efficient. The addition of salicylate (which cannot be converted to mycobactin and carboxymycobactin) at higher concentrations, over 5 μg mL−1, in the medium makes it toxic for the mutants, although the mechanism for this toxicity is not understood. With these studies, we suggest that the organization of salicylate biosynthesis is different between M. smegmatis (current study) Venetoclax mw and M. tuberculosis (Harrison et al., 2006). Distinct

from mbtI of M. tuberculosis, but in common with pchA of P. aeruginosa, trpE2 is coding for ICS in M. smegmatis. Hence, the conversion of chorismate to salicylic acid in M. smegmatis involves a multienzyme complex consisting of trpE2, entC and entD genes. Taken together, these data conclude that in M. smegmatis, the gene product of trpE2 corresponds to ICS; entC and entD code for salicylate synthase. We thank Overseas Research Studentships (UK) for a research studentship to N.N. We are indebted to Prof. Neil Stoker (Royal Veterinary College, London) for his invaluable suggestions in creating

knockout mutants and generously gifting p2NIL and pGOAL19 vectors. “
“Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus Etomidate plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway.

Fraction D3 was then dialyzed against 10 mM NH4OAc buffer (pH 51

Fraction D3 was then dialyzed against 10 mM NH4OAc buffer (pH 5.1) before chromatography on a 2.5 × 20 cm column of carboxymethyl (CM)-cellulose (Sigma) in 10 mM NH4OAc buffer (pH 5.1). After elution of unadsorbed proteins, the adsorbed proteins were eluted successively

using 10 mM NH4OAc buffer (pH 5.1) containing 50, 150 and 1000 mM NaCl. Fraction C3 eluted with 150 mM NaCl was dialyzed against 10 mM phosphate buffer (pH 7) before chromatography on a 1 × 15 cm column of Q-Sepharose (GE Healthcare) in 10 mM phosphate buffer (pH 7). After removal of unadsorbed proteins (fraction Q1), adsorbed proteins were desorbed with a 0–0.4 M NaCl gradient in 10 mM phosphate buffer (pH 6). The first adsorbed fraction (Q2) was then subjected to Navitoclax mw gel filtration on a Superdex 75 HR 10/30 column (GE Healthcare) in 0.2 M NH4HCO3 buffer (pH 8.5) using an AKTA Purifier (GE Healthcare). The second fraction (SU2) with a molecular mass of 29 kDa constituted purified hemolysin, which was designated as schizolysin. The assay was carried out as follows: to 0.1 mL of a 2% suspension of rabbit erythrocytes were added 250 μL 0.15 M NaCl and 50 μL test sample. After incubation in a water bath at 37 °C for 15 min, the mixture was centrifuged at 900 g for 5 min. The A540 nm was

then read. One hundred percent hemolysis was defined as OD540 nm of hemoglobin released from erythrocytes treated with 0.1% Triton X-100. One hemolysin unit (HU) was defined as the amount of hemolysin eliciting 50% hemoglobin release (Ngai & Ng, 2006). Schizolysin was subjected EX 527 in vitro to sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) (Laemmli & Favre, 1973) and gel filtration on a calibrated fast protein liquid chromatography (FPLC)-Superdex 75 HR 10/30 column (GE Healthcare) to determine its molecular mass. Its N-terminal sequence was determined by Edman degradation using a Hewlett-Packard amino acid sequencer. The sequence similarity analysis was performed using blast software against the NCBI protein database. The hemolysis inhibition tests to investigate inhibition of schizolysin-induced hemolysis by various carbohydrates were conducted in a similar manner to the hemolysis test. The results would indicate whether schizolysin interacts with any carbohydrate(s) on the erythrocyte membrane to exert its hemolytic action. A 20-μL aliquot of a water-soluble stock solution Fenbendazole of different carbohydrates (400 mM) was added to 250 μL of 0.15 M NaCl and 25 μL of schizolysin with 16 HU. The mixture was allowed to stand for 30 min at room temperature and then mixed with 100 μL of a 2% rabbit erythrocyte suspension. After incubation in water bath at 37 °C for 15 min, the remaining activity was detected. To investigate inhibition of schizolysin-induced hemolysis by various metal chlorides, the stock solutions of different metal chlorides were individually mixed with hemolysin solution and 250 μL of 0.15 M NaCl to achieve a final metal ion concentration of 5 and 10 mM, respectively.

