Varicella-zoster virus (VZV), the cause of varicella and zoster, is divided into five major clades and four provisional clades, the latter of which have been rarely reported worldwide to date. We present a varicella outbreak by the provisional clade VI within an Indian couple in Germany returning from a trip to Amsterdam. To the best of our knowledge, this is the first case of varicella by the VZV clade VI described in Germany, but the disease was acquired in The Netherlands. Varicellovirus within the subfamily Alphaherpesvirinae and the Herpesviridae family, causes two distinct diseases. Primary infection results in typical signs of varicella also termed as chickenpox, and zoster is caused by endogenous reactivation after the virus has established lifelong latency in trigeminal and dorsal root ganglia. As a function of geographic distribution, VZV has been separated into the five major clades 1–5 confirmed by full-genome sequencing. In addition, there have been proposed four novel clades VI to IX still to be confirmed by further complete sequences [Varicella-zoster virus (VZV), a member of the genus equences . These pequences , 3. The A married couple from India, a 32-year-old man and a 25-year-old woman, was admitted to the dermatologic clinic in July 2010 since both had developed varicella. The woman was pregnant at week 34 of gestation. In both patients, an immunodeficiency was not known. One week before admission, the patients had visited a friend who was also coming from India, but he was a permanent resident in Amsterdam, The Netherlands, at that time. Two days after the meeting, the index case had developed a vesicular exanthema, a typical clinical sign of varicella. However, information on the possible source of the virus (contact person) and the vesicular characteristics of the exanthema was not available.Seven days after the contact with the index case, the Indian couple was admitted to the hospital. The man presented with diffuse exanthema consisting of vesicles with a narrow erythematous rim as well as a vesicular, erosive enanthema of the oral mucosa. As a striking finding, there were single large bullous lesions, measuring up to 8 mm in diameter. Additionally, the patient suffered from fever and adynamia. The woman showed multiple papulovesicular skin lesions. Overall, the male patient showed an initially severe course of varicella, whereas the female patient had a relatively mild course.® Anti-VZV/immunoglobulin (Ig) G and Anti-VZV/IgM  at an early point of time, 5 days after the first cutaneous lesions appeared. IgM und IgG were negative in the man, whereas the woman's IgM was positive and IgG was negative. Unfortunately, a second serum sample from the convalescence phase was not analyzed. The molecular-biological characterization of the virus using restriction fragment length polymorphism analysis of the open reading fames (ORF) 38 and 54 [PstI+BglI−. Further characterization of ORFs 1, 21, 22, 50, 54, and 60 by the scattered single nucleotide polymorphism (SNP) method [Routine blood testing revealed hepatopathy with elevated serum concentration of alanine aminotransferase (2.5x of reference) for the man. Testing of vesicle fluid for VZV DNA using polymerase chain reaction  was posiThe patients received acyclovir intravenously at a dosage of 5 mg per kg body weight 3 times daily for 7 days. The man was treated additionally with systemic antibiotics. Zinc sulphate gel was used as topical antiseptic therapy. Gynecological examination of the woman showed a regular pregnancy. Both patients recovered without complications. Subsequently, the female patient gave mature birth to a healthy child.Varicella is a common highly infectious disease with an average age of manifestation differing due to geographical location. In Europe and North America, varicella is seen in 90% of the cases before the age of 10 years . HoweverSince the incubation period of varicella is up to 21 days, it is beyond doubt that the Indian couple described in this paper acquired varicella during their trip to The Netherlands. Both patients developed the disease within one week after VZV exposure to the index case. The novel provisional VZV clade VI was identified as the etiological agent of varicella. The provisional clade VI is considered to be a combination of the European clade 1 or 3 with the African/Indian clade 5 or clade 4 that has been found predominantly in countries of South and Central America . Till noTwo years ago, we observed a varicella outbreak in Indian students in Magdeburg caused by the African/Indian VZV clade 5 . ClinicaThe patients have given their informed consent for the paper published.Malgorzata Kolesnik and Bernd Bonnekoh contributed equally to this paper.


We quantify the false-negative diagnostic rate of septic arthritis using Gram-stain microscopy of synovial fluid and compare this to values reported in the peer-reviewed literature. We propose a method of improving the diagnostic value of Gram-stain microscopy using Lithium Heparin containers that prevent synovial fluid coagulation. Retrospective study of the Manchester Royal Infirmary microbiology database of patients undergoing synovial fluid Gram-stain and culture between December 2003 and March 2012 was undertaken. The initial cohort of 1896 synovial fluid analyses for suspected septic arthritis was reduced to 143 after exclusion criteria were applied. Analysis of our Gram-stain microscopy yielded 111 false-negative results from a cohort size of 143 positive synovial fluid cultures, giving a false-negative rate of 78%. We report a false-negative rate of Gram-stain microscopy for septic arthritis of 78%. Clinicians should therefore avoid the investigation until a statistically significant data set confirms its efficacy. The investigation's value could be improved by using Lithium Heparin containers to collect homogenous synovial fluid samples. Ongoing research aims to establish how much this could reduce the false-negative rate. We find that synovial fluid Gram-stain microscopy as used at present is of no value in the diagnosis of septic arthritis, with a false negative diagnosis rate of 78%, as we show by analysing microbiological data collected during 9 years at the Manchester Royal Infirmary.Our result is consistent with previous values reported in the literature, which show a false-negative rate of 25–50% –3, and hAlternative diagnostic techniques remain underexplored, but a proposed modification to existing techniques is the use of Lithium Heparin containers to collect the synovial fluid. By preventing coagulation of the fluid, Lithium Heparin tubes can produce more homogenous samples for microscopic examination. Research is now being undertaken to quantify how much this modification would reduce the false-negative rate of synovial fluid Gram-stain microscopy in the clinical diagnosis of septic arthritis.To quantify the false negative rate of Gram-stain microscopy of synovial fluid in diagnosing septic arthritis at the Manchester Royal Infirmary compared with values reported in the peer-reviewed literature.To identify means for improving the diagnostic value of Gram-stain microscopy for the diagnosis of septic arthritis.The false negative rate for Gram-stain microscopy reported in the literature is 25–50% –3. In thA search of the Manchester Royal Infirmary (MRI) microbiology database for all patients undergoing synovial fluid culture between December 2003 and March 2012 was performed. During this period, the microbiology investigation method remained unchanged: Gram-stain microscopy and microbial culture on direct plates (for 48 hours) and in enrichment broth (for up to 5 days) were performed after centrifugation of synovial fluid received in a sterile sample pot.This study assumed that synovial fluid culture is the gold standard for diagnosis of septic arthritis; therefore only patients with synovial fluid culture positive for growth of microorganisms were included. Positive cultures from both native and prosthetic joints were included. Care was taken to eliminate cultures that could have arisen due to contamination of samples. The following exclusion criterion was defined: patients with synovial fluid cultures grown from Brain-Heart-Infusion enrichment broth (BHI) or from direct culture which grew coagulase-negative Staphylococci, Diphtheroids, alpha-haemolytic Streptococci, or fungi were excluded unless successive cultures in these patients corroborated these results. Multiple investigations on the same patient were included as the purpose of this study was to correlate Gram-stain microscopy and synovial fluid culture. Correlation of these results allowed calculation of the false negative rate of Gram-stain microscopy.A positive microscopy result was the one that was reported as positive for presence of microorganisms in the Gram film, while a negative result reported absence of microorganisms on microscopy. As the inclusion criterion was a positive synovial fluid culture, a false negative result was defined as a Gram-stain microscopy report negative for presence of corroborating microorganisms.n = 143 positive synovial fluid culture results.Retrospective analysis of the microbiology database yielded 1896 synovial fluid analyses between December 2003 and March 2012. Of these, only 295 (15.5%) were positive cultures. Of the 295 positive cultures, 146 results were excluded according to the exclusion criteria outlined in the methods section. Gram-stain microscopy reports were unavailable in 6 cases of the remaining 149 results and these were excluded as correlation of the two tests was not possible. This gave a final cohort size of Of 143 positive synovial fluid cultures, Gram-stain microscopy was negative in 111 cases. This gives a false negative rate of 78% for Gram-stain microscopy.Septic arthritis is an orthopaedic emergency. Early diagnosis and intervention is essential to limit the associated morbidity and mortality. Delayed treatment of septic arthritis can lead to rapid destruction of the joint articular cartilage resulting in pain, stiffness, loss of function, overwhelming sepsis, and death. Reports in the literature suggest case-fatality to be as high as 11% . IrreverJoint aspiration for synovial fluid analysis and culture is the standard investigation in suspected septic arthritis. Aspirates are sent for Gram-stain microscopy, culture, and polarised light microscopy; the latter of which can diagnose the crystal arthropathies gout and pseudogout, important differential diagnoses in suspected septic arthritis. Gram-stain microscopy of synovial fluid can rapidly confirm a diagnosis of septic arthritis but carries