Limited data are available regarding correlates of regular sexually transmitted infections (STIs) and HIV screening among female sex workers (FSW) in Sub-Saharan Africa. In this study, we aimed to assess the frequency of regular syphilis and HIV screening and the psychosocial correlates associated with screening among FSW in Uganda.This cross-sectional correlational study was conducted among 441 FSW, aged 17–49 years. We enrolled FSW through peer referrals and ascertained self-reported data on number of serological tests for HIV, syphilis and other STIs in the prior 12 months using an interviewer-administered questionnaire. In addition, we assessed attitudes, norms, social influences and self-efficacy towards 3-monthly Syphilis and 6-monthly HIV testing. We estimated the correlates of regular STI and HIV testing using negative binomial regression.Of the respondents 420 (95.2%) reported to have ever taken an HIV test with 297 (67.4%) testing two or more times in the prior 12 months. Over half of the respondents (59%) reported ever taking a syphilis test with only 62 (14.1%) reporting testing three or more times in the prior 12 months. After adjusting for socio-demographics, attitude and norms, high perceived self-efficacy was associated with a 33% increase in the likelihood of repeated HIV testing  while low perceived confidence was associated with a 25% decrease in the likelihood of repeated HIV testing . Similarly low attitudes and norms were associated with a decrease of 52.6%  and 47%  in the likelihood of repeated syphilis testing respectively.Compared to HIV, uptake of repeated syphilis testing was very low. Correlates of HIV testing include; perceived self-efficacy amidst barriers and perceived confidence for HIV and low attitudes and accepting norms for syphilis. Health campaigns should emphasize overcoming barriers to HIV testing while promoting attitudes and norms including integration of serological syphilis testing and other STIs into HIV services. The burden of HIV and other sexually transmitted infections (STIs) is disproportionately high among female sex workers globally , 2. FemaThe disproportionate HIV burden in African sex workers is engendered by STI co-infection. Evidence shows that in 2014, STI prevalence ranged from 24.9% to 77.5%; between half and two-thirds of FSW have a curable STI at any one time, 30% have reactive syphilis serology and 10% or more have an active genital ulcer . SimilarThe World Health Organization (WHO) recommends STI screening for FSW every 3–6 months and HIV testing every 6–12 months . RegularIn this study, we aimed to assess the frequency of STI and HIV screening and the psychosocial correlates associated with screening among FSW in Uganda. Psychosocial factors including behavioural intentions, attitudes, social norms, perceived efficacy, perceived severity and normative beliefs among others have received recognition as predictors of health behaviour –34. DeriBetween July and October 2018, we conducted a cross-sectional survey of 441 FSW in the municipalities of Arua, Mbale, Mbarara and Kampala   represenPrior to participant recruitment, a mapping exercise was conducted to gain an understanding of time, hot spots, typologies, sex work procurer connections and territorial management in each municipality. From the mapping exercise, the main typologies based on venue were: street, lodge, and bar/club, and brothel . Sex work hotspots constituted the primary sampling units (PSUs). Based on the different typologies, lists were drawn to form a sampling frame for each municipality. In Kampala, we randomly selected 20 PSUs, consisting of brothels, lodges, streets and bar/club. In Mbarara, Arua and Mbale, we randomly selected between 10 and 15 PSUs consisting mainly of lodges, street and bar/club. Between 7 and 10 participants were recruited from each PSU in each municipality.Sampling began with 2–3 FSW at each PSUs, identified through key informants . The recruits were given a brief training on peer recruitment and 2–3 paper coupons. Each coupon contained an identification number, contact information of the research team and the duration of the survey in the municipality. Only respondents who presented the coupon after verification and met the eligibility criteria were consented to participate in an interviewer-administered questionnaire. The questionnaire is included in the supplemental methods . All respondents provided written consent in their local language.The primary outcome was count of STI and HIV testing in the prior 12 months, defined as number of serological tests for HIV, syphilis and other STIs in the prior 12 months. The major independent variables were categorized as attitude or motivators, self-efficacy, moral norms or normative and social influences. These categories were derived from constructs of the Integrated Change Model , 37. AttThe Kaiser–Meyer–Olkin measure verified the sampling adequacy for the analysis, KMO of 0.90, and all KMO values for individual items were greater than 0.85, well above the limit of 0.5 . Bartlett’s test of sphericity X2 435) of 5669.642, p < 0.001, indicated the correlations between the items were large enough for PCA. Nine factors had eigenvalues over Kaiser’s criterion of 1 and all together explained 66.175% of the variance. The items that clustered on the same components suggested that the factors represented Intention to test for HIV in the next 6 months (IN-HIV), Intention to test for syphilis in the next 3 months (IN-SY), attitude towards 6 monthly HIV testing (AT-HIV), Attitude towards 3 monthly syphilis testing (AT-SY), social approval of 6 monthly HIV testing (SN), Moral norm towards testing every 3 months for syphilis (MN_SY), Moral norm towards testing for HIV every 6 months (MN-HIV), Perceived efficacy to go for 3 monthly syphilis checkups (SE-SY) and Perceived efficacy to go for 6 monthly HIV testing (SE_HIV). Using Cronbach’s α statistic, we assessed the reliability of item scales and computed scale medians  using negative binomial regression (for over dispersed count data). We used a likelihood ratio test of alpha = 0 (dispersion parameter) to assess model fit and found no evidence of over-dispersion. Crude and adjusted prevalence ratios (aPR) and 95% confidence intervals (CI) were estimated. Statistical analyses were performed using SPSS version 23.0  and Stata version 12.0 .The median age of the participants was 26 years  Table . Just ovA significant number  of respondents reported past medical history of syphilis  compared to 21  for HIV , condom use practices, as well as self-efficacy, the correlates of repeated syphilis testing were: attitude, moral norm and town location , condom use practices, as well as attitude, moral norms, and social norms, the correlates of repeated HIV testing were: perceived self-efficacy amidst barriers (SEB_HIV), confidence and town location . The findings of the study show that 95.2% of the FSW reported ever testing for HIV with 67.4% testing two or more times in the last 12 months preceding the survey. Relatedly, 59% reported ever testing for syphilis with only 14.1% testing three or more times in the last 12 months. The Health Organization (WHO) recommends 3–6 monthly screening for STIs like syphilis and 6–12 monthly HIV testing strategy , 39–42 aNot much is documented on testing rates for syphilis. However, the high testing rates for HIV compared to syphilis could partly be explained by attitudes, accepting norms perceived self-efficacy and regular HIV outreach services for FSW. We observed that compared to HIV, FSW had low scores on attitudes, accepting norms and self-efficacy towards 3-monthly syphilis testing. Among the FSW, high scores on perceived self-efficacy amidst barriers  was associated with an increase of 33% in the likelihood of regular HIV testing while low confidence was associated with a decreased of 25%. The study findings are consistent with research evidence that fear of positive results, perceptions of stigma and discrimination, perceived lack of privacy and confidentiality are barriers to regular uptake of HIV testing among FSW , 30 but Reasons cited for not screening for syphilis included; having no signs and symptoms, having never thought about it, not aware that you can screen, presence of self-medication in case of signs and symptoms, consistent condom use, not knowing where to obtain the service and costs. Some of the reasons cited above point to limited knowledge about syphilis and screening practices. Furthermore, in Uganda programming tends to focus on HIV both at policy and service delivery with regard to serological screening. For example, while not specific to FSW there exists a national policy guidelines on HIV testing services , but synThe findings of this study support the theorization that psychosocial factors: attitude, norms and perceived self-efficacy are determinants of health behavior. Also significant in this study was town location, compared to FSW in Kampala, residing in Arua was associated with a decrease of 33% in the likelihood of syphilis screening but an increase of 28% in the likelihood of HIV screening. Location is described in literature to influence access to and utilization of services , 46. TheThere are some study limitations related to the design and representativeness. The participants were recruited from the municipalities of Kampala, Mbarara, Mbale and Arua and as such may not be representative of all the municipalities and communities where FSW work in Uganda. It was observed during data collection that these sites have a regular active moonlight HIV Counseling and Testing outreach program. The study design was cross-sectional and findings could differ over a period of time. In addition, social desirability bias in reporting HIV testing behavior, the lifetime HIV testing prevalence of 95% among FSW was high compared to less than 60% reported in a recent Kampala study. However, our findings support the model that low attitudes and norms decrease the chances of regular syphilis screening while high perceived self-efficacy amidst barriers increases the chances of regular HIV testing among FSW. Overcoming barriers and promotion of attitudes and norms should be key in STI and HIV promotional campaigns and programs. At clinical level there is need to emphasize the integration of STI testing into HIV services.Compared to HIV, the uptake of repeated syphilis screening was low among town-based FSW in Uganda, and was significantly positively associated with low attitudes, norms and town location. The psychosocial correlates of repeated HIV testing were perceived self-efficacy and confidence. Health campaigns should emphasize overcoming barriers to HIV testing while promoting attitudes and norms including integration of serological syphilis testing and other STIs into HIV services.Additional file 1. Questionnaire for female sex workers; Quantitative survey among sex workers in Uganda on STI and HIV screening intentions, practices and psychosocial predictors.


