Aedes aegypti, OX513A, has already reached the stage of field evaluation. Sustained releases of OX513A Ae. aegypti males led to 80% suppression of a target wild Ae. aegypti population in the Cayman Islands in 2010. Here we describe sustained series of field releases of OX513A Ae. aegypti males in a suburb of Juazeiro, Bahia, Brazil. This study spanned over a year and reduced the local Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 81% (95% CI: 74.9-85.2%) based on ovitrap indices compared to the adjacent no-release control area. The mating competitiveness of the released males  was similar to that estimated in the Cayman trials , indicating that environmental and target-strain differences had little impact on the mating success of the OX513A males. We conclude that sustained release of OX513A males may be an effective and widely useful method for suppression of the key dengue vector Ae. aegypti. The observed level of suppression would likely be sufficient to prevent dengue epidemics in the locality tested and other areas with similar or lower transmission.The increasing burden of dengue, and the relative failure of traditional vector control programs highlight the need to develop new control methods. SIT using self-limiting genetic technology is one such promising method. A self-limiting strain of  Aedes aegypti. We released transgenic ‘sterile’ male mosquitoes in Itaberaba, a suburb of Juazeiro, a Brazilian city. Sustained release of these males, whose offspring typically die before adulthood as a consequence of the transgenic modification, strongly suppressed the target wild population—by 80–95% according to different measures. These data are consistent with previous releases in the Cayman Islands, suggesting that differences between the two locations, including the environment or wild mosquito strain, made little difference. Mathematical models suggest that this degree of suppression would be highly effective in preventing epidemic dengue.Dengue is a major mosquito-borne disease, increasing in prevalence and severity; there are no specific drugs or licensed vaccine. It is primarily transmitted by one mosquito species,  Aedes aegypti (L.).Dengue is second only to malaria as most important mosquito-borne disease. Unlike malaria and other major infectious diseases, dengue is increasing in incidence and severity, currently inflicting 50–390 million or more cases per year worldwide , 2. DengDengue was reintroduced in Brazil in 1981 , after being almost entirely absent for at least 20 years following DDT-based vector control. Brazil now has serotypes 1–3 circulating throughout the country; in addition serotype 4 was recently detected in several states  concludeThere are no specific drugs or licensed vaccine for dengue, so efforts to reduce transmission depend entirely on vector control . HoweverAe. aegypti [The Sterile Insect Technique (SIT) is a genetic control system based on the release of large numbers of radiation-sterilised insects. These mate with wild insects of the same species and thereby reduce the reproductive potential of the wild pest population, as they produce no or fewer viable offspring due to the radiation-induced presence of lethal mutations in their gametes , 12. Tho aegypti , 21.Ae. aegypti, OX513A, was shown to be able to compete successfully for wild mates, furthermore sustained release of OX513A males suppressed a wild population of Ae. aegypti [Ae. aegypti population in this environment. The fourth core activity, which will be described in detail elsewhere, related to community engagement and regulatory activities.After extensive laboratory development and testing, field testing of engineered insects has begun, with encouraging results. In particular, in the Cayman Islands a self-limiting strain of  aegypti , 22. We Ae. aegypti and thus the area supported a relatively high and stable year-round population that is atypical of fluctuating seasonal populations that are broadly prevalent in the region. This provides a highly challenging environment, terms of mosquito population, in which to evaluate OX513A technology. Baseline monitoring was initiated in July 2010 using ovitraps which revealed the presence of Ae. aegypti and absence of Ae. albopictus in the whole of Itaberaba.The study was conducted in the Itaberaba suburb of the city of Juazeiro, Bahia in the semi-arid North East of Brazil , both treated and control sites were in the same suburb and, consequently, had similar characteristics. The site consisted predominately of housing of relatively low social economic status and was identified by local public health officials as having high dengue incidence. A dependence on stored water (due to irregular services for piped water) and high human densities provided ideal habitats for -1 . Control area density was slightly lower with an estimated 143 people ha-1 (1341 houses with 5726 population) (Human population density was comparable in treated areas A and B with an estimated 165 people haulation) . JuazeirPrior to establishment of the transgenic OX513A line in the mass rearing facility and subsequent open releases, regulatory approvals were obtained from the appropriate Brazilian national regulatory body, Brazilian National Biosafety Technical Commission (CTNBio), for the import permit from the UK to University of São Paulo : Extrato de Parecer (EP) 2.031/2009) and for the containment facility for rearing the strain at Biofábrica Moscamed Brasil (DOU: EP 2.577/2010). Approval for releasing OX513A males in the environment was granted in 2010 by CTNBio for five sites, including Itaberaba, around Juazeiro, Bahia (DOU: EP 2.765/2010).From its inception the project sought to adopt full transparency with a vigorous and proactive community engagement campaign. In addition to national (CTNBio) regulatory approval, consent and support came from regional  and local community leaders . Implementation included communication via local media , community meetings, printed information (posters and leaflets), school presentations, carnival parades, use of small vans with loudspeakers and social media (websites and blogs). Dedicated door-to-door campaigns and ongoing contact with field technicians working in the community provided face-to-face interaction on an individual basis, allowing specific questions to be addressed and for direct feedback and concerns to be aired. Full description of the community engagement will be reported elsewhere.Ae. aegypti with the OX513A insertion were used during this study [Ae. aegypti, as the observed levels of resistance were comparable to levels in the New Orleans susceptible reference strain [Ae. aegypti lines before being transferred to Biofábrica Moscamed Brasil (www.moscamed.com), Juazeiro City.Transgenic is study . The sene strain  used as e strain , 22. The2 laboratory specifically adapted and approved for the purpose. Mass rearing insectaries were maintained at 27°C (+/- 2), 70–90% relative humidity and a 12 hr day/night cycle. A colony of homozygous OX513A was established producing eggs to supply male mosquito production for release. Mosquitoes destined for release were reared to pupae where they were mechanically sorted to remove females [Subsequent to obtaining regulatory approval for field release, production of male mosquitoes was conducted at the Moscamed facility in an 84m females , 25. For females .Ae aegypti from adjacent populated untreated area. For details see Male pupae were aliquoted into release devices (RD) where they eclosed to adults over 24–48 hr before release . For details see Statistical analyses were performed using R freeware . Population size was estimated using Petersen-Lincoln test  as an esAll experiments with animal blood were carried out in accordance with the guidelines of the Ethical Principles for Experiment on Animals adopted by Sociedade Brasileira de Ciência de Animais de laboratório (SBCAL) and approved by the Institutional Ethics Review Committee (Comissão de Ética no Uso de Animais—CEUA)-Universidade de São Paulo, protocol #022/11.The community engagement protocol was approved by the Institutional Review Board in Human Research (Comissão de Ética em Pesquisa com Seres Humanos do Instituto de Ciências Biomédicas/USP) and Comissão Nacional de Ética em Pesquisa—CONEP, protocol #1115.-1 week-1 were released during that period in the treated area  and OX513A (O) males and the relative mating success, such that C = PW/(O(1-P)) where P is proportion of sterile mating (= proportion fluorescent larvae) [By hatching the eggs from ovitraps and scoring the resulting larvae for fluorescence, we could identify which had an OX513A father—and had therefore inherited a copy of the OX513A transgene, which carries a fluorescent marker—and which had not . By comp larvae) , 28. As  larvae) , followiWe next developed rearing and distribution systems in Brazil, building on Oxitec’s prior experience from field release in other countries , 22, 30.-1 week-1, to achieve a target 50% mating fraction, assuming mating competitiveness and wild populations remained the same.In addition to refining rearing methods and associated assessment of the field performance of the released mosquitoes, population suppression was a key endpoint of the release program as this would validate the technology in a Brazilian setting. Sterile-male methods such as the OX513A male release control strategy will successfully suppress the target population if a sufficient proportion of females mate sterile males. This threshold mating fraction required for local elimination in the absence of immigration can be estimated from models of mosquito population dynamics  as 13–57Up to 11th February 2012 we released into areas A and B , comprisActual release numbers, observed mating fraction (% fluorescent larvae), and ovitrap index—a measure of population density—are presented in Mating competitiveness is a key performance measure for released males; estimates of net field mating competitiveness of the released males ranged between 0.0004–0.047 . Mean coAe. aegypti which typically disperse (mean distance travelled) 30–100m as reviewed by Silver et al. [-1 week-1 . This corresponds well with the target release rates predicted from the rangefinder.Although the exact turning point is difficult to pinpoint, it is clear that suppression of the target population, relative to the untreated control area, began at the same time or even just before reducing the area treated (13 Feb 2012). This, combined with ongoing immigration of sterile males from the adjacent Area A, presumably explains that suppression was seen also in Area B. The majority (66%) of area B is < 100m from release points used in area A, and therefore falls under the flight rage of males r et al. . FurtherAe. aegypti adults in the trial area [Ovitraps provide only an indirect measure of the adult population density, reduction of which is the key target for vector control purposes. Though we consider that changes in ovitrap metrics provide good indications of changes in adult density within an area, they provide a poor guide to absolute number . To provial area , exploitAe. aegypti adult standing crop from an average of 418 ha-1 (95% bootstrap CI: 307–532), prior to January 2012, to 20 ha-1 (95% bootstrap CI: 10–29) in area A from May 2012 onwards. As discussed above, this 95% reduction represents an independent and likely more accurate, assessment of the impact on absolute adult population than ovitrap index, and is the most pertinent measure in relation to impact of intervention with regard to reducing the risk of disease transmission.These experiments provide clear evidence of a reduction in the standing crop of adults . We obseAe. aegypti by sustained releases of OX513A male Ae. aegypti. We diminished Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 78% (95% CI: 70.5%-84.8%) based on ovitrap indices compared to the adjacent no-release control area. We estimated mating competitiveness of the released males to be 0.030 (95% bootstrap CI: 0.020–0.040).In this study we demonstrate effective control of a wild population of Ae. aegypti dispersal is relatively short, with most published mean dispersal distances falling between 30–100 m; estimates for OX513A males also fall in this range [Cochliomya hominivorax) [Ceratitis capitata) [These results are similar to the 82% (95% bootstrap CI:69.7–90.0%) suppression achieved by release of OX513A males in a similar study in Grand Cayman . In bothis range . Effectsnivorax) , 34 and apitata) , 36. It apitata) . This isapitata) , 37, andapitata)  that havMean fluorescence ratio corresponding with period when suppression was observes was estimated to be 43% (95% bootstrap CI: 34.8–52.1%). This falls within model predictions of 13–57% (19) as did the Grand Cayman suppression (12%); variations in that threshold may indicate somewhat different population dynamics, as might be expected between two very different areas.The target release rate sufficient to achieve suppression should be proportional to the initial wild population. This may be assessed in terms of the ratio of release to wild males. Alternatively the mating fraction achieved represented a more direct impact of a given release rate without need to account for potential differences in mating competitiveness. Results from this and previous  studies et al. estimated a disease transmission threshold in relation to pupae per person-1 (as a proxy for adult mosquito population), ambient temperature and herd immunity [et al. (2000), we estimate that average pupae person-1 decreased in our treated area from 0.7 pre-treatment to 0.04 post-treatment, which in their model would be sufficient to prevent epidemic transmission under these conditions, or indeed under the most adverse conditions modelled for a naive population with 0% seroprevalence. The long-term goal for vector control should be to suppress below the transmission threshold even given low herd immunity [This study comprised exclusively entomological endpoints. What would be the impact of such striking reduction of vector population density on dengue transmission? Focks immunity , 39. Forimmunity –our dataimmunity S1 Text(DOCX)Click here for additional data file.S1 DatasetOvitrap data including results for screening larvae for expression of fluorescence, numbers of OX513A males released, direct adult trapping data including recapture of marked OX513A males and data presented in Figs (XLSX)Click here for additional data file.S1 Fig-1, solid circles = 2 traps house-1. Adult BG Sentinel trap distribution for the period 10/07-25/09/2012 is also shown (open diamonds). Area A = 5.5 Ha, B = 5.5 Ha, C = 8.5 Ha, D = 34.5.Ovitrap distribution is shown for the period 21/11/2011–19/09/2012; open circles = 1 trap house(TIF)Click here for additional data file.S2 FigWeekly numbers of adult OX513A males released per hectare.(TIF)Click here for additional data file.S3 FigArea A = Red, Area B = Blue, Area C = Yellow.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableAe. aegypti over the release period. φ Sex ratio was different for aspiration (0.45) and for BG-Sentinel traps (0.69).Details of the calculations of mating competitiveness and wild population of (DOCX)Click here for additional data file.S3 Table% Reduction expressed as reduction in relative ovitrap index from before and after suppression. Upper and lower 95% CI values given in parenthesis.(DOCX)Click here for additional data file.