an inherent false negative rate between 25 and 50% –3. Our sAn extensive literature review published by Swan et al. in 2002  highlighThe significant morbidity and mortality associated with septic arthritis demand a rapid and accurate investigation capable of confirming or negating the diagnosis. The high sensitivity (75–95%) of synovial fluid culture makes it a useful investigation for confirmation of septic arthritis, but as culture takes 24–48 hours or more to complete, its usefulness in directing initial management is limited. At the time of writing the only paper analysing the sensitivity and specificity of Gram-stain microscopy was published by Shmerling in 1994 . The maiWith Gram-stain microscopy still relied upon in the diagnosis of septic arthritis, it is striking that in the intervening 20 years since Schmerling's research to determine the accuracy of this test very few further studies have since challenged its diagnostic value or suggested improved alternatives. Regardless of the initial accuracy of Shmerling's findings, Gram-stain microscopy in our department has a false negative rate of 78% for diagnosis of septic arthritis, significantly higher than that reported in the literature (25–50%). One explanation for this could be that the actual false-negative rate for Gram-stain microscopy is higher than that previously documented by Shmerling, and this calls into questioning the value of the test as a diagnostic tool.It is worth emphasising that the diagnosis of septic arthritis remains first and foremost clinical, and “patients with a short history of a hot swollen and tender joint with restriction of movement should be regarded as having septic arthritis until proven otherwise” .An important limitation of our study is that our results were not correlated clinically. This study assumed that synovial fluid culture is the gold standard for diagnosis of septic arthritis despite the fact that this investigation carries an inherent false-negative rate of 5–25% . It is pGood aseptic technique will reduce the incidence of sample contamination, but the main target area to improve sensitivity of Gram-stain microscopy should be the method used to collect and process the synovial fluid.In our department, synovial fluid is currently sent for Gram-stain, microscopy, and culture in a Sterilin sterile pot. Although synovial fluid under normal circumstances contains no clotting factors, acute inflammatory conditions such as septic arthritis, osteoarthritis, or crystal arthropathies can cause local soft tissue swelling. As a result of increased local vascular permeability, clotting factors can become present in synovial fluid, causing coagulation of the fluid in the container before it has reached the lab for analysis . Due to Research into varying methods of synovial fluid collection already exists. Brannan and Jerrard recommended the use of tubes containing EDTA anticoagulant for collection of synovial fluid for microscopy . The useIn making recommendations for collection of synovial fluid, ease of use must also be taken into account. It is for this reason that we suggest the use of a 2.0 mL polystyrene Lithium Heparin bottle such as the one marketed by Teklab for microscopy and for crystal analysis. The Lithium Heparin bottle we suggest using is manufactured aseptically and the Lithium Heparin solution is sterile; however the product is not assembled in a formally sterile environment. The manufacturers are able to sterilise the product after assembly but this does not rule out the small possibility of contamination of the product during assembly, which could result in false-positive Gram-stain microscopy results. An end-sterilised Lithium heparin container in any event would be advisable, so as to avoid inadvertent cross-contamination to any synovial fluid subsequently deposited into another container for culture, if the arthrocentesis syringe needle is not changed between samples. Changing needles on syringes is best avoided, due to the risk of needlestick injury, and this step would not be required if a sterile Lithium heparin bottle is used. For synovial fluid culture, there is evidence available proposing direct collection into blood culture bottles, thus reducing manipulation of the sample and so reducing risk of contamination –18. MoreArthrocentesis should be performed using aseptic technique.Synovial fluid samples for Gram-stain microscopy should be sent in a sterile Lithium Heparin container.For culture, synovial fluid should be inoculated into suitable blood culture bottles, taking care to avoid introducing contamination during this step.We suggest evaluation of these recommendations in our Orthopaedics, Rheumatology, and Accident and Emergency Departments. A prospective audit will be undertaken to ascertain whether this reduces the false-negative rate for Gram-stain microscopy in septic arthritis.


However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro . We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.Human follicular fluid, considered sterile, is aspirated as part of an  Follicular fluid, which surrounds the oocyte during From September 2007 to November 2008, couples commencing fully stimulated IVF cycles at Wesley-Monash IVF in Brisbane, Australia were invited to enrol in this study. Thirty-six follicular fluid specimens were randomly selected for testing and included equal numbers of clear and blood-stained fluids.Ethical approval was obtained from the review boards of Uniting Care Health, Human Research Ethics Committee and Queensland University of Technology Human Ethics Committee. All patients provided informed written consent for their follicular fluids to be used in this study.Follicular fluid was collected by the IVF clinicians at the time of oocyte retrieval as previously described 2 or anaerobically at 37°C for the isolation and identification of microorganisms.Isolation and identification of microbial species from the 36 human follicular fluid specimens were performed as previously described DNA extraction was performed on 1 mL aliquots of each follicular fluid specimen, and then the extracted DNA used as a template for 16 S rRNA PCR. The preparation of PCR products for sequencing was performed as per the Australian Genome Research Facility instructions for the preparation of purified DNA . AGRF sequenced each purified PCR product using a Big Dye 3 sequencing technology (BDT) labelling sequencing platform. The sequence, obtained in a FASTA format was entered into the Basic Local Alignment Search Tool  for identification of clinical isolates.Twelve frozen follicular fluid aliquots were thawed, and 1 µl of each cultured on a range of solid agar plates and in thioglycollate broth containing estradiol and progesterone , at concentrations of 375 µg/L and 800 µg/L respectively  From 24 follicular fluid specimens 1 mL of follicular fluid was aliquoted aseptically into 1.7 mL microcentrifuge tubes and incubated at 37°C aerobically. These 24 specimens were vortexed and subcultured daily: 1 µL of the follicular fluid was subcultured, onto horse blood agar and anaerobic blood agar plates (Oxoid) and incubated aerobically and anaerobically at 37°C. After (24 hours) incubation, the number of colonies on the horse blood agar were counted and expressed as the number of colony forming units (CFU)/mL of follicular fluid.From the same 24 follicular fluid specimens 300 µL was added to a sterile 13 mm coverslip in a well of a 24-well microtiter plate for the biofilm assay. Triplicate microtiter plates  were prepared and subsequently incubated in aerobic and anaerobic conditions at 37°C. Two coverslips were stained as described below, and the bacteria from the third coverslip were inoculated onto media as described in Methods 1.9. The follicular fluid specimens were incubated within the microtiter plates and left undisturbed for ten days, to mimic the average period of development of follicular fluid within a maturing ovarian follicle Biofilms were viewed by light microscopy using an Olympus BX41 light microscope , and images captured with a MicroPublisher 3.3 RTV camera  and QCapture Pro software . Biofilms were examined under total magnifications of ×100 and ×400.Images of the biofilms also were acquired by a TCS SP5 confocal laser scanning microscope  equipped with a Leica HCX PL APO CS ×10 objective, and a Leica HCX PL APO CS × 63 oil immersion objective (NA 1.4). To visualise the Congo red and carbol fuschin signal, the excitation wavelength was set at 561 nm, and the fluorescence emission was detected between 567–668 nm. A series of z stack images were acquired through a volume of 60 µm and the images analysed using LAS AF (Leica Microsystems).et al.Biofilm maturity was graded based on the presence of key characteristic and structural features described previously by Simmons Lactobacillus gasseri and Bacteroides fragilis). 300 µL of each of the follicular fluid specimens or ATCC overnight broth cultures was added to a single sterile 13 mm coverslip in each well of a 24-well microtiter plate. Plates were performed in dulplicate so that one set was grown for 5 days, and the other 10 days. Glass coverslips were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 24 hours at 4°C. After primary fixation, the coverslips were washed three times for 10 minutes in 0.1 M cacodylate buffer and post fixed for one hour in either 1% Osmium tetroxide or 1% Osmium tetroxide with 0.15% ruthenium red. Post-fixation, coverslips were twice washed in water and dehydrated in an ascending ethanol series ) before drying with 100% hexamethyldisilazane (HMDS) (Sigma Aldrich). Specimens were mounted on aluminium stubs with adhesive carbon tape and then sputter coated with 10 mm of gold (Leica SCD005 sputter coater). Examination of samples was performed using a FEI Quanta 3D Focused Ion Beam SEM, operating at 10 kv.SEM was used to visualise biofilm production for four of the follicular fluid specimens and two ATCC controls (The third coverslip (from Methods 1.7) was rinsed with sterile PBS; a sterile swab was used to remove the biofilm from the coverslip surface. This bacterial suspension was made in thioglycollate broth; a 1 µL calibrated inoculating loop was used to subculture the biofilm onto horse blood agar and anaerobic blood agar plates (Oxoid). Plates were incubated aerobically at 37°C for 24–48 hours and at 37°C for up to 7 days. Isolated bacterial species were identified as previously described Lactobacillus species. Propionibacterium spp. (n = 5), Peptostreptococcus spp. (n = 2), or Salmonella enterica (n = 1) were isolated in the remaining specimens. Two bacterial species were isolated