Both sensors were successfully applied as stability indicating method for the analysis of carbachol in presence of its metabolite choline, in ophthalmic preparations, in plasma and urine showing good recovery percentage values. MWCNTs based sensor showed some advantages over graphene sensor regarding lower limit of detection (LOD), longer life time and higher selectivity towards carbachol. Statistical comparison of the proposed sensors with the official method showed no significant difference for accuracy and precision.Several emerging nano scale forms of carbon are showing great promise in electrochemical sensing such as graphene and multi-walled carbon nanotubes (MWCNTs). Herein we present an ecofriendly method to fabricate long life and sensitive ion selective sensors based on graphene and MWCNTs nanocomposites with no need for volatile organic solvents. Both sensors were fabricated, for the analysis of carbachol in ophthalmic solutions, plasma and urine where ion- association complex was formed between cationic carbachol and anionic Sodium tetra phenyl borate (NaTBP) in a ratio 1:1. Both sensors were evaluated according to the IUPAC recommendation data, revealing linear response in the concentration range 10 Science, outlining the isolation of a single atomic layer of graphite . It. It18]. Therefore, in toxicity cases, a method to selectively analyze carbachol in ophthalmic solutions, in presence of its degradation product, and in urine and plasma is required. Moreover, analytical methods are needed to ensure that the concentration of carbachol in pharmaceutical preparations remain at therapeutically active levels. A review of the literature shows that several methods have been developed for the determination of carbachol, including fluorescence, proton magnetic resonance spectroscopy, potentiometry, indirect UV-VIS spectrophotometry, IR spectroscopy, ion chromatography, and the official colorimetric method ,30,31,32Among electrochemical sensors, potentiometric sensors are considered as an important class, which transduces the activity of a certain analyte dissolved in a solution into an electrical potential . To the In our proposed method, we demonstrate for the first time eco-friendly, long-life potentiometric nanocomposite sensors for the accurate, precise, and selective determination of carbachol in ophthalmic solutions, in presence of choline, and in biological fluids. The proposed sensors can be considered as cheap, easy, and fast tools for monitoring urine and serum concentration of carbachol in cases of oral intoxication without the need for tedious and time-consuming extraction methods. The effect of incorporating nanoscale particles on potential stability, considering response time and shelf life of the proposed sensors, was found to be superior to ordinary scale sensors.3 as a bridge electrolyte). Jenway digital ion analyzer  was used for potentiometric measurements. pH glass electrode no. 924005-BO3-Q11C  was used for pH adjustment. A Bandelin Sonorex magnetic stirrer and heater, model Rx 510 S , were used.Ag/AgCl double-junction was used as external reference electrode ; 3.0 M KCl saturated with AgCl as an inner filling solution and 10% KNO3), MWCNTs , choline, carbachol, sodium tetraphenylborate (NaTPB) were purchased from Sigma Aldrich . Urea, 2-chloroethanol, potassium carbamate, triethylamine, sodium chloride, potassium chloride, calcium chloride hydrate, paraffin oil, and benzalkonium chloride were purchased from El Nasr Company . Miostat Intraocular Solution (labeled amount of 0.01% carbachol), manufactured by Alcon Laboratories, INC. , was obtained from the local market. Jestryl ampoules (labeled amount 0.00025 g carbachol/mL) manufactured by veb ankerwerk Rudol stadt thur chem. pharm fabric  was obtained from the local market.All chemicals and reagents used in this study were of analytical grade, and water was bidistilled. Graphene nanoplatelets  was prepared by dissolving 0.1826 g of carbachol in bidistilled water and then filling to the volume of 100 mL using bidistilled water. Working solutions starting from 1 × 10−7 to 1 × 10−3 M were prepared by suitable dilution from the fresh stock solution (1 × 10−2 M) using bidistilled water.Carbachol fresh stock solution . The paste was firmly packed into a plastic tube cavity with a diameter of 3.5 mm with extreme care to prevent formation of air gaps. A copper wire with a diameter of 1 mm was inserted into the other end to obtain appropriate electrical contact. After assembly, the sensors were finally conditioned by soaking each one separately in 10−2 M carbachol solution overnight before measurements.In a mortar, 20 mg ion-association complex and 300 mg graphene and MWCNTs for sensors 1 and 2, respectively, were mixed and homogenized into a smooth paste while using paraffin oil as binder in the ratio 60%:40% −7 to 1 × 10−2 M) into six 100-mL beakers. The measured electromotive force (emf) values within ± 5 mV and ± 3 mV were recorded for sensors 1 and 2, respectively. Washing was done between measurements using bidistilled water for both sensors. Calibration graphs were obtained by recording emf values against negative logarithmic carbachol concentration, and the regression equation for each sensor was computed for the linear part of the curve.The calibration of the proposed sensors was achieved by separately transferring 40-mL aliquots of carbachol throughout the concentration range (1 × 10−4 M) of Miostat and 10 ampoules (1.38 × 10−3 M) of Jestryl were quantitatively transferred separately into a 100-mL volumetric flask and filled to the mark with bidistilled water. Suitable dilutions were prepared for each volumetric flask to obtain concentration of 8.25 × 10−6 M of Miostat and 1.38 × 10−6 M of Jestryl. The recovery values of carbachol using the fabricated sensors were determined from the corresponding regression equations.The content of 10 vials  recommendation data  and are Fabrication of the proposed sensors using graphene and MWCNTs as electrode materials had a great, pronounced effect on sensitivity, stability, and the response time compared to ordinary scale-based sensors. A comparison between the proposed sensors and sensors investigated by the published ordinary scale method  is shown−5 M and 10−6 M carbachol working solutions were used over the pH range 3–10 using diluted solutions of HCl and NaOH. The results showed that both sensors exhibited constant potentials over pH range 4–8, as shown in Two different concentrations were used to study the effect of pH on both sensors. 10The separate solution method was used to get the potentiometric selectivity coefficients of the proposed sensors using the following rearranged Nikolsky–Eisenman equation :A, B = [(EB − EA)/S] + (1 − ZA/ZB)] Log [A], where EA is the electrode potential of 10−4 M carbachol solution, EB is the electrode potential of 10−4 M solution of the interferent ion, and S is the slope of the calibration curve. Commercially available ophthalmic solutions of carbachol were found to contain sodium chloride, potassium chloride, calcium chloride, sodium acetate, sodium citrate, and benzalkonium chloride. The effect of these interferents on the response of the proposed sensors was studied. Inorganic cations, such as Na+, K+, and Ca+2 were studied. These ions can be found in plasma and urine besides ophthalmic solutions. The influence of benzalkonium chloride as interferent was investigated as it is used as a preservative and wetting agent in pharmaceutical preparation. The effect of 2-chloroethanol, urea, triethylamine, and potassium carbamate were also investigated as they are precursors used during carbachol synthesis. The results of the selectivity coefficients, as shown in −3 and 0.2 × 10−4 for sensors 1 and 2, respectively.Log Kpot The determination of carbachol using the proposed sensors was successfully achieved in Miostat and Jestryl pharmaceutical solutions, and the results showed excellent recoveries between 99.74% and 100.98%, as shown in The results of recovery of carbachol in presence of increasing concentrations of choline showed the excellent selectivity of the method. The mean recovery percentages of carbachol in presence of increasing percentage of its degradate (10%–90%) were 101.70 and 101.12 for sensors 1 and 2, respectively. The detailed results are summarized in t-test and F-test at p-value equal to 0.05, as shown in t and F values were less than the critical values, indicating that there was no significant difference between the mean and variances of both methods.The results of the fabricated sensors were statistically compared with results of the official pharmacopoeial method  using thThe present study originated from the fact that carbachol is a positively charged quaternary ammonium choline ester that acts as a cationic exchanger, which reacts with NaTPB as an anionic exchanger. It has been found that NaTPB is the optimum anionic exchanger due to its suitable grain size and low solubility. The proposed structural formula of the ion-association complex formed is shown in The ratio between carbachol and the anionic exchanger was found to be 1:1, as proven by elemental analysis. The high selectivity of the method was achieved by utilizing this ion-association complex, and excellent selectivity coefficients were proven using the separate solution method.The proposed sensors have the benefit of using nanocomposites, which are considered as excellent candidates for dielectric enhancement. Furthermore, graphene and MWCNTs have got interesting physicochemical properties, such as high surface area, high rigidity, high tensile strength, and high electrical conductivity . In the In addition, the proposed sensors were found to offer long lifetime as sensors 1 and 2 were stable for up to 1.5 and 2 months, respectively. Our method is also considered eco-friendly compared to other methods that use toxic hazardous VOCs. Analysis of carbachol in presence of varying concentration of its metabolite choline was successfully demonstrated with excellent recovery values.Although graphene is reported to have a little bit more effectiveness in conductivity enhancement than carbon nanotubes , our stuBoth proposed sensors were also demonstrated as a promising and reliable tool for the determination of carbachol existing in ophthalmic preparations and for the rapid on-site analysis of carbachol levels in plasma and urine in cases of carbachol intoxication.A comparison was carried out between our proposed method and the official method, and the results demonstrated that there was no significant difference between the proposed method and the official one.