The protective effect of Erythropoietin (EPO) analogue rHuEPO on acute renal injury induced by exhaustive exercise had been reported. The purpose of this study is to probe into the protective effect of EPO on chronic renal injury induced by repeated exhaustive exercise for four weeks.Eighty adult male Sprague–Dawley rats were used in this experiment. The animals were randomly allocated to one of four groups: control (C), exhaustive exercise test (ET), ET plus EPO pre-treatement (ET+EPO) and ET+EPO plus LY294002 pretreatment (ET+EPO+LY).P < 0.01), and the pathological changes of kidney cell were much less compared with those of rats without EPO intervention. When LY294002, a specific inhibitor of phospholipids phthalocyanine inositol 3-kinase, was added to the EPO treated rats, the injury changes of renal cell were becoming more pronounced.Compared with the rats in control group, there was considerable damage in kidney cells in rats of ET group as revealed by histological and ultrastructural examinations. However, treatment with EPO during the training, the exhaustive running distance was significant increased (The protective effect of EPO on chronic renal injury induced by repeated exhaustive exercise was demonstrated in the present study. We proposed that the effect could be due to inhibiting the cell apoptosis and blocking the formation of interstitial fibrosis via activation of the PI3K/Akt pathway, thus plays role in the endogenous protection of the kidney injury. Erythropoietin (EPO) belongs to members of multi-functional cytokine super-family and provides protection for various organs -3. In thHigh-intensity work, military training and competitive sports will inevitably cause overload or hypoxia. Long-term high-intensity exercise or overload that exceeds the endurance of the body will cause qualitative change from physiologic stimuli to pathologic stimuli. Eventually, the systemic function will be affected, such as rhabdomyolysis and injuries of heart, liver and neural system. Kidney is one of the vulnerable organs suffered from the high-intensity exercise or overload training, manifesting as cystorrhexis, hematuria, proteinuria, acute renal failure etc. Therefore, to prevent and treat the kidney and other organs injury resulted from overtraining are crucial tasks in sports medicine, modern military medicine and clinical medicine. The present study was designed to determine the histological and ultrastructural changes in kidney cell of the rats after relatively long exhaustive exercise, evaluate the preventive and therapeutic effects of EPO on such a pathological conditions to provide empirical evidence for the application of EPO clinically.Eighty male Sprague–Dawley rats  were used in this experiment. The animals were purchased from Zoology Institution of Hunan Agricultural University and raised in room temperature with natural illumination and ventilation. Their general health state and activity were monitored closely during the experiment. The experimental protocol for this study was pre-approved by the Hunan Normal University Institutional Review Board and in accord with the Public Health Service policy on the use of animals for research .All the rats went through the adaptation training for one week by running on a horizontal-treadmill  at a speed of 15–30 m/min for 15–20 min every day. They were grouped randomly into 4 groups of 20 animals: control group (C), exhaustive exercise group (ET), exhaustive exercise + rhEPO group (ET+EPO) and exhaustive exercise+ rhEPO + LY294002(PI-3- kinase inhibitor)group (ET+EPO+LY). Rats of group (ET+EPO) received intraperitoneal (ip) injection of 2000 u/kg rhEPO 30 minutes prior to the exercise every Monday, Wednesday and Friday. With the same schedules and EPO administration, rats of group (ET+EPO+LY) had an extra ip injection of 100 mg/kg LY294002 one hour prior to exercise. The over-training model was based on the modified exercise protocol proposed by Somani . Animals3) and placed on the microscope slide immersed with glutaraldehyde fixative. The tissues were fixed with 2% paraformaldehyde - 2.5% glutaraldehyde for two h at 4°C, washed for three times (10 min each) with buffer solution of sodium cacodylate (pH 7.2). After dewetting, the tissues were fixed by 1% H2OSO4 for 2 h at 4°C and washed again, fixed with 0.1 m buffer solution of sodium cacodylate and preserved at 0°C for electron microscopy examination. Same procedurals were applied for rats in control group except that the samples were collected one day after adaptation training.Twenty-four h after the exhaustive exercise, rats were anaesthetized by ip injection of 10% chloral hydrate (0.3 ml/100 g) and sacrificed by incising of the heart. The kidney was removed immediately for specimens. Nephridial tissue between cortex and medulla was dissected out and fixed with 10% paraformaldehyde. The tissue was sliced into small pieces .The specimens were gradient dehydrated under 4°C with ethyl alcohol, 50% for 10 min, 70% overnight, 90% 10 min and 100% 15 min twice, and replaced at room temperature with propylene epoxide for 15 min twice, 1:1 propylene epoxide and resin for 1 h, 1:4 propylene epoxide and resin for 1 h and pure resin soakage for 2 h, followed by embedding in pure epoxy resin Epon812 and polymerized in thermostat, 35°C for 16 h, 45°C for 8 h, 55°C for 14 h and 60°C for 48 h. The samples were trimmed and cut into serial sections of 60 nm positioned by azure-cyanine staining optical microscope. The sections were double stained with acetic acid uranium and lead nitrate and studied by a Hitachi H2600 Transmission Electron Microscope.P < 0.05 was considered statistically significant.Data were analyzed by using statistical software SPSS . All data are presented as mean ± SD. one-way ANOVA followed by Bonferroni test was used for comparisons. P < 0.05).Before exercise, all rats were physical normal and appeared active with bright eyes and lustrous hairs. At the end of each exhaustive exercise as group ET, the rats appeared listless with gloomy eyes. They were short of breath and unstable at feet with chest and belly touching the ground, no response to sound and light stimulation, some serious cases even showed conjunctival hemorrhage. All these symptoms were more evident with each passing training day, running shorter and shorter. In the recovery phases after exercise, diet was less and less, the time to free move longer and longer. Rats in group ET+EPO tolerated better to the exhaustive exercise compared with those in group ET as showing in changes of running distance Table . At the P < 0.05, and P < 0.01 between 2nd week and 4th week), while rats of ET+EPO group running much longer than rats of ET group in the training of 3rd week (P < 0.05) and 4th week , capsular space (Cs), electron density and the podocyte protuberance (Pp) were clearly visible and evenly distributed Figure A, the thHowever, sample of ET group revealed notable pathological changes in glomerular endothelial cells, manifested as swollen, paired and vaulted cytoplasm, stripped basilar membrane with fused endothelial cells Figure B, 3B. BeCompared with ET group, samples of ET+EPO group of the rats showed much less ultrastructure abnormalities in renal cells. The endothelial cells (Ec) and renal podocyte protuberance (Pp) were more evenly distributed, the thickness of basilar membrane (Bm) was more identical with clearer boundary Figure C, while Samples of ET+EPO+LY group showed apparently damage in kidney cells but lighter than those in ET group of the rats. The podocyte revealed microvilli alteration, mesenchyme fibrosis Figure D and pleThe ultrastructure of epithelial cells of distal convoluted tubule in control group was integrated and distinguishable Figure A. The miThe general conditions was strongly affected by the long last repeated exhaustive exercise as shown in ET group of the rats. The symptoms of the exhaustive exercise were evident and more serious with the continuation of the training days, running shorter, dieting less, body weight decreasing, etc. The running distance was significantly shorter. A considerable damage in kidney cells were presented in histological and ultrastructural examinations. However, treatment with EPO during the training, the general conditions were much better and the pathological changes in kidney cells were remarkable less compared with the ET rats without EPO intervention. The findings suggested a severe chronic renal injury were induced by four-week exhaustive exercise and such a pathological process was counteracted by the EPO intervention. In agreement, similar results had been reported with acute exhaustive exercise ,13.Normally, the blood flow in the kidney is about 25% of the cardiac output. When exercise, the metabolic rate of muscle is increased and more blood supply required. In exhaustive situation, blood flow redistribution among the organs occurs due to the emergency response, which is activated by increasing in secretion of adrenaline and rennin angiotensin. Consequently, the renal artery contracted and the renal blood flow decreased, eventually the renal injury aggravated, especially when the exercise lasts and the intensity increase further on. This incomplete ischemia state forms so-called “exercise induced renal ischemia”, which then turns to “ischemia-reperfusion (I/R)” when blood supply regained after cessation of the exercise ,15. It wAn intact filtration barrier and proper function of tubular are critical for kidney in maintaining stability of the internal environment. The ultrastructure alternations of renal cells as we found in ET rats were pronounced e.g. disorganized three layers structure of glomerular filtration membrane, mesenchyme fibrosis and cell apoptosis, etc. Since the glomerular endothelial cells are the first layer of filtration barrier, which directly contacts blood flow, affected by the changes in blood component, harmed by hypertransfusion, high filtration and high pressure in the glomeruli , this paThe morphological abnormalities of glomerular basement membrane (GBM) are another characteristic of renal injury, including unnatural thickness (thinner or thicker), losing of completeness, cracking and immune complex deposition etc., which presented almost all in the present samples of the ET rats. The glomerular podocyte foot process composes glomerular hiatus membrane which is an important filtration barrier for large molecules protein . When wiHypoxia can cause kidney tissue atrophy and fibrosis ,22. In aStudies showed that EPO could reduce the cerebral ischemia, diminish the cerebral infarction area ,23, and Renal tubular injury and fibrosis are two common paths to various causes of renal chronical disease . As to tWhen the pathway was blocked by PI3K specific inhibitor LY294002 as we applied in the rats of ET+EPO+LY group, the tolerance of kidney cell to the chronic ischemia and hypoxia was weakened considerably so that the apoptosis cells and interstitial fibrosis produced, which might be the explanations of the present finding that the renal tissue injury was aggravated by the application of LY294002. On the other hand, the protective effect of EPO could be speculated as the activation of PI3K/Akt pathway, and thus increase the kidney resistant to injury and alleviate the secondary damage from chronic ischemia and hypoxia.The protective effect of EPO on chronic renal injury induced by repeated exhaustive exercise demonstrated in the present study could be due to the inhibition of cell apoptosis and interstitial fibrosis formation by activating PI3K/Akt pathway, thus plays role in the endogenous protection of the kidney injury. The EPO effect against exhaustive exercise-induced chronic renal injury demonstrated in the present in vivo study in rat points to the potential interest of EPO in such a clinical situation.The authors declare that they have no competing interests.XXL, CHJ and SLQ conceived the study, established the design and carried out the experimental work. CHJ and ZQL participated in the data analysis and provided critical comments on the study design and manuscript. XXL and CHJ drafted this manuscript. All authors read and approved the final manuscript.Xixiu Lin and Chonghe Jiang are co-authors.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/130/prepub