Aim. The present investigation was designed to evaluate antiarthritic potential of fractions of hydroalcoholic extract from leaves of P. alba. Materials and Methods. Plumeria alba L. leaves were extracted with hydroalcohol (30 : 70) to obtain hydroalcoholic extract of P. alba. This extract was further fractionated with solvents ethyl acetate and n-butanol to obtain EAPA and BPA, respectively. These fractions were tested against formaldehyde and Freund's complete adjuvant (FCA) induced arthritis. Arthritis assessment, paw volume, body weight, motor incoordination, and nociceptive threshold were measured. On day 21, the animals were sacrificed and histopathology was done. Results. The 100 and 200 mg/kg doses of EAPA and BPA caused a significant (P ≤ 0.05–0.01) reduction in paw swelling in both models. Erythrocyte sedimentation rate (ESR) and spleen weight decreased significantly (P < 0.01) in arthritic rats treated with extracts. There was significant (P < 0.05) improvement in thymus weight in EAPA treated rats whereas significant (P < 0.01) improvement was also seen in haemoglobin level (Hb) in diclofenac treated group. Motor incoordination and nociceptive threshold were also significantly (P ≤ 0.05–0.01) improved. Conclusion. The present study suggests that Plumeria alba L. has protective activity against arthritis and supports the traditional use of P. alba for rheumatism and other inflammatory diseases. Inflammatory diseases like rheumatism are a major and worldwide problem. Rheumatic arthritis (RA) is a chronic, inflammatory systemic autoimmune disease that affects mainly Western countries and is more common in females than in males . AlthougThe modern drugs both steroidal and nonsteroidal anti-inflammatory drugs (NSAIDS) like etoricoxib and disease modifying antirheumatic drugs (DMARDSs) like methotrexate sulphasalazine, hydroxychloroquine, and leflunomide are used for the amelioration of the symptoms of the disease; however, they offer only temporary relief and also produce adverse effects . Because Plumeria L. (Apocynaceae) originated from Central America and its different species are now distributed in the warmer regions of the world including India. The genus is represented by topical trees or shrubs, frequently cultivated as ornamental and medicinal plants [ Plumeria species are widely used in gastric ulcer and rubefacient in rheumatism, asthma, piles, gonorrhoea, blood disorders, and tumours [Genusl plants . In Ayur tumours .Plumeria alba L., commonly called white Champa, is 4.5 m high, cultivated in Indian gardens and popularly called “peru” in Tamil [ Plumeria alba shows the presence of iridoids, tannins, and alkaloids [β sitosterol, scopotein, iridoids, isoplumericin, plumeride, plumeride coumerate, and plumeride coumerate glucoside [in Tamil . The plain Tamil . The milin Tamil . Moreovein Tamil , whereasin Tamil . Leaves in Tamil . The aerin Tamil . The roolkaloids . The plalucoside , 17. Plumeria alba were evaluated in animals to substantiate and expand its clinical applications.As far as we know, there have been no reports on the antiarthritic activity of this plant in animals so far. In this study fractions of hydroalcoholic extract of Plumeria alba were collected from Chaudhary Devi Lal Park, in Yamuna Nagar, Haryana, India, February 2011, and identified by Dr. H. B. Singh, Head of Raw Materials Herbarium and Museum (RHMD), New Delhi. A voucher specimen of the plant (reference no. NISCAIR/RHMD/CONSULT/2012-13/2047/55) has been deposited at the herbarium for future reference.The leaves ofThe air-dried leaves (2 kg) were thoroughly washed under running tap water so as to remove any type of contamination, air-dried in shade, powdered in grinder, and passed through sieve of mesh size of no-40. The extraction was done with aqueous ethanol (30 : 70) mixture by hot Soxhlet extraction method and the extract was concentrated in a rotary evaporator under reduced pressure, yielding 720 g (36%) of crude hydroalcoholic extract (HAPA). The dried crude extract was preserved in airtight glass container at 4°C–8°C.Initially, HAPA was partition-fractionated with 1 : 1  of n-hexane and ethanol (50%); the mixture was shaken vigorously and kept for about 30 min to make the two layers separate. The upper layer consisting of n-hexane was removed and concentrated in a rotary evaporator to obtain n-hexane extract (HPA). The same procedure was repeated with the bottom layer by using equivalent volume of other solvents like dichloromethane, ethyl acetate, n-butanol, ethanol, and water to obtain DCMPA, EAPA, BPA, EPA, and APA, respectively.Phytochemical analysis was performed using standard procedures to identify chemical constituents as described by Khandelwal . PrecoatμL was injected into the HPLC instrument (MAKE). HPLC analysis was performed on a Hypersil C 18 150 × 4.6 mm (DRDC/AD/36) column (MAKE) with a UV detector. The mobile phase consisted of methanol : acetonitrile : isopropyl alcohol: 40 : 20 : 20. The flow rate was 1.0 mL/min and the detection wavelength of the UV detector was set at 228 nm. The column temperature was set at 30°C.Stigmasterol and extracts were accurately weighed and dissolved in methanol to produce a solution containing 1.90 mg/25 mL. The sample was run for 20.00 min, and volume of 5.00 Formaldehyde , FCA , and diclofenac . Solvents used for extraction and chemicals used for phytochemical analysis were of analytical grade procured from approved organization.The dosages of EAPA and BPA used in this study were 100 and 200 mg/kg per day, which are the optimal dosages ascertained in our previous study [The reference anti-inflammatory drug diclofenac was dissolved in normal saline for the study. The drug solution was freshly prepared and administered orally at dose 4 mg/kg in volumes not exceeding 10 mL/kg.Healthy Sprague-Dawley rats (200–300 g) of either sex, purchased from a disease free animal house of the National Institute of Pharmaceutical Sciences and Education Research, Mohali, Punjab, India, were housed in the animal house, Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra, Haryana, India. The animals were fed with commercially available feed and were maintained under standard conditions of temperature (25°C ± 5°C), relative humidity (55 ± 10%), and 12/12 h light/dark cycle. They were transferred to the laboratory twelve hours prior to the experiments and given only water ad libitum. In all the experiments, the animals were kept in cages with raised floors of wide mesh, to prevent coprophagy. The animals were housed and cared for in accordance with the Federal Government Legislation on Animal Care. The experiments were also authorized by the Institutional Animals Ethical Committee (IAEC) for Animal Care (register number: 562/GO/02/a/CPCSEA) and were in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Guidelines, India.Sprague-Dawley rats were divided into five groups of six animals each. Baseline recording of the paw volume was made by using plethysmometer  . Group I(a) Induction of Arthritis. Adjuvant arthritis was induced in rats according to the method described by Newbould, 1963, with slight modification [ Mycobacterium tuberculosis in mineral oil) was then injected into the subplantar tissue of the right posterior paw. The volume of the injected , primary response and uninjected , secondary response hind paws was measured on alternate days from 2 to 21 days after the adjuvant injection. Unilateral inflammatory edema of the ipsilateral paw peaking at around days 6–8 was indicative of successful induction of adjuvant arthritis.fication . In this(b) Experimental Design. Grouping of animals is the same as prescribed in formaldehyde-induced nonimmunological arthritic model. Group I received Tween 80 , vehicle control. Group II received Tween 80 , FCA control. Group III received EAPA . Group IV received BPA . Group V received diclofenac sodium .Thirty minutes after administration of vehicle/drug, arthritis was induced by subplantar injection of FCA. This was designated as day 0. After immunization with FCA, all groups were maintained on vehicle/drug treatment for 20 more days . AntiartPhotographs of the arthritic rats were taken on day 21 with a digital 10-megapixel (Panasonic DMC-FS42) camera. Inflammation of each paw was graded blindly by the same person for all rats on day 21 according to the extent of erythema and oedema of the joints, using the criteria , 25 as fThe paw volume of both hind paws was measured just before FCA injection on day 0 and on alternate days after 2 to 21 days using a plethysmometer.Motor incoordination was evaluated by Rotarod apparatus as described earlier . Rats weThe apparatus consists of a hot plate on which the rats were placed for testing (Eddy's hot plate method) . Pain thThe rats were sacrificed with ether on the 21st day, the spleen and thymus were removed, and the weight of the organs was recorded and corrected for 100 g body weight.On the 21st day, blood was withdrawn from each animal through the retroorbital plexus into a test tube containing anticoagulant (5% EDTA) and haematological parameters were determined. The red blood cells (RBC) were determined by the method of Huxtable ; white bμm thick) were stained with haematoxylin and eosin. An experienced pathologist , unaware of the different drug treatment, evaluated the slides under light microscope for the presence of hyperplasia of synovium, inflammatory cells, fibrosis, and destruction of joint space.After sacrifice on the 21st day, ankle joints were removed from the hind paw, weighed, and fixed for 24 h in 10% formalin. After decalcification in 5% formic acid, processed for paraffin embedding, tissue sections  in the fractions Figures  and 2.Administration of 2 per cent formaldehyde on days 1 and 3 showed ankle joint swelling in the injected limb of all the animals. The joint swelling increased up to 4 days and then starts decreasing. EAPA and BPA significantly suppressed the joint swelling when compared with arthritic control between day 4 and day 10 after formaldehyde treatments . Even thFreund's complete adjuvant in rats induced significant inflammatory response characterized by paw swelling in both injected  and noninjected  paws. The response was biphasic, consisting of acute and polyarthritis phase. The acute manifestation of disease was corresponding to days 0–8 characterized by inflammation of ipsilateral paw, followed by polyarthritis phase corresponding to 9–21 days, characterized by involvement of contralateral paw .P < 0.01) for both fractions EAPA and BPA (100 and 200 mg/kg), respectively, as well as reference drug compared with the FCA-arthritic animals  in body weight from day 1 to day 21 as compared to arthritic-control rats. Rats treated with diclofenac (4 mg/kg) also showed significant improvement in body weight throughout the study as compared to arthritic-control animals , respectively, showed significant increase  decreased the primary lesions and secondary lesions seen after 10 days of study, when