Adverse effects have been reported with all antiretrovirals and a

Adverse effects have been reported with all antiretrovirals and are one of the most common reasons for discontinuation of treatment [2–4]. Some adverse events, such as gastrointestinal problems and hypersensitivity,

occur rapidly, within the first few months of starting treatment, while other adverse events, such as cardiovascular disease and pancreatitis, can take much longer to develop [5–7]. Such long-term adverse events can influence the durability of a regimen. Combination antiretroviral therapy (cART) Selleckchem Caspase inhibitor regimens most often include a nonnucleoside reverse transcriptase inhibitor, such as efavirenz or nevirapine, or a ritonavir-boosted protease inhibitor, such as lopinavir [8,9]. cART regimens with durability as well as virological efficacy are required in order to achieve long-term virological

STA-9090 solubility dmso suppression and to maintain CD4 cell counts at a level that significantly reduces the risk of morbidity and mortality. Many cohort studies have compared the short-term and long-term efficacies of different cART regimens [10–14], but less is known about the durability of different regimens, particularly in patients who have started a cART regimen more recently. If a regimen is virologically effective, durability can then be measured as the time to discontinuation of the regimen because of treatment failure or toxicity, or the rate at which changes occur

in potential markers of toxicity, such as liver transaminases and cholesterol. The aim of the study was therefore to compare the long-term durability of nevirapine-based cART regimens with those of efavirenz- or lopinavir-based cART regimens based on the time to discontinuation and the development of any serious clinical adverse events once virological suppression had been achieved and after at least 3 months on the drug to exclude discontinuations because of early-onset very potentially treatment-limiting toxicities that each of the three drugs may cause. The EuroSIDA study is a prospective, observational pan-European study of 16 599 HIV-1-infected patients from across Europe, Israel and Argentina. The study has been described in detail previously [15]. In brief, patients were enrolled into eight cohorts from May 1994. At each follow-up visit, details on all CD4 cell counts and HIV RNA measurements since the last follow-up visit are recorded as well as the date of starting or stopping any antiretroviral drug, the use of any prophylaxis against opportunistic infections, the date and type of development of any AIDS-defining illnesses, non-AIDS-defining illness or opportunistic infections, and death. Data are collected from the centres through follow-up forms at 6-monthly intervals and the database updated accordingly.

Thirty (79%) agreed that yes if they wanted to talk to the pharma

Thirty (79%) agreed that yes if they wanted to talk to the pharmacist then they are easy to contact. In response to being asked how they feel the pharmacist communicates concerns to staff, 27 (71%) viewed that

this is communicated in a helpful way, selleck kinase inhibitor 5 (13%) felt that the communication was more of a reprimand, and 6 (16%) gave a neutral response. When asked in their experience do they think the pharmacist is assertive enough when communicating clinical issues that really matter, 32 (84%) were positive about the pharmacist trying hard to communicate the necessary message, and 6 (16%) were neutral. Of the 21 that responded to the question asking what would be the one thing that pharmacists on the ward can do to improve their communication skills, 10 related to a theme of more pharmacists on the ward spending more time with patients. One respondent replied ‘Don’t tell off juniors’. In general, the overall results of this small scale survey can be interpreted as suggesting that clinical pharmacists are considered approachable, the majority of clinical staff feel that issues are raised