−7 M. Such sensors are very promising candidates as alternatives to sensors utilizing VOCs. They are simply and rapidly prepared using cheap, green, and nontoxic materials. Moreover, they offer the advantage of low LOD and high selectivity.In this work, we have demonstrated two nanocomposite-based sensors that can be applied for simple, rapid, and low-cost determination of carbachol in ophthalmic preparations, in presence of its metabolite choline, and in plasma and urine. The sensors gave precise and accurate results and can therefore be used for monitoring carbachol down to a concentration level of 1 × 10


Sargassum aggregations using both ship-deck observations and satellite sensor observations at three resolutions . Both datasets reported that in summer, Sargassum aggregations were mainly observed off Brazil and near the Caribbean Islands, while they accumulated near the African coast in autumn. Based on in situ observations, we propose a five-class typology allowing standardisation of the description of in situ Sargassum raft shapes and sizes. The most commonly observed Sargassum raft type was windrows, but large rafts composed of a quasi-circular patch hundreds of meters wide were also observed. Satellite imagery showed that these rafts formed larger Sargassum aggregations over a wide range of scales, with smaller aggregations (of tens of m2 area) nested within larger ones (of hundreds of km2). Match-ups between different satellite sensors and in situ observations were limited for this dataset, mainly because of high cloud cover during the periods of observation. Nevertheless, comparisons between the two datasets showed that satellite sensors successfully detected Sargassum abundance and aggregation patterns consistent with in situ observations. MODIS and VIIRS sensors were better suited to describing the Sargassum aggregation distribution and dynamics at Atlantic scale, while the new sensors, OLCI and MSI, proved their ability to detect Sargassum aggregations and to describe their (sub-) mesoscale nested structure. The high variability in raft shape, size, thickness, depth and biomass density observed in situ means that caution is called for when using satellite maps of Sargassum distribution and biomass estimation. Improvements would require additional in situ and airborne observations or very high-resolution satellite imagery.The present study reports on observations carried out in the Tropical North Atlantic in summer and autumn 2017, documenting  Sargassum C. Agardh  are causing serious threats to coastal ecosystems , including Caribbean Islands coastal areas were others floating vegetations were scarce.As Sargassum during these two cruises, and highlight the large-scale Sargassum distribution in the Tropical North Atlantic, for two different seasons of the same year   was mainly recorded in the Sargasso Sea and in the central Atlantic, areas where satellite imagery does not detect Sargassum, at least not at 1 km resolution.The MODIS AFAI composites computed for the duration of both cruises (24 days during June-July 2017 and 18 days during October 2017) give an overview of the occurrence of ame year . MODIS Ac cruise  and in tc cruise . In Juneo -35°E) . These sSargassum aggregation distribution and structure at 1–100 km scale  and highlights small and isolated Sargassum filaments  were compared with the higher resolution images provided by the OLCI sensors (300 m) over the same zone and on the same day . In bothSargassum detected with VIIRS and MODIS were in fact formed by a front of small parallel filaments aligned with the wind direction , located downwind of the filament, probably representing one or several large Sargassum patches. Two of these small areas of high Sargassum coverage pixels (~2 pixels) detected by OLCI , a few meters apart (5–10 m), were observed at this location during the West Atlantic cruise  were available. Here, we compared OLCI and MSI images acquired over the south of Guadeloupe Island at only two days interval . MODIS i Islands . Sargasss region  because  patches . Drop-shI sensor . These Mf Type 4  and Typef Type 4 .Sargassum raft shapes, observed from the ship deck during the two cruises and classified according to our typology are consistent with historical data mostly collected in the Sargasso Sea ([Sargassum rafts such as those observed during the West Atlantic cruise (S9). This observation may thus be unusual, but caution is needed before drawing conclusions as raft thickness, if observed, was rarely recorded in historical reports. In addition, most of our sampling stations were targeted where satellite indexes showed significant aggregations. This differs from previous works [The sso Sea . It is easier to use for any observer, whether scientists or not, thanks to a numbered scale and drawings. It could be included in the already available Sargassum reporting website [Sargassum quantity. Although raft types can be variable because of changes in current and wind, the available Sargassum quantity will be rather stable for a duration shorter than growing and sinking timescales. We thus argue that this typology can provide useful information for Sargassum biomass estimations. Nevertheless, to accurately estimate the quantity through the types, additional information is needed, such as windrows spacing, average width and length, and frequency of patches. The best means to access this information would be airborne imagery.The typology proposed here goes beyond simpler descriptions already proposed in literature ,61,62 an website ,63. MoreSargassum aggregation occurs across a wide range of scales. At small scale (<1 km), floating Sargassum probably aggregate under wind induced drift and current  to formnt sizes . At largth e.g., . Satellith e.g.,  clearly Sargassum windrows  were ubiquitous, consistent with the significant wind speed occurring at most stations [Sargassum were not intertwined. Even in large dense rafts (type 4–5), air bubbles from scuba divers easily dispersed the Sargassum, suggesting that disaggregation could be a rapid process. For example, we observed a disintegrating large raft under a wind of 3.1 m s-1 at station Y04b  were observed at wind speeds of 4–11 m s-1. This may differ from historical observations [Sargassum rafts were observed horizontally disaggregated during light wind conditions, possibly due to the relaxation of the Langmuir cells maintaining the windrow pattern [Sargassum rafts into the water column. This disaggregation seems to appear only under high wind and rough sea conditions , and can lead to Sargassum being mixed into the water column down to a depth of about 1 m  over the whole Tropical North Atlantic, from the Caribbean islands to the African coast [Sargassum was not only present off Brazil and near the Caribbean islands during spring and summer but extended eastward as far as the African coast. In October, the highest density of Sargassum aggregations was observed off Guinea and Sierra Leone. This distribution was validated by Sargassum in situ observations made during the two cruises in spring and autumn 2017. Furthermore, the UNEP Abidjan convention reported beaching of Sargassum along the shorelines of Africa in the winter of 2011 [Sargassum distribution mapped with MERIS [Sargassum distribution mapped with MODIS in the western part of the Tropical North Atlantic from 2011 to 2015 [In the present study, the an coast . The lar of 2011 . This dith MERIS , and wit to 2015 .Sargassum spatial extent observed in June-July 2017 is one of the highest observed since 2011 and seems to be intermediate between the ranges observed in 2014 and 2015 [Sargassum biomass stranding on the Caribbean coast in 2017 . In 2018, massive strandings have also been reported since January [Sargassum spatial range ever recorded since 2011, with quantities that exceed the 2015 and 2017 records [Sargassum abundance since 2011 [In the Western Tropical North Atlantic, the and 2015 . This is January . Satelli records ,70. Thisnce 2011 .Sargassum presence and abundance in the Tropical North Atlantic is most likely linked to both transport and growth processes. [Sargassum distribution pattern at the seasonal scale. Adding a simple Sargassum growth representation in their model, they were able to maintain a realistic high abundance in the tropics.ocesses.  