compared to arthritic control. Normal animals kept under experiment did not show paw swelling.The time dependent changes in the hind paw volumes of rats are shown in Figures Rotarod method was used to evaluate motor incoordination by determining the mean fall-off time. When compared to vehicle control group, FCA treatment group showed consistent decrease in fall-off time from day 0 to 8 and then showed improvement up to day 14 and consistent fall-off time thereafter. With treatment of extracts  and diclofenac (4 mg/kg), initially there was decrease in fall-off time from day 2 to 6; then increase in fall-off time was observed up to 12 days; after that, consistent fall-off time was observed throughout the study .P < 0.05, P < 0.01) improved paw withdrawal latency was observed which was maintained till the end of study , BPA (200 mg/kg), and diclofenac (4 mg/kg) showed decrease in paw withdrawal latency from day 2 to 8; then significant (of study .P < 0.01) inhibited in rats treated with EAPA (100 mg/kg) and BPA (200 mg/kg) as compared to FCA treated rats. However, only treatment with EAPA and diclofenac attenuated the decreased weight of thymus, significantly (P < 0.05 and P < 0.01), respectively.In the experiment, the mean spleen weight was increased whereas there was decrease in thymus weight of the FCA treated rats as compared to vehicle control group . It was P > 0.05) in red blood cells and white blood cell counts of EAPA (100 mg/kg), BPA (200 mg/kg), and diclofenac (4 mg/kg) treated groups compared to the FCA-arthritic animals (P < 0.01) low with both fractions and standard reference drug as compared to FCA-arthritic rats. Further haematological studies revealed significantly (P < 0.01) more hemoglobin level only in animals treated with diclofenac (4 mg/kg).Haematological assessment indicated no significant difference  was based on the previous studies conducted by our research group [Various animal models are used in the preclinical research in inflammatory arthritis , 33. Forch group . The locch group . In the α), interleukin-1 beta (IL-1β), and platelet derived growth factor (PDGF) in the pathogenesis of RA [The standard drug diclofenac sodium prevented the adjuvant induced arthritis which is consistent with previous studies –41. The is of RA , 45. Tre14C-glucose and 14C-leucine was reduced in inflammation whereas anti-inflammatory drug treatment has improved the decreased absorption capacity [The severity of arthritis was expressed as arthritic score is the index of joint inflammation. Arthritic score is the sum of four paws after immunization and values before immunization . Arthritcapacity , 51. In Several studies were conducted to evaluate hyperalgesia by using arthritic rats. Eddy's hot plate is a quantitative method for determination of hyperalgesia related to behaviours , 52. TheSpleen is a vital organ serving as the available source of cells and antibody formation, known to be involved in immunological response in adjuvant arthritis . In the The significant low level of ESR in the EAPA, BPA, and diclofenac treated arthritis rats indicates anti-inflammatory potential. The ESR is indirect method for the measurement of inflammation in the body. Erythrocytes move closer, stack up in a group, due to protein production in inflammation, become denser, and settle faster that increases the erythrocyte sedimentation rate . In variHistopathological studies of the paws contribute towards the treatment of arthritis with EAPA and BPA despite evidence of pathology in the arthritic animals treated with the reference drug. P. alba could be due to the presence of flavonoids, alkaloids, terpenoids, glycosides, saponins, tannins, and steroids detected after preliminary phytochemical screening of the fractions. Nonspecific antiarthritic activity may be due to the combined effect of the different phytoconstituents present.The antiarthritic effect of fractions (EAPA and BPA) of hydroalcoholic extract of2 and prostaglandins due to similar effect of diclofenac.The result of the present study suggests that antiarthritic potential of EAPA and BPA may be due to the protection of synovial membrane, vascular permeability, and prevention of cartilage destruction, thereby improving the health status through haematinic properties. The effect may be due to the inhibition of phospholipase A P. alba possess antiarthritic activity in Sprague-Dawley rats. The present study predicts that the P. Alba provides pharmacological rationale for the traditional use of the plant against inflammation disorders like rheumatoid arthritis.It also demonstrates its beneficial effect during recovery from arthritis by including Hb, ESR, and body weight along with clinical signs including paw oedema, thermal hyperalgesia, and histopathological examination. The study established that the ethyl acetate and n-butanol fractions of P. alba in inflammatory disease.However, further studies are needed to identify and isolate the possible phytoconstituent(s) involved in the antiarthritic activity, which would facilitate the use of


There was a strong correlation among sensor attributes  and different N-levels. It was concluded that tillering stage is most prominent stage to predict crop yield as compared to the other stages by using sensor attributes. The algorithms developed for tillering and booting stages are useful for the prediction of N-application rates for wheat crop. N-application rates predicted by algorithm developed and sensor value were almost the same for plots with different levels of N applied.The experiment was planned to investigate the tractor mounted N-sensor  to predict nitrogen (N) for wheat crop under different nitrogen levels. It was observed that, for tractor mounted N-sensor, spectrometers can scan about 32% of total area of crop under consideration. An algorithm was developed using a linear relationship between sensor sufficiency index (SI Precision agricultural practices have significantly contributed to the improvement of crop productivity and profitability. It enhances farm input use efficiency and reduces environmental impacts . Today, Plants normally contain 1–5% nitrogen by weight. Nitrogen generally has more influence on crop growth, yield, and quality than any other nutrient commonly provided as fertilizer to crops. Many farmers often use uniform rates of N fertilizers based on expected yields  that could be inconsistent from field-to-field and year-to-year depending on factors that are difficult to predict prior to fertilizer application. Also, farmers often apply fertilizer N in doses much higher than the blanket recommendations to ensure higher crop yields. Large temporal and field-to-field variability of soil N supply restricts efficient use of N fertilizer when broad based blanket recommendations are used , 4. A miZea mays L.). Remotely sensed NDVI can provide valuable information regarding in-field N variability and significant relationships between sensor NDVI and maize grain yield have been reported.Recently, optical sensing of crop canopy spectral reflectance from ground, aircraft, and satellite-based platforms on Normalized Difference Vegetation Index (NDVI) has been proposed to identifying the crop nitrogen (N) deficient portions in the fields. These instruments has the potential to provide a fast, inexpensive, and accurate estimate of plant biomass production and grain yield prior to harvest, which would be beneficial for crop breeders , 14. MarLeaf color charts for proper N-management have been recommended in many countries but there are certain issues in their adoption by the farmers. Green seeker, Crop circle, Crop spec, and N-sensor are on-the-go sensing devices to achieve variable-rate application of N fertilizer according to site-specific field conditions. In the present study, the responses of N-sensor  under variable levels of N availability were investigated for wheat crop.to calibrate the tractor mounted N-sensor for estimation of nitrogen and chlorophyll available in wheat crop;to develop an algorithm for N-sensor based on SPAD meter algorithm.The tractor mounted N-sensor is one of the sensor technologies which measures the level of light reflectance by crop canopies using spectrometers. But, there is a need to calibrate and develop an algorithm for N-application for locally recommended varieties of wheat. It can measure crop reflectance characteristics and calculate NDVI for development of algorithms to calculate optimal N application rates. In this paper, an algorithm is developed for converting sensor measurement into N-application. The sensor algorithm is based on a SPAD algorithm published by Varvel et al.  that outField experiments to investigate the tractor mounted N-sensor for wheat crop under variable nitrogen application rates were carried out at the Research Farm of the Department of Farm Machinery and Power Engineering, Punjab Agricultural University, Ludhiana. Soil of experimental field was sandy loam in texture and normal in pH, EC. For experiment, the university recommended PBW550 wheat variety was selected. It was raised with 6 different nitrogen levels, that is, 0, 30, 60, 90, 120, and 150 kg N/ha. The crop was sown on 21 November 2012. The experiment was carried out in randomized complete block design with three replications . The nitThe N-sensor was mounted on the front of a multiutility vehicle (MUV) developed in the department  at 1.6 mN-sensor consists of two diodes array spectrometers, One pass of the field scan was done for each plot in the wheat fields. For each measurement, the field scan recorded spectral information at all wavebands from 600 to 1100 nm with 10 nm intervals. N-sensor scans were done at Tillering (Growth stage: 55DAS or 28 for N-sensor), Booting (growth stage: 85DAS or 36 for N-sensor) and ear emergence (growth stage: 115DAS or 50 for N-sensor) stages of crop growth.The nitrogen and biomass status of crop were also mapped during scanning of crops at different growing period based on spectral reflectance measurement.d is length of the sensor rig (2.0 m), h is height of sensor rig, X1 is inner point of sensed area, X2 is outer point of sensed area, y is width of sensed area, m (X2 − X1), and h is height of sensor mounting, 1.6 m.The area scanned by the N-sensor depends on the height of mounting over the tractor. In present case, N-sensor was mounted at 1.6 m height from the ground. Equations :(1)X1=dX1 = 2.831 m and X2 = 4.143 m, y = X2 − X1 = 1.32 m, total width on both sides of tractor = 8.28 m,  and total width of scan by spectrometers = 2.64 m.From , X1 = 2h) of 1.6 m, it is scanning width of (y) 1.32 m or 2.64 m on both sides of a tractor covering total width 8.28 m. It means spectrometers can scan about 32% of total area of crop under consideration.When sensor was mounted at height  were determined from the N-sensor spectral data collected in the range of 400–1050 nm measured during the field calibration.The data for various parameters like total chlorophyll and nitrogen content were measured