Although hypercoagulable states are most often associated with venous thromboses, arterial thromboses are reported in protein C, protein S, antithrombin deficient patients and in those with factor V Leiden, components of hereditary thrombophilia. Because these arterial thromboses  mostly affect young persons, aged below 45 years, it is important to test and treat these thrombophilic defects. Because the relation thrombophilia – arterial thromboses is still under debate, due to conflicting data, this article is a review of studies published in literature regarding the implication of the above–mentioned thrombophilic defects in cerebral infarcts. Most patients with arterial stroke have diabetes, hypertension, hyperlipidemia, smoking or valvular heart disease as underlying risk factors. When such risk factors are absent, one should suspect hypercoagulable states as causative. The inherited hypercoagulable syndromes primarily affect veins and only rarely cause arterial thrombosis. The predilection to venous as opposed to arterial stroke may be partly due to the differing mechanisms of thrombosis on the venous and arterial sides of the circulation and within various organs. Stasis is a major predisposing factor in venous circulation whereas endothelial damage is a more important predisposing factor in arterial circulation. Because there are still conflicting data regarding the implications of protein C, protein S, antithrombin deficiencies and factor V Leiden mutation in arterial stroke, the present article is a review of the published studies debating this issue. Regarding the cerebral infarctions, the implications of protein S, C, and antithrombin deficiencies is still on debate; more certain is the implication of factor V Leiden. Half of the ischemic strokes are unexplained by conventional risk factors and the genetic predisposition has been deemed responsible for some of these risks.  shows thCADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy; CARASIL, cerebral autosomal recessive arteriopathy with subcortical infarcts and leucoencephalopathy; HERNS, hereditary endotheliopathy with retinopathy, nephropathy, and stroke; MELAS; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes; Stroke means different pathological processes, all of them having as an end point the focal cerebral ischemia. 85% of the strokes are ischemic and 15% hemorrhagic. Even among the patients with ischemic stroke, a number of pathologically different processes are responsible, as cardioembolism, large artery disease with arteriolosclerosis and thromboembolism, and small artery disease. The physiopathological mechanisms and the genetic influences responsible could be different for different stroke subtypes. Two family studies showed an association with large artery and small artery strokes, but not with cardioembolic stroke ,3. The aThe prevalence of protein C, S and antithrombin 3 in ischemic stroke varies up to 23% in different studies .   In an ample meta–analysis published in 2003, reports of cases and studies regarding the deficiencies of proteins S, C, antithrombin, and factor V Leiden in ischemic strokes were mentioned . ProteinProtein S deficiency has been associated with cerebral arterial ischemia more often than has protein C deficiency. However, conflicting reports limit the reliability of this association. The meta-analysis describes case–reports and studies published until 2003 regarding this subject: they reported a frequency of this deficiency of 13,8% 5/36), 19% (8/35), 23% (19/98) in patients aged less than 45 years and  6% (4/66) in patients aged less than 60 years [, 19% 8/. In the Antithrombin deficiency has only rarely been associated with stroke. The meta–analysis presents case–reports and studies regarding the association of this deficiency with stroke. Different studies show frequencies of 5% 3/66), 8% (5/60), but there were studies that reported just one case of the 36 or 329 cases studied. It is worth mentioning that all studies included patients aged less than 45–55 years, and the study that reported a frequency of 5%, all antithrombin deficiency patients had suffered carotid artery territory strokes [6, 8%  is the most common inherited coagulopathy associated with stroke. The meta–analysis reports a prevalence of the mutation of 10% (3/30), 12,3%(10/81), according to different studies and 2 studies that included stroke patients aged younger than 45 years with cryptogenic cerebral ischemia found a prevalence of 12% and 15,9%, respectively.In 2004, a meta–analysis of all the studies made until January 2003, was published in English–language journals and it was related to the investigation of any candidate gene for ischemic stroke in humans. In order to maintain genetic homogeneity, only studies in white adults were included. Data from 120 case–control studies were included. Pooled odds ratios with 95% confidence intervals (CIs) were included. Of the 32 genes studied, 15 polymorphisms were identified. Statistically significant associations with ischemic stroke were identified only for factor V Leiden Arg506Gln , but not for the other 3 deficits [A study performed in Turkey and published in 2005, studied 29 children with ischemic stroke and 20 with intracerebral hemorrhage, all of whom were compared with 20 controls. The authors found no evidence of an association between factor V Leiden mutation and ischemic stroke or intracerebral hemorrhage. The conclusion was that factor V Leiden mutation did not seem to be associated with a risk of cerebrovascular disease . A studyThe risk of a stroke is high for persons with factor V Leiden combined with other vascular risk factors, such as smoking and contraceptive use. A study investigated the prevalence of these mutations in 468 patients with an acute stroke or transient ischemic attack (TIA) before the age of 60 and in a healthy control population individually matched for age and gender. A significant interaction between the factor V Leiden, smoking, and risk of stroke in women was found: female smokers without the factor V Leiden had a somewhat increased risk of stroke of 2.6  compared with nonsmoking non–carriers of the factor V Leiden. No such interaction was observed in men .Nowadays, there are many studies about the association between patent foramen ovale and thrombophilic defects that lead to a high incidence of ischemic strokes, especially in young persons.Patent foramen ovale (PFO) is present in  25% of the general population, but in most cases, it does not affect health. But, this anomaly represents a possible cause of ischemic embolic neurological events in young patients ,14. The One study that included 97 young patients with PFO and 160 age–matched control subjects found that the combination of either factor V Leiden or prothrombin G20210A and PFO was associated with a 4.7–fold  increased risk of cerebral ischemia in young patients. No statistically significant association was found for protein C, S or antithrombin deficiency . Of the It was presumed that the presence of inherited thrombophilic defects could induce the potential risks and reduce the benefits of percutaneous closure of patent foramen ovale . A studyDealing with a case of cerebral infarction with few risk factors for arteriosclerosis, the screening for thrombophilia should be made, especially when patients are aged below 45 years, have a personal history of recurrent thrombosis without precipitating factors, thrombosis in unusual sites, thrombosis during pregnancy, a positive family history of thrombosis. A positive result asks for initiation of the adequate anticoagulant treatment.


To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS).The anterior halves of whole eyeballs from chickens on embryonic (E) days E10 to E21 and Japanese quail on embryonic days E8 to E17 were fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence. Propidium iodide (PI) was used to detect the nuclei of osteocytes in scleral ossicles. FESEM and EDS were then used to show areas of mineralization and to identify differences in the elemental composition of different regions of the ossicles.Using Giemsa as a fluorescence stain, it was possible to observe the detailed morphology and development of both chicken and quail scleral ossicles. In chickens, bone microporosities first became visible at E15. Each microporosity contained a single nucleus, likely that of an osteocyte. The amount of carbon in ossicles steadily decreased during embryogenesis and post-hatching, while the concentration of oxygen showed a distinct increase over this time period. Calcium and phosphate levels in the ossicles increased gradually during embryonic and post-hatching stages.A novel approach to study the development and mineralization of avian scleral ossicles during embryogenesis is presented. This methodology was validated by studying two different species, both important models for avian developmental research. Scleral ossicles are rings of overlapping trapezoid-shaped membrane bones, which are embedded in the sclera surrounding the cornea beneath the conjunctival zone in the eyes of non-mammalian vertebrates -3. The bTo date, the morphology and development of scleral ossicles have only been studied by bright-field microscopy, based on Alizarin Red S staining of ossified material in whole mount preparations of small vertebrates ,8-11. AlGiemsa is a classic stain for bone marrow, gastric tissue, and peripheral blood smears . Giemsa To help maintain the shape of the cornea, it is important for scleral ossicles to be mineralized and hard, with high plate strength. Bone strength is determined not only by the volume of bone tissue and its microarchitectural organization, but also by the degree of mineralization in the bone matrix ,19. MineAll animals were used in accordance with the ARVO Statement for the Use of Animals for Ophthalmic and Vision Research. Fertilized eggs from White Leghorn chickens were purchased from Nelson Poultry Farms Inc.  and fertilized Japanese quail eggs were purchased from B&D Game Farm . Before shipping to our laboratory, both sets of eggs were collected and stored at 15 °C for 1 to 3 days after laying. Both types of eggs were stored at 15 °C for no more than a week after arriving at our laboratory. These fertilized eggs were incubated at 38 °C and 45% humidity from embryonic day (E) 0, the day on which the eggs were put into the incubator. The anterior half of the eye was dissected from chicken eggs at embryonic ages E10 to E21 and from quail eggs from E8 to E17 and rinsed in sterile PBS . Before staining, adherent tissues on the surface of scleral ossicles were gently removed using a blunt knife. The lens and iris were then removed.Immediately after dissection, the anterior half of each eyeball, consisting of only the cornea and surrounding limbus, the conjunctiva, and the sclera, was fixed for 1 min in 100% methanol at room temperature, with mild agitation, and then immediately stained in Giemsa solution  for 5 min. The tissues were subsequently destained with