Finally, we have shown that the selleck kinase inhibitor planting area necessary for the cell population to maintain the “”feeling”" of belonging to a single body, roughly corresponds to the outer diameter of a mature interstitial circle (Figure 7c). Exceeding this critical diameter leads to the loss of structure and breakdown to a macula; however, even in such a case the body is self-inhibited as to lateral spreading. This may perhaps be understood as the last remnants of its “”feeling of integrity”"; the results of our computer simulations suggests that even this seemingly complex effect may be produced by the interplay of mere two signals. Conclusions

Some isolates of Midostaurin chemical structure Serratia sp. produce

colonies exhibiting finite growth and clone-specific appearance, which is easily evaluated thanks to their conspicuous coloration. The shape and patterning of developing colonies and other multicellular bodies is easily malleable by experimental conditions. The appearance of a developing colony results from (i) its internal morphogenetic potential   (ii) the character of neighbor bodies and their overall distribution on the dish.   A simple formal model is proposed, based on two morphogenetic signals generated by the bodies, one of them spreading through the substrate and the other through the gas phase. The model can simulate some of our experimental results, namely: 1. 1. The development of colonies exhibiting finite growth and both rimmed and rimless patterns, the difference between the former and the latter being in the intensity of signal production and/or sensitivity towards the signal(s).   2. 2. Dependence of colony size upon the number of colonies sharing common morphospace, and development of confluent colonies from closely

planted inocula of a rimmed strain.   3. 3. The phenomenon of “”critical planting area”" which must not be exceeded should a colony develop a typical rimmed pattern.   Our observations are thus consistent with bacterial colonies behaving, in some aspects, as true multicellular bodies whose patterning is controlled by positional information; the nature of the relevant signals remains to be established. Methods Strains, media and culture much conditions The strain Serratia rubidaea here labeled R (rimless “”wild type”" phenotype for the purpose of this study), as well as E. coli strain 281, were obtained from the collection of the Department of Genetics and Microbiology, Faculty of Sciences, Charles University. The R strain, originally described as S. marcescens, has been determined as S. rubidaea on the basis of metabolical markers and gyrB gene sequencing (A. Nemec, National Health Institute, Prague, personal communication). The remaining three Serratia sp.

As a result, EEM has been widely applied to the fabrication of ultraprecise mirrors used in synchrotron radiation facilities and EUVL [1]. However, further improvement of the figure correction system is needed because larger optical devices with more complicated figures are now required. For example, ultraprecise X-ray mirrors with a length of 400 mm have become necessary [7]. Ellipsoidal mirrors are also gaining increasing attention in the field of soft X-ray microscopy [8]. To improve the characteristics of stationary spot machining

in EEM, we propose an improved method of flowing a fluid including particles. In particular, nozzle-type EEM utilizes a jet flow, which has been investigated in various fields such as water jet machining, water jet cleaning [9], and surface reforming with cavitation [10]. In these studies, selleck compound the shape BGB324 in vivo of the aperture and the structure of the channel in the nozzle are optimized to form a variable flow from the nozzle. The method used to simulate the fluid flow has also been improved. The behavior of a jet flow can be predicted and effectively used to develop functional nozzles. In this study, we propose a nozzle structure to further improve the properties of stationary spot machining in EEM. The structure can concentrate the fluid after it flows from the nozzle aperture. A fluid simulation is carried out to clarify the advantageousness of the proposed structure. Then, the nozzle is fabricated and tested

to confirm the simulation results. Methods Fluid simulations In nozzle-type EEM, to transport particles to the workpiece surface and remove them from the surface, a high-shear flow is required on the surface. The removal area and removal rate depend on the velocity distribution of the fluid in contact with the surface. The shape of the distribution can be controlled by changing the nozzle specifications MYO10 such as the width, velocity, angle, and stand-off distance, where the stand-off distance

is defined as the length between the nozzle outlet and the workpiece surface. In previous studies, the fluid channel of the nozzle was straight, and its aperture was rectangular or circular, as shown in Figure 1a [4]. The pressurized fluid flows from the nozzle toward the fluid in a tank. In this case, it is commonly considered that the flow diverges after exiting from the aperture since the jet flow is in a strongly turbulent state. To satisfy both the smallness and removal rate required in stationary spot machining, the stand-off distance is selected to be short. Minute stationary spot machining with a spot size of 500 μm in diameter has been realized for a stand-off distance of less than 300 μm [4]. Figure 1 Structure of nozzles used to generate high-shear flow on the workpiece surface in elastic emission machining. (a) Straight-flow nozzle. (b) Focusing-flow nozzle. In this study, the generation of a focusing flow is applied to EEM. Figure 1b shows the concept of a focusing flow.