in laboratory at different growth stages like tillering, booting, and ear emergence of crop and at the same time N-sensor data was also recorded. The chlorophyll content was estimated according to the method of Hiscox and Israelstam  using diBy establishing a reference strip, sensor readings can be collected under near perfect conditions . These readings are considered the optimum sensor reflectance value, a value at which no additional N is required. From these sensor readings a “Sufficiency Index” (SI) can be calculated using the following equation:a0, a1, a2 coefficients represent the intercept, linear, and quadratic terms, respectively, and the response portion of the equation can be solved for N rate as follows:rate can be found asrate that corresponded to the maximum SISPAD measurement is 150 kg N ha−1 (app) for any SISPAD measurement below maximum (max SISPAD) can then be found as follows:sensor  NDVI) into SISPAD measurements for input into the SPAD algorithm is illustrated. Solari et al. [SPAD and SIsensor illustrating its general form asb0, b1 coefficients represent the y intercept and linear terms, respectively. Using the same data set, the following specific equation was determined for SINDVI. Equation (sensor. After substituting (R and SIR terms can be solved for using the following equations:SPAD) and N fertilizer rate at tillering and booting stages of wheat crop are shown in NDVI) and variation in relative chlorophyll meter (SISPAD) readings for data collected at tillering and booting stages are given in The procedures for development of sensor algorithm for translating sensor readings into N applications  are outlramework ; the genamework [SISPAD=a0found asNratemaxg N ha−1 . An appri et al.  demonstr form asSISPAD=b0 form asSISPAD=b0follows:NrateSISPfound asNratemax form asSISPAD=b0follows:Napp=NratFor Tillering Stage. From Figures  Figures  SISPAD=−For Booting Stage. Again from Figures  Figures  SISPAD=0nts from  results app (N-application) rate can be determined through input of SIsensor values into the simplified equations  values measured during the calibration of N-sensor and second on the basis of algorithm developed in quations  and 14)app (N-apr) among variables were determined by the CORR procedure in the SAS system .Various data sets were statistically analyzed by general linear model (GLM) procedure by using SAS software 9.3. All possible pairs of treatment means were compared at 5% level of significance. Pearson's correlation coefficients , biomass data of the crop, and NDVI calculated from the sensor spectral data were established with N and chlorophyll content in the leaves of crop. Results are discussed regarding the N-recommendation for wheat crop on the basis of sensor values and algorithm developed in the study.P < 0.001).P < 0.001).The relationships between sensor biomass values across N application rates at different growth stages are shown in P < 0.001). This is in accordance with many studies conducted to recommend N on the basis of NDVI calculated from crop reflectance data.The NDVI calculated from sensor spectral data across N application rates at different growth stages are shown in The correlation coefficients corresponding to the relationships between NDVI calculated from N-sensor data, biomass value given by sensor, sensor value (SN) with plant N concentration, chlorophyll content of leaves at three growth stages, and yield were obtained from experiments. These relationships have been discussed in the following subheads.Pearson correlation coefficients (PCC) between Lab N and N-sensor attributes were more at tillering stage as compared to the values at booting and ear emergence stages of crop growth . The PeaPearson correlation coefficients between N-sensor attributes and Lab Chl were ranging from 0.61 to 0.82 at all growth stages of crop. The Pearson correlation coefficient between sensor value and Lab Chl was higher for both tillering and booting stage, that is, 0.82 and 0.81. The correlation between sensor biomass and Lab Chl decreased with the growth stages. The coefficient values were 0.79, 0.79, and 0.72 for tillering, booting, and ear emergence stages, respectively. The sensor NDVI value correlation with Lab Chl was having values of PCC 0.75, 0.67, and 0.61 at tillering, booting, and ear emergence stages, respectively . PolynomCorrelation between yield and sensor attributes at different growth stages of crop is shown in app rate) as a function of different N-treatments using N-sensor algorithm developed for tillering and booting stages is shown in N-application (Napp) rates on the basis of N-sensor values (SN) during tillering and booting stages of wheat crop are shown in N-application (NIt is clear that N-application rates based upon algorithm and sensor value methods were in consonance. Both of the methods predicted almost the same application rates for plots with different levels of N applied.For tractor mounted N-sensor, spectrometers can scan about 32% of total area of crop under consideration.Algorithms were developed for N-application at tillering and booting stages of crop growth.There was a strong correlation among sensor attributes and different N-levels, that is, 0, 30, 60, 90, 120, and 150 kg N/ha.Study indicates that the relationship among different sensor attributes is poorly correlated with Lab Chl data as compared with Lab N data.It was concluded that there is a good correlation between sensor attributes and yield at all growth stages of the crop. But tillering stage is most prominent stage to predict crop yield as compared to the other stages by using sensor attributes.The algorithms developed for tillering and booting stages are useful for the prediction of N application rates for wheat crop.N-application rates predicted by algorithm developed and sensor value were almost the same for plots with different levels of N applied.


To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF agents was performed along with in vivo AF (anti-settlement) screening tests. Pre-characterization of different cholinesterases (ChEs) forms present in selected tissues of important biofouling species was performed to confirm the predominance of AChE, and an in vitro AF test using pure AChE activity was developed. Eighteen cyanobacteria strains were tested as source of potential AF and AChE inhibitor agents. Results showed effectiveness in selecting promising eco-friendly AF agents, allowing the understanding of the AF biochemical mode of action induced by different compounds. This study also highlights the potential of cyanobacteria as source of AF agents towards invertebrate macrofouling species.Effective and ecofriendly antifouling (AF) compounds have been arising from naturally produced chemicals. The objective of this study is to use cyanobacteria-derived agents to investigate the role of acetylcholinesterase (AChE) activity as an effect and/or mode of action of promising AF compounds, since AChE inhibitors were found to inhibit invertebrate larval settlement Biofouling is the process by which a range of micro- and macroorganisms attach to natural and artificial underwater surfaces, constituting a diverse settled community and creating serious problems for the maritime industry worldwide . As virtBalanus amphitrite, acting on the cholinergic neurotransmission mechanism [Mytilus species [Pollicipes pollicipes [M. galloprovincialis in particular has been the target of several ChEs characterization studies, usually performed in the gills, the most appropriate organ for biomonitoring studies [Mytilus sp. was found in the foot [M. galloprovincialis [Pollicipes pollicipes, ChEs’ characterization in the peduncle was recently performed, showing AChE properties as given by substrate preference and specific inhibitors [Compounds that might act as neurotransmission disruptors have been described as both promising AF and fouling-inducing agents to invertebrate species . The setechanism . In addiechanism ,12,13, rechanism . ChEs chechanism ,16,17. Cechanism ,19. Inveechanism ,21. Giveechanism ,24,25,26 species  and recellicipes . M. gall studies ,30. Howethe foot  which waincialis , others incialis ,30,31. Rhibitors . ConsideMytilus galloprovincialis, goose barnacle Pollicipes pollicipes and the acorn barnacle Perforatus  perforatus and investigate the role of AChE activity inhibition as an effect and/or mode of action of promising AF compounds. To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF/fouling inducing agents was performed along with in vivo AF (anti-settlement) screening tests. Cyanobacteria were selected as source of a wide range of secondary metabolites with recognized bioactivity on distinct biological responses including AF properties [Considering this, the aim of this study is to perform the characterization of ChEs in specific tissues of important marine invertebrate species of biofouling communities in the NE Atlantic, mussel operties ,33.M. galloprovincialis as expected, since a previous study on ChE expression pattern have shown that in M. edulis, a congeneric species, the highest ChE activity was found in the foot tissue [ChEs activity was observed in the foot of t tissue .M. galloprovincialis ChEs degraded preferentially acetylthiocholine (AcSCh), followed by acetyl-β-methylthiocholine (AbSCh) and propionylthiocholine (PrSCh)  A, while Km) and maximal velocity (Vm) for each substrate were obtained after adjusting the specific activity of each of the four substrates at a range of concentrations, to the best hyperbola through a hyperbolic regression , so the lowest Km value indicates that Vm is reached with less substrate concentration, so having more affinity.Michaelis constant . AbSCh and PrSCh had a similar Vm value  and BuSCh presented the lowest value of Vm (0.85 ± 0.03 nmol min−1 mg protein−1). The higher Km values were observed with PrSCh (36.07 ± 5.29 μM) followed by AcSCh (22.18 ± 0.75 μM) and then AbSCh (14.04 ± 1.12 μM). The lowest value corresponding to BuSCh (2.89 ± 0.88 μM). This is in accordance with expectancies, since it is a non-specific substrate hydrolysed by all ChEs. The second highest Vm value occurred for PrSCh and AbSCh, with values close to each other, indicating that the ChE(s) present are able to hydrolyse these two substrates specific of PrSChE and AChE, respectively. For BuSCh, Vm value was very low, indicating that this substrate is poorly hydrolysed by the ChE(s) present on this species. However, taking into account Km values , despite BuSCh being poorly hydrolysed, it has the highest affinity for the ChE(s) present. These results suggest that the ChE(s) present on M. galloprovincialis foot muscle have predominant hydrolytic characteristics of both AChE and PrChE of vertebrates.The highest iso-OMPA), a specific inhibitor for BuChE, no inhibition of ChE activity was observed at any concentration tested , a non-specific inhibitor, only inhibited ChE activity in this species at the higher concentration tested, 400 μM A. Eserinn tested B. Contran tested