The level of lactate in amniotic fluid may provide useful clinical information when assessing progress of a woman’s labour and if so, a rapid, reliable method to assess amniotic fluid lactate is required in order to be clinically relevant. However, measuring lactate levels in amniotic fluid, using portable, handheld lactate meters may be less accurate than reference laboratory instruments designed to measure lactate levels in aqueous solutions. Prior to conducting a large study, we assessed recruitment, consent and sampling procedures, and the accuracy of a handheld lactate meter to measure lactate in amniotic fluid. We compared amniotic fluid lactate results obtained using the hand held Lactate Pro (Arkray) to results obtained using reference laboratory methods ABX Pentra 400 (Horiba).We recruited 35 nulliparous women during their antenatal hospital visits and tested amniotic fluid samples collected from 20 labouring women. The handheld Lactate Pro meter was found accurate from 9–20 mmol/L with a Passing & Bablok regression of y = 0.18 + 0.97x (95% CI 0.76–1.45). Amniotic fluid lactate results remained reliable in the presence of potential contaminants commonly encountered during labour; obstetric lubricant, blood and meconium.The measurement of amniotic fluid lactate using the Lactate Pro meter was reliable compared to reference laboratory methods for measuring lactate levels in amniotic fluid. The pilot study enabled the refinement of information, recruitment, consenting and sampling procedures prior to commencing a large cohort study. Lack of progress in labour (dystocia) is one of the most commonly occurring problems requiring intervention during labour. In manyCurrently dystocia is diagnosed clinically by observing factors such as slowing of cervical dilation, slowing of descent and rotation of the fetal head, and changes in strength, duration and frequency of uterine contractions. In the Measurement of lactate in amniotic fluid of labouring women may be a surrogate biochemical marker for labour dystocia-9 based If amniotic fluid lactate is relevant in the clinical management of dystocia, it is essential that a rapid and accurate test for amniotic fluid lactate is available. Although measurement of lactate using laboratory based spectrophotometric and fluorometric assays is possible this is not clinically useful in diagnosing dystocia due to time constraints in the clinical setting of active labour. The Lactate Pro is a small, portable hand held device used to process blood samples at the point of care which is relatively inexpensive. It has the potential to be used in rural and remote settings where laboratory technology may be unavailable due to costs and /or operator skill and has been found to be easy to operate and accurate,14. ManuThis report describes an assessment of a handheld meter (the Lactate Pro) for measuring amniotic fluid lactate, prior to conducting a large prospective study. The primary objectives of the pilot amniotic fluid lactate study were to assess the consenting processes for recruiting women to amniotic fluid lactate research and evaluate methods of amniotic fluid collection. We also aimed to determine the clinical aspects of relevant confounding factors in the measurement of lactate in amniotic fluid by contaminants, and determine the concordance between bedside measurement of lactate using the Lactate pro hand held meter and reference laboratory biochemical measurements.Women who booked to give birth at a tertiary teaching hospital between October 2011 and January 2012 were considered for inclusion. Nulliparous women with a live, singleton cephalic fetus and a spontaneous onset or induction of labour at 37–42 weeks gestation were eligible to participate. A final decision regarding inclusion was made when the woman was in labour to ensure the fetus remained in a cephalic presentation and was not compromised, as evidenced by routine admission cardiotocography. Women were excluded if they had a known fetal anomaly or a recognised contraindication to vaginal birth at the onset of labour. Three women with a singleton fetus with polyhydramnios undergoing elective amniocentesis in the third trimester of pregnancy were also recruited, in order to collect amniotic fluid under sterile conditions. The Study Protocol was approved by New South Wales Northern Network Human Research Ethics Committee 11/140 through the National Ethics Application Form Submission AU/19E1A08.Recruitment took place at antenatal visits at or after 36 weeks gestation at the Outpatient Department of a public tertiary level teaching hospital. When a woman who had consented to participate in the study arrived at hospital in labour, or for an induction, the research midwife was notified by staff. The research midwife confirmed that all inclusion criteria and none of the exclusion criteria were present and the woman was enrolled into the study, after giving verbal confirmation of her consent to participate.Amniotic fluid samples were collected from labouring women at the following defined time points after arrival at the hospital. On admission if membranes were already ruptured; at spontaneous rupture of membranes during labour; at the time of artificial rupture of membranes; at routine vaginal assessment of labour progress ; prior to augmentation of labour; and at the time of birth. Amniotic fluid was not collected if a woman had not ruptured her membranes.Non-invasive methods for collecting amniotic fluid were trialled by the research midwife. It became apparent that amniotic fluid was most easily collected at the time of vaginal examination as the fluid pooled in the cupped, gloved, palm of the examining clinician. Results were obtained either by touching the primed Lactate Pro test strip directly to the pooling fluid during the examination or, by collecting some of the pooling fluid in a needle-less syringe. Alternatively, where women had copious flowing amniotic fluid, lactate was measured by applying the primed test strip directly to a freshly applied perineal pad. In collections where a syringe was used, if sufficient fluid was available, after testing with the Lactate Pro, a portion of the sample was sent to the laboratory for reference testing. In contrast, studies by Wiberg Itzel et al. (2008) obtained amniotic fluid by inserting a silicon catheter into the labouring uterus, via the vagina. We did not adopt or test this method of obtaining amniotic fluid as we aim to determine if amniotic fluid lactate, obtained at the time of routine examination by non-invasive methods, is a potential biomarker for clinicians to assess progress of labour.Amniotic fluid specimens were tested at the time of collection using the Lactate Pro analyser according to manufacturer’s instructions. Briefly, a test strip was inserted into the Lactate Pro, ensuring correct orientation. The device, with test strip in situ was dipped in a vertical position into the amniotic fluid sample. Results, available within 1 minute were recorded by the research midwife. In most cases, clinical staff remained blinded to the result, except for teaching purposes where the research midwife shared the results with clinical staff. Amniotic fluid lactate results were never considered in clinical decision making.Amniotic fluid lactate results were compared to validated reference laboratory techniques for measuring lactate in aqueous fluids, the ABX Pentra 400  using the following methods.g for 2 minutes. The supernatant was removed and stored at −20°C until analysis by the ABX Pentra 400, courtesy of the Clinical Chemistry Laboratory at South Eastern Area Laboratory Services, Randwick.At the time of point of care testing with the Lactate Pro, where sufficient amniotic fluid sample was available, amniotic fluid was collected via syringe and inserted into sodium fluoride preservative tubes (Becton Dickinson). Within one hour of collection amniotic fluid samples were centrifuged at 8000×In order to determine if the sodium fluoride preservative used in sample preparation for the AbX Pentra 400 affected lactate levels, a minimum of 0.6 mL of saline was added to the sodium fluoride tubes, then spiked with lactate substrate (62 mmol/L) from the LDH12 kit  to yield estimated low, high and midrange values. The selected values were based on previous lactate studies,15,16. SSamples of amniotic fluid collected under sterile conditions were spiked with lactate and levels measured using both the Lactate Pro and AbX Pentra 400. Baseline biological lactate concentration was determined by testing the amniotic fluid samples with the Lactate Pro meter. A minimum of 0.6 mL of amniotic fluid was then spiked with lactate to yield a predetermined lactate concentration  above baseline. Each concentration was prepared and tested in triplicate. At the time of spiking, samples were tested using the Lactate Pro meter, and the sample centrifuged and stored until laboratory analysis by the AbX Pentra, as described above  which may confound amniotic fluid lactate results, samples of amniotic fluid were prepared containing 1%, 5%, 10% and 20% [weight per volume (w/v) or volume per volume v/v)] of the potential contaminants. The samples were tested for baseline lactate concentration and prepared in triplicate. Glove lubricant was not tested at 20% (v/v) as the mixture was too viscous for analysis by the Lactate Pro Table .As unintended delays may occur between the collection and testing of samples in the clinical environment, we performed a time series to determine the effect of time delays (on lactate concentration). When sufficient quantity of sample was available, after ascertaining baseline amniotic fluid lactate at point of care, a drop of the sample remaining in the collecting syringe was re-tested after 5, 15 and 30 minutes to identify possible degradation of lactate. Lactate stability studies were extended to 24 hours after collection of two amniotic fluid samples collected under sterile conditions (one spiked with 10 mmol/L lactate and one sample unspiked); the spiked, non-centrifuged amniotic fluid samples were stored overnight at 4°C, then tested after 1, 5, 15 and 30 minutes at room temperature.The lactate concentrations in saline samples with and without fluoride preservative were almost identical Table . While aThere was close concordance between results of lactate levels on the two analysis systems . Minor variation was apparent; mostly at high lactate concentration ≥ 10 mmol/L), which are at the upper end of the Lactate Pro analysis range as the Lactate Pro has a narrower analysis range than the AbX Pentra Table . A Passi0 mmol/L,Amniotic fluid lactate increased in the presence of whole blood (which has its own lactate load) and decreased in the presence of other contaminants as the ratio of contaminant to amniotic fluid increased Table . In the In total, 35 women were recruited to the study and 90 attempts were made to collect amniotic fluid from 20 labouring women who gave birth during the study timeframe. Amniotic fluid was not able to be collected from 15 women as nine arrived in hospital in advanced labour, three had reduced liquor volume diagnosed by ultrasound, two had not given birth at the time of data analysis and one woman gave birth with intact membranes. A lactate result was measured in 65/90 attempts to collect amniotic fluid. Mucous samples appeared to prevent the capillary action of the Lactate Pro test strip and no results were obtained from mucousy samples. Nor were results obtained at the time of artificial rupture of membranes performed to induce labour on 10 occasions; despite the procedure, sufficient amniotic fluid for sampling was not detectable at that time. Several other factors impacted on successful readings for example, one ‘Lo’ result was obtained from a sample collected directly after a woman exited the bath (presumably bathwater contamination). Two ‘Lo’ results were attributable to operator error (failure to prime the test strip by inserting it into the meter prior to collecting the sample). Outlier ‘Lo’ results were removed from the analysis.Results show the handheld Lactate Pro meter to be reliable measuring lactate in amniotic fluid samples collected and tested in controlled conditions. A Passing & Bablok regression line applied to the lactate measures shows the Lactate Pro meter accurate from 9–20 mmol/ when measuring lactate in amniotic fluid collected under sterile conditions, compared to results from the reference analyser. A Passing Bablok regression was chosen due to the small sample size, as it allows measurement error (imprecision) in both the X and Y variables, does not assume measurement error is normally distributed, and is robust against outliers. The result compares favourably with correlation reported by Tanner et al. comparin.Our results support previous findings by others who have assessed the Lactate Pro in amniotic fluid lactate research,9,15 altAlthough the Amniotic Fluid Lactate™ monitoring system is now available on the market prior to conducting clinical trials to demonstrate the efficacy of amniotic fluid lactate in obstetric clinical decision making, we remaThis small pilot study establishes proof of principle that the Lactate Pro meter, a commercially available product validated to measure lactate in whole blood, is a reliable measure of lactate in amniotic fluid, in samples collected from women during labour after spontaneous or artificial rupture of membranes. Moreover, we found women interested and willing to participate in amniotic fluid lactate research, although it will be necessary to reduce selection and sampling bias in future amniotic fluid lactate research studies, in order to achieve valid results about the association of lactate results with labour dystocia.All authors declare that they have no competing interests.All the authors had full access to all of the data  in the study. BH and SKT initiated the study, participated in its coordination, drafted the manuscript and gave final approval of the version to be published. BH, WR & JI designed the study protocol, BH obtained ethical approval, recruited women, collected specimens and JI & BH performed sample validation studies and drafted the manuscript. MM conducted reference laboratory tests. JI and MBT performed statistical analysis. All authors commented on and approved the final manuscript.