Pina and Pinb alleles, similar to soft lines. Differences in dough and bread-making properties between lines classified as hard and soft on the basis of molecular data appeared to be of less significance than the differences between lines classified as hard and soft on the basis of physical analyses of grain texture. Values of relative grain hardness at the increased nitrogen fertilization level were significantly higher. At both fertilization levels the NIR parameter determining grain hardness was significantly positively correlated with the wet gluten and sedimentation values, with most of the rheological parameters and bread yield. Values of this parameter correlated with quality characteristics in a higher degree than values of particle size index.The influence of grain hardness, determined by using molecular markers and physical methods (near-infrared (NIR) technique and particle size index—PSI) on dough characteristics, which in turn were determined with the use of a farinograph and reomixer, as well as bread-making properties were studied. The material covered 24 winter wheat genotypes differing in grain hardness. The field experiment was conducted at standard and increased levels of nitrogen fertilization. Results of molecular analyses were in agreement with those obtained by the use of physical methods for soft-grained lines. Some lines classified as hard  appeared to have the wild-type  Wheat grain quality depends on many traits, among which the most important include, e.g., protein content, composition of high-molecular glutenin subunits and grain hardness. In breeding, selection of high-quality wheat is based mainly on the Zeleny sedimentation test, the falling number and the protein content. In most European countries as well as in Canada and the USA grain hardness is also determined during the early stages of breeding.i.e., static methods, such as measurement of the so-called microhardness adopted to cereal materials [Grain hardness is not a clearly defined parameter. It may generally be formulated as a resistance to plastic strain and cracking at a force concentrated on the surface of a given body. Several techniques are employed to determine grain hardness, aterials , as wellaterials , particlaterials , or pearaterials . Hardnesaterials ,6 or Peraterials . Differeaterials –11.Ha) located at the short arm of chromosome 5D [puroindolines a and b . Soft-grained wheats have the wild allele of the Pina gene and accumulate both puroindolines on the surface on starch granules, whereas medium- and hard-grained wheats have mutated alleles at Pinb and have reduced amounts or no puroindoline b on starch granules. The effect of puroindolines on dough and breadcrumb properties is ascribed to their affinity to lipids [Ha locus does not explain all the variations observed in grain hardness of wheat populations [Several studies indicate that grain hardness is controlled by the main hardness locus  technique is of greatest interest to breeders, because it is easy, rapid and non-destructive, but it only provides a relative evaluation of hardness ,10,25,26The aim of this study was to evaluate the dependence between grain hardness and dough rheology as well the relationship of baking properties of winter wheat breeding lines with hardness of grain.The material for the present study covered 24 winter wheat genotypes with significantly varied grain hardness. On the basis of the grain hardness determined by using the NIR technique and particle size index (PSI), the studied genotypes were divided into three groups: Hard-, soft- and medium-grained .Pina-D1a, Pina-D1b, Pinb-D1a and Pinb-D1b had been identified using primer sets described by Lillemo et al. [et al. [Pina-D1a and Pinb-D1a, whereas hard-grained lines in most cases (7 out of 11) had one mutated allele: Pinb-D1b  or Pina-D1b (line 35), but four lines were identified to have the same set of alleles as soft-grained (Pina-D1a and Pinb-D1a) alleles as well as two lines with one mutated allele were detected.The studied wheat genotypes of different grain hardness were subjected to molecular analyses. In our earlier studies , puroindo et al.  and Li e [et al. , respect-grained . Among tHa locus were additionally applied in the present study: Xgwm190 [Xgwm205 and Xgwm358 [Xgwm190 , Xgwm205 , Xgwm358  provided the same size of DNA amplified products  in all soft-grained lines, whereas in hard- and medium-grained groups, the same markers produced different products  markers linked to the  Xgwm190 , Xgwm205products . In FiguMean values of grain hardness and quality parameters assessed using the NIR system, farinograph, mixograph and test baking are presented in i.e., area under centre line up to peak (IHTP), time 1–2 (RM4), peak time (RM6) and bandwidth at 10 min (RM11). GE interaction was insignificant for all bread quality parameters  and peak height (RM8). Among the baking parameters, only bread yield variation was significant. The level of fertilization had a significant effect on most analyzed quality parameters. Exceptions included the following farinograph parameters: Dough consistency, water absorption, dough stability time and reomixer peak height. The genotype-environment (GE) interaction was significant for PSI, most farinograph parameters (except for dough consistency) as well as for four mixing parameters, rameters .F statistics. For the comparison of hard and soft lines, two kinds of contrasts (C1 and C2) were constructed, because composition of hard and soft groups of lines established on the basis of physical and molecular analyses was not the same: In C1, the hard group consisting of 11 lines  was contrasted with the soft group, which consisted of eight lines , whereas in C2 the hard group included nine lines  and the soft group 15 lines   .At both fertilization levels hard-grained wheat types, in comparison to soft-grained wheat types (C1contrast), were characterized by a significantly higher protein content, wet gluten, Zeleny sedimentation value, and a significantly lower starch content .Estimates of contrasts between levels FL1 and FL2 for all the sets of lines indicated that a higher level of nitrogen fertilization caused a significant increase in values of NIR parameters defining grain hardness, protein content and wet gluten as well as Zeleny sedimentation value, and a reduction of starch content. Differences between the values of PSI parameters on FL1 and FL2 appeared to be insignificant, whereas NIR values were significantly higher on FL2 .Estimates of C2 contrast showed significant differences between H and S groups of lines at both fertilization levels only in NIR values, whereas in the case of the other characteristics tested, differences were significant only on FL2. Moreover, differences in protein content were insignificant both on FL1 and FL2 .Hard-grained lines had significantly higher values of water absorption and dough stability than soft-grained wheat types at both the fertilization levels. The rest of the farinograph parameters were similar for both groups of lines except for dough softening, which was significantly higher for soft-grained lines at FL1 . Different results were obtained for dough stability: An estimate of C1 contrast showed better dough stability for soft-grained lines, whereas C2 showed the same for hard-grained ones. For the rest of farinograph parameters differences estimated in C2 contrast were markedly important only at FL1 or FL2 fertilization levels .The following four reomixer parameters were generally higher at the lower fertilization level: IHTP, RM4, RM6 and RM11. The maximum peak height (RM8) was similar for both fertilization levels and both groups of genotypes.Hard-grained lines were assessed to have significantly higher values of mixing parameters  at both fertilization levels , similar to values observed in our earlier studies [In this study, the relationships between grain hardness and some quality parameters were studied in winter wheat breeding lines examined under field conditions at two levels of nitrogen fertilization. Grain hardness was determined by the PSI and NIR spectroscopy; values of these two parameters were negatively significantly correlated  was significant for the grain hardness evaluated by PSI parameter and for some farinograph and mixograph characteristics. Similar results were reported by Tsilo and co-workers  who recoPina and Pinb, alleles, similar to soft lines. Lines with no clearly determined grain hardness had wild-type or mutated alleles of Pin-genes. Moreover, differences in dough and bread-making properties between lines classified as hard and soft on the basis of molecular data appeared to be not so clear as the differences between lines classified as hard and soft on the basis of physical analyses of grain texture. This indicates that the hardness was controlled not only by Ha locus, but also by other regions of the genome in the studied grain material. Similar observations have been reported in several other studies [Ha locus may be ineffective.In the present study, grain hardness was evaluated by physical methods supplemented by molecular analysis, which permitted a better characterization of the studied materials. The results of molecular analyses were in agreement with those obtained by the use of physical methods only in the case of soft-grained lines. Some lines classified by physical methods as hard appeared to have the wild-type  studies ,12,19–24In our study, the relationship between grain hardness and other quality parameters were evaluated. Correlation coefficients between grain hardness determined both by PSI and NIR methods and studied traits at both levels of nitrogen fertilization were significant for the following sedimentation value and mixing parameters: IHTP and bandwidth at 10 min. Among the parameters describing baking quality, grain hardness appeared to be significantly correlated only with bread yield. No association was found between grain hardness and other final baking quality parameters, such as loaf volume or crumb grain. This may be a result of the influence of other components of grains, which were not determined in this study. Hruskova  reportedr = 0.445, p < 0.01) at the lower fertilization level, whereas PSI values were significantly associated with protein content  only at the increased nitrogen fertilization level. This suggests that these two traits may be independent, as shown previously by Bushuk [et al. [i.e., grain hardness and PC, in a population of inbred lines derived from a cross between hard and soft wheat genotypes, which may indicate that loci contributing to these traits are linked to each other. Tsilo et al. [In the present study, values of grain hardness measured by NIR technique correlated more frequently with dough properties