from 6/24 (25%) specimens, and five of these (83%) contained Lactobacillus spp. Only 3/24 (12%) follicular fluid specimens contained three bacterial species and Lactobacillus spp. was isolated from each of these specimens. Lactobacillus spp. and Propionibacterium spp. were the most prevalent isolates detected in follicular fluid, isolated from 51% and 14% specimens respectively  of the specimens, of which 7/15 (47%) contained ectively . Only 133–>106). However, differences were observed in the growth patterns of bacteria cultured in thioglycollate broth (with and without hormones) for 5/12 follicular fluid specimens tested (Lactobacillus spp. were recovered from 7/12 (58%) thioglycollate broths. For one specimen , after which the number of cultivable bacteria reached a plateau and the numbers for these bacteria remained constant for the remaining 27 weeks (103–106). Furthermore, after 5–8 days incubation, only a single bacterial species in pure culture could be isolated from all follicular fluid specimens. With the exception of S. enterica, all of the bacterial species isolated after eight days were Gram-positive . Viable Gram-positive bacteria could be recovered from these follicular fluids, which were incubated in vitro for 28 weeks.All follicular fluid specimens cultured in vitro after incubation at 37°C for 18/24 (75%) of follicular fluids. Of these biofilms, 14 were monomicrobial and four were polymicrobial as determined by the number of different colony types identified by traditional microbiological culture. The bacterial species L. gasseri, L. crispatus, Bifidobacterium longum, S. agalactiae, S. anginosus and S. entericus, if initially present in follicular fluid, were always recovered from the biofilm well sub-cultures after 10 days of in vitro culture. Other bacterial species, including CoNS, Peptostreptococcus spp. and E. coli, were only cultured from the original follicular fluid specimens. In 16/24 (66%) of the follicular fluids tested, the primary culture demonstrated polymicrobial colonisation, but only a single species was detected after 10 days of incubation within each well of the microtiter plate. In addition, if only a single species was detected in the primary follicular fluid culture, the same single species was always detected in the microtiter well culture. The facultative anaerobic species, Staphylococcus spp., Streptococcus spp. and Lactobacillus spp., were isolated following incubation under both aerobic and anaerobic conditions. In contrast, the strict anaerobes Bifidobacterium spp., and Peptostreptococcus spp. were isolated only when the follicular fluid specimens were incubated anaerobically.The 24 follicular fluid specimens were tested to determine their ability to form biofilms on glass coverslips in wells of a 24-well plate. Biofilms formed Lactobacillus spp. biofilms observed for follicular fluids incubated both under aerobic and anaerobic atmospheric conditions. Variations in the architecture of mature biofilms was observed, ranging from flat homogenous cell layers through to cell clusters, microcolonies and towers with channelling and significant quantities of amorphous extracellular material between the more complex heterogeneous structures  was used to visualize the surface topography of biofilms grown ycocalyx . The prein vitro. These results demonstrate that bacterial species that colonise follicular fluid, or gain access to the follicular fluid at the time of oocyte retrieval, may be a relevant focus of infertility investigations in couples with idiopathic infertility or repeated adverse IVF treatment outcomes.Follicular fluids collected at the time of trans-vaginal oocyte retrieval were found to harbour bacteria and continued to support the bacterial growth in vitro, elevated levels of estradiol and progesterone in thioglycollate broth media enhanced the growth of the high numbers of CFUs of Lactobacillus spp., which were originally present within follicular fluid specimens. This is consistent with Lactobacillus spp. growth in the lower genital tract. At puberty, as estrogen levels increase, the vaginal pH drops and Lactobacillus spp. dominate the vaginal microflora Lactobacillus spp. decline Lactobacillus spp. increases L. crispatus, L. gasseri or L. jensenii) were predominant. These results are consistent with the findings reported in this paper, as these species were the only lactobacilli isolated from follicular fluid (collected prior to ovulation when estradiol levels were highest), although Bifidobacterium spp. was also isolated from the hormone-supplemented media. Both genera can metabolise carbohydrates from glycogen degradation in response to elevated estradiol levels, making the female genital tract a niche environment, enhancing the ability of the lactobacilli to persist and protect the genital tract epithelium from opportunistic infection Bifidobacterium spp., whilst traditionally accepted as members of the normal regional flora of the gastrointestinal tract, have more recently been detected in the vaginal flora of healthy women Our study demonstrated that in vitro as they do in vivoDuring an IVF treatment cycle, hormones including follicle stimulating hormone and human chorionic gonadotropin are administered to trigger the simultaneous maturation of multiple ovarian follicles. This results in the production of elevated levels of estradiol and progesterone within the follicular fluids collected from women undergoing trans-vaginal oocyte retrieval for IVF at levels approximately eight and three times higher, respectively, than those of women with normal cycles Propionibacterium spp., Streptococcus spp. and E. coli) were isolated from follicular fluid. The hyaluronidase virulence factor of these species could lead to poor quality cumulus cells via enzymatic hyaluronan degradation, decreased steroid hormone synthesis and ultimately to detrimental effects of these more pathogenic bacteria within the follicle. Our findings indicate that high concentrations of steroid hormones within ovarian follicular fluid can influence bacterial growth in a species-dependent manner.Studies have demonstrated that the hormone concentration within maturing follicles is significantly higher than that found in the systemic circulation, as it is governed by the hyaluronan composed cumulus-oocyte complex in vivo and may facilitate continual microbial colonisation of the follicular fluid. Gurgan et al. S. aureus, S. agalactiae and L. monocytogenes, as these species did not survive in vitro for longer than four days. However, Gurgan et al. Candida albicans for up to 15 days post-inoculation. These results are inconsistent with those reported here, where predominantly Gram-positive species survived, most likely as a result of the composition of the follicular fluid, which may have been significantly altered by the filtration and centrifugation prior to inoculation. To the best of our knowledge, our study is the first continuous follicular fluid culture experiment lasting 28 weeks, indicating that follicular fluid is an excellent growth medium for microorganisms.We demonstrated that follicular fluid supported the survival of viable bacteria for a period of at least 28 weeks. This growth occurred without the addition of supplemental nutrients or the removal of metabolic waste products; such processes that would occur in vitro when cultured on vaginal epithelial cells in vivo on the surface of intrauterine devices Many of the bacteria that we detected within follicular fluid have previously been shown to form biofilms in vitro incubation. This may be due to competition amongst the lactobacilli themselves, or because of alterations in adhesion. It has been reported that some bacterial species can co-colonise the surface of urogenital tract cells already colonised by lactobacilli; however, after lactobacilli are established, they can cause other species of bacterial cells to detach from the epithelium, which may offer a defence mechanism by preventing attachment of pathogens to the epithelium in vitro biofilms reported in this study might therefore be due to the lactic acid and H2O2 production by Lactobacillus spp.The majority of mature biofilms in the present study revealed only a single cultivable species after 10 days, even when two different species of lactobacilli were initially isolated from the follicular fluid prior to Studies of biofilms have reported that bacteria can exist in a viable, but non-cultivable state in vitro development of various grades of biofilms in human follicular fluid  present in the early stage of biofilm formation prior to permanent attachment to the conditioning film. Alternatively, the follicle may have been colonised by non-biofilm forming bacterial species. Further studies examining the effect of these biofilm bacterial species on each other would therefore, be beneficial to our understanding of the impact of single species and polymicrobial biofilms on reproductive health outcomes.in vitro growth of some microbial species; (2) follicular fluid supports the long-term survival of microorganisms; and (3) bacteria isolated from follicular fluid can form biofilms in vitro. Further characterisation of the microorganisms detected in follicular fluid and their metabolites will increase our understanding of the effects of follicular fluid microorganisms on oocyte quality, on IVF fertilisation and pregnancy rates, and on early pregnancy events. Knowledge of the microorganisms present within this anatomical niche may lead to an improved understanding of ovarian resistance to infection and also on IVF outcomes.The findings reported in this paper have shown that (1) the ovarian steroid hormones (estradiol and progesterone) are capable of modulating the