Many arachnids possess adhesive pads on their feet that help them climb smooth surfaces and capture prey. Spider and gecko adhesives have converged on a branched, hairy structure, which theoretically allows them to adhere solely by dry (solid-solid) intermolecular interactions. Indeed, the consensus in the literature is that spiders and their smooth-padded relatives, the solifugids, adhere without the aid of a secretion.Grammostola rosea) by attaching them to a bending beam with a known spring constant and filming the resulting deflection. Individual spider setae showed a lower static friction at rest (26%±2.8 SE of the peak friction) than single gecko setae . This may be explained by the fact that spider setae continued to release fluid after isolation from the animal, lubricating the contact zone.We investigated the adhesive contact zone of living spiders, solifugids and mites using interference reflection microscopy, which allows the detection of thin liquid films. Like insects, all the arachnids we studied left behind hydrophobic fluid footprints on glass . Fluid was not always secreted continuously, suggesting that pads can function in both wet and dry modes. We measured the attachment forces of single adhesive setae from tarantulas (This finding implies that tarsal secretions occur within all major groups of terrestrial arthropods with adhesive pads. The presence of liquid in an adhesive contact zone has important consequences for attachment performance, improving adhesion to rough surfaces and introducing rate-dependent effects. Our results leave geckos and anoles as the only known representatives of truly dry adhesive pads in nature. Engineers seeking biological inspiration for synthetic adhesives should consider whether model species with fluid secretions are appropriate to their design goals. Climbing animals use a vast array of attachment strategies to scale vertical and inverted surfaces Arachnids have attracted little attention from researchers in the field of biological adhesion, despite representing a wide diversity of adhesive morphologies. Some mites have hairy, or “fibrillar”, adhesive pads Here we investigate the adhesive feet of selected arachnids  for the presence of fluid secretions. We used two independent approaches. First, we imaged the footpad-substrate interface directly with interference reflection microscopy (IRM). This technique allows the visualization and characterization of very thin fluid films through transparent substrates such as glass Grammostola rosea  generated on average a higher shear force (290 µN±30 SE) than setae from the gecko . Spider setae had more and larger spatulae  than geckos , but even when normalized by potential contact area (number of spatulae * spatula area), the tarantula setae generated approximately twice as much force.Fluid had a significant effect on the attachment performance of fibrillar adhesives. Individual setae from the tarantula  and we observed that they continued to release fluid even after being isolated from the animal. In this species, fluid may flow through the setal stalk, but more work is needed to establish how the fluid ultimately wets individual spatulae.The properties of arachnid fluid secretions parallel those of insect footpad secretions, where a persistent hydrophobic fluid is left behind on glass Gromphadorholaelaps mite and the glass cover slip substrate  setae generated more shear and adhesive force per potential contact area than Thecadactylus (gecko) setae, and that individual gecko setae maintained higher levels of static friction, whereas spider setae slid relatively easily.Comparative measurements of adhesive forces from fluid-secreting spider setae and dry gecko setae demonstrate that there are consequences for attachment performance when a fluid is introduced into the footpad-substrate interface. We found that individual Grammostola foot adhering to glass showed a significant amount of fluid developing over time as the animal attempts to remain attached, upside-down, to the cover slip substrate , and the silk did not appear to sustain a tensile load . We consider it unlikely that these silk-like secretions contribute significantly to attachment force. It remains an open and interesting question how the evolution and development of these setae might be related to abdominal spinnerets.Previous investigators observed a silk-like material secreted from the tarsi of the theraphosid raphosid . We did Arachnids remain a large, and largely understudied, group of organisms from which we can draw biological and technical insights into the general principles of adhesion. From an evolutionary standpoint it is clear that tarsal secretions are widespread among arthropods; such secretions can have important ecological consequences, for instance in chemical communication Grammostola rosea spiders were obtained from a pet supplier . Salticus scenicus spiders and Balaustium murorum mites were collected in Cambridge, UK. Gromphadorholaelaps schaeferi mites were collected from live Madagascar hissing roaches kept in our laboratory in the Dept. of Zoology, University of Cambridge, UK. Gluvia dorsalis solifugids were collected in Évora, Portugal. Cupiennius salei spiders were provided by Dr. Friedrich Barth from his laboratory colony in the Dept. of Neurobiology, University of Vienna, Austria. All spiders and solifugids were individually housed and fed on a diet of crickets and water. Mites were re-released after the experiments ended.Thecadactylus rapicauda geckos were obtained from California Zoological Supply  and cared for by the Office of Laboratory Animal Care at the University of California, Berkeley, where they were fed on a diet of crickets and water.Thecadactylus were conducted in the Dept. of Integrative Biology at the University of California Berkeley, in accordance with Animal Use Protocol R137 .All experiments involving arachnids were performed in the Zoology Dept. at the University of Cambridge, in accordance with the Animals (Scientific Procedures) Act of 1986. Experiments involving Grammostola rosea, Salticidae: Salticus scenicus), one mite (Laelapidae: Gromphadorholaelaps schaeferi), and one solifugid  attaching to the underside of a glass cover slip with an upright microscope. We filmed movies of the contact using a high-speed HotShot PCI 1280 B/W digital video camera , and a 10-bit B/W QICAM digital camera . In the event that animals were not able or motivated to remain attached while inverted to the coverslip for the duration of imaging, we allowed them to climb across an inclined coverslip for a sustained period of time before imaging the fluid left behind.We viewed three live specimens each from two species of spider (Theraphosidae: max−Imin)/(Imax+Imin)] were compared with the contrasts from similar-sized droplets of calibration fluids (water-glycerol mixtures and immersion oil) with known refractive indices. As the interference patterns of water-glycerol droplets with steeper gradients are damped by the optical resolution of the microscope Interference reflection microscopy (IRM) allowed us to measure the refractive index, contact angle and volume of fluid left behind on the glass. Fluid droplets as deposited by the animals were too small to conduct IRM measurements, so we aggregated multiple droplets from each footprint by dragging a fine glass rod with spherical tip across the surface . Images To determine whether the footprint secretions were hydrophobic or hydrophilic, we deposited small water droplets (5–30 µm in diameter) onto the glass cover slip using an ultrasonic humidifier  and observed whether the footprint droplets dissolved into them.Grammostola rosea  were harvested from living animals and mounted to insect pins using 5-minute epoxy . Each pin was clamped into a pin vice and fixed to a three-dimensional DC motor stage , which drove the seta through pre-programmed shearing motions 0.5–2 mm in amplitude, at set velocities (0.5–4 mm/s). Setae were dragged across a smooth glass substrate glued to the tip of a 58.9 mm length of tungsten wire with a 0.1 mm radius. The spring constant of this bending beam was determined to be 0.46 N/m. The movement of the beam was captured using a Redlake PCI 1000 B/W high-speed video camera , and digitized using ProAnalyst Lite  to yield peak shear and adhesive force, remaining shear and adhesive force after a one-second pause, and the velocity of the seta.Individual setae from the claw tufts and tarsi of Thecadactylus rapicauda, using a smooth silicon substrate fixed to a 46 mm steel wire with radius 0.06 mm and spring constant of 0.079 N/m, at velocities between 0.4–2.8 mm/s (0.5–2 mm sliding distance). Details can be found in An analogous method was used to measure single seta forces in the gecko Video S1Cupiennius salei seta sliding across glass. As the seta is dragged across glass, it deposits fluid. More fluid appears to be deposited during fast movements than during slow ones.(MOV)Click here for additional data file.Video S2Grammostola rosea fluid before and after exposure to water vapor. The secretion does not dissolve in water, indicating its hydrophobic nature.(MOV)Click here for additional data file.Video S3Evidence for a biphasic fluid in arachnids. Hydrophilic droplets underneath the foot of the mite Gromphadorholaelaps schaeferi evaporate quickly once they reach the edge of the pad, much like the volatile hydrophilic component of biphasic adhesive secretions seen in insects.(MOV)Click here for additional data file.Video S4Grammostola setae at various stages of wetting. Close up of Grammostola rosea setae as the animal clings to the underside of the glass. Dark areas are continuous layers of trapped fluid. Camera initially shows distal setae completely covered in fluid, then scans proximally along claw tuft to show setae where less fluid has accumulated and individual spatulae are still visible.(MOV)Click here for additional data file.Video S5Time lapse video of a Grammostola rosea foot sliding across glass. Adhesive setae leave behind clusters of minute fluid droplets, while other setae secrete silk.(MOV)Click here for additional data file.