D, as evin tested .iso-OMPA are capable of inhibiting M. galloprovincialis ChE activity, suggesting that these ChE(s) do not have typical characteristics of vertebrates BuChE. These results are in good agreement with the results obtained with the different substrates, as BuSCh was the most poorly hydrolysed substrate. On the other hand, eserine and BW284C51 both inhibit extensively ChEs’ activity on this species. Comparing these two inhibitors, and considering that eserine inhibits all ChEs, and BW284C51 only inhibits AChE, it is evident, at low inhibitor concentrations, that there are other enzymes that hydrolyse AcSCh, most likely other esterases (CoEs). This suggests that about 9% to 10% of the ChE activity observed, with AcSCh as substrate, is probably due to CoEs activity. Conversely, at high concentrations (>25 μM) of eserine and BW284C51, there were no significant differences between ChE activity with those inhibitors, suggesting that ChE(s) present on this species have inhibitory characteristics of vertebrates AChE.Results show that neither Cu(II) nor M. edulis foot, the highest ChEs activity was also observed using AcSCh as substrate, being PrSCh the second most hydrolysed [iso-OMPA [In drolysed . Moreoveiso-OMPA , which iMytilus spp. have only one pharmacological form of ChE [A previous study, using the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC) digestion, followed by electrophoresis, and characterization by inhibition techniques, suggests that m of ChE . ConsideP. pollicipes ChEs activity was similar for all substrates at low concentrations (<0.04 mM)  and PrSCh (3.68 ± 0.53 nmol min−1 mg protein−1), and the lowest value occurred with AcSCh (1.99 ± 0.13 nmol min−1 mg protein−1). The highest Km value was observed with PrSCh (3716.93 ± 1227.18 μM), followed by AbSCh (96.81 ± 28.37 μM) and, the lowest value was observed with AcSCh (22.97 ± 7.08 μM)  present showed the lowest affinity for this substrate , indicating that this substrate is not the preferred one for the ChE(s) present on this species. AcSCh displayed the lowest Vm although ChE(s) had the highest affinity for it . For AbSCh, it was observed a relatively low Km and one of the highest values of Vm. BuSCh did not adjust to hyperbolic regression due to the low activity observed at all concentrations, suggesting that this substrate is poorly hydrolysed by ChE(s). ChE(s) present in this species were able to hydrolyse both AbSCh and PrSCh suggesting that ChE(s) have hydrolytic characteristics similar to both AChE and PrChE from vertebrates.0.04 mM) B. From 07.08 μM) . BuSCh dubstrate . These rIso-OMPA, specific for BuChE, caused no inhibition at any concentration tested  only inhibits it slightly. On the other hand, eserine and BW284C51 both inhibit extensively ChE activity. Comparing these two inhibitors, considering that eserine inhibits all ChEs and BW284C51 only inhibits AChE, and that there were no significant differences between their activities at any inhibitor concentration tested, it is suggested that all ChEs present on this species, if more than one, have inhibition characteristics of vertebrates’ AChE. The remaining activity observed suggests that there are others enzymes that hydrolyse AcSCh, most likely CoEs. This leads us to believe that about half of the activity observed in this species is probably due to CoEs. Similarly to the results from this study, ChE(s) activity in the peduncle of the same species also showed preference for AcSCh and PrSCh as substrate, and poor BuSCh hydrolysis capacity [iso-OMPA did not inhibit these ChE(s), contrarily to eserine and BW284C51 that widely inhibit their activity. However, previous results indicate that BW284C51 inhibits these ChEs more effectively than eserine, contrarily to our results, where no significant differences between eserine and BW284C51 inhibition levels were found at all concentrations tested [P. pollicipes capitulum soft tissues and in peduncle muscle are not exactly the same isoenzymes.Regarding ChE inhibitors, Cu(II) A caused n tested C. In then tested D, specifP. perforatus ChE activity, using different substrates, showed that the highest activity was observed with AcSCh and AbSCh, followed by PrSCh, and finally BuSCh, which had the lowest activity at all concentrations tested (Vm was obtained for the substrates AcSCh (27.89 ± 2.03 nmol min−1 mg protein−1) and AbSCh (23.78 ± 1.94 nmol min−1 mg protein−1), followed by PrSCh (13.17 ± 0.46 nmol min−1 mg protein−1), being the lowest value observed for BuSCh (6.10 ± 0.46 nmol min−1 mg protein−1) . Lower values were obtained for AcSCh (235.92 ± 44.43 μM), AbSCh (220.62 ± 51.54 μM) and PrSCh (279.12 ± 45.37 μM). Similarly to what was observed for the other species, results show that ChE(s) in this species have the highest affinity for AcSCh, a non-specific substrate hydrolysed by all ChEs. AbSCh had a Vm value close to AcSCh and Km values were also very close to each other, suggesting that ChE(s) of this species have also a higher preference for AbSCh. PrSCh presented Km and Vm values close to the former two substrates, suggesting that the ChE(s) present show similar affinity for the three substrata. Contrarily, BuSCh is poorly hydrolysed given its low Vm and high Km, which indicate low affinity for the enzyme. The results suggest that the ChE(s) of this species have predominant hydrolytic characteristics of both AChE and PrChE from vertebrates, as it was the case of the two former species.s tested C. Inhibiotein−1) . The higIso-OMPA, the specific inhibitor for BuChE, did not inhibit significantly ChE activity at any concentration tested  there were no significant differences between the activities, suggesting that all ChEs presents on this species have inhibition characteristics of vertebrates’ AChE. However, the significant differences in the remaining activity observed after using eserine or BW284C51 at lower concentrations suggests that there are others enzymes that hydrolyse AcSCh, most likely CoEs, at a rate about 6%–7% of the total activity observed. Identical results were also observed in this study for M. galloprovincialis and P. pollicipes. Also, in the grass shrimp, Palaemonetes pugio, ChEs present have the properties of vertebrates AChE, hydrolysing both AcSCh and AbSCh; the hydrolysis of BuSCh was minimal; and the ChEs were inhibited by eserine and BW286C51 but not by iso-OMPA [Echinogammarus meridionalis and the shrimp Atyaephyra desmarestii, similar results were also obtained [Regarding ChE(s) inhibition, Cu(II) presented low inhibition and only at 200 μM (NOEC = 100 μM) . Eserinen tested C. BW284Ciso-OMPA . Concernobtained . This suin vivo antifouling (anti-settlement) tests using mussel plantigrade larvae exposed to crude extracts of 18 different cyanobacterial strains showed significant bioactivity in some organic and aqueous extracts  when compared to DMSO control. No significant differences were found between DMSO control and the negative control with filtered seawater . Regarding aqueous extracts, significant settlement inhibition was observed for Cyanobium sp. LEGE06068 , Leptolyngbya sp. LEGE06070 , Nodularia sp. LEGE06071 , Synechocystis sp. LEGE06083 , Synechocystis sp. LEGE07073 , Microcoleus chtonoplastes LEGE07092  when compared to filtered seawater control. Positive control (CuSO4) was highly responsive to prevent mussel plantigrade larvae settlement (F = −83.75 p < 0.001). No mortality was observed in any of the conditions tested including the positive control.Results from the extracts . Organicin vitro screening test was based on pure AChE activity. Inhibition of AChE activity in vitro by cyanobacteria organic crude extracts was observed for Synechocystis sp. LEGE06079 , and Microcoleus sp. LEGE07076  compared to DMSO control , Synechocystis sp. LEGE06083 , Synechocystis sp. LEGE07073 , Leptolyngbya sp. LEGE07075 , Microcoleus sp. LEGE07076  and Cylindrospermopsis raciborski LEGE99043  caused significant inhibition of AChE activity  caused AChE activity induction, compared to filtered seawater control. Positive control (eserine) was highly responsive regarding AChE activity inhibition .Considering the first part of this work demonstrating that the ChEs present on the selected tissues of three important biofouling species used have mainly hydrolytic characteristics of AChE and are inhibited by specific inhibitors of AChE, the  control A. Among activity B; and Cyin vivo anti-settlement responses and in vitro potential effect responses based on a neurotransmission-related parameter (AChE activity) involved in the processes of invertebrate settlement [in vivo anti-settlement tests indicated that selected cyanobacterial strains showed significant bioactivity on both organic and aqueous crude extracts, confirming their great AF potential towards invertebrate settlement. Concerning the in vitro AChE activity tested, results were also responsive by discriminating cyanobacteria extracts with AChE inhibition and induction potential.In this study, ttlement  were obtttlement , and whittlement . The resSynechocystis sp. LEGE06083 and Synechocystis sp. LEGE07073) showed bioactivity for both anti-settlement and AChE activity inhibition. This suggests that the two activities might be related, and that the AChE activity disruption observed with the crude extracts of these strains might be responsible for the unsuccessful adhesion. Crude extracts of the genus Synechocystis were also found to induce acute toxicity to Artemia salina nauplii, to affect embryonic development of Paracentrotus lividus and to completely inhibit the embryogenesis in M. galloprovincialis [Synechocystis sp. extracts by inducing apoptosis in eukaryotic cells and causing inhibition of Gram-positive bacteria [Synechocystis sp. LEGE06079, however with no relation with settlement impairment. So, these results seem to indicate the presence of bioactive metabolites on these strains, and probably indicating that different promising compounds with AChE activity inhibition mode of action were acting when different activities were found. As it was expected, not all settlement inhibition was explained by an effect on AChE activity, as modes of action of AF compounds are diverse. Several cyanobacterial extracts analyzed were highly competent in the prevention of settlement with no effect on AChE activity. It is the case of all organic extracts that significantly inhibited settlement, and for instance, the aqueous extract of Nodularia sp. LEGE06070 with 95% of settlement inhibition and no effect on AChE activity.Considering the combined responses of the two tests, two cyanobacterial aqueous extracts  by aqueous extracts of Leptolyngbya sp. LEGE07075 and Leptolyngbya sp. LEGE07080 were able to inhibit the growth of other cyanobacteria  [P. lividus pluteus larvae at higher extract concentration (1.6 mg mL−1) [Nostoc sp. LEGE06077 affects the growth of other marine cyanobacteria species like Synechocystis salina, freshwater species such as Microcystis aeruginosa and the microalgae