An analysis is carried out to study the steady two-dimensional stagnation-point flow of a nanofluid over a stretching/shrinking sheet in its own plane. The stretching/shrinking velocity and the ambient fluid velocity are assumed to vary linearly with the distance from the stagnation point. The similarity equations are solved numerically for three types of nanoparticles, namely copper, alumina, and titania in the water-based fluid with Prandtl number Pr = 6.2. The skin friction coefficient, Nusselt number, and the velocity and temperature profiles are presented graphically and discussed. Effects of the solid volume fraction φ on the fluid flow and heat transfer characteristics are thoroughly examined. Different from a stretching sheet, it is found that the solutions for a shrinking sheet are non-unique. Stagnation-point flow, describing the fluid motion near the stagnation region of a solid surface exists in both cases of a fixed or moving body in a fluid. The two-dimensional stagnation-point flow towards a stationary semi-infinite wall was first studied by Hiemenz , who useAll studies mentioned above refer to the stagnation-point flow towards a stretching/shrinking sheet in a viscous and Newtonian fluid. The present paper deals with the problem of a steady boundary-layer flow, heat transfer, and nanoparticle fraction over a stagnation point towards a stretching/shrinking sheet in a nanofluid, with water as the based fluid. Most conventional heat transfer fluids, such as water, ethylene glycol, and engine oil, have limited capabilities in terms of thermal properties, which, in turn, may impose serve restrictions in many thermal applications. On the other hand, most solids, in particular, metals, have thermal conductivities much higher, say, by one to three orders of magnitude, compared with that of liquids. Hence, one can then expect that fluid-containing solid particles may significantly increase its conductivity. The flow over a continuously stretching surface is an important problem in many engineering processes with applications in industries such as the hot rolling, wire drawing, paper production, glass blowing, plastic films drawing, and glass-fiber production. The quality of the final product depends on the rate of heat transfer at the stretching surface. On the other hand, the new type of shrinking sheet flow is essentially a backward flow as discussed by Goldstein  and it sMany of the publications on nanofluids are about understanding of their behaviors so that they can be utilized where straight heat transfer enhancement is paramount as in many industrial applications, nuclear reactors, transportation, electronics as well as biomedicine and food. The broad range of current and future applications involving nanofluids have been given by Wong and Leon . NanofluThe nanofluid model proposed by Buongiorno  was veryy > 0 driven by a stretching/shrinking surface located at y = 0 with a fixed stagnation point at x = 0 as shown in Figure Uw (x) and the ambient fluid velocity U∞ (x) are assumed to vary linearly from the stagnation point, i.e., Uw (x) = ax and U∞ (x) = bx, where a and b are constant with b > 0. We note that a > 0 and a < 0 correspond to stretching and shrinking sheets, respectively. The simplified two-dimensional equations governing the flow in the boundary layer of a steady, laminar, and incompressible nanofluid are (see [Consider the flow of an incompressible nanofluid in the region are (see )andsubject to the boundary conditionsu and v are the velocity components along the x- and y- axes, respectively, T is the temperature of the nanofluid, μnf is the viscosity of the nanofluid, αnf is the thermal diffusivity of the nanofluid and ρnf is the density of the nanofluid, which are given by Oztop and Abu-Nada [where Abu-Nada ρCp)nf is the heat capacity of the nanofluid, knf is the thermal conductivity of the nanofluid, kf and ks are the thermal conductivities of the fluid and of the solid fractions, respectively, and ρf and ρs are the densities of the fluid and of the solid fractions, respectively. It should be mentioned that the use of the above expression for knf is restricted to spherical nanoparticles where it does not account for other shapes of nanoparticles [μnf has been approximated by Brinkman [μf containing dilute suspension of fine spherical particles.Here, φ is the nanoparticle volume fraction,  can be expressed in a simpler form by introducing the following transformation:η is the similarity variable and ψ is the stream function defined as u = ∂ψ/∂y and v = -∂ψ/∂x, which identically satisfies Eq. 1. Employing the similarity variables (6), Eqs. 2 and 3 reduce to the following ordinary differential equations:where subjected to the boundary conditions (4) which becomeη, Pr(= vf/αf) is the Prandtl number, and ε is the velocity ratio parameter defined asIn the above equations, primes denote differentiation with respect to ε > 0 for stretching and ε < 0 for shrinking.where Cf and the local Nusselt number Nux, which are defined asThe physical quantities of interest are the skin friction coefficient τw and the surface heat flux qw are given bywhere the surface shear stress μnf and knf being the dynamic viscosity and thermal conductivity of the nanofluids, respectively. Using the similarity variables (6), we obtainwith x = U∞x /νf is the local Reynolds number.where Ref"(0) and θ'(0), where all profiles satisfy the boundary conditions (9) asymptotically but with different shapes. The effects of the solid volume fraction of nanofluid φ and the Prandtl number Pr are analyzed for three different nanofluids, namely copper (Cu)-water, alumina (Al2O3)-water, and titania (TiO2)-water, as the working fluids. Following Oztop and Abu-Nada [φ ≤ 0.2) in which φ = 0 corresponds to the regular fluid. The thermophysical properties of the base fluid and the nanoparticles are listed in Table ε, as presented in Table f"(0) for φ ≠ 0 are also included in Table m = 1 in Yacob et al. [2O3 and TiO2. This is due to the physical properties of fluid and nanoparticles , see Table Numerical solutions to the governing ordinary differential Eqs. 7 and 8 with the boundary conditions (9) were obtained using a shooting method. The dual solutions were obtained by setting different initial guesses for the missing values of Abu-Nada  or KhanaAbu-Nada , the vallumina AlO3-water,lumina AlO3-water,b et al. . These tf"(0) and -θ'(0) with ε are shown in Figures ε and nanoparticle volume fraction φ. These figures show that there are regions of unique solutions for ε > -1, dual solutions for c ε< ε ≤ -1 and no solutions for ε <c ε< 0, where c εis the critical value of ε. Based on our computation, c ε= -1.2465. This value of c εis in agreement with those reported by Wang [f"(0) and -θ'(0) are stable and physically realizable, while the second solutions are not. The procedure for showing this has been described by Weidman et al. [f"(0) is zero when ε = 1. This is due to the fact that there is no friction at the fluid-solid interface when the fluid and the solid boundary move with the same velocity. The value of f"(0) is positive when ε < 1 and is negative when ε > 1. Physically positive value of f"(0) means the fluid exerts a drag force on the solid boundary and negative value means the opposite. We notice that ε = 0 correspond to Hiemenz [ε = 1 is a degenerate inviscid flow where the stretching matches the conditions at infinity [The variations of  by Wang  and Bach by Wang ,10. Furtn et al. , Merkin n et al. , and vern et al. , so thatφ for three different of nanoparticles: copper (Cu), alumina (Al2O3), and titania (TiO2) with ε = 0.5. These figures show that these quantities increase almost linearly with φ. The presence of the nanoparticles in the fluids increases appreciably the effective thermal conductivity of the fluid and consequently enhances the heat transfer characteristics, as seen in Figure 2 nanoparticles due to domination of conduction mode of heat transfer. This is because TiO2 has the lowest thermal conductivity compared to Cu and Al2O3, as presented in Table 2O3 is negligible. The thermal conductivity of Al2O3 is approximately one tenth of Cu, as given in Table 2O3 is its low thermal diffusivity. The reduced value of thermal diffusivity leads to higher temperature gradients and, therefore, higher enhancement in heat transfers. The Cu nanoparticles have high values of thermal diffusivity and, therefore, this reduces the temperature gradients which will affect the performance of Cu nanoparticles.Figures φ = 0). The terms first solution and second solution refer to the curves shown in Figures f"(0) and -θ'(0) compared to the second solution. Figures The samples of velocity and temperature profiles for some values of parameters are presented in Figures 2O3), and titania (TiO2) in the water-based fluid with Prandtl number Pr = 6.2, to investigate the effect of the solid volume fraction parameter φ on the fluid and heat transfer characteristics. Different from a stretching sheet, it is found that the solutions for a shrinking sheet are non-unique. The inclusion of nanoparticles into the base water fluid has produced an increase in the skin friction and heat transfer coefficients, which increases appreciably with an increase of the nanoparticle volume fraction. Nanofluids are capable to change the velocity and temperature profile in the boundary layer. The type of nanofluids is a key factor for heat transfer enhancement. The highest values of the skin friction coefficient and the local Nusselt number were obtained for the Cu nanoparticles compared with the others.We have presented an analysis for the flow and heat transfer characteristics of a nanofluid over a stretching/shrinking sheet in its own plane. The stretching/shrinking velocity and the ambient fluid velocity are assumed to vary linearly with the distance from the stagnation point. The resulting system of nonlinear ordinary differential equations is solved numerically for three types of nanoparticles, namely copper (Cu), alumina (AlThe authors declare that they have no competing interests.NB and AI performed the numerical analysis and wrote the manuscript. IP carried out the literature review and co-wrote the manuscript. All authors read and approved the final manuscript.


The aim of this study was to assess and compare the ability of the automatically and continuously measured pulse pressure variation (PPV) obtained by an IntelliVue MP monitor and stroke volume variation (SVV) measured by FloTrac™/Vigileo™ to predict fluid responsiveness in septic shock patients.We conducted a prospective study on 42 mechanically ventilated septic shock patients. SVV, PPV and other hemodynamic data were recorded before and after fluid administration with 500 ml of 6% tetrastarch. Responders were defined as patients with an increase in cardiac index ≥15% after fluid loading.r = 0.96, P < 0.001). Twenty-seven (64.3%) patients were classified as fluid responders. PPV and SVV were significantly higher in responders than in nonresponders . There was no difference between the area under the receiver operating characteristic curves of PPV (0.983) and SVV (0.99). The optimal threshold values to predicting fluid responsiveness were 10% for PPV  and 10% for SVV .The agreement (mean bias ± SD) between PPV and SVV was -0.59 ± 1.72% (Figure The automated PPV, obtained by the Intellivue MP monitor, and the SVV, obtained by FloTrac™/Vigileo™, showed comparable performance in terms of predicting fluid responsiveness in mechanically ventilated patients with septic shock.