showed tSargassum inside the Sargasso Sea either in June-July or in October 2017, at least as far as 30°N. This agrees with the very low abundance to quasi-absence of Sargassum recorded in situ during the 2017 West Atlantic cruise.MODIS composites did not detect in situ and satellite observations were rare in our study, for several reasons. Firstly, the timing of the stations was not decided on the basis of satellite overpass but of the presence of Sargassum. Secondly, cloud coverage was high and observations not always available because of the satellite temporal resolution. Thirdly, because Sargassum distribution is not homogeneous, a full match-up between in situ and satellite observations would require the in situ observation of the entire pixel area  observations or using airborne imagery, in order to increase the number of match-ups, should make it possible to bridge in situ and satellite observations.Match-ups (i.e. observation of the same area at the same time) between xel area . For 1kmin situ observations within a ± 3 days windows seem consistent with in situ observations in terms of Sargassum aggregation structure and abundance. Weak and narrow Sargassum filaments  observed with OLCI are consistent with the in situ observation of Type 2 Sargassum rafts observed 2 days earlier, while bright pixels , mainly observed at the front of filaments, probably correspond to the presence of Sargassum patches at the front of the rafts, as often observed in situ (type 4–5). However, no large aggregation extending over several km as suggested by MODIS and VIIRS observations were observed in situ. In situ and higher resolution satellite imagery suggested that these “very large rafts” are in fact formed of several, nested smaller scale Sargassum aggregations. Although these equivalences between in situ and satellite observations have to be considered with caution because of the high variability in raft shape and size, we think that they can help to better interpret future Sargassum satellite observations in terms of Sargassum quantity and aggregation structure.Nevertheless, satellite observations made in the same areas as Sargassum species, in many places worldwide  mesoscale structure. However, OLCI observations are less frequent: only 45% of the stations during the two cruises were covered using this sensor , but only in coastal areas and only every 5 days. To get the full picture of Sargassum aggregations, we are lacking 0.5–10 m observations, which can be acquired through dedicated images from aircraft, drones, or very high-resolution satellite sensors such as SPOT (1.5–6 m) or Pléiades (0.5–2 m). A better understanding of aggregation and disaggregation processes and dynamics would also benefit from a higher temporal resolution and spatial coverage, especially for the high spatial resolution sensor MSI.Satellite and airborne imagery is widely used to detect and map floating marine vegetation at the sea surface, including benthic and pelagic de  for MODIS, 1125 m2 (~25 m x ~45 m) for VIIRS and 180 m2 (~20 m x ~9 m) for OLCI. Environmental conditions are also a constraint on Sargassum detection. Highly diffusive atmospheric conditions such as the presence of aerosols in high quantity  can hamper Sargassum detection by decreasing their signal in the NIR [Sargassum rafts , can lead to large gaps in satellite images that drastically reduce the satellite sensors’ nominal spatial coverage and frequency of observation . During Sargassum algae from space is also not straightforward. Algae indexes used to detect Sargassum do not allow discrimination of Sargassum from other floating vegetations, from surface cyanobacterial and phytoplankton blooms, or from material such as plastic debris or oil slicks , and no blooms of Cyanobacteria (Trichodesmium spp.) were observed during the two cruises, allowing greater confidence to be attributed to Sargassum identification in this area  that thatSargks . Neverty e.g., ,30). Oth. OthSargy e.g., ,43)..SargassuSargassum biomass present in the Atlantic and available for strandings in coastal areas, but the nesting of spatial scales makes this estimation complex. Comparison between observations at various scales  within a satellite pixel  box is mainly <0.1% (<6% for a 1 km-size MODIS pixel), with a biomass density in the surface layer mainly ranging from 0 to 2 g.m-2 (up to 100 g.m-2 within a 1 km-size MODIS pixel) [Sargassum biomass was made in situ for 2 rafts of Type 4 and Type 5 during the West Atlantic cruise. Sargassum was collected with a 1m2 sieve from below during scuba diving. Then the collected mass was weighed on the ship. We recorded biomass density ranging from 5.26 ± 1.59 (S12) to 7.60 ± 0.77 kg.m-2 (S9) for the upper 0.5 m layer, which is close to the 3.34 ± 1.34 kg.m-2 measured in situ by [Sargassum thalli ranging from a tiny air bladder to a 1-m fragment with axes, blades and bladders. In addition, the distribution and density of the thalli within the water column were heterogeneous. The biomass of the surface layer, like that estimated by satellite, may thus not be representative of a whole raft and only represent a a minimum estimation [Sargassum thalli composition and density in order to better estimate their variability and impact on biomass estimation.A challenge for remote-sensing is now to quantify the s scales  to Fig 7l  ,39. OnceSargassum algae were carried out in the North Atlantic Ocean allowing the observation of numerous Sargassum rafts of various types offshore and near the coast. These observations led us to define a five-class typology that simplifies and standardizes Sargassum raft descriptions. These two cruises were routed using satellite observations enabling us to draw recurrent parallels between in situ and space-based Sargassum observations. However, match-ups (i.e. observation of the same area at the same time) between satellite and in situ observations were very difficult to achieve, mainly because of (1) the high cloud cover, (2) the difficulties of coordinating cruise stations and satellite overpass, and (3) the differences in observation scales (a few meters around the ship vs pixel size). A full comparison between satellite and in situ observations would require detailed information about observed raft types, such as the windrows width, spacing and patch frequency and size, over the whole satellite pixel area, which is not possible from a ship. This kind of information could be obtained and detailed comparisons made using airborne imagery or very high resolution satellite images and would be essential to validate satellite estimations of Sargassum fractional coverage and biomass. Other parameters limit Sargassum detection and quantification from space, such as the submerged depth of Sargassum rafts and the raft thickness. Nevertheless, the comparison made in this work between data from satellite sensors and in situ observations, showed that free and easy-access satellite imagery datasets with various spatial, spectral and temporal resolutions provided by the MODIS, VIIRS, and the new OLCI and MSI satellite sensors (i) are able to successfully detect Sargassum abundance consistently with in situ observations, and (ii) are powerful tools to map the Sargassum distribution at large scale , and to describe the (sub-)mesoscale structure of Sargassum aggregations, as well as the Sargassum raft shapes, sizes and patterns of distribution near the coast.In 2017, two cruises dedicated to the study of S1 FigSargassum maps computed using the (A) FAI and (B) MSI-MFAI floating algae index.Comparison between MSI  Zooms of A and B are presented in C and D respectively. Large stripe pattern in surface reflectance is observed across the FAI-derived map (A), caused by sensor parallax effect [Sargassum aggregations (red arrows show Sargassum aggregation poorly visible on the FAI-derived map compared to the MSI-MFAI one).x effect . This pr(TIF)Click here for additional data file.S2 FigSargassum.For each station, the following information is reported see : cruise (PDF)Click here for additional data file.S3 FigMatches are made within a +/- 3 days interval and in an area of 0.1° x 0.1° around the station. For the three stations, the following information are reported (see Tables (PDF)Click here for additional data file.S1 TableTransatlantic cruise (station Y07b) (Patch Width (PW) = 50m and Patch Length (PL) = 250m).For these estimations, Type 1 is characterized by one Sargassum thalli of width SW and length SL each SS meters. Types 2, 3 and 4 are characterized by Sargassum windrows with a width ranging between 10 and 30 cm and windrow spacing varying between 10 and 2 m . For Types 3 and 4, we considered patches of 1 mdiameter each 10 m for Type 3 and 4 m diameter each 50 m for Type 4. For Type 5, we considered only one big patch of a size similar to the Type 5 raft encountered during the (TIF)Click here for additional data file.


Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine . The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed. Oenococcus oeni is the bacterial species most frequently responsible for malolactic fermentation (MLF) in wine , massively introduced to wine usually at the end of the alcoholic fermentation  production can also contribute to Cerning, . Indeed O. oeni adaptation to wine, we examined both EPS production and eps gene expression in several strains able to produce such polymers and representative of the species diversity. The eps gene expression was analyzed along growth and in the presence of different stresses, as well as in wine. This highlighted the conditions possibly stimulating EPS formation. Based on these results, we then examine production and inoculation methods leading to better malolactic starter survival, and verify that these methods preserve wine quality.In order to better understand the role of EPS in The list of strains studied and their origin are indicated in Table O. oeni AWRI_B429 (ACSE00000000) and IOEB-S277 (AZKD00000000). Putative promoter elements were identified by comparison with promoters previously described for O. oeni .Protein and DNA sequence were recovered from the complete genome sequence of The strains were grown in either Grape Juice medium or Semi Defined medium (SMD). The Grape Juice medium contained: commercial red grape juice, 250 g/l, yeast extract, 5 g/l and Tween 80, 1 ml/l. The Grape Juice medium could be supplemented or not with sucrose (10 g/l) and, for solid medium, agar (20 g/l) was added. The medium was sterilized for 20 min at 121°C, 1 bar.TM), 10 g/l, sodium acetate, 3.4 g/l, KH2PO4, 1 g/l, MgSO4, 7 H2O, 0.1 g/l, MnSO4, 4 H2O, 0.1 g/l, ammonium citrate, 2.7 g/l, bactotryptone, 5 g/l, D/L-malate, 3 g/l, yeast nitrogen base (Difco TM), 6.7 g/l, adenine, uracil, thymine, guanine, 5 mg/l each, and a carbohydrate . The carbohydrate solutions were prepared as 10× solutions and were sterilized 20 min at 121°C, 1 bar, while the base was prepared as a 2× solution and sterilized by filtration (0.2 μm cut off).The composition of the SMD medium was: casamino acids . This wine was then modified to produce wines with distinct alcohol content or pH, by ethanol or H3PO4 addition. The total acidity, sugar, malic acid, SO2, pH, and alcohol were analyzed by SARCO laboratory , using the official methods or those recommended by the International Organization of Viticulture and Wine (OIV).The red wine used for stress application was a Merlot of 2011  and the white one, a Sauvignon Blanc of 2010 . A Merlot wine of 2012 from appellation Pessac Leognan, Bordeaux, France, was used for survival tests and malolactic rate measurements . Laboratory wine was also obtained by fermentation of a Cabernet sauvignon must with The liquid medium was gently stirred before sampling and the absorbance (600 nm) was measured.To visualize the bacterial capsule, 10 μl of cell suspension were deposited on a microscope slide and mixed with 20% nigrosine aqueous solution and let to dry (5 min). Afterwards, 10 μl of 1% crystal violet was added and the slide was examined under an Olympus BX51 microscope . The capsule appeared as a white halo around the cells.In the case of plate growth assays, 5 ml of NaCl (9 g/l) were added and the colonies were teared-off and solubilized with a rake. This suspension or, in the case of liquid cultures, the whole culture medium, was centrifuged , and the pellet was removed. Three volumes of Ethanol-HCl 1 N (95-5) were added to the supernatant to precipitate the polysaccharides. The tubes were let to stand for 24 h at 4°C. Then, they were centrifuged , and the pellet was washed with ethanol (80% vol), centrifuged again, dried for 20 min at 65°C and dissolved in distilled water. The amount of neutral polysaccharides was determined by the anthrone sulfuric acid method, using glucose as the standard . The glucose and sucrose concentrations were determined by anion exchange chromatography, as described by Dimopoulou et al. .600nm = 0.6), the culture broth was distributed into 10 ml tubes and centrifuged ; then, the supernatant was removed and replaced by fresh Grape Juice medium displaying or not a specific stress, or by red or white wine at 25°C. For ethanol shocks, pure ethanol  was added to grape juice medium at a final concentration of 2, 5 or 10% (v/v). For acid shock, the pH of the Grape juice medium was adjusted to 3 using H3PO4 1M. For cold shock, the cells were transferred to cold Grape Juice medium (4°C) and then kept at 4°C until the end of the experiment. The stress was applied for 3 or 24 h. The control condition consisted of the bacterial cells harvested at mid exponential growth phase (OD600nm = 0.6) by centrifugation  and then, re-suspended in fresh Grape Juice medium at 25°C. Cell population was measured before and after the incubation in the different media, through serial dilutions and CFU counts after growth on solid Grape Juice medium. Cell viability was measured based on fluorescent labeling and microscopy according to previous protocol  and once with NaCl 0.9%. Between the washing steps, the cells were collected by centrifugation .The pellet was re-suspended into 1 ml of an extraction buffer specifically designed for environments rich in polyphenols (Grape Juice and Wine), containing Tris-HCl, pH 8.0, EDTA 300 mM, NaCl 2M, CTAB (cetyltrimethylammonium bromide) 2%, PVPP (polyvinylpolypyrrolidone) 2%, spermidine 0.05% and β-mercaptoethanol 1%. The cells were then transferred into a suitable Fast Prep tube (FP120 Instrument) and vortexed  with sterile glass beads (0.1 mm diameter). After centrifugation , 500 μl of the supernatant was transferred into an equal volume of chloroform, to dissolve the hydrophobic particles and separate the proteins and nucleic acids. The mixture was vortexed for 10 min and centrifuged . The upper aqueous phase was transferred into an equal volume of isopropanol to precipitate the ribonucleic acid . The RNA pellet was recovered by centrifugation , washed twice with 75% ethanol (prepared with DEPC-treated water), dried and finally taken-up into 20 μl of DEPC water. Samples were then treated with DNase as indicated by the manufacturer . The absence of chromosomal DNA was confirmed by checking the absence of amplification of the malolactic enzyme gene, mleA . The samples were stored at −80°C, for a maximum period of 1 month. The cDNA were then synthesized using the iScript cDNA synthesis kit (Bio-Rad), as recommended by the manufacturer.Cells were harvested by centrifugation , then the cell pellet was washed twice with PBS buffer  to check PCR specificity. Ranges of cDNA of known concentrations were analyzed by qPCR to construct “standard” curves, characterized by a regression coefficient (R2) and the PCR efficiency (e = 10slope)−1(−1/). The validity criteria were R2 ≥ 0, 985 and 0.85 ≤ e ≤ 1. Relative gene expression was calculated using the ΔΔCT method and every PCR was realized in triplicate  contained 10 μL of IQ SYBR Green Supermix and 5 pmol/ml of each primer. Primers , centrifuged and then washed. Industrial maltodextrins were added as cryoprotector and the cells were freeze dried at laboratory scale. The freeze dried cultivable population was determined as previously described  into the indicated wine, previously sterilized by filtration (0.2 μm cut-off). After 48 h of incubation at 28°C in the wines, the survival rate was determined by serial dilutions and colony counts as previously described  supplemented with bacterial Energizer nutrient  as recommended by the manufacturer, and in the presence of sucrose (5 g/l) or not. After 17 h at 28°C in this acclimation medium, the bacteria were transferred (2 × 102) and kept for 2 months at 10°C for stabilization. The tests were then performed according to International Organization for Standardization. Panelists were required to give a liking score on products using a 10-point hedonic scale for the descriptors of aromatic complexity, fruit intensity, bitterness, astringency and mouthfeel perception. The panel consisted of 18 trained laboratory researchers of ISVV, Bordeaux University.Distinct merlot wines were selected to perform sensory analysis. At the end of MLF, the selected wines were sulfured . The EPS specific production level (EPS produced/absorbance change in the same time) is reported in Table eps2 gene cluster formed part of the lowest producers, even at low pH or in the presence of ethanol. For the strains displaying an eps2 gene cluster, the decrease of medium pH to 4 did not significantly modify the specific production, while a more acidic medium (pH 3.5), ethanol (5%) or sucrose (1%) addition induced a significant increase of the soluble EPS specific production level, even if distinct behaviors were observed among the studied strains  displayed both a complete r Figure  and a dsu et al. . All theu