distilled water for 30 min. Throughout this process, the samples were kept in constant gentle movement with a rotary shaker set at 1 revolution/s. while wrapped in aluminum foil to prevent photo bleaching and loss of fluorescence. To compare with Giemsa staining, the anterior halves of separate chicken eyeballs were stained with Alizarin Red S. In the current study, the Alizarin Red S staining protocol was modified from the procedure described previously ,8. BriefAfter staining, the anterior halves of chicken and quail eyeballs were transferred to glass slides with the cornea facing down, covered with a coverslip, and imaged using a fluorescence stereomicroscope (Leica MZ16F) equipped with filters .The chicken scleral ossicles were imaged using a Carl Zeiss LSM700 microscope  with the following parameters: excitation with a 488 nm laser line from an argon ion LASER, and a BP 505–530 nm emission filter. Plan-Neofuar 40×/1.3 DIC objective with the airy unit set to 1 and an optical slice interval of 1.7 µm.The anterior halves of chicken anterior eyeballs (E16 and E20) stained with Giemsa, as described above, were then stained with propidium iodide . Briefly, a 5 µM PI staining solution was made by diluting the 1 mg/ml (1.5 mM) stock solution in 2× SSC . After the Giemsa destaining step, the anterior halves of the chicken eyes were incubated in the PI solution (200 µl) for 30 min at room temperature, rinsed twice with SSC for 1 min, and then viewed by confocal microscopy (Carl Zeiss LSM700 system) . The par2; Oxford Instruments, Bucks, United Kingdom). For EDS, the accelerating voltage was 15 kV, the spot size was 4.5 (0.70 nA), and data were collected over 120 s. Silicon was used for quantum optimization of the EDS. The weight percent (Wt %) of elements were directly calculated and given by the Nova NanoSEM 430 system when analyzing the ossicles.Individual trapezoid-shaped ossicles were dissected from the eyes using a stereomicroscope and freed from other adherent tissues. For cross-sectional imaging and analysis, individual ossicles were bisected parallel to the corneal-scleral interface plane. Ossicles were oriented and mounted on aluminum SEM stubs with carbon adhesive tape to image and analyze the ossicle surface or cross-sections. Imaging and elemental analysis were performed using a field emission scanning electron microscope, Nova NanoSEM 430  equipped with an X-Max Large Area Analytical energy dispersive spectroscopy (EDS) silicon drift detector  from three separate experiments. Statistical analyses were performed using Student’s Appendix 1 . Finally, to allow comparison of these Giemsa images with those from an independent bone stain technique, chicken scleral ossicles also were stained with Alizarin Red S  and phosphorus (P) on the surface of embryonic chicken ossicles were distinctly lower than those in the interior region detected in cross sections . Data coData in In this study, we developed a simple, rapid, sensitive, consistent technique to visualize developmental morphology and monitor the mineralization of avian scleral ossicles using Giemsa staining and FESEM/EDS. Giemsa bone staining is more sensitive than Alizarin Red S, allowing earlier detection of osteogenesis during development (compare Giemsa staining in To our knowledge, the embryonic morphology and development of scleral ossicles in Japanese quail had not been described previously. One earlier publication described a morphological analysis of the scleral ossicles and their modal number, but only in ages of adult quail . Using GIn birds, scleral ossicles are neural crest-derived membrane bones ,24. NeurOsteocytes are the most abundant cells in bone and the only cells embedded in the calcified bone mineral matrix , occupyiThe increased staining intensity using Giemsa fluorescence with increasing developmental age is consistent with data presented in Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial component of mature bone tissue synthesized by osteocytes ,31. The In conclusion, the work presented here represents a novel approach for studying the development and mineralization of avian scleral ossicles. Details and entire features of the developmental morphologies of both chicken and quail ossicles were documented using Giemsa as a very rapid fluorescent stain for ossicle development and mineralization. Moreover, FESEM and EDS supported Giemsa staining findings concerning the presence of a calcified matrix and expanded our quantitative and qualitative knowledge of mineralization within discrete regions of avian scleral ossicles related to developmental stages.


Distribution, extent, composition and structure of this bed were assessed with side scan sonar, remotely operated vehicles, and SCUBA. The mean rate of CaCO3 production was estimated from in situ growth assays at 1.07 kg m−2 yr−1, with a total production rate of 0.025 Gt yr−1, comparable to those of the world's largest biogenic CaCO3 deposits. These gigantic rhodolith beds, of areal extent equivalent to the Great Barrier Reef, Australia, are a critical, yet poorly understood component of the tropical South Atlantic Ocean. Based on the relatively high vulnerability of coralline algae to ocean acidification, these beds are likely to experience a profound restructuring in the coming decades.Rhodoliths are nodules of non-geniculate coralline algae that occur in shallow waters (<150 m depth) subjected to episodic disturbance. Rhodolith beds stand with kelp beds, seagrass meadows, and coralline algal reefs as one of the world's four largest macrophyte-dominated benthic communities. Geographic distribution of rhodolith beds is discontinuous, with large concentrations off Japan, Australia and the Gulf of California, as well as in the Mediterranean, North Atlantic, eastern Caribbean and Brazil. Although there are major gaps in terms of seabed habitat mapping, the largest rhodolith beds are purported to occur off Brazil, where these communities are recorded across a wide latitudinal range (2°N - 27°S). To quantify their extent, we carried out an inter-reefal seabed habitat survey on the Abrolhos Shelf (16°50′ - 19°45′S) off eastern Brazil, and confirmed the most expansive and contiguous rhodolith bed in the world, covering about 20,900 km Although CCAs are recognized as the most important component of Abrolhos reefs The global distribution of rhodolith beds is highly discontinuous, with larger concentrations recorded off southern Japan, western Australia and the Gulf of California, as well as in the Mediterranean and along Norway, Ireland, Scotland, northeastern Canada, the eastern Caribbean and Brazil 3 fluxes of the tropical Southwestern Atlantic hinges on accurate quantification on the extent and CaCO3 production of these large rhodolith beds. The aim of this work was thus to determine the size and CaCO3 production of the rhodolith beds within the Abrolhos Shelf. This meso-scale system may provide valuable insights on the overall role of the massive rhodolith beds of the western tropical and subtropical South American shelf. To accomplish this task, surveys using side scan sonar (SSS), remotely operated vehicles (ROVs) and mixed-gas diving, as well as in situ assays to estimate CaCO3 production were perfomed. Our analysis confirms the overwhelming importance of this CaCO3 bio-factory for the Southwestern Atlantic Ocean CaCO3 cycles.Understanding the CaCO2 which represents at least 45% of the entire Abrolhos Shelf  Penrose , Lithophyllum stictaeforme (Areschoug) Hauck , Mesophyllum engelhartii (Foslie) Adey , Sporolithon episporum (Howe) Dawson (RB 525149), Neogoniolithon brassica-florida (Harvey) Setchell et Mason (RB 525150), and Lithothamnion crispatum Hauck . They are recognized as widespread taxa in tropical and temperate waters. Lithothamnion crispatum was the most abundant species within multispecific rhodoliths, as well as the most common species forming monospecific rhodoliths  than those measured at 45 m depth  .The growth rates determined based on 3 production was estimated at 1.0±0.7 kg m−2 yr−1, with a remarkable spatial variation, with values ranging between 0.3 to 2.7 kg m−2 yr−1 .Together with kelp beds, seagrass meadows, and coral reefs, rhodolith beds are one of Earth's largest macrophyte-dominated benthic communities. As expected from such a widely distributed and highly fragmented habitat type that spans from polar to tropical regions of the main ocean basins, rhodolith beds vary considerably in terms of constituent species and physiognomy  2, an area comparable to that estimated for coral reefs of the Caribbean  and the Great Barrier Reef  Mussismilia spp.) are restricted mostly to shallow waters and do not contribute much to carbonate deposition in larger reef blocks in the deeper parts of the Bank, where rhodoliths thrive. This contrasts with the situation observed in the reefs that occur in the inner shelf, where corals and CCA form the main framework of mushroom-shaped pinnacles that emerge from the bottom (10–25 m) and reach the surface.The world's largest rhodolith beds have been suggested to occur off Brazil −1) are similar to those reported from other studies. Extension rates of rhodolith-forming species are generally 0.5–1 mm yr−1 under a wide range of field and laboratory conditions  3 m−2 yr−1) that are within the range recorded for temperate rhodoliths Growth rates of rhodoliths from Abrolhos Shelf  are similar to those reported for tropical reef environments  in the rhodolith banks suggest that their thicknesses are similar to those of most modern coral reefs. The wide latitudinal span of rhodolith beds along the eastern South American coast (2°N - 27°S) and the extension of some beds, such as the one reported herein, with thousands of square kilometers, support the idea that CCA plays critical biological and physicochemical roles in the South Atlantic.Our CaCO3 minerals 2 (pCO2) is projected to reduce marine calcification 40% by year 2100 and 90% by 2300 2 and temperature will have major implications for carbon dynamics, from the carbonate chemistry of the water column to the ability of the oceans to uptake atmospheric CO2The high-magnesium calcite produced by CCA is the most soluble form of the common CaCO3 production by these beds is comparable to that of the largest biogenic CaCO3 deposits in the world. The gigantic CaCO3 bio-factory reported herein from the Abrolhos Shelf alone accounts for approximately 5% of the world's total carbonate banks. Although the main stressor (ocean acidification) is not manageable at regional scales, a broad array of threats to the Abrolhos' rhodolith beds can be managed and deserves immediate attention. These include sedimentation from land-based sources and large scale dredging and mining. Although the accumulation rate of rhodoliths may appear rapid on a geological time scale (∼1 m 103 yr), it is very slow on a human time scale (∼1 mm yr−1). Therefore, rhodoliths should be regarded as non-renewable resources and valuable providers of critical ecosystem services.The importance