Olfactory groove meningioma is a demanding therapeutic problem involving two medical specialties, otolaryngology and neurosurgery. The use of transnasal endoscopic (TNE) approach to the tumour has been proved effective in many publications. Three patients with meningiomas localized in olfactory groove were treated in 2011 and 2012 by the otolaryngologist-neurosurgeon team using TNE approach and neuronavigation. The diagnosis was based on MR and CT images. In all patients after tumour removal an endoscopic anterior cranial fossa floor reconstruction was performed using homogeneous cartilage or titanium mesh and Hadad-Bassagasteguy flap. During postoperative period in all patients lumbar drainage was used. There were no cerebrospinal fluid leakage episodes. No recurrence was observed in 22, 12 and 8 months of follow-up, respectively. The authors describe otolaryngological and neurosurgical aspects of TNE approach to anterior cranial fossa with special regard to possible radical resection (according to Simpson) and reconstruction of the bony postoperative defect. TNE is a feasible operative method in olfactory groove meningioma management due to good tumour visibility, lack of brain traction, limited neurovascular structure manipulation and acceptable risk of neurological deficiencies when compared to open approach. Cosmetic aspect and short hospitalization is also of great importance. Meningiomas are benign tumours of the central nervous system originating in meningothelial cap cells of arachnoid tuberculations and characterized by diverse growth dynamics. Cerebral falx region, cranial vault, sphenoid bone wings, perisellar region and olfactory groove are the most common primary locations for this tumour. Adults between 40 and 60 are most likely to be diagnosed with meningioma with higher prevalence in females when compared to males. Primary location implies occurrence of certain symptoms, as well as, the method of treatment , 2, 8, 9Localized inside olfactory groove, meningiomas are scarcely symptomatic or present with non-specific, common symptoms (e.g. headache) or even symptoms overlooked by the patients . Progress in tumour size implies more serious disturbances connected with impression on optic nerves or chiasm , frontal lobe  or seizures , 9.Typical treatment entails surgical resection via external approach: frontal/bifrontal, pterional , 8, 9. TIn the available literature we have found only three papers describing series of patients with tumours of such location treated with transnasal endoscopic approach (TNE) . Taking into consideration small number of these reports we would like to share our experience in this method of treatment.The records of three patients  treated by our team in 2011 and 2012 have been analyzed. The patients were 52, 57, 66 years of age, respectively. In each patient the leading symptom was unilateral anosmia observed within 1–2 years before the treatment. Two patients had a history of several episodes of seizures, one with concomitant verbal and logical contact deterioration. One patient presented with vertigo and nausea several days before the admission. However, none of the patients complained of very common in such a case oppressive headache. The diagnosis was based on CT and MR imaging Figs. . All of o optics the cribrose palate was unveiled. In each case the middle turbinate was excised but nasal septum remained intact which later served for reconstruction. After visualization of the cribrose palate by means of neuronavigation the tumour was localized  leakage due to the impression of the tumour in the defect in the skull base  with tracheal intubation on a patient in supine position with the head fixated and extended by 15°. During the whole operation controlled hypotension was induced with the mean blood pressure of 90 mmHg. The first step was local nasal mucosa decongestion with a solution of epinephrine . Then, using the endoscope with 0zed Fig.  and mucoo, 45o and 70o). Crista gali or falx cerebri were not approached during the operation. In the meantime a portion of the fat tissue, fragment of the fascia lata and quadriceps muscle was harvested. The reconstructive stage was performed by the otolaryngologist. The postoperative cavity was firstly filled with pieces of fat and muscle tissue, then with TachoSil and finally the fascia. We used tissue adhesive between the two layers. In the first patient, in which the defect in the skull base was the largest, for better reconstruction the titanium mesh was used. In the remaining two cases the “dense part” of the skull base was reconstructed with a fragment of the septal cartilage. Finally, the wound was covered with pedicled nasoseptal flap. In each case the multilayer reconstruction was performed according to the scheme: fat tissue-tissue adhesive-cartilage/mesh-fascia-tissue adhesive-nasoseptal flat-nasal packing. Tumour sizes and the reconstructive material are shown in the table.The next stage of the operation was led by a neurosurgeon under a microscope using an automatic nasal retractor. Once the skull base was unveiled with the assistance of neuronavigation the centre of the tumour was identified and served as the target for drilling. Once the tumour was visualized, the neurosurgeon precisely assessed its margins, then separated it from the surrounding tissues, dissected from the matrix and removed it. Brain tissues remained intact. We tended to remove the tumour in one piece which was achieved in 2 out of 3 cases. In each case the resection was assessed as total. Haemostasis was achieved by means of bipolar diathermy. No significant bleeding was observed in all cases. The control of the postoperative cavity was performed endoscopically . In order to visualize the base of anterior cranial fossa (olfactory groove), as well as, the sellar region we considered it vital to resect the middle turbinate. However, contrary to the approach via sphenoid bone to the olfactory groove, it is not necessary to remove the posterior part of the nasal septum , 10. Lacdoscopes , 4, 10.A crucial condition of the total resection is the removal of the starting point of the tumour. Many authors emphasize that in all of the patients the aim of the operation remains total or near-total resection >95 %) of the tumour , 2, 11. 5 % of th2) requires multilayer closure. In the first stage, the cavity after resection was filled with fat and muscle tissue and fragment of fascia lata using underlay method (between the matrix and the bone). The packing was then joined with adhesive tissue. Secondly, a fragment of nasal cartilage or a titanium mesh in one case, was placed superficially (case number 1—the aperture size exceeded 7.5 cm2). Finally, the wound was covered with a TachoSil  and a vascularised mucosal flap. Introduction of local pedicled flap based on the posterior vessels of the nasal septum containing mucoperiosteum and mucoperichondrium (Hadad-Bassagasteguy flap) significantly improved robustness of the multilayer bung [The defect in the skull base (usually sized 2–5 cmyer bung –6, 11. Tyer bung  reservedTransnasal endoscopic approach constantly gains popularity as a feasible and effective therapeutic method for olfactory groove meningioma. Up to date there have been 30 reported cases in available literature , 10, 14.


Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro- and antitumourigenic properties have been attributed to NO.The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated.in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression.Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer. Progressive tumour growth and survival is dependent on angiogenesis, the development of a functional blood supply to provide nutrients and remove waste products. Without the concomitant growth of new blood vessels or cooption of existing capillary networks, tumours cannot expand beyond a minimal size or metastasise to distant sites . Angiogein vivo (A plethora of angiogenic stimuli induce the production of nitric oxide (NO), an important inter- and intracellular signalling molecule synthesised from arginine by NO synthases (NOS). Of the three isoforms of NOS, specifically inducible (iNOS), endothelial (eNOS) and neuronal, iNOS produces the greatest amounts of NO and is associated with a number of pathologies  was shown to induce an acute and sustained reduction in human tumour blood volume, providing clinical evidence that inhibition of NOS has tumour antivascular effects , and complemented with fluorescence microscopy.In this study we have investigated the role of tumour cell-derived iNOS on growth and angiogenesis in a model of glioma, which clinically represents one of the most vascularised human cancers  fetal calf serum. The antisense iNOS stable-transfected cell lines (subsequently termed AS lines) were produced by transfection with the pciNOS500 plasmid using the poly L-ornithine method (μg ml−1). For the generation of the pciNOS500 plasmid, the fragment of murine iNOS (5707–6247 bp), previously shown to be an effective antisense target (BamHI restriction enzyme and subcloned into the BamHI site in pcDNA 3.1 (+)/hygro vector .The rat glioma C6 cell line  was maintained in Nutrient Ham's F-10  culture medium containing 2 m−1 TNF-α, 100 U ml−1 IFN-γ, 5 μg ml−1 LPS for 24 h), and from tumour homogenates of AS7 and C6 tumours .Cell extracts were generated from stably transfected antisense cell lines  and parental C6 cells with cytokine stimulation  was determined using a dye-binding assay, in which the concentration of the eluted dye is linearly correlated to the cell number (μl fixation solution (10% (v/v) formalin in 5% (w/v) polyvinylpyrrolidone) for 5 min. The wells were then washed three times with water and air-dried. The cells were stained with 600 μl of filtered 0.04% (w/v) Coomassie Blue R250 (Sigma) in 25% (v/v) ethanol/12% (v/v) acetic acid for 1 h, washed three times with 600 μl of 10% (v/v) ethanol/5% (v/v) acetic acid and allowed to dry. Cell growth was quantified by eluting the stain in 600 μl filtered solution of 1 M potassium acetate in 70% ethanol for 1 h and measuring absorbance at 620 nm using a Titertek Multiscan microtitre plate reader .The growth of C6 and AS7 cells under normal culture conditions or after cytokine stimulation .All procedures were performed in accordance with the local ethical review panel, the UK Home Office Scientific Procedures Act 1986 and the UKCCCR guidelines (in vivo using susceptibility contrast MRI (R2* (s−1) of water in the surrounding tissue. Changes in tissue R2* caused by USPIO particles are dependent on blood volume; hence, an estimate can be made by measuring the absolute changes in R2* following administration of USPIO particles. As USPIO particles probe for blood vessels that are functional at the time of injection, and not necrotic areas or vessels with static blood flow, a USPIO-induced measurement of ΔR2* can provide a valuable non-invasive imaging biomarker associated with tumour angiogenesis (R2* of size-matched (∼0.8 cm3) AS7 and C6 tumours before and after the intravenous administration of 2.5 mg Fe kg−1 of the USPIO contrast agent feruglose  through a lateral tail vein. Apparent R2* maps were calculated on a voxel-by-voxel basis from the MGRE image data sets, and the average apparent R2* relaxation rates were calculated for each tumour for a region of interest encompassing the whole tumour image but excluding any surrounding skin and muscle. Having determined the ΔR2* values (R2*post-USPIO−R2*pre-USPIO), estimates of the fractional tumour blood volume  were then derived as previously described  1 min before excision of the tumour (μm) of each C6 and AS7 tumours were then visualised for Hoechst 33342 uptake by fluorescence microscopy using appropriate filters . To assess vessel density, additional frozen tumour sections were first rehydrated with PBS, and then incubated with 2% BSA/5% goat serum in PBS for 30 min at room temperature. After washing with PBS, sections were incubated with rat anti-mouse PECAM monoclonal antibody  for 1 h at room temperature, and then washed extensively in PBS/0.1% Tween-20. Biotinylated anti-rat immunoglobulins  were then applied onto the sections and incubated for 1 h at room temperature. Following extensive washing in PBS/0.1% Tween-20, sections were incubated with fluorescein streptavidin (1 : 100) in the dark for 30 min, washed with PBS/0.1% Tween-20 and mounted using Vectashield mounting medium (Vector Laboratories). Endothelial