et al. . In the ed Table .eps2 gene clusters were examined for the presence of putative promoters and terminators , supplemented or not with sucrose. Sucrose alone does not support growth of most With strain AWRI_B429, in the absence of sucrose, no significant change in EPS concentration was observed, in SMD as in Grape Juice medium Figure .A capsule was present around the two bacterial strains in all studied media and at all growth stages examined (not shown). However, the method used did not enable to measure the capsule thickness and to visualize if a growth phase was more favorable for capsule formation.eps genes was thus studied: two genes of the eps2 gene cluster, i.e., the priming glycosyltransferase (wobA) and the flippase (wzx) genes as well as the dextransucrase gene (dsrO). The phosphoglucomutase (pgm) and the lactate deshydrogenase (ldh) genes were studied as controls of the central metabolism  indicated that gyrA displayed the highest stability among 4 genes proposed .The relative expression level of some  Figures , 2C. By ldh gene was slightly more expressed during active growth, while the pgm gene showed a slightly higher expression during the stationary growth phase. The eps genes in the cluster eps2 were expressed at a level in the same order of magnitude as gyrA (relative expression levels between 0.4 and 10). Furthermore, the genes wobA and wzx were co-expressed in the case of strain IOEB_S277 and slightly less expressed during the active growth phase than during the stationary phase. In the case of strain AWRI_B429 the genes were not co-expressed: wobA showed the same profile as with strain IOEB_S277 but wzx was always more expressed and the expression ratio wzx/wobA varied from 2 to 5 depending on the growth phase or the medium considered, suggesting the existence of a second promoter in the eps2 gene cluster, between wobA and wzx.Quite similar gene expression profiles were obtained in the absence as in the presence of sucrose Figures . The ldhdsrO was significantly less transcribed than the other eps genes and its expression level was almost not modified along growth in liquid media. Furthermore, its expression level did not change in the presence of sucrose  and would be always moderately ready to produce dextran (constant expression of dsrO).The gene  Figures . Accordieps gene expression was modulated by single stresses relevant to wine or during winemaking: acidity, low temperature and different alcohol concentrations , or during direct inoculation into white and red wine , as those associated with EPS synthesis . Ethanol, on the other hand, slightly stimulated the expression of some of the studied genes, in a dose-dependent manner. The wines slightly stimulated the expression of pgm and dsrO, but decreased that of genes in eps2 cluster, and had little effect on ldh expression.Three hours after medium change, the studied gene expression levels were weakly modified factor 2 maximum). The expression of  maximum.wzx and dsrO and moderately that of the central genes pgm and ldh. The gene wobA was almost unaffected. These modifications are not observed in tested wines except for the case of dsrO in white wine. The expression ratio wzx/wobA, increased whatever the medium change and the increase was particularly high in red wine . The tests were made in glucose + fructose as in glucose + fructose + sucrose containing growth media. Ethanol (5%) addition in the medium or a cold incubation (4°C), for 3 or 24 h just before freeze drying, didn't give the expected results (not shown). More acidic growth media were not assayed as they always led to much lower final biomass yields.Previous work has shown that strains producing dextran in the presence of sucrose added in industrial growth media were more tolerant to freeze drying  were first produced and freeze-dried. Then, they were acclimated during 17 h into diluted wine supplemented or not with sucrose (5 g/l). EPS formation during the 17 h of acclimation was undetectable . However 1 week incubation in the same conditions led to significant production of EPS, only when sucrose was present, in the case of strains in AWRI_B429 and IOEB_S277 families. After the 17 h of acclimation, the strains were inoculated into a Merlot red wine. The survival rate and the fermentation kinetics were followed . Whatever the characteristics of the tested laboratory wine, the use of sucrose in the acclimation medium improved the survival rate of strain S28 and had slightly positive but non-significant effect on that of the two other strains (not shown).The produced Merlot wines were finally tasted in order to verify that the addition of sucrose in the acclimation medium didn't affect the sensory quality of the final product Figure . On the The same wines were quantified for thirty two esters recognized as key aromatic compounds of MLF  all along growth in the tested media supplemented with or not with sucrose. The need for EPS formation may thus be permanent in the conditions studied. However, the low soluble EPS concentrations observed and the inability to measure the thickness of the EPS capsule make it difficult to determine if EPS are produced more actively during a specific growth phase. Furthermore, many single constraints relevant in wine modify the corresponding mRNA concentrations particularly after 24 h. The absence of significant change in certain glycosyltransferase genes expression after short period of time of stress application is in accordance with previous transcriptomic studies (Olguín et al., eps genes are not really involved in the immediate response to stress, but rather in adaptation to new environmental conditions (Guzzo et al., eps and pgm gene expression, may allow a better availability of the isoprenoid lipid carrier and the nucleotide sugars precursors. This may lead to increased polysaccharide production by an increased pool of glucosyltransferase (Levander et al., O. oeni, suggesting that these polymers are not needed for the consolidation of the colonies, unless very adhesive molecules are formed in such conditions.In this paper, we show that the O. oeni AWRI_B429, the presence of multiple potential promoters and terminators in the same cluster is in contrast to what described for most eps operons in lactic acid bacteria (Stingele et al., eps gene confirmed the independent transcription of the priming glycosyltransferase and the flippase genes. This may modify the EPS biosynthetic pathway limiting step and may induce difference in EPS structure or size, in response to medium change. Experiments may be carried out in the future to examine this specific point. In this specific strain, its seems that, after medium change, the cells need more EPS but they may also need different EPS or even both, as shown for cyanobacteria (Ozturk and Aslim, In dsrO is not induced by sucrose, contrarily to what described in Leuconostoc mesenteroides or other glycoside hydrolase in lactic bacteria (Neely and Nott, O. oeni behave similarly Lactobacillus sakei or Weisella confusa (Bounaix et al., Leuconostoc mesenteroides NRRL_1299 for minor dextransucrases (Dols et al., O. oeni, the constitutive expression of dsrO is quite surprising and suggests that O. oeni must always be prepared to produce dextran and that dextran is very useful for the bacteria. The previously demonstrated protective role of the produced dextrans during freeze drying (Dimopoulou et al., dsrO also constitute a selective advantage during the acclimation step: sucrose addition into the acclimation medium increased most freeze-dried strains survival to inoculation in wine, and enabled faster MLF, even though the dextran concentration produced is very low. Several bacterial EPS were shown to be involved in bacterial protection (Ionescu and Belkin, The dextransucrase gene Interestingly the acclimation in the presence of sucrose also improved the sensory attributes of wine. Since specific wine polysaccharides, especially mannoproteins, have been proven to modify wine astringency and sucrosity (Taira et al., O. oeni strains are equipped with at least one EPS biosynthetic pathway and the eps genes are expressed at significant levels whatever the growth conditions, points out the importance of these polymers for the species. These polymers may form part of the tools that render O. oeni able to grow in various niches like grapes, must and wine. The gene dsrO appears as a promising simple and original tool, because dextran production pathway is less energy demanding than the other EPS biosynthetic pathway (Hanson et al., The fact that all the MD, VM, JC, and MD-L designed the experiments. MD, JR, OC, CM-S, and NI made the experiments. MD and MD-L prepared the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Marfan’s syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in the extracellular matrix protein fibrillin-1. Orofacial characteristics