of the Southwestern Atlantic rhodolith beds, particularly those of the Abrolhos Bank, has been largely underestimated. The total annual CaCOin situ assays on growth rate of rhodolith-forming CCA.Benthic surveys were carried out on the Abrolhos Shelf , off the eastern tropical coast of Brazil  between Side Scan Sonar surveys included 20 cross-shelf transects between 10 and 110 m depths . An Edge−2), and dimensions of rhodoliths at all 52 sites.A Seabotix® LBV 150S2 ROV equipped with a color video camera and a pair of scaling lasers (5 cm apart) was used to validate sea-bottom features recorded with SSS. Footage was recorded for at least 40 minutes in each deployment, covering the main benthic features at each site. A total of 52 sites identified by SSS as rhodolith bed  were valSixty rhodoliths were collected at each site. Immediately after collection, the top surface of each specimen was photographed to record the color of CCA thallus, which was used to estimate the percent surface area with live tissue (i.e. percent live surface area). Photographs were analyzed using Coral Point Count with Excel Extensions software Identification of CCA species composing the external surface of rhodoliths was conducted using pieces of fertile material submitted to both light and scanning electron microscopy (SEM). Samples were decalcified in 10% nitric acid, dehydrated in ethanol, embedded in resin, and sectioned (5–10 µm thick) for observation under a light microscope. Specimens examined with SEM were air dried and mounted on aluminum stubs with a double-sided conductive tape. Stubs were sputter coated with gold and investigated with a Zeiss EVO 40 SEM at 15 kV. The species were identified according to recent studies of CCA taxonomy in situ in May 2009, rhodoliths from the central area of the Abrolhos Shelf were selected from 20 and 45 m depths (ten rhodoliths from each depth strata). Each rhodolith was tagged with thin plastic tape and stained in an aerated 0.025% (w/v) alizarin red seawater solution for 24 h, in order to create a detectable band (time line) on the epithelial margin layer of the CCA crust. Rhodoliths were replaced at each corresponding site in a 1×1×0.5 m open-topped enclosure among other unstained individuals. After six months rhodoliths were recovered and sectioned. The distance from the stained region to the newly grown epithelial margin layer was measured (−1).Calcification rates of rhodoliths were determined measured . At leas−2 yr−1) was obtained from mean density, dimension, and percent live surface data, together with the estimates of annual growth of rhodolith-forming CCA. It was taken into account that 99% of CCA mass obtained after calcinations process (72 h at 400°C) correspond to CaCO3. Calculations were performed as follows.The mass of CCA accumulated (g mV) determined by the equation:Rx, Ry, and Rz are the shortest, intermediate, and largest radii, respectively.Rhodoliths have an ellipsoid form, with volumes (V′) was obtained by subtracting initial from final volumes with the following equation:g is rhodolith extension in cm after one year. Values obtained by this method were previously validated by comparing with volume estimates from liquid volume displacement, with a strongly positive correlation between the two methods . Based on in situ assays, the increase in rhodolith thickness was estimated at 1.5 mm yr−1 down to 40 m depth and 1 mm yr−1 between 40 m and 110 m.Increase in rhodolith volume after one year (−33 mass on a rhodolith bed was estimated for a one-year period (M) with the following equation:3 production rate  was obtained with the following equation:−2). The total CaCO3 production by rhodoliths was calculated using a 3-D integral of production rates measured for the Abrolhos Bank.Density (d) is defined as mass/volume, with CCA density estimated at ∼1.56 g cm14C) was determined in samples from the core of three typical rhodoliths collected at 65 m depth. After sawing the rhodolith into two halves, a square piece of 2 mm core of rhodolith was removed and observed by scanning electron microscopy , 48 (67 m depth), and 59 (31 m depth). Note that most of the substrate was covered by live CCA (shades of red) forming rhodoliths.(MOV)Click here for additional data file.


In France, the 2009 A(H1N1) influenza epidemic occurred between September 2009 and January 2010. Sparking widespread controversy, it was intensely reported in the media. Despite therapeutic inefficacy, antibiotic consumption and viral respiratory infections are positively correlated, particularly in France, where antibiotic overconsumption is well-known. We first determined the period when media coverage was high, and then compared, during this period, observed outpatient antibiotic consumption to estimated outpatient antibiotic consumption “without media attention”.To evaluate media coverage, two online databases were consulted: Factiva and Europresse. To quantify outpatient antibiotic consumption, we used data on reimbursements of outpatient systemic antibiotics from the computerized databases of the two main National Health Insurance agencies. Influenza-like syndromes data came from the French GPs Sentinelles Network. Weekly time-series of antibiotic consumption were modeled by autoregressive moving-average models with exogenous inputs and interventions. Analyses were computed for the entire series and by age group .Media coverage was intense between April 2009 and January 2010. No effect on total outpatient antibiotic consumption was observed during the whole mediatic period. However, during the epidemic in France (September 2009-January 2010), we found an antibiotic underconsumption for the entire series, 0–5 and >60 years. Additionally, at the beginning of the pandemic, when cases were still outside France (June 2009-August 2009), we found an antibiotic overconsumption for patients >16 years.The early period of A(H1N1) virus circulation compared with seasonal influenza or an overdeclaration of ILS cases might explain the antibiotic underconsumption observed during the period of active A(H1N1) virus transmission in France. At the pandemic onset, when uncertainty was high, the overconsumption observed for individuals >16 years might have been caused by alarmist media reporting. Additional analyses are needed to understand the determinants of antibiotic consumption during this period. Streptococcus pneumoniae is one of the best-known bacteria that has spread during the past 20 years, and, more recently, Enterobacteria producing extended-spectrum β-lactamase has become a major worry S. pneumoniae antibiotic resistance was observed in France, which also has one of the highest rates of community antibiotic consumption, more than three-fold higher than the Netherlands During the last few decades, the emergence and dissemination of multidrug-resistant bacterial strains have become a major public health issue worldwide In April 2009, the A(H1N1) virus emerged in the USA and Mexico. It spread throughout the world very quickly, resulting in outbreaks in more than 200 countries and claimed the lives of more than 18,000 persons http://www.insee.fr). The results are presented as weekly rates of antibiotic prescriptions per 1,000 inhabitants.The French National Health Insurance (NHI) program covers all medical care provided by outpatient and private practice physicians and pharmacies. We obtained aggregated 2000–2010 data on all antibiotics prescribed, dispensed by outpatient and private pharmacies, and reimbursed computed from anonymous computerized individual files in the databases of the two main NHI agencies that cover salaried workers and the self-employed (>85% of the French population). Each file contains drug-related information, prescription date, patient’s sex, year of birth and region of residence. The study concerned only systemic antibiotics  used in the community. Demographic data were obtained from the French National Institute for Statistics and Economic Studies  Weekly influenza-like syndrome (ILS) incidence was provided by the French Sentinelles Network  and Europresse (http://www.europresse.com). These online databases provide access to newspaper articles of many French and international newspapers. We conducted a search on articles published in two major national daily newspapers, Le Monde and Le Figaro, between January 2009 and August 2010, in order to retrieve and count the number of A(H1N1) influenza-related articles published in these two newspapers. Results are presented as the number of articles published in these two newspapers per week.Two online databases were used to assess media coverage of the A(H1N1) pandemic: Factiva  influenza would have been without media coverage 1 to c7) and six for the rest of the time (r1 to r6)) to the ARMA model identified in step 1. The ILS incidence was added to that model using a simple transfer function (linear function). This allows estimating an average effect of the ILS incidence during the entire period. To select the models, we first tested the independence of the residual series (Ljung & Box test) and its Gaussian distribution (Shapiro-Wilk test). When two or more models were identified, we used Schwartz information criterion and choose the one which minimize that criterion. Every step of model identification is detailed in §1 and 2 in file S1.The intervention ARMAX models were built in two steps. The first concerned the 2000–2002 period, before the first public health campaign, considering antibiotic consumption during it as the baseline. Because of seasonal fluctuations, a trigonometric function had to be estimated and removed to render the residual series in a stationary mode. We then fitted an autoregressive moving-average (ARMA) model to the observed data. The second step evaluated the impact of the public health campaigns on antibiotic consumption between October 2002 and March 2009. As those campaigns were held every year at the same period of the year since 2002, we assumed that they did not modify seasonal fluctuations or change the ARMA model structure, but affected only the means. We added 13 dummy variables   influenza media coverage had to be known  . We then® 9.1, R2.13.1 and Excel® 2010 softwares.Analyses of the entire series and by age group  were computed with SASA total of 530 articles on the A(H1N1) virus were released between January 2009 and August 2010. According to our criteria, media coverage was intense during the 42 weeks beginning in April 2009 (week 18) and ending in February 2010 (week 8) , with 50The link between viral infection and antibiotic consumption has now been very well-documented in numerous studies The data used in this study were provided by two different sources. Antibiotic-prescription reimbursements came from NHI agencies, assuring reliable and thorough data recording during the pandemic. ILS-incidence data was provided by the Sentinelles Network. This network estimates ILS incidence relying on declaration of general practitioners, without virological confirmation. During the