structures were subsequently visualised by fluorescence microscopy at 450–490 nm. Whole tumour sections were scanned using a computer-controlled motorised scanning stage  driven by Stage-Pro  on an IX70 fluorescence microscope . Digital images from both AS7 and C6 tumour sections were acquired using the same exposure time and composite images were synthesised. The digital images were analysed using analySIS software . Fluorescent particles were detected above a constant threshold for all the composite images, and the area of the tumour section with fluorescein and Hoechst 33342 fluorescence was determined and expressed as a percentage of the whole tumour section. Additional formalin-fixed tumour sections were cut and stained with haematoxylin and eosin to assess tissue viability of AS7, AS12 and C6 tumours.Mice bearing size-matched AS7 and C6 tumours were injected intravenously with 15 mg kg165 expression using a murine VEGF ELISA kit . In addition, tumours were excised, weighed, cut into small pieces and maintained in culture medium for 4 days, following which the secretion of VEGF165 by the tumour explants into the culture medium was also determined by ELISA , compared with parental C6 cells, and this level of inhibition of iNOS activity remained similar at the later time point of 48 h  than that of C6 tumours  derived from these data was 1.4±0.1% and 0.9±0.2% for C6 and AS7 tumours, respectively.Susceptibility contrast-enhanced MRI using the USPIO contrast agent feruglose was used to interrogate any differences in magnitude and distribution of fractional blood volume between size-matched C6 and AS7 tumours. Calculated Δurs only . The meaTumour perfusion was also assessed by quantifying the uptake of the fluorescent perfusion marker Hoechst 33342. This is a nuclear dye that, on injection, stains endothelial cells and cells immediately adjacent to tumour blood vessels. The time from injection to euthanisation was limited to 1 min to prevent excessive diffusion of the dye beyond the cells lining perfused blood vessels within the tumour. Thus, Hoechst 33342 uptake reports only on functionally perfused vessels, and also provides validation of the MRI data. Composite images of Hoechst 33342 uptake showed that, as with MRI data, AS7 tumours had fewer perfused areas compared with C6 tumours, and that these regions were restricted mainly to the tumour periphery . The perIn vitro studies of AS7 and C6 cells showed that both cell lines produced similar levels of VEGF165. Induction of iNOS with cytokines for 24 h resulted in a significant 1.5-fold upregulation of VEGF165 in both AS7 and C6 cell lines  associated with tumours, and because of local imbalances of angiogenic factors and or intermittent blood flow, some vessels within the tumour may collapse and cease to function. Although these vessels will stain positive for endothelium and contribute to the vessel density measurements, they will not necessarily contribute to tumour growth  by using an antisense approach that NO derived from tumour cell expression of iNOS is a key modulator of tumour growth and angiogenesis, and (ii) the ability of susceptibility contrast MRI to non-invasively assess the effects of tumour-derived iNOS on vascular development and function in C6 gliomas


This work analyzes several drift compensation mechanisms in wireless sensor networks (WSN). Temperature is an environmental factor that greatly affects oscillators shipped in every WSN mote. This behavior creates the need of improving drift compensation mechanisms in synchronization protocols. Using the Flooding Time Synchronization Protocol (FTSP), this work demonstrates that crystal oscillators are affected by temperature variations. Thus, the influence of temperature provokes a low performance of FTSP in changing conditions of temperature. This article proposes an innovative correction factor that minimizes the impact of temperature in the clock skew. By means of this factor, two new mechanisms are proposed in this paper: the Adjusted Temperature (AT) and the Advanced Adjusted Temperature (A2T). These mechanisms have been combined with FTSP to produce AT-FTSP and A2T-FTSP Both have been tested in a network of TelosB motes running TinyOS. Results show that both AT-FTSP and A2T-FTSP improve the average synchronization errors compared to FTSP and other temperature-compensated protocols (Environment-Aware Clock Skew Estimation and Synchronization for WSN (EACS) and Temperature Compensated Time Synchronization (TCTS)). Clock aet al. [The work of Vig  demonstret al.  demonstret al. [As is described in Section 3.1, there are several types of oscillators. The common Crystal Oscillator (XO) does not compensate for its frequency on temperature, providing a clock stability of ±20 ppm at 25°C . On the other hand, the Temperature Compensated Crystal Oscillators (TCXO) are able to manage temperature variations in order to enclose the errors within a certain range. For example, the Kyocera KT3225T TCXO improves the frequency stability to ±5 ppm. However, the enhancement obtained by TCXO requires more complexity, energy consumption and economic costs. For the previously given models, prices are US$3.10@3K for the TCXO and US$0.25@2K for the XO model. For compensating temperature with an XO, it is mandatory to include a temperature sensor, e.g., a thermistor probe with a cost of US$0.06@2K, which sum up to a total of US$0.31@2K. Therefore, as Yang This work proposes an innovative correction factor to obtain a more precise clock skew, based on temperature variations between the global and the local clocks. With this adjustment, two new synchronization approaches for WSNs are proposed: the Adjusted Temperature FTSP (AT-FTSP) and the Advanced Adjusted Temperature FTSP (A2T-FTSP). Experiments have been carried out in a network of TelosB (Rev.b) motes running TinyOS. Both AT-FTSP and A2T-FTSP improve the average synchronization error with respect to FTSP under temperature variation scenarios.2.synchronization error concept, which shows the differences between the local clock of the nodes compared to a global clock.Synchronization requirements change depending on the final application. Nevertheless, all synchronization mechanisms require the exchange of a minimal amount of messages between nodes. The number of messages is determined by the synchronization protocol used. The quality of the synchronization is measured using the 2.1.In the last 10 years, many works have been published in the WSN area. Most time synchronization approaches in the scientific literature can be grouped into two main fields: The first group finds its foundations in obtaining the synchronization through the acquisition and the processing of data  to estimet al. proposed the Reference Broadcast Synchronization Protocol [Elson Protocol  (RBS). Tet al. proposed the Timing-sync Protocol for Sensor Networks (TSPN) [With the goal of improving the RBS protocol, Ganeriwall s (TSPN) . It useset al. [The Flooding Time Synchronization Protocol (FTSP) was proposed by Maróti et al. . It is bBesides the above mentioned mechanisms, a great amount of protocols have been proposed in the literature to synchronize a WSN as, for example, the Reliable Slotted Broadcast Protocol (RBSP), which is based on the concept of broadcast time slots.2.2.The use of temperature in WSN synchronization protocols is a field of study with high relevance ,6,12. Siet al. [frequency error estimation, which determines the clock skew of the local oscillator related to the reference node of the network for every given measured temperature. The authors assume that temperature remains constant for every clock skew estimation. After obtaining the clock skew for a pair of nodes in a given temperature, a new clock skew with a new temperature value is then computed. When the complete range of temperatures has been explored, TCTS begins with the synchronization phase. If a certain temperature is not computed, the calibration process restarts again to include that new skew information. Thus, the node carries out a realistic adjustment of the frequency. Once the relationship between the frequency error and the obtained temperature is learned, the node increases the time between synchronization intervals. This allows an improvement of the synchronization period, minimizing the number of synchronization periods and, thus, optimizing the energy consumption. Schmid et al. provide simulation results during a 96 h period. For the first 24 h, TCTS is in the calibration mode. At the end of the simulation, 95% of the errors lie within ±4 tics of a 32 khz oscillator. Results show a clock stability of <0.07 ppm for a 16 h test with no resynchronization.Schmid et al.  propose et al. [et al. describe a highly accurate clock skew estimation. The authors propose an additional information-aided multi-model Kalman filter (AMKF) algorithm to dynamically compensate for the clock skew. The great benefit of EACS is the possibility of using it as a component of any conventional WSN synchronization protocol based on the clock skew estimation. This allows the update of the local clock with local information before the clock resynchronization is carried out, which improves the lifetime of the mote by increasing the synchronization period. The EACS algorithm builds up an initial table of each node of the network based on the correlation between temperature and clock skew. A Kalman filtering is applied to obtain the theoretical relationship between those components. This procedure has very large computational requirements, which does not make it a suitable method for low cost WSN. Simulation results show that the error is always below 2 ms for the whole 8, 000-s simulation. Experimental results with a Mica2 testbed provide an error of 8 ms for a 7,200-s test duration. The authors do not provide any measure of frequency stability.EACS was proposed by Yang et al. . Taking The above mentioned proposals improve the behavior of protocols as, for example, FTSP, under temperature variations. However, both approaches (i) have high computational costs and energy consumption, (ii) require a large amount of memory per node to store the relationship between temperature and clock skew, (iii) cannot manage new temperatures without a recalibration process and (iv) are constrained to always use the same reference node. Any change in these restrictions triggers the need for a new calibration.3.etc.Nowadays, many electronic devices are controlled by periodical signals. These signals are produced by electronic components, known as oscillators. They are used in a great range of devices, e.g., digital clocks, smartphones, electrical appliances, computers, vehicles, airplanes, clock skew or clock drift.The utilization of oscillators involves synchronization problems that have to be solved in order to optimize the performance of electronic devices. The oscillation frequency may vary due to different reasons. This fact produces a variation between the real oscillation frequency and the nominal frequency (which is the frequency given by the manufacturer). The above mentioned variation is usually defined as the In nature, all the elements have a resonance frequency that allows their atoms to have an oscillatory movement. Several elements can be found in nature for creating a periodical signal, but among all of them, quartz crystals are the most used ones because of their resonance capability with a fixed frequency. The oscillation frequency is mainly due to the cut of the crystal, the temperature and the voltage applied.A quartz crystal can be cut at different angles. However, the most commonly cut is the right-angled one. Tuning fork oscillators are an example of a right-angled cut. This kind of oscillator is commonly used in most WSN motes, because of its good relation between accuracy and low cost.3.1.The resonance frequency of a crystal unit can vary with temperature. Depending on the method of dealing with the frequency vs. temperature characteristic, Vig  classifiTuning-fork oscillators, which are a particular case of Crystal Oscillators (XO), are the most commonly used ones in WSNs. However, they are very sensitive to temperature variations. Their clock stability is about ±20 ppm. This kind of oscillator has a quadratic dependency on temperature, which enables the application of a correction factor to improve the clock accuracy. Consequently, it is possible to reduce temperature effects by means of software mechanisms, making them a perfect solution for low-cost WSN motes.TCXO and OCXO are less used in WSNs, due to their economic costs, footprint and energy consumption. TCXO crystal units use the information provided by an internal thermistor to generate a correction voltage that is applied to a voltage-variable reactance in the crystal network. The reactance variations produce frequency changes proportional to the temperature changes. In TCXO, the clock stability is in the range of 0.5–5 ppm. Their internal components to compensate the frequency with temperature raise their price to about US$1–100 . In OCXOFinally, there are certain microcomputer-controlled oscillators available with “self-temperature sensing”, which compensates for temperature variations in the frequency. These oscillators are known as MCXO , with a clock stability of 0.05–2 ppm. The use of a microcomputer increases their size and economic costs , making4.et al. [Castillo-Secilla et al.  demonstrUsing a star-based WSN topology with one root and four standard motes, experiments have demonstrated the influence of temperature variations in the clock skew of a tuning-fork oscillator. The root mote is responsible for the synchronization of all other motes. Once the experiment has started and all the motes are synchronized, a base station receives the local time of each mote in order to generate statistical data about the synchronization error.et al. [In the work of Maróti et al. , the topet al.  was forcThe root mote sends synchronization beacons cyclically, with a 5 s period. The base station broadcasts another beacon to obtain the local time of each mote in a 1 s frequency rate. The linear regression is computed with three elements to obtain a good temporal proximity between data.