appropriately by pharmacists, and they also feel the pharmacists are assertive. However, comments captured during the survey such as ‘… I am usually very busy and don’t always appreciate the interruption’, communication from the pharmacist ‘can feel rushed’, and ‘when I’m busy and stressed it can definitely feel like I’m being told off’ suggest there may be an opportunity to improve communication skills. This baseline assessment demonstrates that further research across more hospital BYL719 trusts and geographical locations is warranted to ensure that our results do not just reflect the culture in our trust, and to enable a fuller picture to emerge. 1. Howe H, Wilson K. Modernising Pharmacy Careers Programme Review of Post-Registration Career Development of Pharmacists and Pharmacy Technicians. Background

paper. Medical Education see more England. July 2012. Veronica Smith University of Stirling, Stirling, UK What are the key barriers and facilitators for individual community pharmacists supporting people affected by dementia? When asked what they could do for people affected by dementia; most concerns were about medication management, followed by formal referral to the General Practitioner (GP). Community pharmacists may be the only health professional people affected by dementia regularly visit; they are ideally positioned to support them with medicines management and health advice. Recent policy initiatives are concerned with the role community pharmacists play, as part of the team of health professions providing support to people affected by dementia. The aims of this research are to identify what relationship community pharmacists have with people with dementia and their caregivers.

5 h, and examined the distribution of labeled profiles in relatio

5 h, and examined the distribution of labeled profiles in relation to presynaptic terminals. The results show a good ultrastructural preservation of the tissue, notably membrane structures, allowing unambiguous recognition of pre- and postsynaptic density, synaptic vesicles, mitochondria, GPCR Compound Library etc. (Fig. 3E), comparable with that seen after traditional tissue fixation (Panzanelli et al., 2011). Gephyrin immunogold labeling was prominent in profiles forming symmetric synaptic contacts with axon terminals enriched in synaptic vesicles. This intense immunoreactivity points to excellent preservation

of antigenicity owing to the brief post-fixation. We have assessed the suitability of the ACSF perfusion protocol for RNA purification compared with fresh-frozen tissue, and tested mRNA integrity by qPCR analysis. Experiments were performed in triplicate, using tissue from 2–3 mice per condition. Figure 3F illustrates that high-quality RNA can be purified from brain samples perfused with ACSF. Furthermore, the results demonstrate that RNA extracted from ACSF-perfused mice is compatible with qPCR analysis. By comparison with fresh brain samples, the expression level of four selected genes encoding synaptic proteins remained unaltered (Table 2), giving the opportunity to

study brain morphology and gene expression in parallel. For proof-of-principle that optimal biochemical and immunohistochemical analyses can be performed using tissue blocks taken from Verteporfin the same brain following ACSF-perfusion, we performed Western blotting and immunohistochemistry with tissue from an ACSF-perfused mouse. Each method was compared Linsitinib molecular weight with standard tissue preparations [fresh tissue for Western blotting and sections from perfusion-fixed

brain (4% paraformaldehyde) for immunoperoxidase staining]. In Western blots, we investigated the expression of Tau, APP and Reelin in cerebral cortex and hippocampus. As illustrated in Fig. 4A–C, no difference in relative abundance of Tau, APP or Reelin was observed in fresh-frozen and ACSF-perfused tissue, and proteolytic fragments of Reelin were readily detected, with clear differences in abundance between cortex and hippocampus. In parallel, we stained for Reelin in the hippocampal formation in sections that were pretreated with pepsin, prior to incubation with primary antibodies (Doehner et al., 2010). Immersion-fixation (3 h) of ACSF-perfused tissue allowed detection of Reelin immunoreactivity in hippocampal interneurons and neuropils with similar intensity and high signal-to-noise ratio as in perfusion-fixed tissue (Fig. 4D and E). We have shown previously that the detection of postsynaptic proteins of GABAergic synapses, in particular gephyrin and various GABAAR subunits, is markedly improved in weakly fixed tissue, in particular when derived from living brain slices (Schneider Gasser et al., 2006).