and Hardy et al. [3, 22] found that calcium uptake was decreased in hypochlorhydric subjects, whereas other studies did not observe any effect [23–25]. Only during fasting conditions calcium uptake was decreased among patients using PPIs [2, 22] and among achlorhydric patients [23, 26]. Furthermore, some in vitro [6, 7] and in vivo [5] studies suggested that PPIs could inhibit the osteoclastic proton pump and thereby reduce bone resorption. Conversely, short-term omeprazole treatment did not alter osteoclast or osteoblast function in paediatric users [27]. Moreover, no selleck chemicals llc significant differences were

observed in BMD among postmenopausal women using acid suppressants (PPIs and H2RA), while in men, even lower cross-sectional bone masses were observed [28]. In addition, the most recent study performed by Targownik et al. [29] showed that both chronic PPI use and high daily doses of PPIs were not associated with osteoporosis or accelerated

BMD loss. Several observational studies that investigated the association between duration of acid suppressant use and fracture risk found discrepant results as well [8, 10–12]. Both Yang et al. and Targownik et al. [8, 10] found that fracture risk increased with longer durations of PPI use. In contrast, members of our group found results which are similar to the present study (i.e. PPI use for a duration ≤1 year is associated with the highest fracture risk) this website using the same database as Yang et al. [11]. Moreover, our sensitivity analysis, in which we resembled the definitions of Yang et al., did not support a duration-of-use effect. Additionally, Kaye et al. [12] who also used the GPRD database did not find any association between the number of PPI prescriptions and hip fracture. The reasons for these discrepancies remain unclear. There are alternative explanations for the small, overall 1.2-fold increased risk among current users of acid suppressants. These include the inability of the current and previous studies, to measure (or only partially measure) alcohol consumption, smoking history and low body mass index. All these factors are associated

with an increased risk of fracture [30–32]. Besides, PPIs are often used for the eradication of Helicobacter pylori [33], which may be associated with an increased risk Cobimetinib of osteoporosis [34]. In addition, PPIs are associated with the onset of Clostridium difficile [35], which may be an alternative explanation for the increased risk of fracture. Finally, celiac disease, which is associated with the onset of reflux oesophagitis [36], has recently been associated with an increased risk of both osteoporosis and fracture [37]. Nevertheless, we were unable to fully adjust for these three potential confounders, because PHARMO RLS has missing data of diagnoses determined outside the hospital. Our study has several strengths. As we used a population-based design, our study represents the entire population of the Netherlands.

The protocol was approved by the institutional ethics committees and this study was carried out according to the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The eligibility criteria were histologically proven unresectable colorectal adenocarcinoma; adequate bone marrow, liver, and renal function; Eastern Cooperative Oncology Group (ECOG) performance status (PS) <2; age >20 years at the time of enrolment; and expected survival selleck time >12 weeks. Any

previous chemotherapy (only 1 regimen was allowed) must have been completed at least 28 days before enrolment. Postoperative adjuvant therapy was not counted as prior chemotherapy. Patients with multiple malignancies, this website comorbidities that could influence the outcome, prior radiotherapy, pregnancy or lactation, symptomatic peripheral neuropathy, or a history of serious drug hypersensitivity were excluded. Written informed consent was obtained from all of the subjects. Treatment schedule An implantable port and a disposable

pump were employed so that chemotherapy could be administered on an outpatient basis. An outline of the administration method for mFOLFOX6 therapy, in which the dose of oxaliplatin was reduced from 100 mg/m2 to 85 mg/m2, is shown in Figure 1. A 5-HT3 antagonist and a steroid were administered as premedication. A 2-hour intravenous infusion of oxaliplatin plus l-leucovorin was followed by bolus intravenous injection of 5-FU, after which 5-FU was administered by continuous infusion for 46 hours. An