A(H1N1) pandemic, they declared that the epidemic threshold was crossed week 36–2009. Another surveillance network (GROG), relying on samples to estimate virologically confirmed influenza number of cases, declared the start of the influenza epidemic in France week 47–2009. This discrepancy suggests there might have been an overdeclaration of ILS by general practitioners in September and October 2009, leading to an overestimation of ILS incidence by Sentinelles Network. Some studies suggest that the increase in ILS in September and October 2009 was caused by respiratory viruses other than influenza (eg rhinovirus) http://inatheque.ina.fr) devoted to A(H1N1) indicated the same media period (data not shown).To determine the period of A(H1N1)-influenza–related media coverage in France, we only examined articles from two national newspapers available in two online databases, but no local newspaper coverage, thereby probably underestimating the overall coverage of this period. However, first, because our objective was not to quantify media coverage but to determine the trend and identify the period of most intense media coverage, we considered that examination of only two national daily newspapers was sufficient; second, the comparison with weekly television-news time . The observed impact of the campaigns between 2002 and 2009 were very different depending on the series; however, another analysis of time series until June 2010 yielded estimated values in accordance with our predicted ones . For our predictions, we tried to adhere closely to the observed tendency, and used two scenarios to estimate forecasts and their 95% CI limits to avoid falsely significant results.Media attention was strong for almost 10 months (42 weeks). During that long period, no change in overall antibiotic consumption was observed, which might reflect a loss of power because prediction 95% CI, and thus the final used, widened with the time. When considering a shorter media-coverage period (18 weeks), corresponding to high A(H1N1)-virus circulation in France, underconsumption was estimated for the global antibiotic consumption, and for subjects 0–5 and >60 years old. This observed underconsumption reached −14%. Considering the antibiotic-consumption profile during 2009–2010, no antibiotic-prescription peak was seen when the ILS incidence was at its maximum, which had not been observed in any previous years. Several hypotheses can be made to explain this model-estimated result. First, as previously mentioned, the ILS incidence might have been overestimated; then adjusting to it led to an overestimation of expected antibiotic use, and consequently an observed “underconsumption” as the predicted number was higher than the observed one. Second, due to the early arrival of the pandemic influenza virus in France (September 2009) compared to seasonal influenza (December–January), general practitioners’ offices might have been less saturated than usual during the A(H1N1) period of activity as classical winter viruses were not circulating yet, generating more careful antibiotic prescription Finally, over the 6-week period without A(H1N1) virus circulating in France, overconsumption which varies from 1% to 20%, was observed for individuals >16 years. We here only assessed what would have been the antibiotic consumption “without media attention” and thererefore cannot draw any formal conclusion on the link between media attention and the observed overconsumption for individuals >16 years during this period. However, subjects 16–60 years old might have been affected by the omnipresent media coverage, because unlike seasonal influenza, it corresponds to the age category for which deaths of people without any risk factors were seen. It has already been previously documented that media coverage can influence medical care and drug consumption In conclusion, despite media attention and uncertainty about health effects, no upsurge of antibiotic prescriptions was observed for the whole study period, and in particular when the pandemic virus circulated in France. However, early during the pandemic, when the uncertainty was high, increased antibiotic use for individuals >16 years was observed. Alarmist media reporting might have heightened the worries of those individuals, resulting in increased antibiotic use.Figure S1# for age-group series.Weekly number of antibiotics prescriptions*: fit of the model and predictions for both scenarios *per 1,000 inhabitants. #Scenario 1 corresponds to the “stationary” hypothesis and scenario 2 to the “evolutionary” one.(TIFF)Click here for additional data file.Figure S2# of intervention functions for age-group series.Estimated and predicted changes for both scenarios# Scenario 1 corresponds to the “stationary” hypothesis and scenario 2 to the “evolutionary” one.(TIFF)Click here for additional data file.Text S1Identification of intervention ARMAX models.(PDF)Click here for additional data file.


GAPDH, Actin and EEF2 had tissue specific variations, whereas several ribosomal genes were among the most stably expressed genes in vitro. Our results confirm that two or more reference genes should be used in combination for differential expression analysis of large-scale data obtained from microarray or next generation sequencing studies. Therefore context dependent reference gene sets, as presented in this study, are required for normalization of expression data from diverse technological backgrounds.Transcriptome experiments are performed to assess protein abundance through mRNA expression analysis. Expression levels of genes vary depending on the experimental conditions and the cell response. Transcriptome data must be diverse and yet comparable in reference to stably expressed genes, even if they are generated from different experiments on the same biological context from various laboratories. In this study, expression patterns of 9090 microarray samples grouped into 381 NCBI-GEO datasets were investigated to identify novel candidate reference genes using randomizations and Receiver Operating Characteristic (ROC) curves. The analysis demonstrated that cell type specific reference gene sets display less variability than a united set for all tissues. Therefore, constitutively and stably expressed, origin specific novel reference gene sets were identified based on their coefficient of variation and percentage of occurrence in all GEO datasets, which were classified using Medical Subject Headings (MeSH). A large number of MeSH grouped reference gene lists are presented as novel tissue specific reference gene lists. The most commonly observed 17 genes in these sets were compared for their expression in 8 hepatocellular, 5 breast and 3 colon carcinoma cells by RT-qPCR to verify tissue specificity. Indeed, commonly used housekeeping genes  During the last decade, there has been remarkable progress in the identification of human cells' transcriptome blueprint through small or large-scale quantitative gene expression studies. Since the gene expression is the major determinant of the protein abundance in the cell, transcriptome analysis experiments have been widely applied to reveal the molecular mechanisms of various cellular conditions. Depending on the cell fate, expression levels of genes vary. Although it seems straightforward to assess these variations through Real Time quantitative PCR or microarrays, there is a continuing and confusing debate on what basis these variations should be considered as deviations from normal physiology. Therefore, there is a need for compilation and comprehensive analysis of a gene of interest across several experiments from different sources. A gene's expression data must be scaled in a comparable platform. Various normalization methods are available to scale these data within the same experiment, yet it becomes problematic to compare arrays of different sources without using references.ACTB, GAPDH, TBP) were differentially expressed in different pathological stages of hepatocellular carcinoma Arabidopsis from the Genevestigator database and developed an online tool, Ref-Genes, that can be used to search for genes with minimal standard deviation across a chosen set of arrays Although most genes show variable expression depending on cellular context, tissue of origin or treatment conditions, some genes are constitutively expressed in all cells in all conditions. These constitutively expressed genes are required for the maintenance of the basal cellular functions such as metabolism, gene expression, protein synthesis and cell signaling Although publicly available microarray or next generation sequencing (NGS) experiments were used to generate lists of candidate reference genes, novel statistical approaches for testing accuracy of a reference gene are still needed. Herein, we aimed to confirm the reliability of available housekeeping gene sets using randomization as well as to determine other invariably expressed gene sets based on Receiver Operating Characteristic (ROC) curves for classifiers under large number of experimental conditions and across a wide panel of tissue types.in vitro.Our method provides reference gene lists for global and cell-type specific normalization of transcriptome data. Gene lists are scored based on their expression stability, and classified according to the Medical Subject Headings (MeSH) associated with the transcriptome study that was published and indexed by National Library of Medicine. Gene lists are provided in the supporting dataset . RT-qPCRHousekeeping/reference genes should exhibit relatively constant expression levels when compared to non-housekeeping genes. To identify the consistency of the so-called housekeeping genes across large-scale experiment sets, the gene expression data were downloaded from the NCBI Gene Expression Omnibus (NCBI-GEO) database iG and a predefined CV threshold t, the ratio of GEO datasets, where CV is less than t, Ratiot(iG), is calculated as the measure of stable expression. Ratiot(iG) is the ratio of GEO datasets, in which a gene exhibits a coefficient of variation less than t. The Ratiot(iG) value, by itself, is not a sufficient measure to identify statistically significant reference genes. A gene that has a small enough CV value can get a perfect ratio even if it is observed in only a single GEO dataset. Therefore, a new parameter, PO(iG), calculated as the percentage of datasets that each gene has been observed in at least once, was used to adjust Ratiot(iG) parameter (t(iG) in the context of PO(iG) allowed for accurate normalization of a large set of microarray data, since it takes into account the information about the differences in probe/clone composition of the arrays. We assessed the utility of these measures by implementing a simple threshold based classifier and computing the sensitivity and specificity of this classifier using a published reference gene set as the ground truth (Methods). Two sets of reference genes were generated. First list contains reference gene lists with a CV