A grant from the NIH that funded the last author's work for the article is incorrect. The grant number is: R01-EY19304.


A 46-year-old woman presented with a painless palpable mass in the left breast for two weeks. She had no nipple discharge and no familial history of breast carcinoma. Physical examination revealed a 4.5-cm circumscribed, movable mass in the left upper outer quadrant. The overlying skin was normal. The axillary lymph node was not enlarged.Mammograms revealed a 4.5 cm, well-circumscribed mass without calcification at 3 o’clock in the left breast . UltrasoFine-needle aspiration biopsy (FNAB) of the mass showed benign epithelial cells which could be either fibroadenoma or phyllodes tumour. The patient underwent a wide excision of the mass. She made an uneventful recovery, and a simple mastectomy was planned.At gross examination, the specimen contained a circumscribed mass measuring 4.5 cm in diameter with grayish-white trabeculated cut surface . MicroscPhyllodes tumour, previously described by Johannes Muller in 1838 as cystosarcoma phyllodes . PhyllodPreoperative diagnosis of phyllodes tumour with FNAB is controversial because fibroadenoma is frequently diagnosed due to the substantial cytologic overlap similar to our presented. Occasionally, false-positive diagnosis of carcinoma is also made ,5. MultiTreatment of phyllodes tumour requires complete removal of the tumour with wide margins if the tumour is small and a simple mastectomy may require if the tumour is large. Local recurrence occurs in approximately 20% of cases if the tumour is incompletely excised ,7. Routi


Sir,Mansonella perstans and apologize for the inadvertent error. The only reported case of pleural effusion due to M. perstans was of Kahn in 1983.[This is in reference to the letter by an esteemed reader. I appreciate the interest shown by him regarding our case. We regret that the case reported by him was wrongly quoted in our article as caused by We did not consider pleural biopsy as the diagnosis was firmly established by repeated demonstration of microfilariae in pleural fluid, and the patient denied consent for pleural biopsy. The literature shows that pleural biopsy does not always demonstrate microfilariae.3The method used for demonstration of microfilariae in pleural fluid was hematoxylin and eosin stain on smears prepared from the sediment of pleural fluid after centrifuging at 2000 rpm for 15 min. Pleural fluid samples were collected at night time or in early morning.


We analyze the laminar motion of incompressible fluids in self-acting thrust bearings with spiral grooves with inner or external pumping. The purpose of the study is to find some mathematical relations useful to approach the theoretical functionality of these bearings having magnetic controllable fluids as incompressible fluids, in the presence of a controllable magnetic field. This theoretical study approaches the permanent motion regime. To validate the theoretical results, we compare them to some experimental results presented in previous papers. The laminar motion of incompressible fluids in bearings is described by the fundamental equations of fluid dynamics. We developed and particularized these equations by taking into consideration the geometrical and functional characteristics of these hydrodynamic bearings. Through the integration of the differential equation, we determined the pressure and speed distributions in bearings with length in the “pumping” direction. These pressure and speed distributions offer important information, both quantitative (concerning the bearing performances) and qualitative , for the laminar and permanent motion regime. Incompressible fluid motion in “self-acting thrust bearings with spiral grooves” (SATBESPIG) is a complex motion in thin layers –5 boundeSome authors have developed comparative theoretical and experimental studies concerning different aspects of spiral groove bearings functioning in the laminar regime, with equations for their functioning using magnetic fluids, cavitation phenomenon, and the geometry of the bearings with spiral/axial multiples grooves.In , 9, the The diversification and optimization of the geometry of the bearings with spiral or axial multiples of grooves were approached in –12. In , the autWe consider the theoretical results as a first step in our intention to approach the functionality of the hydrodynamic bearings in general and of the thrust bearings with spiral grooves in particular in the stationary/nonstationary turbulent regime. The viscous compressible/incompressible fluid motion can be approached by taking into consideration the thermo-viscous-inertial phenomena ensemble that exists in bearings.ψ from Figures r) without taking into consideration the effects of the inertial forces.The SATBESPIG Figures  and 2 ara1 is the width of the spiral channel, measured on the circle arc contour; (ii) a2 is the width of the spiral threshold, measured on the circle arc contour; (iii) Δθ is the center angle corresponding to a channel—threshold pair; (iv) β0 is the generator angle of the logarithmic spiral, which describes the form of the cannels; (v) β1 and β2 are the input and output angles, respectively, in and from the channel ; (vi) ω1 is the bearing angular rotation speed; (vii) ri and re are the inner and external radius of the bearing, respectively; (viii) rc is the radius marking the zone of the spiral channels; (ix) r is the current radius of the bearing; (x) h1 is the lubrication film height over the bearing channels; (xi) h2 is the lubrication film height over the bearing thresholds; (xii) W is the bearing load (the weight); and (xiii) by definition, α = a1/(a1 + a2).In Figures θ = 2π/np, where np is the number of pairs channel—thresholds, a1 = αrΔθ, a2 = (1 − α)rΔθ, a = a1 + a2 = rΔθ, and ω2 = 0 (the grooved surface is fixed).With the geometrical dimensions in y, r, θ) and local  coordinate systems used for the motion study.To study the mathematical model, the coordinate systems and the control volume must be determined so as to define the speed distributions and the fluid mass flow rate , 5, 15. Next, we start from the fact that the incompressible fluid laminar motion, between the two quasiparallel surfaces of the bearing, is described by the following speed profiles:u(y) is the fluid speed component in the x (or θ) direction, (ii) vn(y) is the fluid speed component in the normal direction y, (iii) w(y) is the fluid speed component in the z (or r) direction, (iv) vψ(y) is the fluid speed component in the ψ curb direction, (v) vξ(y) is the fluid speed component in the ξ direction, (vi) p is the pressure in the fluid, (vii) h is the height of the lubrication film, and (viii) η is the fluid dynamical viscosity.In –1e), , The speed distributions, given by –1e), ar, ar1e), ψ .Using relation , the flu Figures  and 5, fentation , 5:(3)ℳθ is infinitely small, meaning that there exist an infinite number of spiral channels. Given the relations ,,θ is infψ direction can be expressed as follows:τ is the time, and the fluid mass m, contained in the control volume Vol, is given by the relationThe fluid mass conservation in the elations  and 7),,ψ directfollows:∂∂τ(m)+∂∂For the permanent motion regime,  becomesUsing nondimensional variables , 14, 15,For the stationary motion regime,  becomes ψ direction, the fluid film h varies rapidly from h1 to h2, at the frontier r≅rc  the flow regime through bearing , (ii) the value of the rapport h1/h2, (iii) the fact that we take (or not) into consideration the inertial forces, and (iv) the fluid type [In the  Figures  and 5. Tuid type , 17.psupp. is the supply pressure of the lubrication fluid and patm. is the atmospheric pressure), we obtain the mathematical relations for the pressure distributions in the SATBESPIG:h = h2. Considerh = h1.Integrating differential equation  and obseTBESPIG:p(ψ) ≅ph2Considerp(ψ) ≅psuh = h2. Considerh = h1.In a similar way, for the SATBESPIG with exterior pumping , we obtap(ψ)≅ph2+Considerp(ψ) ≅psuph1 and ph2. If we do not take into consideration the influence of the inertial forces, then the pressures ph1 and ph2 are equal. Thus, ph1 ≡ ph2, Δp = ph2 − ph1 = 0.In the relations –15), al, al15), ψ direction and the extreme pressures ph1 and ph2, we must analyze the liquid motion in the fluid film existing between the quasiparallel surfaces of the SATBESPIG [ph1 and ph2) exist on all the surfaces of the SATBESPIG in the ψ direction, but the maximum effect is concentrated in the zone of the radial step, r≅rc , al, al22), Qψ will be established using differential equation 28) are cequation  or (28),solution , 5.QψI and QψII algebraic solutions, for QψI and QψII using medium  values for the physical and geometrical dimensions [QψI < 0 and QψII > 0. Thus, the mathematical relation for the calculus of the fluid volumetric flow rate Qψ isQψII isThe numerical evaluation of mensions , 2 of (2quations  or 28)..QψI anThe established mathematical relations allow the numerical calculation of the pressure and speed distributions for several SATBESPIG with inner or external pumping in permanent and laminar regimes. For these numerical calculations we used two different computer programs: one for the SATBESPIG with inner pumping and the other for the SATBESPIG with external pumping.re = 90 mm, rc = 57.15 mm, ri = 45 mm, np = 10, α = 0.615, β0 = 17°, ρ = 905 kg/m3, η = 10−4 Pa·s, psupp. = 101325 N/m2, n = 900 rot/min, α0 = 6/5, ε = 2/15, γ = 1/5, δ = 1/10, β = 2/15, and the parameters h1 and h2 as written in the figures.In Figures rc = 77.85 mm.h1, h2, and n written in the figure.In h1, h2, n, ri, and re [In all our studies, the main constructive geometrical and functional parameters for the calculation of these bearings were  and re , 5.rc dimension. The First Variant is the SATBESPIG from rc = 57.15 mm), and the Second Variant has rc = 70.29 mm.n and for all the analyzed rapports h1/h2, the First Variant SATBESPIG with inner pumping had superior hydrodynamics performance compared to the Second Variant SATBESPIG. Thus, high pressures in bearings can be realized with an appropriate dimension of the bearing surface, especially the dimension of the grooved surface, meaning the parameters ri, rc, and re.For all the rotation per minute (r.p.m.) ranges ri, rc, and re can be realized by increasing the number of the rotation per minute n. The modification of the rapport h1/h2 has a small influence.Generally speaking, increasing other hydraulics performance of the SATBESPIG