may be useful in identification of the syndrome. Severe periodontitis is sometimes observed in MFS patients, but no in-depth information has been reported in Italian groups of growing subjects with MFS. The aim of this study was to analyze the periodontal condition on a group of growing subjects affected by MFS, in comparison with a typically developed control group.A group of 16 subjects with diagnosed MFS were recruited from the Centre for Rare Diseases for Marfan Syndrome and Related Disorders of Tor Vergata University Hospital. The Marfan Group (MG) was compared with a Control Group (CG) composed by 20 nonsyndromic subjects. The periodontal clinical parameters like Marginal Gingival Thickness (GT), Plaque Index (PI), Bleeding On Probing (BOP) and Modified Periodontal Screening and Recording (PSR) were assessed.The mean value of PI in MG was 59%, instead in CG it reached 21%. Analysis showed a significant difference between MG and CG also for the BOP. In MG the mean value of BOP attained 36% and in CG it reached 16%. A statistical significant difference of distribution of PSR index between the two groups was found for all sextant examined.Patients with Marfan syndrome reveal a higher presence of plaque and consequently a generalized inflammation in the oral cavity when compared with a control group. Marfan’s syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in the extracellular matrix protein fibrillin-1; its prevalence has been estimated as 1 in 5–10,000 individuals . MFS wasClinical manifestations typically involve the cardiovascular, skeletal, and ocular systems. The diagnosis is based on a combination of major and minor clinical features, described by Berlin classification system in 1986 . A revisOrofacial characteristics may be useful in the identification of the syndrome, consisting in: long and narrow face, maxillary/mandibular retrognatia, temporomandibular joint alterations, high arched palate, dental crowding, posterior crossbite and sleep disorders . It is kThe periodontal ligament (PDL) is a specialized connective tissue composed of collagen and elastic system fibers and situated between the cementum covering the root of teeth and the alveolar bone socket .Furthermore, severe periodontitis is sometimes observed in MFS patients, but no in-depth information has been reported in growing subjects with MFS.Previous studies revealed a strong relationship between periodontal diseases and pathophysiology of cardiovascular complications in MFS patients .To our knowledge, few studies were conducted on animals  or adultThe aim of this study was to analyze the periodontal condition in a group of growing subjects affected by MFS, in comparison with a control group.The study protocol was approved by the Ethical Committee of the University of Rome Tor Vergata (Protocol number: 4544/2017) and an informed consent was signed by the parents of the patients involved.For the actual preliminary study a group of 16 Italian subjects with genetic assessment of MFS, were recruited from the Centre for Rare Diseases for Marfan Syndrome and Related Disorders of Tor Vergata University Hospital, from November 2017 to April 2018 and, evaluated in the Departments of Orthodontics of the same Institution. The inclusion criteria were: genetic assessment of Marfan Syndrome, Caucasian ancestry, no post pubertal vertebral maturation (CS5–CS6).The Control Group (CG) was composed by 20 subjects  and was collected from the Departments of Orthodontics of Tor Vergata University Hospital, using the following inclusion criteria: Caucasian ancestry, good occlusion, no previous orthodontic treatment, no deciduous stage of dentition, no post pubertal vertebral maturation (CS5–CS6). All the subjects in the control group were non- syndromic subjectsExclusion criteria for both MG and CG were: presence of orthodontic treatment, cleft lip and/or palate, other genetic diseases. The study teeth included both maxillary and mandibular arches. Locally, teeth with restorations within 1 mm of the gingival margin, crowded, ectopically positioned, or with history of surgical procedures, were excluded.A single examiner performed all clinical measurements using a plane-faced dental mirror and Michigan O probe with Williams markings coded at 1, 2, 3, 5, 7, 8, 9, and 10 mm. Intra-examiner reliability was performed by repeating the clinical measurements on three randomly-selected patients one week later.The periodontal clinical parameters were assessed in the following order: 1.Marginal gingival thickness (GT) was assessed by visibility of the out-line of the probe through the marginal gingiva  ; 2.The plaque index (PI) was recorded based on the scale of  3.Bleeding on probing (BOP) were recorded using a manual probe at six points  on a right upper molar, an upper incisor, a left upper molar, a right lower molar, a lower incisor and a left lower molar; 4.Modified Periodontal Screening and Recording (PSR)  indices P < 0.05 were considered significant. Descriptive statistics were used to describe both sample groups (SG and CG) in terms of age, sex and gingival thickness. Statistical significance of differences between groups for qualitative variables were assessed using the Chi-squared test; t-test for unpaired data was applied for assessing the comparison of the quantitative variables between groups.All statistical analyses were performed with the aid of statistical software . Values of A total of 9 males and 7 females (mean age of 9.4 ± 2.3 years) with confirmed MFS were evaluated in the Department of Orthodontics of Tor Vergata University. 8 males and 12 females with a mean age of 10.0 ± 2.6 years composed the Control Group. About the Marginal gingival thickness, in the MG, 5 subjects showed thick marginal gingiva and 11 subjects showed thin marginal gingival thickness. Similar results were found in the CG, in which 6 subjects showed thick marginal gingiva and 15 subjects showed thin marginal gingival thickness.T-test for unpaired data showed a statistical significant difference (P < 0.05) between the two groups.The mean value of PI registered in the MG was 59% instead in the CG it reached 21%. P < 0.05) between MG and CG also for the BOP evaluated for all the subjects in both groups. In MG the mean value of BOP attained 36% and in CG it reached 16%. Analysis showed a significant difference , in particular in types III, IV, and VIII .It was well reported that periodontitis is frequently observed in MFS patients . This geMFS patients may be susceptible to periodontopathic bacteria, that invade from the periodontal area with a connective tissue abnormality.Moreover, periodontitis may influence the pathophysiology of cardiovascular complications in MFS patients . Judge &The present study analyzed for the first time the association of MFS and gingival inflammatory condition in young subjects.Our study, unlike the previous ones, considered a group of growing subjects, in order to evaluate possible early correlations between periodontitis and Marfan’s syndrome. The results evidenced that already at an early age in MFS subjects a plaque index of 59% and a bleeding on probing attaining 36% is possible to be observed.Periodontal disease among children and adolescents consists mainly of gingivitis . The preIn a study carried out by Suzuki et al. in 2013, that analyzed a group of 40 Marfan subjects and 15 control group’s subjects, MS patients presented periodontitis more frequently than control group subjects at the same age. In addition, patients with MS presented significantly severe periodontitis, compared to control group subjects .The data from our study show that patients with Marfan syndrome have a gingival inflammatory condition statistically significant higher than patients in GC. Furthermore, the presence of pockets up to 4 mm has also been detected, especially on permanent teeth .Considering the increased risk of cardiovascular complications suffering from Marfan syndrome, early diagnosis is essential to the success of treatment and prevention of infection. To combat the severity and progression of periodontal disease among persons with these systemic diseases, a preventive strategy should be implemented that includes more frequent than 6 months professional oral hygiene, chlorhexidine mouth rinse, antibiotic therapy when indicated, fluoride gel, and/or professionally applied fluoride varnish .To the best of our knowledge, this is the first study analyzing the periodontal index in a group of growing subjects affected by MFS. Based on our data, patients with Marfan syndrome reveal a higher presence of plaque and consequently a generalized inflammation in the oral cavity. Moreover, no signs of severe periodontal attachment loss on multiple teeth were on growing subjects with MFS.10.7717/peerj.6606/supp-1Supplemental Information 1Raw data analysed for the studyClick here for additional data file.