threshold of 0.12 and with various sensitivity values while the second list is built with sensitivity of 0.5 for a range of CV threshold values. The average changes in the rank of each gene in each GEO dataset (GDS) was computed based on the ratio of the standard deviation to the mean and termed as its coefficient of variation value (CV) see . CV, as arameter . Ratiot(t(iG) than that of randomly selected genes. In order to compare the expression behaviors of reference genes to that of random genes, we first analyzed the largest previously reported housekeeping datasets et al. was used in our analysis. The 566 housekeeping genes in this dataset were compared to five different randomly selected sets of non-housekeeping genes having the same mean rank distribution as that of the housekeeping gene set.Housekeeping/Reference genes should exhibit relatively constant expression levels and their average rank change should be lower than that of remaining genes. Hence, we assumed that the candidate reference genes should have lower CV and higher Ratiot = 0.5, t = 0.1, t = 0.05, and t = 0.01 and minimum percentage of occurrences PO = 75%, 50%, 25% and 5%. When coefficient of variation, CV, was less than 0.5 (t = 0.5), percentile-ranked GEO datasets showed high Ratiot values for nearly all of the analyzed genes (housekeeping or not), for all PO values. At lower CV thresholds, t = 0.1, t = 0.05 and t = 0.01, housekeeping genes had significantly higher Ratiot values than those of random gene sets for all PO values. The randomization approach allowed us to test an optimum range of t and PO values that can discriminate between reference and non-reference genes. Graphs of Ratiot at PO = 50% with four different CV thresholds  curve in t values were fixed at 75% and 0.90 respectively. The accuracy of this classifier in predicting reference genes was assessed in comparison with the previously reported 566 housekeeping gene set Our primary goal in this study was to define a novel reference gene set that can be used for both global and cell type-specific normalization of expression experiments. For this purpose, a classifier that can be used to identify novel reference genes was built. Based on our analysis with the known 566-housekeeping gene set, coefficient of variation (CV) measure was set as the variable for building the classifier while minimum percentage of occurrence (PO) and RatiotRatio values should still be considered for their potential as a reference gene. CV, Ratio and Percentile Rank values are provided for each gene in the supporting hyperlinked dataset. The global reference genes were given in this list under the category of All with CV of 0.12, sensitivity equal or over 0.5, specificity of 0.97 and minimum percentage of occurrence of 0.75  associated with their experimental data, published and indexed by National Library of Medicine. Of the 381 GEO datasets analyzed in this study, 341 were associated with 272 different medical publications and 264 of these publications were associated with a total of 5754 MeSH terms AARS, ACTB, CFL1, EEF2, GAPDH, GSTO1, H2AFZ, HBXIP, RPL30, RPL41, RPL7, RPN2, RPS10, RPS17, RPS3A, SOD1, TPT1) were assessed by RT-qPCR in 16 different cell lines consisting of 8 Hepatocellular Carcinoma (HCC) , 5 Breast Cancer , and 3 Colon Cancer  cell lines. Housekeeping/reference genes are expected to have high expression and low variability in expression levels between cells. Therefore, in RT-qPCR amplification, they should have low threshold cycle (Cq <30) and low standard deviation. All of the tested reference genes met this requirement for Cq values and standard deviations . Genes were ranked by their stability based on their CV. Comparison of the expression values by analysis of variance (ANOVA) identified RPL30 as the most stable gene with lowest variance within and between the Liver, Breast and Colon Carcinoma cell line groups, followed by RPL41, RPS10 and CFL1. The variance within each of these three groups was low for RPS3A, RPS17, RPL7, ACTB, H2AFZ, and HBXIP reference gene expression. However, the variance between the three groups was relatively high. This implied that these reference genes are more suitable for normalization of cell lines from single tissue-origin than cell lines with different tissue-origins. GSTO1, TPT1, RPN2, SOD1 showed the highest variability.Among the large panel of identified reference genes, 17 genes were selected for experimental validation . Expressviations . The besviations  A–C. In EEF2, GSTO1, RPN2 and TPT1, which have a standard deviation value above the mean standard deviation (stdev = 1.42). The selected reference genes were ranked according to their overall stability values across 16 different cell lines, determined by geNorm and NormFinder  when comparing Liver, Breast and Colon Carcinoma cell lines. An even more accurate normalization can be achieved if RPL7 and RPS3A are also included as reference genes. .In order to determine the optimal number of reference genes for accurate normalization, pairwise variation VRPS13, RPL27, RPS20 and OAZ1. For each gene the coefficient of variation (CV) of its expression and the maximum fold change was calculated to identify genes with the minor variation in expression et. al. published an updated new housekeeping gene list based on analysis of RNA-seq data Even the most frequently used reference genes are subject to differential regulation under specific treatments or between different cell lines or tissues. Therefore, new reference gene sets should be determined instead of using traditional housekeeping/reference genes that are themselves prone to differential regulation. The use of two or more housekeeping genes for normalization can improve the reliability of normalization RPL30 and RPL41 are good reference genes for comparing all cell lines regardless of their origin. Previously, 451 housekeeping genes that were expressed in all of the 19 distinct normal human tissue types studied, were shown to have variable expression levels among different tissues and ribosomal genes, which were among the most stable genes, were suggested as reference genes suitable for normalization purposes Our reference gene lists were dominated by the ribosomal protein genes and genes that are involved in maintenance of basal cellular activities such as translation and metabolism. Especially, iG in a sample ie, a single rank value, r(iG.j)e was computed  is a function, which gives the mean normalized expression value of gene g in sample e and G is the set of all genes.In order to normalize expression values, percentile ranking have been applied. For each gene computed . For a gComputation of coefficient of variation value for each gene in each GEO dataset and identification of candidate reference genes1G.....mG} be the set of genes and S  =  {1e.....ne} be a GEO dataset of n samples. For gene iG in experiment je, a single rank value, r(iG.je), was computed. Given a set S with n experiments, we had at most n rank values for a gene. The average amount of change in the rank of gene iG, in set S was then computed by the Coefficient of Variation CV(iS.G) as given below in Let G =  {iG in each GEO dataset S.In this analysis, we computed a coefficient of variation value for each gene t, observed in as many experiments as possible. In order to account for platform related differences, for each gene iG and threshold t, we computed a ratio. Ratiot(iG) as follows:Next, we identified candidate reference genes with coefficient of variation values below a predefined threshold, fPO(r) be the function that gives the number of genes with tRatio greater than a given r and occur in at least in PO% of the datasets (Let datasets .(6)fPO(r), varying r from 0 to 1, for housekeeping genes and randomly selected non-housekeeping genes.We plotted and compared the graph of t of the datasets as housekeeping genes. All these analyses are performed at a fixed percentage of occurrence of 75% and with a fixed Ratiot of 0.9. The ground truth genes that are identified as housekeeping genes by this classifier are true positives (TP) whereas, the ground truth genes that are identified as non-housekeeping genes are false negatives (FN). Similarly, genes not in the ground truth set but identified as housekeeping genes are false positive genes (FP) and genes not in the ground truth set identified as non-housekeeping genes by the classifier are true negatives (TN). Using these definitions, sensitivity is given by TP/(TP+FN) and specificity is computed as TN/(TN+FP).We use the housekeeping gene set published by Eisenberg et al. Cell lines were obtained from the following sources and validated by STR analysis: HepG2 (ATCC HB-8065), FOCUS RNA was extracted from 8 Hepatocellular Carcinoma cell lines , 5 Breast Carcinoma cell lines  and 3 Colon Carcinoma cell lines  with NucleoSpin Total RNA Isolation Kit and the concentration and purity of total RNA from each cell line was measured by using a NanoDrop Spectrophotometer (NanoDrop Technologies). Reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit (Fermentas) from 2 μg RNA with oligodT primer.(−1/slope)-1. Real-time quantitative PCR assays were performed in duplicate for each candidate gene using 1 μl of 1∶100 diluted cDNA template with DyNAmo SYBR Green qPCR Kit (Finnzymes) on BioRad iCycler Real-Time qPCR System. The following program was used: Initial denaturation at 95°C 15 min amplification for 45 cycles (95°C 15 s followed by 57°C 30 s and 72°C 30 s), and final extension at 72°C 10 min. Melting curve analysis was done for each run, in addition to agarose gel electrophoresis, to confirm the amplicon size and presence of a single gene-specific peak free from any primer-dimer or genomic DNA amplification.Primers for 17 selected housekeeping genes were designed using the Primer3 software  was calculated as the ratio of standard deviation to the mean relative expression. geNorm and NormFinder software were used for stability analysis.BioRad iCycler was programmed to set the Cq threshold on a fixed level. Cq values generated by BioRad iCycler system were transformed into quantities  according to Vandesompele Supporting Information S1Reference Gene Lists.(ZIP)Click here for additional data file.Supporting Information S2Tables S1–S5. Table S1. Kolmogorov–Simirnov Test results for the hypothesis comparing pair-wise equivalence of the ratio distribution of housekeeping gene set to the ratio distribution of random sets of genes excluding the housekeeping genes. Table S2. Kolmogorov–Simirnov Test results for the hypothesis comparing pair-wise equivalence of the ratio distribution of random sets of genes, excluding the housekeeping genes. Table S3. Stability values of 17 reference genes calculated by NormFinder and geNorm. Table S4. Stability values of 13 genes, with standard deviation lower than 1.42, calculated by NormFinder and geNorm. Table S5. Real-time quantitative PCR primers.(PDF)Click here for additional data file.Supporting Information S3ROC Curves.(ZIP)Click here for additional data file.