Nannochloropsis sp. [Leptolyngbya sp. LEGE06070 was previously found to induce toxic effects on Artemia salina [Cyanobium strains were found to inhibit microalgae (Nannochloropsis sp.) and bacterial (Pseudomonas putida NB3L) growth, and also induced a decrease in P. lividus larvae growth [Some of the cyanobacteria extracts that showed anti-settlement potential in this study were previously found to produce effects on other different endpoints. Namely, organic extracts of  salina) , and alsmg mL−1) ; the orgpsis sp. ; aqueousa salina ; Cyanobie growth . This alin vitro AChE activity with the in vivo anti-settlement tests in the screening approach seems to be valuable in the assessment of promising AF compounds; one assessing a selected phenotypic target (settlement), integrating the effect of many possible compounds on various pathways, and the other assessing the effects on a specific mechanism that may be involved in settlement inhibition, the inhibition of AChE.Considering this, further studies on the chemical purification and elucidation of the active principles of the selected bioactive strains, with AF properties, should be conducted to possibly obtain new natural effective AF agents. In addition, the combination of the All experiments were conducted in accordance with ethical guidelines of the European Union Council (Directive 2010/63/EU) and the Portuguese Agricultural Ministry (Portaria nr. 1005/92 of 23 October) for the protection of animals used for experimental and other scientific purposes.M. galloprovincialis (mussel), P. pollicipes (goose barnacle) and P. perforatus (acorn barnacle). All species are abundant in the intertidal zone, occur all year round and are easy to capture. They have also in common an initial planktonic period in their life cycles, after which settlement and metamorphosis into adult sessile organisms occurs.This study has focused on three main biofouling invertebrate species of the Portuguese coast, Specimens were collected in the intertidal rocky shore, during low tide in Memória beach, Matosinhos, Portugal , and immediately transported to the laboratory in controlled conditions.M. galloprovincialis, the whole capitulum for P. pollicipes, and the whole soft tissues for P. perforatus. M. galloprovincialis 2 to 3 feet were pooled per sample. For P. pollicipes, the capitula of 2 to 3 animals, and for P. perforatus the soft tissues of 5 animals were homogenized on ice in 1 mL of K-phosphate buffer 0.1 M  at pH 7.2 using an ultraturrax  and, when necessary, an ultrasonic homogenizer  was used for short cycles to avoid warming up and degradation of the sample. Homogenates were centrifuged at 6000 rpm for 3 min at 4 °C . Supernatants were stored at −80 °C until further analyses.Selected tissues from each invertebrate species containing the adhesive organs (mussels) and/or the majority of nervous tissues (barnacles) were isolated in refrigeration. The anterior part of foot muscle, in the case of −1.Protein determination of samples was performed in triplicate using bovine serum albumin (BSA)  as standard  in a micFor ChE characterization according to the preference for a specific molecule upon which the enzyme acts (substrate), three specific substrates were used: acetyl-β-methylthiocholine (AbSCh) (SIGMA A3271), specific for AChE; butyrylthiocholine (BuSCh) SIGMA B3253), specific for butyrylcholinesterases (BuChE); propionylthiocholine (PrSCh) (SIGMA P2880), specific for propionylcholinesterases (PrChE); and a non-specific substrate, acetylthiocholine (AcSCh) (SIGMA A5751). Reaction solutions were prepared for each substrate at 13 concentrations ranging from 6.0 μM to 24.62 mM ,25 with 3, specifIso-OMPA (SIGMA T1505), a BuChE specific inhibitor; and Cupper (II), a non-specific inhibitor. Iso-OMPA solutions were prepared in 100% ethanol, while for the other inhibitors ultrapure water was used. Test concentrations of inhibitors ranged from 6.25 to 200 μM of eserine and BW284C51, 0.25 to 8 mM of iso-OMPA and 12.5 to 400 µM on assays with copper(II) [Three inhibitors were used for ChE characterization according to the response to selective inhibitors: eserine (SIGMA E8375), a non-specific reversible inhibitor; BW284C51 (SIGMA A9013), an AChE specific reversible inhibitor; pper(II) ,25,43. F−6 E m−2 s−1. After 60 to 90 days of growth, the cyanobacterial cells were collected and lyophilized. The biomass from each cyanobacterial strain was repeatedly extracted with warm (<40 °C) CH2Cl2/MeOH (2:1) and the solvents removed in vacuo and/or under a N2 stream [Eighteen cyanobacteria strains from Portuguese estuaries, intertidal rocky beaches and freshwater systems (LEGE culture collection) were cultured and up-scaled in aerated Z8 medium  under la2 stream .After the organic extraction, the remaining biomass was subjected to aqueous extraction (ultra-pure water), decanted and centrifuged at 4600 rpm for 15 min. The resulting supernatant was freeze-dried, weighed and stored at −20 °C.−1) in dimethyl-sulfoxide (DMSO) and aqueous extracts in ultra-pure water.Just before the tests, organic extracts were dissolved .These tests aim to investigate the ability of organic and aqueous extracts of cyanobacteria strains to inhibit the settlement of −1. M. galloprovincialis plantigrade larvae were exposed to test solutions in 24-well microplates for 15 h in the darkness, to maximize byssal threads production. Four well replicates were used per condition with five larvae per well. A negative control with filtered seawater was included in all tests, as well as a positive control with 5 μM CuSO4 (a potent AF agent). A second negative control (C DMSO) was used only in the organic extracts assays, including filtered seawater with 0.1% DMSO, to control the effect of the solvent.Test solutions were obtained by dilution of the cyanobacteria extracts stock solutions in filtered seawater to a concentration of 30 µg mLAt the end of the exposure period, the presence/absence and number of byssal threads produced by each individual was assessed for all the conditions.in vitro screening tests aim to test the hypothesis that some extracts of cyanobacteria strains act as AF agents through ChE inhibition.These Electrophorus electricus Type V-S  and crude extracts of cyanobacteria as inhibition/induction agents. Briefly, 250 μL of the reaction solution containing 30 mL of phosphate buffer, 1000 μL of the reagent dithiobisnitrobenzoate (DTNB) 10 mM (acid dithiobisnitrobenzoate and sodium hydrogen carbonate in phosphate buffer) and 200 μL of acetylcholine iodide 0.075 M was added to 50 μL of pure acetylcholinesterase enzyme (0.25 U/mL) and 3 μL of each cyanobacteria extract  in quadruplicate. The optical density was measured at 412 nm in a microplate reader during 5 min at 25 °C. A negative control with ultra-pure water instead of extract was included in all assays as well as a positive control with 20 mM eserine (a potent AChE inhibitor). A second negative control (C DMSO) was used in the organic extracts assays including ultra-pure water with 0.1% DMSO, to account for the possible effect of the solvent. Blank conditions were also included containing 50 μL of phosphate buffer instead of pure AChE.AChE activity was determined as previously described , using ain vivo anti-settlement tests and in vitro AChE activity were analysed using a one-way analysis of variance (ANOVA) followed by a multi-comparisons Dunnett test (p < 0.05). When ANOVA presuppositions were not met in the data, a non-parametric test, the Kruskal-Wallis on ranks, followed by the Dunn’s test, were used. ChEs activity kinetics for the different substrates was obtained according to Michaelis-Menten model [Km and Vm, for each substrate, were obtained through a hyperbolic regression analysis using the software STATISTICA 12. Km (μM) is the concentration of substrate at which half the active sites are filled, providing a measure of the substrate concentration required for significant catalysis to occur. Vm (nmol min−1 mg protein−1) represents the number of substrate molecules converted into product by the enzyme at full substrate saturation [.All data concerning ChEs activity characterization, en model . A non-lturation .In the case of data from ChEs’ characterization using inhibitors, Dunnett’s test was used to compare each treatment with the control and to determine the values of the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) . The sofM. galloprovincialis foot, P. pollicipes capitulum soft tissues and P. perforatus whole soft tissues have predominantly hydrolytic characteristic of vertebrates AChE and PrChE, and are inhibited by specific inhibitors of vertebrates AChE. This suggests that ChE isoenzymes of these three species have characteristics of both vertebrates AChE and PrChE. Considering this and the role of AChE in larval settlement, in vitro AChE activity inhibition was used along with an in vivo anti-settlement test in a screening approach for promising AF agents using cyanobacteria strains as a source of bioactive secondary metabolites. Results from this study contribute to a better understanding of the nature and identity of these biofouling invertebrate species ChEs that will be helpful to further studies using ChEs activity on these species. Also, the optimization of the in vitro ChEs assays will be helpful for further AF and fouling-inducing screening approaches, and for the understanding of modes of action that are responsible for the AF activity induced by different compounds, including cyanobacteria-derived products. The combination of in vivo and in vitro responses proved to be a good strategy in the search for eco-friendly AF compounds.This study allowed to establish that ChEs present on