oral steroid was administered for 3 days from day 2 after the start of therapy. The duration of one cycle was 2 weeks. Figure 1 Schedule for mFOLFOX Therapy. With each treatment cycle, administration was only started after confirming that all of the following criteria had been fulfilled. (1) Hematological toxicity: leukocyte count >3,000/mm3 buy Abiraterone and platelet count >75,000/mm3.   (2) Non-hematological toxicity: Grade 2 or less according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC), and Grade 1 or less for peripheral neuropathy.   (3) Even if these conditions for treatment were met, administration could be postponed at the investigator’s discretion (e.g., for a rapid decrease of the leukocyte count/platelet count, occurrence of jaundice, etc).   If any of the criteria were not met, treatment was postponed. The subsequent course could be postponed for up to 21 days (excluding the scheduled day of starting administration). If administration could not be commenced during this period, the study was discontinued. Discontinuation of therapy Administration was continued until any of the following criteria for discontinuation were fulfilled. (1) The patient was judged to have progressive disease (PD), including clinical PD.   (2) Adverse events occurred that made further administration difficult.

Construction of exoF::TnphoA fusion To generate plasmid-borne exoF::TnphoA fusions, plasmid pD82, a cosmid clone carrying the S. meliloti exoF gene and surrounding region of the genome [26], was introduced into the S. meliloti exoF::TnphoA fusion

strain Rm8369 [27]. This construct was subsequently www.selleckchem.com/products/abc294640.html transferred into E. coli strain MT607, by triparental conjugation using E. coli strain MT616 as the mobilizer. Transconjugants were selected on LB KmTc, and the nature of the fusion was confirmed by testing for inability to confer YMA mucoidy on the exoF::TnphoA mutant Rm7055. The resulting construct was named pD82 exoF::TnphoA. Biochemical assays Alkaline phosphatase activity of exoF::TnphoA fusions in S. meliloti strains was measured according to the method of Brinkmann and Beckwith [46]. Cells were grown to an OD600 of 0.7. 1 ml of culture was washed twice in 1 M Tris-HCl (pH 8.0), and resuspended in 1 ml 1 M Tris-HCl (pH 8.0). The OD600 of this cell suspension was then measured. Following

a 10 min equilibration period at 37°C, 50 μl of 4 mg/ml p-nitrophenyl phosphate (NPP) was added to start the reaction. check details The reaction was allowed to continue for 11 min at 37°C before being stopped by the addition of 50 μl of 1 M K2HPO4. The cells were pelleted and 50 μl of the supernatant was diluted in 450 μl of 1 M Tris-HCl (pH 8.0) and OD420 was measured. Units (U) of alkaline phosphatase activity were calculated using the formula: (1) Assuming a molar coefficient of 16,000 for p-nitrophenyl phosphate, 1 U is equal to 0.062 nmol of NPP hydrolyzed per min at a cell OD600 of 1. Therefore: (2) For PHB assays, 50 ml cultures were grown at 30°C to stationary phase in YMB. Cells were harvested and washed in 0.85% NaCl solution before resuspension in 50 ml 0.85%

NaCl. PHB was extracted from a 2 ml fraction of this suspension and the remaining 48 ml was used for cell dry weight determination by incubation of the pellet at 60°C until the pellet was dry and no further loss in mass was recorded. PHB content was measured by the method of Law and Slepecky [47] and expressed as a percentage of total cell dry weight. All glassware was washed in hot chloroform and ADP ribosylation factor rinsed in ethanol before use, to eliminate plasticizers. A standard curve was constructed by dissolving known quantities of PHB (Sigma) in hot chloroform to a final volume of 1 ml. The chloroform was allowed to evaporate before addition of 10 ml of H2SO4 and PHB was processed as described elsewhere [47]. Carbon starvation assay Saturated TY cultures were washed twice to remove traces of nutrients, and were subcultured 1:50 into carbon-free M9 medium. These cultures were incubated at 30°C, shaking at 180 rpm. Viable cell counts were monitored at weekly intervals by plating on TY agar. Samples at t = 0 were each given a relative value of 1, and all subsequent samples are compared to this starting value. Values recorded are the means from triplicate cultures.