μs.Between time 0 s and 800 s, the temperature was fixed for all the network. For this period, the average synchronization error was about 1.90 At 800 s, a temperature variation of 15°C is provoked in just one of the motes of the network. As can be observed in μs.The test ends with an average synchronization error of 4.10 These results show a decrease of the performance of about 215.78%.The experiment has a duration of 0.5 h, obtaining the following results:The obtained results show a clear influence of temperature in the synchronization error. This is because of the change in the frequency of the oscillator due to temperature variations. The frequency change affects the clock skew estimation, and thus, the average synchronization error increases. These results enforce the postulates of Vig , who sta5.5.1.The motes used in this work have been TelosB (Rev. b) . These mThanks to the work of Maróti and Sallai ,18, an aThat external oscillator is classified within the tuning-fork  family. f0 is the nominal frequency, β is a constant, known as the temperature coefficient, T is the ambient temperature and T0 is the reference temperature (25°C). In real scenarios, the actual frequency (f′0) is slightly different when compared to f0 for a given temperature, T0. This deviation is known as frequency tolerance. In the CMR200T oscillator, f0 = 32,768 Hz, β = −0.034 ± 0.006 ppm, and the frequency tolerance is about ±20 ppm. This working interval is mainly due to the effects of the cut of the crystal.This work proposes two new approaches to obtain a more precise clock skew, taking into consideration the temperature variations . IncludiThe TelosB model includes the Sensirion SHT11 temperature sensor , which a5.2.Adjusted Temperature FTSP (AT-FTSP). Based on The first approach proposed in this work is called Working on clock skew value of FTSP, a new clock skew value is obtained:By using the correction factor proposed in AT-FTSP is to be used in those environments where the clock of the reference node cannot be . For example, AT-FTSP is the perfect option for a scenario where the temperature sensor of the reference node is broken, miscalibrated or is known to provide Byzantine errors. On the contrary, when the temperature of the reference node is available, A2T-FTSP, which is described below, can be used to incorporate that information in the clock skew computation process.5.3.Advanced Adjusted Temperature FTSP (A2T-FTSP). If the temperature in the root mote is known, a more precise clock skew in the local mote can be obtained. The only requirement is to have temperature sensors, both in the root and the local motes.The second approach is called clock skew of the root mote and the one of the local mote can be compared. Thus, the clock skew value of the local mote can be improved compared to AT-FTSP and FTSP approaches.By using clock skew value due to temperature effects is determined by Thus, the n ≃ βr. Using the clock skew of As the first approximation and in order to reduce the complexity of the formulation, it is possible to assume that βBefore providing accuracy results, it is possible to analyze both proposed methods from a computational point of view. It can be concluded that AT-FTSP is a less complex method than A2T-FTSP, and therefore, it is more suitable for low computational power nodes. Besides, A2T-FTSP requires one to send the temperature of the reference node in each synchronization packet, reducing the bandwidth of the communication channel. However, experimental results shown in Section 7 permit one to evaluate both methods with analytical data, taking accuracy into account.6.6.1.This section describes the different technologies used in this work: WSN protocols, operating system and WSN motes. A small review of these technologies is shown based on different works and WSN surveys.i.e., ambient monitoring, medical monitoring, security, among others. These applications can be used in real-time scenarios, and thus, it is necessary to ensure the correct performance of the network through two approaches: by optimizing the lifetime of the WSN and by using WSN synchronization protocols.Generally, a WSN is a group of low-cost and battery-powered motes. A WSN has a great range of applications, IEEE802.15.4[IEEE 802.15.4 stack is developed for its use with the TinyOS [Nowadays, the research community has a great range of low cost wireless communication technologies. For this work, E802.15.4 has beene TinyOS  operatinAs a WSN mote, the TelosB model has been selected. The TelosB platform is equipped with a Texas Instruments 16-bit MSP430 microcontroller with an internal DCO working at 1 MHZ and an external 32 kHZ oscillator mainly used by peripherals, one of which is the Sensirion SHT11 temperature sensor . In orde6.1.1.Both AT-FTSP and A2T-FTSP are based on the concept of FTSP, and thus, the configurable parameters  are basiWSN synchronization protocols are designed to determine a global network clock. In order to achieve this, the local clock of the mote has to be obtained with the lowest accumulated error. Maróti and Ganeriwal ,10 demonrfxlink as the communication driver. In the TinyOS tree, this library is located in /tinyos/lib/rfxlink. CC2420xActiveMessageC library has been used for the required data encapsulation, while keeping compatibility with CC2420x stack.There are several FTSP implementations, but, none of the published ones use the timers of TelosB motes in TinyOS to obtain microsecond accuracy The original version of FTSP for TinyOS only supports millisecond accuracy In this work, the FTSP protocol implementation for TinyOS has been improved. With this improvement, microsecond accuracy has been reached, and the results of AT-FTSP and A2T-FTSP can be compared with FTSP in microseconds. To do so, several tasks have been performed. FTSP implementation for TinyOS is based on IEEE 802.15.4 by using the CC2420 communications stack. The above mentioned stack has been developed to provide support in motes with the Texas Instruments CC2420 radio. By default, the stack for the CC2420 does not support MAC time-stamping with microsecond accuracy, and thus, it is necessary to modify it. The CC2420x stack of TinyOS can be used. This communication stack uses the As has been described in Section 5.1, the microsecond accuracy is obtained by using the internal DCO of the MSP430, which controls the timer where the local time is stored. TinyOS is responsible for the dynamic recalibration of the clock of the DCO, adjusting its working frequency (increasing or reducing it) according to the external 32 Khz oscillator frequency.6.1.2.One central mote to obtain all the synchronization data from the network, with the goal of storing the information in a file to process it with MATLAB.One mote the broadcasts beacon frames based on the BEACON_RATE parameter.Thirteen motes flashed with FTSP, AT-FTSP or A2-FTSP One of them is the root mote, which keeps the global time of the network. Between the final nodes, only three nodes (randomly selected per test) suffer from the variation of temperature, as can be seen in Section 7.The experimental IEEE 802.15.4 network is composed of:6.1.3.In order to ensure the reliability of the extracted information, each configuration of the network has been executed for 3,000 s.To determine the average synchronization error, the sequence number of each packet has been used. Packets with the same sequence number are grouped to determine the average synchronization error in each group . Once thOther parameters with statistical relevance have been obtained: variance, typical deviation and maximum error.7.This section describes the experiments carried out with temperature variations. These variations demonstrate a better performance of the new approaches compared to FTSP.Low temperature: Cases in which the temperature range is between 9°C–22°C.Intermediate temperature: In this range, the temperature is near the nominal value of the CMR200T oscillator. The temperature range is between 22°C–32°C.High temperature: The last temperature range encloses temperatures between 22°C–40°C.Three temperature-based cases of study have been considered for the experiments:Several ranges of temperature are used to study the performance and behavior of the proposed approaches. In order to obtain a good relationship between synchronization and energy consumption, a BEACON_RATE of 30 s has been selected. This rate is appropriate to test the effects of temperature in relation to the oscillator frequency. A slower synchronization rate would not clearly show the frequency variations due to temperature, and also, the energy consumption would be highly penalized. The linear regression uses three elements to obtain information about offset and clock skew, using temporally closer values.7.1.This section shows the results obtained with the configuration parameters as detailed in Section 7. All the proposed methods are affected by a temperature variation in the range of 9°C–22°C.The tests have been carried out for an elapsed time of 3,000 s. A temperature variation is created at instant 1,000 s, where the temperature has been decreased from 22°C to 9°C.At the end of this subsection, a linear graphic is shown. This one see  shows th7.1.1.μs.7.1.2.μs. After the temperature change, the average synchronization error increases more slowly compared to FTSP, reaching an average synchronization error of 1.9 μs, until time 1, 700 s. Ending that instant, the average synchronization error decreases back to the initial values. This behavior demonstrates a good performance. The final average synchronization error is 1.558 μs. The obtained speedup improves in 104.17% the results of FTSP.During the first 1,000 s of the test, a good performance of AT-FTSP is observed, with an average synchronization error of 1.1 7.1.3.μs to 1.459 μs. These results show a clear improvement, obtaining a speedup of 118% over FTSP.The graphical chart of A2T-FTSP see  shows a The inclusion of the temperature see  to obtaiTemperature-based methods improve the average synchronization error between 104.17% and 118% compared to FTSP.After analyzing the tests with a temperature variation between 22°C and 9°C with three elements in linear regression and a beacon rate of 30 s, it can be concluded that:7.2.Once research with low temperatures has ended, new tests with intermediate temperature values are carried out. Temperatures in the interval 22°C–32°C have been considered to be intermediate values.Each test starts by fixing the temperature to 22°C during 1, 000 s. After that, a temperature variation is forced, until the temperature reaches 32°C. This variation creates a modification in the clock skew of the oscillator, due to the change of the frequency, and thus, this affects the performance of the synchronization protocols. The final results are summarize in 7.2.1.In μs. A temperature variation is created at instant 1, 000 s. The influence on the synchronization error is evident, and it can be observed that the average synchronization error increases drastically at 1,100 s, 1,250 s and 1,500 s. After instant 1,500 s, the average synchronization error stabilizes to 4.624 μs. At the end of the experiment, the average synchronization error is about 3.582 μs.For the first 1, 000 s, a stable behavior can be observed, obtaining an average synchronization error of 1.488 These results show the great influence of temperature in FTSP, and once again, new approaches are required to improve this issue.7.2.2.