8 As previously stated, there is ample evidence of pharmacist-run

8 As previously stated, there is ample evidence of pharmacist-run and physician/nurse-run travel health clinics in the Selleckchem RG7204 literature.4,5,9 None have described a multidisciplinary team approach that our travel health clinic has as part of an ambulatory care outpatient clinic affiliated with a hospital. The team

consists of an infectious disease physician, a nurse, and a pharmacist affiliated with a college of pharmacy. Additionally, pharmacy students enrolled in their advanced pharmacy practice rotations are involved with the clinic. Prior to the start of their rotations, all students participate in a travel health class as part of the pharmacy curriculum. The primary role of all team members includes provision of travel-related education to patients; the pharmacist and pharmacy students emphasize vaccine-related adverse effects, the nurse is responsible for administration of immunizations, and the physician performs any necessary physical assessment. The clinic has operated once a week by both an appointment-only as well as a walk-in system since August 2009 when the

local health department could no longer administer travel vaccines due to budgetary cuts. The clinic is certified to administer the Yellow Fever vaccine. The clinic is fee for service and does not accept insurance at this time for services rendered. No consultation fee is charged if the patient receives injectable immunizations. If only oral medications Talazoparib chemical structure are prescribed a nominal consultation fee is charged. At each appointment an individualized risk assessment is performed by the team for each patient following completion of a screening form. Information such as the patient’s medical history, current medications, immunization records, travel destinations on the itinerary, Orotidine 5′-phosphate decarboxylase and the planned length of stay are reviewed prior to making any recommendations. The CDC Travelers’ Health web site,10 the CDC’s Yellow Book,11and theWHO web site12 are used as references for travel health recommendations. Recommendations are made by the pharmacist and nurse and are reviewed by the infectious diseases physician for accuracy

and confirmation. An individualized counseling session is conducted with the pharmacist and pharmacy students in a private examination room that is based upon the itinerary, the immunizations to be administered, malaria prophylaxis (if appropriate), and personal protective measures. Each counseling session focuses on health promotion and disease prevention that may last up to 30 minutes depending upon the traveler’s individual needs. Personal protective measures reviewed include mosquito/tick bite protection, traveler’s diarrhea, personal safety, and sexually transmitted diseases. All patients receive user-friendly handouts developed by the pharmacist and pharmacy students educating them about the medications and vaccines prescribed.

18 This study investigated Taiwanese physicians’ and nurses’

18 This study investigated Taiwanese physicians’ and nurses’ Epacadostat clinical trial knowledge of malaria, yellow fever, and dengue fever. The results can help government and medical care systems promote the professional development of travel medicine and enhance the quality of travelers’ health care. This study represents a cross-sectional questionnaire survey of physicians and nurses interested in travel medicine. The Training Center for Travel Medicine from the National Taiwan University held three nationwide one day seminars on travel medicine in the northern, southern, and eastern part of Taiwan from April to September of 2008.

The seminars were promoted in hospitals interested in travel medicine nationwide and advertized on internet websites. These seminars were also supported by the Center for Disease Control of Taiwan and the Taiwan Association of International Health. Participants were mainly hospital-based physicians and nurses. The questionnaire and consent forms were administered to all participants prior to the seminars and were returned PD0332991 in vivo before

the start of the seminars. All the study procedures were approved by the Ethical Committee of the National Taiwan University Hospital. The self-administered, single-choice questionnaire included four parts and started with an assessment of general background information. The remaining three parts included 17 questions regarding knowledge of the epidemiology, preventative medications for malaria (6 questions), yellow fever (4 questions), dengue fever (5 questions), and vaccine information for yellow fever (2 questions). The questionnaire was based upon personal practice experiences and designed after a careful literature review. Five experts tested the content

validity, while the face validity was tested by two physicians and three nurses. The scores from the knowledge of each disease were summed by assigning each correct answer one point. Data management and statistical analyses were performed using SPSS 11.0 software. 2-hydroxyphytanoyl-CoA lyase Frequency distributions described the demographic data. The chi-square test was used to compare the percentage of correct answers between physicians and nurses for each question, and the t-test was used to compare the overall scores between the two groups. A p value less than 0.05 was considered statistically significant. A total of 289 health-care providers (86 physicians and 203 nurses) who were interested in travel medicine were given the questionnaire, and all responded. After eliminating four incomplete questionnaires, 285 were included in the final analysis (85 physicians and 200 nurses). The mean age was 37.4, and no health-care provider had received any prior certification in travel medicine.