Biophys J 84(4):2508–2516PubMed Croce R, Muller

MG, Caffarri S, Bassi R, Holzwarth AR (2003b) Energy transfer pathways in the minor antenna complex CP29 of photosystem II: a femtosecond study of carotenoid to chlorophyll transfer on mutant and WT complexes. Biophys J 84(4):2517–2532PubMed Daum B, Nicastro D, Austin J II, McIntosh JR, Kuhlbrandt W (2010) Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea. Plant Cell 22(4):1299–1312PubMed de Bianchi S, Dall’Osto L, Tognon G, Morosinotto T, Bassi R (2008) Minor antenna proteins CP24 and CP26 affect the interactions between photosystem II subunits and the electron transport rate in grana membranes of arabidopsis. Plant Cell 20(4):1012–1028PubMed Dekker JP, Boekema EJ (2005) Supramolecular organization of thylakoid membrane proteins in green plants. Biochim Biophys Acta 1706:12–39PubMed Dunahay TG, RNA Synthesis inhibitor Staehelin LA, Seibert M, Ogilvie PD, Berg SP (1984) Structural, biochemical and biophysical characterization JNK signaling inhibitors of four oxygen-evolving photosystem II preparations

from spinach. Biochim Biophys Acta 764:179–193 Durrant JR, Hastings G, Joseph DM, Barber J, Porter G, Klug DR (1992) Subpicosecond equilibration of excitation energy in isolated photosystem II reaction centers. Proc Natl Acad Sci USA 89:11632–11636PubMed Engelmann ECM, Zucchelli G, Garlaschi FM, Casazza AP, Jennings RC (2005) The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid photosystem II. Biochim Biophys Acta 1706(3):276–286PubMed Georgakopoulou S, van der Zwan G, Bassi R, van Grondelle R, van Amerongen H, Croce R (2007) Understanding the changes in the circular dichroism

of light harvesting for complex II upon varying its pigment composition and organization. Biochemistry 46(16):4745–4754PubMed Germano M, Gradinaru CC, Shkuropatov AY, van Stokkum IH, Shuvalov VA, Dekker JP, van Grondelle R, van Gorkom HJ (2004) Energy and electron transfer in photosystem II reaction centers with modified pheophytin composition. Biophys J 86(3):1664–1672PubMed Goral TK, Johnson MP, Brain APR, Kirchhoff H, Ruban AV, Mullineaux CW (2010) Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation. Plant J 62(6):948–959PubMed Gradinaru CC, Pascal AA, van Mourik F, Robert B, Horton P, van Grondelle R, Van Amerongen H (1998) Ultrafast evolution of the excited states in the chlorophyll a/b complex CP29 from green plants studied by energy-selective pump- probe spectroscopy. Biochemistry 37:1143–1149PubMed Gradinaru CC, van Stokkum IHM, Pascal AA, van Grondelle R, Van Amerongen H (2000) Identifying the pathways of energy transfer between carotenoids and chlorophylls in LHCII and CP29. A multicolor, femtosecond pump-probe study.

2004; Su et al. 2006; Jiang et al. 2008; Zhou et al. 2010; Luo et al. 2013a). The limestone-dominated regions in Southwestern China are undergoing rapid changes due to the central government’s, ‘‘Great Western Development’’ plan (Zhou and Grumbine 2011). Under population and development pressures, severe limestone desertification has occurred on more than half the total limestone areas in China (Jiang et al. 2008).

Environmental degradation in these regions has made sustainable Selleck Liproxstatin-1 development and poverty alleviation more difficult. Many Dendrobium species, including D. catenatum, can also be grown, though may not be the optimal condition, on bare limestone rocks, so its cultivation can help to alleviate rock desertification. Social benefits Growing, tending and harvesting economic forests are labor intensive. This can be difficult for people in Yachang where a large proportion click here of young laborers have migrated to coastal regions to seek

better incomes. Elders, women and children remain in the villages. Similarly, the industrial scale artificial cultivation operations described above, which demand very large initial investments (Table 1) and somewhat complex management, exclude the participation of villagers with limited education and financial means, other than perhaps being employed as cheap labor. The proposed restoration-friendly orchid cultivation, with proper training and appropriate small loans, can be adopted by the marginalized populations of older and female rural residents in orchid hotspots because it