Glossina palpalis gambiensis Vanderplank and Glossina tachinoides Westwood The cotton belt in Mali/Burkina Faso is among the eco-zones with the highest potential for agriculture and livestock development in West Africa. In this zone, most humans live in rural settings and the development of more sustainable and profitable livestock and mixed-farming systems is mainly constrained by African animal trypanosomosis (AAT), transmitted by the riverine tsetse flies Glossina pallidipes Austen from Zulu Land in South Africa Glossina austeni Newstead from Unguja Island, Zanzibar Glossina morsitans centralis Machado from the Okavango delta in Botswana The Pan African Tsetse and Trypanosomosis Eradication Campaign (PATTEC) recognised that, for tsetse eradication to be sustainable, it requires an area-wide approach where the control effort is directed against an entire pest population within a circumscribed area G. p. gambiensis is mainly linear along the hydrological network during the dry season, flies can also disperse perpendicular to the river systems, especially during the rainy season, although accurate field data are scarce As riverine tsetse populations are mainly confined to suitable vegetation along the hydrological network in the subhumid savannah, it was suggested that the “river basin” could be used as the unit of operation in AW-IPM G. p. gambiensis were released in tributaries of two river basins , in close proximity to the adjacent basin (Niger). Attempts were made to recapture the released flies in the savannah between the river basins and, in one site, well within the adjacent river basin.To clarify the role of river basins in structuring tsetse populations, genetic studies were initiated in various areas in West Africa. These studies indicated considerable gene flow between riverine tsetse populations belonging to different river basins, and hence, these populations cannot be considered isolated G. p. gambiensis colony maintained at the Centre International de Recherche-Développement Sur l'Elevage en Zone Subhumide (CIRDES), Bobo Dioulasso, Burkina Faso since 1972. Flies were sterilised with a dose of 110 Gy in a 137Ce source. Both sterile male and female flies were marked with a dot of acrylic paint on the thorax, with a different colour for each week. The flies were transported in carton release containers (dimensions 115×90×50 mm) at a density of 100 flies per box with chartered light aircraft from Bobo Dioulasso to Bamako, Mali, arriving at destination between 7:30 and 10:30 a.m. Upon arrival at the airport, the flies were immediately transported by car to the different release points (RPs). RP 1 and 2 were located on tributaries of the river Senegal and RP 3 and 4 on tributaries of the river Bani  and a constant mortality rate μ of 0.1 (0.07–0.14) for the entire observation period (28 d). The confidence intervals below corresponded to the values giving the same maximal correlation coefficient using this mortality range. Similar mortality rates were observed for this strain of During the entire monitoring period, a total of 589 sterile male flies (1.8% recapture rate) and 327 sterile female flies (1.4% recapture rate) were trapped. In addition, a total of 18 wild males and 16 wild females were caught . Mean survival upon arrival at the RPs was 70% and 85% for the sterile male and female flies, respectively. Percentage of nonfliers were 0% and 7%, respectively. In one of the two sites where traps were placed also in the adjacent Niger basin (RP2), one marked female fly was recaptured on the other side of the watershed .2/d and 0.101 km2/d (D = λ2/2). With an average mortality rate of 0.02  and using the mean estimation of λ, the calculated average dispersal distances were 1,268 m for female and 501 m for male flies.The estimations of λ were very similar between batches and sites, but surprisingly, it was higher in females  than males , corresponding to diffusion coefficients D of 0.304 kmSince the average distance between the release point RP 2 and the traps deployed in the adjacent Niger Basin was ∼3 km, the probability that one fly might reach one of the traps was 0.086, for λ = 780 m and μ = 0.1 . As 5,17G. p. gambiensis can disperse between river basins in Mali. The dispersal data are comparable to the measured diffusion coefficients in savannah areas, and flies were recaptured in traps deployed at 2 km distance from the river forest in Burkina Faso The data presented in this paper indicate that 110 Gy-treated flies  were capable of crossing the watershed between adjacent river basins in Mali. Although only one fly was recaptured in the adjacent river basin, it is proof of principle that G. p. gambiensis populations from different river basins cannot be considered isolated from one another. Barriers to prevent reinvasion would have to be established between eradication blocks should governments involved in the PATTEC initiative plan a sequential eradication strategy using the rolling carpet approach G. p. gambiensis and G. tachinoidesG. m. centralis in the Okavango delta in Botswana G. p. gambiensis belt from Ghana to Senegal with a view to determining the most appropriate locations for establishing barriers to prevent reinvasion, taking into account the suitability and fragmentation of vegetation between the river basins.These data corroborate results from population genetics studies indicating that in West Africa


Given the rapidly expanding list of inflammatory conditions for which tumor necrosis factor-α inhibitors are receiving FDA approval, the incidence of tuberculosis in this patient population has increased. Despite heightened awareness by physicians, the diagnosis of tuberculosis can remain challenging, given that extrapulmonary sites of infection are more frequently involved. We present a case of pulmonary and peritoneal tuberculosis in a gentleman being treated with a tumor necrosis factor-α inhibitor and discuss the diagnostic challenges of establishing the diagnosis.The association between the use of tumor necrosis factor- We report a case of both pulmonary and peritoneal TB in a patient being treated with adalimumab that illustrates some of the challenges associated with making this diagnosis in this patient population.The number of tuberculosis (TB) cases in the United States (USA) has been falling since the peak in 1992. However since 1992 there has also been a gradual increase of the percentage of total TB cases that have involved extrapulmonary sites of infection. Furthermore, the introduction of the tumor necrosis factor- (TNF-) A previously healthy 28-year-old man with a history of extensive plaque psoriasis on treatment with subcutaneous adalimumab for over two years presented to the infectious diseases clinic with a two month history of low-grade fevers, myalgias, and a severe nonproductive cough. The patient had been treated with a course of doxycycline followed by azithromycin approximately one month earlier, causing temporary improvement in his symptoms. The patient had emigrated from Vietnam 11 years previously. The patient's grandfather had been treated for pulmonary mycobacterium tuberculosis (MTB) in Vietnam 15 years previously, and the patient had been treated for latent tuberculosis with nine months of isoniazid in 2004. The patient lived in Washington, DC, worked in a healthcare setting, was a nonsmoker, had no pets, and had never received a pneumococcal vaccine. Outpatient evaluation by a pulmonologist three weeks previously included unremarkable laboratory studies and clear chest X-ray. On evaluation in our clinic, the patient was noted to be nontoxic appearing and able to speak in full sentences. His temperature on initial evaluation was 37.2°C, blood pressure 109/73 mmHg, heart rate 85 beats per minute, and respiratory rate 14 breathes per minute. His lung examination was unremarkable with clear breath sounds bilaterally without rhonchi or wheezes. A computed tomography (CT) scan of the chest performed the same day showed a 1.1 cm lobular opacity in the right lower lobe, a moderate right pleural effusion , hilar lOn hospital admission, his temperature was 39°C, blood pressure 121/87 mmHg, heart rate 95 beats per minute, and respiratory rate 22 breathes per minute, with an oxygen saturation of 97% on ambient air. Physical exam was notable for bilateral crackles in his lungs and a diffuse psoriatic rash. A CT scan of the abdomen and pelvis performed with intravenous contrast revealed diffuse nodularity of the omentum and peritoneal lining , raising On the second hospital day, the patient underwent an unremarkable bronchoscopy and a thoracentesis which revealed serosanguinous pleural fluid. The pleural fluid was exudative with an 80% lymphocytic predominance. No acid fast bacilli (AFB) were seen. The pleural fluid adenosine deaminase (ADA) was elevated at 110.3 u/L. A subsequent percutaneous peritoneal biopsy was performed revealing bloody peritoneal fluid. Grocott's methenamine silver stain, AFB stain, Fite's stain, and mucin stains on the fluid were negative. Surgical pathology revealed necrotizing granulomatous inflammation with no AFB seen on Auramine-rhodamine stain. The patient was empirically started on isoniazid, rifampin, pyrazinamide, and ethambutol based on the imaging and pathology results. The patient underwent an abdominal laparoscopy one week after admission in an attempt to make a definitive diagnosis. This revealed extensive peritoneal and omental studding measuring 2 to 3 mm in size. A modest amount of bloody ascites was present. Pathology revealed granulomatous inflammation and one AFB on Fite's stain but no AFB on Auramine-rhodamine stain. Growth of MTB complex was noted from the percutaneous peritoneal biopsy after 16 days, and subsequently all other culture specimens showed growth by day 30. Sensitivities revealed no antimicrobial resistance. The patient was continued on his four drug MTB therapy, but the rifampin was discontinued after 2 weeks due to drug-induced hepatitis. Once his hepatitis had resolved, the rifampin was restarted without further transaminitis. He completed 2 months of directly observed therapy with isoniazid, rifampin, pyrazinamide, and ethambutol and a further 7 months of isoniazid and rifampin. He has made a full recovery.In 2011, a total of 10,521 new MTB cases were reported in the United States (USA). This incidence of 3.4 cases per 100,000 population is the lowest number of reported cases since reporting began in the USA in 1953 [The peritoneum is reported as the sixth most common site of involvement for extrapulmonary MTB . The symA variety of imaging modalities can detect ascites and omental thickening seen in TBP. Ascites is seen radiographically in 30%–100% of cases . UltrasoSanai and Bzeizidocumented abnormal chest radiography in 38% of 1002 cases they reviewed . PleuralCulture of the tuberculous bacilli from an intra-abdominal clinical specimen is diagnostic for TBP. However mycobacterial culture of ascitic fluid was positive in only 35% of 446 patients reviewed from 22 studies [The current gold standard for establishing the diagnosis of TBP is obtaining mycobacterial cultures from either the peritoneal or omentum. Open laparoscopy has been shown to be superior to percutaneous peritoneal biopsy for obtaining these culture specimens, since laparoscopy allows for macroscopic visualization of the peritoneum. Laparoscopic examination can reveal whitish nodules scattered throughout the peritoneum along with ascites, thickened omentum and thickened bowel loops, or yellowish nodules with adhesions . Histoloα inhibitor in 1998, there has been a noticeable rise in the incidence of tuberculous infections in this patient population [α in controlling the spread of mycobacterial infections. MTB infectionsresult in activation of macrophages which, as a result, secret proinflammatory cytokines including TNF-α, which is vital for granuloma formation [α promotes further macrophage activation, which in turn stimulates phagocytosis of the bacilli [α inhibitors interfere with the formation of granulomas required to contain certain infections, there is a subsequent increased susceptibility to develop infections due to fungi and mycobacteria in this population. Furthermore, TNF-α inhibitors have also been shown to cause T-cell death and monocytopenia, both of which are also necessary for granuloma formation and further reducing immunity against such infections [Since the approval of the first TNF-pulation  with a gpulation . This hiormation . TNF-α p bacilli . Necrosifections .