, 2008) Our results suggest that Archaea occupy a significant po

, 2008). Our results suggest that Archaea occupy a significant portion of the prokaryotic communities in aged Mn crusts and sandy sediments. The microbial communities on/within basaltic glass and rocks on the seafloor have been well studied (Fisk et al., 2003; Lysnes

et al., 2004; Mason et al., 2007; Einen et al., 2008; Santelli et al., 2008); however, little is known about those on well-developed Mn crusts on the aged seafloor. For the first time, we analyzed the composition and diversity of Archaea and Bacteria on an selleck aged Mn crust (Fig. 3 and Table 1). The archaeal clones recovered from the Mn crust were affiliated with MGI Crenarchaeota (Delong, 1992; Fuhrman et al., 1992) and with the pSL12-related group (Barns et al., 1996) (Fig. S2a). MGI includes the chemolithoautotrophic ammonia-oxidizing archaeon Nitrosopumilus maritimus (Könneke et al., 2005). The pSL12-related group may also include ammonia oxidizers as inferred by the analysis of 16S rRNA and archaeal amoA genes (Mincer et al., 2007; Kato et al., 2009b). Several microdiverse phylogenetic clusters within MGI have been defined in previous reports (Massana et al., 2000; selleck chemical Takai et al., 2004;

Durbin & Teske, 2010). Our MGI clones recovered from the overlying seawater were affiliated with the MGI-γ (Fig. S2a). Those from the Mn crust and sediment samples were affiliated with other MGI clusters such as the α, η–κ–υ, ι and ɛ–ζ–θ clusters (Fig. S2a). In the case study of the South Pacific Gyre (Durbin & Teske, 2010), the relative abundance of the MGI-α in the archaeal clone libraries has been high in the overlying seawater and those of the MGI-η and –υ have been high in the libraries from the sediments. Although it

is unclear what kinds of factors are responsible for the relative abundance of each MGI PDK4 cluster among deep-sea environments, these differences may reflect differences in geography, environmental characteristics and/or experimental procedures (such as the DNA extraction methods and the PCR primers used). In contrast to Archaea, diverse bacterial phylotypes were detected in the Mn crust, sediment and seawater samples. All analyses, i.e., Chao1 species estimates and the Shannon index (Table 1) and rarefaction curves (Fig. S3), indicated that the community diversity of Bacteria in the crust sample was comparable to or higher than that in sediment and overlying seawater. In addition, the diversity of Bacteria was higher in all samples than those of Archaea (Table 1). The bacterial diversity of the Mn crust was comparable to or higher than those of seafloor basaltic rocks reported previously (Lysnes et al., 2004; Mason et al., 2007; Santelli et al., 2008), suggesting that aged Mn crusts provide a habitat for diverse Bacteria as in basaltic rocks. Bacterial phylotypes dominated in all libraries (75.3–94.3% of the total clone numbers; Fig. 3).

Taken together, the results suggest that resveratrol protects aga

Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt, GSK-3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3-K/Akt signaling

pathway, subsequently downregulating expression of GSK-3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats. “
“Many aspects of retinal physiology are modulated by circadian clocks, but it is unclear whether clock malfunction impinges directly on photoreceptor survival, differentiation or function. Eyes from wild-type (WT) and Period1 (Per1) and Period2 (Per2) mutant mice (Per1Brdm1Per2Brdm1) were examined for structural (histology, in vivo Selleck Enzalutamide imaging), phenotypical (RNA expression, immunohistochemistry) and functional