requires non-intensive labor and smaller initial investments than shade house operations (Table 1). As mentioned above, these medicinal orchids command a high market value and can be harvested non-destructively for up to a decade or more in some species, allowing rural farmers to gain financial independence. Potential pitfalls and possible ways to overcome them There are three major potential pitfalls that may prevent the realization of the intended benefits of restoration-friendly cultivation. Firstly, seedlings of Oxaprozin inappropriate genetic provenance are used such that species level genetic diversity is reduced or location adaption is lost or broken (Vallee et al. 2004; McKay et al. 2005). As a general guideline we recommend that local sources should be used to preserve and restore possible local adaptations, as has been practiced at several locations where restoration-friendly cultivation has started (unpublished data). This is especially important for species with relatively wide natural distributions, such as D. catenatum, which are found in China and Japan, from the warm temperate region such as Zhejiang province to the subtropical Yunnan, Guizhou, Guangxi and Guangdong provinces. Population genetic studies revealed significant differences among populations across D. catenatum’s distribution range (Ding et al. 2008).

WM, JO, AM-S have made substantial

contributions to patients sample collection. IM has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, drafting the manuscript and revising it critically for important intellectual content. He has also given final approval of the version to be published.”
“Background Pituitary adenomas are common lesions and represent 20% of all primary brain tumors[1, 2]. The epidemiological studies PI3K inhibitor have demonstrated that nearly 20% of the general population harbor pituitary adenomas[3, 4]. Pituitary adenomas are broadly classified into two groups[5]. In the first category are those that secrete excess amounts of normal pituitary hormones and present with a variety of clinical syndromes depending on the types of hormones secreted. Meanwhile, some macroadenoma may present with pressure symptoms, often increase in size if untreated, and in some rare cases they may cause symptoms related to mass

effect in which the optic nerves and chiasm are compressed[6, 7]. The second category of pituitary adenomas is nonfunctioning adenomas that do not secrete any known biologically active pituitary hormones. Patients can also suffer hypopituitarism secondary LY294002 mouse to compression of the normal functioning pituitary gland[8]. In the treatment of pituitary adenomas the goal is to remove the tumor mass or arrest further growth and when present normalize hormonal hypersecretion. Transsphenoidal surgery is established as one of the most reliable treatment modalities. This modern microsurgical technique can reduce tumor mass to protect surrounding structures from potential compression, and achieve the endocrinological cure of the symptoms caused by hormone secreting tumors. Long term tumor control rates after transsphenoidal excision alone vary from 50 to 80%[9]. However, in some cases, many patients are already in poor physical condition caused by extended production of the excess pituitary hormones, and general anesthesia itself sometimes brings a certain risk for them. Also, they

often show invasion to surrounding structures including cavernous sinus. And for these types of pituitary adenomas, incomplete tumor resection or recurrence as a result of tumor invasion into ID-8 surrounding structures is quite common[10]. In recent years, gamma knife radiosurgery(GKRS) has emerged as an important treatment modality in the management of secretory pituitary adenomas with its high efficacy. Radiosurgical treatment may deliver a high dose to the adenomas with high accuracy and may not influence the nearby neural structures to induce neurological defect[11]. Recently, more and more reports have detailed treatment results for secretory pituitary adenomas with GKRS, and there have been a number of reports of GKRS as a primary treatment for secretory pituitary adenomas[12].

Proteins DnaK, CH60, and EF-Tu are among the most abundant cellular proteins found in bacteria, including those possessing no flagellum. It is unlikely that these proteins would interact with FliX in a specific manner. Furthermore, when washing the sepharose bead complexes with phosphate buffer containing NaCl ranging from 0.3 to 2.65 M, these three proteins were readily released to the washing buffer throughout the salt gradient, whereas no FlbD or FliX protein could be washed off even with the highest salt strength

used. The co-occurrence of FliX and FlbD in the sepharose bead complexes demonstrates that FlbD indeed directly interacts with FliX inside of Caulobacter cells, and that the affinity between the two proteins is remarkably high. RAD001 mw We did not observe any other major specific component of the FlbD-FliX complex, although we cannot rule out the possibility that there might be transiently associated proteins, which are not detectable by the method described here. Figure 1 Proteins bound to the sepharose beads coated with histidine-tagged FliX.