α inhibitors. Through 2002, the FDA reported an MTB incidence of 144 per 100,000 patients treated with infliximab and 35 per 100,000 patients treated with etanercept [α inhibitors, using data from a large registry of biological treatments used in patients with rheumatoid arthritis [In 2004, the Adverse Event Reporting System of the FDA highlighted the increased incidence of MTB in patients being treated with TNF-anercept . Adalimuanercept . A largerthritis . Adalimurthritis . α blockade. Etanercept only binds soluble TNF-α, while adalimumab and infliximab bind soluble, transmembrane, and receptor-bound TNF in an irreversible manner [α in tuberculosis immunity thereby reducing the incidence of tuberculosis [One possible explanation for the difference in MTB incidence with these agents is related to their degree of TNF-e manner . It is trculosis .α inhibitors, especially in extrapulmonary locations, it often remains challenging to definitively establish this diagnosis. Clinicians need to maintain a high index of suspicion and should aggressively pursue tissue specimens for microbiologic culture and histology. Our case highlights a patient who had both pulmonary and peritoneal TB who did not present with the classic pulmonary or peritoneal symptoms. However, a high index of suspicion by the evaluating physician prompted appropriate radiographic and surgical evaluation, which ultimately revealed the underlying mycobacterial infection.In conclusion, while it has been well established that there is an increased risk for reactivation of TB in patients being treated with TNF-


Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, environment and disease. The effects of the field environment on plant hormone-specific metabolite profiles are largely unknown. Few studies have analyzed useful phenotypes generated by introducing single or multiple gene events alongside the non-engineered wild type control at field scale to determine the robustness of the genetic trait and its modulation in the metabolome as a function of specific agroecosystem environments.We evaluated the influence of genetic background  and environments  on the metabolomic profile of isogenic reverse genetic mutations and selected mulch based cropping systems in tomato fruit. Net photosynthesis and fruit yield were also determined.NMR spectroscopy was used for quantifying metabolites that are central to primary metabolism. We analyzed both the first moment (means) of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, and the second moment (covariances) of responses by creating networks that depicted changes in correlations of paired metabolites. This particular approach is novel and was necessary because our experimental material yielded highly variable metabolic responses that could not be easily understood using the traditional analytical approaches for first moment statistics.High endogenous spermidine and spermine content exhibited strong effects on amino acids, Krebs cycle intermediates and energy molecules (ADP + ATP) in ripening fruits of plants grown under different agroecosystem environments. The metabolic response to high polyamine genotypes was similar to the response to hairy vetch cover crop mulch; supported by the pattern of changes in correlation between metabolites. Changes in primary metabolites of genotypes mutated for the deficiency of ethylene or methyl jasmonate were unique under all growth conditions and opposite of high polyamine genotype results. The high polyamine trait was found to dominate the low ethylene and low jasmonate mutations under field conditions. For several metabolites low ethylene and low methyl jasmonate genotypes had an inverse relationship. Collectively, these results affirm that interactions between metabolite pathways and growth environments are affected by genotype, and influence the metabolite quality of a crop.This study portrays how metabolite relationships change, both in mean and in correlation, under different genotypic and environmental conditions. Although these networks are surprisingly dynamic, we also find examples of selectively conserved associations.The online version of this article (doi:10.1007/s11306-016-1037-2) contains supplementary material, which is available to authorized users. Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, biodiversity, environment and disease. Thus, to mention a few examples, metabolomics has contributed to mapping plant cell types  of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, as well as the second moment (covariances) of response by creating networks that depicting changes in correlations of paired metabolites.Previous research investigating metabolomic patterns have focused on the first  moment, i.e. changes in mean values, whereby observed metabolic mean values and differences between mean values are presented to describe the metabolomic state or change in state of the organism under study in response to an exogenous or genetic manipulation. We also investigated changes in the second  moment, covariances of metabolites; how the covariances responded to experimental treatment  in a fruit specific manner for enhanced metabolic activity and accumulate polyamines spermidine and spermine in a fruit ripening-specific manner , which have higher processing quality, longer vine life and higher lycopene content  gene under CaMV 35S promoter to constitutively suppress the production of ripening hormone ethylene , which produces half the ethylene than the control 556AZ line and has considerably longer shelf-life   homozygous for the indicated genetic event(s) were tested alongside the azygous control line (556AZ): Five genotypes  in 2006, and an additional line 2AS-2HO was added to the field trial in 2007. The codes used for each tomato line (genotypes) are summarized in Supplementary Table 1. These included three types of engineered genotypes of tomato with the modulated fruit shelf-life, fruit texture and metabolic/nutrient activity of tomato fruit. Thus, individual genetic events included tomato lines engineered with yeast SAM decarboxylase gene under ripening-specific E8 promoter  under ambient mid-day conditions of light, temperature, humidity and CO2 concentration, using a broad-leaf cuvette. Measurements were made on July 18, Aug 01, Aug 17 and Sep 07 in 2006 and on Aug 02, Aug 14, Aug 27, and Sep 12 in 2007. Leaf temperatures ranged from 26 to 30 °C across all measurement dates. Mean values of the leaf to air difference in water vapor pressure ranged from 1.2 to 1.8 kPa for the different measurement dates. Photosynthetic photon flux density exceeded 1200 µmol m−2 s−1 for all measurements, and the CO2 concentration external to the leaves was controlled at 370 µmol mol−1. Leaves selected for measurement were recently fully expanded upper canopy leaves fully exposed to light, and were found to have the highest rates of any leaves on the plants. On each date, gas exchange was measured on one leaf of each genotype in each replicate plot. The order of measurements of genotypes within mulch treatments was random, and the mulch treatments were measured such that the mean time of day was equalized. Measurements were completed in about 2 h on each date.Single leaf COIn 2006, pink and red fruit  were harvested from the five-plant subplots from each replication on two dates, the first harvest took place on Sep 12, and the final harvest on Sep 22. In 2007, the five-plant subplots were harvested from three replicates at two times. For the first harvest, all the pink and red fruits were harvested Sep 6 to 9. The same plants were harvested for a second time on Sep 25 to 28. The number and weight of fruit per plant and average fruit weight were calculated both for the early harvest and for the total of the two harvests.g for 15 min and the supernatants assayed  following the manufacturer’s instructions, in a final volume of 20 μl. The final cDNA products were diluted tenfold prior to use in real-time RT. Quantitative RT-PCR (Q-PCR) was performed with gene-specific primers (Supplementary Table 3), which were designed using Primer3 software. The Q-PCR conditions were the same as before . RNA quantification and quality were determined using a spectrophotometer and native agarose gel electrophoresis, respectively. First strand cDNA synthesis was performed using the SuperScriptUnivariate analyses were performed with analysis of variance (ANOVA) using the mixed model procedure of SAS version 9.2 . The model for analyzing fruit yield variables, photosynthesis variables, polyamines and metabolites had mulch, genotype and mulch by genotype interaction treatments as fixed effects. Year and field block nested within year were random effects for the fruit yield analyses, year and block within year and sampling date within year were random effects for the photosynthesis analyses, and year and fruit stage were random effects for the polyamine and metabolite analyses. Principal components analysis (PCA) was performed on the metabolite data set after standardization to mean zero and standard deviation one. Two ANOVA’s were performed, one for each of the first two components using mulch, genotype and mulch by genotype interaction treatments as fixed effects.Metabolites were organized into groupings of amino acids, organic acids, sugars, and energy metabolites in order to facilitate analysis of overall trends within groups. Each metabolite was standardized to mean = 0 and standard deviation = 1 to give equal weighting to each metabolite. An ANOVA was then performed on the standardized metabolic data by group with genotype or mulch treatment as a fixed effect.2 concentration) and metabolite variables. Univariate Pearson correlations between these variables were also calculated.Canonical