than those determined by PSI. In addition, correlation coefficients between NIR, farinograph and reomixer parameters were significant at both fertilization levels, whereas with PSI, this was more frequently found at only one level—FL1 or FL2. The association between grain hardness and protein content was not clear; NIR values correlated significantly with protein content  at 69 kg·ha−1 P, 120 kg·ha−1 K and 187 kg·ha−1 N. The experiment was established in a complete blocks design in two replications on plots of 5 m2, with a row spacing of 12.5 cm. Seeds were sown on 29 September 2008, while harvest was performed on 28 July 2009.The field experiment was conducted in the season of 2008/2009 on an experimental plot in Choryń near Leszno  on light brown soil using two fertilization levels: Standard (FL1) at 69 kg·haGrain hardness was determined using two methods: (1) By measurement PSI  with theThe NIR technique was also used to determine protein content, starch content, wet gluten and Zeleny sedimentation value.DNA extraction: Leaves of 14-day-old wheat plants were used for genomic DNA extraction (Promega Kit). The extracts were diluted to 100 ng/mL and stored at −20 °C.Ha locus were used for better characterization of the studied wheat lines: Xgwm190 [Xgwm205 and Xgwm358 [2, 1.0 unit of Taq DNA polymerase, and 50 ng of genomic DNA. The samples, denatured at 94 °C, were submitted to 45 cycles of 1 min denaturation at 94 °C, 1 min annealing at 55–60 °C (depending on primer Tm), and 2 min elongation at 72 °C, with a final extension of 10 min at 72 °C at the end. Microsatellite alleles were detected on the 3130 Genetic Analyzer .Puroindoline identification in the studied lines had been performed earlier . In the  Xgwm190 , Xgwm205The rheological properties of dough were analyzed on a microscale using farinograph and Reomixer. Ten g flour samples of 14% moisture were analysed.Farinograph analysis was made using a type E farinograph  in a Mixer S10. Five rheological parameters were recorded: Consistency, water absorption, dough development time, dough stability and dough softening at 10 min. The farinograph quality number was recorded as well. Measurements were taken in accordance with a procedure described in the AACC Standard 54–21.02  and ICC Dough mixing was measured using a Bohlen Reomixer , which is a planetary pin mixer similar to the Mixograph  with a 10 g flour capacity, a mixing speed of 88 rpm and data recording at 10 points/s –37. The 3) and crumb grain number according to the Dallmann scale [Baking quality was evaluated on the basis of test baking, in which bread yield in comparison to the flour used (%), loaf volume  were excluded from the analysis.The results of the observations and measurements were statistically processed using 2-way analysis of variance. The hypotheses claiming a lack of influence of fertilization level, genotype and interaction of genotype × fertilisation level (GE) were verified. Contrasts between fertilization levels F1 and F2 and between groups of lines with hard and soft grain were estimated and tested by Ha locus may not be fully effective. Grain hardness of winter wheat breeding lines was higher at an increased nitrogen fertilization level than at the standard fertilization level. Differences in values of NIR and PSI parameters between hard and soft wheat types were more marked at standard fertilization.The obtained results suggest that selection of wheat for good quality based only on molecular markers connected with the Values of NIR parameter determining grain hardness correlated with quality characteristics at a higher degree than values of particle size index. The NIR correlated with wet gluten and sedimentation value as determined by NIR technique. A significant association was also found between NIR and farinograph parameters such as dough consistency, water absorption capacity and dough stability time, and some reomixer characteristics: Area under center line, time 1–2, peak time and bandwidth at 10 min. A significant relationship was ascertained between NIR and bread yield. This means that grain hardness evaluated by NIR technique may be used as a criterion in breeding selection of winter wheat breeding lines with improved quality.


Cellulitis, a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin, is a common lesion in children, usually responsive to systemic antibiotic therapy. However, an unusual course of healing or some nontypical features should call the treating physician to consider and investigate for other diagnoses that might prevent unnecessary treatment and alleviate parental stress. We present a case of posttraumatic fat necrosis, demonstrating some pitfalls in the process of diagnosis. Posttraumatic fat necrosis of the subcutaneous fat tissue can occur following a fall, blunt injury, surgery, and minor procedures such as injections . It is mA 9-year-old girl was admitted to the Emergency Department 4 days after a blunt trauma in her left leg. On admission the patient had no fever. On physical examination, there was an area of edema, warmth, erythema, and tenderness with indistinctive margins on the anteromedial aspect of the left leg.The rest of the physical examination was normal except for obesity and her history was unremarkable. X-ray of the leg was normal. She was clinically diagnosed as having cellulitis and was discharged with oral amoxicillin/clavulonic acid. However, edema, warmth, erythema, and tenderness of the leg persisted for 2 more days, denoting no improvement and the patient was readmitted 2 days later to the Pediatrics Department. Oral antibiotic was replaced with intravenous therapy (amoxicillin/clavulanic acid 75 mg/kg/day). 3/μL , hemoglobin 13.0 g/dL, platelets 292 × 103/μL, C-reactive protein 94.1 mg/dL, erythrocyte sedimentation rate 67 mm at 1 hour, blood chemistry: normal. Laboratory findings were white blood cell count 13.6 × 10The antibiotic therapy was changed 4 days later to cloxacillin (100 mg/kg/day) and ampicillin (100 mg/kg/day) because of unsatisfying improvement.Gradual improvement occurred over the next two days; edema, warmth and erythema became mild and the tenderness disappeared. The patient was discharged 6 days after her admission with oral cephalexin (35 mg/kg/day). Physical examination on a scheduled outpatient visit three days after discharge revealed erythema and induration of the skin of the left leg without tenderness or edema laboratory findings were within normal range. Ultrasound of the left shin demonstrated swelling of the soft tissue in the anteromedial aspect with a number of hypoechoic collections, the biggest one was 0.9 × 2.13 × 2 cm, margins were indistinctive with unclear fluid, blood supply was not increased, and the bones were intact, thus excluding the possibility of osteomyelitis . At thisTwo months after admission, MRI demonstrated edema of the subcutaneous fat medially to the tibial bone with thickening of the subcutaneous septations and unclear boundaries. There was no involvement of bone cortex or marrow and no enhancement was evident . This piFollow-up ultrasound 3 month after trauma showed resolution of soft tissue edema with organization of echogenic (fatty) lesion surround by hypoechoic lesion . Posttraumatic fat necrosis is quite prevalent, especially in the pediatric age group where minor trauma is a normal and common event, making this population more prone to this condition . AlthougMost cases of posttraumatic fat necrosis appear as a palpable lump or as a local depression , 4. SubcThe index case appeared with edema, warmth, erythema and tenderness—a clinical picture similar to cellulitis. Our patient had a prolonged duration of symptoms uncharacteristic of cellulitis. In a patient with cellulitis, a symptomatic improvement is expected within 24–48 hours of the beginning of therapy and a visible improvement within 72 hours. Persistence of symptoms or signs after the expected time prompts further evaluation and imaging of the affected area to rule out other possible diagnoses such as subcutaneous fat necrosis, tumors such as lymphoma, sarcoma, chloroma and early stages of necrotizing fasciitis and other rare infections. The appearance of our patient resembles that of earlier descriptions with gender distribution, typical location of lesion and recent history of trauma, and is different in that there is lack of a lump or depression at the site of the lesion. An alternative diagnosis that could explain these differences could be cellulitis with an extension to deeper layers, also known as infectious panniculitis. Primary infection can result from a pathogen introduced directly into the subcutaneous tissue through a penetration of the skin . However


Aim. This paper investigated the pathogenesis and treatment strategies of acute pancreatitis (AP) in pregnancy. Methods. We analyzed retrospectively the characteristics, auxiliary diagnosis, treatment strategies, and clinical outcomes of 26 cases of patients with AP in pregnancy. Results. All patients were cured finally. (1) Nine cases of 22 mild acute pancreatitis (MAP) patients selected automatic termination of pregnancy because of the unsatisfied therapeutic efficacy or those patients' requirements. (2) Four cases of all patients were complicated with severe acute pancreatitis (SAP); 2 cases underwent uterine incision delivery while one of them also received cholecystectomy, debridement and drainage of pancreatic necrosis, and percutaneous jejunostomy. One case had a fetal death when complicated with SAP; she had to receive extraction of bile duct stones and drainage of abdominal cavity after induced abortion. The other one case with hyperlipidemic pancreatitis was given induced abortion and hemofiltration. Conclusions. The first choice of MAP in pregnancy is the conventional therapy. Apart from the conventional therapy, we need to terminate pregnancy as early as possible for patients with SAP. Removing biliary calculi and drainage is supposed to be considered for acute biliary pancreatitis. Lowering blood lipids treatment should be applied to hyperlipidemic pancreatitis or given to hemofiltration when necessary. Acute pancreatitis in pregnancy (APIP) is rare and occurs in approximately 1 in 1,000 to 3 in 10,000 births .The commonest reasons of APIP are biliary disease and congenital or acquired hypertriglyceridemia, which can occur during any trimester but over a half occurs during the third trimester, and very rarely APIP is associated with preeclampsia-eclampsia or HELLP syndrome , 5. PregAPIP often presents as an acute abdomen and can have a lethal effect on the mother and the fetus. It is important to be aware that APIP may be more severe, posing a survival threat even in the youngest patients. We present 26 cases of APIP who were admitted from March 1997 to December 2009 in our hospital, in order to investigate the pathogenesis and treatment strategies.The diagnosis was based on a clinical diagnosis and grading criteria of AP established by the pancreatic group of the Surgical Society of the Chinese Medical Association for those cases before 2007 , 10 and Among the 26 APIP patients, there were an average age of 28.35 ± 5.00 (22–38) years, a mean length of hospital stay of 12.39 ± 10.00 days, and mean pregnancy weeks of 25.74 ± 17.00 . There were 3 cases with overeating fast food before APIP onset. The main clinical symptom was as follows: upper abdominal pain, nausea, and vomiting in 15 cases, ectopic pain of left lower back in 5 cases, and abdominal distension in 6 cases. The main outcome