Purified FliX-His was conjugated to sepharose beads prior to incubation with cell lysis of LS107. The bead complexes were boiled with the sample buffer and were subject to SDS-PAGE analysis. The identities of the five major bands were determined by mass spectrometry. Interaction between FlbD and FliX is required for stabilizing each other in Pifithrin-�� chemical structure vivo The finding that FlbD and FliX form high affinity in vivo complexes motivated us to examine whether the two proteins depend on each other for existence. We assayed the half-life of each protein in

a wild-type Caulobacter strain (LS107), a strain bearing a deletion in fliX (JG1172), and a strain having a Tn5 insertion in flbD (SC1032). Chloramphenicol was added to cell cultures at mid-log phase to inhibit protein synthesis, and the protein contents of FlbD and FliX were analyzed periodically. In strain LS107, both FlbD and FliX were stable; 2-hydroxyphytanoyl-CoA lyase neither exhibited significant reduction in concentration following 45 min of exposure to chloramphenicol (Figure 2). In contrast, after 45 min, less than 40% of FlbD remained in strain JG1172. Likewise, a similar decrease in FliX level was evident in SC1032 cells. These results indicate that FlbD has a reduction in stability in the absence of FliX, and vice versa. Figure 2 Stability assays of FliX and FlbD. Samples were periodically removed from cell cultures after the addition of chloramphenicol. Cell pellets were analyzed by SDS-PAGE followed by immunoblotting using anti-FlbD (upper panels) and anti-FliX (lower panels) antibodies. Site-directed mutagenesis of FliX To learn more about the interaction between FliX and FlbD, we performed site-directed mutagenesis with fliX and investigated the effects of mutations on FlbD activity. Both FlbD and FliX homologs are present in dozens of α-proteobacteria species that possess polar flagella.

Neutralization of clostridial or streptococcal circulating toxins by the use of intravenous immune globulin has shown promising results but there are no data to support a strong recommendation for its regular use in patients with gas gangrene [20]. Adjunctive hyperbaric oxygen therapy has been suggested for patients with aggressive soft tissue infections and has been shown to increase survival in animal model and in humans but no prospective controlled trials have been contacted in humans so far. Better definition of necrotic tissue facilitating more precise debridement and its bacteriostatic effects on clostridia both in vivo and in vitro is the rationale for the use of hyperbaric oxygen therapy in

patients with gas gangrene Lenvatinib order [21, 22]. In most of the patients with limb preservation after see more gas gangrene, a residual function of the affected limb was present. In half of them functionality of the limb was characterized as normal. Patients with limited function of the preserved limb had generally longer duration of hospitalization. This might be at least in part because these patients, as our case, needed several

interventions following initial surgery until the limb re-attained as much as possible of its functionality. This prolongation of hospital stay is well balanced by the invaluable benefit of functional limb salvage. Whether the preservation of the limb makes postoperative recovery more severe is essentially the question whether amputation offers better control of the infection compared with adequate debridement. Again there is no evidence that amputation controls better the infection compared with adequate debridement. However, it is plausible that amputation may achieve margins that are wider and clearer

of infection if it is compared with an inadequate debridement in order to “”save”" the limb [15, 16]. In conclusion, physician and emergency medicine personnel should always maintain high index of suspicion for necrotizing infections in illicit drug users presenting with soft tissue infections. Early surgical debridement, antimicrobial treatment and intensive care monitoring may lead to survival with limb salvage in carefully selected patients. Consent Written informed consent was obtained from the patient for publication Carnitine dehydrogenase of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. References 1. Bryant AE, Stevens DL: Clostridial myonecrosis: new insights in pathogenesis and management. Curr Infect Dis Rep 2010,12(5):383–91.PubMedCrossRef 2. Bryan C: Gangrene bug killed 35 heroin users. WJM 2000, 173:82–83.CrossRef 3. Stevens : Clostridial Myonecrosis and other Clostridial Diseases. In Cecil Textbook of Medicine. Volume chapter 334. 21st edition. Edited by: L Goldman, JC Bennett. Philadelphia: WB Saunders; 2000:1668–1673. 4.