μs has been obtained. From instant 1, 000 s until the end of the test, the temperature variation increases the average synchronization error to 1.934 μs.Attending to the ordinate axis, a great improvement with respect to FTSP can be observed, both in average synchronization error and behavior. During the first temporal interval , an average synchronization error of 1.267 μs. This result improves the average synchronization error of FTSP by about 55.6%.The final average synchronization error is about 2.302 7.2.3.The results obtained with A2T-FTSP can be studied in μs, with an almost completely constant behavior during all of this period. As is programmed, at instant 1,000 s, a temperature variation is created, going from 22°C to 32°C. At instants 1,200 s and 1,500 s, a slight increase in the average synchronization error is observed. However, once A2T-FTSP reaches instant 1, 500 s in the test, a great improvement in the average synchronization error can be observed, ending the test with an average synchronization error of 1.712 μs, which is 109.96% better than FTSP.A2T-FTSP has a great performance from the beginning of the test. For the first 1,000 s of the experiment, the average synchronization error is about 1.123 As CMR200T oscillator (25°C). As a consequence, the frequency variations are minimal, obtaining similar results between the new methods.The behavior differences between the new proposals are minimal see . This isBy using both AT-FTSP and A2T-FTSP, an improvement in the average synchronization error is obtained, ranging from 55.60% to 109.22% compared to FTSP.Analyzing the results with a temperature variation between 22°C and 32°C, it can be concluded that:7.3.The following paragraphs show the experimental results obtained with the parameters described in Section 7. These tests are affected by a temperature variation from 22°C to 40°C. The results of the above mentioned tests are shown in 7.3.1.μs. Once instant 1, 000 s is reached, the temperature is quickly increased to 40°C. In the above-mentioned μs at instant 1, 300 s is shown. After this peak, the average synchronization error decreases, until it stabilizes to 10 μs for the rest of the experiment. The average synchronization error of this test is about 7.432 μs.The results obtained with FTSP (high temperature range) are shown in 7.3.2.μs.AT-FTSP see  shows a μs. In a short period of time, the proposed clock skew module works to obtain a better synchronization error, minimizing its value to 3.679 μs for the second interval .A temperature variation at instant 1,000 is induced. This change is shown in μs, and this means an improvement of 159.40% with respect to FTSP.This test ends with an average synchronization error of 2.685 7.3.3.Once both FTSP and AT-FTSP have been analyzed, this set of experiments end with the results of A2T-FTSP for high temperature ranges.μs). Nevertheless, after applying a temperature variation on the nodes, the average synchronization error increases drastically, producing a peak average synchronization error of 4.25 μs. After this situation, A2T-FTSP starts to apply the new clock skew method, decreasing the average synchronization error gradually, until the end of the test, where the error returns to similar values compared to the first temporal interval. The test ends with an average synchronization error of 2.609 μs. The improvement with respect to FTSP is clear, amounting to 184.86%.For the temporal range without temperature change , A2T-FTSP has a good behavior and a better average synchronization error . Taking into account these values, the clock stability is, approximately, 0.37 ppm. However, for very large periods of resynchronization, the authors obtained < 0.07 ppm (4 ms of error in a 16 h period).Schmid et al.  carried On the other hand, the proposed AT/A2T-FTSP methods have obtained similar values in clock stability terms as TCTS for large resynchronization periods. TCTS provided very good results under simulation; however, real-world scenarios include many uncertainties that modify the results from the ideal simulation. Those variations from the simulation results to the real-world experiments have been clearly stated in EACS, where simulation provided an error of 2 ms, while in the experimental setup, that error went up to 8 ms. In this work, all the results are obtained in a real environment and not a simulated one. Nevertheless, the real-world results from AT/A2T are in the same range as the simulation results from TCTS. It is worth noting that neither AT nor A2T require any calibration period, meanwhile TCTS uses 24 h to obtain a good calibration phase.Besides, as the proposed methods use a 1 MHZ timer compensated for by the 32 KHZ oscillator, microsecond accuracy can be achieved. For the sake of comparison, errors are provided in terms of 1 MHZ clock ticks in These results clearly show the accuracy of the proposed methods and outperforms the previous approaches for the experimental setup described in this work. The simplicity of the proposed methods makes these less computationally demanding than the previous approaches. Besides, the proposed methods do not require any calibration period for temperature compensation. Finally, they are able to deal with dynamic reference node swapping, which was not feasible in previous approaches.9.In this work, two new methods to obtain a more precise clock skew based on temperature for synchronization protocols in WSNs have been proposed. Oscillators are highly influenced by temperature changes. The influence of temperature variations in synchronization protocols has been demonstrated using the Flooding Time Synchronization Protocol (FTSP). The obtained results clearly show the influence of temperature and reveal the necessity of creating new synchronization approaches based on temperature.Taking into account temperature variations in the clock skew estimation, two new FTSP-based approaches have been developed and tested: AT-FTSP and A2T-FTSP Both approaches use the tuning-fork equation to determine the real frequency of the oscillator according to the ambient temperature. With this information, the clock skew estimation is improved to obtain a more precise value, minimizing the effects due to temperature.Taking into account the temperature in the computation of the clock skew minimizes the error due to frequency variations. This work has shown the reliability of the new proposed temperature methods for a large range of temperatures (from 9°C to 40°C).μs and A2T-FTSP gets about 1.459 μs. These results mean an improvement between 104.17% and 118%.Using the new temperature-based methods in the range of temperature from 9°C to 22°C permit one to improve the average synchronization error. AT-FTSP provides an average synchronization error of 1.558 CMR200T oscillator (25°C). As a consequence, the frequency variations are minimal, obtaining similar results between the new methods. For the above-mentioned interval, AT-FTSP and A2T-FTSP improve about 55.60% (2.302 μs) and 109.22% (1.706 μs) compared to FTSP (3.582 μs).For the temperature interval, 22°C–32°C, the behavior differences between the new proposals are minimal. This is mainly due to the thermal interval used, which is really close to the nominal frequency of the μs) and 184.86% (1.349 μs) with respect to FTSP (7.432 μs).High temperatures ranges largely penalize the good behavior of the synchronization mechanisms. This is a direct consequence of the big influence of heat. Once again, AT-FTSP and A2T-FTSP improve the average synchronization error, obtaining 159.40%  and TCTS (0.37 ppm). The new methods have fewer computational costs and a better performance, which make them a suitable option to be used in WSNs. Furthermore, it is worth noting that all the obtained results come from real-world experiments and not from simulations.The new methods improve other proposals based on temperature. In order to compare to other temperature-based proposals (EACS and TCTS), results in terms of tics and ppm for <95% of the errors have been obtained. In tics, AT-FTSP and AT2-FTSP offer similar results  to the TCTS proposal (±4.16). Besides, the results in ppm show a clear improvement of AT-FTSP and AT2-FTSP  The proposed methods can be applied in any synchronization protocol based on the clock skew concept.The average synchronization error can be improved, taking into account the temperature. The increase of source code lines is minimal compared to the benefits obtained in terms of synchronization.K, and also, the basic thermistor (US$ 0.06@2K) is needed to measure the temperature, which makes a total of US$ 0.31. On the other hand, a self-compensated oscillator with temperature (TCXO) can be used to adjust the frequency of the oscillator, for example, the KT3225T oscillator, which is a TCXO model with the same characteristics of CMR200T, but whose cost is about US$ 3.10@3K. With the proposed methods, the hardware cost of the motes can be reduced by simply using software mechanism to compensate for the drift of the frequency.In this work, the used oscillator is the Citizen CMR200T, which has a cost of US$ 0.25@2The basic mechanism (AT) is suitable for environments where the temperature of the reference node is not available . It requires low computational power. Besides, it uses less bandwidth, as no temperature is sent in the synchronization packets.The advanced adjustment mechanism (A2T) takes into account both the local temperature and the reference node temperature. It provides better accuracy than the AT method, although it has higher computational requirements and a higher bandwidth usage.To summarize the above lines, the main contributions of this article are below:10.et al. [This work will be improved by testing the proposed methods in multi-hop scenarios and/or other time synchronization protocols. It is thought that they will behave properly, because the proposed mechanisms do not depend on the routing protocol, although further testing is required. Lenzen et al.  have demNew experiments are planned. These new experiments will use very large synchronization periods in order to compare these methods under the same scenarios as the ones used in TCTS and EACS.Finally, these mechanisms are to be designed in simulation platforms, like OMNeT++, which is one of the most commonly used simulators for WSNs. This will allow much more theoretical experiments during longer periods of time.


According to Guyton, the difference between mean systemic filling pressure (Pms) and right atrial pressure (RAP) is the venous pressure gradient (VP). This is proportional to venous return and cardiac output (CO). According to the Frank-Starling law a fluid challenge successfully increases the stroke volume if the preload increases in the ascending part of the curve. The aim of this study was to assess the significance of the analogue of the Pms (Pmsa) measured with the Navigator™ , the central venous pressure (CVP) (as a surrogate of RAP) and the VP during a fluid challenge.A prospective observational study was performed in postsurgical patients. Patients were monitored with a central venous catheter, an arterial line, a calibrated LiDCO™plus  and the Navigator™. A 250 ml fluid challenge was used to maximise the stroke volume (SV). Data were recorded before and after the fluid challenge which was given over 5 minutes. A positive response to the fluid challenge was defined as either a stroke volume or CO increase of 10% or more.Twenty-five fluid challenges in 14 patients were observed. In seven cases (28%), the fluid challenge increased SV (and CO) by ≥10% (Table Our study demonstrates that the Navigator™ may be used to monitor the effect of fluid challenges by assessing the change in VP to the challenge.