for the same dimensions r≅rc. The nondimensional pressures change, but not linearly with the channel length, from the input, where pimput≅psupp., up to the pressure ph1 and from the pressure ph2 to the output pressure, where poutput≅psupp..The nondimensional speeds increase (and decrease) from the bearing input to the bearing output, with a jump at the radial step p = ph1 − ph2 theoretically tends to zero when the rapport h1/h2 → 1 and thus when the radial step vanishes or when ph1≅ph2. The case ph1≅ph2 appears only when we do not take into consideration the inertial effects.The pressure jump Δp depends not only on the rapport h1/h2 but also on the value of the n and on the bearing geometry .The pressure jump Δri, re, h1/h2, n,…), the SATBESPIG with exterior pumping gives lower pressures than the bearing with inner pumping.For the same constructive geometrical and functional parameters . Some of the mathematical relations established above can be used to approach the theoretical functionality of the SATBESPIG having magnetic controllable fluids  as incompressible fluids, in the presence of a controllable magnetic field [The comparative analysis between the theoretical and experimental results shows good correlation, especially at low ic field –20.The analysis of the theoretical results allows some conclusions to be drawn concerning the laminar motion of incompressible fluids in some models of a SATBESPIG. We analyzed two variants of the SATBESPIG with inner pumping, which are called the First Variant and the Second Variant below. The difference between these bearings is the


Graphical results are presented and discussed quantitatively with respect to the influence of pertinent parameters, such as solid volume fraction of nanoparticles (φ), magnetic field parameter (Ha), buoyancy effect (Gr), Eckert number (Ec), suction/injection parameter (fw), Biot number (Bi), and slip parameter (β), on the dimensionless velocity, temperature, skin friction coefficient, and heat transfer rate.We examine the effect of magnetic field on boundary layer flow of an incompressible electrically conducting water-based nanofluids past a convectively heated vertical porous plate with Navier slip boundary condition. A suitable similarity transformation is employed to reduce the governing partial differential equations into nonlinear ordinary differential equations, which are solved numerically by employing fourth-order Runge-Kutta with a shooting technique. Three different water-based nanofluids containing copper (Cu), aluminium oxide (Al Magnetohydrodynamic (MHD) boundary layer flow of an electrically conducting viscous incompressible fluid with a convective surface boundary condition is frequently encountered in many industrial and technological applications such as extrusion of plastics in the manufacture of Rayon and Nylon, the cooling of reactors, purification of crude oil, textile industry, polymer technology, and metallurgy. As a result, the simultaneous occurrence of buoyancy and magnetic field forces on fluid flow has been investigated by many researchers –5. In th2O3, CuO, and TiO2), nitride ceramics , carbide ceramics , semiconductors, carbon nanotubes, and composite materials such as alloyed nanoparticles or nanoparticle core-polymer shell composites. It is well known that conventional heat transfer fluids, such as oil, water, and ethylene glycol, in general, have poor heat transfer properties compared to those of most solids. Nanofluids have enhanced thermophysical properties such as thermal conductivity, thermal diffusivity, viscosity, and convective heat transfer coefficients compared with those of base fluids like oil or water [On the other hand, with the advent of nanofluids, there has been wide usage of recently discovered smart fluid in many industrial and biomedical applications. Nanofluid concept is employed to designate a fluid in which nanometer-sized particles are suspended in conventional heat transfer base fluids to improve their thermal physical properties. Nanoparticles are made from various materials, such as metals , oxide ceramics  are the velocity components of the nanofluid in the x- and y-directions, respectively, T is the nanofluid temperature, U∞(x) = ax is the free stream velocity , T∞ is the free stream temperature, g is acceleration due to gravity, λ is the slip coefficient, μnf is dynamic viscosity of the nanofluid, ρnf is density of the nanofluid, knf is thermal conductivity of the nanofluid, σnf is electrical conductivity of the nanofluid, (ρcp)nf is heat capacity at constant pressure of the nanofluid, and βnf is volumetric expansion coefficient of the nanofluid which are defined as [φ is the nanoparticle volume fraction (φ = 0 correspond to a regular fluid), ρf and ρs are the densities of the base fluid and the nanoparticle, respectively, βf and βs are the thermal expansion coefficients of the base fluid and the nanoparticle, respectively, kf and ks are the thermal conductivities of the base fluid and the nanoparticles, respectively, (ρcp)f and (ρcp)s are the heat capacitance of the base fluid and the nanoparticle, respectively, and σs and σf are the electrical conductivities of the base fluid and the nanofluid, respectively.Assuming a Boussinesq incompressible fluid model, the continuity, momentum, and energy equations describing the flow can be written asfined as , 22(3)μη is the similarity variable and ψ is the stream function defined asη, fw is a constant with fw > 0 representing suction rate at the plate surface, fw < 0 corresponds to injection, fw = 0 shows an impermeable surface, λ = 0 represents highly lubricated surface, and λ = ∞ corresponds to a normal surface. The local Reynolds number (Rex), Grashof number (Gr), Hartmann number (Ha), Prandtl number (Pr), Eckert number (Ec), slip parameter (β), and Biot number (Bi), are defined asCf and the local Nusselt number Nu, which are expressed asτw is the skin friction and qw is the heat flux from the plate which are given byf′(η), θ(η), f′′(η), and θ′(η), respectively. In order to simplify the mathematical analysis of the problem, we introduce the following dimensionless variables:fined asu=∂ψ∂y,  given byτw=μnf∂u∂essed asCf=τwρfU∞oducing (f′′′+(1−φions aref(0)=fw, oducing (f′′′+(1−φions aref(0)=fw, φ = 0 is regular fluid.Physically realistic numerical values were assigned to the pertinent parameters in the system in order to gain an insight into the flow structure with respect to velocity, temperature, skin friction, and Nusselt's number. The results were presented graphically in Figures φ), Eckert number (Ec), Grashof number (Gr), and the suction/injection parameter (fw) causes an overshoot of the fluid velocity towards the plate surface hence decreasing both the momentum boundary layer thickness and the fluid velocity. From the physics of the problem, an increase in the magnetic field intensity leads to an increase in the Lorentz force which is a retarding force to the transport phenomena. This retarding force can control the nanofluids velocity which is useful in numerous applications such as magneto hydrodynamic power generation and electromagnetic coating of wires and metal. We also note that the fluid velocity at the plate surface increases with an increase in the slip parameter (β). This is in agreement with the fact that higher β implies an increase in the lubrication and slipperiness of the surface. Figures φ, Bi, and Ec leads to an increase in both the fluid temperature and the thermal boundary layer thickness. This can be attributed to the additional heating due resistance of fluid flow as a result of the magnetic field, the presence of the nanoparticle, the increased rate at which the heat moves from the hot fluid to the plate and the additional heating as a result of the viscous dissipation.Figures fw and β increase. On the other hand, it is evident that surface slipperiness and suction affect the temperature of the fluid inversely. This is clearly seen from 2O3, and TiO2) used, with Cu-water exhibiting the highest increment. This is as expected, since Cu-water moves closer to the plate surface leading to an elevation in the velocity gradient at the plate surface. As expected, increasing Ha, Gr, Ec, and fw leads to an increase in the skin friction coefficient, while an increase in β reduces the skin friction coefficient as shown in Figures φ, Bi, and fw as seen in Figures 2O3 exhibiting the highest increment. The converse is seen with increasing Ha as shown in Figures β), but the reverse is observed with the increase in the magnetic field intensity (Ha), nanoparticle volume fraction (φ), Eckert number (Ec), Grashof number (Gr), and the suction/injection parameter (fw). Both the temperature and the thermal boundary layer thickness are enhanced by increasing the magnetic field intensity (Ha),nanoparticle volume fraction (φ), Eckert number (Ec), and the intensity of Newtonian heating (Bi), while the cooling effect on the convectively heated plate surface is enhanced by increasing the velocity slip (β) and suction parameter (fw). The problem of hydromagnetic boundary layer flow of an incompressible electrically conducting water-based nanofluids past a convectively heated vertical porous plate with Navier slip boundary condition was studied. The governing nonlinear partial differential equations were transformed into a self-similar form and numerically solved using shooting technique with a fourth-order Runge-Kutta-Fehlberg integration scheme, putting into consideration the enhanced electrical conductivity of the convectional base fluid due to the presence of the nanoparticles. Our results showed that the fluid velocity increases, while the local skin friction decreases with the increase in the slip parameter (