characteristics. PS-341 order Transcriptional levels of selected cone genes [red/green opsin (Opn1mw), blue cone opsin (Opn1sw) and cone arrestin (Arr3)] and one circadian clock gene (RORb) were quantified by real-time polymerase chain reaction. Although there were no changes in general retinal histology or visual responses (electroretinograms) between WT and Per1Brdm1Per2Brdm1 mice, compared with age-matched controls, Per1Brdm1Per2Brdm1 mice showed scattered retinal deformations by fundus inspection. Also, mRNA expression levels and immunostaining of blue cone opsin were significantly reduced in mutant mice. Especially, there was an alteration in the dorsal–ventral patterning of

blue cones. Decreased blue cone opsin immunoreactivity was present by early postnatal stages, and remained throughout maturation. General photoreceptor differentiation was retarded in Metalloexopeptidase young mutant mice. In conclusion, deletion of both Per1 and Per2 clock genes leads to multiple discrete changes in retina, notably patchy tissue disorganization, reductions in cone opsin mRNA and protein levels, and altered distribution. These data represent the first direct link between Per1 and Per2 clock genes, and cone photoreceptor differentiation and function. “
“When we make hand movements to visual targets, gaze usually leads hand position by a series of saccades to task-relevant locations. Recent research suggests that the slow smooth pursuit eye movement system may interact with the saccadic system in complex tasks, suggesting that the smooth pursuit system can receive non-retinal input. We hypothesise that a combination of saccades and smooth pursuit guides the hand movements towards a goal in a complex environment, using an internal representation of future trajectories as input to the visuomotor system. This would imply that smooth pursuit leads hand position, which is remarkable, as the general idea is that smooth pursuit is driven by retinal slip.

Individuals also make significantly shorter journeys of less than

Individuals also make significantly shorter journeys of less than 5 weeks, and were more likely to visit the TAVC more than 30 days before departure than in the past. Only 24% of the Mecca travelers accepted the recommended dTP vaccine. Possible reasons for this low acceptance are that most of these

travelers do not come to our clinic for health advice, but for a vaccination that is necessary to obtain a visa. Other reasons can be the costs of the vaccinations, and that people are not informed about the possible risks and recommended vaccinations prior to their visit to us. Communication is often difficult because of language barriers. In univariate analysis, women, second-generation Muslims, and older people were significantly more likely to accept dTP vaccination than Buparlisib men and younger people. In multivariate analysis, the variable second-generation Muslims was no longer significant, and younger

see more people were significantly more likely to accept dTP. Schlagenhauf and colleagues also found that women are significantly more likely to obtain pretravel advice.6 Another predictor for dTP acceptance in our study is health. The more unhealthy people are, the more likely it is that they will accept the recommended vaccinations. Looking at the specific disorders, individuals with heart or vascular disorders, those with liver and gastrointestinal disorders, and those with other disorders were significantly more often likely to accept the dTP vaccine. Apparently, the more vulnerable people’s health, the more they are willing to protect themselves from other diseases. The reason that, independently, younger PAK5 people are more likely to accept recommended vaccinations is possibly because they are better informed, and communication is easier because

there are no language barriers. In conclusion, only a quarter of Mecca travelers who visit a travel clinic for their mandatory meningitis vaccination also take other, recommended, vaccinations. Women, younger people, and less healthy people are more likely to follow recommendations. To improve uptake, which in this scenario would be more people accepting recommended vaccinations, Islamic organizations that provide Mecca travelers with travel advice should be better informed, not only about the required vaccinations, but also about recommended vaccinations and other health advice. We thank Dr Lothar D.J. Kuijper, Vrije Universiteit Amsterdam, for his support of this study. The authors state they have no conflicts of interest to declare. “
“Travelers to countries where rabies is endemic may be at risk of rabies exposure. We assessed rabies immunization of travelers attending a travel clinic in Thailand. The medical charts of international travelers who came for preexposure (PrEP) or postexposure (PEP) rabies prophylaxis at the Queen Saovabha Memorial Institute (QSMI), Bangkok, Thailand between 2001 and 2011 were retrospectively reviewed.