measures included clinical manifestation and auxiliary examination in the treated group and the control group. The etiology included biliary pancreatitis in 20 cases, hyperlipidemic pancreatitis in 3 cases, and, an unknown reason in 3 cases (mild cases). Results were expressed as Mean ± SD.9/L and a percentage of leukocyte with that of 85.22 ± 6.03%. B-ultrasound showed pancreas enlargement, decreased echo, and peripancreatic, pelvic, and abdominal cavity effusion in all patients. Among the 13 cases with biliary tract disease cholecystitis, cholecystolithiasis and bile duct stone were found in 4 cases while cholecystitis and cholecystolithiasis in 4 cases, cholecystolithiasis, and bile duct stone in 3 cases, choledocholithiasis in 3 cases, and congenital choledochal cyst in 1 case. CT scan displayed pancreas enlargement with partial necrosis, fuzzy boundary, disappearance of peripancreatic fat space, and ascites in 4 cases with SAP. The 4 cases with SAP, ERCP demonstrated obvious dilation of bile-pancreas duct and lower bile duct stones which were extracted in 1 case. Blood-gas analysis revealed respiratory alkalosis and metabolic acidosis in all SAP patients. The average content of triglyceride , total cholesterol , and blood amylase were 5.46 ± 6.14 mmol/L, 7.25 ± 4.47 mmol/L, and 563.96 ± 582.60 U/L for all APIP cases, respectively.All patients had high blood and urine amylase levels, blood amylase 563.96 ± 582.60 U/L  and urine amylase 7761.82 ± 3396.00 U/L . Six cases had hyperglycemia with the maximum blood glucose contents of 26 mmol/L. The majority of the 26 cases had a leukocyte increase with a mean number of 15.32 ± 7.27 × 10We used the conventional therapy and enhanced fetal monitoring and miscarriage prevention with gastrointestinal decompression, ECG monitoring, inhibition of gastric acid secretion (pantoprazole and omeprazole), trypsin secretion (octreotide and somatostatin), and activity (aprotinin) on the basis of fasting, anti-inflammation , fluid infusion, spasmolysis, and lowering blood lipid. Some scholars reported that octreotide and somatostatin can be effective in controlling some APIP patients and do not induce any malformation and do not affect foetal development, but do not have a large-scale clinical validation , 14. We + weeks pregnancy diagnosed as biliary SAP complicated with acute pulmonary injury and diabetes mellitus deteriorated after the conventional therapy and then had uterine incision deliveries (neonatus died). Another case of 30-week with biliary pancreatitis took ERCP sphincterotomy and drainage of abdominal cavity after induced abortion because of a fetal death a week later. There was 1 case with hyperlipidemic pancreatitis (blood triglyceride level >26.54 mmol/L) at 25-week pregnancy which received induced abortion and hemofiltration because of threatened abortion (neonates died). All patients were cured and discharged.We carried out conventional therapy including nutritional support, correction of water-electrolyte imbalance and acid-base disturbance, and protection of organ function as well as administration of insulin for hyperglycemia. Cases with late pregnancy were forced to be terminated without considering whether the fetus could survive. We would end pregnancy for those who had unsatisfied therapeutic efficacy. Biliary-pancreatic surgery should be given for those with biliary pancreatitis conditionally to eliminate the predisposing factor of bile reflux. 1 case of 35-week pregnancy diagnosed as biliary SAP with MOF symptoms including hyperthermia, dyspnea, tachycardia and oliguria and deep yellow and turbid ascites with the higher amylase of 8280 U/L as well as intrauterine fetal distress received uterine incision deliveries and cholecystectomy, debridement and drainage of pancreatic necrosis, and percutaneous jejunostomy. In this study, there was 1 case of 30The relationship between AP and pregnancy is not quite clear. It's generally believed that APIP results from a synergy effect of several factors. The incidence of gestational pancreatitis in this series was one in 6,790 pregnancies . APIP shAnother pathogenesis is hyperlipoidemia in pregnancy that can result in the microcirculation disturbance and hyperlipoproteinemia. As a consequence, an amount of fatty acid derived from degradation of blood triglyceride by high pancreatic lipase causes APIP by pancreatic ischemia and necrosis resulted from capillary thrombus and breakdown of vessel wall. The placental lactogen produced by syneytiotrophoblast in pregnancy can disassociate fat notably and release a sum of free fatty acids which cause acute adipose infiltration of acinar cells and fat embolism of pancreatic vessel that lead to pancreatitis and necrosis , 21. In The fact that the augmented uterus compresses pancreatic and biliary duct is a key factor for the development of APIP. Terminating pregnancy is to eliminate the compression on one side and not taking the fetus into account; the fetus for treatment on the other side. Many great medications can be applied so that for enriching treatment options. The majority of cases with MAP in pregnancy can be cured by the conventional therapy with a good prognosis. But it remains controversial whether surgery is required or not. We think that the pancreatic capsule incision, partial pancreatectomy, and peripancreatic and peritoneal drainage can be used for cases with severe infection, necrosis, and peripancreatic, and peritoneal, effusion. If treated conservatively, pregnant patients with an biliary pancreatitis appear to have a high recurrence rate. Early surgical intervention is appropriate and safe and does not increase the length of the hospital stay. Since cholelithiasis is an important predisposing factor for APIP while biliary surgery has very few impacts on pregnant women and fetus with the development of medicine, some scholars suggest that women of late marriage should receive cholecystectomy before pregnancy as well as pregnant women at the mid trimester . Endoscopy is a great breakthrough for biliary APIP to eliminate bile duct stone and reduce the reflux of pancreatic duct by sphincterotomy and ENBD placing with better therapeutic efficacy than the conventional therapy and decreased mortality . The treData from our study indicates that MAP is not absolutely a sign for neither pregnancy termination nor cesarean section but aggravating SAP is. But in China, the national single child per couple policy has profound influence in each family. A couple can only have one child in their lifetime. The Chinese people must obey this rule; anyone violating it will face severe punishment; the most common punishment is fine. When APIP occurrs, the pregnant women and their other family members will pay much attention to the fetal development and would rather terminate pregnancy when the adverse factors may affect the fetus. In this group, induced abortion was conducted in 5 cases of the above nine MAP patients during 12–34 weeks while uterine incision deliveries were done in another 4 MAP cases during 33–37 weeks because of two main reasons: (1) pregnancy progressively aggravated the AP condition; (2) there was fear of some therapeutic drugs which can affect fetal development. This phenomenon is relatively common in China. We found many patients were treated with aprotinin, octreotide and sandostatin, spasmolytics, cephalosporin antibiotics, and some kind of traditional Chinese medicines. Among of them many drugs cannot get large-scale using in clinical validation for pregnant women, but the doctors have to use because of no other drugs can choose to cure those APIP patients. However, unless the fetus is too young or the labour goes well, we should consider saving lives of pregnant women at first and terminate pregnancy as soon as possible once the situation deteriorates or no relief occurrsFor APIP, most scholars advocate non-surgical treatment except for those following cases: (1) pancreatic abscess or infected effusion; (2) associated with other serious complications such as gastrointestinal perforation; (3) the situation deteriorates after active treatment of 2-3 d . There w


Chemotherapy research highly prioritizes overcoming the multidrug resistance (MDR) in human tumors. Liposomal formulation of fluoxetine, as a fourth generation chemosensitizer, was constructed and characterized for percent entrapment, release profile, morphology, particle size, zeta potential and stability. Liposomes were prepared using different active loading techniques. The influence of different formulation variables such as loading methodology, type of main lipid, addition of PEGylated lipid and cholesterol percentage was evaluated to achieve required entrapment efficiency, in vitro release behavior and stability. The studied parameters had significant effect on physicochemical characteristics of the nanocarriers. High fluoxetine encapsulation efficiency (83% ± 3%) and appropriate particle size (101 ± 12 nm) and zeta potential (-9 ± 2 mv) were achieved for PEGylation liposomes composed of DSPE-PEG, DSPC and cholesterol at respective molar ratio of 5:70:25. An in vitro fluoxetine release of about 20% in 48 h was observed from optimum formulation. Atomic force microscopy (AFM) studies confirmed homogeneous distribution of particles and spherical shape with smooth surface. The optimum formulation was stable for 9 days when incubated at 37 °C. The results of this study are very encouraging for application of the developed fluoxetine liposomal formulation in drug-resistant tumor models.  Chemotherapy is the most powerful therapeutic tool against advanced and disseminated cancers. However, in the battle against cancer, development of multiple drug resistance (MDR) is one of the major hurdles against successful chemotherapy. As the term implies, MDR refers to a cross-resistance to a wide variety of structural and functional distinct drugs or chemicals, thereby rendering the tumor unresponsive to most chemotherapeutic options -3. The mThe well-established role of P-gp in MDR has led to a great deal of research centered on agents that reverse or modulate P-gp activity in solid tumors. Many compounds with P-gp inhibitory activity, which are sometimes referred as “chemosensitizers”, have been identified or synthesized to address this issue . First gFluoxetine (FLX) is a highly active and selective serotonin reuptake inhibitor used for clinical depression for more than two decades . Recent In spite of high potency and specificity, a major confounding factor in use of free form of a chemosensitizer is the fact that besides exerting an effect on P-gp function in cancerous cells, it also has a profound effect on pharmacokinetics and cytotoxicity of the anticancer drugs concurrently administered . The phaA great deal of these shortcomings of chemosensitizers may be tackled by administering these agents using nanoparticulate delivery systems . It shoutheir potential for entrapping both hydrophilic and lipophilic drugs, and their colloidal size which make them useful for various applications , 22. Recently, Ong and his coworkers prepared liposomal formulation of FLX . The preMaterialsFluoxetine (FLX), 98% pure, was a kind gift from Dr. Abidi Pharmaceutical Co. . Distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), purified egg phosphatidylcholine (EPC) and distearoylphosphatidylethanolamine-poly(ethyleneglycol)2000 (DSPE-PEG2000) were obtained from Lipoid GmbH (Germany). Cholesterol (Chol), potassium chloride, citric acid, sodium hydroxide, disodium