Ovarian steroid cell tumours (not otherwise specified) are rare neoplasms of the ovary and are classified under lipid cell tumours. Their diagnosis can be considered as one of exclusion. Histopathologically, the tumour should carefully be evaluated for microscopic features of malignancy, but it is essential for the clinician and the pathologist to remember that in these tumours, pathologically benign histomorphology does not exclude the possibility of clinically malignant behaviour. Our case study focuses on the comparative findings in a postmenopausal female diagnosed with an ovarian steroid tumour (not otherwise specified). A careful correlation between clinical and surgical evaluation and microscopic analysis is necessary, as is a regular followup. The spectrum of ovarian neoplasms covers an extremely wide range of tumours. The best recognized of these are the surface epithelial cell tumours. Amongst the less common variants, lipid or steroid cell tumours comprise an important category, although they account for only 0.1% of all ovarian tumours [The origin of steroid cell tumours has long been a matter of controversy and debate. Their nomenclature as such arises from their resemblance to steroid hormone secreting cells   and theyThe following report focuses on a case of a steroid cell tumour (not otherwise specified) diagnosed in a 54-year-old postmenopausal female. The case was significant keeping in mind the short duration of complaints, the age of the patient, lack of any overt androgenic manifestations, and discrepancies between clinicopathological findings and light microscopic features.A 54-year-old multiparous postmenopausal female presented in the outpatient section of the department of obstetrics and gynecology in our hospital, with a short history of pain in the abdomen (in the epigastric region) for two months, associated with abdominal distention and loss of appetite for fifteen days. There were no other associated complaints. Perabdominal examination revealed distention of the abdomen. Pervaginal examination did not yield definite findings, with a vague fullness being detected in both fornices. Uterine size could not be made out. A CT scan was carried out which showed a heterogenous, solid-cystic, right adnexal mass , associaThe patient underwent an exploratory laparotomy, with total abdominal hysterectomy, bilateral salpingo-oopherectomy and sampling of pelvic and mesenteric lymph nodes and omentum. Imprint/scrape smears were prepared peroperatively from the tumour surface, hemidiaphragm, liver, and peritoneum and from what appeared to be deposits on the omental and colonic surface. Fluid samples were also collected from the subdiaphragmatic spaces and para-colic gutters and pouch of Douglas. We did not attempt to assess the tumour intraoperatively with frozen sections. The resected specimens were sent to the histopathology section of the department of pathology, while the smears and fluid samples were received in the cytopathology section and were processed as per the protocol. A firm yellow-coloured tumour was seen at one end of the right ovary, 6 × 5 × 4 cms in size, with a nodular/bosselated surface. The cut surface was solid with occasional small cystic spaces, yellowish and with focal areas of haemorrhage. There was capsular extension with presence of satellite nodules. The gross appearance is demonstrated in  The rest of the ovary was occupied by a smooth cystic cavity, 4 × 3 cms, containing watery fluid. The right fallopian tube was grossly normal, as were the left tube and ovary. An intramural leiomyoma, 1.5 cms in diameter, was detected in the posterior and lower segment of the uterus. Lymphnode and omental samples were also processed.On evaluation of the light microscopic features, the tumour areas showed a bimodal cell population; there were large, round to polyhedral cells with vacuolated cytoplasm as well as smaller cells which had eosinophilic granular cytoplasm . Some deThe tumour tissue was also sent for immunohistochemical analysis for calretinin but the result was negative.The associated cystic area in rest of the ovary turned out to be a simple serous cyst. Other histopathological findings included chronic cervicitis, an atrophic endometrium with endometritis, myometritis, and an intramural leiomyoma, salpingitis, and a simple serous cyst in the left ovary. Reactive changes were noted in the mesenteric lymph node, while the sample of pelvic lymphnode yielded only fibrofatty tissue. No tumour cell deposits were detected on light microscopy in the sections of lymphnode and omentum.The cytologic samples (fluid and imprint smears) did not yield any significant findings.The histopathological features supported a diagnosis of a Lipid (Steroid) cell tumour (not otherwise specified) of the right ovary with serosal nodules. The patient was treated postoperatively with chemotherapy and is on regular followup.The most important factor to be determined in lipid/steroid cell tumours of the ovary is whether the tumour has malignant features or not. In one of the major studies done on lipid cell tumours of the ovary , certainIt is interesting to note that in the case under discussion, the patient had ascites and on gross examination, there was evidence of capsular extension and satellite nodules.In contrast to the clinical findings, the microscopic appearance did not reveal any prominent finding in favour of malignancy. Mitosis was not noted, there was no haemorrhage or necrosis, tumour size was less than 7 cms in all dimensions, and only grade 1 atypia was observed. This tumour thus had a benign microscopic appearance. It is known that pathologically benign steroid cell tumours can behave in a clinically malignant fashion . Therefo Another unusual finding in this case is the apparent absence of androgenic manifestations. However, it is possible that the changes were very subtle and not clinically evident. Some of the other pathological entities that have to be excluded are leydig cell tumors, luteinized granulosa cell tumours, clear cell carcinomas, and metastatic renal cell carcinomas. Reinke's crystals can usually be demonstrated in leydig cell tumours. Granulosa cell tumours can have prominent luteinized areas but typical foci of the tumour can be identified. A Periodic acid-Schiff stain can be used to demonstrate the presence of glycogen in clear cell carcinomas and metastatic renal cell carcinomas, which is absent in steroid cell tumours.A calretinin stain was performed in this case but was found to be negative. These tumours are usually positive for inhibin although it is not specific for this tumour. A recent study analysed ovarian sex-cord stromal tumours for calretinin positivity . All theA careful correlation between clinical findings and histopathology is always essential, as is amply demonstrated by this particular case. Moreover, the necessity of a regular followup must be stressed upon, with the aim of detecting a possible recurrence or evidence of distant metastasis.


The successful purification, crystallization and first data collection to 1.8 Å resolution of the putative transfer protein TraN from the Gram-positive conjugative plasmid pIP501 are reported. Enterococcus faecalis are presented. Native crystals diffracted to 1.8 Å resolution on a synchrotron beamline. The crystals belonged to space group P21, with unit-cell parameters a = 32.88, b = 54.94, c = 57.71 Å, β = 91.89° and two molecules per asymmetric unit.Conjugative plasmid transfer is the most important route for the spread of resistance and virulence genes among bacteria. Consequently, bacteria carrying conjugative plasmids are a substantial threat to human health, especially hospitalized patients. Whilst detailed information about the process has been obtained for Gram-negative type-4 secretion systems, little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. The successful purification and crystallization of the putative transfer protein TraN from the G+ conjugative model plasmid pIP501 of  Escherichia coli or Agrobacterium tumefaciens) make use of type-4 secretion (T4S) systems, multi-protein complexes which are dedicated to the intercellular transport of proteins or protein–DNA complexes  of the transfer operon from the G+ conjugative plasmid pIP501. TraN is the second potential transfer protein of this system to be crystallized. A sequence-similarity search within G+ and G− T4S systems has not revealed any related proteins. Previously performed electrophoretic mobility shift assays  mark TraN as a dsDNA-binding protein. Its possible functions include a role as an accessory protein for the pIP501-encoded relaxase TraA or as a transcription regulator adjusting the frequency of expression of key pIP501 transfer proteins. The high-resolution structure will be key to elucidating the function of TraN.2.traN was cloned into the 7×His-tag expression vector pQTEV , and E. coli BL21-CodonPlus (DE3)-RIL  competent cells were transformed with the recombinant construct pQTEV-traN. Large-scale expression was performed in 500 ml LB medium supplemented with 100 µg ml−1 ampicillin. TraN expression was induced at an OD600 of ∼0.6 by addition of 1 mM IPTG and expression continued for 3 h at 310 K. Cells were harvested and immediately frozen at 253 K. TraN expression levels were monitored by SDS–PAGE .GE Fig. 1a.M HEPES pH 7.6, 75 mM Na2SO4 (buffer A). 2 U DNAse , phenylmethanesulfonylfluoride and benzamidine  were added. The cell suspension was vigorously mixed  and kept on ice for 30 min. The solution was sonicated  and centrifuged for 30 min at 281 K and 15 000g. Pellet and supernatant fractions were analysed by SDS–PAGE. The pellet was applied to a second extraction step with 20 ml buffer A. The TraN-containing supernatant fractions were pooled and loaded onto a HisTrap FF 1 ml column  for affinity purification . The purity of TraN was assessed by SDS–PAGE . Imidazole was removed by buffer exchange during concentration .TraN cell pellets were first lysed in 40 ml 25 mon Fig. 1b. The pGE Fig. 1a. Imida−1 was subjected to an adapted differential scanning fluorometry buffer optimization screening  PEG 3350] and No. 72 , were selected for microbatch pH/PEG/protein concentration optimization matrices. A constant protein drop volume of 1 µl and different protein stock concentrations were used.His-tagged TraN was initially set up with an Index screen at a stock concentration of 4.8 mg mlvia matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometry (MS) measurements  to confirm the integrity of TraN in the crystals. The crystals were dissolved in 10 µl pure H2O. The MS analysis showed that TraN was significantly smaller than the original His-tagged construct. Two peaks of equal height were visible with molecular masses of 14 222 and 14 478 Da (data not shown), implying two cleavage sites with a difference of two amino acids. The His tag, including the TEV cleavage site and linker, amounts to a molecular weight of 3213 Da , likely owing to the inherent anisotropy of the data.The crystal used for the data collection belonged to space group n Table 2. The datMOLREP  experiments. The native data presented here will be used in the refinement of a preliminary model obtained from these efforts.