There is evidence that consumption of foods containing dietary fiber decreases the risk of colorectal cancer (CRC). Whole grains contain dietary fiber, as well as a range of micronutrients and bioactive compounds, but the association between the consumption of whole grains and the risk of CRC remains less studied. The aim of the present study was to investigate the association between whole-grain bread consumption and CRC incidence among Norwegian women, using data from a prospective cohort study (the Norwegian Women and Cancer Study). Dietary intake was estimated from the food-frequency questionnaires of 78,254 women in the cohort (median age: 55 years), and these women were then followed up for CRC incidence. During the 9 years of median follow-up, 795 women were diagnosed with CRC . Associations between whole-grain bread consumption and the risk of CRC  were investigated using Cox proportional hazards regression models. When compared to the low consumption group, the hazard ratio for CRC was 0.89 : 0.72–1.09) for the high consumption group and 0.86 (95% CI: 0.72–1.02) for the medium consumption group in a multivariable model. Overall, no association between whole-grain bread consumption and CRC was found. Colorectal cancer (CRC) is the second most common cancer in women and the third most common in men worldwide . There iDiet is believed to play a role in the development of CRC . AccordiBread is a staple food in Norway, accounting for 21% of energy intake ,23, and n = 90,592) were eligible for inclusion in the present analysis. The reason for including enrollment questionnaire, first and second follow-up questionnaires, is that these questionnaires had the same questions about bread consumption (the bread questions in the NOWAC questionnaire have varied over time). For each participant, information from only one questionnaire was used in the analyses.The Norwegian Women and Cancer (NOWAC) Study is a nationwide prospective cohort study consisting of approximately 172,000 women. The study has been enrolling women for 15 years, with the first wave of enrollment taking place in 1991. All study participants complete a mailed questionnaire at enrollment, and follow-up questionnaires are sent approximately every 6th year after enrollment. NOWAC participants who answered an enrollment questionnaire, first follow-up questionnaire, or second follow-up questionnaire between 2002 and 2006 (n = 4887), as well as those who had follow-up <1 year (n = 649). We further excluded those with International Classification of Diseases for Oncology (ICD-O) tumor behavior code 6  (n = 1) or ICD-O code 9  (n = 16) and those with a daily energy intake <2.5 MJ (n = 1061) or >15.0 MJ (n = 153). Women with missing information on any of the following variables were also excluded: all three questions on bread consumption  (n = 1124), self-reported weight (n = 2582), self-reported height (n = 279), smoking status (n = 1380) and smoking history (n = 198). We further excluded those with body mass index (BMI) <13.0 kg·m−2 (n = 3) or >60.0 kg·m−2 (n = 4). Finally, one woman who returned the questionnaire in 2007 was excluded. Thus a total of 78,254 women, which included three questionnaire subcohorts  were included in the present analyses.We excluded women diagnosed with any invasive cancer prior to the start of follow-up . Information on education was taken from the enrollment questionnaire for the entire study sample. All other exposure data used in the analysis were taken from the questionnaire the participants completed during the study period, BMI was calculated from self-reported weight and height, both of which have been validated . InformaAll of the considered questionnaires included a semi-quantitative food frequency questionnaire (FFQ), which covered food items commonly eaten in Norway, but not the total diet. A validation study found that the FFQ’s ability to rank NOWAC participants was good for foods eaten frequently . The SpeParticipants reported their average consumption of foods during the last year in the FFQ by ticking fixed frequencies. Knowledge about Norwegian food habits was the basis when the frequency options, including the number of frequency options, were chosen. Participants reported the portion size of a food consumed in natural units (for example banana), household units (for example tablespoons) or deciliters. We multiplied the mean of the frequency option by the portion size to calculate the consumption of each food (in g). For the uppermost category of a food, the lowest value in the frequency option was used . The Norwegian Weight and Measurement Table  was used−1, 5–7 slices·week−1, 2–3 slices·day−1, 4–5 slices·day−1 and ≥6 slices·day−1). “Seldom” covers more than zero consumption, but <1 slice week−1. Study participants who reported a “zero/seldom” consumption of bread, were categorized as consuming no bread in our analyses. One slice of whole-grain bread was estimated to weigh 40 g and one slice of partly refined bread to weigh 30 g [Participants reported their consumption of bread in slices of different bread types . Moreover, six different frequency options were included  for vital status , and to the Cancer Registry of Norway for cancer incidence.CRC cases were classified according to the International Classification of Diseases, Seventh Revision (ICD-7) by the Cancer Registry of Norway and further classified according to anatomic subsite: CRC (ICD-7 code 153.0–154.0); colon cancer (ICD-7 code 153.0–153.9); proximal colon cancer ; distal colon cancer (ICD-7 code 153.2–153.3: descending and sigmoid colon); and rectal cancer (ICD-7 code 154.0). Cancers of the colon with codes 153.4 (recto-sigmoid junction), 153.6 (appendix), and 153.9 (unspecified location) were only included in the analyses for total colon cancer. Anal canal tumors (ICD-7 code 154.1) were not included in the analyses. Moreover, only cases with ICD-O tumor behavior code 3  were classified as CRC.Descriptive characteristics of the participants at the start of follow-up are presented as median values with 5th and 95th percentiles or as percentages. We used Kruskal-Wallis equality of populations rank test and Spearman’s rank correlation coefficient for examining differences in the distribution of baseline characteristics between the whole-grain bread consumption groups. The associations between whole-grain bread consumption and incidence of total CRC, total colon, proximal colon, distal colon, and rectal cancer were investigated in Cox proportional hazards regression models with age as the time scale. All models were stratified by the questionnaire subcohorts. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated.Start of follow-up was set as the age of the woman at the time the questionnaire was returned. Person-years were calculated from the start of follow-up to age at diagnosis of any cancer (except non-melanoma skin cancer), emigration, death, or the end of follow up (31 December 2013), whichever occurred first.A Cox proportional hazards regression model (full model) was established with whole-grain bread consumption as the exposure and adjusted for predefined potential confounders for CRC selected according to a literature review . Two additional models are presented: an age-adjusted model  and an energy-adjusted full model (adjusting for everything in the full model plus energy).n = 72,175) did not change the HRs for total CRC and the colorectal subsites in the age-adjusted and the full model, respectively. Due to this, the missing information on physical activity level (n = 6079), and the moderate power in the study, physical activity level was not included in the full model.Adjusting for physical activity level (−1), low (14/34 g·day−1), medium (100 g·day−1) and high (180/240 g·day−1). A zero/seldom consumption group was created since whole-grain bread consumption is common in Norway [Whole-grain bread consumption was modelled as a categorical variable; the groups were classified as zero/seldom (0 g·dayn Norway  and ther−1); and potato consumption . Furthermore, the following variables were modelled continuously: BMI (kg·m−2); red meat consumption (g·day−1); processed meat consumption (g·day−1); fiber from foods other than whole-grain bread (g·day−1); calcium from food (mg·day−1); alcohol consumption (g·day−1); energy intake (kJ·day−1); height (cm); folate from foods other than whole-grain bread (µg·day−1); fruit consumption (g·day−1); and vegetable consumption (g·day−1).The following variables were modelled categorically: hormone replacement therapy ; smoking ; physical activity level ; duration of education ; area of residence ; an indicator variable for period of birth ; partly refined bread consumption , women with <7 years of education (n = 289) and >29 years of education (n = 26)) affected the associations. Finally, we excluded all CRC cases with follow-up ≤1 year to eliminate the influence of pre-existing disease on the estimates of the associations for total CRC and the colorectal subsites.Additional analyses were performed to examine the robustness of the associations for total CRC and the colorectal subsites. First, we ran analyses using the full model less one variable  to determine if the removal of this variable affected the results. We also performed separate analyses using the full model plus one variable  to determine if the addition of this variable affected the results. Then we examined the effect of using fruit, vegetables, and potatoes instead of fiber from foods other than whole-grain bread in the full model. We also investigated whether adding education  were inAnalyses were carried out using STATA  version 14.0.During the 717,482 person-years of observation, 795, 577 (73%) and 218 (27%) incident cases of CRC  and colon  and rectal cancer, respectively, were reported in the study sample. Among the colon cancers, 316 (55%) were proximal, and 193 (33%) were distal; the rest were cancer of the recto-sigmoid junction/appendix/unspecified location. Median follow-up was 9 (range 1–11) years, and the median age at CRC diagnosis was 65 (range 48–86) years.Those eating more whole-grain bread reported less smoking and a higher physical activity level . The med−1. The consumption of crisp bread and breakfast cereals, two other foods which may contain whole grains and therefore may confound the results of our analyses, were low: median consumption was 4 (range 0–74) g·day−1 for crisp bread and zero/seldom (range 0–72) g·day−1 for breakfast cereals in all the whole-grain bread consumption groups.The range in whole-grain bread consumption was 0–240 g·dayThe median consumption of whole-grain bread was the same in the total sample and among CRC cases . Also, tWhen investigating the associations between whole-grain bread consumption and incidence of total CRC and cancer at the colorectal subsites in the three different models , overall no association between whole-grain bread consumption and CRC was found . When con = 74,165) did not substantially alter the HRs for total CRC and the colorectal subsites when added to the age-adjusted and the full model, respectively. When comparing the high, the medium and the zero/seldom consumption groups with the low consumption group after excluding all CRC cases with follow-up time ≤1 year in the full model, the HRs for total CRC were 0.89 (95% CI: 0.72–1.10), 0.86 (95% CI: 0.72–1.03) and 0.88 (95% CI: 0.68–1.14), respectively. Furthermore, when performing the same comparisons for proximal colon cancer in the full model, the HRs were 0.82 (95% CI: 0.59–1.15), 0.76 (95% CI: 0.58–1.01) and 0.80 (95% CI: 0.54–1.19), respectively. Since excluding all CRC cases with follow-up time ≤1 year hardly changed the HRs for total CRC and the colorectal subsites in the three models, these CRC cases were included in the analyses. No interaction effects were found between whole-grain bread and red meat or processed meat in the full model for total CRC or any of the colorectal subsites.Running analyses using the full model less one variable , using the full model plus one variable (height/area of residence/period of birth/partly refined bread/folate from foods other than whole-grain bread), or using fruit, vegetables and potato consumption instead of fiber from foods other than whole-grain bread in the full model did not alter the HRs substantially for total CRC or any of the colorectal subsites. Education  adjusted models and the two multivariable models were mostly minor. Nevertheless, residual confounding cannot be ruled out, for example due to measurement errors in the investigated confounders. A specific concern may be raised with regard to physical activity level. This was the only covariate with a noticeable fraction of missing values; furthermore it is well-known that it is challenging to accurately assess physical activity by questionnaires . We alsoA previous meta-analysis indicated that there may be an inverse association between the consumption of whole grains and risk of CRC . Howeverversus distal) [versus first quartile) when examining the association between the consumption of whole-grain products  and proximal colon cancer among women, and adjusted IRR of 0.98  and 0.88  for distal colon cancer [It has been discussed whether the pathology and risk factors of colon cancer differ by colon subsites  ,44,45,46n cancer . The conn cancer . Our sign cancer .Different results have been observed when examining whole grain consumption in relation to rectal cancer ,15,18. OIn conclusion, overall, no association between whole-grain bread consumption and CRC was found among the women in the NOWAC Study. Our study does not support the findings of a previous meta-analysis, which indicated that there may be an inverse association between the consumption of whole-grains and the risk of CRC . However