This study aimed to investigate the effect of interstitial flow magnitude in combination with the vascular endothelial growth factor (VEGF) concentration on 3D microvascular network formation. Human umbilical vein endothelial cells (HUVECs) were cultured in a series of interstitial flow generated by 2, 8, and 25 mmH2O. Our findings indicated that interstitial flow significantly enhanced vascular sprout formation, network extension, and the development of branching networks in a magnitude-dependent manner. Furthermore, we demonstrated that the proangiogenic effect of interstitial flow application could not be substituted by the increased VEGF concentration. In addition, we found that HUVECs near vascular sprouts significantly elongated in >8 mmH2O conditions, while activation of Src was detected even in 2 mmH2O conditions. Our results suggest that the balance between the interstitial flow magnitude and the VEGF concentration plays an important role in the regulation of 3D microvascular network formation in vitro.Hemodynamic and biochemical factors play important roles in critical steps of angiogenesis. In particular, interstitial flow has attracted attention as an important hemodynamic factor controlling the angiogenic process. Here, we applied a wide range of interstitial flow magnitudes to an  Angiogenesis is the phenomenon that new blood vessels are formed from existing blood vessels, which occurs under physiological and pathological conditions such as tissue regeneration, ischemic disorder, and tumor growth.Various biochemical factors which promote microvascular network formation have been recognized.in vivo and in vitro experiments.et al.et al.In addition to biochemical factors, hemodynamic factors become increasingly important in regulation of angiogenic processes. ECs covering the luminal surface of blood vessels are constantly exposed to hemodynamic factors such as fluid shear stress and pressure due to blood shear flow and plasma interstitial flow.in vitro 3D angiogenesis models that mimic in vivo microenvironments. In particular, the precise control of fluid in a microfluidic device led to the investigation of angiogenic processes affected by directional interstitial flow with monitoring angiogenic cell behaviors in real time. Vickerman and Kammet al.et al.Microfluidic devices have been used for development of 2O pressure differences. Although there is increasing evidence that the interaction between ECs and stromal cells, such as pericytes and fibroblasts, is important during vascular formation,in vitro model which is composed of only ECs to focus on the direct effect of interstitial flow on the angiogenic response of ECs. First, we analyzed the process of microvascular network formation in the series of interstitial flow conditions. We then investigated microvascular network formation in the combination of interstitial flow and the VEGF concentration focusing on VEGF flux. Furthermore, we explored a mechanism that can explain the effect of interstitial flow on angiogenesis.Here, we investigated the effect of interstitial flow magnitude on 3D microvascular network formation. Specifically, we utilized microfluidic devices to control a series of interstitial flow magnitudes, which was generated by 2, 8, and 25 mmH2O conditions corresponding to the mean velocities of 0.07, 0.7, and 4.3 μm/s during the first 24 h of interstitial flow experiments, respectively , while cells were also cultured in static conditions as control experiments. Cells were monitored by phase-contrast microscopy until day 5 to clarify the effect of interstitial flow magnitude on 3D network formation. The representative phase-contrast images showed that HUVECs formed vascular sprouts extending into collagen gel on day 1 only in interstitial flow conditions . In particular, the number of detached cells was the greatest in the condition of higher interstitial flow with a lower VEGF concentration such as 25 mmH2O conditions with 3.3 ng/ml VEGF , Invitrogen) and mouse antihuman PECAM monoclonal antibody (Millipore) for 2 h. Nuclei were also stained with 4′,6-diamidino-2-phenylindole  for 10 min. Z-stack fluorescence images were obtained using a confocal laser-scanning microscope .Next, morphological changes of ECs were visualized by immunofluorescence staining. In particular, we investigated the expression of Src which functions as a molecular switch of the intracellular signaling pathway during the angiogenic process, including EC proliferation and migration.Furthermore, the effect of interstitial flow on the morphological changes of ECs in a monolayer was investigated using the confocal images of PECAM. The shape index was calculated to quantify the degree of EC alignment in response to interstitial flow. This value was calculated using the area and perimeter of ECs, and the formula is given asFinally, we analyzed the structures of 3D networks after 5 days of interstitial flow loading. ECs were fixed as described above, and F-actin and nuclei were stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen) for 2 h and DAPI for 10 min, respectively. Fluorescence images were obtained with a confocal microscope and used for quantitative analyses. The length of networks was determined as the length from the cell monolayer to the network tip. The area of degraded gel was measured using two-dimensional (2D) projected images of F-actin. The network density was calculated using 2D projected images of F-actin according to the following formula:μM CI3 or 10 μM GM6001 for 1 h before interstitial flow experiments. After loading 8 mmH2O interstitial flow for 24 h, vascular sprouts were photographed using a phase-contrast microscope. The length of vascular sprouts was measured using ImageJ. In addition, the number of vascular sprouts formed between trapezoidal PDMS posts was counted. Because 0.08% DMSO was included in culture medium during inhibition experiments, DMSO at the same concentration was also added in culture medium of a control group.Calpain is an intracellular calcium-activated cysteine protease, thereby inducing MMP expression needed for angiogenic sprouting.p < 0.05 was considered to indicate statistical significance.Data are presented as the means ± standard deviation (SD) for each group. Statistical analysis was performed using SPSS software (IBM). At least three independent experiments were performed for all analyses. Comparisons among three or more groups were performed by one-way analysis of variance (ANOVA) followed by posthoc pairwise comparison testing using Tukey's method. Two-way ANOVA was only performed to determine the effect of effective VEGF supply on microvascular network formation. A value of supplementary material for the details of interstitial flow analysis.See the