Am J Trop Med Hyg 85: 105–112 by Hartung et al, the first and corresponding author's affiliation and current address are incorrect. TK Hartung's affiliation should be listed as Department of Medicine, College of Medicine, University of Malawi, Blantyre, Malawi and Department of Respiratory Medicine, Victoria Hospital, Kirkcaldy, United Kingdom. His current address should be Department of Respiratory Medicine, Victoria Hospital, Hayfield Road, Kirkcaldy, KY2 5AH Kirkcaldy, United Kingdom. E-mail: thomas.hartung@nhs.net.In Am J Trop Med Hyg 85: 303–308 by Jutla et al, the affiliation of some of the authors was incorrectly listed in the print version of the article. The acronym for the Water and Environmental Research, Education and Actionable Solutions Network should have been listed as WE REASoN.In


This special issue focuses on the pleural space, a unique region of the human body affected by some of the earliest described maladies in medical science yet remaining a mystery in both purpose and function. The two pleural spaces, defined by the bony thorax, diaphragm, and mediastinum, are each occupied by the lungs. Consequently, the pathophysiologic disorders of the space involve not only benign and malignant disorders that take the form of solid masses or effusions, but also gaseous disorders as well. Ongoing air leakage through the surface of a lung will result in collapse and, if ongoing, will result in a fatal pneumothorax. These are some of the most common problems encountered in the pleural space and are treated, along with other maladies, with tube access of the pleural space. Although a common procedure, it requires technical precision and judgment to avoid potentially disastrous complications, as detailed in this special issue. Embryologic development of the pleural structures, discussed in this special issue, results in planes of dissection and paths of least resistance that can result in unusual and unexpected patterns of air accumulation when the air leaks within the lungs. The net negative pressure within the pleural space and the positive pressure in the pulmonary arterial circulation and the lymphatic circulation maintain a dynamic equilibrium in pleural space, the net effect being a minimal amount of fluid at any given time despite a high flow from the visceral to parietal pleural surfaces. Perturbation of this delicate balance results in fluid accumulation. Because of the extraordinary number of factors involved in maintaining this balance, it can be very difficult to diagnose the reason for the fluid buildup. Within the context of the overall clinical picture, the diagnosis is often rendered by analysis of the fluid itself. As described in this special issue, there are many tests and criteria accessible to the clinician to diagnose the etiology of the fluid accumulation. When the fluid is caused by a malignancy or noninfectious benign process, it is typically necessary to intervene and stop the fluid accumulation. The most common approach remains pleurodesis, affecting a symphysis between the visceral and parietal pleural surfaces for the purpose of obliterating the space. Using talc for this purpose remains one of the most common, and arguably best, techniques and is described in this special issue.When the fluid does accumulate and becomes infected, it is called an empyema, and this too can easily escalate into a fatal condition without prompt and appropriate treatment. Although drainage, as first described by Hippocrates over 2000 years ago, remains a critical element of the treatment, there are now multiple tools and techniques available to the clinician to combat this common and lethal disorder. A review of this important topic is covered in this special issue. Although the pleural space is highly resistant and resilient in the face of infectious challenges, the ultimate clearance and recovery most often hinges on full lung expansion with pleural-pleural apposition. When an empyema is not drained early enough, the lung can become encased in a fibrous peel that precludes reexpansion and results in a chronic space. Ideally, the fibrous peel can be surgically resected to reexpand the lung and fill the space. At times, however, the lung will no longer fill space, and this results in a challenging and deadly situation that warrants surgical intervention. As described in this special issue, this can take the form of opening the space to allow full drainage to obliterating the space by filling it with healthy vascularized tissue and/or collapsing the chest wall to meet the lung surface.The pleural space is a fascinating and enigmatic region of the body maintained in balance by a symphony of homeostatic mechanisms. Perturbation of this balance by benign or malignant processes can lead to a myriad of problems, unique to the pleural space. Addressing these problems requires an understanding of the embryology, anatomy, and physiology and then combining this knowledge with sound judgment, scientific analysis, meticulous technique, and, at times, a dash of detective work. We hope you enjoy this special issue as it explores these topics. Joseph S. FriedbergJoseph S. FriedbergTakashi NakanoTakashi Nakano


It is becoming increasingly clear that the tumour microenvironment has a very important role in tumour progression and drug resistance. Many different cell types within the tumour stroma have an effect on tumour progression either in a positive or in a negative way. Mesenchymal stem cells (MSCs) are a distinct population of cells that have been linked with tumour growth. Mesenchymal stem cells can home to tumours where they modulate the immune system and facilitate tumour growth, angiogenesis and metastasis. Recent studies have shown that MSCs also have an important role in the resistance to various anti-cancer drugs. This mini-review provides an overview of the functional properties of MSCs in tumour progression and drug resistance. They are a relatively easy accessible pool of stem cells that have the ability to differentiate into various cell types like osteoblasts, chondrocytes, fibroblasts and adipocytes. Mesenchymal stem cells are characterised as plastic adherent cells that express the markers CD105, CD90, CD73 and lack expression of markers CD45, CD34, CD14, CD11b, CD79d HLA-DR . Well esd HLA-DR . Many cye homing . In addiMesenchymal stem cells are, in addition to other cells in the tumour microenvironment, increasingly identified as an important population of cells that modulate tumour progression and drug sensitivity. The broadly described physiological functions of MSCs in immune suppression have also been linked with tumour progression. It has been shown that in co-cultures of MSCs, breast cancer cells and peripheral blood mononuclear cells MSCs were able to shift the balance of Th1/Th2 cytokines in favour of Th2 cytokines resulting in the ability of the breast cancer cells to evade the immune system , or it can be the result of the tumour microenvironment protecting tumour cells against treatment (extrinsic resistance). The tumour microenvironment can promote drug resistance in a passive way, by preventing penetration of drugs into the tumour or in an active way by secreting protective cytokines or changing gene transcription within the tumour cells to override the cytotoxic effects of anti-cancer agents . The use of a trans-well system indicated that factors that give chemoresistance are secreted by the BMSCs and that no direct cell–cell contact is needed  in response to the cisplatin. The PIFAs that were identified are 12-S-keto-5,8,10-heptadecatrienoic acid (KHT) and 4,7,10,13-hexadecatetraenoic acid (16 : 4(n−3)). 12-S-keto-5,8,10-heptadecatrienoic acid is the oxidised metabolite of 12-S-hydroxy-5,8,10-heptadecatrienoic acid (12-S-HHT) which is a fatty acid that is formed as a by-product in the production of thromboxane A2 synthesis within the arachidonic acid (AA) pathway. Metabolism of AA leads to the production of a series of active fatty acids derivatives also known as eicosanoids including 12-S-HHT and KHT. Very little is known about 16 : 4(n−3), it is an omega-3 fatty acid and it has been described to be produced by some marine algae , which is needed to protect these long-lived cells against toxic compounds and reactive oxygen species . One hypin vitro cultured MSCs or injected supraphysiological concentrations of MSCs, making it difficult to draw conclusions about endogenous levels of MSCs and their role in tumour progression and therapy resistance. Furthermore, clinical data on the role of MSCs in tumour progression is still lacking. Results from Most studies use in vitro and in mouse models. As MSCs have such diverse ways to enhance tumour progression, inhibiting these cells in a clinical setting will be challenging. The potent homing of MSCs to tumours is already explored to selectively target tumours. However, it should be kept in mind that these genetically engineered MSCs still contain the numerous intrinsic mechanisms that can promote tumour growth, metastasis and drug resistance indicating that using them as vehicles to target tumours may have severe side effects if not used very carefully.A remarkable characteristic of the role of MSCs in tumour progression is its broad versatility and plasticity. Mesenchymal stem cells have been implicated in promoting almost every hallmark of cancer including angiogenesis, metastasis, anti-apoptosis, pro-survival and evasion of immune system. Mesenchymal stem cells can promote tumour growth and drug resistance either via close contact with the tumour cells or via systemic mechanisms involving secreted factors. Targeting MSCs as part of anti-cancer therapy can significantly inhibit tumour growth and metastasis and increase therapy response Despite the enormous amount of data present on the tumour-promoting properties of MSCs there are also reports that MSCs can inhibit tumour growth (see Taken together, MSCs are very important regulators of tumour growth and key modulators of treatment response. This makes these cells an attractive target for therapy, which deserves further fundamental and translational studies.


Malignant proliferating trichilemmal tumour is a rare cutaneous malignant neoplasm usually occurring in elderly women. We present a case of malignant trichilemmal tumour in a young lady of 26 years of age with a previous history of proliferating trichilemmal tumour at the same site. Malignant proliferating trichilemmal tumour (MPTT) is a rare cutaneous tumour predominantly affecting the scalp, eyelids, neck and face. A review of the literature reveals MPTT to be a neoplasm of the older age group.2 We presA 26-year-old lady presented with a history of swelling behind her right ear of seven months’ duration. There was no history of any discharge or constitutional symptoms.She gave a history of a similar swelling in the same region (right post-auricular) for which she underwent excision and split-skin grafting nine months ago. On histopathology, it was found to be a proliferating trichilemmal tumour. The lesion recurred within two months of excision and increased to the present size in seven months. There was a rapid increase in size in the last two months. The patient also gave a history of another swelling in the right parieto-occipital region which was excised two years ago and was diagnosed as a benign trichilemmal tumour.On examination, there was a 2.5 × 2 cm hemispherical swelling in the right post-auricular region, with an overlying scar of the previous split-skin grafting . It was Under general anaesthesia with endotracheal intubation, patient in the supine position with the head turned towards the left, a rhomboid-shaped incision was made around the tumour. Wide excision was done giving a 2 cm clearance all around. The underlying mastoid was shaved as a precautionary measure . A transGross examination of the excised skin showed a centrally positioned nodule measuring 2.2 cm in diameter just beneath the skin. Grossly, the overlying skin, circumferential and deep resected margins were not involved by the lesion.Histopathological examination of haematoxylin and eosin (H and E)-stained sections from the skin showed a tumour located in the dermis. The tumour cells were arranged as islands, in lobular and diffuse pattern Figures  and 5a. Final diagnosis on histopathological examination was malignant proliferating trichilemmal tumour. One year after the surgery, patient is doing well with no recurrence .et al., and its biological behaviour still remains unpredictable.[Malignant proliferating trichilemmal tumour is an extremely uncommon tumour with differentiation towards hair and/or follicle. The termdictable. There ardictable. In a studictable.Proliferating trichilemmal tumour is a benign neoplasm that may be multiple as noticed in our case and it can rarely undergo malignant transformation in a step-wise manner starting with an adenomatous stage of the trichilemmal cyst to an epitheliomatous stage of the PTT evolving into carcinomatous stage of the MPTT. This tumIt may be difficult to distinguish MPTT from squamous cell carcinoma and trichilemmal carcinoma (TLC) which are known to occur at the same site.10 HistolMalignant proliferating trichilemmal tumour may further resemble TLC on histology. Trichilemmal carcinoma is a rare malignant skin tumour occurring in older individuals with a predilection to sun-exposed areas. Trichilemmal carcinomas have been reported in solar keratosis, scars of burns, following high-dose radiation exposure and in post-transplant states. Trichilemmal carcinoma often occurs as a papular, nodular or exophytic solitary lesion measuring less than 2 cm in size. Histopathologically, TLC demonstrates a lobular proliferation centred on pilosebaceous structures and is composed of clear cells that are characterized by clear cytoplasm and prominent nucleoli. Trichilemmal carcinoma usually shows continuity with the epidermis or follicular epithelium as a pagetoid interface, exhibits trichilemmal keratinization and dermal invasion.–13Based on size, clinical details and histology a diagnosis of MPTT was suggested in the present case. Malignant proliferating trichilemmal tumour usually arises as a subepidermal tumour in women over 60 years of age, while the present case report highlights its occurrence in a young lady. HistopatTreatment modalities differ for TLC and MPTT and a review of the literature reveals the former to be a malignant neoplasm with indolent behaviour, hardly ever resulting in recurrence or metastasis. Surgery Malignant proliferating trichilemmal tumour can rarely occur in the young especially, in an individual with pre-existing proliferating trichilemmal tumour. Since it follows an aggressive course, it is essential to distinguish it from other similar-looking neoplasms for an appropriate therapy.


CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor  VZV is a human pathogen that spreads to children as varicella or chicken pox and re-emerges later as zoster or shingles Ross, . VZV is Varicella infection, within its natural human host, spreads from the nasopharynx via infection of a limited number of T cells that home to the skin epidermis : XBP1 splicing and a greatly expanded ER  or VZV ORF62 (pCMV_IE62) under the CMV promoter as described previously  to form ΔCT values which were then used to calculate averages and standard deviations between triplicate measurements. Subsequent ΔΔCT values were calculated by differences between averages of VZV infected ΔCT values or tunicamycin treated ΔCT values with the average uninfected ΔCT values. Uncertainties correspond to propagation of errors using standard deviations between the uninfected and infected or tunicamycin averages.Total RNA was extracted from uninfected, tunicamycin treated and VZV infected fibroblast cells in six well plates at the given time points using the RNEasy mini kit (Qiagen). RNA quality and quantity was assayed by UV spectroscopy using a NanoDrop spectrometer. Both A260/A280 and A260/A230 ratios were within 20% of 2.0 and infected cells from a six well plate well (6.5 sq cm) yielded approximately 3 μg of RNA in 60 μl. Further, the RNA was electrophoresed in an Agilent Bioanalyzer 2100 (Agilent) and yielded RIN values within 20% of 10. Polyadenylated RNA was converted to cDNA using anchored Oligo(dT) primers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) to yield approximately 20 ng of cDNA. The entire cDNA sample from one well of cells was mixed into 1 ml of 1× diluted Power SYBR Green Master Mix (ABI) and split into all wells of a SA Biosciences UPR PCR array (Life Technologies) with a multichannel pipettor (25 μl per well). The measurements were carried out in triplicate using cDNA from three of the original six wells in the plate for all types of samples using a Model 7000 real time PCR instrument (ABI). The resulting PCR results were processed using the SDS 1.2.3 software (Applied Biosystems). CTo confirm measurements from the UPR specific PCR array, several RT-PCR measurements were carried out using the following primers: BiP: forward 5′-CCC CAA CTG GTG AAG AGG AT-3′ and reverse 5′-GCA GTA AAC AGC CGC TTA GG-3′; DNAJB9/ERDj4: forward 5′-ACA TCT GTG ACT TGC GTT GC-3′ and reverse 5′-TGG GCA ATA AAA CCA TTT CC-3′; CREBH: forward 5′-GGG AGA CGA GCT GTG AGC-3′ and reverse 5′-TGT CTG AGT GTC GGT TCC TG-3′; PERK: forward 5′-GCC TAA GGA GGT AGC AGC AA-3′ and reverse 5′-GGG ACA AAA ATG GAG TCA GC-3′.Murine MAb antibodies to VZV gE (3B3) and IE62 (5C6) produced in our laboratory were used in addition to a rabbit polyclonal antibody to LC3B (Santa Cruz Biotech sc-28266).Samples of infected and uninfected cells were prepared for confocal microscopy by methods described previously  have been described in earlier papers in which we were investigating VZV glycoprotein biosynthesis  was obtained in powder form from Molecular Probes (D-273) and dissolved (0.7 mg/ml) in ethanol  was added to warm cell culture medium; this medium was applied to live cells for 30 min, then rinsed 2× with PBS and processed for fluorescent microscopy as described above.DiOCWithin cell culture, VZV is entirely cell associated with no release of cell-free virus . This 96 well plate consists of 84 wells containing primers to the 3′ Untranslated Region (UTR) of transcripts associated with the UPR and the remaining 12 wells containing primers to housekeeping genes and PCR and cDNA quality control wells. Table T values were normalized so that in each case the housekeeping gene transcripts measured CT average was 16 and then the triplicate measurements were averaged and standard deviations computed to generate ΔCT. Differences between the uninfected ΔCT and those associated with TM treated and VZV infected cell transcript measurements were then calculated to form the final measurements ΔΔCT listed in Table CHOP (pink). By contrast, only 43 of the UPR genes are upregulated in VZV infected cells. In particular, those genes most upregulated such as CREB3L3/CREBH (light blue) are more upregulated than after TM treatment. VZV infected cells also upregulated the LIPID transcripts AMFR/gp78 and INSIG (green) while downregulating a number of ERAD components such as UBXN4/erasin and EDEM3 (red). These differences will be considered by group in the subsequent sections.Gene transcripts were measured in uninfected human fibroblasts, tunicamycin (TM) treated fibroblasts and VZV infected fibroblasts. Each measurement was done in triplicate. The measured CPERK, IRE1α and ATF6 but also two CREB proteins (CREB3/LUMAN and CREBH) as well as primers to the Golgi resident proteases S1P and S2P that activate AT6 and the CREB proteins by cleavage  than in TM treated cells . However, the downregulation of PERK in VZV infected cells was not confirmed.In order to confirm the results of the UPR specific PCR array, we carried out qPCR measurements using primers to K Figure  in TM trHSPA5/BiP and SIL1/BaP; five DNAJ HSP-40 homologs including DNAJB9/ERdj4 and DNAJC10/ERdj5; twelve wells contain primers to transcripts encoding ER lumen folding components including CALR and CANX; three components of the folding chaperonin complex and finally four components in the ER membrane including RPN1 and SEC63. Many of these transcripts encode proteins which assist secretory protein folding but also sense misfolded proteins in the ER . However, we also reassessed the regulation of the ER-co-chaperone DNAJC10/ERdj5 and found a correlation with the UPR-specific array (data not shown).VZV infected cells exhibited very uneven transcription of folding chaperones (FOLD) while TM treatment robustly upregulated transcription of these chaperones particularly P Figure . Measures Figure  and founOS9, PPIA and SELS along with a number of FOLD transcripts), trimming of mannose residues prior to recognition by E3 ubiquitin ligases (EDEM1 and EDEM3), recognition of misfolded proteins by E3 ubiquition ligases , exportation to the cytosolic side of the ER membrane (SEC62) where the VCP/p97 complex poly-ubiquitinates protein substrates before extracting/clipping the protein from the membrane to be ultimately degraded in the cytosol by the proteasome  in VZV infected cells as compared to the value in TM treated cells . We therefore conclude that expression of a single VZV glycoprotein gene in cells is sufficient to activate the CREBH arm of the UPR.In 2011, we found that transfecting cells with VZV glycoprotein genes led to increased autophagosome production and inflation of the ER. Transfection with VZV IE62 led to neither increased autophagosomes nor a larger ER. Therefore, we measured by qPCR whether CREBH and BiP transcription was increased by transfection of a glycoprotein vs. a non-glycoprotein that is also the major transactivator encoded by VZV. Transfection with a plasmid encoding VZV gE under the CMV immediate early promoter led to approximately 10% of transfected cells Figure  while tr Figures  whereas CREBH. At the same time, VZV infected cells showed decreased transcription of genes associated with ERAD and apoptosis. We hypothesize that this transcriptional profile is compatible with the infected cell attempting to accommodate the influx of viral glycoproteins by greatly increasing the capacity of the ER. For example, increased transcription of AMFR (gp78) and INSIG is associated with a decrease in cholesterol synthesis via degradation of HMG-CoA reductase, an enzyme necessary for cholesterol synthesis  The choice of primers in the PCR array vs. those used in the qPCR measurements or (ii) the asynchronous nature of VZV infection. In general, there was better agreement between tunicamycin treated values as measured by the UPR-specific PCR array and individual qPCR assays. The biggest differences were observed in values measured from VZV infected cells. The latter scenario suggests that asynchronous VZV infection may play a role; for example, the input virus is always extremely low, such that some cells within a monolayer will remain uninfected even at 72 hpi. This same scenario may explain why we have observed similar unexpected differences in experiments to measure protein expression, for example, BiP. Because of increased VZV-induced autophagy, we predicted increased BiP production in infected cells. However, we have observed variable changes in BiP protein expression following VZV infection. Nevertheless, the main conclusions of this report were confirmed by both assays, namely, the significant upregulation of CREBH as well as the more modest upregulation of the cholesterol regulator INSIG.A key question is whether the infected cell is responding to abundant viral glycoprotein expression through normal mechanisms or alternatively, does the virus encode its own proteins which manipulate the UPR. Recently, Burnett et al found that HSV ICP0 transactivated elements of the UPR and in turn was transactivated by the UPR itself via an ERSE promoter element in the HSV ICP0 gene (Burnett et al., CREBH and BiP transcription. We observed abundant VZV gE protein in the ER/Golgi after transfection. In contrast, transfection with VZV IE62, a non-glycosylated viral protein, did not lead to increased transcription of either transcript. Obviously, the IE62 protein never enters the ER/Golgi. These results confirm and expand our 2011 report that transfection with VZV glycoprotein genes resulted in increased ER size and increased autophagosome production. The UPR is known to upregulate autophagy (Yorimitsu et al., In further support of our hypothesis that abundant expression of VZV glycoproteins contributes to the activation of the UPR in a specific way that leads to an enlarged ER and increased autophagosome production, we found that transfection of the VZV gE gene led to increased CREBH and cholesterol synthesis regulation transcription and diminished ERAD transcription. The transcription patterns appeared to correlate with increasing ER capacity secondary to increasing viral glycoprotein synthesis in the infected cell. Of importance, the CREBH data were totally unexpected, based on all prior VZV research, and would never have been uncovered in the absence of the UPR array data described in this