ethylenediaminetetraacetate (EDTA), sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, ammonium sulfate, chloroform, methanol, and HPLC-grade acetonitrile were supplied by Merck . Solubility of FLXThe solubility of FLX was investigated in 300 mM EDTA disodium salt, ammonium sulfate, or sodium citrate salt solutions (pH=4). The pH values of these salt solutions were adjusted with sodium hydroxide or hydrochloric acid. The salt solutions were added to FLX powder. Each sample was vortexed for 5 min and incubated at 25 °C for 12 h. The drug precipitate was separated from the supernatant by centrifugation . The FLX concentration in the supernatants was quantified by high-performance liquid chromatography (HPLC) as described in section 2.4.5.Liposomes preparationFLX was remote-loaded as previously described for other weak basic drugs . Empty lLiposome characterizationThe resulting ELs were dialyzed for 12 h at room temperature against the external buffer to establish a pH gradient across the liposome membrane. FLX in buffer was then mixed and incubated with ELs at 60 °C for 1 h. Residual nonencapsulated FLX were removed by exhaustive dialysis against phosphate-buffered saline (PBS) for 12 h at 4 °C. The prepared liposomes were characterized with respect to encapsulation efficiency (EE), size distribution, zeta potential, stability, morphology and in vitro release kinetics.Estimation of encapsulation efficiencyEE of liposomes was determined using the dialysis technique for separating the non-entrapped drug from liposomes. Small aliquot of liposomes (50 μL) was diluted in 950 μL methanol, and then it was subjected to sonication until complete liposomes disruption. Quantitative determination of FLX was performed by a validated HPLC method developed in our laboratory (See section 2.4.5). Lipid components did not have any interference with the estimation of FLX. The EE was calculated from the ratio of the FLX content of the liposomes and the amount of FLX used for preparation and expressed as a percentage.Determination of particle size and zeta potential The mean hydrodynamic diameter and polydispersity of liposomes were determined by the dynamic light scattering technique (DLS) in a Zetasizer Nano ZS  equipped with a 633 nm laser source. The analysis was performed at 25 °C and a scattering angle of 90° after the appropriate dilution with PBS. Each value given is the average of three measurements.Zeta potential of the liposomes was measured using electrophoretic light scattering by a Malvern Zetasizer Nano ZS. The measurement was performed at 25 °C after appropriate dilution with distilled water. All of the measurements were repeated at least three times.Stability of formulationsThe stability of liposomal formulations was evaluated at 4 °C and 37 °C. At intervals aliquots of samples were withdrawn for EE, particle size and zeta potential measurement.In-vitro drug releaseThe in vitro drug release of FLX encapsulated in different liposomal formulations was determined using dialysis tubing (molecular weight cutoff of 12 KDa). Liposomal solutions (0.5 mL) were placed in dialysis bag and immersed in 20 mL of release medium (PBS), under 100 rpm magnetic stirring at 37 °C. At predetermined time intervals, 1 mL samples were withdrawn and immediately replaced with an equal volume of the fresh medium. The concentration of FLX in the samples was measured by HPLC as described below (See section 2.4.5). The cumulative percentage of drug release was calculated and plotted versus time. Analysis of FLX by HPLC The amount of FLX in nanoformulations and in release medium was determined by reverse-phase HPLC. The chromatographic separation was accomplished on a PerfectSil C18 column,  using an isocratic mobile phase that consisted of acetonitrile and water  as the eluent at a flow rate of 1 ml/min . The analyte was detected with the ultraviolet detection  at the wavelength of 227 nm. The column was kept at room temperature. A linear response was observed in the range of 100–1000 ng/mL, with a correlation coefficient, of 0.9995. The coefficients of variation for the inter-day and intra-day assays were found to be less than 5.0%. Imaging of liposomes by atomic force microscopyThe shape and morphology of empty and drug-loaded liposomal formulations were observed using a NanoWizard®II atomic force microscope . Before AFM imaging, nanoformulations were diluted with distilled water and one drop of diluted dispersion was mounted on the glass slides, air-dried and scanned by the AFM. AFM was operated at room temperature with image resolution of 512 pixels × 512 pixels at a scan speed from 0.9 to 1.2 Hz in air. Statistical analysis Statistical analysis was performed using SPSS software . Data are expressed as mean ± SEM. Student›s t test was used to compare each variable between two groups. The analysis of variance (ANOVA), which was followed by the post-hoc test, was used when more than two groups were compared. For all of the tests, the differences of P < 0.05 were interpreted as statistically significant. Active loading techniques provide a versatile method to prepare liposomes with encapsulated cargo, provided that factors influencing drug loading, release properties and formulation stability are systematically studied. To the best of our knowledge, no efforts have been made to evaluate effect of formulation parameters such as lipid composition, presence of PEG and Chol/main lipid ratio on physicochemical characteristics of FLX nanoliposomes. In our study, EE, as a key parameter in liposomal drug delivery, was chosen as one of the major parameters to be optimized. Besides efficient loading, release of the drug which is a critical factor for a drug delivery system, was also studied.Based on our preliminarily studies, encapsulation of FLX in preformed liposomes led to significant increase in vesicle size  and within a few hours aggregate of drug molecules and lipidic components appeared suggesting the occurrence of a phase separation phenomenon which was in agreement with the previous report of interaction of FLX with pure as well as cholesterol containing phosphatidylcholine membranes . TherefoSolubility of FLX in different salt solutionsin-vivo drug release from liposomal nanocarriers  modification on the liposomal surface is known to be effective in increasing formulation stability, controlling release rate, prolonging blood circulation time and preventing carrier uptake by the reticuloendothelial system. These properties made PEGylated liposomes an attractive platform to improve the therapeutic index of a variety of drugs. The previous studies have suggested that 1–5 mol% DSPE-PEG2000 is commonly added to liposomal formulations and 5 mol% DSPE-PEG2000 has been reported as the sufficient and optimal concentration  and 39. The drug release rate of conventional and PEGylated EPC- and DMPC-liposomes were almost comparable. In case of DPPC and DSPC liposomes, the release rate of the drug was higher in conventional liposomes than that of stealth carriers. It seems that characteristics of main lipid can markedly influence the capacity of PEG coated liposomes in modulating drug release rate. PEGylated liposomes containing FLX were evaluated for physical stability at 37 °C. The physical stability was evaluated by monitorin vesicle size as previously described. PEGylated DSPC liposomes (F10) were stable for about 9 days at 37 °C, and retained about 80% of their initial drug content over 48 hours. The PEG chains on the nanocarrier surface have a higher volume of hydrated layer owning to their hydrophilicity. This can contribute to improved colloidal stability through both steric repulsion by PEGylated lipids on the surface of the particles as well as electrostatic repulsion by the ions in the hydrated layer thus retarding the increase in the nanoparticle size .Effect of cholesterol percentage on physicochemical characteristics of FLX liposomesin-vivo behavior of the carrier. Chol is a common component of liposomes, controlling membrane permeability, providing rigidity to the membrane, stabilizing the bilayer structure and improving plasma stability  was studied under AFM for morphological evaluation. The results showed uniform, homogenous and spherical shape vesicles with smooth surface . No aggrLiposome stabilityThe physical and chemical stability of F10 Formula were evaluated at 5 °C, 25 °C and 37 °C for two weeks. The stealth DSPC liposomes loaded with FLX were stable for at least two weeks at room and refrigerator temperatures as the particle size and the EE of the liposomes did not change significantly during this period (data not shown). The data presented in in-vitro characteristics. Nanosized PEGylated DSPC liposomes would be promising delivery systems for FLX in the treatment of drug-resistant tumors. Further in-vivo studies in tumor models need to be warranted in order to derive the feasibility of these formulations to improve the therapeutic efficacy.This study contributes to the understanding of the different formulation parameters on FLX loading into liposomes. The studies presented here suggested that the counter ion, the main lipid, the Chol percentage and addition of PEG lipid play important roles in drug loading efficiency, stability and release kinetics of FLX loaded liposomes. Formulation of highly stable nanoliposome constructs of FLX was prepared and characterized regarding various


Allergic heredity plays a major role in the development of allergic rhinitis. In addition the introduction of food may influence the risk of subsequent allergic disease. The aim of this study was to analyse early risk factors and protective factors for allergic rhinitis at preschool age. Data were obtained from a prospective, longitudinal study of a cohort of children born in the region of western Sweden in 2003 and 8,176 families (50% of the birth cohort) were randomly selected. The parents answered questionnaires at 6 and 12 months and at 4½ yr of age. The response rate at 4½ yr was 4,496, i.e. 83% of the 5,398 questionnaires distributed at 4½ yr. At 4½ yr of age, 5.5% reported symptoms of allergic rhinitis during the last year. In the multivariate analysis, independent risk factors for allergic rhinitis were: allergic sensitisation to food allergens at 4½ yr , recurrent wheeze at 4½ yr , doctor-diagnosed eczema at 4½ yr , parental rhinitis , eczema first year  and male gender . The risk was reduced with fish introduction before 9 months . In conclusion, we found that previous and present allergic disease, heredity and male gender increased the risk of allergic rhinitis at 4½ yr of age. The introduction of fish before the age of 9 months reduced the risk. Allergic rhinitis is not a common disease at pre-school age, but at this age symptoms of allergic rhinitis start to appear. The prevalence at 4–5 yr has been reported to vary between 5%  and 9.6%There is currently considerable interest in the effects of fish and fish products, especially omega-3 fatty acids, on the development of allergic disease . The timWe have previously described a positive influence on the prevalence of eczema following the early introduction of fish  and, in In this paper, the aim was to investigate the prevalence of allergic rhinitis at 4½ yr of age in a western Sweden population and to analyse early risk factors and protective factors for allergic rhinitis at preschool age.Data were obtained from a prospective, longitudinal cohort study of children born in the region of western Sweden in 2003. The region has 1.5 million inhabitants, one sixth of the Swedish population. It comprises urban, rural and coastal areas and the largest city is Gothenburg, with 500,000 inhabitants.Eight thousand one hundred and seventy-six families (50% of the birth cohort) were randomly selected. After written informed consent was obtained, the parents answered questionnaires at 6 and 12 months and at 4½ yr of age. The response rate at 6 months of age was 69%, while it was 60% at 12 months of age. The response rate at 4½ yr was 4496, i.e. 83% of the 5398 questionnaires distributed. This equals 55.0% of the families that were initially contacted. After supplementation with data from the Swedish Medical Birth Register (MBR), the database consists of 4171 infants with data from all questionnaires and the MBR. Details relating to the questionnaires at 6 and 12 months have been published previously , 17. DatThe questions relating to allergic rhinitis in the questionnaire were: (i) ‘Has your child experienced sneezing, runny nose, nasal blockage or red and itching eyes after contact with: furry animals, leafing or grass?’; (ii) ‘Has your child experienced these symptoms during the last 12 months?’ and (iii) ‘Has your child been diagnosed with hay fever or allergic rhinitis by a doctor?’‘Current allergic rhinitis’ was defined as the group of infants whose parents answered yes to (i) and (ii) and/or had been diagnosed by a doctor (iii).Questions concerning important covariates were:Allergy testing: ‘Has your child undergone allergy testing (skin prick test and/or blood test)? (Yes/No). If yes: What has the test shown positive reactions for? (Multiple answers are possible): Dog, cat, horse, rabbit, house dust mite, egg, milk, fish, wheat, peanut, soy bean, timothy grass, birch, mugwort, moulds or pea.’ (Asked at 4½ yr of age).Fish: ‘When did you start the introduction of fish? (Age in weeks)’ (Asked at 12 months of age).Neonatal antibiotics: ‘Was your baby admitted to a neonatal ward because of problems after birth? (yes/no). If yes, did your baby get penicillin or other antibiotics? (yes/no).’ (Asked at 6 months of age).Medical treatment during pregnancy: ‘Did you as mother take any medications during pregnancy? (yes/no). If yes, please specify.’ (Asked at 6 months of age).Eczema at one yr: ‘Has your baby had eczema? (Asked at 12 months of age).Eczema at 4½ yr: ‘Has your child been diagnosed with eczema by a doctor after 12 months of age?, ‘Has your child had eczema the last 12 months?’ and ‘Have you treated the eczema with cortisone ointment the last 12 months?’ (Asked at 4½ yr of age).Rural childhood: Responding ‘In the country’ as opposed to ‘City center’, ‘Suburban area’ or ‘Other densely populated area’ to the question ‘Where do you live?’ (Asked at 6 months of age).χ2 test were used and risks were estimated using the Mantel–Haenszel common odds ratio (OR) estimate. In the multivariate analysis, binary logistic regression was used and OR with 95% confidence intervals (CI) were calculated.In the univariate statistical analysis, 2 × 2 tables with the In the multivariate analysis, all significant variables with a p-value < 0.01 were initially included. This cut-off level was chosen to avoid problems with multiple testing. Although not reaching this level, the variable ‘antibiotic treatment first week of life’ (p = 0.032) was included since there is evidence that exposure can lead to an increased risk of subsequent asthma , 18. SinThis resulted in a model containing parental allergic rhinitis, parental eczema, parental asthma, rural residence first year, maternal medication during pregnancy, treatment with broad-spectrum antibiotics first week of life, any bottle feeding first week of life, cat in the home first year, fish introduction before the age of 9 months, fish consumption >1 ×/month first year, recurrent wheeze at 12 months of age, eczema first year, doctor-diagnosed food allergy first year, gender, maternal smoking during pregnancy, parental education, breast-feeding, doctor-diagnosed eczema at 4½ yr, recurrent wheeze at 4½ yr, doctor-diagnosed food allergy at 4½ yr and parental report of positive allergy testing for food or inhalant allergens.The multivariate analysis was performed with a forward stepwise logistic regression with these variables.The attributable fraction, AF, was calculated using the formula: spss v. 17 statistical package was used for statistical calculations.The The study was approved by the Ethics Committee, University of Gothenburg.At 4½ yr of age, 5.5%  reported symptoms of allergic rhinitis during the last year, i.e. current allergic rhinitis. Of these, 30% also reported their symptoms to be doctor-diagnosed. In the total study population the prevalence of doctor-diagnosed allergic rhinitis with symptoms during the last year, was 1.7% .Of the 246 infants with current allergic rhinitis, 68% were boys and 81% had a positive parental history of allergy or asthma. Seventy-three per cent reported only pollen as a trigger factor, 13% reported that symptoms were triggered by furry animals and 14% by both furry animals and pollen . Among tConcurrent allergic manifestations and wheezing at preschool age were common among the children with allergic rhinitis: eczema 37%, food allergy 16% and recurrent wheezing 25%.The results of the univariate analyses are presented in In order to elucidate whether the small proportion of children not being introduced to fish before 9 months differed from the rest of the population, we performed a stratified analysis. 83.6% of infants had been given fish before 9 months of age, and 16.4% not. Of the infants with a late introduction of fish, 6.6% had a doctor's diagnose of food allergy compared to 3.4% of the early introducers. They also more often had parents with allergy . However, the effect of early fish introduction was significantly protective in both strata, i.e. there was no significant interaction .In the multivariate analysis, independent risk factors for current allergic rhinitis were: positive food allergy test at 4½ yr, recurrent wheeze at 4½ yr, doctor-diagnosed eczema treated with topical steroids at 4½ yr, parental rhinitis, eczema first year and male gender. The introduction of fish before the age of 9 months reduced the risk .Fish introduction before the age of 9 months was protective against reported positive allergy test (skin prick or blood), both for inhalant  and food allergens .The attributable fraction for not introducing fish before 9 months of age was calculated as 10.5% (95% CI 3.4%–15.2%).The main finding in this study was that positive allergy test, recurrent wheeze, eczema, heredity and male gender increased the risk of allergic rhinitis at 4½ yr of age. The risk was reduced if fish was introduced before the age of 9 months.Our definition of allergic rhinitis is very similar to the definitions in the ISAAC study  and of KThe main explanation for the relatively low figure of 40% with positive allergy test is most likely that many children with symptoms had not yet undergone allergy testing. It is also possible that some children with symptoms of allergic rhinitis do not yet test positive at this young age . This isAllergic rhinitis, and especially doctor-diagnosed allergic rhinitis, is not common at preschool age. In our material, only 75 infants 1.7%) had a doctor-diagnosed allergic rhinitis, while 246 (5.5%) reported symptoms compatible with allergic rhinitis. The prevalence of allergic rhinitis is comparable to that seen in previous studies .7% had a, 15. FroNot surprisingly, allergic heredity and own allergic disease emerged as the by far strongest risk factors for allergic rhinitis. This emphasises the strong hereditary component in this disease and is in consonance with several previous studies , 6. FurtLike earlier studies, we find that rhinitis at preschool age often occurs concomitantly with wheeze and eczema. In the present study, recurrent wheeze at age 4½ yr was an independent risk factor, as found in also some other studies . AlreadyIn addition, male gender appeared as an independent risk factor for allergic rhinitis at preschool age. This is not found consistently although it has been seen in some other studies .In consonance with several other studies we did not see any effect of maternal smoking or short breast-feeding , 6.A protective effect of the early introduction of fish on allergic rhinitis at preschool age seen in this study is supported by previous reports of a reduced risk of allergic disease following a high fish intake during pregnancy and during infancy , 11, 25.The composition of fatty acids in the diet (omega-3) and its implications on the developing immune system and the Th1/Th2 balance has been discussed as a possible pathophysiological mechanism. However, it is possible that the effect of fish is mediated not only via omega-3 fatty acids but also by other components of fish. In the present follow-up and in our 1-yr follow-up on eczema , we noteThe timing of introduction of different foods, with a window of opportunity during the first year of life, seems to be of importance for the maturing immune system , 9. In aOther possible confounding factors often discussed are parental allergic disease and early allergic manifestations in the child itself. These factors could influence the timing of introduction of solid foods. We have tried to adjust for this difference by controlling for parental rhinitis, asthma and eczema, as well as for wheezing, eczema and doctor-diagnosed food allergy in the child itself. The introduction of fish before the age of 9 months was still an independent protective factor for allergic rhinitis at preschool age. Furthermore, we found no significant interactions, i.e. the effect of early fish introduction was protective irrespective of heredity or own allergic disease.There is also a possibility that the small proportion of infants with a delayed introduction of fish in some other way can differ from the rest of the population. Therefore, we tested potential confounding variables indicative of life-style in the multivariate model. In doing that, we found no effects of adherence to vaccinations or maternity care, AD-vitamin supplementation, vegetarian life-style and housing. However, even with these adjustments, there is always a risk of residual confounding.The effects of early fish introduction were similar as those reported earlier for recurrent wheezing at 4½ yr of age  .The attributable fraction of 10.5% implies that approximately one tenth of the cases with current allergic rhinitis at preschool age could statistically be attributed to the introduction of fish after the age of 9 months.Another early exposure with possible impact on the maturing immune system is the exposure to a farming environment in early childhood, with a suggested protective effect on allergic disease , 20, 30.A weakness of the study is that the data were collected by postal questionnaires. As a result, the endpoint is not objectively measured. The strengths are the large size of the birth cohort and the good response rate of 70% initially and the current 83% in this follow-up. In all, we have data from all the questionnaires relating to more than four thousand children. Moreover, as reported earlier, the material appears to be largely representative of the population .Positive allergy test, recurrent wheeze, eczema, heredity and male gender were independent risk factors for allergic rhinitis at age 4½ yr. We also found that the introduction of fish before the age of 9 months reduced the risk of allergic rhinitis at preschool age.