correlation analysis (CCA) was performed to explore multivariate correlations between whole plant variables  for individual metabolite pairs, following the Fisher r-to-z transformation, and show only large changes in correlation (for P < 0.05). Pearson correlations were calculated using the R statistical software . We did not include correlation changes involving the uncharacterized “B” metabolite. We color-coded the networks, with blue representing increases in correlation  and red representing decreases in correlation . The thickness of the lines joining the metabolites was used to represent the magnitude of the change , with the thinnest line representing a change of about 0.1 in correlation and the thickest a change of about 0.7 in correlation. An important point to remember when looking at the figures is that they do not represent the correlations themselves, rather they represent changes in correlation. Thus, two metabolites that are highly correlated in both subsets of data would not be linked in the figure because their correlation did not change.To determine which changes in correlations were the important ones to depict in our networks, we established a confidence interval for the hypothesis of no change in correlation  or more generally, using approaches such as false discovery rate .We are aware that analyses repeated in different ways on the same data set leads to correlated test statistics. The ySAMDC), tomato E8 (SlE8) and tomato lipoxygenase (SlLOX) in different tomato genotypes grown in BP (black plastic mulch) and HV (hairy vetch mulch) are shown in Supplementary Fig. 1. As expected, lines 8 and 10, carrying the ySAMDC transgene, expressed ySAMDC transcripts. However, line 20 had higher expression of ySAMDC transcripts than its parent line 8. Specific effects of the BP versus HV were evident: ySAMDC transcript expression in line 20 (G20) was several-fold enhanced under BP . To ascertain if this BP effect was due to an effect on the SlE8 promoter used to drive the expression of the chimeric ySAMDC construct, the accumulation of SlE8 transcripts was also quantified under BP and HV. The pattern of SlE8 transcript accumulation in BP-grown lines 8 and 10 was similar to the pattern of ySAMDC transcripts . In fact, BP-grown line 12 had higher expression of SlE8 transcripts than when grown under HV , and in contrast these transcripts in control azygous line 5 accumulated maximally under HV. SlLOX transcripts were suppressed in both line 12 and its sibling, genotype 20, as expected while mulch systems had little differential effect. HV mulch had a more enhancing effect on SlLOX expression in line 5 (G5) and line 8 (G8). In general, the relative accumulation levels of SlE8 transcripts were considerably lower than ySAMDC and SlLOX transcripts.We first ascertained the stability of the introduced genes in tomato plants in the field and/or their biochemical phenotype. In addition to the engineered lines mentioned above, we developed and tested additional tomato lines with double genetic events by making backcrosses between one high polyamine line (line 10) and ethylene-suppressed line 2 , as well as another high polyamine line (line 8) and meJAS-deficient line 12  with an azygous line as a control (description in Supplementary Table 1). Q-PCR data of three genes—yeast SAM decarboxylase . There were no significant effects for any of these variables among genotypes 5, 8, 12, and 20.Constitutive suppression of ethylene was found to result in higher net photosynthesis and stomatal conductance in line 2 than in the azygous control (Supplementary Table 5). This was true also for its sibling line 4 (crossed with high polyamine line 10), which behaved like parent 2 for all variables. In contrast, line 10 with fruit-specific high polyamine content had the lowest net photosynthesis, stomatal conductance and fruit yield. This phenotypic trait was, as expected, absent from line 4, likely because of the differential regulation of the genetic events introduced, suggesting that this fruit-specific event was not transferred to vegetative parts of the tomato plant. In all genotypes, the differences in net photosynthesis were found unrelated to internal COTomato fruit yield was higher in plants grown in hairy vetch or black polyethylene mulch than in rye mulch or bare soil Fig. , in lineP values >0.05, results not shown). Although this analysis did not adequately resolve genotype and mulch system treatments, we further explored the data set, looking at the variables independently using univariate ANOVA, and then with metabolite networks.Principal components analysis (PCA) was performed on the metabolite data set after standardization to zero mean and unit standard deviation. PCA decomposed the metabolome data set to two principal components with 36 % of the explainable variation described by the first component and 20 % of the explainable variation described by the second component. Other components each described less than 10 % of this variation. The first principal components axis was most positively correlated with the metabolites histidine (HIS), asparagine (ASN), phenylalanine (PHE), and threonine (THR) (eigenvectors > +0.25) and most negatively correlated with glucose (bGLC) and fructose (FRU) (eigenvectors < −0.08) . In contrast genotype 2 had higher levels of inositol and adenosine than either genotypes 8 and 10. Genotype 10 had higher levels of choline and mNAM than all other genotypes. Sibling 20 was more similar to its parent genotype 8 for valine, citrate, succinate, and adenosine, but more similar to genotype 12 for asparagine. Sibling 4 was more like its parent genotype 10 for valine, inositol, and adenosine, but more like its parent genotype 2 for choline and mNAM.Significant univariate effects due to agroecosystem environment, created by different mulches, on amino acids, organic acids, sugars, and energy fruit metabolites are summarized in Supplementary Table 7. The hairy vetch (HV) mulching system led to higher levels of selected amino acids , and organic acids  and mNAM, and lower levels of the sugars (glucose and fructose). Like HV, rye mulch (Rye) also increased glutamate and threonine but its effect on aspartate was greater than HV, the black polyethylene (BP) mulching system, and bare soil (BS) . In contrast to HV and Rye systems, both glucose and fructose, and NUC2, were higher under BP and BS environments.P < 0.05) among standardized amino acid and organic acid means for both mulch and genotype, but not for means of standardized sugar or energy metabolites (Supplementary Tables 6 and 7). Plants grown in the HV mulch system had higher overall amino acid and organic acid levels, whereas plants grown in the BP mulch system had the lowest levels . Univariate correlations between plant and fruit metabolite variables were generally low . Thus, there was little linkage between plant performance and fruit metabolism in these genotypes and mulching systems.changes in correlations between metabolite pairs among subsets of data. A listing of the correlation between all pairs of metabolites for each data subset is presented in Supplementary Table 8. The data were partitioned into subsets of interest for this study, namely, (a) genotypes whose fruit have low  versus high polyamine content (Spd and Spm) , (b) normal  versus deficient (lines 12 and 20) meJAS content, and (c) mulch system in which the plants were grown (two categories: bare and black polyethylene versus hairy vetch and rye cover mulches).When we used PCA and WCNA . The least stable metabolite pairs are colored darkest red  in the heat map while the most stable are yellow . The most stable was the pair, TRP-SUCR, with the smallest standard deviation of 0.109, and the least stable was AMP-Nucl2, with the highest standard deviation of 0.518. Metabolites that had the most stable associations with other metabolites were citrate and formate  and sucrose . Correlational relationships amongst these metabolite pairs with low standard deviations were least disturbed by perturbations caused by differences in genotype or mulching system and represent the most stable linkages between metabolites in this experiment. In contrast, metabolite pairs with large standard deviations illustrate pairs whose linkage changed most in contrasting treatments; analyzed in more detail below.Figure Cytoscape (v. 3.1.1)  generated via transgenic intervention, and by the agroecosystem environment. Our data on metabolic changes reinforce the need to account for agroecosystem effects on ‘novel’ and other engineered genotypes for variables like fruit yield, metabolite/nutrient content and longevity. Studies like ours contribute information necessary for agricultural sustainability by identifying genotypes that have robust agronomic and other desirable traits under different agroecosystems  versus under different agroecosystems in the field. Such a comparison proved quite revealing, when data generated in the present study were compared to those using GH-grown tomatoes  trait was dominant in the background of methyl jasmonate deficiency in maintaining higher levels of several amino acids  under field conditions. Similarly, the high polyamine trait was found to be dominant for Krebs cycle intermediates—citrate, succinate and fumarate—and energy-related metabolites (ATP + ADP) under field conditions but not under greenhouse conditions. Collectively, these results affirm that specific interactions which exist between a particular metabolite pathway and growth environment are affected by the genotype being tested, and will therefore influence the metabolite quality of a crop.Arabidopsis mutants and transgenic crops have shown involvement of polyamines in plant resistance to drought, salinity and heat  the precursors of polyamine biosynthesis are themselves amino acids—arginine (ARG) and ornithine (ORN)—whose decarboxylation reactions form putrescine (Put), which is subsequently converted step-wise to spermidine (Spd), spermine (Spm) and thermo-spermine (T-Spm); (2) studies with Many of these metabolites were affected by genotype in our analyses. Univariate analyses showed alanine and succinate levels were increased in high polyamine genotypes, suggesting an accelerated movement of carbon through polyamines into Krebs cycle along with generation of ATP + ADP, which were also increased in high polyamine genotypes. Network analysis also showed alanine and succinate as hubs of metabolic changes as genotype changes from low to high polyamine accumulation Fig. a. AlthouFinally, our data analysis highlights the distinctive fluidity of metabolite networks associated with agroecosystem environment. Not only were metabolic profiles different in the field (this experiment) versus greenhouse (previous experiments) environments, but modification of the field agroecosystem had a profound effect on metabolites. The traditional culture of tomatoes in black polyethylene mulch increases soil temperature, and accelerates vegetative growth and the advent of reproductive development leading to higher relative growth rates of fruit Below is the link to the electronic supplementary material.