Background. This study investigated an association between systemic absolute neutrophil count (ANC) and albuminuria in elderly Chinese people. Methods. A cross-sectional study was conducted on 2265 participants attending a routine medical examination in Minhang District as part of a Platform of Chronic Disease program. Their drug history, waist circumference, height, blood pressure, fasting blood glucose, ANC, and urine albumin levels were recorded. This study conformed to the requirements of the STROBE statement. Results. Of the 2265 subjects, 1254 (55.4%) were diabetic and 641 (28.3%) had albuminuria. The mean ANC of patients with diabetes comorbid with macroalbuminuria was significantly higher than that of both the nondiabetic patients and patients with diabetes with lower levels of albuminuria; the latter 2 groups had statistically similar ANC. ANC significantly and positively correlated with levels of urine albumin. Based on multivariate analysis, with each 109/L increase in ANC, the increase in rates of macroalbuminuria was significant but not in rates of albuminuria positivity. Based on areas under the receiver operating characteristic curve, ANC was the strongest factor predicting macroalbuminuria. Conclusions. Elevated ANC was associated with macroalbuminuria in diabetes, indicating that neutrophil-mediated inflammation may be involved in the exacerbation of albuminuria. Albuminuria  is defined as an abnormally high amount of albumin in the urine and is a strong sign of early renal damage –4. In tyInflammation and endothelial dysfunction may be key mechanisms in the development of albuminuria –13. HoweAbsolute neutrophil count (ANC) is also a sensitive marker of inflammation, and the test for it is simple and inexpensive. Recently, clinical studies have shown a link between neutrophils and the development of diabetic retinopathy –18. AlthWe hypothesized an association between ANC and UA levels. Like the world's human population in general, China's is aging and we chose to study the elderly. Here, the UA creatinine ratio (UACR) was used as a measure of albumin excretion. Since overnight UACR correlates well with UA excretion , this stThis community-based, cross-sectional study focused on the clinical risk factors of albuminuria. We performed the study with outpatients of our hospital in Shanghai, from March to August of 2010. All community-dwelling participants aged ≥60 years, including those residing in private residences and personal care homes, were recruited to a Platform of Chronic Disease (PCD) prevention program for a routine medical checkup. Participants who could not come to the prevention center were interviewed in their place of residence by trained personnel. Those who refused to join the study were not included in this analysis. The participants provided informed consent.All questionnaires and procedures were approved by the Institutional Review Board in our hospital for research with human subjects. The identification of diabetes was based on the diagnostic criteria recommended by the American Diabetes Association in 1997. Potential subjects included those with a history of past diabetes or who had newly received a diagnosis by oral glucose tolerance test [9/L or serum creatinine (SCr) ≥141 μmol/L. Ultimately, 3003 subjects were selected for this study. Further 738 subjects had missing UA data, because they did not collect morning urine samples.In total, 3053 subjects aged between 60 and 91 years (median age: 70 y) were considered for the study. The following subjects were excluded: 28 subjects presenting with painful urination, fever, cough, or skin damage; 5 subjects who were incontinent and not capable of caring for themselves; and 12 subjects with white blood cell (WBC) counts ≥10 × 10Therefore, 2265 subjects were included in the final analysis.Blood samples were collected at 8:15 a.m. after a 12-hour fast. The subjects underwent a glucose tolerance test with a 75 g oral glucose overload. Only water was permitted during the test period and no physical exercise was taken. The blood samples were collected by EDTA tube before and at 2 h after the tolerance test. After centrifugation, samples were stored at −80°C for subsequent analysis. Plasma biochemical parameters were measured in duplicate by standard enzymatic methods. Glucose, cholesterol, triglycerides, SCr, and alanine aminotransferase (ALT) levels were measured using a Dimension Vista analyzer (Siemens AG). WBC, ANC, and ANC/WBC ratio values were measured using an automated 5-category hematology analyzer (Japanese Sysmex XS1000i).+) at an albumin-to-creatinine ratio (ACR) >0 mg/g; otherwise the results were accepted as negative . Microalbuminuria, or a moderate increase in UA, was identified as an ACR from 30 to 299 mg/g. Macroalbuminuria was defined as ACR ≥300 mg/g.Participants were asked to collect a random morning spot urine sample in a clean, lidded container. All urine samples were assessed centrally in a single reference laboratory at Fifth Hospital, Shanghai, China. Samples were centrifuged at 1500 ×g for 10 min and then stored at −80°C prior to analysis. Further analysis was performed with a Beckman Coulter Array 360 chemistry analyzer. Test results were considered positive for albuminuria . Hypertension was defined as systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg.2 (kg/m2).The following measurements were recorded for all subjects: height, weight, waist circumference , and hip measurement  by experienced nurses in the PCD program. Body mass index (BMI) was calculated as weight/heightU tests and chi-squared tests were used for analysis of continuous variables and categorical variables, respectively. The ANC levels of the following subgroups of patients were calculated and compared by analysis of variance: nondiabetic; diabetic without albuminuria; diabetic with low-level albuminuria (0 < UA < 30 mg/g); diabetic with microalbuminuria (UA 30–299 mg/g); and diabetic with macroalbuminuria (UA ≥ 300 mg/g).Mann-Whitney P ≤ 0.05.The least significant difference was used for intergroup comparisons. All subjects were categorized into 5 groups according to the quintile of ANC levels (I–V). Multivariate logistic regression analysis was used to investigate an independent association between ANC and UA. Differences were considered significant at Receiver operating characteristic (ROC) curves were plotted for factors predicting macroalbuminuria. Areas under the ROC (AUC) curve ≥0.5 were considered statistically significant .+) were older, with higher BMI and waist-to-hip ratio, SBP, DBP, SCr, ANC, WBC, and ANC/WBC compared with the 1624 persons testing negative for UACR (UA−).The data analysis included 2265 participants aged between 60 and 91 years (median: 70 y) with a male-to-female ratio of 56.8 to 43.1 . CompareP < 0.0001). Patients with macroalbuminuria had significantly higher ANC and ANC/WBC counts than did the patients in the other 4 groups (P < 0.001). There was no statistical difference between the other 4 groups.As described in Subjects and Methods, all subjects were classified as follows: nondiabetic; diabetic without albuminuria; diabetic with low-level albuminuria; diabetic with microalbuminuria; or diabetic with macroalbuminuria . ANC levP < 0.0001). Compared with quintile I of ANC, quintile V of ANC had the highest OR value  indicated that ANC levels significantly correlated with risk of macroalbuminuria  followed closely by FBG (AUC = 0.5876) and SBP . Thus ANC and FBG were the most meaningful diagnostic indices for macroalbuminuria.To investigate interactions among ANC, FBG, SBP, and SCr, they were grouped in separate analyses. ANC × FBG × SBP  and ANC × FBG × SBP × SCr (Model 3) indicated 1.67% and 3.12% increase, respectively, in the predictive value for macroalbuminuria, compared with Model 1 (ANC × FBG), but these increases were not statistically significant , interleukin- (IL-) 1β, IL-6, and C-reactive protein, as well as excessive production of superoxide and reactive oxygen species [Although establishing a causal mechanism for the association between ANC and albuminuria was beyond the scope of this study, the underlying link may concern vascular damage. In clinical and laboratory studies, elevated neutrophil counts were found associated with increased levels of tumor necrosis factor- (TNF-)  species –23. Base species , 25. Und species . These r species .In the present study, compared with the other 4 quintile ANC groups, the highest level of the ANC group had the highest OR for the risk of macroalbuminuria and not quintiles II or III. This shows that the higher the ANC level, the more likely the presence of macroalbuminuria, reflecting an overtly inflammatory status in patients with macroalbuminuria , 27. TheThere are several limitations to our study. First, the study design was cross-sectional and therefore we were not able to confirm a causal link between ANC and macroalbuminuria. Second, the inclusion of participants attending a routine health checkup in one community represents a potential source of selection bias, which should be considered when interpreting the results. Third, information regarding other traditional risk factors for albuminuria, such as heavy exercise , could nThis clinical study revealed an association between elevated systemic neutrophil counts and macroalbuminuria. High levels of UA may indicate an overt inflammatory state. The potential effect of neutrophil-mediated inflammation in the progression of macroalbuminuria warrants further research.