Salmonella strains and thereby reveal differences not apparent at the gene sequence level.The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 isolate (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of transposon insertions or had very few. For three of these nine genes, part of the annotated gene lacked transposon integrations . At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes  are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential.A transposon Tn5 derivative was used to construct mutants in Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene contributions to fitness among syntenic homologs. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology. When a library of transposon (Tn) integrations is created in a bacterial genome, some insertions are not recovered in the resulting pool of mutants, either because the insertion is in an essential gene or because the gene is required in the media used to grow the bacterium. This fact has been exploited extensively to identify genes under selection when growth conditions are changed-3.Salmonella.Another potential utility of such data, that is explored here, is to compare different strains, serovars, and species to reveal apparent orthologs that have very different levels of fitness in different strains. We perform the first experiments to quantitate this phenomenon in Salmonella enterica serovar Typhimurium strain ATCC 14028 (STM1) and in Salmonella enterica serovar Typhi Ty2 (STY1) after growth in rich media (Luria Broth). Our datasets were compared to each other and to a series of other published data on the fitness of mutations in Salmonella [Escherichia coli (ECO)[htrAaroC and aroD [We used high-throughput sequencing to determine the location of tens of thousands of integration sites of a Tn5 derivative in the genome of lmonella  and Eschli (ECO)-10, incland aroD .Differences in the selective pressure on apparent orthologs in the related genomes  are of interest because they likely reflect differences in the systems that interact with these otherwise functionally similar genes or their products.S. Typhimurium ATCC 14028 (STM1) and two in S. Typhi Ty2 (STY1), using the EZ-Tn5 < KAN-2 > Promoter Insertion Kit (Epicentre Biotechnologies) (see methods), and grown in Luria broth  beyond the primer. The remainder of the sequence was mapped to the genome to determine the transposon integration site.Salmonella sequence due to the random DNA shearing used in the sequencing protocol. To reduce bias due to preferential PCR of some fragments, duplicate identical shear events were removed. The remaining reads for each transposon integration site were used to determine the number of different “shear events” for that transposon. This filtering resulted in the mapping of 234,152 and 53,556 Tn integration locations, with 2,827,876 and 313,585 unique shear events in STM1 and STY1, respectively. The average density of integrations into the genome was one every 20 bases with an average of about 12 shear events per site in STM1, and one every 90 bases with an average of about 6 shear events per site in STY1.Each Tn integration site was generally represented by multiple reads, and these reads usually varied in the length of the priA (primosome assembly), and a gene with an essential region, ftsN (involved in cell division). Regions that were essential in Luria broth are identifiable in this plot as having no transposon integrations. Near-essential regions have a lower than average number of integration sites, usually accompanied by a lower number of shear events.The transposon libraries used in these experiments were constructed and grown in rich media. Those regions of the genome with rare or absent transposon integrations include regions that are essential or under strong selection in rich media. A sizeable subset of these regions should also be essential under all growth conditions. An example of a profile of transposon integrations is shown in FigurecytR, adjacent to ftsN, the negative strand contains far more transposons; this is the strand in which the heavily expressed antibiotic resistance marker of the transposon is oriented away from ftsN, likely making these integrations less disruptive. Many other examples of general selection and strand-specific selection are seen in Additional fileOur data also provide information on the orientation of each transposon, which can be informative. For example, in FigurehtrA-aroC- and aroD- mutant STY2 were derived from two selections: a single passage on a solid medium (i) and six passages in Luria broth (ii).The transposon frequency analyses for all genes in STM1  and STY1 are presented in Additional fileE. coli, S. Typhimurium and S. Typhi, over 60% of protein coding genes are syntenic and have over 95% amino acid sequence identity[Between identity. There aidentity-10  and three had average amounts of transposon insertions (murBftsN and degS) indicating that mutants were not under selection in LB in this isolate. Two other genes did not meet the “selected” threshold in our STY1 assay  and one gene, folA, was not found to be selected in the published STY2 data[We ranked all yejM, a putative hydrolase; ftsN, which encodes a cell division protein; and murB, a UDP-N-acetylenolpyruvoylglucosamine reductase. FigureftsN gene as an example. These genes can apparently be disrupted in certain locations without losing their essential function. Thus, because of the high density of transposon integration data, we were able to reveal those cases where only part of the gene is essential.Visual inspection of Additional filetrpS, encodes a tryptophanyl-tRNA synthetase. It may tolerate transposon insertions in STM1 and STY because of the presence of a distant paralog (trpS2), which does not exist in ECO. TrpS2 may substitute the TrpS function, although it is only 28% identical[The fourth of the six cases, dentical.degS, a serine endoprotease. This gene was under strong selection in STY2[htrA mutant, a paralog of degS [degS was somewhat diminished, but not enough to qualify the gene for the “selected” category, suggesting at least some effect of the lack of a functional HtrA in STY2. However, in STM1 there is no evidence for any degree of transposon underrepresentation in degS, and it seems likely that another paralog, as yet undefined, can perform the proteolytic activity of DegS in this strain, if needed.The fifth gene essential in ECO and not under strong selection in STM1 is  in STY2. That st of degS . In our folK was somewhat underrepresented in transposon insertion frequency in STM1, but not to a degree that warranted inclusion in the “selected” category.Finally, folA gene, involved in folate biosynthesis, was strongly selected in STM1 and STY1 but did not show as much selection in STY2, especially after six passages in LB. Lastly, yrfF and gpsA are two genes essential in ECO that did not meet the “selected” threshold in our STY1 assay. Both these genes were under strong selection in STM1 and in STY2 after six passages in LB, indicating that mutations cannot be maintained for many passages in this medium. The yrfF (igaA) gene encodes an intracellular growth attenuator protein; and gpsA encodes a NAD(P)H-dependent glycerol-3-phosphate dehydrogenase. IgaA has been described as essential in S. Typhimurium unless there are additional mutations in the RcsCDB system, because it acts as a repressor of this system[igaA is positively regulated by Lon and negatively modulated by Hnr (MviA) through the transcriptional regulator RpoS[S. Typhi Ty2 carries a defect in the rpoS gene[igaA in STY.There are three other genes that are essential in ECO and STM1, but seemed not strongly selected in our STY1 survey and/or STY2. The s system,15. Exprtor RpoS. S. TyphpoS gene,18, whicSalmonella  but not essential in ECO were identified . Given the conservation of these genes between Salmonella and E. coli, these are particularly interesting targets for future studies to determine their exact function.The list of essential genes in ECO is stringent, generally including the inability to obtain a viable deletion mutant in rich medium. In contrast, the measure available from transposon integrations in STM1 and STY reveals genes that were under strong selection, but not necessarily essential. A group of 159 such genes that are under selection in all Salmonella-specific genes) yielded only two strongly and consistently selected genes: STM14_5442 and STM14_2872, which both encode putative cytoplasmic proteins. However, Santiviago et al. reported successful knockout mutations in both of these genes in this same strain, so they are likely not essential[The approximately 900 genes shared by STM1 and STY that have no synteny in ECO  is a response regulator which post-transcriptionally modulates RpoS levels[stpA, which encodes a 53% identical paralog of hns, also showed a greater tolerance for transposons in STY than in STM1. Unlike hns, viable hnr mutants can be obtained in S. Typhimurium[hnr show reduced growth rate because of an increased RpoS stability, which increases transcription of genes involved in growth arrest and resistance to a variety of stresses[hnr mutations seen in STM1 might be due to the non-advantageous phenotype of slow cell division in these mutants when they are in competitive growth.Genes that were under stronger selection in STM1 than in STY when the transposon libraries were grown in LB are depicted in Table present, no selerpoSgene,18. Mutae viable, and hnrS levels. Intereshimurium, even thhimurium. Mutantsstresses. The selpotF; an excisionase; a cation transport regulator chaB; the L,D-carboxypeptidase A ldcA; the phosphoribosylaminoimidazole-succinocarboxamide synthase purC; and cpxP, a periplasmic repressor of the envelope stress response pathway. Viable