report.In summary, even though both tunicamycin treatment and VZV infection induced an UPR, the profiles of UPR related genes were different after the two analyses. The UPR in VZV infected cells exhibited greatly increased The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Asarum heterotropoides (EOAH) on depression-like behaviors and brain expressions of CRF, 5-HT, and TH in mice challenged with stress.Psychological stressors may cause affective disorders, such as depression and anxiety, by altering expressions of corticotropin releasing factor (CRF), serotonin (5-HT), and tyrosine hydroxylase (TH) in the brain. This study investigated the effects of essential oil from Male ICR mice received fragrance inhalation of EOAH  for 3 h in the special cage capped with a filter paper before start of the forced swimming test (FST) and tail suspension test (TST). The duration of immobility was measured for the determination of depression-like behavior in the FST and TST. The selective serotonin reuptake inhibitor fluoxetine as positive control was administered at a dose of 15 mg/kg (i.p.) 30 min before start of behavioral testing. Immunoreactivities of CRF, 5-HT, and TH in the brain were also measured using separate groups of mice subjected to the FST.EOAH at higher doses (1.0 and 2.0 g) reduced immobility time in the FST and TST. In addition, EOAH at a dose of 1.0 g significantly reduced the expected increases in the expression of CRF positive neurons in the paraventricular nucleus and the expression of TH positive neurons in the locus coeruleus, and the expected decreases of the 5-HT positive neurons in the dorsal raphe nucleus.These results provide strong evidence that EOAH effectively inhibits depression-like behavioral responses, brain CRF and TH expression increases, and brain 5-HT expression decreases in mice challenged with stress. Physical and psychological stressors are thought to cause affective disorders including depression and anxiety ,2. The nStudies in laboratory animals and humans have demonstrated that inhalation of essential oils can produce antidepressant and anxiolytic effects exposed to stress by modulating the central nervous system. For examples, inhalation of lemon oil reduced anxiogenic and depressant effects in an elevated plus-maze test and a forced swimming test (FST) by modulating serotonergic and dopaminergic pathways in mice brain . In addiAsarum heterotropoides Fr. Mandshuricum has been shown to be effective for reducing anxiety and for the relief of pain [Asarum heterotropoides can produce wide spectrums of action including anti-inflammatory effect [ of pain ,10. Furty effect ,12 and ay effect  and antiy effect .Asarum heteropoides (EOAH) on depression–like behaviors in the FST and tail suspension test (TST). A role for brain corticotropin releasing factor (CRF), serotonin (5-HT), and tyrosine hydroxylase (TH) expression in EOAH effects is also explored.The present study was carried out to investigate the effects of inhalation of essential oil from ad libitum for the study. All animal experiments were carried out in accordance with the animal care guidelines of the National Institute of Health (NIH) and approved by the Institutional Animal Care and Use Committee of Daegu Hanny University.Male mice of ICR strain  at 5 weeks after the birth were housed under a controlled temperature (23 ± 2°C) and humidity (50 ± 10%) with a 12 h light–dark cycle (lights on at 8:00 h). Food and water were made available Asarum heterotropoides (1.2 kg) were purchased from Daegu Yangnyeongsi  in Daegu, South Korea. The pulverized Asarum heterotropoides were used to extract essential oil at the room temperature with n-hexane (5 L x 2) for 24 h. The extracts (EOAH) were filtered and evaporated in the vacuum at 40°C to remove n-hexane. The final concentration of essential oil was 22.5 g. The specimen of this plant material (RCBROM-AEM2012) is deposited at the Research Center for Biomedical Resources of Oriental Medicine, Daegu Haany University. EOAH was analyzed for the determination of chemical composition using GC-MS (Agilent Technologies 7890A/5975C insert XL Mass Selective detector) with injection volume 1 μl . The identification of EOAH was carried out by comparing their relative retention time and mass spectra to those generated by chromatographic analysis of standards and W9N08.L library and literature.The dried and chipped roots of n = 5); EOAH 0.25 g (n = 5); EOAH 0.5 g (n = 5); EOAH 1.0 g (n = 5); EOAH 2.0 g (n = 5). Mice weighing between 25 and 30 g at the start of the experiment were used. The mice were pretreated with inhalation of EOAH  or saline (control group). The inhalation dose of essential oil was chosen based on previous studies [n = 7) as positive control or saline  by intraperitoneal injection 30 min before the start of behavioral testing. An injection volume was 0.3 ml/30 g body weight.To evaluate the effects of EOAH on depression-like behaviors, the mice were randomly divided into the following 5 groups: Control ; Control (n = 9); EOAH 0.5 g (n = 9); EOAH 1.0 g (n = 9). At the end of the FST, mice were sacrificed and brains were prepared to measure the expressions of 5-HT, TH, and CRF, respectively. Mice in normal group were not allowed to have the FST. Immunohistological study began with transcardial perfusion of heparinized phosphate-buffered saline  for 30 s followed by perfusion of 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4) for 10–15 min. Brains were removed and allowed to stand in the same fixative by overnight, followed by cryoprotected in 30% sucrose solution in PBS. The individual brain was embedded in OCT compound and serially sectioned on a cryostat  at 30 μm thickness by the coronal plane, and then collected in PBS using free floating methods. The individual primary antibody includes anti-5HT , anti-CRF , and anti-TH  was incubated in a cocktail solution  for 72 h at 4°C, respectively. After three time rinses in PBS, the sections were placed in Vectastain Elite ABC reagent  for 2 h at room temperature. Following a further rinsing in PBS, the tissue was developed using diaminobenzadine  as the chromogen. The images of 5-HT-, CRF-, and TH-immunoreactive neurons were captured using a DP2-BSW imaging system  and measured as described by others [The expressions of 5-HT, TH, and CRF were measured using different groups of mice. Mice were divided 4 groups: Normal , pentadecane (6.78%), 2,3,5-trimethoxytoluene (5.54%), 4-(chloromethyl) cyclohexene (3.36%), myristicine (3.27%), sesamin (3.24%), and kakoul (2.63%).4,20 = 3.19, p < 0.05). Post hoc tests revealed that mice receiving saline (control) had significantly greater immobility time than those treated with EOAH during the FST (p < 0.05). When mice were exposed to the TST, inhalation of EOAH  significantly decreased immobility time . Post hoc tests revealed that mice receiving saline had significantly greater immobility time than those treated with EOAH during the TST (p < 0.05). Similarly, administration of fluoxetine markedly reduced immobility time at both the FST and TST compared to saline group  significantly reduced immobility time in the FST , inhalation of EOAH (1.0 g) increased 5-HT expression in the dorsal raphe nucleus. A one-way ANOVA analysis revealed a significant main effect of EOAH inhalation . Post-hoc comparisons indicated that there was a significant enhancement in the 5-HT activity in the dorsal raphe nucleus of the 1.0 g EOAH group compared with the control group (p < 0.01). Group 0.5 g EOAH mice tended to have greater 5-HT activity in the dorsal raphe nucleus than the control group, but these differences were not statistically significant.Changes in the expression of 5-HT-immunoreactive neurons were evaluated in mice exposed to the FST following inhalation of EOAH Figure A-D. As sp < 0.001), there was an increase in 5-HT expression in those receiving inhalation of EOAH (0.5 and 1.0 g). A one-way ANOVA revealed a significant main effect of the treatment on the expression of CRF-immunoreactive neurons in the paraventricular nucleus . Post hoc tests indicated that inhalation of EOAH (0.5 and 1.0 g) induced marked increases in the expression of CRF-immunoreactive neurons in the paraventricular nucleus compared with saline-treated mice . Post hoc tests indicated that inhalation of EOAH (0.5 and 1.0 g) induced marked increases in the expression of TH-immunoreactive neurons in the locus coeruleus compared with treatment of saline (p < 0.001, Figure The expression of TH in the paraventricular nucleus was shown after inhalation of EOAH Figure A-D. SimiThe present results demonstrated that the inhalation of EOAH effectively attenuated depression-like behavior, increases in brain expressions of CRF and TH, a decrease in brain expressions of 5-HT in response to 6 min of forced swimming or immobilization stress. Behavioral responses to the FST and TST are consistent with previous findings ,4. The FAsarum heterotropoides [Pharmacologically, a variety of anti-depressants and related-compounds with potential anti-depressant activity reduce the duration of immobility in the FST and TST ,23. Therropoides , produceropoides .Depression-like behaviors induced by stressors have been linked to endogenous systems of CRF and monoamines including 5-HT and NE ,30. PharMost importantly, results showed that EOAH at the dose of 1.0 g significantly reduced the expected increases in the expression of CRF positive neurons in the paraventricular nucleus, the expected decreases in the expression of TH in the locus coeruleus and the 5-HT positive neurons in the dorsal raphe nucleus in mice subjected to forced swimming stress. Also, EOAH at the dose of 1.0 g reduced a significant increase in immobility time in the FST and TST. Based on a role for endogenous systems of CRF, 5-HT, and NE in the brain, one possible mechanism where EOAH could diminish depression-like behaviors is by affecting brain levels of 5-HT, CRF, and TH. Recently, it has been proposed a hypothetical model that might explain how inhalation of volatile essential oils activates brain areas . In brieIn conclusion, our results suggest that the reduced stress response produced by inhalation of EOAH is most likely mediated via an activation of serotonergic system and an inhibition of corticotropinergic and catecholaminergic system in the brain.