mutants were obtained in this same Typhimurium strain for six of these genes[At least 26 additional genes appeared to be under strong selection in STM1 but not in either STY. This class includes, among others, the putrescine ABC transporter se genes  and, interestingly, has been reported as essential in this species. In STM14_1358 and STM14_1498.L, levels of selection in STM1 were similar to the levels in both STY. In the other two cases, there was no selection in at least one of the two STY isolates.Integrations in genes that are thought to be pseudogenes in STY and intact in STM1, or vice versa, were inspected. Of approximately 60 putative pseudogenes annotated in the S. Typhi Ty2, four were strongly selected in at least one of the STY: eda, a keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase; astA, an arginine succinyltransferase; t2152 (STM14_0843), a putative glycosyltransferase involved in cell wall biogenesis; and t3548 (STM14_4894), a putative cytoplasmic protein. The eda gene is the only case showing a strong selection in both STY1 and STY2.Of approximately 200 pseudogenes in S. Typhi Ty2, an RNA-seq analysis of the transcriptome was recently published[In ublished and conchtrA [aroCaroD, and htrA [aro genes, which encode enzymes involved in the shikimate biosynthesis pathway, render bacteria auxotrophic for the aromatic amino acids p-aminobenzoate (pABA) and 2,3-dihydroxybenzoate[aro mutations also result in the inability to produce ubiquinone and menaquinone, leading to respiration defects[htrA mutant presents more susceptibility to oxidative stress than the wild type[In our study, 53,556 transposon integration sites for STY1 were determined. In a previous work, 370,000 insertion sites were identified in STY2. Over 100 genes showed a difference in fitness between these two strains. Some of these differences may be attributable to the growth conditions used in the two studies. Our STY1 data are from a single LB growth passage whereas the STY2 data were from L-agar supplemented with aromatic compounds as well as from six passages in LB. Furthermore, STY2 is an attenuated strain, CVD908-and htrA . Deletiobenzoate. These a defects, and in  defects, unless ild type.htrA and one each in aroC and aroD in STY1. The number of transposons in htrA was close to the average random transposon insertion frequency (15.9) whereas aroC and aroD showed selection in both STY1 and STY2. The apparent selection of aroC and aroD in STY2 is explained by the fact that these genes were knocked out in this strain, resulting in a much smaller gene remnant as transposon target area. The htrA mutation may explain at least some differences between the two strains of STY, such as the selection of the periplasmic protein HlpA and the strong selection of DsbA after six passages in LB in STY2, but not in our STY1 study. In E. coli, the skp  degP (htrA in STM) double mutant is lethal[dsbA degP double mutant shows reduced growth[We found 17 transposons in s lethal and the d growth.Salmonella Pathogenicity Island 2 (SPI-2) genes[ssaIssaHssaRssaTsifB) but not in STY2. Some other SPI genes were interesting from a regulatory point of view. The hilC and rtsA SPI-1 regulators were selected in STY1, but not in STY2[rtsB, whose product represses the master regulator of the flagellar regulon, flhDC [STY1 showed selection in some 2) genes,33. Some in STY2. RtsA isn, flhDC .S. Typhimurium and involve over 60 genes integrated in a hierarchy of controlled transcription[flgM gene encoding the anti-sigma 28 negative regulator of the synthesis of the flagellar filament, was selected in broth media, but not on LB agar. FlgM binds to the sigma 28 factor to prevent transcription of late flagellar genes before the completion of HBB structure[flgCflgJfliIfliK, and fliO were selected only when bacteria were grown on an agar surface. FlgC is one of the structural rod components and FlgJ is the capping protein of the rod which also possesses muramidase activity[fliT, which encodes a protein that acts as a repressor of flagella biosynthesis[flhE showed selection on agar growth and after six passages in LB in STY2[et al. reported that flagellar genes were regulated in a surface-specific manner[flgM and fliT; and selection for genes necessary for flagellar biosynthesis when bacteria are grown on agar.Flagellar genes show different patterns of selection comparing growth in LB broth under aeration  versus on LB agar (STY2). Flagella and motility are highly regulated in cription. The flacription. The flgtructure. In contactivity. FliI isactivity. FliK reactivity. FliO isactivity. These fynthesis, was selynthesis. The fla in STY2. Althoug in STY2. In agrec manner. Overallin vitro growth in LB using an insertion-duplication mutagenesis (IDM) strategy based on a temperature-sensitive integration plasmid[S. Typhimurium LT2 in LB, of which 71 were already known to be essential in E. coli [We compared our data , indicating that the vast bulk of integration sites were largely randomly distributed. However, hotspots, where Tn5 integration is preferred, have been reported. GoryshiYWRanC-t. The dasSalmonella Typhimurium strain 14028 and two strains of S. Typhi Ty2. We also found potential differences in essentiality of homologous genes between Salmonella and E. coli  and S. Typhi Ty2 strain JSG624 (STY1) provided by Ferric Fang . Bacterial cells were grown in LB medium containing 1% Bacto tryptone (Difco), 0.5% Bacto yeast extract , and 1% NaCl, supplemented with kanamycin at 50 μg/ml, when necessary.The strains used in this study were Salmonella cells were made competent by standard methodology. Briefly, cells were grown in LB with shaking at 37°C to logarithmic phase, then washed three times with cold 10% glycerol and concentrated 250 fold in 10% glycerol. Transposome mixtures were prepared mixing 2 μl glycerol, 2 μl EZ-Tn5 < T7/KAN-2 > transposon, and 4 μl EZ-Tn5 transposase. After 3 h of incubation, 1 μl of this mixture was mixed with 50 μl of competent cells and 1 μl TypeOne restriction inhibitor. Transformation was performed at 2.5 kV using 0.2 cm electrode gap cuvettes and a Bio-Rad MicroPulser at EC2 setting. Transformed cells in each cuvette were resuspended in 1 ml of LB and incubated for 1 h at 37°C. After incubation, reaction aliquots were joined, complexity was determined by cell counts of various dilutions on LB agar containing kanamycin, and the remainder was grown overnight at 37°C in LB broth supplemented with kanamycin. Stocks of the different transposon mutant libraries containing 20% glycerol were prepared from the overnight cultures.et al. [The entire procedure is illustrated in Additional fileet al. . SubsequThe beginning of each read primer contained a code that defined the particular transposon library used. The codes are listed in Additional fileE. coli K-12 were identified by Blast searches of annotated genes against each other in the different genomes. Synteny was determined based on at least one of the two adjacent genes also being a best hit and being adjacent in other genomes. The assigned unique gene numbers differ for all the genome annotations, so all assigned gene numbers and gene symbols in these five genomes are reported in the Additional fileBest hits between Typhimurium 14028, Typhimurium LT2, Typhi CT18, Typhi Ty2 and 2. The published STY2 data[Because the distribution and number of transposons is not identical between STM1 and STY1, the data were processed to express the ratio of the number of transposon integrations in each gene and intergenic regions versus the number of integrations in that region that would be expected if all the integrations were randomly distributed. The ratio of observed divided by expected numbers were then expressed as a logTY2 data were rec2 ratios (observed number of insertion events/expected number of insertion events) among all 3,907 genes shared between these genomes were ranked from those with the lowest density of transposons to those with the highest density. Ranks in STM1 and both STYs were subsequently subtracted to identify those genes with the biggest difference in rank order of transposon density.To identify those genes that had large differences in fitness among STM1, STY1 and STY2, all logSalmonella. We arbitrarily defined “selected” genes as those having a sum of the two percentile ranks less than 30% .The percentile rank of the number of transposons per base and the number of reads per base (shear events) were calculated for each putative ortholog in each experiment in The authors declare that they have no competing interests.RC constructed the transposon libraries, and developed the high-throughput sequencing strategy. She helped to write the manuscript and prepare the tables and figures. CF performed the sample preparation for sequencing. SWC coordinated and directed the sequencing project. XQX implemented the trimming and mapping strategy for counting transposons and shear events. He implemented the plots of transposon and shear events across the STM1 genome. BA provided four of the transposon libraries used for screening. HLAP advised on some steps of the project. SP was involved in Typhimurium annotation and manuscript writing. MM devised the sequencing and mapping strategies, helped to analyze the data, and wrote the manuscript. All authors read and approved the final manuscript.Table S1. Transposon libraries (EZ-Tn5 < T7/Kan-2>) assayed.Click here for fileSupplemental Methods. Detailed explanation of protocols.Click here for fileFigure S1. Diagram of sequencing protocol.Click here for fileFigure S2. Transposon insertion frequency across the S. Typhimurium 14028 s genome.Click here for fileTable S2. Essentiality surveys and transposon data.Click here for fileTable S3. STY1 complete transposon data.Click here for file